A sulfate-reducing bacterial agent and its application

Abstract

The present invention relates to a sulfate-reducing bacteria agent and its application. The sulfate-reducing bacteria is Massilia timonae YJY, which is named as Massilia timonae YJY and has a preservation number of CGMCC NO.25091. Through enrichment culture, separation and purification, a single strain of sulfate-reducing bacteria is obtained, and the degradation effect of sulfate is verified. Through domestication, its degradation ability and tolerance are improved, and the product of action is clarified. The strain and its bacterial agent are put into an activated sludge anaerobic tank / anoxic tank, which can effectively tolerate high loads or inhibit the impact of harmful substances, and can use sulfate in a biochemical system as the only carbon and nitrogen source to thoroughly and harmlessly reduce sulfate to ensure the normal operation of the biochemical system. The strain with high-efficiency sulfate reduction provided by the present invention lays a material foundation for the research on biological treatment of wastewater containing sulfate substances, and has a high application value.

Description

Technical Field

[0001] The invention belongs to the field of environmental microorganisms and wastewater treatment, and specifically relates to a high-efficiency sulfate-reducing bacterial agent and application thereof. Background Art

[0002] Sulfate-reducing bacteria (SRB) are a group of prokaryotic microorganisms that use organic compounds (chemoheterotrophic) or inorganic compounds (chemoautotrophic) as electron donors to produce sulfide. It is not a taxonomic unit, but a general term for microbial groups with the same function. They are distributed in five phyla of the Bacteria and Archaea domains, so some literature also calls them SRP (Sulfate-reducing prokaryotes). However, since the abundance and number of species of sulfate-reducing archaea are far less than those of bacteria, they are still customarily called SRB.

[0003] SRB was first discovered by Dutch biologist MW Beijerinck in 1895 when studying water softening. With the understanding of the toxicity and production process of hydrogen sulfide and the continuous analysis of the causes of metal corrosion, SRB has fully entered the mainstream biological community. Therefore, early research on SRB mainly focused on how to prevent SRB from forming biofilms in oil production environments and biocorrosion caused by such biofilms. However, since most SRBs require an anaerobic environment (at least a low redox potential) for growth, it was not until the anaerobic microbial culture technology and molecular biology-based analytical methods matured that research on SRB began to make major breakthroughs. Since the 1960s, the metabolic mode of SRB with sulfate as a substrate has attracted people's attention. Many reports in the 1990s summarized various special life processes in which SRB participate, enriched the theory of heterotrophic sulfate reduction, and improved people's understanding of this type of special life. At present, the sulfate-reducing bacteria isolated by domestic and foreign scholars generally have low reduction capabilities and can generally grow at sulfate concentrations <1500mg / L. There are few reports of strains resistant to high concentrations of sulfate. It is necessary to continue to search for sulfate-reducing bacteria with strong tolerance and high reducing ability, so as to obtain good application in wastewater with high concentration of sulfate pollution. Summary of the invention

[0004] The purpose of the present invention is to provide a highly efficient sulfate-reducing bacteria agent and its application, which can be applied to the bio-enhanced treatment of sulfate wastewater to help degrade sulfate substances in the wastewater biological treatment system.

[0005] Another object of the present invention is to provide application of the strain in treating sulfate wastewater.

[0006] In order to achieve the technical purpose of the present invention, the technical solution of the present invention is as follows:

[0007] A highly efficient sulfate-reducing bacteria agent, wherein the highly efficient sulfate-reducing bacteria is Massilia timonae, named Massilia timonae YJY, the strain is currently deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.25091, and the gene sequence of 16SrDNA of the strain is shown in SEQ ID No: 1.

[0008] Furthermore, the highly efficient sulfate-reducing bacteria has the ability to degrade sulfate in the absence of carbon and nitrogen sources.

