JP2010227858A - Method of treating fat-containing wastewater by lipase secretion microorganisms capable of propagation/fat splitting under weak acid conditions, grease trap cleaning method and fat-splitting agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively treat wastewater containing high-concentration fat and oil like kitchen wastewater by highly efficiently degrading fat and oil by microorganisms. <P>SOLUTION: The fat and oil are degraded by lipase secretion microorganisms capable of propagation/fat-splitting under the same weak acid conditions as the inside of a grease trap. By using the method of treating the fat-containing wastewater, oil accumulated in the grease trap is removed inside a trap tank. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for treating fat-containing wastewater by a lipase-secreting microorganism capable of growing and decomposing oil under mildly acidic conditions, a method for purifying grease traps, and an oil-degrading agent.

Grease traps, which are processing equipment that removes oil contained in kitchen wastewater from the food service industry by solid-liquid separation, are sources of malodors and pests, and labor and costs associated with maintenance, such as recovery, transportation, and cleaning of the separated oil In view of the above, the establishment of a revolutionary technology that eliminates the oil in the grease trap is eagerly desired by the industry, particularly the restaurant industry. Accordingly, attempts have been made to apply a technique for decomposing oils and fats using microorganisms. Many of such attempts are to isolate microorganisms having an excellent ability to decompose fats and oils, to examine the ability to decompose fats and oils, and to actually apply them to grease traps (for example, Patent Documents 1 to 6). In fact, many microbial preparations for grease traps containing oil-degrading bacteria are commercially available.
However, kitchen wastewater in the food service industry usually contains high concentrations of fats and oils of 1 g / L or more, and when high, 10 g / L or more, and the residence time of the wastewater in many grease traps is about 10 minutes. short. For this reason, it is difficult to treat oil only in the grease trap, and so far, the normal hexane value of 30 mg / L, which is an emission target set by many local governments, is always achieved only by treatment in the grease trap. No fat-degrading microorganisms that can be achieved have been reported.
Patent Documents 3 to 5 actually exemplified above show only demonstration examples using a flask or a water tank. Moreover, in patent document 2 and 6, the fats-and-oils decomposition tank is provided in addition to the grease trap, and it decomposes | disassembles in it. Further, in these examples, only examples in which fats and oils having a concentration of 30 to 300 mg / L, which is much lower than normal waste water, are decomposed are shown. In Patent Document 1, an example of processing in a grease trap is shown, but the result of continuous operation is not shown, and processing is performed only up to an order of magnitude higher than the aforementioned discharge target value. Results are shown. Therefore, in order to process the oil only in the grease trap, it is necessary to further improve the decomposition efficiency by microorganisms.
In addition, many commercially available microbial preparations are effective in decomposing fats and oils in laboratory culture, but do not show the expected decomposing effects even when put in a grease trap tank. Therefore, it is necessary to develop a microorganism exhibiting a high growth ability and fat / oil decomposition ability even in an actual grease trap.

JP-A-11-75832 Japanese Patent Laid-Open No. 2003-24051 JP 2003-38169 A JP 2004-242553 A JP 2008-144202 A JP 2008-220201 A

The present invention has been made in view of the above circumstances, and provides a method and an oil decomposing agent for effectively treating oil-containing wastewater by a lipase-secreting microorganism capable of growing and degrading oil under mildly acidic conditions. The first object is to treat the wastewater containing high concentration fats and oils such as kitchen wastewater effectively.

In many cases, fats and oils-containing wastewater is weakly acidic at a pH of about 5.5 to about 6.0 because a part of the oils and fats is hydrolyzed by the action of microorganisms and the like. Therefore, the present inventor considered that a lipase-secreting microorganism capable of growing and decomposing even under weakly acidic conditions effectively removes oil in the actual oil-containing wastewater and has a high purification effect of the grease trap.
To this end, the present invention is characterized by a method for treating fat and oil containing wastewater by decomposing oil and fat with a lipase-secreting microorganism capable of decomposing and growing oil and fat even under mildly acidic conditions (claim 1). Furthermore, in the method for treating fat and oil-containing wastewater according to claim 1, the lipase-secreting microorganism is Burkholderia arbolis (claim 2). Further, the method for treating oil-containing wastewater according to claim 1 or 2 is characterized in that the oil accumulated in the grease trap is removed in the trap tank to purify the grease trap (claim 3).
Further, the present invention is characterized by an oil and fat decomposing agent containing a lipase-secreting microorganism capable of decomposing and growing oil and fat even under weakly acidic conditions. Furthermore, in the oil and fat decomposing agent according to claim 4, the lipase-secreting microorganism is Burkholderia alboris (claim 5).
If the above-described method and fat removing agent according to the present invention are used, it is also possible to decompose and purify the oil in a grease trap, which is a facility for treating kitchen wastewater containing a high concentration of fat.

