A desulfurization composite bacteria with high desulfurization characteristics and its application

Abstract

The present invention discloses a desulfurization composite bacterium with high desulfurization properties and its application. The desulfurization composite bacterium of the present invention is composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1. The desulfurization composite bacterium of the present invention has high desulfurization properties and can be used to treat wastewater with a sulfide content of 40 to 150 mg / L (such as freshwater aquaculture wastewater), providing a healthy growth environment for aquatic animals and improving aquaculture efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and wastewater treatment, and in particular relates to a desulfurization composite bacterium with high desulfurization performance and application thereof. Background Art

[0002] With the rapid development of my country's aquaculture industry, the environmental problems associated with high-density, intensive aquaculture are becoming increasingly prominent. Excessive fertilization leads to an overabundance of nutrients in the water, and this nutrient accumulation can trigger a series of environmental and ecological problems. Excessive feeding, on the one hand, leads to direct economic losses due to the loss of bait, and on the other hand, the decomposition of bait deposited at the bottom of the aquaculture pond consumes significant amounts of oxygen, degrading the local substrate environment. In short, large amounts of leftover bait, dead plants and animals, excrement, and fertilizer accumulate in the pond sediment, easily generating harmful substances such as ammonia, organic acids, sulfides, and methane, which harm aquaculture production.

[0003] Sulfide is a harmful product of high-density, large-scale feeding aquaculture methods that damage the aquaculture ecological environment and weaken the self-purification ability of water bodies. Bottom sulfide is one of the important factors affecting the aquaculture environment and an important indicator for measuring the quality of the bottom environment. Moreover, hydrogen sulfide itself, as a strong reducing agent, can affect the normal metabolism of most aerobic microorganisms and algae, thereby weakening the self-purification ability of water bodies. In addition, sulfide can combine with hemoglobin in the blood of farmed organisms to produce sulfhemoglobin, generating chocolate-like black blood, which reduces the oxygen-carrying capacity of the body's blood. Sulfide has a strong irritating and corrosive effect on the gill tissue of farmed organisms, causing coagulative necrosis of the tissue, resulting in difficulty breathing and even suffocation. The toxicity of sulfide to fish and shrimp is mainly through the mucous membrane on the gill surface and the Na in the tissue + or Cu in blood 2+ They combine to form substances with strong stimulating effects, inhibiting the occurrence of certain enzymatic reactions and causing harm to the body.

[0004] At present, there are few studies on the screening of freshwater desulfurization bacteria. Therefore, the isolation and screening of desulfurization bacteria with high desulfurization rates are of great significance for the treatment of aquaculture wastewater, maintaining a healthy aquaculture environment, and improving aquaculture benefits. Summary of the Invention

[0005] In order to better treat aquaculture wastewater, maintain a healthy growth environment for aquatic animals, and improve aquaculture benefits, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a desulfurization composite bacterium with high desulfurization properties, wherein the desulfurization composite bacterium is composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1.

[0007] The Priestia megaterium A-1 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC NO.33038 and the deposit date December 12, 2024.

[0008] The Exiguobacterium acetylicum C-1 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC NO.33039 and the deposit date December 12, 2024.

[0009] Preferably, the 16S rDNA sequence of the Priesteria gigantea A-1 is shown in SEQ ID NO: 1, and the specific sequence is as follows:

[0010]

[0011] Preferably, the 16S rDNA sequence of Exiguobacterium acetylicum C-1 is shown as SEQ ID NO: 2.

[0012] The specific sequence is as follows:

[0013]

[0014] Preferably, in the desulfurization composite bacteria, the effective viable bacterial count ratio of the Prioccludinium giganteum A-1 and the Exiguobacterium acetylicum C-1 is 5:1 to 1:5, for example: 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0015] Furthermore, the effective viable cell count ratio of the Priesteria megaterium A-1 to the Exiguobacterium acetylicum C-1 is 1:3.

[0016] In a second aspect, the present invention provides a method for culturing the desulfurization composite bacteria described in the first aspect, the culturing method comprising using a culture medium, the concentration of sulfide in the culture medium being 1.0 to 3.0 mg / mL, for example: 1.0 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL, 3.0 mg / mL.

