CN113528374B

CN113528374B - Lysis strain, sludge reduction treatment agent and application thereof

Info

Publication number: CN113528374B
Application number: CN202110623974.5A
Authority: CN
Inventors: 黄瑛; 张中玉; 迟宝岩; 段欣欣
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2023-04-21
Anticipated expiration: 2041-06-04
Also published as: CN113528374A

Abstract

The invention relates to a lysis strain, a sludge reduction treatment agent and application thereof, wherein the lysis strain is geobacillus @Geobacillus sp.) THE preservation number of THE THE-4 is CCTCC M2021512. Is obtained by separating and screening high-temperature domesticated mature sludge, the lysis strain and a sludge reduction treatment agent containing the lysis strain can be applied to sludge reduction treatment, and the lysis strain is bacillus terreusGeobacillus sp.) THE THE-4 can utilize organic matters in THE sludge to grow and reproduce, and enzyme generated in THE metabolic process of THE strain can accelerate THE hydrolysis of extracellular polymers and THE cracking of sludge cells, so as to achieve THE purpose of sludge lysis.

Description

Lysis strain, sludge reduction treatment agent and application thereof

Technical Field

The invention belongs to the field of biological sludge treatment, and particularly relates to a lysis strain, a sludge reduction treatment agent and application thereof.

Background

Biological wastewater treatment plants (WWTPs) have been used worldwide for treating municipal wastewater. Although the organic matter in the wastewater can be effectively removed, a large amount of surplus sludge is generated. With the increasing popularity of sewage treatment plants, the population is increasing, and the construction of new sewage treatment plants and the stricter sewage treatment requirements are such that the sludge yield will continue to increase. Thus, the treatment and disposal of sludge has become an increasingly significant challenge in the field of environmental engineering.

In conventional sewage treatment plants, there are mainly two methods for reducing the residual amount of sludge, namely a wastewater line and a sludge line. The in-situ sludge reduction technology is an important means for disposing sludge from the source, the common method is to recycle the backflow sludge after lysis to the main flow bioreactor for further biodegradation, and the physical, chemical and biological means are utilized to minimize the amount of sludge discharged outwards by the whole sewage treatment system, so that the method has good application prospect. Physical and chemical methods can result in additional energy consumption, high costs, secondary pollution, and other economic costs. In contrast, biological pretreatment shows absolute advantages in accelerating the sludge hydrolysis process.

Extracellular Polymers (EPS) are an important component of the matrix of sludge flocs, consisting of a number of organic substances such as organic macromolecules like polysaccharides, proteins, humic acids, uronic acids, lipid compounds, etc. The sludge reduction by adopting the biological pretreatment method is to add hydrolase into the sludge, accelerate the hydrolysis of extracellular polymers under the catalysis of the hydrolase, destroy the microbial cell structure and dissolve insoluble substances in the sludge. However, the direct addition of hydrolase has the problems of higher cost and more severe reaction conditions, and the actual large-scale application is difficult. Therefore, in order to improve the industrial application of the biological pretreatment method, it is necessary to separate and screen out a strain capable of reducing the sludge, and the hydrolysis of extracellular polymers is accelerated by enzymes generated by the strain in the metabolic process of the sludge, so as to achieve the purpose of sludge reduction.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a lysis strain, a sludge reduction treatment agent and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the technical scheme is as follows:

a lysis strain is Geobacillus sp THE-4, belongs to Geobacillus sp, and is preserved in China center for type culture Collection, with a preservation address of university of Wuhan, china, with a preservation time of 2021, 5 months and 11 days, and a preservation number of CCTCC M2021512.

Further, THE 16S rDNA sequence of THE Geobacillus sp.) THE-4 is shown in SEQ ID: 1.

Further, the lysis strain is obtained by separating and screening from high-temperature domesticated mature sludge.

Further, the high-temperature domesticated mature sludge is obtained by domesticating sewage sludge at 55-80 ℃.

