A rapid enrichment method of sulfur-oxidizing functional bacteria and its application in in-situ bioaugmentation deodorization

Abstract

The application discloses a rapid enrichment method of sulfur-oxidizing bacteria and application of the method in in-situ biological strengthening deodorization, and is directly started from activated sludge of a sulfur-containing wastewater treatment system, and directional enrichment is carried out in an enrichment culture medium containing sodium thiosulfate as the only sulfur source by setting a step-by-step elevated sodium thiosulfate concentration gradient, and the time for generation is determined quickly by gradient concentration domestication and an iodine-starch method, so that high-activity sulfur-oxidizing bacteria can be obtained within 10 days, the method is simple to operate, has a short enrichment period, does not need separation and purification and artificial compounding, has high enrichment efficiency, the bacteria originate from indigenous bacteria, the obtained bacteria have a complete metabolic pathway from sodium thiosulfate to sulfate, have better metabolic flexibility and robustness, have stable activity, do not need domestication after being returned, have a remarkable deodorization effect, and have a good engineering application prospect.

Description

Technical Field

[0001] This invention relates to the field of sulfur-containing wastewater treatment and biological deodorization technology, specifically to a method for rapid enrichment of sulfur-oxidizing functional bacteria and its application in in-situ biological enhanced deodorization. Background Technology

[0002] Sulfur-containing wastewater (such as mineral processing wastewater, municipal sewage, aquaculture wastewater, and petrochemical wastewater) commonly produces hydrogen sulfide (H2S) odor during treatment. H2S not only has a strong, pungent odor but is also toxic, posing a threat to the environment and human health. Therefore, developing efficient, economical, and environmentally friendly deodorization technologies is of great significance. Traditional deodorization technologies mainly rely on physical adsorption and chemical oxidation. For example, while activated carbon adsorption can reduce odor substances, it is easily saturated, requires frequent replacement, is costly, and can easily cause secondary pollution; chemical oxidation methods are prone to secondary pollution, have high operating costs, and are difficult to consistently meet standards.

[0003] Biological deodorization relies on sulfur-oxidizing microorganisms to deodorize sulfides (S) 2- ) Directional oxidation to sulfate (SO4) 2- Sulfur oxidizing bacteria (SOC) have advantages such as being green, sustainable, and having low operating costs. However, existing technologies related to SOC have the following shortcomings:

[0004] Most studies isolate and purify single sulfur-oxidizing strains from the environment. However, the sulfur oxidation capacity of single strains is limited, they are sensitive to environmental conditions, and chemoautotrophic bacteria grow slowly and have long culture cycles. In addition, from the perspective of metabolic mechanism, the sulfur oxidation of single strains usually depends on only one main pathway (such as the Sox or Dsr pathway), resulting in insufficient metabolic flexibility and unstable treatment efficiency in practical applications.

[0005] To compensate for the limitations of single strains, some studies have constructed complex bacterial communities by combining multiple isolated and purified single strains in specific proportions. This approach still requires tedious steps such as enrichment, streak plating, multiple rounds of purification, strain identification, single-strain amplification, and optimization of the compounding ratio. From sampling to obtaining a functional bacterial community, it usually takes several weeks to several months. The operation is complex and time-consuming, and the artificial combination disrupts the metabolic synergy network that the bacterial community has evolved over a long period of time under natural conditions.

[0006] A recently published patent application (CN120888431A) uses an upflow reactor to continuously supply a nutrient solution containing Na2S•9H2O for the enrichment of sulfur-oxidizing bacteria, with Thiomonas arsenitoxydans (relative abundance 40%) as the dominant bacterium. This method has the following limitations: ① it depends on a specific reactor configuration, resulting in high equipment investment; ② it requires continuous nutrient supply, making operation complex; ③ the enrichment period is as long as 30-60 days; ④ the enriched bacterial community is used for biofilm formation in a biotrickling filter, rather than for in-situ enhancement. Summary of the Invention

[0007] The purpose of this invention is to provide a rapid enrichment method for sulfur-oxidizing bacteria and its application in in-situ bio-enhanced deodorization, so as to solve the problems of complex operation process, long cycle, dependence on specific equipment, and poor adaptability of existing technologies.