[0017] Preferably, the basic formula of the culture medium is as follows:

[0018] Glucose 5.0 g, KH2PO4 1.0 g, K2HPO4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl2 0.01 g, NH4Cl 0.4 g, NaHS 5.0 g, distilled water 1000 mL, pH 6.8-7.2.

[0019] Preferably, the culture method further comprises inoculating the desulfurization composite bacteria into synthetic wastewater.

[0020] Furthermore, the basic formula of the synthetic wastewater is: 3.0 g of glucose, 1.0 g of KH2PO4, 3.0 g of Na2S, 1000 mL of distilled water, and a pH value of 6.8-7.2.

[0021] In a third aspect, the present invention provides a bacterial agent comprising the desulfurization composite bacteria described in the first aspect.

[0022] Preferably, the effective viable count of Priesteria gigantea A-1 in the bacterial agent is 1×10 8 ~9×10 9 CFU / mL, for example: 1×10 8 CFU / mL, 3×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 3×10 9 CFU / mL, 6×109 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL.

[0023] Alternatively, the OD of Priesteria gigantea A-1 in the bacterial agent 600 =0.6~1.0, for example: 0.6, 0.7, 0.8, 0.9, 1.0.

[0024] Preferably, the effective viable count of Exiguobacterium acetylicum C-1 in the bacterial agent is 1×10 8 ~9×10 9 CFU / mL, for example: 1×10 8 CFU / mL, 3×10 8 CFU / mL, 6×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 3×10 9 CFU / mL, 6×10 9 CFU / mL, 8×10 9 CFU / mL, 9×10 9 CFU / mL.

[0025] Alternatively, the OD of Exiguobacterium acetylicum C-1 in the bacterial agent is 600 =0.6~1.0, for example: 0.6, 0.7, 0.8, 0.9, 1.0.

[0026] Preferably, the bacterial agent may also include non-oxygenous photosynthetic bacteria such as Rhodopseudomonas (such as Rhodopseudomonas palustris), green non-sulfur bacteria, etc.

[0027] In a fourth aspect, the present invention provides a wastewater treatment agent, which comprises the desulfurization composite bacteria described in the first aspect or the bacterial agent described in the third aspect.

[0028] Preferably, the wastewater treatment agent may also include a water purification agent, such as polyaluminum chloride, polyacrylamide, activated carbon or diatomaceous earth.

[0029] In a fifth aspect, the present invention provides use of the desulfurization composite bacteria described in the first aspect or the bacterial agent described in the third aspect in preparing a wastewater treatment agent.

[0030] In a sixth aspect, the present invention provides use of the desulfurization composite bacteria described in the first aspect, the bacterial agent described in the third aspect, or the wastewater treatment agent described in the fourth aspect in treating wastewater.

[0031] Preferably, the sulfide concentration in the wastewater is ≥40 mg / L.

[0032] Furthermore, the sulfide concentration in the wastewater is ≤150 mg / L, for example: 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L.

[0033] Furthermore, the sulfides include but are not limited to H2S, HS - 、S 2- , soluble sulfides and acid-soluble metal sulfides.

[0034] Furthermore, the sulfide includes H2S, HS - 、S 2- Any one or a combination of two or more.

[0035] Preferably, the desulfurization composite bacteria, the bacterial agent or the wastewater treatment agent is added to the wastewater to remove the sulfide.

[0036] Preferably, the wastewater is aquaculture wastewater, more preferably freshwater aquaculture wastewater.

[0037] Preferably, the inoculation amount of the desulfurization composite bacteria is 1-5%, for example: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%.

[0038] The terms “include” or “comprising” described in the present invention are open-ended expressions, which include the specified components or steps described, as well as other specified components or steps that will not be substantially affected.

[0039] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0040] Beneficial effects of the present invention:

[0041] The desulfurization composite bacteria of the present invention are composed of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1, have high desulfurization properties, can be used to treat wastewater with a sulfide content of 40 to 150 mg / L (such as freshwater aquaculture wastewater), provide a healthy growth environment for aquatic animals, and improve aquaculture efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The colony morphology and Gram staining images of the strains are shown, where A is Priesteria gigantea A-1 and B is Exiguobacterium acetylicum C-1;

[0043] Figure 2 The figure shows the effect of different bacterial solution ratios on the desulfurization performance of composite bacteria;

[0044] Figure 3 The effect of different C / S ratios on the desulfurization performance of composite bacteria;

[0045] Figure 4 The effect of different pH on the desulfurization performance of composite bacteria;

[0046] Figure 5 The effect of different temperatures on the desulfurization performance of composite bacteria;

[0047] Figure 6 This is a comparative growth curve of the sulfide degradation of the composite bacteria and a single strain.

