CN111073840B - A kind of sludge degrading bacteria and its application - Google Patents

Abstract

The invention relates to the technical field of environmental microorganisms, in particular to a sludge-degrading bacterial species and its application. The invention provides a kind of Serratia marcescens SN-H1, the preservation number of which is CCTCC NO: M 2018652; the Serratia marcescens SN-H1 can effectively improve the removal rate of VSS in wastewater Or the degradation rate of organic matter in sludge; it can be used for the reduction and stabilization of sludge, municipal sludge, river bottom sludge, etc. in the process of organic wastewater treatment, and has high application value.

Description

A kind of sludge degrading bacteria and its application

technical field

The invention relates to the technical field of environmental microorganisms, in particular to a sludge-degrading bacterial species and its application.

Background technique

Sludge is a by-product of the sewage treatment process, and is an extremely complex heterogeneous body composed of organic matter, microbial cells, inorganic particles, and colloids. In recent years, although many sludge treatment technologies have been applied to various types of sewage treatment around the world and can effectively remove the organic matter in the sludge, a large amount of sludge is still generated and cannot be treated in time. According to statistics, the average annual production of wet sludge in my country is 240 million tons, of which only urban sewage treatment plants produce more than 40 million tons of wet sludge each year. 80 million tons. The sludge composition is complex, which contains a large number of refractory substances, pathogenic microorganisms, parasite eggs and heavy metals and other toxic and harmful substances. Therefore, reducing sludge production has become a research hotspot, and finding more economical and environmentally friendly alternatives to sludge degradation has received more and more attention.

At present, the main methods of sludge disposal at home and abroad include sanitary landfill, sludge composting, agricultural use and sludge incineration. The essence and difficulty of sludge degradation is the cracking and degradation of flocs composed of sludge organic components, microorganisms and extracellular polymers (EPS). In order to further degrade and utilize the sludge, release the encapsulated organic matter and increase the content of dissolved organic matter, researchers have also developed many sludge degradation methods to crack EPS and internal microorganisms, such as chemical methods such as ozonation, peroxidation, etc. Acetic acid oxidation, alkali addition, etc.; physical methods include ultrasonic treatment, heat treatment, etc.; biological methods include adding microbial agents, enzyme preparations, micro-animal predation, etc.; mechanical methods include sludge concentration, high-pressure homogenization, etc.; combined treatment methods include ozone A series of treatment methods such as chemical and ultrasonic combined treatment. Among them, these physical methods and chemical methods are limited by natural conditions, the production energy consumption is relatively large, and they are corrosive to the equipment to a certain extent, which will bring high cost and secondary pollution problems. In the biological method, adding microbial inoculants to degrade sludge mainly utilizes the growth of microorganisms and their secreted enzymes to hydrolyze the degradable components in the sludge, so as to achieve sludge reduction. Microbial sludge degradation technology has received more and more attention in recent years due to its advantages of good economic benefits, convenient operation and no pollution, and has become a new type of sludge reduction solution.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a sludge degrading strain and its application, which are used to solve the problems in the prior art.

In order to achieve the above-mentioned purpose and other related purposes, a first aspect of the present invention provides a sludge-degrading bacterial species, which is identified as Serratia marcescens, and is preserved in the China Type Culture Collection Center CCTCC, the name of the bacterial species. It is Serratia marcescens SN-H1, the preservation date is 2018.9.25, and the preservation number is CCTCC NO: M2018652.

The Serratia marcescens SN-H1 is characterized in that it is a Gram-negative bacterium, short rod-shaped, with a dark red round colony shape, neat edges, and a smooth and moist surface.

The Serratia marcescens SN-H1 contains the gene sequence shown in SEQ ID NO.1.

The Serratia marcescens SN-H1 was cultured in the sludge liquid medium for four weeks, and the VSS removal rate was over 48%.

The VSS removal rate is obtained by inoculating the bacterial liquid into the sludge liquid medium for a certain period of time and then detecting it by the following method.

