JP2006230332A - New microorganism and method for treating organic sludge therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective and easily treatable microorganism for reducing and utilizing excessive sewage sludge or the like as resources, and to provide a method for treating organic sludge such as sewage sludge with the microorganism. <P>SOLUTION: This new gram-positive microorganism which belongs to Brevibacillus bacteria, produces a protease, and has an organic sludge-degrading ability. The microorganism is preferably a microorganism having a reducing dissolution ability of at least 10% for floating solid contents having a sewage sludge weight concentration, especially Brevibacillus sp. KH3 strain as an accession number FERM P-20399. The microorganism is used to dissolve the organic sludge, thereby efficiently treating the organic sludge. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a novel microorganism capable of decomposing organic sludge and a method for treating sewage sludge, various biological sludges or organic sludge using the microorganisms.

With the spread of sewerage, the amount of sewage sludge generated in the country tends to increase year by year. Sewage and wastewater are usually treated by the activated sludge method, but the amount of surplus sludge generated at that time continues to increase year by year, and sewage surplus sludge is ranked first in the proportion of industrial waste in Japan. is occupying. According to the report of the Japan Sewage Works Association, the amount of sludge generated in the sewage treatment plants in fiscal 2002, up to 430 million m ³ or more, the effective utilization rate was 60%. On the other hand, the remaining 40% is disposed of in landfills, but it has become difficult to secure landfill sites every year, and the remaining years are estimated to be 3.9 years in Japan. In addition, waste such as surplus sludge, animal manure and other organic sludge is recognized as an important measure as well as expansion of the recycling frame as a resource circulation.

In order to efficiently carry out the treatment of sludge with microorganisms, search for beneficial microorganisms has been attempted. For example, JP 2000-139449 (Patent Document 1) belongs to the genus Bacillus and is alkaline. Japanese Unexamined Patent Application Publication No. 2002-125657 (Patent Document 2) discloses a microorganism having the ability to decompose sludge under conditions, and a microorganism belonging to Bacillus subtilis that decomposes proteins contained in organic sludge and biological sludge. Japanese Patent No. 2003-235547 (Patent Document 3) discloses a microorganism belonging to the genus Pseudomonas that has the ability to decompose and extinguish organic substances in organic waste and sewage sludge. Japanese Patent Application Laid-Open No. 2004-267127 (Patent Document 5) discloses a microorganism belonging to the genus Rhodobacter that is useful for environmental purification such as wastewater treatment. Disclosed are microorganisms belonging to the genus Bacillus that are useful for the treatment of products. However, it cannot always be said that it is sufficient for practical use industrially.
JP 2000-139449 A JP 2002-125657 A JP 2003-235547 A JP 2003-245066 A JP 2004-267127 A

A technique for reducing excess sludge using a thermophilic bacterium belonging to the genus Bacillus has also been proposed (Non-Patent Document 1). However, in order for this thermophilic bacterium to act on growth and sludge, a high temperature such as 65 ° C is necessary, and in order to satisfy this condition, a large amount of expenses such as operating costs and capital investment are inevitable. There is.
Susumu Hasegawa “Sludge Reduction and Occurrence Prevention Technology” pp. 248-270, NTS (2000)

An object of the present invention is to find an efficient and easy-to-handle microorganism for reducing and recycling sewage surplus sludge and to provide a method for treating organic sludge such as sewage sludge using the microorganism. There is.

The invention described in claim 1 is a novel Gram-positive microorganism belonging to the genus Brevibacillus, which produces a proteolytic enzyme and has an ability to decompose organic sludge.

And invention of Claim 2 concerns the microorganisms which further limited the functional characteristic among the microorganisms of Claim 1, and the floating solid content whose weight concentration of a sewage sludge is 25% is 30-50 degreeC, The novel microorganism according to claim 1, which has a lytic ability capable of being reduced by 10% or more after 48 hours of culture.

The invention of claim 3 relates to a strain of Brevibacillus sp. KH3 deposited at the Patent Organism Depositary of National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-20399. As long as it has substantially the same characteristics as this strain, the mutant is also included in the invention of claim 3.

Furthermore, another aspect of the present invention is a method for treating organic sludge, characterized in that organic sludge is solubilized using the microorganism according to any one of claims 1 to 3.

In the sludge reduction treatment using the strain of Brevibacillus sp. KH3 of the present invention, it is not necessary to incorporate a new apparatus into the sewage treatment system in order to reduce excess sewage sludge generated in the conventional activated sludge method. And since this strain grows in the temperature range of 30 ° C to 50 ° C, the utility costs for culturing can be put into practice relatively inexpensively and in industries such as reducing the amount of excess sewage sludge and improving the standard activated sludge method. The above usage can be expected.

