CN114456982A

CN114456982A - Bacillus brevis and application thereof in degradation or digestion of sludge

Info

Publication number: CN114456982A
Application number: CN202210202283.2A
Authority: CN
Inventors: 刘圣鹏; 高素芹
Original assignee: Qingdao Weilan Saide Biotechnology Co ltd
Current assignee: Qingdao Weilan Saide Biotechnology Co ltd
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-05-10
Anticipated expiration: 2042-03-03
Also published as: CN114456982B

Abstract

The invention relates to a Brevibacillus brevis (Brevibacillus sp) capable of degrading or digesting sludge, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 24389.

Description

Bacillus brevis and application thereof in degradation or digestion of sludge

Technical Field

The invention relates to a Brevibacillus brevis (Brevibacillus sp.), in particular to a Brevibacillus borstelensis capable of degrading or digesting sludge, belonging to the technical field of environmental microbiology.

Background

At present, more than 80% of sewage treatment plants in the world adopt an activated sludge method for sewage treatment, and according to the statistics of data published in 2015 of the environmental protection department of China, the daily sewage treatment capacity of China is not lower than 1.7 billion cubic meters, and the sludge yield is not lower than 15 million tons. Therefore, the excess sludge yield and the increase speed in China are not small.

Statistics shows that the equipment investment is about 30-40% of the total investment in the establishment of a sewage treatment plant, the cost for treating the sludge is about 20-50% of the total operation cost after the establishment, and the cost and the investment for treating the sludge are very high. Meanwhile, the sludge contains a large amount of toxic and harmful substances, and if the sludge is not effectively treated, secondary pollution to soil, underground water and the like is very easy to cause, and the public health and environmental safety are directly threatened, so that the problem needs to be effectively solved. In view of the characteristics of large sludge yield, high production speed, high treatment and operation cost, great environmental hazard and the like, sludge treatment and disposal become an increasingly prominent problem and have attracted more and more attention and research.

At present, sludge treatment modes at home and abroad are many, and common sludge treatment methods generally comprise sanitary landfill, incineration, land utilization, composting and the like.

(1) Sanitary landfill

The sanitary landfill method is common in China, the required cost is low, the operation is simple, the effect is quick, and a landfill site is used for landfill, so that the required site is large, toxic organic matters and inorganic matters in sludge can permeate into soil, and secondary harm is caused to land resources and the environment.

(2) Land utilization

The sludge can be used as fertilizer for gardens and woodlands after effective sterilization and deodorization; after the sludge is injected into the damaged barren land, the barren land can be improved due to the fact that the flocculent structure of the sludge has certain viscosity and a large amount of organic and inorganic matters are contained; the sludge is constructed into the artificial wetland, the diversity of the ecological system is more stable by utilizing the solar energy, and the resource utilization of the sludge is improved.

(3) Sludge incineration

Sludge incineration allows efficient sludge reduction, a process which is common in europe and japan. The heat energy and the electric energy can be recovered in the incineration process, and the generated slag can be made into new coal or building materials. According to statistics, before 2016, the amount of sludge incinerated in China accounts for 3% of all sludge treatment technologies, and the amounts of sludge incinerated in the United states, Japan and European Union account for 22%, 68% and 20-40% respectively, and the sludge incinerating technology in China is relatively less in application because of worrying about the air quality problem in China and is determined by the technology, the national conditions and the economy of China.

(4) Aerobic composting

The sludge composting is that sludge and some plant straws are piled together, external conditions are manually controlled, a large number of microorganisms in the sludge are utilized for fermentation, organic matters are converted into humus, the volume and the quality of the sludge are reduced, partial odor taste and pathogens are eliminated, and the sludge compost can also be used as organic fertilizer required by plants. However, the compost is long in time demand, and heavy metals and organic pollutants are difficult to remove fundamentally.

In the face of the severe situation of the existing sludge treatment, the way of the kettle bottom paying is to reduce the generation of sludge from the source and treat the sludge from the sludge reduction angle. First, the content of organic and inorganic substances in the sludge is reduced, and the growth of microorganisms in the sludge is inhibited to reduce the amount of excess sludge.

