阅读说明：本技术 一种溶胞菌株、污泥减量处理剂及其应用 (Cell-dissolving strain, sludge reduction treatment agent and application thereof ) 是由 黄瑛 张中玉 迟宝岩 段欣欣 于 2021-06-04 设计创作，主要内容包括：本发明涉及一种溶胞菌株、污泥减量处理剂及其应用,所述溶胞菌株为土芽孢杆菌(Geobacillus sp.)THE-4,保藏编号为CCTCC M 2021512。是从高温驯化成熟的污泥中分离筛选得到,所述溶胞菌株和包含所述溶胞菌株的污泥减量处理剂能够应用于污泥的减量处理中,本发明分离筛选溶胞菌株为土芽孢杆菌(Geobacillus sp.)THE-4能够利用污泥中的有机质生长繁殖,菌株的代谢过程中产生的酶能够加速胞外聚合物的水解和污泥细胞的裂解,达到污泥溶胞的目的。(The invention relates to a cell dissolving strain, a sludge reduction treatment agent and application thereof, wherein the cell dissolving strain is Geobacillus (Geobacillus) ((Bacillus subtilis)) and the sludge reduction treatment agent is prepared from Bacillus subtilis (Bacillus subtilis) Geobacillus sp. ) THE preservation number of THE THE-4 is CCTCC M2021512. The cell lysing strain is separated and screened from sludge matured by high-temperature acclimation, the cell lysing strain and a sludge reduction treatment agent containing the cell lysing strain can be applied to sludge reduction treatment, and the cell lysing strain is Geobacillus: ( Geobacillus sp. ) THE THE-4 can utilize organic matters in THE sludge to grow and reproduce, and THE enzyme generated in THE metabolic process of THE strain can accelerate THE hydrolysis of extracellular polymers and THE cracking of sludge cells, thereby achieving THE purpose of sludge cell lysis.)

1. A cell-lysing strain, which is characterized by comprising Bacillus terreus (Geobacillus sp.) THE THE-4 is preserved in China center for type culture Collection with a preservation number of CCTCC M2021512.

2. The lysate strain of claim 1, wherein the Geobacillus bacterium (C.), (C.), (C.Geobacillus sp.) THE 16S rDNA sequence of THE THE-4 is shown in SEQ ID:1 is shown.

3. The lysate strain of claim 1, wherein the lysate strain is isolated and screened from a high temperature acclimated mature sludge.

4. The lysate strain according to claim 3, wherein the high-temperature acclimated mature sludge is obtained by acclimating sewage sludge at 55-80 ℃.

5. The lysate strain of claim 1, wherein the method of activation of the lysate strain comprises the steps of:

mixing the lysate strain Geobacillus (Geobacillus sp.) Inoculating THE-4 in liquid culture medium, and performing constant temperature shaking culture for activation to obtain OD₆₀₀Then the seed liquid is added into the seed liquid of 1.0-1.2, and then the centrifugal separation is carried out, and the thalli are collected.

6. THE lysate strain of claim 5, wherein THE liquid medium contains 1-5% of Geobacillus sp (THE-4); the temperature of shaking culture is 50-85 deg.C, shaking rate is 40-200rpm, the number of times of shaking culture at constant temperature is 2-3 times, and each shaking culture time is 6-20 h.

7. A sludge reducing agent comprising the lysate strain according to any one of claims 1 to 6.

8. Use of the sludge-reducing agent according to claim 7 for sludge reduction treatment.

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

step one, mixing the lysis strain Geobacillus (B.terreus) (C.terreus)Geobacillus sp.) Uniformly blowing and beating THE thalli of THE THE-4 with sterile water to obtain a bacterial liquid;

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

10. The method for sludge reduction treatment using a lysate strain according to claim 9,

in the first step, the OD of the bacterial liquid₆₀₀The value is 0.4-0.8, the adding amount of the bacterial liquid in the step two 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 1-36 h.

Technical Field

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

Background

Biological wastewater treatment plants (WWTP) have been used worldwide to treat municipal wastewater. Although effective in removing organic matter from wastewater, a large amount of excess sludge is produced. With the increasing popularity and population of sewage treatment plants, the construction of new sewage treatment plants and the stricter sewage treatment requirements, the production of sludge will continue to increase. Therefore, the treatment and disposal of sludge has become an increasingly significant challenge in the field of environmental engineering.

