JP5900948B2 - Microbial preparation and waste liquid treatment method - Google Patents

Description

  The present invention relates to a microbial preparation using a microorganism having an ability to decompose fats and oils and a waste liquid treatment method.

  Wastewater discharged from food processing factories and commercial facilities kitchens contains a large amount of oil and fat, so if there is a risk of significantly hindering the function of the piping equipment to sewers or septic tanks, a grease trap will be added to the piping equipment. It is obliged to install (grease interceptor). However, such a grease trap may cause problems such as deterioration of function due to accumulation of fats and oils, blockage of drain pipes, generation of malodors, and outflow of fats and oils.

  Therefore, in order to solve the above-mentioned problems, biological treatment methods using microorganisms having oil-degrading ability (oil-decomposing microorganisms) have been proposed, and microbial preparations containing these oil-degrading microorganisms have also been developed. ing.

For example, Patent Document 1 discloses a microbial preparation for oil degradation using Rhodotorula pacifica (FERM P-21121) and Cryptococcus laurentii (FERM P-21122). According to the document, it is described that a high oil and fat decomposition effect can be obtained even in an environment of low nitrogen and low phosphorus concentration, and various oils and fats can be efficiently decomposed even at a low temperature of 30 ° C. or lower.

JP 2008-142042

  However, when the biological treatment method as described above is applied to wastewater with a high fat concentration, the pH of the wastewater becomes acidic due to fatty acids generated when fats and oils are biodegraded by fat-degrading microorganisms. The tendency is particularly strong in the grease trap, and the grease trap drainage shows pH 3.5 to 4.5.

  On the other hand, most of the conventionally known oil-degrading microorganisms have an optimum pH of the oil-degrading ability of around 8, so that the oil-degrading ability cannot be sufficiently exhibited in the low pH environment as described above. There was a problem.

  The present invention has been made in view of the problems as described above, and the object of the present invention is to prepare a microorganism preparation and a waste liquid treatment using a microorganism that can exhibit an excellent ability to decompose oils and fats even under low pH conditions. It is to provide a method.

The microorganism preparation for decomposing oils and fats according to the present invention, which has been made to solve the above-mentioned problems, contains at least one of Raoultella planticola 232-2 and Raoultella planticola NBRC 14939. It is characterized by doing.

In addition, the waste liquid treatment method according to the present invention includes Raoultella planticola ( Raoultella planticola ) 232-2 strain and Raoultella planticcola ( Raoultella planticola ) in waste liquid containing oil and fat and having a pH range of 3.5 to 9. By adding at least one of the NBRC14939 strains, the oil and fat in the waste liquid is decomposed.

  The above-mentioned Lao Ulterra plantic cola can exhibit excellent oil and fat decomposition ability in a wide pH range of about 3 to 9. Therefore, according to the microorganism preparation for decomposing oil and fat of the present invention containing this microorganism and the waste liquid treatment method of the present invention using this microorganism, a grease trap or the like that has been difficult to perform sufficient oil and fat decomposition with a conventionally known microorganism Even in an acidic environment, it is possible to perform highly efficient oil and fat decomposition.

The graph which shows the fats-and-oils decomposition ability in pH4 of R. planticola 232-2 strain | stump | stock and a commercially available oil-degrading microorganism. The graph which shows the fats-and-oils decomposition | disassembly ability of R. planticola 232-2 strain in various pH. The graph which shows the fats and oils decomposition ability of R. planticola 232-2 strain in various temperature. The graph which shows the fats and oils decomposition ability of R. planticola 232-2 strain in various fats and oils density | concentrations. The graph which shows the fats and oils decomposition ability in pH4 of R. planticola NBRC14939 strain and R. planticola 232-2 strain. The graph which shows the fats and oils decomposition ability in various temperature and various pH of R. planticola NBRC14939 strain.

  The present inventors searched for a microorganism that can exhibit an excellent fat-and-oil decomposition ability even under low pH conditions, and as a result, separated one strain from the soil. When this strain was tested for morphological observation with an optical microscope, catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F), the following results were obtained. It was.

  The results of physiological and biochemical tests using the bacterial identification kit (API20E, manufactured by BioMerieux) for the same strain are as follows.

  Furthermore, the results of additional tests conducted based on the results of the physiological and biochemical tests are as follows.

  The base sequence of 16S rDNA (16S rRNA gene) of the strain is as shown in SEQ ID NO: 1 in the sequence listing.

From the above test results and nucleotide sequences, the present inventors have the above strains were identified as Raoultella Planty Cola (Raoultella planticola), it was designated by the same strain and Raoultella planticola 232-2. This strain is deposited under the accession number NITE P-981 with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation.

