Composite microbial agent with desulfurization function and application thereof

Abstract

The present application relates to the field of microorganism, disclose a kind of composite microbial inoculant with desulfurization function and its application.The composite microbial inoculant includes: Thiobacillus thioparus and Gordonia alkanivorans;Wherein, the ratio of the viable bacterial count of Thiobacillus thioparus and Gordonia alkanivorans is 1:(0.05-5).The composite microbial inoculant provided by the present application can efficiently degrade sulfide, improve the desulfurization rate, and the desulfurization operation is simple, low in cost and free from secondary pollution.

Description

Technical Field

[0001] This invention relates to the field of biological desulfurization technology, specifically to a composite microbial agent with desulfurization function and its application. Background Technology

[0002] Sulfur-containing wastewater, a byproduct of numerous industries such as dyes, petrochemicals, medicine, pesticides, and metallurgy, contains sulfides, its main component which poses significant harm to the environment and human health. First, sulfides in water can produce hydrogen sulfide gas, which not only has a strong, foul odor but also pollutes the atmosphere, accelerates acid rain formation, and disrupts the balance of ecosystems. Second, sulfides react with metal ions (such as iron ions) in water to form black sulfide precipitates, turning the water black and disrupting photosynthesis in aquatic ecosystems, threatening the survival of aquatic organisms. Furthermore, sulfides are toxic and can accumulate through the food chain, posing a threat to human and other biological health. Long-term exposure to sulfur-contaminated wastewater can lead to respiratory illnesses, skin irritation, and nervous system damage. Therefore, treating sulfur-containing wastewater to minimize the concentration of sulfides in emissions is crucial for protecting human health and environmental safety.

[0003] Currently, domestic and international methods for treating sulfur-containing wastewater can be broadly categorized into physicochemical and biological methods. Physicochemical methods mainly include acid recovery, alkaline absorption, chemical precipitation, electrochemical methods, and oxidation methods. However, physicochemical methods generally suffer from drawbacks such as high requirements for equipment sealing and corrosion resistance, high investment costs, high energy consumption, difficult operation, and significant risks of secondary pollution. Biological treatment of sulfur-containing wastewater, also known as biological desulfurization, utilizes the metabolic processes of microorganisms to convert sulfides in wastewater into elemental sulfur. In comparison, this method has significant advantages such as low cost, low energy consumption, mild reaction conditions, and no secondary pollution, and is gradually becoming a research hotspot. However, the desulfurization rate of using single microorganisms to treat sulfur-containing wastewater is limited, making it difficult to meet the needs of large-scale industrial applications. This has prompted researchers to develop composite microbial agents to treat sulfur-containing wastewater more efficiently and stably. Summary of the Invention

[0004] The purpose of this invention is to overcome the problem of low desulfurization rate in existing biological desulfurization technologies and to provide a composite microbial agent with desulfurization function and its application.

[0005] To achieve the above objectives, the first aspect of the present invention provides a composite microbial agent with desulfurization function, the composite microbial agent comprising: Thiobacillus thioparus and Gordonia alkanivorans, wherein the ratio of the viable number of Thiobacillus thioparus to the viable number of Gordonia alkanivorans is 1:(0.05-5).

[0006] The second aspect of this invention provides the application of the aforementioned composite microbial agent in biological desulfurization.

[0007] A third aspect of the present invention provides a method for treating sulfur-containing water, the method comprising: inoculating a compound microbial agent into the sulfur-containing water for fermentation, wherein the compound microbial agent is the aforementioned compound microbial agent.

[0008] The beneficial effects of the present invention through the above technical solution include at least the following:

[0009] Compared to traditional single-biodegradation methods for sulfur-containing wastewater, this composite microbial agent utilizes the synergistic effect of two microbial enzyme systems to more efficiently degrade sulfides, improving the desulfurization rate. It effectively addresses the problems of toxicity, corrosiveness, odor, severe environmental pollution, and difficulty in recovering sulfur components from sulfur-containing wastewater. The composite microbial method for degrading sulfur-containing water provided by this invention is simple to operate, low in cost, and poses no risk of secondary pollution, demonstrating both environmental and economic feasibility.

