Method for treating oil-containing wastewater

Abstract

The present invention provides a method for treating oil-containing wastewater, the method comprising causing dead cells of microorganisms that degrade oil or a component derived from the dead cells to act on oil-containing wastewater.

Description

Method for treating wastewater containing oil and fat

[0001] The present invention relates to a method for treating oil-containing wastewater, and more particularly to a method for treating oil-containing wastewater using killed cells of oil-decomposing microorganisms.

[0002] Wastewater discharged from food processing plants, commercial kitchens, and the like contains large amounts of oil and fat. Pressure flotation is commonly used as a method for treating such oil-containing wastewater. Pressure flotation generates fine bubbles in wastewater, and the buoyancy of the bubbles is used to float solids in the wastewater, separating the liquid from the solids in the wastewater. However, wastewater treatment using pressure flotation has many challenges. For example, a large amount of electricity is required to generate the bubbles and operate the pressurizing device, and the separated oil mud must be separately disposed of as industrial waste, resulting in high running costs. In light of this, methods using oil-degrading microorganisms have been proposed as an alternative method for treating oil-containing wastewater (Patent Documents 1 and 2).

[0003] The treatment of oil-containing wastewater using oil-degrading microorganisms has many advantages over the pressurized flotation method, such as reducing the amount of waste sludge. However, there are also challenges with the treatment of oil-containing wastewater using microorganisms.

[0004] For example, many oil-degrading microorganisms are aerobic, so aeration is required to keep these microorganisms alive. Therefore, energy costs remain a challenge in wastewater treatment using microorganisms. Another challenge is the need to add large amounts of oil-degrading microorganisms to combat competition with indigenous bacteria in wastewater. Furthermore, the need to maintain the microorganisms in a viable state places significant constraints on the storage, transportation, formulation, and wastewater treatment conditions of the microorganisms. Furthermore, after being added to wastewater, oil-degrading microorganisms begin to metabolize oils and fats through steps of survival and proliferation. Therefore, there is room for improvement in terms of immediate effectiveness.

[0005] JP 2003-116526 A JP 2011-160713 A

[0006] In light of this background, an object of the present invention is to provide a novel means capable of solving the problems associated with the treatment of oil-containing wastewater using microorganisms.

[0007] As a result of intensive research into the above-mentioned problems, the present inventors have found that when dead cells of oil-degrading microorganisms or the components derived from the dead cells themselves are applied to oil-containing wastewater, (1) the concentration of normal hexane in the wastewater can be quickly reduced, (2) the amount of suspended solids in the wastewater can be reduced, (3) the biochemical oxygen demand (BOD) in the wastewater can be reduced, (4) oil-degradation in the wastewater progresses even in the presence of other microorganisms, and (5) oil-degradation and a reduction in the amount of waste sludge can be achieved without the need for air supply. In addition, the inventors have discovered that (6) the change in BOD value does not decrease monotonically but increases once and then decreases, making it possible to prepare a treated solution of oil-containing wastewater with an increased BOD value compared to before treatment, (7) oil-degrading microorganisms can be efficiently grown using oil-containing wastewater that was originally the target of treatment (e.g., wastewater containing residual oil (dark oil) discharged from an oil refinery), and (8) oil-containing wastewater can also be efficiently treated using killed cells of oil-degrading microorganisms grown in oil-containing wastewater. Based on these findings, the inventors have conducted further research and have completed the present invention. Specifically, the present invention is as follows.

[0008] [1] A method for treating oil- and fat-containing wastewater, comprising treating the oil- and fat-containing wastewater with killed cells of oil- and fat-degrading microorganisms or components derived from the killed cells. [2] The method according to [1], wherein the oil- and fat-degrading microorganisms are killed in the stationary phase. [3] The method according to [1] or [2], wherein the method comprises killing two types of oil- and fat-degrading microorganisms. [4] The method according to [3], wherein the two types of oil- and fat-degrading microorganisms are a bacterium and a fungus. [5] The method according to [4], wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica. [6] The method according to any one of [1] to [5], wherein the oil- and fat-degrading wastewater is treated with killed cells of oil- and fat-degrading microorganisms or components derived from the killed cells under anaerobic conditions. [7] An agent for treating oil- and fat-containing wastewater, comprising killed cells of oil- and fat-degrading microorganisms or components derived from the killed cells. [8] The agent according to [7], wherein the oil- and fat-degrading microorganisms are killed in the stationary phase. [9] The agent according to [7] or [8], which contains killed cells of two types of oil- and fat-degrading microorganisms.

[10] The agent according to [9], wherein the two types of oil- and fat-degrading microorganisms are a bacterium and a fungus.

[11] The agent according to

[10] , wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

[12] The agent according to any of [7] to

[11] , which is added to oil- and fat-containing wastewater under anaerobic conditions.

[13] The agent according to [7] to

[12] , which is used to improve the efficiency of methane fermentation of oil- and fat-containing wastewater.

[14] A method for improving the efficiency of methane fermentation of oil- and fat-containing wastewater, which comprises adding the agent according to any of [7] to

[12] to oil- and fat-containing wastewater to decompose the oils and fats contained in the oil- and fat-containing wastewater.

[15] A method for producing an agent for treating oil- and fat-containing wastewater, which comprises culturing oil- and fat-degrading microorganisms in oil- and fat-containing wastewater, and killing the oil- and fat-degrading microorganisms.

[16] The method according to

[15] , wherein the oils and fats contained in the oil-containing wastewater are at least one selected from the group consisting of residual oil, waste oil from processed oils and fats, waste oil from food manufacturing, waste oil from the food service industry, and household waste oil.