The composition of sludge liquid culture medium is:

Wet sludge (water content is 84%, VSS content is 64% of the dry weight of the sludge, both are mass fractions) and water is added to adjust the concentration of the sludge (dry weight) to 10g/L, and the pH value is 7.0 to 7.5.

Measurement steps:

(1) centrifuge the sludge liquid culture medium to retain the solid;

(2) Bake the solid sample to a constant weight in an oven at 105°C, and grind it into powder for subsequent use;

(3) weigh the weight of the crucible after drying, and denote it as m ₀ ;

(4) Weigh the powdered sample in step 2, the weight of the sample is recorded as M _sample , put it in the crucible in step 3, put it in a muffle furnace and heat it to 550 ° C for 240 min, and wait for the temperature in the furnace to decrease Take it out to below 100°C, cool it to room temperature in a desiccator and weigh the total weight, denoted as M;

(5) Calculate the VSS content of the sample according to the weighing data and calculation formula ①, that is, VSS%:

VSS%=[1-(M-m ₀ )/M _sample ]×100% ①

(6) Calculate the VSS removal rate according to the calculation formula ②:

[VSS ₍₀₎ %-VSS ₍₂₈₎ %]/VSS ₍₀₎ %×100% ②

VSS ₍₀₎ % is the initial VSS content of the sludge broth without the inoculated strain,

VSS ₍₂₈₎ % is the VSS content of the sample taken on the 28th day from the date of inoculation of the strain.

After four weeks of culture, the VSS removal rate was the VSS removal rate obtained by sampling 28 days from the date of inoculation of the strain.

The second aspect of the present invention provides the application of Serratia marcescens SN-H1 in sludge degradation.

The sludge includes sludge in the organic wastewater treatment process, municipal sludge, river bottom sludge and the like.

The organic wastewater includes livestock and poultry breeding wastewater, industrial organic wastewater, domestic wastewater, landfill leachate, biogas slurry, and the like.

A sludge degradation method, comprising at least the following steps:

(1) the seed liquid of Serratia marcescens SN-H1 is inoculated into sludge and mixed;

(2) Under the conditions of suitable temperature, pH value and DO value (dissolved oxygen content), aerobic cultivation is carried out to degrade the sludge.

The inoculation amount of the inoculation is 1-10% of the sludge volume, preferably 2-5%;

The temperature is 20～40℃, preferably 30℃;

The pH value is 6-8, preferably 7-7.5;

The DO value is 3-7 mg/L, preferably 3-5 mg/L.

As mentioned above, the sludge degrading bacteria of the present invention and its application have the following beneficial effects:

(1) Efficiently degrade protein, cellulose, starch and other organic matter in sludge, effectively improve the removal rate of VSS in wastewater or the degradation rate of organic matter in sludge, and at the same time have the ability to remove TN and COD in sludge mixture; (2) After expanded cultivation, it can be applied to the reduction and stabilization treatment of sludge, municipal sludge, river bottom sludge, etc. in the process of organic wastewater treatment, which has high application value;

(3) High treatment efficiency, good economic benefits, convenient operation and no pollution.

Description of drawings

Figure 1 shows the phylogenetic tree map of the sludge degrading strain SN-H1 of the present application.

FIG. 2 shows the removal rate of VSS in the sludge liquid by the sludge degrading strain SN-H1 of the present application.

Fig. 3 shows the situation of sludge degradation in the treatment of livestock and poultry breeding wastewater by the sludge degrading strain SN-H1 of the present application;

Fig. 4 shows the situation that the sludge degrading strain SN-H1 of the present application degrades the river bottom sludge;

Figure 5 shows the degradation of sludge in kitchen waste biogas slurry by the sludge degrading strain SN-H1 of the present application.

Detailed ways

The embodiments of the present invention are described below by specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all use conventional techniques in molecular biology, biochemistry, analytical chemistry, recombinant DNA technology and related fields in the technical field. These techniques are well described in the existing literature.

A first aspect of the present invention provides a Serratia marcescens, which is identified as Serratia marcescens SN after being separated and purified from a sludge mixture in a sewage treatment tank. -H1.

This strain has been deposited in the China Center for Type Culture Collection on September 25, 2018, and the deposit number is CCTCC NO: M 2018652.