[0010] In a preferred embodiment of the present invention, the desulfurization efficiency of the composite microorganisms is further improved by selecting a suitable ratio of viable counts of Thiobacillus thiogenes and Gordonella alkali-eating bacteria, the fermentation conditions for degrading sulfur-containing water, and the culture medium. The desulfurization rate in the sulfur-containing water after fermentation is over 95%, and the fermentation cycle is only about 5 days. Attached Figure Description

[0011] Figure 1 This is a growth curve of Thiobacillus thiocyanate in Example 1;

[0012] Figure 2 This is the growth curve of *Gordonella alkali-eating* in Example 1;

[0013] Figure 3 This is a line graph showing the desulfurization rate over time when the compound microbial agent of Example 1 is used to treat sulfur-containing water. Detailed Implementation

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] The first aspect of this invention provides a composite microbial agent with desulfurization function, the composite microbial agent comprising: Thiobacillus thiogenes and Gordon's alkali-eating bacteria, wherein the ratio of viable counts of Thiobacillus thiogenes and Gordon's alkali-eating bacteria can be 1:(0.05-5), preferably 1:(0.2-1), more preferably 1:(0.45-0.55. The viable count can be tested by the dilution plating method, and the unit is "CFU".

[0016] According to the present invention, the thiobacillus can be any common thiobacillus, and the alkali-eating Gordon's bacterium can be any common alkali-eating Gordon's bacterium. Preferably, the strain number of the thiobacillus is ACCC NO.10288, and / or the strain number of the alkali-eating Gordon's bacterium is CICC NO.20663. The inventors of the present invention have discovered that the desulfurization effect can be further improved by combining thiobacillus and alkali-eating Gordon's bacterium from specific sources.

[0017] According to the present invention, the composite microbial agent can exist in various forms, such as anhydrous solid form, aqueous solution form, or suspension form. The aqueous solution form includes a concentrated form prepared with water and a form prepared directly to the required concentration on-site.

[0018] A second aspect of the present invention provides the application of the aforementioned composite microbial agent in biological desulfurization (especially desulfurization products, such as sodium sulfide).

[0019] A third aspect of the present invention provides a method for treating sulfur-containing water, the method comprising: inoculating a compound microbial agent into the sulfur-containing water for fermentation; wherein the compound microbial agent is the aforementioned compound microbial agent.

[0020] In this invention, the amount of the composite microbial agent used, relative to each gram of sulfur, can achieve a viable count of 10. 6 -10 10 CFU, preferably 10 8 -10 9 CFU.

[0021] In this invention, the sulfur-containing water body may include sulfides. Preferably, the sulfides can be normal salts, including sodium sulfide (Na₂S) and / or potassium sulfide (K₂S), more preferably sodium sulfide. It is understood that microbial desulfurization mainly converts sulfur ions in sulfides into elemental sulfur. Therefore, the cations in sulfides do not participate in the desulfurization process. Thus, besides Na₂S used in this invention to test desulfurization capacity, the microbial agent of this invention can also be used to treat sulfur-containing acid salts (HS-S) containing other types of sulfides (such as sulfur acid salts). - ), sulfur neutral salts (S 2- ) and polysulfides (S n 2- It can be used to desulfurize water bodies such as [list of water bodies], and it can also achieve very good results.

[0022] According to the present invention, the concentration of sulfur in the sulfur-containing water body can be 0.001-10 mol / L, preferably 0.1-1 mol / L. As long as the concentration of sulfur in the water body does not cause all the bacterial agents of the present invention to be inactivated or die, the bacterial agents of the present invention can be used for desulfurization treatment of water bodies.

[0023] In this invention, the composite microbial agent can be activated and expanded before being mixed into the sulfur-containing water body for fermentation. The total concentration of viable bacteria after activation and expansion can be 10-1. 6 CFU / mL or higher, preferably 10 7 -10 8 CFU / mL.

