CN111073840B - Sludge degradation strain and application thereof - Google Patents
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Abstract
The invention relates to the technical field of environmental microorganisms, in particular to a sludge degradation strain and application thereof. The invention provides Serratia marcescens Serratia marcocens SN-H1 with the preservation number of CCTCC NO: m2018652; the Serratia marcescens Serratia marcocens SN-H1 can effectively improve the removal rate of VSS in wastewater or the degradation rate of organic matters in sludge; can be used for the reduction and stabilization treatment of sludge, municipal sludge, river sediment and the like in the organic wastewater treatment process, and has higher application value.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to a sludge degradation strain and application thereof.
Background
Sludge is a by-product of sewage treatment process, and is an extremely complex heterogeneous body composed of organic matters, microbial cells, inorganic particles, colloids and the like. In recent years, although many sludge treatment technologies have been applied to various types of sewage treatment around the world, organic matters in sludge can be effectively removed, but a large amount of sludge is generated and cannot be treated in time. According to statistics, 2.4 million tons of wet sludge are produced in China every year, wherein the annual wet sludge production of only urban sewage treatment plants reaches more than 4000 million tons, and the annual wet sludge production of urban sewage treatment plants in China reaches 6000-8000 million tons in 2020 predicted. The sludge has complex components, contains a large amount of refractory substances, pathogenic microorganisms, parasitic ova, heavy metals and other toxic and harmful substances, is easy to cause secondary pollution to the environment if being treated improperly, and brings a serious challenge to the living environment of human beings. Therefore, reducing sludge production has become a research focus, and finding more economical and environmentally friendly alternatives to sludge degradation is becoming more and more important.
At present, the main methods for sludge treatment at home and abroad comprise sanitary landfill, sludge composting, agricultural use, sludge incineration and the like, but the problem of sludge treatment is not fundamentally solved due to site limitation, high infrastructure and operation management costs and the like. The essence and difficulty of sludge degradation is the cracking and degradation of flocs composed of sludge organic components, microorganisms, and Extracellular Polymeric Substances (EPS). In order to further degrade and utilize the sludge, release encapsulated organic material and increase the content of dissolved organic material, researchers have also developed a number of sludge degradation processes to lyse EPS and internal microorganisms, such as chemical processes like ozonation, peracetic acid oxidation, alkalinization, etc.; physical methods include ultrasonic treatment, heat treatment, and the like; the biological method comprises adding microbial agent, enzyme preparation, and microfauna predation; mechanical methods include sludge concentration, high-pressure homogenization and the like; the combined treatment method comprises a series of treatment modes such as ozonization and ultrasonic combined treatment. Among them, these physical methods and chemical methods are limited by natural conditions, and have large production energy consumption, certain corrosiveness to equipment, and high cost and secondary pollution problems. The microbial agent is added in the biological method to degrade the sludge, and the growth of the microorganisms and the enzymes secreted by the microorganisms are mainly utilized to hydrolyze degradable components in the sludge, so that the sludge reduction is realized. Due to the advantages of good economic benefit, convenient operation and no pollution, the microbial sludge degradation technology has received more and more attention in recent years and becomes a novel sludge reduction solution.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a sludge degrading strain and its application, which are used to solve the problems of the prior art.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a sludge degrading strain identified as Serratia marcescens (Serratia marcescens) deposited in the chinese center for type culture collection CCTCC, with the strain name of Serratia marcescens SN-H1, the preservation date of 2018.9.25, the preservation number of CCTCC NO: m2018652.
The Serratia marcescens Serratia marcocens SN-H1 is characterized in that the Serratia marcescens is gram-negative bacteria and short rod-shaped, the colony form is deep red and round, the edge is neat, and the surface is smooth and moist.
The Serratia marcescens SN-H1 contains a gene sequence shown in SEQ ID NO. 1.
The Serratia marcescens Serratia marcocens SN-H1 is cultured in a sludge liquid culture medium for four weeks, and the VSS removal rate is more than 48%.
The VSS removal rate is obtained by inoculating a bacterial liquid into a sludge liquid culture medium for culturing for a certain time and detecting by adopting the following method.
The sludge liquid culture medium comprises the following components:
adding water into wet sludge (the water content is 84%, the VSS content is 64% of the dry weight of the sludge, and the mass fraction of the water and the VSS content is the mass fraction) to adjust the concentration of the sludge (dry weight) to be 10g/L and the pH value to be 7.0-7.5.