[0009] The present invention is characterized by killing the oil-degrading microorganisms. Killing the microorganisms allows them to be easily formulated into a powder, eliminating constraints on processes such as concentration, storage, and transportation. Furthermore, while using live microorganisms can result in the disadvantage of increased waste due to the proliferation of the added microorganisms, the present invention eliminates this disadvantage because the microorganisms are killed. Furthermore, the present invention does not involve competition between the oil-degrading microorganisms and resident bacteria. Therefore, the microbial network formed by resident bacteria is not affected. Furthermore, the present invention does not require the provision of air supply or other conditions that allow the oil-degrading microorganisms to function. Additionally, the present invention allows for the rapid achievement of the desired effect after addition to wastewater. Therefore, the present invention enables the treatment (purification) of oil-containing wastewater in a low-cost, simple, rapid, and environmentally friendly manner. Furthermore, the present invention allows the production of an agent for treating oil-containing wastewater using oil-containing wastewater. Therefore, companies that discharge large amounts of oil-containing wastewater, such as oil refineries and food manufacturers, can use the present invention not only to treat oil-containing wastewater but also to produce an agent for treating oil-containing wastewater.

[0010] Figure 1 shows the results of confirming the fat-degrading ability of samples obtained by subjecting fat-degrading microorganisms to various killing treatments and the effectiveness of the killing treatments by spreading the samples on OLIA medium or SCDA agar medium. Figure 2 shows the change over time (0 to 72 hours) in the appearance of the culture solution when killed cells (72 hours of stationary phase) prepared by autoclaving wastewater containing starch, which is difficult to treat with microorganisms, are added. Figure 3 shows the change over time (0 to 48 hours) in the appearance of the culture solution when killed cells (3 days of stationary phase) prepared by heating at 70°C for 2 hours are added to wastewater containing high concentrations of fats, oils, and organic matter.

[0011] The present invention will be described in detail below.

[0012] 1. Method for treating oil- and fat-containing wastewater The present invention provides a method for treating oil- and fat-containing wastewater (hereinafter sometimes referred to as the "treatment method of the present invention"), which comprises treating oil- and fat-containing wastewater with killed cells of an oil- and fat-degrading microorganism or a component derived from the killed cells.

[0013] The oil-containing wastewater in the treatment method of the present invention is not particularly limited as long as it contains oil and fat, and may be any wastewater. Examples of the oil and fat contained in such wastewater include, but are not limited to, residual oil, processed oil and fat waste oil, food manufacturing waste oil, food service industry waste oil, and household waste oil.

[0014] Residual oil refers to oil primarily composed of free fatty acids (also referred to as "dark oil") that is produced after the deacidification step in the oil and fat refining process at oil refineries and the like. Residual oil can be used for the production of biodiesel fuel, etc. In the treatment method of the present invention, when the oil and fat contained in the oil-containing wastewater is residual oil (dark oil), the wastewater to be treated is the small amount of dark oil remaining after the majority of the residual oil is recovered from the liquid composition containing the residual oil. Processed oil and fat waste oil refers to waste oil produced in the process of producing processed oils and fats such as margarine and shortening. Food production waste oil refers to waste oil produced in the process of producing foods such as confectionery, instant noodles, and fish paste products. Food service industry waste oil refers to waste oil produced in the process of producing food and beverages at restaurants and fast food restaurants. Household waste oil refers to waste oil produced in the process of producing food and beverages in ordinary households.

[0015] In the treatment method of the present invention, the oils and fats are not particularly limited as long as they can be decomposed by oil-degrading microorganisms, and may be either animal or vegetable oils and fats. Examples of oils and fats include, but are not limited to, rapeseed oil (canola oil), cottonseed oil, palm oil, coconut oil, sunflower oil, soybean oil, rice bran oil, palm oil, coconut oil, olive oil, beef tallow, lard, chicken fat, whale oil, and fish oil.

[0016] In the treatment method of the present invention, treatment of oil-containing wastewater means achieving at least one of the following in the oil-containing wastewater: (1) a reduction in the amount of oil and fat, (2) a reduction in the amount of suspended solids (SS), and (3) a reduction in biochemical oxygen demand (BOD). In a preferred embodiment, the treatment method of the present invention can achieve all of a reduction in the amount of oil and fat, a reduction in the amount of SS, and a reduction in BOD in the wastewater.

[0017] In the treatment method of the present invention, the oil-degrading microorganism is not particularly limited as long as it is a microorganism that produces a substance that directly or indirectly decomposes oils and fats. In one aspect, the oil-degrading microorganism may be a bacterium or fungus capable of decomposing oils and fats.

[0018] Bacteria capable of decomposing fats and oils include bacteria of the genus Bacillus, bacteria of the genus Corynebacterium, bacteria of the genus Rhodococcus, bacteria of the genus Burkholderia, bacteria of the genus Acinetobacter, bacteria of the genus Pseudomonas, bacteria of the genus Alcaligenes, bacteria of the genus Rhodobacter, bacteria of the genus Ralstonia, and bacteria of the genus Acidovorax. Examples of suitable bacteria include, but are not limited to, bacteria of the genus Serratia, bacteria of the genus Flavobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Enterococcus, bacteria of the genus Sphingomonas, bacteria of the genus Staphylococcus, bacteria of the genus Rhizobium, bacteria of the genus Tetrasphaera, bacteria of the genus Stenotrophomonas, etc. In one embodiment, bacteria of the genus Burkholderia are preferred, and Burkholderia arboris is preferred.

[0019] Examples of fungi capable of decomposing fats and oils include, but are not limited to, fungi of the genus Yarrowia, Cryptococcus, Trichosporon, Hansenula, Candida, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Penicillium, Rhizopus, Fusarium, and Humicola. In one aspect, fungi of the genus Yarrowia are preferred, and Yarrowia lipolytica is preferred.

[0020] In the treatment method of the present invention, the method for killing fat-degrading microorganisms is not particularly limited as long as the desired effect of the present invention can be obtained, and any method known per se can be used. Examples of methods for killing fat-degrading microorganisms include heat treatment, electromagnetic wave treatment, chemical treatment, centrifugation, and drying treatment. Heat treatment involves heating microorganisms at high temperatures to denature enzymes and structural proteins in the microorganisms, thereby killing the microorganisms. Examples of heat treatment include, but are not limited to, steam sterilization.