Disclosure of Invention

Aiming at the severe form of sludge treatment and the defects of the existing sludge treatment method, the invention provides the brevibacillus brevis and the microbial agent which can degrade or digest organic matters and inorganic matters in sludge and further play a role in reducing the amount of the sludge, and the application thereof.

A Bacillus brevis (Brevibacillus sp.) BV2-4, deposited in China general microbiological culture Collection center with the address of: the microorganism institute of Zhongke institute No. 3, Xilu No. 1, Beijing, Chaoyang, with the preservation number: CGMCC No.24389, the preservation date is: no special mention is made of the strain BV2-4 in Brevibacillus brevis, which is shown in SEQ ID No. 1 at 10/2 in 2022, and the 16S rDNA sequence thereof.

The brevibacillus brevis provided by the invention is obtained by screening environmental sludge, protease, amylase and other enzymes are secreted in the fermentation process of the strain, and the metabolic activity and metabolic products of the brevibacillus brevis can decompose organic matters and inorganic matters in the sludge, so that the digestion and degradation of the sludge are promoted together, and the sludge reduction effect is achieved. 16S rDNA sequence test and alignment are carried out, and the strain is identified as Brevibacillus borstelensis (Brevibacillus borstelensis).

The brevibacillus brevis of the invention has the following characteristics:

(1) multiple groups of experimental tests prove that the activating solution of the strain has a sludge reduction effect of more than 7% on sludge of starch factories and food factories at an experimental temperature of 30 ℃, and the sludge degradation rate is up to 11.1% under the condition of application amount of 1000 ppm;

(2) the strain is high temperature resistant, and the sludge degradation rate in 3 days reaches 12% under the conditions that the temperature is 45 ℃, the dissolved oxygen is sufficient, and the inoculation amount of a strain activating solution is 100 ppm;

(3) the strain has strong stress resistance, the yield of the prepared bacterial powder can reach 92 percent, the yield of the material can reach 96 percent, the production process is mature, the cost is low, the shelf life is long, and the strain is easy to popularize and apply.

The invention also claims a microbial agent containing the brevibacillus brevis, and the microbial agent is preferably solid bacterial powder based on the requirement of convenience in storage and use.

The invention also claims a method for degrading or digesting sludge using the activating solution of brevibacillus brevis or the microbial agent comprising brevibacillus brevis, comprising the step of applying the activating solution of brevibacillus brevis or the microbial agent comprising brevibacillus brevis to sludge.

Preferably, the amount of the activating solution and the microbial agent applied to Brevibacillus brevis is 50ppm or more, more preferably 50 to 2000ppm, and most preferably 100-1000 ppm.

Preferably, the temperature is controlled to be 15-45 ℃ in the process of degrading or digesting the sludge, and preferably 30-45 ℃.

The invention also requires the use of the protected brevibacillus brevis and the microbial agent containing the brevibacillus brevis in the degradation or digestion of sludge.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1 screening and Performance testing of strains

1. Preliminary screening

Collecting sludge of a certain sewage treatment plant, weighing 10g of sludge, transferring the sludge into a 250ml triangular flask filled with 90ml of sterile water, and shaking at 180rpm for 20 min. Then sucking 1ml of the diluent, transferring the diluent into a test tube filled with 9ml of sterile water, and sequentially diluting to 10^-3、10^-4、10^-5、10^-6And 10^-7And respectively sucking 100 mu l of each diluent to a separation culture medium (10% sludge agar culture medium) plate, uniformly coating, and then inversely placing at 30 ℃ for culturing for 24-48h until a single colony grows out. Selecting single colony with different forms and larger hydrolysis ring, transferring to nutrient agar test tube slant, culturing at 30 deg.C for 48-72 hr, and transferring to 4 deg.C refrigerator for storage.

Obtaining 4 strains according to the separation method, wherein the strains are respectively numbered as follows: BV 2-1, BV 2-2, BV 2-3 and BV 2-4.

2. Double sieve

Respectively selecting 1 ring of 4 strains obtained by primary screening in a sterile environment, inoculating the 4 strains in a 250ml triangular flask containing 100ml LB culture medium, culturing for 24h under the conditions of 30 ℃ and 180rpm, and activating to obtain an activation solution.