[0009] Furthermore, the method for screening and acclimating efficient sulfate-reducing bacteria comprises the following steps:

[0010] (1) Enrichment culture of strains

[0011] The following culture medium was used for enrichment culture. The components of the primary screening and secondary screening culture medium were K2HPO4 0.5g, NH4Cl1.0g, Na2SO4 0.5g, MgSO4﹒7H2O 2.0g, sodium lactate 5.0mL, CaCl2﹒2H2O 0.1g, FeSO4﹒7H2O 0.5g, yeast extract 1.0g, distilled water was added to 1000mL, pH was adjusted to 7.0-7.5, high pressure steam sterilization was performed at 121℃ for 20min, and then cooled to 55℃, and ferrous ammonium sulfate 0.5g / L and ascorbic acid 0.1g / L were added;

[0012] (2) Isolation and purification of bacterial strains

[0013] The anaerobic bacteria separation and purification method or the double-layer plate method is used to separate and purify the strains. The enriched culture solution of step (1) is made into a gradient dilution solution, and 0.1 mL of each dilution of the bacterial solution is respectively transferred, and the bacterial suspension is separated into three zones and streaked;

[0014] (3) Domestication of strains

[0015] By continuously increasing the substrate concentration and testing its tolerance.

[0016] Furthermore, the fermentation medium used in the fermentation process is: K2HPO4 0.5g, NH4Cl 1.0g, Na2SO40.5g, MgSO4﹒7H2O 2.0g, sodium lactate 5.0mL, CaCl2﹒2H2O 0.1g, FeSO4﹒7H2O 0.5g, yeast extract 1.0g, add distilled water to 1000mL, adjust pH to 7.0-7.5, sterilize at 121℃ high pressure steam for 20min, cool to 55℃, add 0.5g / L ammonium ferrous sulfate and 0.1g / L ascorbic acid.

[0017] An application of a high-efficiency sulfate-reducing bacterial agent, wherein any of the above-mentioned high-efficiency sulfate-reducing bacterial agents is applied to the treatment of pollutants containing sulfate.

[0018] Furthermore, the pollutant containing sulfate is industrial wastewater, agricultural wastewater, waste residue or sediment.

[0019] Furthermore, the initial sulfate concentration in the sulfate pollutant is no more than 3000 mg / L.

[0020] Furthermore, the amount of the high-efficiency sulfate-reducing bacteria agent added is 2-5% by mass of the pollutants containing sulfate.

[0021] The beneficial effects of the present invention are:

[0022] The sulfate-reducing bacteria of the present invention has a highly efficient sulfate wastewater biodegradation treatment function, can also rapidly degrade sulfate in the absence of a carbon source or a nitrogen source, and can achieve a sulfate degradation rate of more than 80% at an initial concentration of 1000-1500 mg / L within 72 hours of anaerobic culture at a temperature of 30-37°C and a pH of 7.0-7.5, thereby greatly reducing the wastewater treatment cost and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of sulfate-reducing bacteria of the present invention.

[0024] Figure 2 This is a diagram showing the degradation effect of sulfate-reducing bacteria of the present invention.

[0025] Figure 3 This is a comparison chart of the sulfate-reducing bacteria 50000 mg / L sulfate tolerance experiment of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described in detail by way of examples below. The following examples are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be interpreted as limiting the technical solution of the present invention. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies.

[0027] The present application provides a highly efficient sulfate-reducing bacterial agent, wherein the highly efficient sulfate-reducing bacteria is Massilia timonae, named Massilia timonae YJY, and the strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 16, 2022, with a deposit number of CGMCC NO.25091, and the gene sequence of the strain 16SrDNA is shown in SEQ ID No: 1.

[0028] Example 1

[0029] 1. Obtaining strains:

[0030] Sulfate-reducing bacteria was sampled from activated sludge in a sewage treatment plant in Binzhou City, Shandong Province, and a strain of bacteria that efficiently degrades sulfate was obtained through primary and secondary screening. The specific steps are as follows:

[0031] The primary screening and rescreening culture medium is: K2HPO4 0.5g, NH4Cl 1.0g, Na2SO4 0.5g, MgSO4﹒7H2O2.0g, sodium lactate 5.0mL, CaCl2﹒2H2O 0.1g, FeSO4﹒7H2O 0.5g, yeast extract 1.0g, add distilled water to 1000mL, adjust pH 7.0-7.5. Sterilize at 121℃ high pressure steam for 20min, cool to 55℃, add 0.5g / L ammonium ferrous sulfate and 0.1g / L ascorbic acid. The dilution coating-stacked plate sandwich method and streak separation method were used for step-by-step screening and purification, and the bacteria with high efficiency in degrading sulfate were obtained as shown in the attached. Figure 1 .