[0021] In the treatment method of the present invention, the treatment temperature and treatment time used when killing fat / oil-decomposing microorganisms by heat treatment may vary depending on the type of microorganism, etc., but are not particularly limited as long as the temperature and time are capable of killing the microorganisms. The treatment temperature may be, for example, 40 to 200°C (preferably 50 to 121°C, 60 to 110°C, 60 to 105°C, 60 to 100°C, 60 to 95°C, 60 to 90°C, 60 to 85°C, 60 to 80°C, etc.), and the treatment time may be, for example, 15 minutes to 10 days (preferably 30 minutes to 9 days, 30 minutes to 8 days, 30 minutes to 7 days, 30 minutes to 6 days, 30 minutes to 5 days, 30 minutes to 4 days, more preferably 30 minutes to 3 days, 60 minutes to 3 days, 60 minutes to 2 days, 60 minutes to 1 day, 60 minutes to 12 hours, 60 minutes to 6 hours, 60 minutes to 5 hours, 60 minutes to 4 hours, 60 minutes to 3 hours, etc.), but is not limited thereto. In a preferred embodiment of the present invention, the treatment temperature and treatment time used when killing fat-decomposing microorganisms by heat treatment are preferably such that the microorganisms are killed but the activity of proteins contained in the microorganisms that are directly or indirectly involved in fat-decomposition, such as lipase, is not completely inactivated. Examples of the treatment temperature and time include, but are not limited to, a treatment temperature of 40 to 200°C (preferably 50 to 121°C, 60 to 110°C, 60 to 105°C, 60 to 100°C, 60 to 95°C, 60 to 90°C, 60 to 85°C, 60 to 80°C, etc.), and a treatment time of 15 minutes to 10 days (preferably 30 minutes to 9 days, 30 minutes to 8 days, 30 minutes to 7 days, 30 minutes to 6 days, 30 minutes to 5 days, 30 minutes to 4 days, more preferably 30 minutes to 3 days, 60 minutes to 3 days, 60 minutes to 2 days, 60 minutes to 1 day, 60 minutes to 12 hours, 60 minutes to 6 hours, 60 minutes to 5 hours, 60 minutes to 4 hours, 60 minutes to 3 hours, etc.).

[0022] In the treatment method of the present invention, when killing fat-degrading microorganisms by electromagnetic wave treatment, the conditions, such as the wavelength and irradiation time of the electromagnetic waves used may vary depending on the type of microorganism, but it is preferable to use conditions that kill the microorganisms but do not completely inactivate the activity of proteins contained in the microorganisms that are directly or indirectly involved in fat-degradation, such as lipase. Examples of electromagnetic waves include ultraviolet light, gamma rays, and X-rays, but are not limited to these as long as they are electromagnetic waves that can kill fat-degrading microorganisms. Techniques for killing microorganisms using electromagnetic waves are known per se. For example, ultraviolet light with a wavelength of approximately 254 nm (UVC wavelength band) can be used. This is because ultraviolet light in this wavelength band can effectively damage the DNA and RNA of microorganisms. Furthermore, the irradiation time of ultraviolet light can be, but is not limited to, 15 to 180 minutes (preferably 30 to 150 minutes, more preferably 60 to 120 minutes).

[0023] The conditions used for killing fat-and-oil decomposition microorganisms by centrifugation in the treatment method of the present invention may vary depending on the type of microorganism, etc., but it is preferable to use conditions that kill the microorganisms but do not completely inactivate the activity of proteins contained in the microorganisms that are directly or indirectly involved in fat and oil decomposition, such as lipase. As an example, conditions can be used that physically damage the cells by centrifugation, resulting in the death of the microorganisms.

[0024] The drying conditions used when killing fat-decomposing microorganisms by drying in the treatment method of the present invention may vary depending on the type of microorganism, etc., but it is preferable to use conditions that kill the microorganisms but do not completely inactivate the activity of proteins contained in the microorganisms that are directly or indirectly involved in fat-decomposition, such as lipase. A person skilled in the art can set appropriate temperature, humidity, and drying time depending on the type of fat-decomposing microorganism.

[0025] In a preferred embodiment, it is preferable to kill oil-degrading microorganisms in a stationary phase. When preparing killed oil-degrading microorganisms, first, living oil-degrading microorganisms are grown by culturing. Generally, the growth of oil-degrading microorganisms in a culture system is classified into a "lag phase" immediately after the start of culture, a "logarithmic growth phase" in which microorganisms grow exponentially, and a "stationary phase (also called a stationary phase)" in which microbial growth slows or stops due to nutrient depletion in the medium and / or a lack of space in which microorganisms can grow. In the present invention, it is preferable to kill microorganisms in the stationary phase. The culture conditions for oil-degrading microorganisms can be appropriately determined taking into account the type of microorganism, the concentration of the microorganisms finally obtained, the size of the culture vessel used, etc.

[0026] In one aspect, the treatment method of the present invention may use one type of microorganism or two or more types. When multiple microorganisms are used, the combination may be, for example, any combination of the fat-and-oil-decomposing microorganisms described above, and is not limited thereto. In a preferred aspect, two types of microorganisms may be used. In a more preferred aspect, the two types of microorganisms may be a bacterium and a fungus, with Burkholderia arboris being preferred as the bacterium and Yarrowia lipolytica being preferred as the fungus.

[0027] In one embodiment of the treatment method of the present invention, killed cells of oil- and fat-degrading microorganisms can be added to oil- and fat-containing wastewater under anaerobic conditions. In this specification, the term "anaerobic conditions" refers to a state in which there is no oxygen, a state in which there is essentially no oxygen, and a state in which there is not enough oxygen to allow live aerobic oil- and fat-degrading microorganisms to survive and decompose oils and fats. In other words, the treatment method of the present invention can treat oil- and fat-containing wastewater even in a state in which oil and fat decomposition by microorganisms cannot be achieved without the supply of air.