[0024] In this invention, the activation and expansion can be carried out independently in a culture medium. The culture medium can be LB medium and / or PDB medium, preferably LB medium. The LB medium may contain at least one of tryptone, yeast extract, and sodium chloride, wherein the concentrations of tryptone, yeast extract, and sodium chloride may be 5-15 g / L, 3-7 g / L, and 5-15 g / L, respectively.

[0025] In this invention, the activation conditions preferably include: pH value of 6.5-7.5; temperature of 20-35℃, more preferably 24-28℃; and time of 1-4h, more preferably 1.5-2.5h.

[0026] In this invention, the preferred conditions for the expansion culture include: pH value of 6.5-7.5, temperature of 20-35℃, more preferably 24-28℃; and time of 10-40h, more preferably 20-30h.

[0027] In this invention, the activation and expansion can be carried out in a shaker at a speed of 100-200 rpm, preferably 130-170 rpm.

[0028] In this invention, the fermentation conditions preferably include: a temperature of 20-35℃, more preferably 24-28℃, and a time of 80-160h, more preferably 110-130h.

[0029] In this invention, the stirring speed during fermentation can be 100-200 rpm, preferably 120-180 rpm.

[0030] The present invention will be described in detail below through examples and comparative examples. Unless otherwise specified, all methods used in the following examples and comparative examples are conventional; the reagents and materials used are commercially available unless otherwise specified. The specific determination methods and raw materials involved in each example and comparative example are as follows:

[0031] Sulfide concentration determination method: The analysis shall be carried out in accordance with the national standard HJ / T 60-2000, and the analytical method shall be iodometric titration.

[0032] Method for determining viable cell count: dilution plating method;

[0033] Desulfurization rate = (Concentration of Na2S after desulfurization / Initial concentration of Na2S) × 100%;

[0034] Sulfur recovery rate = (number of moles of sulfur in the desulfurized sulfur element / initial number of moles of Na₂S) × 100%. Thiophobic bacteria;

[0035] Growth curve determination method: Add 500 mL of sulfur-containing water with a Na2S concentration of 1000 mg / L to two 1000 mL Erlenmeyer flasks respectively, seal the flasks and sterilize at 121℃ for 20 min. After cooling to room temperature, inoculate Thiobacillus thioformis and Gordonella alkali-eating bacteria at an inoculum of 5 vol.% under aseptic conditions. Co-culture in a constant temperature shaker at 28℃ and 150 rpm for 7 days. Take 5 mL of bacterial solution every 12 h and measure the absorbance at 600 nm in a UV-Vis spectrophotometer.

[0036] Thiobacillus thioparus was purchased from the China Agricultural Microbial Culture Collection Center (ACCC), catalog number ACCCNO.10288. Gordon's alkaline bacteria was purchased from the China Industrial Microbial Culture Collection Center (CICC), catalog number CICCNO.20663. Thiobacillus ferrooxidans was purchased from Shanghai Bio-Net, catalog number SHBCC D24636. The other Thiobacillus thioparus strain used in the comparative example was purchased from Shanghai Bio-Net, catalog number SHBCC D24933. All strains were activated and cultured according to the methods provided by the suppliers, and were stored for future use after three generations of culture.

[0037] LB liquid medium composition: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL distilled water, pH 7. The medium should be sterilized at 121℃ for 20min before use.

[0038] Sulfur-containing water: prepared from Na2S·9H2O, wherein the concentration of Na2S is 1000mg / L.