In a conventional sewage treatment plant, there are mainly two methods of reducing the amount of sludge remaining, namely a wastewater line and a sludge line. The method is characterized in that return sludge after cell lysis is recycled to a mainstream bioreactor for further biodegradation, and physical, chemical and biological means are utilized to minimize the sludge discharged outwards by the whole sewage treatment system, so that the method has good application prospect. Physical and chemical methods 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 Polymeric Substance (EPS) is an important component of a sludge floc matrix and is composed of various organic substances, such as organic macromolecules, such as polysaccharide, protein, humic acid, uronic acid, lipid compounds and the like. The sludge reduction by the biological pretreatment method is realized by adding hydrolase into the sludge, accelerating the hydrolysis of extracellular polymers under the catalysis of the hydrolase, destroying the cell structure of microorganisms and dissolving insoluble substances in the sludge. However, the direct addition of the hydrolase has the problems of high cost, harsh reaction conditions and high difficulty in large-scale practical application. Therefore, in order to increase the industrial applicability of the biological pretreatment method, it is necessary to isolate and screen a strain capable of reducing the sludge, and the hydrolysis of extracellular polymers is accelerated by enzymes produced by the strain in the metabolic process of the sludge, thereby achieving the purpose of reducing the sludge.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a cell-lysing strain, a sludge reduction treatment agent and application thereof, wherein the cell-lysing strain can utilize organic matters in sludge to grow and reproduce, and enzymes 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 cell lysis.

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

the first technical scheme is as follows:

a lysis strain is Geobacillus sp (THE-4), belongs to Geobacillus sp (Geobacillus sp), is preserved in China center for type culture Collection, and has a preservation time of 2021 year, 5 month and 11 days at THE preservation address of Wuhan university, Wuhan, China, and a preservation number of CCTCC M2021512.

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

Further, the lysate strain is obtained by separating and screening sludge matured by high-temperature acclimatization.

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

Further, the method for activating said lysate strain, comprising the steps of:

inoculating Bacillus terreus (Geobacillus sp.) THE-4 into liquid culture medium, and performing constant temperature shaking culture for activation to obtain OD₆₀₀Then the seed liquid is added into the seed liquid of 1.0-1.2, and then the centrifugal separation is carried out, and the thalli are collected.

Further, THE inoculation amount of THE Geobacillus sp (THE-4) in THE liquid culture medium is 1-5%; the temperature of shaking culture is 50-85 deg.C, shaking rate is 40-200rpm, the number of times of shaking culture at constant temperature is 2-3 times, and each shaking culture time is 6-20 h.

The second technical scheme is as follows:

a sludge-reducing agent comprising the said lysate strain.

The third technical scheme is as follows:

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

The technical scheme is as follows:

a method for sludge reduction treatment using said lysate strain, comprising the steps of:

firstly, uniformly blowing and beating thalli of a lysis bacterial strain Geobacillus sp (THE-4) with sterile water to obtain a bacterial liquid;

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

Further, in the step one, the OD of the bacterial liquid₆₀₀The 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 1-36 h.

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

1. THE invention adopts THE sludge domesticated and matured under THE condition of 55-80 ℃ to separate and screen THE lysis bacterial strain, THE lysis bacterial strain Geobacillus sp (THE) THE-4 separated by THE method can invisibly grow in THE sludge to be treated, secrete extracellular enzymes and promote hydrolysis of macromolecular organic matters in THE sludge, so that organic matters in a solid phase of THE sludge are released into a liquid phase for subsequent utilization, and THE lysis bacterial strain Geobacillus sp (THE) THE-4 obtained by separation and screening of THE invention has larger secretion amount of extracellular enzymes and better lysis effect compared with other lysis bacteria.

2. Compared with the traditional physical chemistry decrement technology, the lysis strain and the sludge treatment method provided by the invention have the characteristics of economy, high efficiency, low energy consumption, high lysis efficiency and easily controlled reaction conditions.

Drawings

FIG. 1 is a phylogenetic tree of cytolytic strains;

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

Detailed Description

The invention is further illustrated by the following examples.