[0008] This invention is achieved through the following technical solutions:

[0009] A rapid enrichment method for sulfur-oxidizing bacteria, comprising the following steps:

[0010] S1. Using activated sludge from a sulfur-containing wastewater treatment system as inoculum, the sludge was inoculated into an enrichment medium containing sodium thiosulfate for enrichment culture. The enrichment culture conditions were: temperature 28–32℃, shaking speed 150–200 r / min, and pH 7.5–8.5. The enrichment medium used sodium thiosulfate as the sole sulfur source, and a progressively increasing thiosulfate concentration gradient was set up to directionally enrich the sulfur-oxidizing bacteria. The inorganic basic salt medium formula contained 3.0 g KH₂PO₄, 3.0 g K₂HPO₄, 0.3 g NH₄Cl, 0.2 g MgSO₄·7H₂O, 0.02 g CaCl₂, 2.0 g NaHCO₃, and 1.0 mL of trace element solution per liter. The trace element solution contained 0.5 g / L EDTA, 0.2 g / L FeSO₄·7H₂O, and 0.01 g / L ZnSO₄·7H₂O. MnCl2·4H2O 0.003 g / L, H3BO3 0.03 g / L, CoCl2·6H2O 0.02 g / L, CuCl2·2H2O0.001 g / L, NiCl2·6H2O 0.002 g / L, NaMoO4·2H2O 0.003 g / L;

[0011] S2. During the enrichment process, when the pH of the system decreases rapidly and the OD... 600 When the plateau is reached within 3 days and the iodine-starch method shows a deep purple color, it is determined that the substrate is about to be depleted, and the process is switched to the next concentration gradient for further enrichment.

[0012] S3, when the bacterial community is stably tolerant to sodium thiosulfate concentrations not less than 5 g / L, OD 600 When the value is greater than 1.0, the enrichment and domestication of autotrophic sulfur-oxidizing bacteria is completed.

[0013] Preferably, the sodium thiosulfate concentration gradient in the enrichment culture medium is 2, 4, 6, 8, and 10 g / L, respectively.

[0014] This invention also protects an application of in-situ bio-enhancing of sulfur-oxidizing bacteria obtained by the above method to eliminate the malodor of H2S in sulfur-containing wastewater from the source, comprising the following steps: sulfur-oxidizing bacteria obtained from the activated sludge of the sulfur-containing wastewater treatment system according to the above method are reintroduced into the sulfur-containing wastewater treatment system and subjected to biological oxidation deodorization treatment under aerobic conditions to efficiently oxidize the sulfides in the wastewater into sulfates.

[0015] Preferably, the inoculum amount of sulfur-oxidizing bacteria reintroduced into the sulfur-containing wastewater treatment system is 1-10% (v / v), more preferably 5% (v / v).

[0016] The beneficial effects of this invention are as follows:

[0017] (1) Simple operation and short enrichment cycle: No separation, purification and artificial compounding are required. The active sludge from the sulfur-containing wastewater treatment system is used directly. In the enrichment culture medium with sodium thiosulfate as the only sulfur source, a progressively increasing sodium thiosulfate concentration gradient is set for directional enrichment. The timing of subculturing is quickly determined by gradient concentration acclimatization combined with the iodine-starch method. Highly active sulfur-oxidizing bacteria can be obtained within 10 days, which has a significant advantage over the existing technology cycle of several weeks to several months.

[0018] (2) No need to rely on specific reactor configuration: enrichment can be completed by conventional shake flask batch culture, with low equipment investment and easy to promote and apply.

[0019] (3) Complete synergistic metabolism of the microbial community: During the gradient domestication process, the microbial community autonomously forms a complete synergistic metabolic network, rather than being artificially combined. 16S rRNA sequencing and functional gene analysis showed that the core functional genes of sulfur oxidation (sox, sqr, tsdA, etc.) were enriched synchronously with domestication, and the microbial community has a complete metabolic pathway from thiosulfate to sulfate, with better metabolic elasticity and robustness.

[0020] (4) The enrichment endpoint is flexible and adjustable: users can select the enrichment endpoint (such as 6 g / L or 10 g / L) according to the sulfide load level under actual working conditions, and minimize the enrichment time while meeting the treatment requirements.

[0021] (5) In-situ enhancement strategy: The microbial community comes from the activated sludge of the system to be treated. After being reintroduced, it can quickly play a role without additional acclimatization, avoiding the problems of poor adaptability and low survival rate of exogenous microbial agents, and there is no ecological risk of introducing foreign microorganisms.