20. mu.l of each activating solution was pipetted and inoculated into a plastic bottle containing 200ml of sterilized municipal sludge, and the mixture was subjected to static culture at 30 ℃. And (4) replacing the activating solution with sterile water as a blank, and detecting the centrifugal dehydration rate of the sludge after two days.

The detection method of the centrifugal dehydration rate comprises the following steps: 50ml of sludge is accurately measured and centrifuged for 10min at 6000r/min, and the ratio of the volume of the residual wet sludge after supernatant liquid is poured to the volume of the original sludge represents the centrifugal dehydration rate.

TABLE 1 sludge centrifuge dewatering ratio of each strain

According to the detection results in the table 1, the 4 strains of the primary screening strains already show better solid-liquid separation effect, wherein the centrifugal dehydration rate of the BV2-4 strain is the highest and is 4.08 percent higher than that of the control, so that further identification and test are carried out.

Example 2 identification of strains

1. Experimental methods

1.1 extraction of bacterial genomic DNA

(1) Collecting 1.0X 10 with 2ml centrifuge tube⁹(1ml of bacterial liquid OD₆₀₀1-1.5), centrifuged at 12,000 Xg for 30s, discardedAnd (6) supernatant fluid. The pellet was suspended with 150. mu.l Buffer S to which RNase A had been added.

(2) Add 20. mu.l lysozyme stock solution, mix well, and let stand at room temperature for 5 min.

(3) Add 30. mu.l of 0.25mol/L EDTA (pH 8.0), mix well and ice-wash for 5 min.

(4) Add 450. mu.l Buffer G-A, vortex for 15s, water bath at 65 ℃ for 10 min.

(5) Mu.l of Buffer G-B and 1ml of Buffer DV (precooled at 4 ℃) were added, mixed vigorously and centrifuged at 12,000 Xg for 2 min.

(6) The upper phase was discarded as much as possible, leaving the interphase precipitate and the lower phase. 1ml of precooler DV at 4 ℃ was added, mixed vigorously and centrifuged at 12,000 Xg for 2 min.

(7) The upper phase was discarded and the lower phase was transferred to a filter (filter placed in a 2ml centrifuge tube) and centrifuged at 12,000 Xg for 1 min.

(8) The filter was discarded, 400. mu.l Buffer BV was added to the filtrate and mixed well.

(9) The preparation tube was placed in a 2ml centrifuge tube, the mixture from step 8 was transferred to the preparation tube and centrifuged at 12,000 Xg for 1 min.

(10) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube, 500. mu.l of Buffer W1 was added, and centrifugation was carried out at 12,000 Xg for 1 min.

(11) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube, and 700. mu.l of Buffer W2 was added and centrifuged at 12,000 Xg for 1 min.

(12) In the same manner, 700. mu.l of Buffer W2 was washed once more.

(13) The filtrate was discarded, and the preparation tube was returned to the original 2ml centrifuge tube and centrifuged at 12,000 Xg for 1 min.

(14) The preparation tube was placed in another clean 1.5ml centrifuge tube, and 200. mu.l of Eluent or deionized water was added to the center of the silica membrane and allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12,000 Xg for 1 min.

2. PCR amplification of bacterial genomes

TABLE 2 PCR amplification primer design

Primer name	Sequence of
		27F	5-AGAGTTTGATCCTGGCTCAG-3
1492R	5-CTACGGCTACCTTGTTACGA-3

PCR amplification reaction system

The following ingredients were added to a 0.2ml centrifuge tube:

TABLE 3 PCR amplification reaction System

Flicking and mixing evenly, collecting liquid drops on the tube wall to the tube bottom by instantaneous centrifugation, and carrying out PCR reaction on a PCR amplification instrument, wherein the reaction parameters are shown in Table 4:

TABLE 4 PCR amplification reaction procedure

Pre-denaturation	Denaturation of the material	Annealing	Extension	Final extension	Number of cycles
						95℃，5min	95℃，30s	58℃，30s	72℃，1min30s	72℃，7min	35

After the reaction was completed, 3. mu.l of the PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR-amplified fragment.