[0028] The killed cell-derived component in the treatment method of the present invention refers to a composition obtained by extracting the contents of killed cells after preparing killed cells of fat-degrading microorganisms. A method known per se may be used to extract the contents from killed cells. Generally, when microorganisms are killed by heat treatment or the like, the outer shell of the microorganism is destroyed, making it easy to extract the contents of the killed cells. The extracted contents of the killed cells may be used as the killed cell-derived component as is, or the contents may be concentrated by powdering or the like, and this may be used as the killed cell-derived component.

[0029] In one aspect of the treatment method of the present invention, killed cells of fat-degrading microorganisms and components derived from the killed cells may be used in combination. Alternatively, killed cells of fat-degrading microorganisms and / or components derived from the killed cells may be used in combination with live cells of fat-degrading microorganisms. Examples of embodiments using both killed cells and live cells include the following: (1) Living fat-degrading microorganisms are cultured and allowed to grow. (2) A portion of the grown microorganisms is killed to obtain a mixture of live cells and killed cells of fat-degrading microorganisms. (3) The resulting mixture is added to fat-containing wastewater.

[0030] In the treatment method of the present invention, adding killed bacteria and / or components derived from killed bacteria to oil-containing wastewater achieves a reduction in the amount of oil, a reduction in SS, and a reduction in BOD, but as suggested by Test Example 9 below, it is believed that the BOD will increase initially and then decrease. Therefore, if the goal is to reduce the BOD, it is sufficient to add a sufficient amount of killed bacteria and / or components derived from killed bacteria relative to the amount of oil contained in the wastewater and set a treatment time sufficient for the reduction in the BOD value to reach a plateau. The amount of killed bacteria and / or components derived from killed bacteria to be added and the treatment time can be appropriately determined by one skilled in the art, taking into account the amount of oil contained in the wastewater and other treatment conditions.

[0031] 2. Treatment Agent for Oil-Containing Wastewater The present invention also provides an agent for treating oil-containing wastewater (hereinafter sometimes referred to as "the treatment agent of the present invention"), which comprises killed cells of an oil-degrading microorganism or a component derived from the killed cells.

[0032] The fats and oils, fat-decomposing microorganisms, and methods for preparing the killed cells thereof in the treatment agent of the present invention are the same as those explained in the section on the treatment method of the present invention.

[0033] The amount of killed cells of fat-degrading microorganisms to be incorporated into the treatment agent of the present invention is not particularly limited, but is typically 0.01 to 100% by weight, preferably 1 to 100% by weight, 5 to 100% by weight, 10 to 100% by weight, 15 to 100% by weight, 20 to 100% by weight, 25 to 100% by weight, 30 to 100% by weight, 35 to 100% by weight, 40 to 100% by weight, 45 to 100% by weight, 50 to 100% by weight, 55 to 100% by weight, 60 to 100% by weight, 65 to 100% by weight, 70 to 100% by weight, 80 to 100% by weight, 85 to 100% by weight, 90 to 100% by weight, or 95 to 100% by weight, but is not limited thereto. When killed cells of two or more types of microorganisms are incorporated, the total amount of killed cells of all microorganisms should be within the above-mentioned range.

[0034] The treatment agent of the present invention may contain components other than killed cells of fat-degrading microorganisms. Such components are not particularly limited as long as the desired effects of the present invention can be obtained. Examples of such components include additives that are typically incorporated into microbial preparations. Examples of additives include, but are not limited to, preservatives, stabilizers, and colorants.

[0035] The form of the treatment agent of the present invention is not particularly limited as long as the desired effects of the present invention can be obtained. The form of the treatment agent of the present invention may be, for example, but is not limited to, a solid, particulate, powder, semi-solid, liquid, or slurry.

[0036] The treating agent of the present invention may be used alone or in combination with other preparations that can be used in wastewater treatment. Alternatively, the treating agent of the present invention may further contain live cells of fat-and-oil-degrading microorganisms. When live cells are included in the treating agent of the present invention, it can be produced, for example, by the following steps: (1) Cultivating and growing living fat-and-oil-degrading microorganisms. (2) Killing a portion of the grown microorganisms to obtain a mixture of live and killed cells of the fat-and-oil-degrading microorganisms. (3) Adding further additives to the obtained mixture as necessary to obtain the treating agent of the present invention.

[0037] 3. Method for Improving the Efficiency of Methane Fermentation of Oil-Containing Wastewater As shown in the following examples (e.g., Test Example 9), adding killed cells of oil-degrading microorganisms contained in the treatment agent of the present invention to oil-containing wastewater increases the BOD while reducing oil and suspended solids (SS), thereby converting the oil-containing wastewater into treated water suitable for microbial growth. This aspect of the present invention can also be applied to existing waste recycling technologies. One example is the application of oil-containing wastewater to methane fermentation. Generally, methane fermentation of oil-containing wastewater is considered inefficient because oil and fat act as factors that inhibit the microbial fermentation process. However, treating oil-containing wastewater using the present invention reduces the oil and fat content in the wastewater and makes the wastewater suitable for microbial growth, thereby promoting the maintenance and proliferation of microorganisms involved in methane fermentation in the oil-containing wastewater. Furthermore, because the methanogens responsible for methane fermentation are anaerobic, it is difficult to coexist aerobic oil-degrading microorganisms with methanogens. However, since killed bacteria are used in the present invention, efficient decomposition of fats and oils and methane fermentation can be achieved in an anaerobic environment. Therefore, in one aspect, the treatment agent of the present invention can be used to improve the efficiency of methane fermentation of fats and oils-containing wastewater. Alternatively, the present invention provides a method for improving the efficiency of methane fermentation of fats and oils-containing wastewater, comprising the step of adding the treatment agent of the present invention to fats and oils-containing wastewater and decomposing the fats and oils contained in the fats and oils-containing wastewater.