[0048] Culture deposits for patent procedures:

[0049] Priestia megaterium A-1, deposit number is CGMCC NO.33038, and deposit date is December 12, 2024.

[0050] Exiguobacterium acetylicum C-1, the deposit number is CGMCC NO.33039, and the deposit date is December 12, 2024.

[0051] Depository: China General Microbiology Center (CGMCC).

[0052] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101. DETAILED DESCRIPTION

[0053] The technical solutions of the present invention are described clearly and completely below in conjunction with the embodiments and accompanying drawings. The described embodiments are only a portion of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0054] The culture medium of the present invention is:

[0055] Enrichment medium: glucose 5.0 g, KH2PO4 1.0 g, K2HPO4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl2 0.01 g, NH4Cl 0.4 g, NaHS 5.0 g, distilled water 1000 mL, pH 6.8-7.2, sterilized at 115°C for 30 min.

[0056] Isolation solid culture medium: glucose 5.0 g, KH2PO4 1.0 g, K2HPO4 1.0 g, magnesium chloride hexahydrate 0.8 g, FeCl2 0.01 g, NH4Cl 0.4 g, NaHS 5.0 g, distilled water 1000 mL, agar powder 17 g, pH 6.8-7.2, sterilize at 115°C for 30 min.

[0057] Synthetic wastewater: 3.0 g glucose, 1.0 g KH2PO4, 3.0 g Na2S, 1000 mL distilled water, pH 6.8-7.2; due to the volatilization of sulfide during the preparation process, the sulfide concentration is 90-150 mg / L.

[0058] LB medium: 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, 1 L of distilled water, pH 7.0, sterilized at 121°C for 20 min.

[0059] Example 1 Isolation, screening and identification of desulfurization strains with high desulfurization rates

[0060] Take 10g of Benxi Guanyin Pavilion sediment sample and place it in 90mL enrichment culture medium, place it in a shaker at 30℃ and 180rpm for constant temperature culture for 1 day, take 10mL of enriched bacterial liquid and add it to 90mL enrichment culture medium for culture, and repeat this three times; then take the bacterial liquid for gradient dilution and spread it on the surface of separation solid culture medium, after constant temperature culture at 30℃ for 1-3 days, select different strains with good growth and further purify them on separation solid culture medium, pick single colonies for streak separation, repeat this three times to obtain a single strain; use an inoculation loop to pick a single colony and add it to synthetic wastewater, use methylene blue spectrophotometry to measure the strain with the strongest desulfurization ability, and store this single strain in a -80℃ refrigerator.

[0061] The morphological identification results are as follows Figure 1 As shown, the surface of the screened colonies is moist, smooth, opaque, with complete and regular edges, presenting round protrusions, and the colonies are light yellow.

[0062] Molecular biological identification: DNA from the screened strains was extracted using a bacterial genome extraction kit (Tiangen). PCR amplification of the conserved target fragment was performed using universal primers 27F (SEQ ID NO: 3) and 1492R (SEQ ID NO: 4) for the 16S rDNA gene. The amplified product was excised and recovered from the gel, followed by sequencing analysis. PCR amplification, sequencing, and BLAST comparison revealed that the 16S rDNA gene sequences of the two screened strains showed that one strain, as shown in SEQ ID NO: 1, had a 99.99% similarity to Priestia megaterium. The other strain, as shown in SEQ ID NO: 2, had a 99.99% similarity to Exiguobacterium acetylicum. Therefore, strain A-1 was identified as Priestia megaterium and named Priestia megaterium A-1; strain C-1 was identified as Exiguobacterium acetylicum and named Exiguobacterium acetylicum C-1.