[0032] 2. Identification of sulfate-reducing bacteria strains:

[0033] The 16S rDNA gene of the sulfate-reducing bacteria was cloned and sequenced, and then Blast comparison was performed in GenBank. The results showed that the sulfate-reducing strain was identified as Massilia timonae.

[0034] Example 2

[0035] Degradation characteristics of the strain:

[0036] 1. Degradation rate experiment:

[0037] A 2L system was prepared for acclimatization and degradation testing. The results are shown in the following table:

[0038] Table 1 The first batch of acclimatization and sulfate degradation capacity determination

[0039]

[0040] Table 2 Second batch of acclimatization and sulfate degradation capacity determination

[0041]

[0042]

[0043] Table 3 Determination of sulfate degradation capacity of the third batch of acclimatization

[0044]

[0045] 2. Optimum working temperature:

[0046] The experimental temperatures were 10℃, 25℃, 35℃ and 45℃ respectively, and the results are as follows:

[0047]

[0048] The experimental results of the optimum action temperature in Table 4 show that the optimum action temperature of Masseille timonei is 35°C, and the sulfate degradation in 3 days is 1760.53 mg / L, with a degradation rate of 57.85%.

[0049] Example 3

[0050] Tolerance of strains:

[0051] 5000, 10000, 20000, 30000 and 50000 mg / L sulfate systems were prepared, sulfate-reducing bacteria were gradually added, and the sulfate degradation amount was measured after 5 days. The results are as follows.

[0052] Table 5 The first batch of sulfate tolerance experiments

[0053] <![CDATA[Initial SO4 2- (mg / L)]]> <![CDATA[5th SO4 2- (mg / L)]]> 5d degradation amount (mg / L) 4772.18 3009.43 1762.75 10138.23 7282.85 2855.38 19658.70 18276.28 1382.42

[0054] Table 6 The second batch of sulfate tolerance test

[0055] <![CDATA[Initial SO4 2- (mg / L)]]> <![CDATA[5th SO4 2- (mg / L)]]> 5d degradation amount (mg / L) 13841.30 11476.11 2365.19 20423.21 18569.97 1853.24 33533.56 30893.06 2640.5

[0056] Table 7 The third batch of sulfate tolerance test

[0057] <![CDATA[Initial SO4 2- (mg / L)]]> <![CDATA[5th SO4 2- (mg / L)]]> 5d degradation amount (mg / L) 50656.39 49753.27 903.12

[0058] The results showed that the domesticated sulfate-reducing bacteria could tolerate a maximum sulfate concentration of 50,000 mg / L, and the system produced FeS black precipitate, but the degradation ability decreased with increasing concentration, and the sulfate degradation after 5 days was 903.12 mg / L. Its tolerance can be improved after domestication.

[0059] Example 4

[0060] Application of efficient sulfate-reducing bacteria in treating sulfate wastewater in sewage treatment plants:

[0061] The specific method of sulfate-reducing bacteria is as follows: suspend the sulfate-reducing bacteria in a sulfate-containing water solution, and anaerobically culture for 72 hours at a temperature of 30-37° C. and a pH of 7.0-7.5. The inoculation amount of the sulfate-reducing bacteria is 2-5%.

[0062] The initial concentration of sulfate in the sulfate-containing water is not greater than 3000 mg / L.

[0063] In the absence of carbon and nitrogen sources, sulfate can be rapidly degraded. Under anaerobic conditions of 30-37°C and pH 7.0-7.5, the degradation rate of sulfate with an initial concentration of 1000-1500 mg / L can reach more than 80% within 72 hours of anaerobic culture.

[0064] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sulfate-reducing bacterial agent, It is characterized in that The sulfate-reducing bacteria included Massillarium timonei ( Massilia Timonae ) YJY, the strain is currently deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.25091. The gene sequence of 16SrDNA of the strain is shown in SEQ ID No:

1.

2. Application of a sulfate-reducing bacterial agent, It is characterized in that The sulfate-reducing bacteria agent of claim 1 is used for treating pollutants containing sulfate.

3. The use according to claim 2, It is characterized in that Pollutants containing sulfates are industrial wastewater, agricultural wastewater, waste residues or sediments.

4. The use according to claim 3, It is characterized in that The initial sulfate concentration in the sulfate pollutant is not more than 3000 mg / L.

5. The use according to claim 4, It is characterized in that The amount of sulfate-reducing bacteria added is 2-5% of the mass percentage of the pollutants containing sulfate.

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