The main characteristics of the Serratia marcescens SN-H1 are Gram-negative bacteria, short rod-shaped, dark red round colony shape, neat edges, and smooth and moist surface.

The Serratia marcescens SN-H1 contains the gene sequence shown in SEQ ID NO.1.

The Serratia marcescens SN-H1 was cultured in the sludge liquid medium for four weeks, and the VSS removal rate was over 48%.

The composition of the sludge liquid culture medium is: wet sludge (water content is 84%, VSS content is 64% of the dry weight of the sludge, both are mass fractions), add water to adjust the sludge (dry weight) concentration to 10g/L, pH value 7.0 to 7.5.

The second aspect of the present invention provides the application of Serratia marcescens SN-H1 in sludge degradation.

The sludge includes sludge in the organic wastewater treatment process, municipal sludge, river bottom sludge and the like.

The organic wastewater includes livestock and poultry breeding wastewater, industrial organic wastewater, domestic wastewater, landfill leachate, biogas slurry, and the like. A third aspect of the present invention provides a sludge degradation method, comprising at least the following steps:

(1) the seed liquid of Serratia marcescens SN-H1 is inoculated into sludge and mixed;

(2) Under the conditions of suitable temperature, pH value and DO value (dissolved oxygen content), aerobic cultivation is carried out to degrade the sludge.

The inoculation amount of the inoculation is 1-10% of the sludge volume, preferably 2-5%;

The temperature is 20～40℃, preferably 30℃;

The pH value is 6-8, preferably 7-7.5;

The DO value is 3-7 mg/L, preferably 3-5 mg/L.

Example 1:

Isolation, Screening and Performance Measurement of Sludge Degrading Bacteria SN-H1

The media and components used are as follows:

LB liquid medium: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, pH 7.0-7.5.

LB solid medium: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 15g/L agar powder, pH 7.0-7.5.

Sludge liquid culture medium: wet sludge (water content is 84%, VSS content is 64%, both are mass fractions), add water to adjust the concentration to 10g/L, and pH 7.0 to 7.5.

Sludge solid medium: wet sludge (water content is 84%, VSS content is 64% of the dry weight of the sludge, both are mass fractions), add water to adjust the sludge (dry weight) concentration to 30g/L, agar 20g/L , pH 7.0 to 7.5.

Protease production screening medium: casein 10g/L, 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 15g/L agar powder, pH 7.0-7.5.

Cellulase production screening medium: sodium carboxymethylcellulose 10g/L, 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 15g/L agar powder, pH 7.0～7.5 .

Amylase production screening medium: soluble starch 10g/L, 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract, 15g/L agar powder, pH 7.0-7.5.

After the above media were prepared, they were all sterilized by high pressure steam at 121 °C for 20 min, and used for later use.

Separation and purification: The sludge mixture of the test sample was collected from different sources of sewage treatment tanks, and 5mL of the sludge mixture was taken into 45mL of sterilized LB liquid medium. 24 hours of enrichment culture in medium; after gradient dilution of the cultured enrichment solution, it was evenly spread on LB solid medium; after 24 hours of culture in a 30°C constant temperature incubator, single clones with different colony shapes were picked and streaked. Numbered preservation after purification. A total of 18 strains were obtained through isolation and purification.

Primary screening: The pure strains obtained by separation and purification are inoculated onto the solid medium of sludge by the method of spot connection, and the primary screening is carried out. The strains with better growth on mud agar medium were streaked, purified and numbered for preservation. A total of 7 strains were obtained through primary screening.

Re-screening: Inoculate the pure strains obtained from the primary screening on the protease production screening medium, cellulase production screening medium and amylase production screening medium by spot-joining method to measure the enzyme-producing ability of the strains, so as to carry out Rescreen. After culturing the inoculated plate in a constant temperature incubator at 30°C for 48 to 72 hours, measure the colony diameter (d) and the diameter of the transparent circle (D), and calculate D/d as shown in Table 1, and select the strain with stronger enzyme-producing ability. Streak purified and numbered preservation. A total of 3 strains were obtained after re-screening, and SN-H1 had the highest enzyme-producing ability. Finally, SN-H1 was selected as the follow-up sludge degradation experiment.