[0063] The specific sequence of 16S rDNA of Priesteria gigantea A-1 is as follows:

[0064]

[0065] The specific sequence of 16S rDNA of Exiguobacterium acetylicum C-1 is as follows:

[0066]

[0067] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:3)

[0068] 1492R:5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO:4)

[0069] Example 2 Effect of different bacterial liquid ratios on the desulfurization effect of composite bacteria

[0070] Seed solution preparation: strains Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 were picked and placed in LB medium, cultured at 30°C and 180 rpm for 10-12 hours, and the culture medium was taken to measure the OD value of the solution. 600 OD 600 When the concentration reaches 0.6-0.8, it indicates that the strain has entered the logarithmic growth phase and the seed solution can be used normally. The viable counts of Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 are both 6-8×10 8 CFU / mL.

[0071] The seed solution ratios of Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 were adjusted to A-1:C-1 = 5:1, A-1:C-1 = 3:1, A-1:C-1 = 1:1, A-1:C-1 = 1:3, and A-1:C-1 = 1:5, respectively. Each composite seed solution was inoculated into 100 mL of synthetic wastewater at a 3.0% inoculum. The culture was shaken at 180 rpm and pH = 7 for 24 hours at 25°C. After 24 hours, water samples were taken and the sulfide content in the solution was measured using methylene blue spectrophotometry. The initial synthetic wastewater, cultured under the same conditions and for the same time, was used as a blank control. The desulfurization rate was calculated according to the following formula.

[0072] Desulfurization rate = (sulfide concentration of blank control group - sulfide concentration of water sample) / sulfide concentration of blank control group.

[0073] like Figure 2 As shown, the desulfurization rate of the composite bacteria was above 92% when the ratio of A-1:C-1 was 5:1 to 1:5, and the highest desulfurization rate was 95% when the ratio was 1:3.

[0074] Example 3 Effect of carbon-sulfur ratio (C / S) on desulfurization effect of composite bacteria

[0075] Seed solution preparation: same as Example 2.

[0076] The C / S ratios of the synthetic wastewater were adjusted to 1:1, 3:1, 5:1, 7:1, and 9:1, respectively. A composite seed solution of A-1:C-1 = 1:3 was inoculated with 3.0% inoculum into 100 mL of synthetic wastewater with different C / S ratios. The mixture was cultured at pH = 7, 25°C, and 180 rpm with shaking for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as in Example 2).

[0077] like Figure 3 As shown in the figure, when the C / S ratio is 1:1-9:1, the desulfurization rate of the composite bacteria is higher than 84%; when the C / S ratio is 1:1-7:1, the desulfurization rate of the composite bacteria is higher than 93%; when the C / S ratio is 1:1-3:1, the desulfurization rate is higher than 95%; when the C / S ratio is 3:1, the desulfurization rate reaches 98%.

[0078] Example 4 Effect of pH on Desulfurization Effect of Composite Bacteria

[0079] Seed solution preparation: same as Example 2.

[0080] The pH values ​​of the synthetic wastewater were adjusted to 5.5, 6, 6.5, 7, 7.5, and 8, respectively. The composite seed solution of A-1:C-1=1:3 was inoculated into 100 mL of synthetic wastewater with different pH values ​​at an inoculum size of 3.0%. The culture was carried out at C / S=3:1, 25°C, and 180 rpm with shaking for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as in Example 2).

[0081] like Figure 4 As shown, when the pH value is 6-8, the desulfurization rate of the composite bacteria for wastewater is higher than 85%; when the pH value is 6.5-7.5, the desulfurization rate is higher than 93%; when the pH value is 7.0, the desulfurization rate reaches 97%.

[0082] Example 5 Study on the effect of temperature on the desulfurization effect of composite bacteria

[0083] Seed solution preparation: same as Example 2.

[0084] The temperature of the synthetic wastewater was adjusted to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively. The composite seed solution of A-1:C-1=1:3 was inoculated into 100 mL of synthetic wastewater at different temperatures at a 3.0% inoculum size. The pH was 7, C / S=3:1, and the culture was shaken at 180 rpm for 24 h. After 24 h, water samples were taken and the sulfide content was determined by methylene blue spectrophotometry, and the desulfurization rate was calculated (same as in Example 2).

[0085] like Figure 5 As shown, when the temperature is 15-35℃, the desulfurization rate of the composite bacteria for wastewater is higher than 90%; when the temperature is 20-30℃, the desulfurization rate is higher than 93%; at 20℃, the desulfurization rate reaches 97%.