In addition, there has been no report on an example of oil / fat decomposing ability in microorganisms of the same genus / genus. Therefore, the present inventors further confirmed the ability to decompose oils and fats for Raoultella planticola NBRC14939, which is a standard strain of the above-mentioned Lao Ulterra plantic cola. As a result, initially, the standard strain showed no ability to decompose fats and oils (Table 4). Thereafter, when the standard strain was re-tested several times, the above-mentioned Raoultella planticola 232-2 was tested under acidic conditions. It was confirmed that the oil-and-fat decomposition ability was almost the same as that (Fig. 5). The average fat decomposition rate in the test of FIG. 5 is a in Raoultella planticola 232-2 67.7 ± 9.9 (% ), it was in Raoultella planticola NBRC14939 65.7 ± 10.1 (% ).

The evaluation conditions in the tests of Table 4 and FIG. 5 (both n = 3) are as follows.
Mixed fat (salad oil: lard: beef tallow = 1: 1: 1 (w / w)): 3,000 ppm,
Inoculum : 1% pre-culture of R. planticola 232-2 or R. planticola NBRC 14939 (standard strain)
Culture conditions: 25 ° C, 120 rpm, 24 hours shaking culture,
Medium (g / L): yeast extract 1, urea 0.5, Na ₂ HPO ₄ 0.5, NaCl 0.15, KCl 0.07, CaCl ₂ · 2H ₂ O 0.09, MgSO ₄ · 7H ₂ O 0.1, pH 4,
pH adjustment: Adjust with hydrochloric acid

  The microorganism preparation according to the present invention contains the above-mentioned Lao Ulterra plantic cola. Methods for formulating the microorganism include, for example, Sawao Murao et al., “Applied Microbiology Revised Edition” (1993, Baifukan), Takayuki Uejima “Industrial Enzyme” (1995, Maruzen), or Microbiological Research Method The methods described in the round-table discussion, “Microbiology Experimental Method” (1993, Kodansha) and the like can be used. Specific preparation methods are listed below, but the microbial preparation according to the present invention is not limited to those produced by the following method.

In the case of a liquid preparation, it can be formulated by any of the following methods, for example.
(A) The microorganism is cultured in a general nutrient medium such as a broth medium for about 12 to 36 hours, and if necessary, a pH adjuster or the like is added to prepare a preparation.
(B) Bacteria are collected from the culture of (A) by centrifugation or the like, and suspended in a medium such as physiological saline to a suitable concentration. And if necessary, a pH adjuster etc. are added to this and it is set as a formulation.
(C) The culture of (A) is concentrated to an appropriate concentration by lyophilization or the like, and a pH adjuster or the like is added thereto as necessary to obtain a preparation.
(D) The cells are collected from the culture of (A) by centrifugation or the like, and the cells are suspended in a medium such as a broth medium. And if necessary, a pH adjuster etc. are added to this and it is set as a formulation.
(E) The above (D) is further concentrated to an appropriate concentration by freeze-drying or the like to prepare a preparation.

In the case of a powder formulation, it can be formulated by any of the following methods, for example.
(A) The microorganism is cultured in a general nutrient medium such as a broth medium for about 12 to 36 hours, and if necessary, a pH adjuster or the like is added thereto and dried by freeze drying or the like to obtain a preparation.
(B) The cells are collected from the culture of (a) by centrifugation or the like, and the cells are suspended in a medium such as a solution containing physiological saline or skim milk and sodium glutamate to a suitable concentration. If necessary, a pH adjuster or the like is added thereto and dried by lyophilization or the like to obtain a preparation.
(C) The cells are collected from the culture of (a) by centrifugation or the like, and the cells are suspended in a medium such as a gravy medium, and a pH adjuster is added thereto as necessary, followed by freeze-drying, etc. To dry the formulation.
(D) Fine powders such as fiber waste, sawdust, white clay, diatomaceous earth, etc. are added to the above (a) to (c) to prepare a preparation.

  In addition to the above methods, the above microorganisms may be immobilized with various immobilization materials by a known technique such as a carrier binding method, a crosslinking method, an entrapment method, or a composite method. Further, the microorganisms may be tableted by other known tableting techniques.

  In the waste liquid treatment method according to the present invention, the oil and fat content in the waste liquid is decomposed by adding the above-mentioned Lao Ulterra plantic cola to the waste liquid containing the fat and oil content. As described above, since the microorganism of the present invention can maintain a high oil and fat decomposition ability even in an acidic region, the waste liquid treatment method according to the present invention is suitably applied to, for example, the treatment of the waste liquid in the above-described grease trap. can do. The microorganism used in the microorganism preparation and waste liquid treatment method according to the present invention (hereinafter referred to as “oil-decomposing microorganism according to the present invention”) has a higher decomposition rate at 20 ° C. to 35 ° C., particularly at 30 ° C. to 35 ° C. Therefore, it is possible to suitably cope with a grease trap in which hot water often flows.