The determination step comprises:
(1) centrifuging the sludge liquid culture medium, and retaining solids;
(2) drying the solid sample in an oven at 105 ℃ to constant weight, and grinding the solid sample into powder for later use;
(3) the weight of the dried crucible is weighed as m0;
(4) Weighing the powdery sample in the step 2, and recording the weight of the sample as MSample (A)Putting the crucible in the step 3 into a muffle furnace, heating to 550 ℃, firing for 240min, taking out after the temperature in the furnace is reduced to be below 100 ℃, cooling to room temperature in a dryer, and weighing the total weight as M;
(5) calculating the VSS content of the sample, namely VSS%, according to the weighing data and a calculation formula:
VSS%=[1-(M-m0)/Msample (A)]×100% ①
(6) And calculating the VSS removal rate according to a calculation formula II:
[VSS(0)%-VSS(28)%]/VSS(0)%×100% ②
VSS(0)% is the initial VSS content of the sludge broth without inoculated strain,
VSS(28)% is the VSS content of the sample taken on day 28 from the date of inoculation of the strain.
The VSS removal rate is the VSS removal rate obtained by sampling 28 days from the date of inoculation of the strain.
The second aspect of the invention provides the application of Serratia marcescens SN-H1 in sludge degradation.
The sludge comprises sludge, municipal sludge, river sediment and the like in the organic wastewater treatment process.
The organic wastewater comprises livestock and poultry breeding wastewater, industrial organic wastewater, domestic wastewater, garbage leachate, biogas slurry and the like.
A sludge degradation method at least comprises the following steps:
(1) inoculating the seed liquid of Serratia marcescens Serratia marcocens SN-H1 to the sludge and mixing uniformly;
(2) aerobic culture is carried out under the conditions of proper temperature, pH value and DO value (dissolved oxygen content) to degrade the sludge.
The inoculation amount of the inoculation is 1-10% of the volume of the sludge, and preferably 2-5%;
the temperature is 20-40 ℃, and preferably 30 ℃;
the pH value is 6-8, and preferably 7-7.5;
the DO value is 3-7 mg/L, preferably 3-5 mg/L.
As mentioned above, the sludge degrading strain and the application thereof have the following beneficial effects:
(1) the organic matters such as protein, cellulose and starch in the sludge are efficiently degraded, the removal rate of VSS in the wastewater or the degradation rate of the organic matters in the sludge are effectively improved, and the removal capability of TN and COD in the sludge mixed solution is realized; (2) the culture medium can be applied to reduction and stabilization treatment of sludge, municipal sludge, river sediment and the like in the organic wastewater treatment process after the expanded culture, and has high application value;
(3) high treatment efficiency, good economic benefit, convenient operation and no pollution.
Drawings
FIG. 1 shows a phylogenetic tree map of the sludge degrading strain SN-H1 of the present application.
FIG. 2 shows the removal rate of VSS in sludge liquid by the sludge degrading strain SN-H1 of the present application.
FIG. 3 shows the degradation of sludge in livestock and poultry breeding wastewater treatment by the sludge degrading strain SN-H1 of the application;
FIG. 4 shows the degradation of river sediment by the sludge degrading strain SN-H1 of the application;
FIG. 5 shows the degradation condition of the sludge degradation strain SN-H1 in the application on the sludge in the kitchen waste biogas slurry.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art of molecular biology, biochemistry, analytical chemistry, recombinant DNA technology, and related fields. These techniques are well described in the literature.
The invention provides Serratia marcescens, which is separated and purified from sludge mixed liquor in a sewage treatment pool, and identified to obtain Serratia marcescens SN-H1.
The strain is preserved in China center for type culture Collection in 2018, 9 and 25 months, and the preservation number is CCTCC NO: m2018652.
The Serratia marcescens Serratia marcocens SN-H1 is mainly characterized by gram-negative bacteria, short rod-shaped, deep red and round colony morphology, neat edge and smooth and moist surface.
The Serratia marcescens SN-H1 contains a gene sequence shown in SEQ ID NO. 1.
The Serratia marcescens Serratia marcocens SN-H1 is cultured in a sludge liquid culture medium for four weeks, and the VSS removal rate is more than 48%.
The sludge liquid culture medium comprises the following components: adding water into wet sludge (the water content is 84%, the VSS content is 64% of the dry weight of the sludge, and the mass fraction of the water and the VSS content is the mass fraction) to adjust the concentration of the sludge (dry weight) to be 10g/L and the pH value to be 7.0-7.5.
The second aspect of the invention provides the application of Serratia marcescens SN-H1 in sludge degradation.
The sludge comprises sludge, municipal sludge, river sediment and the like in the organic wastewater treatment process.