The sewage sludge used by the separation and screening of the invention comes from the following sources: nanjing, Qiaobei processing plant A²the/O process reflows the sludge in the sludge pump room; indexes of sewage sludge: the TSS is 13285 +/-146 mg/L; 6003 +/-30 mg/L of VSS; TCOD is 4350 +/-88 mg/L; SCOD is 65 plus or minus 5 mg/L; the pH value is 6.8 plus or minus 0.2;

the sewage sludge used in the lysis test of the invention comes from: a. the²the/O process operates residual sludge from sewage plants, the properties of which are as follows: total Suspended Solids (TSS) is 14685 + -146 mg/L, Volatile Suspended Solids (VSS) is 6021 + -25 mg/L, Total Chemical Oxygen Demand (TCOD) is 4350 + -88 mg/L, dissolved chemical oxygen demand is 65 + -5 mg/L, and pH is 7.0-7.3. Sieving the sludge to remove particles with the particle size of more than 1mm, and then storing in a refrigerator at 4 ℃, wherein the storage period is not more than 1 week.

The reference strain YL-1 belongs to Geobacillus sp, and is preserved in China center for type culture Collection with the preservation address: wuhan university in Wuhan, China; preservation time 2021 year 5 month 8; the preservation number is as follows: CCTCC AB 2021109; the control strain YL-1 was a publicly available strain.

Liquid culture medium: macroelements: nitrilotriacetic acid 100mg, NaCl 8mg, KH₂PO₄ 111mg，MgSO₄·7H₂O100 mg, peptone 1g, yeast extract 1 g; trace elements: na (Na)₂MoO₄·2H₂O 0.025mg，FeCl₃ 0.28mg， CuSO₄ 0.16mg，MnSO₄·H₂O 2.2mg，H₃BO₃ 0.5mg，ZnSO₄·7H₂O 0.5mg，CoCl₂·6H₂O 0.046 mg，CaSO₄60 mg. Adding 1L deionized water, adjusting pH to 7-7.5, placing in autoclave, and sterilizing at 121 deg.C for 20 min.

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

The invention mainly comprises the following parts, namely, separating and screening the domesticated sludge to obtain dominant strains, and identifying the dominant strains; determining the species relationship of the dominant strains; and thirdly, obtaining the optimal process for carrying out sludge reduction treatment by utilizing the screened dominant bacterial strain through process optimization.

Firstly, separating and screening the domesticated sludge to obtain the dominant bacterial strain, and specifically comprises the following steps:

step 1, sludge domestication: acclimating the sewage sludge at the aeration condition of 0.1-1.0vvm and the temperature of 55-80 ℃ for 5-60 days;

step 2, after the domesticated mature sludge is subjected to gradient dilution, a dilution pouring method and a three-zone scribing method are adopted for separation and purification, a strain of a single colony is obtained, and the strain is named and numbered respectively;

step 2.1, pouring the plate: solid medium: macroelements: nitrilotriacetic acid 100mg, NaCl 8mg, KH₂PO₄111mg，MgSO₄·7H₂O100 mg, peptone 1g, yeast extract 1 g; agar 20g trace elements: na (Na)₂MoO₄·2H₂O 0.025mg，FeCl₃ 0.28mg，CuSO₄ 0.16mg，MnSO₄·H₂O 2.2mg，H₃BO₃ 0.5mg，ZnSO₄·7H₂O 0.5 mg，CoCl₂·6H₂O 0.046mg，CaSO₄60 mg. Adding 1L deionized water, adjusting pH to 7-7.5, placing in autoclave, and sterilizing at 121 deg.C for 20 min. And when the temperature is cooled to 50-55 ℃, pouring the mixture into a sterile culture dish in sequence, ensuring that the mixture is fully paved at about 2/3 of the dish bottom, and inversely buckling the flat plate on a sterile table when agar is solidified.

Step 2.2, separation: taking a plurality of sterilized EP tubes, respectively adding 0.9ml of sterile water, taking 0.1ml of sludge domesticated and matured at 65 ℃, adding the sludge into a first tube of sterile water, fully mixing and shaking up, taking 0.1ml of diluent from the first tube to the next sterilized EP tube, mixing and shaking up, so diluting to a fourth tube and a fifth tube, wherein the dilution times are respectively as follows from the first tube: 10^-1、 10^-2、10^-3、10^-4、10^-5(ii) a Dropwise addition 10^-4And 10^-50.1ml of each of the two tubes of the diluent was placed in a corresponding plate and the diluent was coated uniformly with a sterilized coating bar. Will coatPlacing the smeared flat plate on a sterile table for 20-25 min, and pouring the flat plate into an incubator for 12h when the bacterial liquid permeates into the culture medium, wherein the culture temperature is 50-85 ℃. Selecting a single colony by using the sterilized inoculating loop, scribing on a flat plate by adopting a three-region scribing method, repeating for 3-4 times to obtain a purified colony, and totally separating 15 strains of the strain in the embodiment;