3. Recovery of PCR products

The PCR product is recovered by using an AxyPrep DNA gel recovery kit, the specific operation is carried out according to the kit instruction, and the steps are as follows:

(1) the agarose gel containing the desired DNA was cut under an ultraviolet lamp and placed in a clean centrifuge tube and weighed.

(2) 3 gel volumes of Buffer DE-A were added, mixed well and heated at 75 ℃ until the gel mass was completely melted.

(3) Adding 0.5 Buffer DE-B with the volume of the Buffer DE-A, and uniformly mixing; when the isolated DNA fragment was less than 400bp, 1 gel volume of isopropanol was added.

(4) The mixture was transferred to a DNA preparation tube and centrifuged at 12,000 Xg for 1min, and the filtrate was discarded.

(5) The preparation tube was put back into a 2ml centrifuge tube, 500. mu.l of Buffer W1 was added, and the mixture was centrifuged at 12,000 Xg for 30 seconds, and the filtrate was discarded.

(6) The preparation tube was put back into a 2ml centrifuge tube, 700. mu.l of Buffer W2 was added, and the mixture was centrifuged at 12,000 Xg for 30 seconds, and the filtrate was discarded. The cells were centrifuged again at 700. mu.l Buffer W2, 12,000 Xg, for 1min in the same manner.

(7) The prepared tube was placed back into a 2ml centrifuge tube and centrifuged at 12,000 Xg for 1 min.

(8) The preparation tube was placed in a clean 1.5ml centrifuge tube (provided in the kit), 25-30. mu.l deionized water was added to the center of the preparation membrane, and the membrane was allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12,000 Xg for 1 min.

4. Sequence determination and analysis

Taking the PCR products after each strain purification, using a sequencer ABI3730-XL to carry out DNA sequencing, and the 16S rDNA gene sequence determination result of the strain BV2-4 is shown as SEQ ID No. 1.

5. Sequence analysis

The NCBI Blast program is used for comparing the spliced sequence file with data in an NCBI 16S database to obtain species information with the maximum sequence similarity with the species to be detected, namely the identification result, which is identified as Brevibacillus borstelensis (Brevibacillus borstelensis), and the result is shown in Table 5.

TABLE 5 sample NCBI alignment results

Example 3 preparation of Bacillus brevis preparation

1. First order Shake flask activation

Selecting a strain of 1-ring brevibacillus brevis BV2-4 in a sterile environment, inoculating the strain into a 250ml triangular flask filled with 100ml enrichment medium (LB medium), and culturing for 24h under the conditions of 45 ℃ and 180rpm to obtain a primary activating solution.

2. Two stage shake flask culture

In an aseptic environment, respectively transferring the primary activating solution into 50ml to four 1L triangular flasks filled with 450ml LB culture medium, culturing for 24h on a shaking table at 45 ℃ and 180rpm, wherein the pH is not required to be controlled in the process, the fermentation is stopped when the spawn is more than 90 percent and the maturity is more than 90 percent, so as to obtain fermentation liquor, the number of viable bacteria is up to 200 hundred million cfu/ml, the vitality of the thallus is strongest, the residue of fermentation nutrient substances is least, and the number of the stored viable bacteria is less attenuated.

TABLE 6 variation of spore rates and maturity during fermentation

4. Enzyme activity detection

Protease activity detection: centrifuging the fermentation liquid at 10000rpm and 4 ℃ for 30min, collecting supernatant, and determining the neutral protease activity of the fermentation liquid by Folin-phenol color development method, wherein 1ug of tyrosine generated by hydrolyzing casein per minute is 1 enzyme activity unit (U/ml) under the condition that the pH value is 7.2.

Detection of amylase activity: centrifuging the fermentation liquid for 30min at 10000rpm and 4 ℃, collecting supernatant, and measuring the amylase activity of the fermentation liquid by adopting a spectrophotometer method, wherein the enzyme amount required for liquefying 1g of starch per hour is 1 enzyme activity unit (U/ml) under the condition that the pH value is 6.0.