The organic wastewater comprises livestock and poultry breeding wastewater, industrial organic wastewater, domestic wastewater, garbage leachate, biogas slurry and the like. The third aspect of the invention provides a sludge degradation method, which at least comprises the following steps:
(1) inoculating the seed liquid of Serratia marcescens Serratia marcocens SN-H1 to the sludge and mixing uniformly;
(2) aerobic culture is carried out under the conditions of proper temperature, pH value and DO value (dissolved oxygen content) to degrade the sludge.
The inoculation amount of the inoculation is 1-10% of the volume of the sludge, and preferably 2-5%;
the temperature is 20-40 ℃, and preferably 30 ℃;
the pH value is 6-8, and preferably 7-7.5;
the DO value is 3-7 mg/L, preferably 3-5 mg/L.
Example 1:
separation screening and performance determination of sludge degrading bacteria SN-H1
The media and components used were as follows:
LB liquid medium: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and a pH value of 7.0-7.5.
LB solid medium: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract and 15g/L agar powder, and the pH value is 7.0-7.5.
Sludge liquid culture medium: adding water into wet sludge (the water content is 84%, the VSS content is 64%, and the mass fractions are all) to adjust the concentration to 10g/L and the pH value to 7.0-7.5.
Sludge solid culture medium: adding water into wet sludge (the water content is 84%, the VSS content is 64% of the dry weight of the sludge, and the mass fractions of the water and the VSS are all) to adjust the concentration of the sludge (dry weight) to be 30g/L, the agar content to be 20g/L and the pH value to be 7.0-7.5.
Protease screening culture medium: 10g/L of casein, 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast extract, 15g/L of agar powder and 7.0-7.5 of pH value.
Production of cellulase screening culture medium: 10g/L sodium carboxymethylcellulose, 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 15g/L agar powder and a pH value of 7.0-7.5.
Producing amylase screening culture medium: 10g/L of soluble starch, 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast extract, 15g/L of agar powder and the pH value of the agar powder is 7.0-7.5.
The prepared culture medium is sterilized by high pressure steam at 121 deg.C for 20 min.
Separation and purification: collecting sludge mixed liquor of a test sample from sewage treatment pools from different sources, taking 5mL of sludge mixed liquor into 45mL of sterilized LB liquid culture medium, uniformly mixing, and placing in a shaking table at 30 ℃ and 180r/min for enrichment culture for 24 hours; after the cultured enrichment solution is subjected to gradient dilution, uniformly coating the enrichment solution on an LB solid culture medium; after culturing for 24 hours in a constant temperature incubator at 30 ℃, selecting monoclonals with different colony morphologies, and marking, purifying and preserving the monoclonals. A total of 18 strains are obtained by separation and purification.
Primary screening: inoculating the pure strains obtained by separation and purification to a sludge solid culture medium by adopting a point grafting method, performing primary screening, culturing the inoculated flat plate in a constant temperature incubator at 30 ℃ for 48-72 h, selecting strains which grow well on a sludge agar culture medium, scribing, purifying, numbering and preserving. 7 strains are obtained through primary screening.
Re-screening: and inoculating the pure strains obtained by primary screening to a protease production screening culture medium, a cellulase production screening culture medium and a amylase production screening culture medium by adopting a point grafting method, and measuring the enzyme production capacity of the strains so as to perform secondary screening. And (3) culturing the inoculated plate in a constant temperature incubator at 30 ℃ for 48-72 h, measuring the diameter (D) of a bacterial colony and the diameter (D) of a transparent ring, calculating the D/D shown in table 1, selecting a strain with stronger enzyme production capacity, scribing, purifying, numbering and preserving. 3 strains are obtained through re-screening, the SN-H1 has the highest enzyme production capability, and the SN-H1 is finally selected as a subsequent sludge degradation experiment.
TABLE 1 measurement results of enzyme-producing ability of the strains
And (3) measuring the degradation performance of the sludge: inoculating the strain obtained by re-screening into LB liquid culture medium, culturing for 16h in a shaking table at 30 ℃ and 180r/min, taking 2% (volume ratio) bacterial liquid, washing with sterile normal saline, suspending, and inoculating into sterilized sludge liquid culture medium. Culturing in a shaking table at 30 ℃ and 180r/min, sampling periodically every other week to measure the removal rate of VSS in the sludge mixed liquor, and evaluating the sludge degradation performance of the strain compared with a control group without the strain.
