A method for enrichment culture of desulfurizing bacillus in sulfur mine acid wastewater

Abstract

This invention discloses a method for enriching and culturing Campylobacter desulfurized spores in acidic wastewater from sulfur-containing mines, relating to the field of environmental pollution bioremediation technology. The method involves using original water samples of acidic wastewater from sulfur-containing mines or acidic sediment suspensions treated with heat shock at 55°C to 60°C for 20 to 30 minutes as inoculum. It employs nanoscale modified biochar, surface-oxidized with nitric acid or hydrogen peroxide and washed to neutral, as an electron mediator, sulfide adsorption / passivation carrier, and microenvironment buffer platform. Combined with mild heat shock at 55–60°C to selectively activate spores, an optimized lactic acid-ethanol mixed carbon source, sodium molybdate as a selective inhibitor, and a precise gradient acidification acclimatization strategy, this method effectively overcomes the problems of inhibited growth, community imbalance, and low reducing activity of Campylobacter desulfurized spores under strong acid conditions. It also avoids the non-specific killing and toxic inhibition caused by traditional high-temperature heat shock and copper ion addition.

Description

Technical Field

[0001] This invention relates to the field of environmental pollution bioremediation technology, and in particular to a method for enriching and culturing desulfurized Campylobacter spores in acidic wastewater from sulfur-containing mines. Background Technology

[0002] Acidic wastewater from sulfur-containing mines typically has a pH of 2.0–4.5, sulfate concentrations as high as 1000–5000 mg / L, and is accompanied by various heavy metal ions, making its treatment a challenge for mine environmental remediation. Biological methods utilize sulfate-reducing bacteria to reduce sulfate to sulfides, which then react with heavy metals to form insoluble metal sulfide precipitates. This is considered a green technology with the potential for both desulfurization and heavy metal removal. However, traditional enrichment culture methods mainly target non-spore-forming SRBs under neutral conditions, making it difficult to effectively enrich functional bacterial communities with engineering application value under the low pH conditions simulating AMD.

[0003] Recent studies have found that *Campylobacter desulfurization*, due to its ability to form heat- and acid-resistant spores and to perform dissimilatory sulfate reduction within a pH range of 3.0–6.5, has become a potentially advantageous species for treating AMD. However, existing enrichment strategies still have significant shortcomings: conventional methods do not perform mild heat shock pretreatment (55–60℃) on the original samples, failing to effectively inhibit acidophilic non-spore-forming bacteria and selectively activate the germination of target spores, resulting in low enrichment efficiency; secondly, although some studies have attempted to add sodium molybdate to inhibit common sulfate-reducing bacteria, in AMD with a low heavy metal background, the selective pressure of molybdate alone is still insufficient, making it difficult to effectively enrich *Campylobacter desulfurization* resistant to molybdate; furthermore, the H2S generated during the enrichment process accumulates in a free form under acidic conditions, exhibiting strong toxicity to *Campylobacter desulfurization* itself, causing activity decay or even enrichment failure. In addition, existing technologies lack effective in-situ sulfide passivation and electron transport mediators, resulting in a serious disconnect between the laboratory enrichment system and the actual wastewater quality, making it difficult to stably apply the obtained bacterial population to engineering practice. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Take the original water sample of acidic wastewater from sulfur-containing mines or the acidic sediment suspension after heat shock treatment at 55℃ to 60℃ for 20 to 30 minutes as inoculum; The inoculum was inoculated into an enrichment medium with an initial pH of 5.0 to 6.0, wherein the enrichment medium used lactic acid and ethanol as a mixed carbon source and sodium sulfate or potassium sulfate as a sulfur source, wherein the final concentration of sulfate was 1.0 g / L to 5.0 g / L, the total organic carbon concentration of lactic acid and ethanol was 1.0 g / L to 3.0 g / L, and the COD / SO4 ratio was [not specified]. 2- The molar ratio was 2.0 to 3.0, and sodium molybdate was added to obtain the initial enrichment culture system; Under strictly anaerobic conditions, modified biochar was added once to the initial enrichment culture system at a dosage of 0.5 g / L to 2.0 g / L; Gradient acidification and acclimatization culture were carried out. When the culture medium became significantly darker or black metal sulfide precipitates appeared, the bacterial culture was transferred to a freshly prepared culture system with the pH adjusted to the target value and cultured again. This process was repeated until a highly active and abundant culture of desulfurized spore-forming Campylobacter was obtained.