  The microorganism preparation and waste liquid treatment method according to the present invention can be suitably used for the decomposition of animal and plant-derived fats and oils, particularly animal fats and oils, and other fats and derivatives obtained by synthetic methods, and others. It can also be used to decompose various hydrocarbons.

  Hereinafter, test examples conducted for confirming the effects of the microorganism preparation and the waste liquid treatment method according to the present invention will be described.

(Test Example 1)
Using the waste water actually collected from the grease trap tank, the oil and fat decomposing ability at pH 4 of the oil decomposing microorganisms (Example) according to the present invention and the oil decomposing microorganisms (comparative example) of other companies were compared. In addition, as the oil-and-fat decomposing microorganism according to the present invention, the above-mentioned Raoultella planticola 232-2 strain (Accession No. NITE P-981) was used (hereinafter the same in Test Examples 1 to 4). In addition, as the oil-degrading microorganisms of the other companies, a mixed solution of Rhodotorula pacifica FERM AP-21121 and Cryptococcus laurentii FERM AP-21122 culture solution was used.

First, the wastewater collected from a grease trap tank of a Japanese restaurant in Matsumoto City, Nagano Prefecture was adjusted to pH 4.0 with 7% HCl and dispensed into a 1000 mL beaker. 10 mL each of the oil-degrading microorganisms adjusted to 1x10 ⁸ cells / mL was inoculated into this waste water, stirred for 120 hours at 25 ° C using a stirrer, and the time-dependent change in n-hexane extract substance concentration (oil concentration) Was measured. In addition, the measurement of the n-hexane extract material was performed based on Environmental Agency Notification No. 64 Appendix 4 in 1974.

The above results are shown in FIG. The oil-and-oil decomposing microorganisms of other companies of the comparative example showed high oil-degrading ability almost equal to that of the oil-and-oil decomposing microorganisms of the present invention by the treatment for 120 hours at pH 7 separately, but at pH 4 as shown in FIG. Compared with the oil-degrading microorganism ( R. planticola 232-2) according to the invention, the oil-decomposing ability was remarkably low. In addition, the optimum pH of the fat and oil decomposing ability by the microorganism of the comparative example was 8, and the fat and oil decomposing ability of the microorganism of the comparative example in this test example was about 10% of the oil and fat decomposing ability at the optimum pH (pH 8). . On the other hand, the optimum pH of the oil-degrading ability by the oil-decomposing microorganism according to the present invention is 5, but an oil-and-fat resolving power is substantially equivalent between pH 3 and 9. Accordingly, the ability of the microorganisms to decompose fats and oils in this test example was equivalent to the ability of the microorganisms to decompose fats and oils at the optimum pH (pH 5). From this, it was confirmed that the fat-and-oil decomposing microorganism of the comparative example has a large decrease in fat-and-oil decomposing ability at pH 4, whereas the fat-and-oil decomposing microorganism according to the present invention does not decrease in fat and oil-decomposing ability even at pH 4.

(Test Example 2)
The oil and fat decomposing ability at various pH of the oil and fat decomposing microorganism according to the present invention was evaluated.

The oil and fat decomposition ability was evaluated by the following procedure.
(1) 1% of the pre-culture of the oil-degrading microorganism according to the present invention was inoculated into a baffle flask with 100 mL of medium and 3,000 ppm of mixed oil and fat, and cultured with shaking at 30 ° C. and 120 rpm for 24 hours. The composition of the medium and the mixed fat is as follows.
Medium (g / L) _Yeast extract 1, urea 0.5, Na ₂ HPO ₄ 0.5, NaCl 0.15, KCl 0.07, CaCl ₂ · 2H ₂ O 0.09, MgSO ₄ · 7H ₂ O 0.1
pH adjustment_The acid side was adjusted with hydrochloric acid, and the alkali side was adjusted with NaOH.
Oils and fats_salad oil: lard: beef tallow = 1: 1: 1 (w / w)
(2) The autoclave process was performed after completion | finish of culture, and chloroform-methanol extraction was performed in the following procedures.
First, the whole culture solution was put into a separating funnel, the culture flask was washed with 40 mL of a chloroform-methanol (3: 1 (v / v)) solution, and the fats and oils in the culture solution were extracted with the washing solution. Only the chloroform layer (lower layer) was separated, and the aqueous layer (upper layer) was extracted again with chloroform-methanol solution 40 mL. Thereafter, the chloroform layer (lower layer) and the aqueous layer (upper layer) were each transferred to a centrifuge tube, centrifuged at 10,000 rpm for 10 minutes, and then gently returned to a separatory funnel to collect only the chloroform layer (lower layer).
(3) Then, after dehydrating and filtering with anhydrous magnesium sulfate, the filtrate was taken in an eggplant type flask, the solvent was removed under reduced pressure, and the weight of the remaining fat was measured.
(4) As a control group, those not inoculated with the preculture solution were similarly subjected to shaking culture and extraction for 24 hours, and the residual fat weight was measured.
(5) The fat and oil decomposition rate was calculated by the following equation from the amount of residual fat in the control group and the experimental group (inoculated with the preculture solution).
Decomposition rate of fats and oils (%) = [(residual fat weight in control group−residual fat weight in experimental group) / residual fat weight in control group] × 100

