Method for producing anaerobic predatory bacterial consortium, anaerobic predatory bacterial consortium produced by the method, and sludge treatment method using the produced anaerobic predatory bacterial consortium

Abstract

To provide a method for producing an anaerobic predatory bacterial consortium, an anaerobic predatory bacterial consortium produced by the production method, and a method for treating sludge through anaerobic methane fermentation employing the anaerobic predatory bacterial consortium.SOLUTION: There is provided a method for producing an anaerobic predatory bacterial consortium. The method includes: culturing anaerobic sludge in an anaerobic liquid medium that contains a reducing agent and feeder bacteria; withdrawing the culture fluid after turbidity of the liquid medium during culturing decreases; and performing subculturing by inoculating into a fresh liquid medium. There are also provided: an anaerobic predatory bacterial consortium produced by the method; and a sludge treatment method that involves carrying out methane fermentation using recovered sludge as a fermentation feedstock in coexistence of the anaerobic predatory bacterial consortium and methanogenic bacteria.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a method for producing an anaerobic predatory bacteria enrichment system, an anaerobic predatory bacteria enrichment system produced by this production method, and a sludge treatment method by anaerobic methane fermentation using the produced anaerobic predatory bacteria enrichment system. [Background technology]

[0002] The activated sludge process using aerobic bacteria is commonly used for the biological treatment of organic wastewater containing organic matter, excess sewage sludge, food waste, and other organic waste. The activated sludge process treats organic matter through the metabolism of aerobic bacteria, which causes the proliferation of aerobic bacteria. Therefore, in the activated sludge process, it is necessary to remove the excessively proliferated aerobic bacteria as excess sludge. In 2023, the amount of excess sludge generated at sewage treatment plants in Japan will reach 77,280,000 tons per year, accounting for approximately 48% of the sludge discharged as industrial waste (162,676,000 tons per year). Therefore, there is a need to curb the generation and reduce the amount of excess sludge.

[0003] Anaerobic wastewater treatment using anaerobic bacteria has attracted attention because it consumes little energy, produces less carbon dioxide, and can recover methane, which can be used as fuel gas or as a hydrogen source for fuel cells. However, anaerobic bacteria, including methane-fermenting bacteria, grow more slowly than aerobic bacteria. Therefore, maintaining anaerobic bacteria is important for stable anaerobic wastewater treatment using methane fermentation and other methods.

[0004] In recent years, membrane separation, which combines anaerobic treatment with membrane separation technology, has attracted attention because it prevents the outflow of anaerobic bacteria and makes it easy to maintain bacterial mass. The membrane separation method is a wastewater treatment method that combines a reactor tank that performs anaerobic fermentation treatment with a membrane separation tank that discharges only liquid through membrane separation. For example, in Patent Document 1, the present applicant has proposed a wastewater treatment system that includes a reactor tank and a membrane separation tank that ferment low-concentration wastewater containing organic matter using upflow methane fermentation, in which the reactor tank and membrane separation tank are provided with an overflow pipe at the top and a sludge return pipe at the bottom. The reactor tank is provided with a wastewater supply pipe at the bottom and a gas circulation pipe that circulates biogas from the top to the bottom. The membrane separation tank is provided with a discharge pipe for filtered water.

[0005] Patent Document 2 proposes an anaerobic digestion method for recovered sewage sludge, in which recovered sewage sludge, consisting of excess sludge produced in an activated sludge process, is subjected to anaerobically digestion treatment in an anaerobic digestion tank, in which additives containing Fe, Cu, Zn, Mg, etc. are added to the anaerobic digestion tank when the temperature is lower than a predetermined temperature. The method described in Patent Document 2 uses excess sludge as a raw material, which can reduce the cost of treating excess sludge as waste. However, because excess sludge contains a large amount of aerobic bacteria, and bacteria have cell membranes, biofilms, etc. to protect themselves, the anaerobic fermentation of excess sludge takes a long time, which is an issue.