Further, the activation method of the lysis strain comprises the following steps:

inoculating Bacillus terreus (Geobacillus sp.) THE-4 strain into liquid culture medium, and performing constant temperature shaking culture for activation to obtain OD ₆₀₀ 1.0-1.2, and centrifuging to collect thallus.

Further, THE inoculum size of THE Geobacillus (Geobacillus sp.) THE-4 in THE liquid culture medium is 1-5%; the temperature of shaking culture is 50-85 ℃, the shaking speed is 40-200rpm, the times of shaking culture at constant temperature are 2-3 times, and the time of shaking culture each time is 6-20h.

The second technical scheme is as follows:

a sludge reduction treatment agent comprising the lysis strain.

The technical scheme is as follows:

the application of the sludge reduction treatment agent in sludge reduction treatment.

The technical scheme is as follows:

a method for sludge reduction treatment by using the lysis strain comprises the following steps:

step one, uniformly blowing thalli of a lysis strain of Geobacillus (Geobacillus sp.) THE-4 with sterile water to obtain bacterial liquid;

step two, adding bacterial liquid into the organic sludge to be treated, adjusting the pH to 6-10, and carrying out lysis reaction under aeration conditions and stirring conditions.

Further, in the first step, the OD of the bacterial liquid ₆₀₀ The value is 0.4-0.8, in the second step, the adding amount of the bacterial liquid is 10-15% of the volume of the organic sludge to be treated, the temperature of the lysis reaction is 50-85 ℃, the aeration rate is 0.1-1.0vvm, the stirring rate is 50-200rpm, and the time of the lysis reaction is 1-36h.

Compared with the prior art, the invention has the following beneficial effects:

1. according to THE invention, THE mature sludge is domesticated at 55-80 ℃ to separate and screen THE lysis strains, and THE lysis strains of THE Geobacillus (Geobacillus sp.) THE-4 separated by THE method can carry out invisible growth in THE sludge to be treated, secrete extracellular enzymes, promote hydrolysis of macromolecular organic matters in THE sludge, so that organic matters in a solid phase of THE sludge can be released into a liquid phase for subsequent utilization.

2. Compared with the traditional physicochemical reduction technology, the method for the lysis of the sludge has the characteristics of economy, high efficiency, lower energy consumption, high efficiency of the lysis and easy control of reaction conditions, and in the sewage treatment process, the lysed sludge can continuously flow back to a biochemical tank to provide a carbon source for the denitrification process of the sewage, and simultaneously, a large amount of dissolved organic matters released after the lysis can be used for anaerobic digestion of the sludge, so that the resource utilization rate is improved.

Drawings

FIG. 1 is a phylogenetic tree of lytic strains;

FIG. 2 is a graph showing changes in THE activity of THE Bacillus terreus (Geobacillus sp.) THE-4 protease of example 2;

Detailed Description

The invention is further illustrated below with reference to examples.

The sewage sludge used for separation and screening of the invention is derived from: north treatment plant A in Nanjing City ² the/O process returns sludge in a sludge pump house; index of sewage sludge: TSS 13285 + -146 mg/L; VSS 6003+ -30 mg/L; TCOD 4350+ -88 mg/L; SCOD is 65+/-5 mg/L; pH is 6.8 plus or minus 0.2;

the sewage sludge used in the lysis test of the invention is derived from: a is that ² the/O process runs the surplus sludge of the sewage plant, and the sewage sludge has the following properties: total Suspended Solids (TSS) of 14685+ -146 mg/L, volatile Suspended Solids (VSS) of 6021+ -25 mg/L, total Chemical Oxygen Demand (TCOD) of 4350+ -88 mg/L, dissolved chemical oxygen demand of 65+ -5 mg/L, pH of 7.0-7.3. Sieving sludge to remove particles with particle size of more than 1mm, and storing in refrigerator at 4deg.C for no more than 1 week.

Control strain YL-1, belonging to the genus Geobacillus (Geobacillus sp.) deposited at the chinese collection of typical cultures, deposit address: university of martial arts in chinese; the preservation time is 2021, 5 months and 8; the preservation number is: cctccc AB 2021109; the control strain YL-1 is a publicly available strain.