[0038] When the killed cells and / or components derived from the killed cells of the oil-degrading microorganisms contained in the treatment agent of the present invention are added to oil-containing wastewater to increase the BOD value and reduce the oil content and SS, other conditions such as the amount of killed cells and / or components derived from the killed cells to be added and the treatment time can be appropriately determined by a person skilled in the art, taking into consideration the amount of organic matter (oils, proteins, carbohydrates, etc.) in the oil-containing wastewater to be treated, the temperature conditions, etc.

[0039] 4. Method for producing the treating agent of the present invention using oil-containing wastewater The present invention also provides a method for producing the treating agent of the present invention (hereinafter, sometimes referred to as the "production method of the present invention"), which includes the steps of culturing oil-degrading microorganisms in oil-containing wastewater and killing the oil-degrading microorganisms.

[0040] The fats and oils, fats and oil-containing wastewater, fats and oil-decomposing microorganisms, method for killing bacteria, and treatment agent of the present invention in the production method of the present invention are as described above.

[0041] The production method of the present invention is characterized in that fat-degrading microorganisms, which are the active ingredients of the treatment agent of the present invention, are grown in fat-containing wastewater. The culture conditions for growing fat-degrading microorganisms in fat-containing wastewater are not particularly limited as long as the fat-degrading microorganisms can grow, and any conditions may be used. Those skilled in the art can appropriately select appropriate culture conditions for each fat-degrading microorganism. For example, when using Burkholderia arboris and Yarrowia lipolytica as fat-degrading microorganisms, they can be cultured under conditions of 30°C and air supply (0.4 L / min) after adding an appropriate additive such as urea to the wastewater, as described in Test Example 11 below, but are not limited thereto.

[0042] In a preferred embodiment, the grease-degrading microorganisms may be Burkholderia arboris and Yarrowia lipolytica. Also, in a preferred embodiment, the grease-containing wastewater may be residual oil (dark oil)-containing wastewater.

[0043] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0044] Test Example 1: Examination of the proliferation of Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain Using a 5 L fermenter, tap water, urea, an aqueous solution of various metal salts, and canola oil were added, and Burkholderia arboris SL1B1 strain or Yarrowia lipolytica 1A1 strain was inoculated and cultured with stirring at 30°C and air supply (0.4 L / min.). Sampling to count the number of bacteria was performed at the time of inoculation of the microorganisms and 20 hours, 44 hours, and 68 hours after inoculation.

[0045] The number of bacteria in the microorganisms cultured using the fermenter was counted using the following procedure. The cultured samples were thoroughly suspended. Each sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a cone-like rod. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the spread microorganisms were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The average colony count for the two plates was used. The results are shown in Table 1 below.

[0046]

[0047] The bacterial density of Burkholderia arboris and Yarrowia lipolytica reached its maximum at 44 hours after inoculation and remained constant until 68 hours after inoculation, indicating that these microorganisms had reached stationary phase after 44 hours.

[0048] [Test Example 2] Preparation of killed cells of stationary-phase Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain. Burkholderia arboris SL1B1 strain or Yarrowia lipolytica 1A1 strain was cultured in a 5 L fermenter for 71 hours in a culture medium (1.6 L). The resulting culture medium was then heated at 70°C for 30 minutes or 2 hours to kill the microorganisms while maintaining lipase activity. As controls, a condition without heat treatment (untreated) and a condition with autoclaving at 121°C for 15 minutes (autoclaved) were set.

[0049] The lipolytic ability and the presence of general bacteria in each sample were confirmed using the following procedure. 0.1 mL of each sample was taken and dropped onto two plate media, and the bacterial solution was spread on the media using a cone-sized stick. Oil agar medium (OLIA medium) was used to confirm lipolytic ability, and SCDA agar medium was used to measure the number of general bacteria. The plates with the culture spread on them were cultured at 30°C for a maximum of 7 days. Photographs of the oil agar medium (OLIA medium) and SCDA medium on the 7th day are shown in Figure 1.

[0050] When a sample obtained by heat treatment at 70°C for 2 hours was spread on SCDA agar medium and cultured, no colonies were observed after 7 days. Furthermore, when the same sample was spread on OLIA medium and cultured, decomposition of fats and oils was observed. Furthermore, in the case of a sample obtained by heat treatment at 70°C for 30 minutes, only one colony was observed on SCDA agar medium, and fat-decomposing ability was observed in OLIA medium. On the other hand, in the untreated sample, fat-decomposing ability was observed in OLIA medium, and numerous colonies were observed on SCDA agar medium. Finally, in the autoclaved sample, no colonies were observed on SCDA agar medium, and fat-decomposing ability was not observed in OLIA medium. These results demonstrate that heat treatment of a culture solution of fat-decomposing microorganisms at 70°C for 30 minutes to 2 hours can kill the microorganisms while maintaining their fat-decomposing ability.

[0051] Test Example 3: Wastewater treatment (sterilized wastewater) using killed cells prepared from the culture of Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain. Using a 5 L fermenter, 20 mL of killed cells was added to food company wastewater (sterilized; 1.6 L), and the mixture was cultured with stirring at 30°C for approximately 24 hours with air supply (0.4 L / min). Two types of killed cells were used: one that had been heat-treated at 70°C for 2 hours as in Test Example 2, and one that had been autoclaved.

[0052] After 24 hours of cultivation, the normal hexane extractable substance value, BOD value, SS value, and DO value, which are representative indicators of the amount of fat and oil, were measured. These values ​​are shown in Tables 2 to 5 below.

[0053]

[0054]

[0055]

[0056]

[0057] As shown in Tables 2 to 5, when wastewater was treated by adding killed cells of fat-degrading microorganisms, the normal hexane extractables, BOD, and SS values ​​all decreased by 90% or more, indicating that even killed cells of fat-degrading microorganisms have a strong fat-degrading effect. Furthermore, autoclaved killed cells also showed a high fat-degrading effect, similar to killed cells prepared by heat treatment at 70°C for 2 hours. Furthermore, even when the two types of killed cells were treated under air supply conditions (0.4 L / min), the dissolved oxygen (DO) value in the wastewater hardly changed.