It is a graph which shows the change of the medium turbidity when the commercially available microorganism preparation in the comparative example 1 is cultured in the fat and oil containing medium of pH7 (circle mark) or pH6 (square mark). It is a graph which shows the change of the turbidity of a culture medium when the Burkholderia arboris SL1B1 strain in Example 1 is cultured in a fat and oil containing medium of pH 7 (circle mark) or pH 6 (square mark). It is a graph which shows the time-dependent change of CFU, lipase activity, total fatty acid concentration, and ammonium ion concentration of Burkholderia arboris SL1B1 strain in Example 2.

Next, embodiments of the present invention will be described, but the technical scope of the present invention is not limited by these embodiments, and can be implemented in various forms without changing the gist of the invention. . Further, the technical scope of the present invention extends to an equivalent range.
The microorganism used in the present invention is a lipase-secreting microorganism that can grow and decompose oils and fats even under mildly acidic conditions. Such microorganisms can be isolated on an inorganic salt agar medium containing fat and oil as the sole carbon source and adjusted to pH 6. In addition, lipase-secreting microorganisms can be identified because they form a clear zone (halo) around the colony formed on the agar medium. In fact, the SL1B1 strain was isolated by these methods as a microorganism having a high ability to secrete lipase, decompose oils and fats even under mildly acidic conditions. This microorganism was identified as Burkholderia arboris by determination of the base sequence of 16S ribosomal DNA and phylogenetic analysis, and it was received on March 17, 2009 by the National Institute for Product Evaluation Technology Patent Microorganism Depositary. The receipt number is NITE AP-724.
Examples of microorganisms that secrete lipase and decompose fats and oils under mildly acidic conditions include true bacteria, yeasts, and filamentous fungi. Of these, preferred are true bacteria and yeast, and more preferred are Gram-positive bacteria and proteobacteria. Among Gram-positive bacteria, Bacillus bacteria are particularly good. Among the proteobacteria, alpha bacteria, beta bacteria, and gamma bacteria are more preferable. Furthermore, among them, Burkholderia bacteria, Acinetobacter bacteria, Pseudomonas bacteria, Alcaligenes bacteria, Rhodobacter bacteria, Ralstonia bacteria, Acidborax The genus bacteria are particularly preferred along with the Bacillus bacteria belonging to the aforementioned gram-positive bacteria. Furthermore, among these genus of true bacteria, Burkholderia bacteria are most preferable. Of the Burkholderia bacteria, Burkholderia arboris is most preferred. As an example, the above-mentioned Burkholderia arboris SL1B1 strain can be mentioned.
The ability of the microorganism to secrete lipase can be evaluated by measuring the lipase activity of the culture supernatant obtained by centrifugation of the microorganism culture solution. The lipase activity is the amount of p-nitrophenol produced by enzymatic reaction using 4-nitrophenyl palmitate (4-NPP), which is an ester of palmitic acid and 4-nitrophenol, as a substrate. Can be determined by measuring the absorbance at 410 nm. First, 4-NPP (18.9 mg) is added to 3% (v / v) Triton X-100 (12 ml) and dissolved at 70 ° C. to obtain a substrate solution. PH 6 by adding NaOH or HCl to 1 mL of substrate solution, 0.9 mL of ion exchange water and 150 mM GTA buffer (150 mM 3,3-dimethyllactic acid, 150 mM Tris, and 150 mM 2-amino-2-methyl-1,3-propandiol) 1 mL is put into a cell and kept at 28 ° C. for 5 minutes. 0.1 mL of the culture supernatant is added thereto, and the value at 410 nm is measured while stirring. The lipase activity is measured by defining the amount of enzyme that produces 1 μmol of 4-nitrophenol as 1 unit (U), and the unit per 1 mL of culture supernatant is calculated.
The ability of the microorganisms to decompose and consume fatty acids and fatty acids can be evaluated by quantifying the fatty acids contained in the fats and oils remaining in the medium and the free fatty acids generated by hydrolysis by gas chromatography. Specifically, 1 mL of the culture supernatant is acidified with hydrochloric acid, and 2 mL of chloroform is added. After stirring for 2 minutes, the mixture is centrifuged, and 1 mL of the chloroform layer is transferred to another container, and the solvent is evaporated to concentrate. 2 mL of methanolysis solution (methanol: sulfuric acid = 17: 3) is added and heated at 100 ° C. for 2 hours to methylate the fats and oils and free fatty acids. Thereafter, 2 mL of chloroform and 1 mL of pure water are added and stirred, and then the chloroform layer is analyzed by gas chromatography to quantify the fatty acid methyl ester.
As a method for examining the growth ability of microorganisms, there are a method of measuring absorbance (turbidity) (OD660) at 660 nm as an optical density of bacterial cells, a method of measuring colony forming unit (CFU), and the like. In the latter, a certain amount of the stock solution and the diluted solution of the culture solution are spread on the agar medium, and colonies formed by stationary culture are counted.
The microorganism and the oil / fat decomposer may be in a liquid form or a solid form. Examples of the liquid form include a microorganism culture solution itself, a microorganism collected by centrifugation or the like, and dispersed again in water or the like. The solid form can be obtained, for example, by freeze-drying cultured cells, and can be made into powder, granules, tablets, and the like. Moreover, you may fix microorganisms to various support | carriers.
If the fat and oil decomposer contains at least one kind of lipase-secreting microorganism that can be grown and decomposed under mildly acidic conditions such as Burkholderia arboris SL1B1, it will increase the activity of more kinds of microorganisms and microorganisms. Or may contain a substance for maintaining for a long time.
The oil-and-fat decomposition technology and the oil-and-oil decomposer according to the present invention can be applied to the treatment of all wastewater containing oil and fat as well as grease traps. In addition, when applied to a grease trap, a separate decomposition treatment tank may be provided, but it is also possible to introduce an oil and fat decomposing agent or a microorganism directly into the grease trap and perform the decomposition treatment in the grease trap.