[0006] Predatory bacteria are bacteria that can lyse (eat) other living bacteria and use them as a nutrient source for themselves, and all predatory bacteria reported to date are obligate aerobic bacteria. It has been shown that the lytic action of predatory bacteria promotes the solubilization and mineralization of excess sludge, potentially reducing the amount of excess sludge that needs to be treated (Non-Patent Document 1). However, in the investigations conducted by the present inventors, no reduction in sludge volume was observed due to predatory bacteria. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-084530 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-055216 [Non-patent literature]

[0008] [Non-Patent Document 1] Yu, R., Zhang, S., Chen, Z. et al. “Isolation and application of predatory Bdellovibrio-and-like organisms for municipal waste sludge biolysis and dewaterability enhancement.”, Front.Environ.Sci.Eng.,11,10(2017). Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a method for producing an anaerobic predatory bacteria enrichment system, an anaerobic predatory bacteria enrichment system produced by this method, and a method for treating sludge by anaerobic methane fermentation using an anaerobic predatory bacteria enrichment system. [Means for solving the problem]

[0010] The means for solving the problems of the present invention are as follows. 1. Cultivating anaerobic sludge using an anaerobic liquid medium containing a reducing agent and feeding bacteria; A method for producing an anaerobic predatory bacteria enrichment system, characterized in that the culture medium is removed after the turbidity of the liquid medium during cultivation has decreased, and the culture medium is inoculated into a new liquid medium and subcultured. 2. The method for producing an anaerobic predatory bacteria enrichment system according to 1, wherein the feeding bacteria is Escherichia coli. 3. An anaerobic predatory bacteria enrichment system produced by the production method described in 1. or 2. 4. A sludge treatment method characterized by carrying out a methane fermentation treatment using recovered sludge as a fermentation raw material in the presence of an anaerobic predatory bacteria enrichment system produced by the production method described in 1. or 2. and methane fermentation bacteria. 5. The sludge treatment method according to 4, wherein the methane fermentation treatment is carried out by a membrane separation method using a reactor tank and a membrane separation tank. [Effects of the Invention]

[0011] According to the present invention, an anaerobic predatory bacteria enrichment system can be produced. The anaerobic predatory bacteria enrichment system can decompose (metabolize) bacterial cells under anaerobic conditions, and can efficiently carry out anaerobic fermentation treatment using low-molecular-weight organic compounds produced by the decomposition as raw materials. In the case of methane fermentation using recovered sludge as the fermentation raw material, methane gas can be produced more efficiently than conventional methods. The sludge treatment method of the present invention uses recovered sludge as a raw material for methane fermentation, thereby reducing the amount of industrial waste derived from recovered sludge that is ultimately disposed of. The sludge treatment method of the present invention can produce methane gas that can be used as an energy source from recovered sludge, which previously required large amounts of energy for treatments such as drying and combustion. Therefore, adverse effects on the environment can be significantly reduced. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the change in turbidity of a liquid medium during approximately three months of subculture to produce an anaerobic predatory bacteria enrichment system. [Figure 2] Graph showing the change over time in the number of Escherichia coli in liquid medium after approximately 3 months of subculture. [Figure 3] 1 is a graph showing the cumulative amount of methane gas produced in anaerobic digestion test 1. [Figure 4] Graph showing cumulative methane gas production in anaerobic digestion test 2. DETAILED DESCRIPTION OF THE INVENTION

[0013] "Method for producing an anaerobic predatory bacteria enrichment system" The method for producing an anaerobic predatory bacteria enrichment system of the present invention is characterized in that anaerobic sludge is cultivated using an anaerobic liquid medium containing a reducing agent and feeding bacteria, and after the turbidity of the liquid medium decreases during cultivation, the culture medium is removed and inoculated into a new liquid medium for subcultivation.