[0058] As shown in the above-mentioned Test Example 2, killed cells prepared by autoclaving did not exhibit fat-decomposing effect in an oil agar template, but a high fat-decomposing effect was confirmed in this Test Example. Without wishing to be bound by theory, it is believed that fat-decomposing microorganisms not only contain heat-sensitive enzymes such as lipase, but also have other factors (group of factors) that are heat-resistant and have fat-decomposing ability, and therefore even killed cells of fat-decomposing microorganisms prepared under conditions that inactivate lipase maintain a high fat-decomposing effect.

[0059] [Test Example 4] Wastewater treatment using killed cells prepared from the culture solution of Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain and the supernatant of the killed cells, and wastewater treatment using killed cells under conditions of no air supply (sterilized wastewater) 20 mL of killed cells prepared by heat treatment at 70 ° C. for 2 hours was added to a 5 L fermenter in food company wastewater (sterilized; 1.6 L) and 30 ° C., air supply (0.4 L / min.) or 30 ° C., no air supply, and stirred culture for up to 24 hours. In addition, the supernatant (20 mL) of the killed cell culture solution when centrifuged and collected by heat treatment was added to the wastewater, and stirred culture was carried out for up to 24 hours under the conditions of 30 ° C., air supply (0.4 L / min.).

[0060] The normal hexane extractable substance values, BOD values, SS values, and DO values ​​after 24 hours of culture are shown in Tables 6 to 9, respectively.

[0061]

[0062]

[0063]

[0064]

[0065] As shown in Tables 6 to 9, when killed bacteria and killed bacteria supernatant were added to wastewater, the mass of the normal-hexane extract in the wastewater decreased by more than 90% 6 hours after addition. Furthermore, at 24 hours, the BOD and SS values ​​also decreased by more than 90%. Even without air supply, the normal-hexane extract mass decreased by more than 90% from 6 hours, and the BOD and SS values ​​at 24 hours also decreased by more than 90%. Furthermore, since the DO value decreased slightly without air supply, it was thought that the effect was achieved by consuming a small amount of DO in the wastewater without air supply. On the other hand, under air supply conditions, the killed bacteria hardly decreased the DO value, and a slight decrease was observed for the killed bacteria supernatant.

[0066] [Test Example 5] Comparison of wastewater treatment of cultures of Burkholderia arboris SL1B1 and Yarrowia lipolytica 1A1 strains and killed bacteria prepared from the cultures Using a 5L fermenter, livestock company wastewater (sterilized; 1.6L) was added with 20mL of killed bacteria prepared by heat treatment at 70 ° C. for 2 hours, and then cultured for about 24 hours under the conditions of 30 ° C. and air supply (0.4L / min.). Also, as a control, 16mL of a 50-fold diluted culture containing the Burkholderia arboris SL1B1 and Yarrowia lipolytica 1A1 strains was added to wastewater (sterilized; 1.6L), and under the same conditions in a fermenter at 30 ° C. and air supply (0.4L / min.), and cultured for about 24 hours under the conditions of stirring.

[0067] Table 10 shows the normal hexane extractable substance value (%), BOD value (%), and SS value (%) remaining after 24 hours of culture, with the values ​​before the start being set at 100%.

[0068]

[0069] As shown in Table 10, when killed bacteria were added to wastewater, the residual amounts of normal-hexane extract mass, BOD amount, and SS amount all decreased to 10% or less after 24 hours. On the other hand, when a microbial culture solution containing live bacteria was used, each value also decreased, but the residual SS value was only 39%, which was a lower reduction effect compared to killed bacteria. This suggests that the SS value increased as the live bacteria consumed wastewater nutrients and proliferated, resulting in a weakened effect on the SS value.

[0070] Test Example 6 Preparation of Killed Cells of Burkholderia arboris SL1B1 Strain and Yarrowia lipolytica 1A1 Strain in Stationary Phase and Growth Phase Using a 5 L fermenter, tap water, urea, an aqueous solution of various metal salts, and canola oil were added, and Burkholderia arboris SL1B1 strain or Yarrowia lipolytica 1A1 strain was inoculated and cultured with stirring at 30°C and air supply (0.4 L / min.). Sampling to count the number of bacteria was performed at the time of inoculation of the microorganisms and 24 hours, 72 hours, and 140 hours after inoculation.

[0071] The bacterial count of microorganisms cultured using a fermenter was performed using the following procedure. The cultured samples were thoroughly suspended. Each sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plate media. The bacterial solution was then spread onto the media using a cone-like rod. Oil agar medium was used for colony formation of B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation of Y. lipolytica. The plate media on which the microorganisms were spread were cultured at 30°C for at least 3 days, after which the number of colonies formed on the plate media was counted and used as the viable bacterial count. The colony count was the average of the two plate media. The results are shown in Table 11 below.

[0072]

[0073] At 24 hours after seeding, Burkholderia arboris and Yarrowia lipolytica were still growing, suggesting that they were in the growth phase. At 72 hours, however, the bacterial density of both microorganisms reached its maximum, after which no growth was observed. These results indicated that these microorganisms had entered the stationary phase 72 hours after seeding.

[0074] Killed cells in the growth phase (24 hours) and stationary phase (72 hours and 140 hours) were prepared by autoclaving at 121°C for 15 minutes (autoclaving).

[0075] Test Example 7 Comparison of Wastewater Treatment of Culture Solutions of Burkholderia arboris Strain SL1B1 and Yarrowia lipolytica Strain 1A1 and Killed Bacteria Prepared from the Culture Solutions (2) Using a 5 L fermenter, 20 mL of killed bacteria (stationary phase 72 hours) prepared by autoclaving food company wastewater (sterilized; 1.6 L), which is difficult to treat with microorganisms, was added, and the mixture was stirred for approximately 24 hours at 30°C with or without air supply (0.4 L / min). As a control, a culture solution (50-fold diluted, 16 mL) containing the aforementioned Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain was added to wastewater (sterilized; 1.6 L), and the mixture was stirred in a fermenter under the same conditions at 30°C with air supply (0.4 L / min) for approximately 24 hours.