[0086] Example 6 Evaluation of the desulfurization effect of composite bacteria on simulated wastewater

[0087] Seed solution preparation: same as Example 2.

[0088] The composite seed solution of A-1:C-1=1:3 was inoculated into 500 mL of synthetic wastewater at a 3.0% inoculum size. The mixture was cultured at pH=7, C / S=3:1, 20°C, and 180 rpm with shaking for 24 h. Water samples were taken every 6 h to determine the sulfide concentration and solution OD. 600 .

[0089] like Figure 6 As shown in the figure, from 0 to 48 hours, compared with the blank control group, the sulfur content in the wastewater treated with Prioccludin A-1, Exiguobacterium acetylicum C-1, and the composite bacteria was lower than that in the blank control group. The sulfur content was in the following order from high to low: blank control group, Prioccludin A-1, Exiguobacterium acetylicum C-1, and composite bacteria. From 0 to 30 hours, the order of wastewater desulfurization rate was composite bacteria, Exiguobacterium acetylicum C-1, and Prioccludin A-1. After 30 hours of wastewater treatment, the desulfurization rate of the composite bacteria was 87.31%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 85.10%, and the desulfurization rate of Priesteria gigantea A-1 was 65.69%; after 36 hours of wastewater treatment, as the desulfurization rate gradually increased, the difference in desulfurization rate between Priesteria gigantea A-1, Exiguobacterium acetylicum C-1 and the composite bacteria gradually became smaller. At 36 hours, the desulfurization rate of the composite bacteria was 92.99%, the desulfurization rate of Exiguobacterium acetylicum C-1 was 92.99%, and the desulfurization rate of The desulfurization rate of the composite bacteria was 93.45%, and that of P. gigantea A-1 was 84.72%. After 42 hours, the desulfurization rates of the composite bacteria were 98.70%, 98.13%, and 96.21%, respectively. After 48 hours, the desulfurization rates of the composite bacteria were 99.21%, 98.87%, and 97.90%, respectively. Within 48 hours, the composite bacteria could remove more than 99% of sulfides from the wastewater, demonstrating superior desulfurization performance compared to both P. gigantea A-1 and P. gigantea.

[0090] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A desulfurization composite bacteria with high desulfurization properties, characterized in that: The desulfurization composite bacteria consists of Priestia megaterium A-1 and Exiguobacterium acetylicum C-1; The Priestia megaterium A-1 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.33038 and the deposit date of December 12, 2024; The Exiguobacterium acetylicum C-1 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC NO.33039 and the deposit date December 12, 2024.

2. The desulfurization composite bacteria according to claim 1, characterized in that In the desulfurization composite bacteria, the effective viable bacterial count ratio of the Priesteria gigantea A-1 to the Exiguobacterium acetylicum C-1 is 5:1 to 1:

5.

3. A bacterial agent, characterized in that The bacterial agent comprises the desulfurization composite bacteria according to claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that The effective viable counts of Priesteria gigantea A-1 and Exiguobacterium acetylicum C-1 in the bacterial agent were both 1×10 8 ~9×10 9 CFU / mL.

5. A wastewater treatment agent, characterized in that The wastewater treatment agent comprises the desulfurization composite bacteria according to any one of claims 1-2 or the bacterial agent according to any one of claims 3-4.

6. Use of the desulfurization composite bacteria according to any one of claims 1-2 or the bacterial agent according to any one of claims 3-4 in preparing a wastewater treatment agent for sulfide-containing wastewater.

7. Use of the desulfurization composite bacteria according to any one of claims 1-2, the bacterial agent according to any one of claims 3-4, or the wastewater treatment agent according to claim 5 in treating sulfide-containing wastewater.

8. The use according to claim 7, characterized in that The sulfide concentration in the wastewater is 40-150 mg / L.

9. The use according to claim 8, characterized in that The sulfide includes H2S, HS - 、S 2- Any one or a combination of two or more.

10. The use according to any one of claims 7 to 9, characterized in that: The desulfurization composite bacteria, the bacterial agent or the wastewater treatment agent is added to the wastewater.

11. The use according to claim 10, characterized in that The inoculation amount of the desulfurization composite bacteria is 1-5%.

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