[0014] The anaerobic sludge used for the culture is not particularly limited, and anaerobic sludge recovered from an anaerobic treatment tank or the like can be used. The reducing agent is used to maintain a reduced state (anaerobic state) and can be appropriately selected from conventionally known reducing agents depending on factors such as oxidation-reduction potential (ORP) and duration of effect. Examples include iron sulfide, cysteine ​​hydrochloride, sodium thioglycolate, a combination of sodium sulfide, L-cysteine, and DL-dithiothreitol, titanium(III) complexed with citrate or nitrilotriacetate, and reduced glutathione (Patent No. 7349051). The concentration of the reducing agent is not particularly limited as long as it can maintain a reduced state and does not adversely affect the culture, and can be, for example, between 0.01 g / L and 1 g / L.

[0015] In the production method of the present invention, the term "feeding bacteria" refers to bacteria that are preyed upon by anaerobic predatory bacteria. The feeding bacteria are not particularly limited, but examples include Escherichia coli and Pseudomonas aeruginosa, with Escherichia coli being preferred because it is easy to cultivate. The culture conditions are not particularly limited as long as they allow the growth of anaerobic predatory bacteria, and can be, for example, a temperature of 20°C to 50°C and a pH of about 6 to 10. If anaerobic predatory bacteria grow during culture, they will prey on the feeding bacteria, causing a decrease in turbidity. The turbidity (OD 600 ) has decreased to about 0.2 to 0.3, the bacteria are then inoculated into a new liquid medium and subcultured.

[0016] "Anaerobic predatory bacterial accumulation system" By the above-mentioned production method, an anaerobic predatory bacteria enrichment system can be obtained. Anaerobic bacteria grow more slowly than aerobic bacteria and die in the presence of oxygen, making their isolation, culture, and identification extremely difficult. Furthermore, enrichment systems are collections of diverse microorganisms, and isolation is sometimes impossible due to the interdependent exchange of substances between multiple microbial species. Therefore, isolating and identifying anaerobic predatory bacteria from enrichment systems containing multiple anaerobic bacteria is extremely difficult and impractical. While it is possible to deposit mixed microorganisms (enrichment systems) with the National Institute of Technology and Evaluation (NITE), this requires clarifying the taxonomic position of all the microorganisms and providing a viability test method for each microorganism. Therefore, depositing an enrichment system containing multiple anaerobic bacteria is also impractical.

[0017] "Sludge treatment method" The sludge treatment method of the present invention is characterized by carrying out a methane fermentation treatment using recovered sludge as a fermentation raw material in the presence of an anaerobic predatory bacteria enrichment system produced by the above method and methane fermentation bacteria. The recovered sludge is not particularly limited as long as it contains bacteria, and for example, one or more of the following can be used in wastewater treatment using the activated sludge method: primary sedimentation sludge produced in a primary sedimentation basin; excess sludge mainly composed of aerobic bacteria produced in an activated sludge tank; digested sludge mainly composed of anaerobic bacteria produced by anaerobic treatment. Among these, it is preferable to use excess sludge, which is produced in large quantities and requires high treatment costs for incineration, landfilling, etc.

[0018] Recovered sludge contains many aerobic bacteria. Bacteria have cell membranes and cell walls, and may be enclosed in biofilms made of exopolysaccharides (EPS), which are their defense mechanisms. Therefore, bacteria are difficult to metabolize by anaerobic bacteria and are therefore a carbon source that is difficult to use as a fermentation feedstock for methane fermentation.

[0019] In the wastewater treatment method of the present invention, recovered sludge containing a large amount of bacteria is subjected to methane fermentation treatment in the presence of an anaerobic predatory bacteria enrichment system and methane fermentation bacteria, and the aerobic bacteria in the recovered sludge are processed (eaten) by the anaerobic predatory bacteria enrichment system and converted into low-molecular-weight organic compounds that are easily usable as raw materials for methane fermentation.The low-molecular-weight organic compounds produced by the anaerobic predatory bacteria enrichment system are then used as the main fermentation raw materials for methane fermentation. It is obvious to those skilled in the art that when bacteria are consumed, they are decomposed (metabolized) and converted into low molecular weight compounds, and that low molecular weight compounds are easier to use as fermentation raw materials.