In the present invention, organic sludge refers to biological sludge such as raw sludge and surplus sludge discharged from sewage treatment processes such as sewage treatment plants and manure treatment plants, or manufacturing processes or wastewater treatment processes such as food factories and chemical factories. All organic sludge discharged from the plant is included. The microorganism of the present invention is capable of producing a proteolytic enzyme and decomposing these organic sludges, and is scientifically a Gram-positive microorganism belonging to the genus Brevibacillus.

The present inventors have found such microorganisms by screening microorganisms that dissolve sludge from surplus sludge conditioned at 50 ° C. for the purpose of reducing and recycling sewage surplus sludge. That is, using conditioned sludge as a sample, it was applied to a medium in which sterilized sludge was suspended, and those in which halo was observed around colonies of grown bacteria were separated. The isolated bacterium was a Gram-positive gonococcus, and as a result of biochemical and physiological tests and 16S ribosomal RNA gene DNA homology analysis, it was found to be a bacterium belonging to the genus Brevibacillus. These tests were carried out according to the methods and means described in the Bergey's Manual of Systematic Bacteriology 2nd edition (1989).

So far, it has not been reported that bacteria belonging to the genus Brevibacillus solubilize sewage sludge. The bacterial strain isolated and identified by the inventors this time is at a temperature of 30 ° C to 50 ° C.
Dissolve sewage sludge. Biochemical and physiological tests on this strain identified it as Brevibacillus brevis with a 98% probability. Furthermore, in the DNA homology analysis of the 16S ribosomal RNA gene, it was determined with a probability of 92% Brevibacillus sp. RG-30 and Brevibacillus agri. From these results, this strain was judged to be a new species of bacteria belonging to the genus Brevibacillus, so this bacterial strain was named Brevibacillus sp. KH3.

This strain was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on February 14, 2005, under the accession number FERM P-20399.

The microorganism of the present invention is a Gram-positive rod and forms spores. Moreover, it is an aerobic bacterium with good growth on LB medium. And it can culture | cultivate on aerobic conditions at 30-50 degreeC, for example by LB culture medium (It is pH 7.0 containing 10g of bactotryptones, 5g of bacto yeast extract powder, and 5g of sodium chloride per 1000mL of culture media. Further, when cultured in LB medium at 30 ° C. for 18 hours, a round colony having a diameter of about 3 mm and a creamy flat shape is formed. The surface shape of the colony is smooth, opaque and has a butter-like consistency.

[Example 1] Separation and identification of bacteria 200 g of excess sewage sludge was placed in a 500 ml Erlenmeyer flask and cultured with shaking at 50 ° C and 60 rpm for 1 week, and the culture and the newly collected excess sludge were weighed. The mixture was mixed at a ratio of 1: 2 and continuously cultured. Dilute the culture solution (8 times continuous culture) repeated once a week, suspend sterilized surplus sludge, apply to a flat agar medium and culture at 50 ° C. Among them, those that could be confirmed as halo were isolated around the colony. The isolated bacterial strains were subjected to biochemical and physiological properties test (API20E) and carbohydrate fermentation test (API50CHB) to examine the morphological and biochemical and physiological properties of the strain. The results are shown in Tables 1 and 2.

Based on the culture conditions and the results in Tables 1 and 2, the fungus can grow in the temperature range of 30 ° C to 50 ° C and pH 6.0 to 9.0. Gram and spore staining, biochemical and physiological tests As a result of performing [API20E and API50CHB (Biomerieux, France)], it was found that the bacteria had high homology (98%) with the aerobic spore-forming gram-positive bacterium Brevibacillus brevis.

Next, we analyzed the DNA homology of the 16S ribosomal RNA gene, which is used as the most effective molecular marker for the study of microbial evolutionary strains. The 16S ribosomal RNA gene of this strain was amplified by PCR (63f forward primer: 5'-AGGCCTAACACATGCAAGTC-3 ', 1387r reverse primer: 5'-GGGCGGTGTGTACAAGGC-3', reaction conditions: (1) 95 ° C for 5 minutes, ( 2) 95 ° C for 1 minute, (3) 50 ° C for 1 minute, (4) 72 ° C for 1.5 minutes, 30 cycles of (2) to (4), (5) 72 ° C for 5 minutes), PCR products were ligated to plasmid vectors using TOPO XL PCR Cloning Kit (Invitrogen, USA). The plasmid containing the 16S ribosomal RNA gene was inserted into E. coli, and the plasmid was recovered from the cultured E. coli. Then, the sequence reaction was performed with M13 forward primer, M13 reverse primer, r2L primer (5'-CATCGTTTACGGCGTGGA-3 '), or r3L primer (5'-TTGCGCTCGTTGCGGGACT-3'), and DNA sequencer CEQ8000 (Beckman Coulter, USA) The base sequence was determined, and the DNA base sequence of the 16S ribosomal RNA gene of the sludge solubilizing bacterium was obtained. Based on the obtained base sequence of about 1000 bases, sequence matching was performed with BLAST (http://www.ddbj.nig.ac.jp/) which is a typical DNA homology search engine. As a result, as shown in Table 3, the homology was highest with Brevibacillus sp. RG-30 and Brevibacillus agri.

