A heterotrophic-aerobic desulfurization bacterium with high desulfurization rate and its application

Abstract

The present invention discloses a heterotrophic aerobic desulfurizing bacterium with a high desulfurization rate and its application. The present invention screened a strain, Exiguobacterium acetylicum C-1, with a deposit number of CGMCC NO. 33039. The Exiguobacterium acetylicum C-1 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 invention belongs to the technical field of microorganisms and wastewater treatment, and particularly relates to a heterotrophic-aerobic desulfurization bacterium with a high desulfurization rate and an 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 the first aspect, the present invention provides a desulfurization bacterium with a high desulfurization rate, wherein the desulfurization bacterium is Exiguobacterium acetylicum C-1, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCCNO.33039 and a deposit date of December 12, 2024.

[0007] In a second aspect, the present invention provides a method for culturing the desulfurization 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.

[0008] Preferably, the basic formula of the culture medium is as follows:

[0009] 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.

[0010] Preferably, the culturing method further comprises inoculating the desulfurization bacteria into synthetic wastewater.

[0011] 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.

[0012] Furthermore, the carbon-sulfur ratio (C / S) of the synthetic wastewater is 1 to 9, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, and more preferably 1.

[0013] In a third aspect, the present invention provides a bacterial agent comprising the desulfurization bacteria described in the first aspect.

[0014] 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.

[0015] Alternatively, the OD of Exiguobacterium acetylicum C-1 in the bacterial agent is600 =0.6~1.0, for example: 0.6, 0.7, 0.8, 0.9, 1.0.

[0016] Preferably, the bacterial agent may also include non-oxygenous photosynthetic bacteria such as Rhodopseudomonas (such as Rhodopseudomonas palustris) and green non-sulfur bacteria.

[0017] In a fourth aspect, the present invention provides a wastewater treatment agent, which comprises the desulfurization bacteria described in the first aspect or the bacterial agent described in the third aspect.

[0018] Preferably, the wastewater treatment agent may also include a water purification agent, such as polyaluminum chloride, polyacrylamide, activated carbon or diatomaceous earth.

[0019] In a fifth aspect, the present invention provides use of the desulfurization bacteria described in the first aspect or the bacterial agent described in the third aspect in preparing a wastewater treatment agent.

[0020] In a sixth aspect, the present invention provides use of the desulfurization 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.

[0021] Preferably, the sulfide concentration in the wastewater is ≥40 mg / L.

[0022] 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.

[0023] Furthermore, the sulfides include but are not limited to H2S, HS - 、S 2- , soluble sulfides and acid-soluble metal sulfides.

[0024] Preferably, the desulfurization bacteria, the bacterial agent or the wastewater treatment agent is added to the wastewater to remove the sulfide.

[0025] Preferably, the wastewater is aquaculture wastewater, more preferably freshwater aquaculture wastewater.

[0026] Preferably, the inoculation amount of the desulfurization bacteria is 1-5%, for example: 1%, 2%, 3%, 4%, 5%.

[0027] 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.

[0028] 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."

[0029] Beneficial effects of the present invention:

[0030] The Exiguobacterium acetylicum C-1 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The colony morphology and Gram staining of strain C-1;

[0032] Figure 2 The effect of different C / S ratios on the growth of strain C-1;

[0033] Figure 3 The effect of different pH on the growth of strain C-1;

[0034] Figure 4 is the effect of temperature on the growth of strain C-1;

[0035] Figure 5 The effect of different carbon sources on the growth of strain C-1;

[0036] Figure 6 The effect of different C / S ratios on the desulfurization performance of strain C-1;

[0037] Figure 7 The effect of different pH on the desulfurization performance of strain C-1;

[0038] Figure 8 The effect of different temperatures on the desulfurization performance of strain C-1;

[0039] Figure 9 This is the sulfide degradation and growth curve of strain C-1 under optimal conditions.

[0040] Culture deposits for patent procedures:

[0041] Exiguobacterium acetylicum C-1, the deposit number is CGMCC NO.33039, and the deposit date is December 12, 2024.

[0042] Depository: China General Microbiology Center (CGMCC).

[0043] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101. DETAILED DESCRIPTION

[0044] 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.

[0045] The culture medium of the present invention is:

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Example 1 Isolation, screening and identification of freshwater heterotrophic-aerobic desulfurization strains with high desulfurization rate characteristics

[0051] 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.