[0020] The anaerobic treatment method in the sludge treatment method of the present invention is not particularly limited and may be any of completely mixed methane fermentation, upflow methane fermentation, and others, but upflow methane fermentation is preferred because anaerobic bacteria are more likely to form granules. Furthermore, a membrane separation method using a reactor tank and a membrane separation tank is preferred in order to prevent the loss of anaerobic bacteria due to outflow.

[0021] In the present invention, the wastewater to be treated by methane fermentation has an organic matter (VS: Volatile Solid) load of 1 kgVS / m 3 / day or more 20kgVS / m 3 / day or less. VS load is 3kgVS / m 3 / day or more is preferable, 5kgVS / m 3 / day or more is more preferable, and 10gVS / m 3 / day or more is even more preferable. VS load is 20kgVS / m 3 / day, the methane production rate saturates and hardly increases any further. The wastewater to be subjected to methane fermentation treatment contains treated sludge as a fermentation raw material, but it can also contain fermentation raw materials other than treated sludge. [Example]

[0022] "Production of an anaerobic predatory bacteria enrichment system" Excess sludge collected from sewage treatment plant A was used as a source for isolation of anaerobic predatory bacteria. The HM buffer and 0.01% resazurin shown in Table 1 were placed in a vial, sealed with a butyl rubber stopper, and sterilized in an autoclave after purging with nitrogen for 15 minutes. After cooling, the reducing agent shown in Table 2 and Escherichia coli (E. coli HB101, hereinafter also referred to as HB101) were added as bait bacteria to measure OD. 600 =1.5 to obtain a liquid medium. Anaerobic sludge, which is the source of anaerobic predatory bacteria, was inoculated into the liquid medium at a volume of 10% (v / v). The turbidity of the liquid medium decreased (OD 600 Static culture was then performed at 30°C until the OD 600 = 1.5), and the cells were repeatedly inoculated at 10% volume (v / v) and subcultured for approximately 3 months. The turbidity of the liquid medium during subculture was measured using an absorption spectrophotometer. The change in turbidity over time is shown in Figure 1.

[0023] [Table 1] [Table 2]

[0024] Measurement of viable HB101 count by viability q-PCR After approximately 3 months of subculture, 40 μL of enrichment culture was treated with EMA, and DNA was extracted using Cica Geneus DNA extraction reagent and used for viability qPCR measurement. For qPCR measurements, primers designed for the HB101 tbpA gene were used. 2 μL of sample DNA was mixed with PCR reaction solution (5 μL of SYBR Green PCR MAster Mix (Applied Biosystems, CA, USA), 0.2 μL of forward / reverse primer, and 2.6 μL of sterile ultrapure water), and q-PCR was performed using the incalculator method with a CFX Maestro Ver. 1.1 (Bio-Rad). The temperature conditions were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds and 66°C for 1 minute. Figure 2 shows the daily changes in E. coli counts measured by viability qPCR.

[0025] ·result Anaerobic sludge was subcultured for approximately three months in HM buffer supplemented with HB101, and the E. coli turbidity was confirmed to have decreased to approximately 0.2-0.3 after about six days of culture. The decrease in turbidity was confirmed for more than three months even after repeated subculture (Figure 1). The results of viability q-PCR performed 8 days after inoculating E. coli into the enrichment culture after approximately 3 months of subculture confirmed that the number of viable E. coli cells had significantly decreased by the 6th day (Figure 2). These results confirmed that an anaerobic predatory bacteria accumulation system had been obtained.