Liquid medium: macroelements: nitrilotriacetic acid 100mg,NaCl 8mg,KH ₂ PO ₄ 111mg，MgSO ₄ ·7H ₂ O100 mg, peptone 1g, yeast extract 1g; trace elements: na (Na) ₂ MoO ₄ ·2H ₂ O 0.025mg，FeCl ₃ 0.28mg， CuSO ₄ 0.16mg，MnSO ₄ ·H ₂ O 2.2mg，H ₃ BO ₃ 0.5mg，ZnSO ₄ ·7H ₂ O 0.5mg，CoCl ₂ ·6H ₂ O 0.046 mg，CaSO ₄ 60mg. Adding 1L deionized water, adjusting pH to 7-7.5, placing in an autoclave, and sterilizing at 121deg.C for 20min.

Example 1 isolation, screening, identification and use of dominant lysates

The method mainly comprises the following steps of firstly separating and screening dominant strains from domesticated sludge, and secondly identifying the dominant strains; determining the species relation of dominant strains; 3. the optimal process for carrying out sludge reduction treatment by utilizing the screened dominant strain is obtained through process optimization.

1. Separating and screening the acclimatized sludge to obtain dominant strains, wherein the method specifically comprises the following steps:

step 1, domesticating sludge: domesticating sewage sludge under aeration condition of 0.1-1.0vvm at 55-80deg.C for 5-60 days;

step 2, performing gradient dilution on the domesticated and mature sludge, separating and purifying by adopting a dilution pouring method and a three-zone streaking method to obtain single-colony bacterial strains, and naming and numbering the bacterial strains respectively;

step 2.1, plate pouring: solid medium: macroelements: nitrilotriacetic acid 100mg, naCl 8mg, KH ₂ PO ₄ 111mg，MgSO ₄ ·7H ₂ O100 mg, peptone 1g, yeast extract 1g; qiongqiong teaFat 20g trace elements: na (Na) ₂ MoO ₄ ·2H ₂ O 0.025mg，FeCl ₃ 0.28mg，CuSO ₄ 0.16mg，MnSO ₄ ·H ₂ O 2.2mg，H ₃ BO ₃ 0.5mg，ZnSO ₄ ·7H ₂ O 0.5 mg，CoCl ₂ ·6H ₂ O 0.046mg，CaSO ₄ 60mg. Adding 1L deionized water, adjusting pH to 7-7.5, placing in an autoclave, and sterilizing at 121deg.C for 20min. And (3) after cooling to 50-55 ℃, pouring the agar into an aseptic culture dish in sequence, ensuring that the bottom of the aseptic culture dish is about 2/3 of that of the aseptic culture dish, and reversely buckling the flat plate on an aseptic table after agar is solidified.

Step 2.2, separation: taking a plurality of sterilized EP pipes, respectively adding 0.9ml of sterile water, taking 0.1ml of sludge domesticated and matured at 65 ℃, adding the sludge into the sterile water of a first pipe, fully mixing and shaking uniformly, taking 0.1ml of diluent from the first pipe into the next sterilized EP pipe, mixing and shaking uniformly, diluting to a fourth pipe and a fifth pipe in such a way that the dilution factors are respectively as follows from the first pipe: 10 ^-1 、 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 The method comprises the steps of carrying out a first treatment on the surface of the Drop-wise adding 10 ^-4 And 10 ^-5 Each of the two tubes was 0.1ml in each case, and the diluted solution was applied uniformly to the corresponding plate with a sterilized applicator rod. Placing the smeared flat plate on a sterile table for 20-25 min, and inverting the flat plate in an incubator for 12h when bacteria liquid permeates into the culture medium, wherein the culture temperature is 50-85 ℃. Selecting single bacterial colony by using a sterilized inoculating loop, streaking on a flat plate by adopting a three-area streaking method, repeating for 3-4 times to obtain purified bacterial colony, and separating 15 bacterial strains in the embodiment;