TABLE 7 variation of enzyme Activity during fermentation

5. Preparation of microbial inoculum

And (3) mixing the fermentation liquor, centrifuging at 4000rpm for 10min to obtain bacterial sludge, adding calcium carbonate into the bacterial sludge according to the mass ratio of 1:1, uniformly mixing, and drying to obtain 115g of bacterial powder.

1.0000g of bacterial powder was transferred to a 250ml triangular flask filled with 99ml of sterile water and glass beads spread on the bottom, and shaken at 180rpm for 20 min. Then, another 1ml of the diluted solution was aspirated and transferred to a test tube containing 9ml of sterile water, and then sequentially diluted to 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8And 10^-9Respectively suck 10^-7、10^-8、10^-9And (3) adding 100 mu l of each diluent to a nutrient agar plate, uniformly coating, then inverting the nutrient agar plate, culturing for 24-48h at the temperature of 30 ℃ until a single colony grows out, then counting, and taking the average number of the colonies on the plate, namely the bacterial powder quantity.

The bacterial count of the bacterial powder is (305+335+319)/3 multiplied by 10⁹+(32+31+33)/3×10¹⁰＝3.2×10¹¹cfu/g

Yield of bacterial quantity＝(3.2×10¹¹)cfu/g×115g/2×10¹⁰cfu/ml×2000ml＝92％

The yield of the material was 115g/(0.06g/ml × 2000 ml): 95.83%

Example 4 evaluation of sludge degradation Effect in starch factories of Shandong

1. Activation of bacterial species

Selecting 1-ring Brevibacillus brevis BV2-4 strain in sterile environment, inoculating into 250ml triangular flask containing 100ml enrichment medium (LB medium), culturing at 30 deg.C and 180rpm for 24 hr to obtain activated bacteria solution, diluting to OD₆₀₀The concentration of 1 is ready for use.

2. Evaluation test of degradability

Respectively adding 10 mul, 50 mul and 200 mul of activated bacteria liquid (respectively corresponding to the addition amount of 50ppm, 250 ppm and 1000ppm of the activated bacteria liquid) into a mineral water bottle filled with 200g of sludge of a starch factory in Shandong, arranging 1 blank control group for replacing the activated bacteria liquid with sterile water, arranging 3 parallel experiments in each experiment group, placing the blank control group and each experiment group into an incubator at 30 ℃ for static culture, and detecting MLSS after 3 days and 7 days, wherein the MLSS is the suspended solid concentration of the mixed liquid and represents the content of solid in the sludge.

3. Results of the experiment

The results of the evaluation experiments are shown in the following table.

TABLE 8 degradation effect of different amounts of applied bacteria solutions on sludge from starch plants in Shandong province

As is clear from Table 8, when the amount of the applied bacteria liquid was 1000ppm at 30 ℃, the bacteria could degrade MLSS of sludge by 10.63% in 3 days, and even if only 50ppm was added, the bacteria could degrade MLSS of sludge by 7.19 in 3 days, so that the bacteria had a significant digesting effect on sludge. In addition, the sludge degradation rate after 7 days is not much different from that after 3 days, so the strain can basically complete the sludge degradation process within 3 days.

Example 5 evaluation of sludge degrading ability in certain food plant

1. Activation of bacterial species

Selecting 1-ring Brevibacillus BV2-4 strain in sterile environment, inoculating into 250ml triangular flask containing 100ml enrichment medium (LB medium), culturing at 30 deg.C and 180rpm for 24 hr to obtain activated bacteria solution, diluting to OD₆₀₀The concentration of 1 is ready for use.

2. Evaluation experiment of sludge degradation capability

Adding 20 μ l of activated bacteria solution (corresponding to the addition amount of 100ppm) into mineral water bottle containing 200g of sludge from certain food factory, respectively, standing and culturing in 15 deg.C, 30 deg.C, 45 deg.C incubator and 45 deg.C, shaking at 180rpm, detecting MLSS after 3 days, and setting 3 parallel experiments and 1 blank control group with activated bacteria solution replaced by sterile water for each experimental group.

3. Results of the experiment

The results of the evaluation experiments are shown in the following table.