step 3, primarily screening the separated single bacterial colony strains through a hydrolysis ring test, further screening the bacterial strains with larger hydrolysis rings by adopting a sludge lysis test and an extracellular enzyme activity test, and reserving the bacterial strains with higher sludge VSS degradation rate and higher extracellular enzyme activity, namely dominant bacterial strains; THE dominant strain obtained by screening in THE embodiment is THE-4;

hydrolysis ring test: inoculating the cell-dissolving strain separated from the domesticated mature sludge into a skim milk powder solid culture medium, placing the cell-dissolving strain in a constant temperature incubator, and observing the change of a hydrolysis ring after 12 hours of each strain.

The solid culture medium of the skimmed milk powder comprises the following components: macroelements: nitrilotriacetic acid 100mg, NaCl 8mg, KH₂PO₄111mg, MgSO₄·7H₂100mg of O, 1g of peptone, 1g of yeast extract, 20g of agar, 20g of skimmed milk powder and 1L of distilled water; trace elements: na (Na)₂MoO₄·2H₂O 0.025mg，FeCl₃ 0.28mg，CuSO₄ 0.16mg，MnSO₄·H₂O 2.2mg，H₃BO₃0.5mg，ZnSO₄·7H₂O 0.5mg，CoCl₂·6H₂O 0.046mg，CaSO₄60 mg. Adjusting the pH value to 7.0-7.5.

Preliminary dissolution test of sludge: taking a plurality of conical flasks, and respectively adding sludge with a solid content of 1.2-1.5%; respectively culturing the strains subjected to preliminary screening for 12-15h, uniformly blowing the strains with sterile water, respectively inoculating the strains into corresponding conical flasks, carrying out constant-temperature shaking table culture at the temperature of 50-85 ℃ and the oscillation rate of 100rpm, simultaneously carrying out blank control by using ultrapure water as a blank, measuring the content of Volatile Suspended Solids (VSS) of sludge in each conical flask after 24h, and calculating the reduction rate of VSS of the sludge.

Extracellular enzyme activity assay: and respectively inoculating the strains after primary screening into a liquid culture medium according to the inoculation amount of 1%, and determining the activity of the protease by adopting an azocasein method.

Secondly, identifying the dominant strains and determining the species relationship of the dominant strains; the method specifically comprises physiological and biochemical identification and 16S rDNA identification:

1) gram staining microscopy: gram staining microscopic examination is carried out on THE dominant strain THE-4, then oxalic acid crystal violet staining solution is dip-stained for 1min, iodine solution is dip-stained for 1min, 95% ethanol is decolored for 30s, safranine solution is dip-stained for 2min-3min, a cover glass is covered, THE obtained product is placed under a microscope for observation, and THE observation result is as follows: THE thalli of THE THE-4 appeared red, rod-shaped, gram-negative.

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

Through identification, THE dominant strain THE-4 belongs to Geobacillus, and a phylogenetic tree constructed by an adjacent junction method is shown in figure 1.

And thirdly, obtaining the optimal process for carrying out sludge reduction treatment by utilizing the screened dominant bacterial strain through process optimization.

In this embodiment, THE optimum process for sludge reduction treatment using THE dominant strain bacillus terrae (Geobacillus sp.) than-4 is as follows:

step 1, activating and culturing dominant strains:

inoculating dominant strain THE-4 in liquid culture medium according to 1-5% of inoculum size, shake culturing at 50-85 deg.C for 2-3 times for activation to obtain OD₆₀₀Centrifuging in 1.0-1.2 seed liquid, and collecting thallus; the time of each shaking culture is 12-15h, and the shaking speed is 100-120 rpm.