[0039] Preparation Example

[0040] Single colonies of *Thiobacillus thiogenes* and *Gordonella alkali-eating* were inoculated into shake flasks containing LB broth and activated for 2 hours at 26°C and 150 rpm. Then, the activated *Thiobacillus thiogenes* and *Gordonella alkali-eating* were inoculated into shake flasks containing LB broth (1 mL per 100 mL LB broth), and cultured for 160 hours at 26°C and 150 rpm. The absorbance of *Thiobacillus thiogenes* and *Gordonella alkali-eating* was measured every 12 hours for a total of 14 measurements. The changes in absorbance of *Thiobacillus thiogenes* and *Gordonella alkali-eating* over time are shown below. Figure 1 and 2 As shown. By Figure 1 and Figure 2 It can be seen that the growth cycle of both microorganisms consists of four phases: the lag phase, the logarithmic growth phase, the stationary phase, and the death phase.

[0041] The seed culture prepared and expanded according to the above procedure were used for the treatment of sulfur-containing water in subsequent examples and comparative examples. After expansion, the viable cell concentration of the bacterial culture was 10-1. 6 -10 8 Within the CFU / mL range.

[0042] Example 1

[0043] The bacterial cultures of Thiobacillus thiogenes and Gordonella alkali-eating obtained in the preparation example were mixed in a certain proportion to obtain a seed culture of composite microorganisms. In the mixed seed culture, the ratio of the number of viable Thiobacillus thiogenes to the number of viable Gordonella alkali-eating was 1:0.5.

[0044] Sulfur-containing water with a Na2S concentration of 1000 mg / L was prepared using Na2S·9H2O. 500 mL of the solution was accurately weighed and placed in a 1 L beaker, then sterilized at 121℃ for 20 min. The seed culture of the compound microorganisms was then inoculated into the sulfur-containing water. The total amount of both types of bacteria used, based on viable cell count, was 2 × 10⁻⁶. 5 CFU / mL sulfur-containing water. Fermented for 144 h in a fermenter at 26℃ and 150 rpm.

[0045] During fermentation, the desulfurization rate in the fermenter was measured every 24 hours, and the change in desulfurization rate over time was as follows: Figure 3 As shown in Table 2, the results of desulfurization rate (peak value), fermentation time at which the peak desulfurization rate occurs, and sulfur recovery rate at the time the peak desulfurization rate occurs are presented.

[0046] Examples 2-9

[0047] The sulfur-containing water body was treated according to the method in Example 1. The difference is that, as shown in Table 1, the ratio of the number of viable bacteria of the two types in the seed culture of the compound microorganism, the inoculation concentration of viable bacteria, or the fermentation temperature are different from those in Example 1.

[0048] Example 10

[0049] The sulfur-containing water body was treated according to the method in Example 1, except that the culture medium used to prepare the seed culture of the composite microorganisms was different; PDB medium was used instead of LB medium. The PDB medium contained: 200 g / L potato slurry (obtained by cutting, boiling, melting, and filtering potatoes), 20 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 8 mg / L vitamin B1, and deionized water. The pH of the medium was adjusted to 6, and it was sterilized at 121°C for 20 min before use.

[0050] Comparative Example 1

[0051] The sulfur-containing water body was treated according to the method of Example 1, except that Thiobacillus thioproliferators was replaced with an equal number of live bacteria of Gordonella alkali-eating.

[0052] Comparative Example 2

[0053] The sulfur-containing water body was treated according to the method of Example 1, except that *Gordonella alkali-eating* was replaced with an equal number of live *Thiobacillus*.

[0054] Comparative Example 3

[0055] The sulfur-containing water body was treated according to the method of Example 1, except that Thiobacillus thiogenes was replaced with Thiobacillus ferrooxidans in an equal number of viable bacteria.

[0056] Comparative Example 4

[0057] The sulfur-containing water body was treated according to the method of Example 1, except that the alkaline-eating Gordon's bacterium was replaced with another type of sulfur-producing bacterium, numbered SHBCC D24933, with the same number of viable bacteria.

[0058] Table 1

[0059]

[0060] Table 2

[0061]

[0062]

[0063] pass Figure 3 It can be seen that in Example 1, the desulfurization rate increased with the extension of fermentation time from 0 to 120 hours, reaching a peak of 95.78% at 120 hours. The desulfurization rate at 144 hours was 88.91%, slightly lower than that at 120 hours. This indicates that the composite microorganisms in Example 1 have a good ability to treat sulfides in sulfur-containing water bodies, and under these conditions, the desulfurization rate was highest at 120 hours.