As Example 1, 10 g / L canola oil was added to an inorganic salt medium (20 mL) adjusted to pH 7 or pH 6, and Burkholderia arboris SL1B1 strain was inoculated thereto, and cultured with shaking at 28 ° C. . The culture medium was collected over time, and OD660 was measured to examine the ability of the cells to grow at both pHs. Moreover, as Comparative Example 1, a commercially available microbial preparation was cultured under the same conditions, and OD660 was measured.
The measurement results of OD660 are shown in FIG. 1 (Comparative Example 1) and FIG. 2 (Example 1). It can be seen that the commercially available microorganism of Comparative Example 1 can grow at pH 7, but cannot grow at pH 6. In contrast, it is clear that the Burkholderia arboris SL1B1 strain shown in Example 1 can grow well at pH 7 as well as at pH 7.
As Example 2, a 5 L fermenter was used to inoculate the lipase-secreting bacterium Burkholderia arboris SL1B1 strain in an inorganic salt medium (3 L) containing 10 g / L of canola oil, and the mixture was stirred at 28 ° C. and pH 6 and stirred. Cultured. The medium was collected over time, and the total fatty acid amount, the ammonium ion concentration, and the lipase activity were determined by combining CFU, the fatty acid contained in the oil and fat, and the free fatty acid. The result is shown in FIG. It was again shown that the Burkholderia arboris SL1B1 strain consumed ammonium ions (triangles) as a nitrogen source and proliferated vigorously as indicated by an increase in CFU (diamonds) even under pH 6 conditions. Moreover, it was clearly shown by the measurement of lipase activity (open circles) that the lipase was secreted even at pH 6, and the total amount of fatty acids (filled circles) actually decreased and was completely decomposed within 40 hours. That is, 10 g / L of oil was completely removed.
An experiment was conducted in which an oil removing agent containing Burkholderia arboris SL1B1 strain was actually administered to the grease trap. The grease trap uses the facilities of the university co-op, and its configuration is the same as that conventionally known, so a detailed explanation is omitted. However, the outline is composed of three tanks partitioned by plates, and each tank is connected at the bottom. ing. The drainage flows into the first tank, flows through the lower opening to the second tank and further to the third tank, and finally flows out from the third tank. The internal volume is 200 L, and the average residence time of the waste water is 12 minutes. The fat removal agent should be administered immediately after the inflow and outflow of wastewater from the Co-op cafeteria kitchen stopped at night, and water for measurement of normal hexane values should be collected before the addition of the fat removal agent and the next morning. We went just before the inflow and outflow began again. 400 mL of the oil and fat removing agent was put into the first tank every night.

Table 1 shows the results of adding oil removing agent and decomposing oil in the grease trap. When the oil removing agent was not used, the first day when the administration of the oil removing agent was started, the normal hexane value (mg / L) after one week and three weeks after the continuous administration were respectively shown in Comparative Example 2, Example 3, and Examples 4 and 5 are shown. As is apparent from the results shown in Table 1, it was found that the oil and fat removing agent according to the present invention has a significant oil content reducing effect even in the grease trap and is effective for purification of the grease trap.

Claims (5)

A fat and oil-containing wastewater treatment method comprising decomposing oil and fat with a lipase-secreting microorganism capable of decomposing and growing under mildly acidic conditions. The fat-and-oil wastewater treatment method according to claim 1, wherein the lipase-secreting microorganism is Burkholderia arbolis. A method for purifying a grease trap, wherein oil accumulated in a grease trap is removed in a trap tank by the method for treating oil-containing wastewater according to claim 1 or 2. An oil and fat degrading agent containing lipase-secreting microorganisms that can decompose and grow even under mildly acidic conditions. The fat and oil decomposing agent according to claim 4, wherein the lipase-secreting microorganism is Burkholderia arboris.