[0022] (6) Significant deodorization effect: After the enriched bacterial community is reintroduced into the sulfur-containing wastewater, it can efficiently oxidize sulfides into sulfates in a short time, inhibiting H2S generation from the source. Examples show that after adding the bacterial community, the removal rate of sulfides (initial concentration 280 mg / L) exceeds 99% after 12 h, and the relative sulfate generation rate is 75.3% after 36 h, which is more than 19% higher than the system without the addition of bacterial community.

[0023] In summary, this invention starts directly from the activated sludge of a sulfur-containing wastewater treatment system. In an enrichment culture medium with sodium thiosulfate as the sole sulfur source, a progressively increasing sodium thiosulfate concentration gradient is established for targeted enrichment. By combining gradient concentration acclimation with the iodine-starch method to quickly determine the passage time, highly active sulfur-oxidizing bacteria can be obtained within 10 days. The operation is simple, the enrichment cycle is short, no separation, purification, or artificial compounding is required, the enrichment efficiency is high, the bacteria are derived from indigenous species, and the obtained bacteria possess a complete metabolic pathway from thiosulfate to sulfate, exhibiting superior metabolic elasticity and robustness, stable activity, and requiring no acclimation after reintroduction. It also demonstrates significant deodorization effects and has promising engineering application prospects. Attached Figure Description

[0024] Figure 1 The OD values ​​for bacterial growth at different sodium thiosulfate concentrations in Example 1 are shown. 600 (a) and pH change curve (b);

[0025] Figure 2 The graph shows the changes in Alpha diversity during the enrichment of sulfur-oxidizing bacteria in Example 2, where (a) is the Chao1 index and (b) is the Shannon index.

[0026] Figure 3 This is a diagram of the horizontal community structure succession during the enrichment of sulfur-oxidizing bacteria in Example 2;

[0027] Figure 4 This is a graph showing the changes in the relative abundance of sulfur metabolism-related functional genes during the enrichment of sulfur-oxidizing bacteria in Example 2.

[0028] Figure 5 This is a schematic diagram of the sulfur metabolism network of the sulfur-oxidizing bacteria in Example 2;

[0029] Figure 6 This is a response surface plot showing the effect of the interaction of three environmental factors on sulfate formation in Example 3.

[0030] Figure 7 This is a comparison of the concentration of sulfate generated in the system in Example 4 and Comparative Example 1. Detailed Implementation

[0031] The following is a further description of the invention, but not a limitation thereof.

[0032] Example 1:

[0033] Sulfur-oxidizing bacteria were inoculated and enriched using activated sludge from the aerobic tank of a mineral processing wastewater treatment system. The enrichment medium was formulated with inorganic basic salts containing 3.0 g KH2PO4, 3.0 g K2HPO4, 0.3 g NH4Cl, 0.2 g MgSO4·7H2O, 0.02 g CaCl2, 2.0 g NaHCO3, and 1.0 mL trace element solution per liter. The trace element solution contained the following trace elements: EDTA 0.5 g / L, FeSO4·7H2O 0.2 g / L, ZnSO4·7H2O 0.01 g / L, MnCl2·4H2O 0.003 g / L, H3BO3 0.03 g / L, CoCl2·6H2O 0.02 g / L, CuCl2·2H2O 0.001 g / L, NiCl2·6H2O 0.002 g / L, and NaMoO4·2H2O 0.003 g / L. The pH of the culture medium was adjusted to 7.0-7.5.

[0034] 5% (v / v) activated sludge was inoculated into an enrichment medium containing 2 g / L sodium thiosulfate. The culture conditions were 30℃ and 150 r / min.

[0035] Daily monitoring of OD 600 pH was measured, and the consumption of thiosulfate was semi-quantitatively determined using the iodine-starch method. The iodine-starch method was performed as follows: In a white porcelain dropper plate, 1 drop of culture medium, 1 drop of starch indicator, and 1 drop of dilute iodine solution were added sequentially, mixed, and the color was observed. Initially colorless (thiosulfate reduces iodine), the blue color gradually deepened as the substrate was consumed; when it turned deep purple, it indicated that the substrate was about to be depleted.

[0036] When the bacterial community reaches the passage standard (OD) within 3 days 600 When the plateau phase occurs (pH drops rapidly, and the iodine-starch method produces a deep purple color), proceed to the next concentration (increase by 2 g / L sodium thiosulfate). Gradually increase the concentration to 4, 6, 8, and 10 g / L.