This strain is identified by biochemical and physiological tests, has a DNA homology of 98% or less of 16S ribosomal RNA gene, and dissolves sewage sludge as shown in Example 2 below. Therefore, this bacterium was named Brevibacillus sp. KH3 strain (Accession No. FERM P-20399).

[Example 2] Measurement of sludge reduction rate [adjustment of sterilized sludge]
300 g of excess sludge collected from the sewage treatment plant was taken into a 500 ml Erlenmeyer flask, steam sterilized at 121 ° C. for 20 minutes, transferred to a sterilized centrifuge tube, and centrifuged at 4 ° C. and 10,000 × g. The supernatant was discarded, and the remaining precipitate was washed three times with sterilized purified water, and then adjusted so that its weight concentration (w / v) was 25%. The weight concentration of normal sewage sludge is 20 to 30%.

[SS measurement]
The suspended solid (SS) in the sludge was centrifuged at 18,000 × g for 10 minutes, and the supernatant was removed. The precipitate was dried at 105 ° C. for 2 days, and its weight was measured.

[Measurement of sludge reduction rate]
30 ° C in LB liquid medium (Tryptone: 10 g / L, Yeast extract: 5 g / L, NaCl: 5 g / L, pH 7.0)
Brevibacillus sp. KH3 strain pre-cultured at 120 rpm for 16 hours was inoculated into the adjusted sterilized sludge (final bacteria amount of about 10 ⁶
cfu / ml), and each temperature was cultured at 60 rpm in a constant temperature shaker. The sterilized sludge was collected over time, and the change in the weight reduction rate was examined with the SS dry weight occupying the sludge as 100%.

As shown in FIG. 1, the Brevibacillus sp. KH3 strain dissolved sludge at a culture temperature of 30 ° C. to 50 ° C. In addition, the conditioned sludge was hardly dissolved in the culture at 20 ° C. and 60 ° C. (data not shown). The highest sludge reduction rate of 31.3% in this strain was at a culture temperature of 50 ° C. for 120 hours. It can be seen that at a culture temperature of 30 ° C. to 50 ° C., a dissolution rate of at least about 10% to 20% is exhibited in 48 hours. At the same time, growth of this strain is also observed (data not shown), so it is considered that Brevibacillus sp. KH3 strain dissolves sludge and grows using the lysate as a carbon source and nitrogen source.

[Example 3] Proteolytic enzyme produced by Brevibacillus sp. KH3
When the Brevibacillus sp. KH3 strain is applied to a flat agar medium in which sludge is suspended and cultured, a halo in which sludge is dissolved is formed around the formed colonies. That is, this strain is considered to dissolve sludge by releasing a solubilizing factor for sludge outside the cells. Moreover, when this strain is applied to casein or skim milk agar medium, a halo is formed around the colony, suggesting that this solubilizing factor is a proteolytic enzyme.

Then, when the activity of the proteolytic enzyme produced by the KH3 strain was examined, it was found that the highest proteolytic enzyme activity was exhibited when cultured at 50 ° C. for 48 hours as shown in FIG. As a result of examining the supernatant cultured for 48 hours by polyacrylamide electrophoresis, one thick band (molecular weight: about 60 kilodaltons) was observed. Furthermore, the activity of proteolytic enzyme could be confirmed by casein zymography (see Archives of Biochemistry and Biophysics, 319, 211-216 (1995)). From the above results, it was found that the sludge solubilizing factor is a proteolytic enzyme.

INDUSTRIAL APPLICABILITY The present invention can be used for improvement or improvement of microorganisms to be used or a treatment method in a facility that treats organic sludge using an activated sludge method or the like, such as a sewage treatment plant or a factory wastewater treatment facility .

The sludge dissolving ability of Brevibacillus sp. KH3 at each temperature is shown. The activity of the proteolytic enzyme in the supernatant cultured at each temperature and each time is shown.

Claims (4)

A new Gram-positive microorganism belonging to the genus Brevibacillus that produces proteolytic enzymes and has the ability to decompose organic sludge. The novel microorganism according to claim 1, which has a dissolving ability capable of reducing a suspended solid content of 25% by weight of sewage sludge by 10% or more by culturing at 30 to 50 ° C for 48 hours. The novel microorganism according to claim 1 or 2, which is Brevibacillus sp. KH3 strain (Accession No. FERM P-20399). A method for treating organic sludge, comprising solubilizing organic sludge using the novel microorganism according to any one of claims 1 to 3.

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