TABLE 9 evaluation of sludge degrading ability of certain food plant

As is clear from Table 9, in the same condition of the application amount of 100ppm, the sludge degradation rate after static culture at 45 ℃ was 8.9% higher than that after static culture at 15 ℃ for 3 days, and the sludge degradation rate after static culture at 45 ℃ was 1.66% higher than that after static culture, so that the sludge degradation effect was the best when the temperature was high and the dissolved oxygen was sufficient.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Islands Ulmarie science and technology Limited

<120> Bacillus brevis and application thereof in degradation or digestion of sludge

<130> 2

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1429

<212> DNA

<213> Brevibacillus brevis (Brevibacillus sp.)

<400> 1

gtgctgttat aatgttagtc gagcgagtcc cttcgggggc tagcggcgga cgggtgagta 60

acacgtaggc aacctgcccg taagctcggg ataacatggg gaaactcatg ctaataccgg 120

atagggtctt ctctcgcatg agaggagacg gaaaggtggc gcaagctacc acttacggat 180

gggcctgcgg cgcattagct agttggtggg gtaacggcct accaaggcga cgatgcgtag 240

ccgacctgag agggtgaccg gccacactgg gactgagaca cggcccagac tcctacggga 300

ggcagcagta gggaattttc cacaatggac gaaagtctga tggagcaacg ccgcgtgaac 360

gatgaaggtc ttcggattgt aaagttctgt tgtcagagac gaacaagtac cgttcgaaca 420

gggcggtacc ttgacggtac ctgacgagaa agccacggct aactacgtgc cagcagccgc 480

ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg 540

ctatgtaagt ctggtgttaa agcccggggc tcaaccccgg ttcgcatcgg aaactgtgta 600

gcttgagtgc agaagaggaa agcggtattc cacgtgtagc ggtgaaatgc gtagagatgt 660

ggaggaacac cagtggcgaa ggcggctttc tggtctgtaa ctgacgctga ggcgcgaaag 720

cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctag 780

gtgttggggg tttcaatacc ctcagtgccg cagctaacgc aataagcact ccgcctgggg 840

agtacgctcg caagagtgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900

atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ccgctgaccg 960

tcctagagat agggcttccc ttcggggcag cggtgacagg tggtgcatgg ttgtcgtcag 1020

ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat ctttagttgc 1080

cagcattcag ttgggcactc tagagagact gccgtcgaca agacggagga aggcggggat 1140

gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggctggtac 1200

aacgggaagc tagctcgcga gagtatgcca atctcttaaa accagtctca gttcggattg 1260

caggctgcaa ctcgcctgca tgaagtcgga atcgctagta atcgcggatc agcatgccgc 1320

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgggag tttgcaacac 1380

ccgaagtcgg tgaggtaacc gcaaggagcc agccgccgaa agagggacc 1429

Claims

1. A Brevibacillus brevis (Brevibacillus sp.) capable of degrading or digesting sludge is characterized by being preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 24389.

2. A microbial agent characterized in that the effective ingredient comprises the Bacillus brevis of claim 1.

3. The microbial agent according to claim 2, wherein the microbial agent is a solid bacterial powder.

4. A method for degrading or digesting sludge, comprising the step of applying an activated liquid of Bacillus brevis as set forth in claim 1 or a microbial agent as set forth in claim 2 or 3 to sludge.

5. The method according to claim 4, wherein the amount of the activating solution for Brevibacillus and the microbial agent to be applied is 50ppm or more.

6. The method as claimed in claim 5, wherein the activating solution for Brevibacillus brevis and the microbial agent are applied in an amount of 50 to 2000 ppm.

7. The method as claimed in claim 6, wherein the activating solution and the microbial agent are applied in an amount of 100-1000 ppm.

8. The method according to any one of claims 4 to 7, wherein the temperature is controlled to be 15 to 45 ℃ during the degradation or digestion of the sludge.

9. The method according to claim 8, wherein the temperature is controlled to be 30-45 ℃ during the degradation or digestion of the sludge.

10. Use of the brevibacillus brevis of claim 1 and the microbial agent of claim 2 or 3 for degrading or digesting sludge.