Table 1 The results of the determination of the enzyme-producing ability of the strains

Determination of sludge degradation performance: Inoculate the strains obtained by re-screening into LB liquid medium, culture at 30°C, 180r/min shaker for 16h, take 2% (volume ratio) bacterial liquid and centrifuge it with sterile saline for washing , resuspended, and inoculated into sterilized sludge liquid medium. Culture in a shaker at 30°C and 180 r/min, and periodically sample the sludge mixture every other week to measure the removal rate of VSS in the sludge mixture.

Determination steps of VSS content:

(1) After centrifuging the sludge mixture for 10 min at 10000 r/min, the solid is used to measure the VSS content of the sludge;

(2) drying the obtained part of the solid samples to a constant weight in a 105° C. oven, and grinding them into powder for subsequent use;

(3) Put the clean crucible into a 105°C oven and bake it to a constant weight, take it out and place it in a desiccator and cool it to room temperature and weigh it, denoted as m ₀ ;

(4) Weigh the powdered sample in step (2), denoted as M _sample , put it in a crucible with a weight of m ₀ , transfer it into a muffle furnace, heat it to 550 ° C and burn it for 240 min (from the temperature to 550 ° C Start timing), wait for the temperature in the furnace to drop below 100 °C, take it out and put it in a desiccator to cool and weigh the total weight until it reaches a constant weight, denoted as M;

(5) Calculate the VSS content of the sample, namely VSS%, according to the weighing data and calculation formula ①:

VSS%=[1-(M-m ₀ )/M _sample ]×100% ①

(6) Calculate the VSS removal rate according to the calculation formula ②:

[VSS( ₀ )%-VSS _(x) %]/VSS ₍₀₎ %×100% ②

VSS ₍₀₎ % is the initial VSS content of the sludge broth without the inoculated strain,

In VSS( _x )%, x represents the sampling time point, and VSS( _x )% is the VSS content of the sample sampled at the x time point.

It can be seen from Figure 2 that after 4 weeks of culture, the removal rate of VSS by strain SN-H1 reached 48.8%, which was 19.9% higher than that of the control group, which was 40.7%. It shows that the strain SN-H1 has a significant sludge degradation effect.

Example 2: Molecular biological identification of strain SN-H1 CCTCC NO: M 2018652

Strains were identified by 16S rDNA sequence alignment. The genomic DNA of the strain SN-H1 was extracted according to the prior art, and using this as a template, a pair of universal primers (27F, 1492R) were used to amplify the 16S rDNA of the strain. The upstream primer is 27F

(5'-AGAGTTGATCCTGGCTCA-3'), the downstream primer was 1492R (5'-GGTTACCTTGTTACG ACTT-3').

The PCR reaction system (20 μL) was as follows: template DNA 0.5 μL, PCR Taqmix 10 μL, upstream and downstream primers 0.6 μL each, and ddH ₂ O was added to make the reaction system 20 μL.

PCR program: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min 30 s, a total of 30 cycles of the above, extension at 72 °C for 10 min, and finally storage at 4 °C.

Purification and sequencing of PCR products were performed by Shanghai Jie Li Biotechnology Co., Ltd. The DNA sequence of 16S r obtained by sequencing was submitted to NCBI, and the homology sequence alignment analysis was carried out with GenBank through the software, and the phylogenetic tree of the strain was constructed using MEGA6 software (as shown in Figure 1).

The effective sequence length of the gene of strain SN-H1 is 1430bp, see SEQ ID NO.1 in the gene sequence table. After alignment, the sequence is 99.9% homologous with Serratia marcescens (NCBI accession number: AB680131.1) in the NCBI database, belonging to Serratia marcescens, and named Serratia marcescens Serratiamarcescens 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 was taken from the mixed solution of the primary sedimentation tank in the wastewater treatment process of a pig farm. The TSS content of the original solution was 26g/L, the VSS content was 17g/L, and the pH value was 8.5.