[0052] 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.

[0053] Molecular biological identification: DNA of the screening strain was extracted using a bacterial genome extraction kit (Tiangen), and PCR amplification of the conserved target fragment was performed using the 16S rDNA gene universal primers 27F (SEQ ID NO: 2) and 1492R (SEQ ID NO: 3). The amplified product was excised and recovered, and then sequenced and analyzed. PCR amplification, sequencing, and BLAST comparison were performed. The results showed that the 16S rDNA gene sequence (SEQ ID NO: 1) of the screening strain had a high similarity to Exiguobacterium acetylicum, with a similarity of 99.99% to Exiguobacterium acetylicum. Therefore, the strain was determined to be Exiguobacterium acetylicum and was named Exiguobacterium acetylicum C-1.

[0054] The specific sequence is as follows:

[0055]

[0056] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO:2)

[0057] 1492R:5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO:3)

[0058] Example 2 Effect of Carbon-Sulfur Ratio (C / S) on the Growth of Exiguobacterium acetylicum C-1

[0059] Preparation of seed solution: Pick the strain and place it in LB medium, shake and culture at 30℃ and 180rpm for 10-12h, then take the culture medium and measure the OD value of the solution. 600 ;OD 600 When the content 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 content of viable bacteria of Exiguobacterium acetyl C-1 is 6-8×10 8 CFU / mL.

[0060] The C / S ratio of the synthetic wastewater was adjusted to 1:1, 3:1, 5:1, 7:1, and 9:1, and 3.0% of the bacterial strain was inoculated into 100 mL of synthetic wastewater with different C / S ratios. The culture was shaken at pH = 7, 25 °C, and 180 rpm for 24 h. The solution OD was measured after 24 h. 600 .

[0061] like Figure 2 As shown, Exiguobacterium acetylicum C-1 grew better when the C / S ratio was 1:1 to 3:1.

[0062] Example 3 Effect of pH on the Growth of Exiguobacterium acetylicum C-1

[0063] Seed solution preparation: same as Example 2.

[0064] The pH values ​​of the synthetic wastewater were adjusted to 5.5, 6, 6.5, 7, 7.5, and 8, respectively. 3.0% inoculum was inoculated into 100 mL of synthetic wastewater with different pH values. C / S = 1:1, 25 ° C, 180 rpm shaking culture was carried out for 24 h. Samples were taken after 24 h to measure the OD value of the solution. 600 .

[0065] like Figure 3 As shown, Exiguobacterium acetylicum C-1 grows better at a pH of 6.5 to 7.0.

[0066] Example 4: Effect of Temperature on the Growth of Exiguobacterium acetylicum C-1

[0067] Seed solution preparation: same as Example 2.

[0068] The temperature of the synthetic wastewater was adjusted to 15°C, 20°C, 25°C, 30°C, and 35°C, and 3.0% inoculum was inoculated into 100 mL of synthetic wastewater at different temperatures. The pH was 7, C / S was 1:1, and the culture was shaken at 180 rpm for 24 h. After 24 h, water samples were taken to measure the solution OD. 600 .

[0069] like Figure 4 As shown, Exiguobacterium acetylicum C-1 grows better at a temperature of 20-25°C.

[0070] Example 5 Effect of Carbon Source on the Growth of Exiguobacterium acetylicum C-1

[0071] Seed solution preparation: same as Example 2.

[0072] The carbon sources of synthetic wastewater were adjusted to be citric acid, sodium acetate, sucrose, and glucose, and 3.0% inoculum was inoculated into 100 mL of synthetic wastewater with different carbon sources, pH = 7, C / S = 1:1, and cultured at 180 rpm for 24 h. After 24 h, water samples were taken to measure the solution OD. 600 .

[0073] like Figure 5 As shown, Exiguobacterium acetylicum C-1 grew better when the carbon source was glucose or sucrose.

[0074] Example 6 Effect of Carbon-Sulfur Ratio (C / S) on Desulfurization Efficiency of Exiguobacterium acetylicum C-1

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

[0076] The C / S ratio of the synthetic wastewater was adjusted to 1:1, 3:1, 5:1, 7:1, and 9:1, respectively. The culture was inoculated with 3.0% of the bacterial strain into 100 mL of synthetic wastewater with different C / S ratios. The culture was carried out at pH = 7, 25°C, and 180 rpm with shaking for 24 h. After 24 h, water samples were taken to determine the sulfide content by methylene blue spectrophotometry. The initial synthetic wastewater with the same culture conditions and time was used as the blank control group, and the desulfurization rate was calculated according to the following formula.