[0076] The values ​​(%) of normal hexane extractable substances remaining after 24 hours of stirring are shown in Table 12, with the values ​​before the start being set at 100%.

[0077]

[0078] As shown in Table 12, the wastewater used in this study was one in which both microorganisms did not readily grow, and therefore the addition of microbial culture solution did not result in a decrease in the n-hexane extractables value. On the other hand, the addition of killed microbial cells (72 hours of stationary phase) reduced the n-hexane value by approximately 36%. Furthermore, under conditions in which air was not supplied, this effect was even stronger, with the n-hexane value decreasing by 95%. These results demonstrate that killed microbial cells can reduce the n-hexane extractables value even in wastewater in which microorganisms do not readily grow and are therefore difficult to treat with microorganisms, and that this effect is even more pronounced under conditions in which air is not supplied.

[0079] [Test Example 8] Treatment of starch-containing wastewater using killed cells prepared from the culture broth of Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain. Using a 5 L fermenter, 20 mL of killed cells (72 hours of stationary phase) prepared by autoclaving was added to starch-containing wastewater (non-sterilized: 1.6 L), which is known to be difficult to treat with microorganisms, and the mixture was stirred for 24 hours at 30 ° C. without air supply. After 24 hours of stirring, 20 mL of the killed cells was added and stirring was continued. The same procedure was repeated at 48 hours, and the mixture was stirred for a maximum of 72 hours.

[0080] Chemical oxygen demand (CODmn) (manganese method) at the start, 24 hours, 48 ​​hours, and 72 hours is shown in Table 13, and total organic carbon (TOC) is shown in Table 14. Photographs of the culture solution at each point are shown in Figure 2.

[0081]

[0082]

[0083] As shown in Tables 13 and 14, when treatment with killed cells (72 hours in the stationary phase) was continued without air supply, the CODmn and TOC values ​​decreased. It is believed that these decreases are due to the decomposition of starch. Furthermore, as shown in Figure 2, it was observed that the more treatment with killed cells was continued under non-sterile conditions, the more the proliferation of other bacteria was promoted. These results suggest that starch wastewater, which is difficult to treat with microorganisms, can be converted into wastewater in which resident bacteria are activated by pretreatment with killed cells.

[0084] [Test Example 9] High-load food wastewater treatment using killed cells prepared from the culture solution of Burkholderia arboris SL1B1 strain and Yarrowia lipolytica 1A1 strain. Using a 5L fermenter, 20mL of killed cells (stationary phase 3 days) prepared by heat treatment at 70 ° C for 2 hours was added to wastewater (sterilized: 1.6L) containing high concentrations of oils and fats and organic matter, and stirred for 24 hours at 30 ° C without air supply. Next, after 24 hours of stirring, 20mL of the killed cells described above was added, and stirring was continued for 48 hours from the start. The killed cell solution was stored refrigerated and incubated at 70 ° C for about 30 minutes before use and then added to the wastewater.

[0085] The normal hexane extractable substance values ​​before the start and 6 hours, 24 hours, and 48 hours are shown in Table 15, the BOD values ​​before the start and 24 hours and 48 hours are shown in Table 16, and the BOD values ​​before the start and 24 hours and 48 hours are shown in Table 17. Photographs of the culture solution at each point are shown in Figure 3.

[0086]

[0087]

[0088]

[0089] As shown in Tables 15 and 17, when killed bacteria (treated at 70°C for 3 days during the stationary phase) were added to wastewater containing high concentrations of oils and organic matter without air supply, the normal hexane extractables and SS values ​​decreased. On the other hand, the BOD value did not decrease, but rather increased (Table 16). Based on the results of other test examples and this test example, it is believed that the BOD value resulting from the addition of killed bacteria does not decrease monotonically from the start of the addition of killed bacteria (and / or components derived from killed bacteria), but rather increases temporarily after the addition of killed bacteria (and / or components derived from killed bacteria) and then decreases. In this test example, the temporary increase in the BOD value at 48 hours was confirmed, likely due to the relatively high concentrations of oils and organic matter contained in the wastewater being treated. Therefore, if the treatment time of the wastewater with killed bacteria in Test Example 9 were further extended, it is expected that the BOD value would ultimately be significantly reduced from the published value.

[0090] Furthermore, the precipitates (aggregates of fats and oils, polysaccharides, proteins, dead microorganisms, etc.) formed by the aggregation of organic insoluble substances (i.e., SS) present in the wastewater due to the treatment with killed bacteria were completely decomposed at the time point of 48 hours (Figure 3).

[0091] The results of Test Example 9 suggest that treatment of oil-containing wastewater using the present invention temporarily increases the BOD of the oil-containing wastewater. Considering that an increase in BOD indicates an increase in organic matter degradable by aerobic microorganisms and that wastewater containing high concentrations of oils and fats is not an environment suitable for the survival and proliferation of aerobic microorganisms, one aspect of the present invention can be interpreted as a method for converting oil-containing wastewater that is unsuitable for decomposition using microorganisms into a treated oil-containing wastewater solution suitable for decomposition using microorganisms. Based on this aspect of the present invention, the present invention is expected to be used in combination with processes for producing useful substances using microorganisms or processes for decomposing harmful substances. Specifically, for example, the present invention is expected to be applied to convert oil-containing wastewater that is not suitable for bioremediation technology as it is into wastewater suitable for bioremediation technology. Alternatively, although the present invention is characterized by the use of killed bacteria (and / or components derived from killed bacteria), the desired effects of the present invention can be achieved in both aerobic and anaerobic environments as long as the killed bacteria (and / or components derived from killed bacteria) are used. Therefore, the present invention is expected to be useful in combination with processes carried out in anaerobic environments, such as methane fermentation. Briefly explaining the case of methane fermentation as an example, it is believed that by applying the method of the present invention to a composition that contains high concentrations of organic matter such as fats and oils, proteins, and carbohydrates, and as a result, methanogens cannot efficiently produce methane, the composition can be converted into a composition in which the methanogenic reaction mediated by methanogens proceeds more efficiently.