Step 2, uniformly blowing and beating the collected thalli with sterile water to obtain a bacterial liquid for later use; OD of the bacterial liquid₆₀₀0.7-0.8;

step 3, lysis treatment of sludge

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

Example 2: exo-enzyme activity analysis of dominant strain Geobacillus sp THE-4

The residual sludge is organic wastewater containing suspended solids with high concentration, and the main components of the residual sludge comprise some micromolecular soluble organic matters (monosaccharide, amino acid and the like) and difficultly degraded macromolecular organic matters (mainly comprising protein, polysaccharide and lipid compounds), wherein the proportion of carbohydrate accounts for 20 percent 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 the hydrolysis of proteins is more critical. Therefore, the activity of extracellular proteases secreted by bacteria is of crucial importance.

THE dominant strain bacillus terrae (Geobacillus sp.) THE-4 is inoculated into a corresponding liquid culture medium according to THE inoculation amount of 1%, and THE activity of protease is determined by adopting an azo casein method. Taking bacterial liquid at different time points, centrifuging in a desktop centrifuge (4 ℃, 10min, 14,000 Xg), taking supernatant, fully reacting with azocasein serving as a substrate at 50-85 ℃ for 30 min, immediately adding 5% trichloroacetic acid (TCA), fully mixing for 5 seconds by using a vortex oscillator, placing under ice blocks for balancing for 10min, centrifuging, taking supernatant, dropwise adding NaOH until the concentration is 0.4M, and measuring the absorbance value under 440nm by using a UV/VIS spectrophotometer. 1 protease activity unit is defined as the number of enzymes required to hydrolyze soluble casein to 1 micromole tyrosine in a unit time under certain experimental conditions. The research results show that: THE overall enzyme activity of THE dominant strain THE-4 of THE invention tends to be stable, reaches 587.5U/L in 14h, and has average enzyme activity of 455.5U/L in 14h respectively.

Example 3: exploration on sludge dissolving performance of dominant strain Geobacillus sp (Geobacillus sp.) THE-4

THE dominant strain Geobacillus sp (THE-4) and THE control strain YL-1 are respectively inoculated into corresponding solid culture media, activated and cultured at 50-85 ℃ for 12h, THE activated strain is inoculated into a 250ml conical flask containing 50ml of liquid culture medium by using a sterile gun head, cultured at 50-85 ℃ for 12h, THE shaking speed is 120rpm, and THE activation is repeated for 3 times. After the activated and matured thallus is centrifuged by a desk centrifuge (6000rpm, 10min), the thallus is collected and blown by sterile water uniformly. The volume of the sludge dissolved in each batch is 360ml, the volume of the added bacterial liquid is 40ml, a blank control group which is not inoculated is additionally arranged, and 40ml of distilled water is added into the blank control group which is not inoculated, so that the volume of the bacterial liquid accounts for 10% of the total reaction system. Then, stirring and reacting the groups for 36 hours at 50-85 ℃ and pH6.8-7.0 under aeration conditions; 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 VSS of the sludge. The results are shown in Table 1, and the results of the study show that: THE degradation rate of THE sludge VSS in THE blank control which is not inoculated is 4.7%, while THE degradation rate of THE sludge VSS in THE Geobacillus sp (Geobacillus sp.) THE-4 is 20.2%. Compared with a blank control group VSS, THE degradation rate of THE Geobacillus sp (THEE-4) is improved by 15.5%, and compared with a control strain YL-1, THE degradation rate of THE Geobacillus sp (THEE-4) is improved by 12.9%.

TABLE 1

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

Example 4: effect of temperature on lysis

Culturing dominant strain Geobacillus sp (THE-4) in liquid culture medium at 60 deg.C, 65 deg.C and 70 deg.C for 15-18h, and repeatedly activating for 3 times to make OD₆₀₀Between 1.0 and 1.2, centrifugation (6000rpm, 10min) was carried out to collect the cells for subsequent lysis experiments. The temperature of each reactor was 60 ℃, 65 ℃ and 70 ℃. The volume of dissolved sludge is 360ml, the volume of added bacterial liquid is 40ml, and the volume of the bacterial liquid accounts for 10% of the total reaction system. THE degradation rate of VSS of THE Geobacillus sp (Geobacillus sp.) THE-4 at different temperatures is shown in Table 2, wherein THE sludge dissolving effect of THE Geobacillus sp THE-4 at 65 ℃ is THE best, and THE sludge dissolving effect is up to 20.2%.

TABLE 2

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

The embodiments described above are only preferred embodiments of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Sequence listing

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