  The result of the evaluation test (n = 3) of the fats and oils decomposition ability by the above is shown in FIG. The drainage of the grease trap usually shows an extremely low value of pH 3.5 to 4.5. In order to efficiently decompose fats and oils in such wastewater, microorganisms that exhibit a strong ability to decompose fats and oils in such an acidic environment are required. As shown in FIG. 2, the oil-degrading microorganism according to the present invention exhibits excellent oil-degrading activity at pH 3 to 9, and thus can be said to be an oil-degrading microorganism having a very wide application range that can be used in a grease trap and subsequent steps. .

(Test Example 3)
The oil and fat decomposing ability at various temperatures of the oil and fat decomposing microorganism according to the present invention was evaluated.

  The evaluation of the ability to decompose fats and oils was carried out in the same manner as in Test Example 2 except that the medium was adjusted to pH 4 (pH was adjusted with hydrochloric acid) and the culture temperature was variously changed between 10 ° C. and 40 ° C.

  The result of the evaluation test (n = 3) of the fats and oils decomposition ability by the above is shown in FIG. The oil-degrading ability of the oil-degrading microorganisms according to the present invention gradually increases from 20 ° C to 35 ° C, and 3,000 ppm of mixed oil consisting of salad oil: lard: beef tallow = 1: 1: 1 (w / w) is 45% per day. Decomposed ~ 60%.

(Test Example 4)
The fat and oil decomposing ability at various oil and fat concentrations of the oil and fat decomposing microorganism according to the present invention was evaluated.

  Evaluation of the oil and fat decomposition ability was carried out in the same manner as in Test Example 2 except that the medium was adjusted to pH 4 (pH was adjusted with hydrochloric acid) and the oil and fat concentration was variously changed between 1,000 ppm and 10,000 ppm.

  The evaluation result of the fats and oils decomposition ability by the above is shown in FIG. The oil-degrading activity per day of the oil-degrading microorganism according to the present invention was about 55% to 65% when the mixed oil concentration was 1,000 ppm to 5,000 ppm.

(Test Example 5)
The standard strain of Raoultella planticola ( Raoultella planticola NBRC14939) was used as the oil- and fat-decomposing microorganism according to the present invention, and the ability of the microorganism to decompose oil and fat at various pH and various temperatures was evaluated.

  The ability to decompose fats and oils was tested except that the above-mentioned standard strain was used as the fat and oil-degrading microorganism, that the culture medium had two types of pH 4 and pH 8, and that the culture temperature was two types of 25 ° C and 30 ° C. Performed as in Example 2.

The evaluation result of the fats and oils decomposition ability by the above is shown in FIG. As is clear from the figure, Raoultella planticola NBRC14939 exhibited excellent fat and oil degrading activity at both pH 4 and pH 8 under both temperature conditions of 25 ° C. and 30 ° C. In addition, the average of the fats and oils decomposition rate in the test (n = 3) in the figure is 65.7 ± 10.1 (%) at pH 4 and 25 ° C., 50.7 ± 5.4 (%) at pH 4 and 30 ° C., and 64.1 ± at pH 8 and 25 ° C. It was 52.9 ± 15.1 (%) at 6.5 (%), pH 8, and 30 ° C.

Claims (2)

A microorganism preparation for decomposing oil and fat, comprising at least one of Raoultella planticola 232-2 and Raoultella planticola NBRC14939. Containing fats and oils, Raoultella Planty cola pH range of the liquid waste of 3.5~9 (Raoultella planticola) 232-2 strain, and Raoultella Planty Cola (Raoultella planticola) by adding at least one of the NBRC14939 strain, A waste liquid treatment method comprising decomposing oil and fat in the waste liquid.

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