step 3, primarily screening the separated strains with single colonies through a hydrolysis circle test, further screening the strains with larger hydrolysis circles by adopting a sludge lysis test and an extracellular enzyme activity test, and retaining the strains with higher sludge VSS degradation rate and higher extracellular enzyme activity, namely the dominant strains; THE dominant strain obtained by screening in this example was THE-4;

hydrolysis circle test: inoculating the lysis strain separated from the domesticated mature sludge into a skimmed milk powder solid culture medium, placing the skimmed milk powder solid culture medium into a constant temperature incubator, and observing the change condition of hydrolysis circles after each strain is 12 hours.

The solid culture medium of the skim milk powder comprises the following components: macroelements: nitrilotriacetic acid 100mg,NaCl 8mg,KH ₂ PO ₄ 111mg, MgSO ₄ ·7H ₂ O100 mg, peptone 1g, yeast extract 1g, agar 20g, skimmed milk powder 20g, distilled water 1L; trace elements: na (Na) ₂ MoO ₄ ·2H ₂ O 0.025mg，FeCl ₃ 0.28mg，CuSO ₄ 0.16mg，MnSO ₄ ·H ₂ O 2.2mg，H ₃ BO ₃ 0.5mg，ZnSO ₄ ·7H ₂ O 0.5mg，CoCl ₂ ·6H ₂ O 0.046mg，CaSO ₄ 60mg. The pH is adjusted to 7.0-7.5.

Preliminary sludge dissolution test: taking a plurality of conical flasks and respectively adding sludge with the solid content of 1.2-1.5%; after the strains subjected to primary screening are respectively cultivated for 12-15 hours, sterile water is used for blowing and beating uniformly, then the strains are respectively inoculated into corresponding conical flasks, constant-temperature shaking table cultivation is carried out at the temperature of 50-85 ℃ and the oscillating speed of 100rpm, meanwhile, ultrapure water is used as blank control, the Volatile Suspended Solid (VSS) content of the sludge in each conical flask after 24 hours is measured, and the reduction rate of the VSS of the sludge is calculated.

Extracellular enzyme Activity assay: the strains after primary screening are respectively inoculated into a liquid culture medium according to the inoculum size of 1 percent, and the activity of protease is measured by adopting an azo casein method.

2. Identifying dominant strains and determining the species relationship of the dominant strains; specifically comprises physiological and biochemical identification and 16S rDNA identification:

1) Gram staining microscopy: THE dominant strain is THE-4, which is subjected to gram-staining microscopic examination, oxalic acid crystal violet dye liquor is sequentially subjected to dip-staining for 1min, iodine liquor is subjected to dip-staining for 1min,95% ethanol is decolorized for 30s, safranine liquor is subjected to dip-staining for 2min-3min, and a cover glass is placed under a microscope for observation, wherein THE observation results are as follows: THE cells of THE-4 were red, rod-like, and gram-negative.

2) 16S rDNA identification: THE nucleotide sequence of THE 16S rDNA of THE dominant strain THE-4 is shown as SEQ ID 1.

THE dominant strain THE-4 belongs to THE genus Geobacillus and THE phylogenetic tree constructed by adopting THE adjacency method is shown in figure 1.

3. And obtaining the optimal process for sludge reduction treatment by using the screened dominant strain through process optimization.

In this example, THE optimal process for sludge reduction treatment using THE dominant strain Geobacillus sp.) THE-4:

step 1, activating and culturing dominant strains:

inoculating dominant strain THE-4 into liquid culture medium at 1-5% of inoculation amount, shake culturing at 50-85deg.C for 2-3 times for activation to obtain OD ₆₀₀ 1.0 to 1.2, centrifuging the seed liquid, and collecting thalli; the time of each shaking culture is 12-15h, and the shaking speed is 100-120rpm.