[0064] As shown in Table 2, Examples 1-10, using the composite microorganisms of the present invention to degrade sulfides in sulfur-containing wastewater, achieved a desulfurization rate of over 75%, with Example 1 showing the highest desulfurization rate at 95.78%. Comparative Examples 1-2 used only *Gordonella alkali-eating* and *Thiobacillus thioformis*, respectively, while Comparative Examples 3-4 used a different strain each, resulting in significantly lower desulfurization rates compared to Examples 1-10. This indicates that the composite microbial agent of the present invention possesses a strong ability to degrade sulfides. Compared to Examples 4-5, Example 1 differed in the ratio of viable bacteria in the seed culture of the composite microorganisms; compared to Examples 6-9, it differed in fermentation temperature and inoculum size; and compared to Example 10, it differed in the culture medium. Compared to other examples, Example 1 showed a higher desulfurization rate. The sulfur recovery rate followed a similar pattern to the desulfurization rate. This demonstrates that the composite microorganisms of the present invention have a good degradation effect on sulfides in sulfur-containing wastewater; when two specific bacteria are selected and the number of viable bacteria of each bacteria meets a certain ratio, the synergistic effect between the enzyme systems produced by the composite microorganisms can be further improved; in addition, by selecting appropriate fermentation temperature, fermentation time, inoculum size and culture medium, the ability of the composite microorganisms to degrade sulfides in sulfur-containing wastewater can be further improved.

[0065] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A compound microbial agent with desulfurization function, characterized in that, The compound microbial agent includes Thiobacillus thioparus and Gordonia alkanivorans; wherein the ratio of the viable number of Thiobacillus thioparus to the viable number of Gordonia alkanivorans is 1:(0.05-5).

2. The composite microbial agent according to claim 1, wherein, The ratio of viable counts of the *Thiobacillus thiogenes* and the *Gordonella alkali-eating* is 1:(0.25-1), preferably 1:(0.4-0.6); And / or, the Thiobacillus thiogenes is numbered ACCC NO.10288; And / or, the *Gordonella alkali-eating* strain is designated CICC NO.20663.

3. The application of the composite microbial agent according to claim 1 or 2 in biological desulfurization.

4. A method for treating sulfur-containing water, characterized in that, The method includes: inoculating a compound microbial agent into a sulfur-containing water body for fermentation; wherein the compound microbial agent is the compound microbial agent according to claim 1 or 2.

5. The method according to claim 4, wherein, The amount of the compound microbial agent used, relative to each gram of sulfur, results in a viable count of 10. 6 -10 10 CFU, preferably 10 8 -10 9 CFU.

6. The method according to claim 4, wherein, The sulfur-containing water body includes sulfides, preferably, the sulfides are normal salts, including sodium sulfide and / or potassium sulfide, more preferably sodium sulfide.

7. The method according to claim 4, wherein, The concentration of sulfur in the sulfur-containing water body is 0.001-10 mol / L, preferably 0.1-1 mol / L.

8. The method according to claim 4, wherein, The method further includes: before inoculating the compound microbial agent into the sulfur-containing water body, activating and expanding Thiobacillus thioformis and Alkaloidophytes Gordon's in a culture medium, respectively, wherein the culture medium is LB medium and / or PDB medium, preferably LB medium.

9. The method according to claim 8, wherein, The activation conditions include: a temperature of 20-35℃, preferably 24-28℃; and a time of 1-4 hours, preferably 1.5-2.5 hours. And / or, the conditions for the propagation include: a temperature of 20-35°C, preferably 24-28°C; and a time of 10-40 hours, preferably 20-30 hours.

10. The method according to claim 4, wherein, The fermentation temperature is 20-35℃, preferably 22-30℃, and more preferably 24-28℃; And / or, the fermentation time is 80-160 hours, preferably 110-130 hours.

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