And (3) determination of the VSS content:
(1) centrifuging the sludge mixed liquor for 10min at 10000r/min, and measuring the VSS content of the sludge by using the solid;
(2) drying part of the obtained solid sample in an oven at 105 ℃ to constant weight, and grinding the solid sample into powder for later use;
(3) putting the clean crucible into a 105 ℃ oven to be dried to constant weight, taking out the crucible, putting the crucible into a drier to be cooled to room temperature, weighing the crucible, and recording the weight as m0;
(4) Weighing the powdery sample in the step (2) and recording the powdery sample as MSample (A)In the weight of m0The crucible is moved into a muffle furnace, heated to 550 ℃, burned for 240min (timing from the temperature reaching 550 ℃), taken out when the temperature in the furnace is reduced to be below 100 ℃, placed in a drier for cooling and weighed until the total weight is constant, and marked as M;
(5) calculating the VSS content of the sample, namely VSS%, according to the weighing data and a calculation formula:
VSS%=[1-(M-m0)/Msample (A)]×100% ①
(6) And calculating the VSS removal rate according to a calculation formula II:
[VSS(0)%-VSS(x)%]/VSS(0)%×100% ②
VSS(0)% is the initial VSS content of the sludge broth without inoculated strain,
VSS(x) % x represents the sampling time, VSS: (1)x) % is the VSS content of the sample sampled at the x time point.
As can be seen from FIG. 2, after 4 weeks of culture, the removal rate of VSS by the strain SN-H1 reaches 48.8%, which is 19.9% higher than the removal rate of VSS by the control group of 40.7%. The strain SN-H1 has obvious sludge degradation effect.
Example 2: the strain SN-H1 CCTCC NO: molecular biological identification of M2018652
The strain identification adopts a 16S rDNA sequence alignment method. Genomic DNA of strain SN-H1 was extracted according to the prior art and used as template for amplification of the 16S rDNA strain using a pair of universal primers (27F, 1492R). The upstream primer is 27F
(5'-AGAGTTTGATCCTGGCTCA-3'), the downstream primer is 1492R (5'-GGTTACCTTGTTACG ACTT-3').
The PCR reaction (20. mu.L) was as follows: form panel0.5 μ L of DNA, 10 μ L of PCR Taqmix, 0.6 μ L of each of the upstream and downstream primers, and ddH2O to the reaction system was 20. mu.L.
PCR procedure: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 1min and 30s, circulating for 30 times, extension at 72 deg.C for 10min, and storing at 4 deg.C.
Purification and sequencing of the PCR products were performed by Shanghai Jili Biotechnology Ltd. The DNA sequence of 16S r obtained by sequencing was submitted at NCBI, and the strain phylogenetic tree was constructed using MEGA6 software by performing homology sequence alignment analysis with GenBank software (see FIG. 1).
The effective gene sequence length of the strain SN-H1 is 1430bp, and is shown in a gene sequence table SEQ ID NO. 1. After comparison, the sequence has 99.9 percent of homology with Serratia marcescens (NCBI accession number: AB680131.1) of NCBI database, belongs to Serratia marcescens (Serratia marcescens), and is named as Serratia marcescens SN-H1.
Example 3: degradation effect of sludge degrading bacteria SN-H1 on sludge in livestock and poultry breeding wastewater
The livestock and poultry breeding wastewater is taken from mixed liquor in a primary sedimentation tank in the wastewater treatment process of a certain pig farm, the TSS content of stock solution is 26g/L, the VSS content is 17g/L, and the pH value is 8.5.
Inoculating the strain SN-H1 into an LB liquid culture medium, culturing for 16H in a shaking table at 30 ℃ and 180r/min, centrifuging and washing bacterial liquid in a logarithmic phase according to a volume ratio of 5%, and adding the bacterial liquid into a reactor, wherein the control conditions are as follows: the rotation speed of the electric stirrer is 180r/min, the DO value is 3mg/L, the temperature is 30 ℃, and the pH value is 7. Samples were taken every 2d during the reaction, and the removal rate of VSS in the wastewater was measured according to the VSS measurement procedure in example 1.
FIG. 3 shows the degradation of sludge in pig farm wastewater by strain SN-H1 in 24 days, and it can be seen that the maximum value of VSS removal rate by strain SN-H1 reaches 79.6%, which is improved compared with 53.0% of VSS removal rate without inoculation in the control group
50.2 percent. The strain SN-H1 has obvious degradation effect on the sludge in the wastewater of the pig farm.
Example 4: degradation effect of sludge degrading bacteria SN-H1 on bottom mud of river channel
The river sediment is taken from the sediment of a river in Shanghai city, namely, pollutants from life and industry are deposited at the bottom of a river bed through physical, chemical and biological actions to form grey black sludge rich in organic matters and nutrient salts. The water content of the river sediment to be tested is 89%, the content of organic matters in dry basis is 8-9%, and the pH value is 6.5-7.5.