Step 2, uniformly blowing the collected thalli with sterile water to obtain a bacterial liquid for later use; OD of the bacterial liquid ₆₀₀ 0.7-0.8;

step 3, the lysis treatment of the sludge

Adding bacterial liquid into the organic sludge to be treated according to the volume of 10-15%, adjusting the pH value to 6-10, and then carrying out a lysis reaction for 1-36h under the conditions of stirring and aeration at 50-85 ℃; the aeration rate is 0.1-1.0vvm, and the stirring rate is 40-200rpm.

Example 2: extracellular enzyme Activity analysis of THE dominant Strain Geobacillus sp THE-4

The residual sludge is organic waste water containing suspended solids with higher concentration, and the main components of the residual sludge comprise small molecular soluble organic matters (monosaccharides, amino acids and the like) and refractory macromolecular organic matters (mainly including proteins, polysaccharides and lipid compounds), wherein the carbohydrate accounts for 20% of the total organic matters. It was found that during the dissolution of excess sludge, glucosidase, protease and alpha-amylase are closely related to the degradation of extracellular polymers of sludge, and that protein hydrolysis is a more critical factor. Therefore, the activity of the extracellular protease secreted by bacteria is of paramount importance.

THE dominant strain of THE invention, geobacillus sp. THE-4, was inoculated into THE corresponding liquid medium at an inoculum size of 1% and THE protease activity was determined by azocasein method. Taking bacterial solutions at different time points, centrifuging in a desk centrifuge (4 ℃,10min,14,000Xg), taking supernatant and a substrate azocasein to fully react for 30 min at 50-85 ℃, immediately adding 5% trichloroacetic acid (TCA), fully mixing for 5 seconds by using a vortex oscillator, then balancing under ice cubes for 10min, centrifuging, taking supernatant, dripping NaOH until the concentration is 0.4M, and measuring absorbance value at 440nm by using a UV/VIS spectrophotometer. 1 protease activity unit is defined as the amount of enzyme required to hydrolyze soluble casein to 1 micromole of tyrosine per unit time under certain experimental conditions. The research conclusion shows that: THE overall enzyme activity of THE dominant strain THE-4 of THE invention tends to be stable, and reaches 587.5U/L at 14h, and THE average enzyme activity at 14h is 455.5U/L respectively.

Example 3: THE dominant strain Geobacillus sp. THE-4 was investigated for its sludge dissolving performance

THE dominant strain of THE invention, namely THE Geobacillus sp. THE-4 and THE control strain YL-1, are respectively inoculated into corresponding solid culture media, activated and cultured for 12 hours at 50-85 ℃, THE activated strain is inoculated into a 250ml conical flask containing 50ml of liquid culture media by using a sterile gun head, cultured for 12 hours at 50-85 ℃, THE shaking speed is 120rpm, and THE activation is repeated for 3 times. After activated and matured thalli are centrifuged by a desk top centrifuge (6000 rpm,10 min), the thalli are collected and are blown by sterile water uniformly. The volume of the dissolved sludge in each batch is 360ml, the volume of the added bacterial liquid is 40ml, and a non-inoculated blank control group is additionally arranged, and 40ml of distilled water is added into the non-inoculated blank control group, so that the bacterial liquid volume accounts for 10% of the total reaction system. Then stirring each group for reaction for 36h at 50-85 ℃ and pH6.8-7.0 under aeration condition; the aeration rate was 0.2vvm and the stirring rate was 60.+ -.10 rpm. And measuring the VSS concentration in each sample by adopting a national standard method, and calculating the degradation rate of the sludge VSS. The results are shown in Table 1, and the study conclusion shows that: THE degradation rate of sludge VSS in THE unvaccinated blank was 4.7%, while THE degradation rate of sludge VSS in THE Geobacillus sp. Of THE present invention was 20.2%. THE degradation rate of THE Geobacillus sp is improved by 15.5% compared with that of THE blank control group VSS, and is improved by 12.9% compared with that of THE control strain YL-1.