[0037] like Figure 1 As shown, with the gradual increase of sodium thiosulfate concentration, the activity of the bacterial community continuously increased, eventually reaching the highest OD value at 10 g / L. 600 On day 3, the pH exceeded 1.2, and the system pH continued to decrease from 7.0-7.5 to approximately 2.6. The bacterial community maintained good growth during the pH decrease, indicating that it has strong acid tolerance.

[0038] Example 2: 16S rRNA high-throughput sequencing and functional gene analysis

[0039] 16S rRNA high-throughput sequencing and functional gene analysis were performed on initial sludge and microbial samples at different concentration gradients (2, 6, 10 g / L).

[0040] Figure 2 The data showed that during the gradual enrichment of sulfur-oxidizing bacteria, the Chao1 index decreased from 245 in the initial sludge to 11, and the Shannon index also decreased from 2.79 to 0.15, indicating that the high concentration of sodium thiosulfate effectively suppressed non-target microorganisms, and the community structure was significantly simplified. The community structure succession is as follows: Figure 3 As shown, the dominant genera in the initial sludge at the genus level were *Meiothermus* (relative abundance 48.5%) and *Thiobacillus* (11.9%). With increasing sodium thiosulfate concentration, significant directional succession occurred in the community structure. The relative abundance of non-sulfur-oxidizing bacteria (such as *Meiothermus* and *Thiobacillus*) in the initial sludge continuously decreased, while the relative abundance of *Halothiobacillus* increased with increasing substrate concentration, reaching 92.3% at 6 g / L and 97.6% at 10 g / L, becoming the absolutely dominant genus. This genus is a typical chemoautotrophic sulfur-oxidizing bacterium, utilizing reducing sulfur compounds such as sodium thiosulfate and sulfides as electron donors and energy sources. It obtains the energy required for growth by oxidizing inorganic sulfur, making it a key functional group in sulfur metabolism. This further verifies the successful enrichment of the functional bacterial community.

[0041] The relative abundance and evolution of core functional genes for sulfur oxidation are as follows: Figure 4 As shown, the core functional genes for sulfur oxidation (sox series, sqr, tsdA, TST, cys, fccAB, sat, etc.) were significantly enriched with increasing sodium thiosulfate concentration, reaching a peak at 10 g / L. The overall succession pattern fully validates the effectiveness of the gradient enrichment strategy, successfully achieving targeted enrichment of sulfur-oxidizing bacteria. Figure 5 A schematic diagram of the metabolic network of sulfur-oxidizing bacteria is presented, showing that the community retains the metabolic processes from S2O3. 2− To SO4 2− The complete metabolic pathway includes direct oxidation by the sox system and the tsdA-catalyzed production of tetrathionate (S4O6). 2− TST is converted into SO3 2− and SO3 2− Oxidation to SO4 via aprAB / sat / cysNC / cysN / cysD 2− Multiple pathways, among others. The integrity of this metabolic network is not possessed by a single strain.

[0042] Example 3: Determination of Optimal Processing Conditions

[0043] To obtain the optimal deodorization conditions for the microbial community, the effects of temperature, pH, and rotation speed on sulfate formation rate over 24 h were investigated using response surface methodology. The results showed that the influence of each factor on sulfate formation rate was in the order of rotation speed > temperature > pH. From the three-dimensional response surface plot (…),… Figure 6 From the data, the interaction between temperature and pH is not significant, but there is an interaction between temperature and rotation speed, and similarly, there is an interaction between rotation speed and pH. This indicates that rotation speed is the core influencing factor, and its interaction with temperature and pH is significant.

[0044] The optimal culture conditions determined through response surface methodology were: pH=8, rotation speed 200 r / min, and temperature 28.8℃ (adjusted to 30℃ during actual measurement). The predicted sulfate formation rate was 69.23%, and the measured value was 68.85%, which was very close to the predicted value, thus verifying the accuracy of the model.