[0092] Test Example 10: Examination of the growth potential of Burkholderia arboris SL1B1 and Yarrowia lipolytica 1A1 strains using dark oil-containing wastewater. Urea and various metal salts were added to dark oil-containing wastewater in a 5 L fermenter, and the pH was then adjusted to neutral. Burkholderia arboris SL1B1 or Yarrowia lipolytica 1A1 was then inoculated and cultured at 30°C with air supply (0.4 L / min) under agitation. As a control, Burkholderia arboris SL1B1 strain or Yarrowia lipolytica 1A1 strain was inoculated into an aqueous solution of tap water, urea, various metal salts, and canola oil, and cultured with stirring at 30°C with air supply (0.4 L / min.) Sampling for counting the number of bacteria was performed at the time of inoculation of the microorganisms and 44 hours after inoculation.

[0093] The bacterial count of microorganisms cultured using a fermenter was performed using the following procedure. The cultured samples were thoroughly suspended. Each sample was appropriately diluted to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a cone-like rod. Oil agar medium was used for colony formation of B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation of Y. lipolytica. The plates with the spread microorganisms were cultured at 30°C for at least 3 days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 18 (dark oil) and Table 19 (canola oil) below.

[0094]

[0095]

[0096] At 44 hours after inoculation, good growth of Burkholderia arboris strain SL1B1 and Yarrowia lipolytica strain 1A1 was observed in both culture media.

[0097] Test Example 11: Study on the treatment of food oil-containing wastewater using killed microbial cells cultured in dark oil-containing wastewater Killed microbial cells were prepared by heating each microbial culture solution (a microbial culture solution containing dark oil-containing wastewater and a microbial culture solution containing canola oil-containing wastewater) prepared in Test Example 10 at 70°C for 2 hours. Killed microbial cells prepared under these conditions maintained the activity of lipase and other enzymes. The killed microbial cell solution was stored refrigerated and heated at 70°C for approximately 30 minutes immediately before use.

[0098] Using a 5L fermenter, 20mL of each killed bacterial cell (killed bacterial cells of microorganisms cultured in dark oil and killed bacterial cells of microorganisms cultured in canola oil) was added to oil-containing wastewater (sterilized: 1.6L) provided by a food manufacturing company. The wastewater was treated with the killed bacterial cells for approximately 24 hours at 30°C, without air supply, and with stirring. At 24 hours after the start of the action of the killed bacterial cells, an additional 20mL of each killed bacterial cell was added, and wastewater treatment continued until 42 hours after the start of the action of the killed bacterial cells. At each time point, normal hexane extractables (before the start of action, 24 hours, and 42 hours), BOD values ​​(before the start of action and 42 hours), and SS values ​​(before the start of action and 42 hours) were measured. The results are shown in Table 20 (normal hexane extractables), Table 21 (BOD values), and Table 22 (SS values).

[0099]

[0100]

[0101]

[0102] As shown in Tables 20 to 22, the normal hexane extractable substance value, BOD value, and SS value all decreased after 42 hours by adding killed cells of microorganisms cultured using dark oil-containing wastewater.

[0103] According to the present invention, it is possible to treat oil-containing wastewater in a low-cost, simple, rapid, and environmentally friendly manner. In addition, according to the present invention, an agent for treating oil-containing wastewater can be produced using oil-containing wastewater. Therefore, the present invention is useful in fields such as the oil refining industry, food processing industry, and restaurant industry. In addition, according to the present invention, the efficiency of the technology can be improved by using it in combination with a technology for producing useful substances using microorganisms or a technology for decomposing harmful substances.

[0104] This application is based on patent application No. 2023-159071 filed in Japan (filing date: September 22, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for treating oil-containing wastewater, which comprises reacting killed cells of an oil-decomposing microorganism or a component derived from the killed cells with the oil-containing wastewater.

2. The method according to claim 1, wherein the fat-and-oil decomposing microorganism is killed in the stationary phase.

3. The method according to claim 1 or 2, comprising killed cells of two types of fat-decomposing microorganisms.

4. The method according to claim 3, wherein the two types of oil-decomposing microorganisms are bacteria and fungi.

5. The method of claim 4, wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

6. The method according to claim 1, characterized in that killed cells of oil-decomposing microorganisms or components derived from said killed cells are allowed to act on wastewater containing oils and fats under anaerobic conditions.

7. An agent for treating wastewater containing oils and fats, comprising killed cells of an oil-decomposing microorganism or a component derived from said killed cells.

8. The agent according to claim 7, wherein the fat-and-oil decomposing microorganisms are killed in the stationary phase.

9. The agent according to claim 7 or 8, which contains killed cells of two types of fat-decomposing microorganisms.

10. The agent according to claim 9, wherein the two types of oil-decomposing microorganisms are bacteria and fungi.

11. The agent according to claim 10, wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

12. The agent according to claim 7, which is added to oil-containing wastewater under anaerobic conditions.

13. The agent according to claim 7, which is intended to improve the efficiency of methane fermentation of wastewater containing oils and fats.

14. A method for improving the efficiency of methane fermentation of oil-containing wastewater, comprising adding the agent according to claim 7 to the oil-containing wastewater to decompose the oils and fats contained in the oil-containing wastewater.

15. A method for producing an agent for treating oil-containing wastewater, comprising culturing oil-decomposing microorganisms in oil-containing wastewater, and killing the oil-decomposing microorganisms.

16. The manufacturing method according to claim 15, wherein the oil and fat contained in the oil-containing wastewater is at least one selected from the group consisting of residual oil, waste oil from processed oils and fats, waste oil from food manufacturing, waste oil from the food service industry, and household waste oil.