TABLE 1

	THE-4	Control strain YL-1	Blank control group
				Degradation rate of VSS (%)	20.2±0.20	7.3±0.03	4.7±0.05

Example 4: effect of temperature on lysis

Culturing THE dominant strain Geobacillus sp THE-4 with liquid culture medium at 60deg.C, 65deg.C and 70deg.C for 15-18 hr, and repeatedly activating for 3 times to obtain OD ₆₀₀ Between 1.0 and 1.2, centrifugation (6000 rpm,10 min) was performed to collect the cells for subsequent lysis experiments. The temperature of each reactor was 60 ℃,65 ℃ and 70 ℃, respectively. The volume of the dissolved sludge is 360ml, the volume of the added bacterial liquid is 40ml, and the bacterial liquid volume accounts for 10% of the total reaction system. THE degradation rate of THE Bacillus terreus (Geobacillus sp.) THE-4 at VSS at different temperatures is shown in Table 2, wherein THE best sludge dissolving effect of THE Bacillus terreus (Geobacillus sp.) THE-4 at 65 ℃ is up to 20.2%.

TABLE 2

	60℃	65℃	70℃
				Degradation rate of VSS (%)	19.7±0.20	20.2±0.20	15.2±0.20

The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.

Sequence listing

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agggaatctt ccgcaatgga cgaaagtctg acggagcgac gccgcgtgag cgaagaaggt 420

cttcggatcg taaagctctg ttgttaggga agaagaagta ccgttcgaat agggcggtac 480

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tagggggcga gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gtcccttaag 600

tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg ggacttgagt 660

gcagaagagg agagcggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720

accagtggcg aaggcggctc tctggtctgt aactgacgct gaggcgcgaa agcgtgggga 780

gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttaga 840

ggggttaaac cctttagtgc tgtagctaac gcgttaagca ctccgcctgg ggagtacggc 900

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attcagttgg gcactctagg gggactgccg gctaaaagtc ggaggaaggt ggggatgacg 1200

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Claims

1. A lysis strain, characterized by being geobacillus (geobacillus) th-4, preserved in China center for type culture collection (cctcm) 2021512;

the lysis strain is obtained by separating and screening from high-temperature domesticated mature sludge;

the high-temperature domesticated mature sludge is obtained by domesticating sewage sludge at 55-80 ℃;

the sludge acclimation method comprises the following steps: domesticating sewage sludge under aeration condition of 0.1-1.0vvm at 55-80deg.C for 5-60 days;

THE 16SrDNA sequence of Geobacillus (Geobacillus) THE-4 is as set forth in SEQ ID: 1.

2. The lysis strain of claim 1, wherein the method of activation of the lysis strain comprises the steps of:

inoculating THE bacillus terrae (Geobacillus) THE-4 strain into liquid culture medium, and performing constant temperature shaking culture for activation to obtain OD ₆₀₀ 1.0-1.2, and centrifuging to collect thallus.

3. THE lysis strain of claim 2, wherein THE inoculum size of geobacillus (geobacilussp.) th-4 in liquid medium is 1-5%; the temperature of shaking culture is 50-85 ℃, the shaking speed is 40-200rpm, the times of shaking culture at constant temperature are 2-3 times, and the time of shaking culture each time is 6-20h.

4. A sludge reduction treatment agent comprising the lysis strain according to any one of claims 1 to 3.

5. A method of reducing sludge using the sludge reduction treatment agent as claimed in claim 4.

6. A method for sludge reduction treatment using the lysis strain according to any one of claims 1 to 3, comprising the steps of:

step one, uniformly blowing thalli of a lysis strain of geobacillus (Geobacillus) THE-4 with sterile water to obtain bacterial liquid;

7. The method for sludge reduction treatment by using a lysate strain according to claim 6,

in the first step, the OD of the bacterial liquid ₆₀₀ The value is 0.4-0.8, the adding amount of the bacterial liquid in the second step is 10-15% of the volume of the organic sludge to be treated, the temperature of the lysis reaction is 50-85 ℃, the aeration rate is 0.1-1.0vvm, the stirring rate is 50-200rpm, and the time of the lysis reaction is 1-36h.