The strain SN-H1 was inoculated into LB liquid medium, cultured at 30°C, 180r/min shaker for 16h, centrifuged and washed the bacterial liquid in the logarithmic growth phase according to 5% volume ratio, and then added to the reactor to control the conditions. It is: the rotating speed of the electric stirrer is 180r/min, the DO value is 3mg/L, the temperature is 30℃, and the pH value is 7. During the reaction, a sample was taken every 2 days, and the VSS removal rate in the wastewater was determined according to the VSS determination procedure in Example 1.

Figure 3 shows the degradation of sludge in pig farm wastewater by strain SN-H1 within 24 days. It can be seen that the maximum removal rate of VSS by strain SN-H1 reaches 79.6%, which is higher than that of VSS without bacteria in the control group. The removal rate was improved compared to 53.0%

50.2%. It shows that the strain SN-H1 has a significant degradation effect on the sludge in the pig farm wastewater.

Example 4: Degradation effect of sludge degrading bacteria SN-H1 on river bottom sludge

The river sediment is taken from the sediment of a river in Shanghai, that is, the pollutants from life and industry are deposited at the bottom of the river bed through physical, chemical and biological actions, forming gray-black sludge rich in organic matter and nutrients. The water content of the river sediments tested is 89%, the organic matter content in the dry base is 8-9%, and the pH value is 6.5-7.5.

The strain SN-H1 was inoculated into the LB liquid medium, cultured at 30°C, 180r/min shaker for 16h, centrifuged and washed the bacterial liquid in the logarithmic growth phase according to 3% volume ratio, and then added to the reactor to control. The conditions are: the rotating speed of the electric stirrer is 180r/min, the DO value is 3mg/L, the temperature is 30°C, and the pH value is 7. During the reaction, a sample was taken every 12 h, and the VSS removal rate of the river bottom mud was determined according to the VSS determination procedure in Example 1.

Figure 4 shows the degradation of river sediment by strain SN-H1 within 72 hours. It can be seen that the highest value of VSS removal rate of strain SN-H1 is 14.6%, which is 8.9% higher than that of the control group without inoculation. Compared with that, it improved by 64.0%. It shows that the strain SN-H1 has a significant degradation effect on river sediment.

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 the biogas slurry discharged from the biogas fermentation tank of a kitchen waste treatment plant. The TSS content of the raw liquid is 11%, the VSS content is 9.7%, and the pH value is 5.0-6.0.

Strain SN-H1 was inoculated into LB liquid medium, cultured at 30°C, 180r/min shaker for 16h, centrifuged and washed the bacterial liquid in logarithmic growth phase according to 3% volume ratio, and then added to the reactor for control. The conditions are: the rotating speed of the electric stirrer is 180r/min, the DO value is 4mg/L, the temperature is 30°C, and the pH value is 6.5. In the reaction process, a sample was taken every 1 d, and the removal rate of VSS in the kitchen waste biogas slurry was determined according to the VSS determination procedure in Example 1.

Figure 5 shows the degradation of sludge in the kitchen waste biogas slurry by strain SN-H1 within 7 days. It can be seen that the maximum removal rate of VSS by strain SN-H1 reaches 77.2%, which is higher than that of the control group without bacteria inoculation. Compared with the VSS removal rate of 62.5%, an increase of 23.5%. It shows that the strain SN-H1 has a significant degradation effect on kitchen waste.

In summary, the present invention obtained a high-efficiency sludge-degrading strain named SN-H1 through the separation and purification of the LB medium, the primary screening of the sludge agar medium, the re-screening of the enzyme-producing medium, and the verification of the sludge degradation performance. The taxonomic status of the strain was identified as Serratia marcescens by comprehensive analysis of the data of colony and cell morphology, physiological and biochemical characteristics, and 16S rDNA gene sequence determination. The strain was deposited in the China Center for Type Culture Collection (CCTCC), the deposit name is Serratia marcescens SN-H1, the deposit date is 2018.9.25, the deposit number is CCTCC NO: M2018652, and the deposit address is Bayi, Wuchang District, Wuhan City, Hubei Province. Wuhan University Chinese Type Culture Collection Center. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

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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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