[0077] Desulfurization rate = (sulfide concentration of blank control group - sulfide concentration of water sample) / sulfide concentration of blank control group.

[0078] like Figure 6 As shown, when the C / S ratio was 1:1 to 9:1, the desulfurization rate of Exiguobacterium acetylicum C-1 was higher than 82%; when the C / S ratio was 1:1 to 3:1, the desulfurization rate was higher than 90%; and when the C / S ratio was 1:1, it reached 96%.

[0079] Example 7 Effect of pH on the Desulfurization Effect of Exiguobacterium acetylicum C-1

[0080] Seed solution preparation: same as Example 2.

[0081] The pH values ​​of the synthetic wastewater were adjusted to 5.5, 6, 6.5, 7, 7.5, and 8, respectively. 3.0% inoculum was inoculated into 100 mL of synthetic wastewater with different pH values. C / S = 1:1, 25 ° C, and shaking culture at 180 rpm were carried out 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 Example 6).

[0082] like Figure 7 As shown, when the pH value is 5.5-8, the desulfurization rate of wastewater by Exiguobacterium acetylicum C-1 is higher than 80%; when the pH value is 6.5-7.5, the desulfurization rate is higher than 90%; when the pH value is 6.5-7.0, the desulfurization rate reaches 93%.

[0083] Example 8 Study on the effect of temperature on the desulfurization effect of Exiguobacterium acetyl C-1

[0084] Seed solution preparation: same as Example 2.

[0085] The temperature of the synthetic wastewater was adjusted to 15°C, 20°C, 25°C, 30°C, and 35°C, respectively. The inoculum size was 3.0% and inoculated into 100 mL of synthetic wastewater at different temperatures. The pH was 7, the C / S ratio was 1:1, 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 6).

[0086] like Figure 8 As shown, when the temperature is 20-30℃, the desulfurization rate of wastewater by Exiguobacterium acetylicum C-1 is higher than 90%; at 25℃, the desulfurization rate reaches 94%; at 20℃, the desulfurization rate reaches 95%.

[0087] Example 9 Evaluation of the desulfurization effect of Exiguobacterium acetylicum C-1 on simulated wastewater

[0088] Seed solution preparation: same as Example 2.

[0089] The inoculum size was 3.0% and inoculated into 500 mL of synthetic wastewater. The pH was 7, C / S was 1:1, 20°C, and the shaking temperature was 180 rpm. The water samples were taken every 6 hours to determine the sulfide concentration and the solution OD by methylene blue spectrophotometry. 600 .

[0090] The results showed that after 12 hours, Exiguobacterium acetylicum C-1 entered the logarithmic growth phase, after 36 hours, Exiguobacterium acetylicum C-1 entered the stable growth phase, and more than 98% of sulfide could be removed within 48 hours, showing a good desulfurization effect.

[0091] 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 heterotrophic-aerobic desulfurization bacterium with a high desulfurization rate, characterized in that: The desulfurization bacteria is Exiguobacterium acetylicum ( Exiguobacterium acetylicum ) C-1, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.33039 and the deposit date of December 12, 2024.

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

3. The microbial agent according to claim 2, characterized in that The effective viable bacteria count of Exiguobacterium acetylicum C-1 in the bacterial agent is 1×10 8 ~9×10 9 CFU / mL.

4. A wastewater treatment agent, characterized in that The wastewater treatment agent comprises the desulfurization bacteria according to claim 1 or the bacterial agent according to claim 2.

5. Use of the desulfurization bacteria according to claim 1, the bacterial agent according to any one of claims 2-3, or the wastewater treatment agent according to claim 4 in treating wastewater, wherein the sulfide concentration in the wastewater is 40-150 mg / L.

6. The use according to claim 5, characterized in that The desulfurization bacteria according to claim 1, the bacterial agent according to any one of claims 2-3, or the wastewater treatment agent according to claim 4 are added to the wastewater to remove the sulfide.

7. The use according to any one of claims 5-6, characterized in that: The inoculation amount of the desulfurization bacteria is 1-5%.

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