[0045] Example 4: Verification of in-situ biological enhanced deodorization efficiency

[0046] Under optimal culture conditions (pH=8, rotation speed 200 r / min, temperature 30℃), the microbial community enriched in Example 1 was reintroduced at a 5% (v / v) inoculum into the simulated mineral processing wastewater treatment system corresponding to the original sludge (initial sulfide 280 mg / L). The control group did not receive any enriched microbial community and operated naturally with only the original activated sludge. Results are as follows... Figure 7 As shown, the sulfide removal rate in the experimental group exceeded 99% at 12 h, and the relative sulfate formation rate was 75.3% at 36 h, which was 19.34% higher than that in the sludge group (control group) without added bacteria. This indicates that the indigenous bacteria enriched from the target system can quickly and efficiently perform deodorization without acclimatization after reintroduction.

[0047] Comparative Example 1

[0048] Using the same mineral processing wastewater, without the addition of enriching bacteria, and only treating with raw activated sludge, the sulfide removal rate and sulfate formation rate were significantly better than in Example 4. Figure 7 This demonstrates that the autotrophic sulfur-oxidizing bacteria enriched in this invention have excellent deodorizing properties.

[0049] Comparative Example 2:

[0050] Referring to Example 1, the difference is that the sodium thiosulfate concentration was not increased stepwise; the initial concentration was 10 g / L. The resulting OD... 600 No significant increase indicates enrichment failure.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, or simple variations made to the above embodiments within the technical concept and principles of the present invention (including but not limited to adjustments to the sodium thiosulfate concentration gradient, conventional replacements of the culture medium formulation, appropriate relaxation of the iodine-starch method judgment criteria, flexible selection of the enrichment endpoint, and application of the obtained bacterial community to in-situ bioaugmentation treatment of other sulfur-containing wastewater, etc.) shall fall within the protection scope of the present invention.

Claims

1. A method for rapid enrichment of sulfur-oxidizing bacteria, characterized in that, The method includes the following steps: S1. Activated sludge from a sulfur-containing wastewater treatment system was used as inoculum and inoculated into an enrichment medium containing sodium thiosulfate for enrichment culture. The enrichment culture conditions were: temperature 28–32℃, shaking speed 150–200 r / min, and pH 7.5–8.

5. The enrichment medium used sodium thiosulfate as the sole sulfur source, and a progressively increasing thiosulfate concentration gradient was set up to directionally enrich the sulfur-oxidizing bacteria. The inorganic basic salt medium formula contained 3.0 g KH₂PO₄, 3.0 g K₂HPO₄, 0.3 g NH₄Cl, 0.2 g MgSO₄·7H₂O, 0.02 g CaCl₂, 2.0 g NaHCO₃, and 1.0 mL of trace element solution per liter. The trace element solution contained 0.5 g / L EDTA, 0.2 g / L FeSO₄·7H₂O, and 0.01 g / L ZnSO₄·7H₂O. g / L, MnCl2·4H2O 0.003 g / L, H3BO3 0.03 g / L, CoCl2·6H2O 0.02 g / L, CuCl2·2H2O 0.001g / L, NiCl2·6H2O 0.002 g / L, NaMoO4·2H2O 0.003 g / L; S2. During the enrichment process, when the pH of the system decreases rapidly and the OD... 600 When the plateau is reached within 3 days and the iodine-starch method shows a deep purple color, it is determined that the substrate is about to be depleted, and the process is switched to the next concentration gradient for further enrichment. S3, when the bacterial community is stably tolerant to sodium thiosulfate concentrations not less than 5 g / L, OD 600 When the value is greater than 1.0, the enrichment and domestication of autotrophic sulfur-oxidizing bacteria is completed.

2. The method according to claim 1, characterized in that, The sodium thiosulfate concentration gradient in the enrichment medium was 2, 4, 6, 8, and 10 g / L, respectively.

3. The application of the sulfur-oxidizing bacterial community obtained by the method described in claim 1 for in-situ bio-enhancing to eliminate the malodorous H2S in sulfur-containing wastewater from the source, characterized in that, Includes the following steps: The sulfur-oxidizing bacteria obtained from the activated sludge of the sulfur-containing wastewater treatment system according to the method described in claim 1 are reintroduced into the sulfur-containing wastewater treatment system for biological oxidation and deodorization under aerobic conditions, thereby efficiently oxidizing the sulfides in the wastewater into sulfates.

4. The application according to claim 3, characterized in that, The inoculum of sulfur-oxidizing bacteria is 1-10 vol when reintroduced into the sulfur-containing wastewater treatment system.

5. The application according to claim 3, characterized in that, The inoculum size of the sulfur-oxidizing bacteria was 5 vol.

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