Inoculating the strain SN-H1 into an LB liquid culture medium, culturing for 16H in a shaking table at 30 ℃ and 180r/min, centrifuging and washing bacterial liquid in a logarithmic phase according to the volume ratio of 3%, and then adding the bacterial liquid into a reactor, wherein the control conditions are as follows: the rotation speed of the electric stirrer is 180r/min, the DO value is 3mg/L, the temperature is 30 ℃, and the pH value is 7. Samples were taken every 12 hours during the reaction, and the VSS removal rate of the bottom sludge in the river was measured according to the VSS measurement procedure in example 1.
FIG. 4 shows the degradation of the strain SN-H1 on the bottom sediment of the river within 72H, and it can be seen that the maximum value of the removal rate of VSS by the strain SN-H1 reaches 14.6%, which is 64.0% higher than the removal rate of VSS without inoculation in the control group, which is 8.9%. The strain SN-H1 has obvious degradation effect on the bottom sediment of the river.
Example 5: degradation effect of sludge degrading bacteria SN-H1 on sludge in kitchen waste biogas slurry
The kitchen waste biogas slurry is taken from biogas slurry discharged from a biogas fermentation tank of a kitchen waste treatment plant, the TSS content of the stock solution is 11%, the VSS content is 9.7%, and the pH value is 5.0-6.0.
Inoculating the strain SN-H1 into an LB liquid culture medium, culturing for 16H in a shaking table at 30 ℃ and 180r/min, centrifuging and washing bacterial liquid in a logarithmic phase according to the volume ratio of 3%, and then adding the bacterial liquid into a reactor, wherein the control conditions are as follows: the rotation speed of the electric stirrer is 180r/min, the DO value is 4mg/L, the temperature is 30 ℃, and the pH value is 6.5. Sampling every 1d in the reaction process, and determining the removal rate of VSS in the kitchen waste biogas slurry according to the VSS determination step in the embodiment 1.
FIG. 5 shows the degradation condition of the strain SN-H1 on sludge in the kitchen waste biogas slurry in 7d, and it can be seen that the maximum value of the removal rate of the strain SN-H1 on VSS reaches 77.2%, which is 23.5% higher than the removal rate of VSS of a control group without inoculation of bacteria. The strain SN-H1 has obvious degradation effect on the kitchen waste.
In conclusion, the efficient sludge degrading strain is obtained by LB culture medium separation and purification, sludge agar culture medium primary screening, enzyme production culture medium secondary screening and sludge degradation performance verification, and is named as SN-H1. The taxonomic status of the strain is identified through comprehensive analysis of data such as colony and cell morphology, physiological and biochemical characteristics, 16S rDNA gene sequence determination and the like: serratia marcescens (Serratia marcescens). The strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation name of Serratia marcocecens SN-H1, the preservation date of 2018.9.25 and the preservation number of CCTCC NO: m2018652, the preservation address is China type culture Collection of Wuhan university in eight roads in Wuchang district in Wuhan city, Hubei province. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
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<120> sludge degradation strain and application thereof
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<151>2019-08-28
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Claims (8)
1. Serratia marcescens (Serratia marcocens) SN-H1 with the preservation number of CCTCC NO: m2018652.
2. Serratia marcescens (Serratia marcescens) SN-H1 according to claim 1, wherein the Serratia marcescens has a gene sequence shown in SEQ ID No. 1.
3. The Serratia marcescens (Serratia marcescens) SN-H1 according to claim 1, wherein the Serratia marcescens (Serratia marcescens) SN-H1 is cultured in a sludge liquid culture medium for four weeks, and the VSS removal rate is more than 48%.
4. Use of Serratia marcescens (Serratia marcescens) SN-H1 according to any one of claims 1 to 3 for sludge degradation.
5. The use according to claim 4, wherein the sludge is selected from the group consisting of sludge from organic wastewater treatment processes, municipal sludge, and river sediment.
6. The use according to claim 5, wherein the organic wastewater comprises livestock and poultry breeding wastewater, industrial organic wastewater, domestic wastewater, landfill leachate, biogas slurry.
7. A sludge degradation method is characterized by at least comprising the following steps:
(1) inoculating the seed liquid of Serratia marcescens (Serratia marcocens) SN-H1 of any one of claims 1-3 to sludge and mixing uniformly;
(2) aerobic culture is carried out under the conditions of proper temperature, pH value and DO value to degrade the sludge.
8. The method of sludge degradation of claim 7, further comprising one or more of the following conditions:
(1) the inoculation amount of the inoculation is 1-10% of the volume of the sludge;
(2) the temperature is 20-40 ℃;
(3) the pH value is 6-8;
(4) the DO value is 3-7 mg/L.
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