[0026] Anaerobic digestion test 1 Excess sludge was collected from sewage treatment plant B. To eliminate the influence of organic matter other than bacteria in the excess sludge, the excess sludge was centrifuged (2,500 g, 10 min) and the supernatant was removed. The sludge was then washed twice with the mineral salts medium shown in Table 3, and the volume was increased to 2 L with mineral salts medium to obtain the recovered sludge. [Table 3]

[0027] 200 mL of the anaerobic predatory bacteria enrichment obtained above was centrifuged (10,000 g, 10 min) and washed twice with HM buffer. The entire recovered bacterial mass was suspended in HM buffer to a volume of 2 mL. 68 mL of recovered sludge and 2 mL of anaerobic predatory bacteria enrichment were mixed in a 100 mL vial, the gas phase was purged with nitrogen, and the mixture was left to stand at 30°C for 14 days for an anaerobic digestion test. Although the excess sludge was aerobic, a small number of anaerobic bacteria coexisted within the aerobic sludge. Simply converting the aerobic sludge to an anaerobic environment allowed methane fermentation to proceed. During the test period, the amount of biogas produced and the methane gas concentration were measured (GC-BID).

[0028] ·result It was shown that adding the anaerobic predatory bacteria enrichment system increased the amount of methane gas produced from recovered sludge by approximately 15.5% (Figure 3). This is thought to be because the anaerobic predatory bacteria preyed (metabolized) the bacteria in the recovered sludge, producing low-molecular-weight organic compounds, and these low-molecular-weight organic compounds were used as a raw material for efficient methane fermentation. This confirmed that the addition of an anaerobic predatory bacteria enrichment system enabled efficient processing of organic compounds and methane production by methane fermentation bacteria.

[0029] Anaerobic digestion test 2 The anaerobic predatory bacteria enrichment system from sewage treatment plant A, which was produced in experiment 1 and subsequently subcultured for 24 months, and the anaerobic predatory bacteria enrichment system from sewage treatment plant B, which was produced in the same way and subcultured for 4 months using excess sludge collected from sewage treatment plant B, were used. Note that the anaerobic predatory bacteria enrichment system from sewage treatment plant B also showed a decrease in turbidity and viable cell count over time in the liquid medium containing Escherichia coli (E. coli K12), confirming that an anaerobic predatory bacteria enrichment system had been produced. Using these, the recovered sludge washed in the same manner as in Experiment 1 was mixed with the anaerobic predatory bacteria enrichment system, and then cultured for a predetermined period at 30°C and 120 rpm to conduct an anaerobic digestion test. The daily changes in cumulative methane production for each test system are shown in Figure 4. The values ​​for each test system in the graph in Figure 4 represent the rate of increase in methane gas production compared to uninoculated bacteria.

[0030] The system with the anaerobic predatory bacteria enrichment system produced more methane than the uninoculated system without the addition of anaerobic predatory bacteria. Methane production was particularly high in the early stages of methane fermentation (up to 14 days after the start of the test). This confirmed that the anaerobic predatory bacteria in the anaerobic predatory bacteria enrichment system prey on (metabolize) the bacterial cells in the recovered sludge and convert them into low-molecular-weight organic compounds, and that the presence of these low-molecular-weight organic compounds in higher concentrations promotes methane fermentation.

Claims

1. Cultivating anaerobic sludge using an anaerobic liquid medium containing a reducing agent and a feeding bacterium; A method for producing an anaerobic predatory bacteria enrichment system, characterized in that the culture medium is removed after the turbidity of the liquid medium during cultivation has decreased, and the culture medium is inoculated into a new liquid medium and subcultured.

2. 2. The method for producing an anaerobic predatory bacteria enrichment system according to claim 1, wherein the feeding bacteria is Escherichia coli.

3. An anaerobic predatory bacteria enrichment system produced by the method according to claim 1 or 2.

4. A sludge treatment method, comprising carrying out a methane fermentation treatment using recovered sludge as a fermentation raw material in the presence of an anaerobic predatory bacteria enrichment system produced by the production method according to claim 1 or 2 and methane fermentation bacteria.

5. 5. The sludge treatment method according to claim 4, wherein the methane fermentation treatment is carried out by a membrane separation method using a reactor tank and a membrane separation tank.

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