[0006] As a preferred embodiment of the enrichment and cultivation method of desulfurized Campylobacter spores in acidic wastewater from sulfur-containing mines according to the present invention, wherein the molar ratio of lactic acid to ethanol is 1:0.2 to 1:2.

[0007] As a preferred embodiment of the enrichment culture method for Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines according to the present invention, the final concentration of sodium molybdate in the enrichment culture medium is 0.1 mmol / L to 1.0 mmol / L.

[0008] As a preferred embodiment of the enrichment and cultivation method of Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines according to the present invention, wherein: during the gradient acidification and acclimatization cultivation process, the pH of the cultivation system is reduced every 24 to 72 hours, with each reduction being 0.3 to 0.6 pH units, and the final pH is reduced to 3.0 to 4.0.

[0009] As a preferred embodiment of the enrichment and cultivation method of Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines according to the present invention, the strictly anaerobic conditions are achieved by introducing high-purity nitrogen or argon into the headspace of the reaction system and replacing it with a mixed gas with a volume ratio of 80% N2: 20% CO2 for 10 to 15 minutes, and replacing it 1 to 3 times, so that the dissolved oxygen concentration in the system is reduced to below 0.1 mg / L, and then the system is sealed and maintained.

[0010] As a preferred embodiment of the enrichment and cultivation method of desulfurized Campylobacter spores in acidic wastewater from sulfur-containing mines according to the present invention, the modified biochar has a particle size of 20 nanometers to 100 nanometers.

[0011] As a preferred embodiment of the enrichment and cultivation method of desulfurized Campylobacter spores in acidic wastewater from sulfur-containing mines according to the present invention, the modified biochar is biochar that has been surface-oxidized by nitric acid or hydrogen peroxide and repeatedly washed with deionized water until the washing liquid is neutral.

[0012] As a preferred embodiment of the enrichment and cultivation method of desulfurized Campylobacter spores in acidic wastewater from sulfur-containing mines according to the present invention, the dosage of modified biochar is 0.5 g / L to 2.0 g / L.

[0013] The beneficial effects of this invention are as follows: By using nanoscale modified biochar that has been surface-oxidized with nitric acid or hydrogen peroxide and washed to neutrality as an electron mediator, sulfide adsorption / passivation carrier, and microenvironment buffer platform, combined with mild heat shock treatment at 55–60℃ to selectively activate spores, an optimized lactic acid-ethanol mixed carbon source, sodium molybdate as a selective inhibitor, and a precise gradient acidification and acclimatization strategy, this invention effectively overcomes the problems of inhibited growth, community imbalance, and low reduction activity of desulfurized spore-forming Campylobacter under strong acid conditions. It not only avoids the non-specific killing and toxic inhibition caused by traditional high-temperature heat shock and copper ion addition, but also achieves in-situ synergistic passivation of toxic H2S through modified biochar, improving the enrichment efficiency, abundance, and sulfate reduction activity of target bacteria. This provides a stable and reliable bacterial resource and technical path for low-cost and efficient bioremediation of acidic wastewater from sulfur-containing mines. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a flowchart of the enrichment and culture method of desulfurized spore-forming Campylobacter in acidic wastewater from sulfur-containing mines in Example 1. Detailed Implementation

[0016] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0017] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0018] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0019] Example 1, referring to Figure 1 This is the first embodiment of the present invention, which provides a method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines, comprising: Acidic wastewater (pH 3.2, SO42-) taken from the drainage outlet of a copper mine 2- Concentration 2800 mg / L, containing Cu 2+ 5 mg / L) was used as the original sample; 100 mL of the original sample was heat-shocked in a water bath at 60 °C for 25 minutes, and then cooled to be used as the inoculum; The inoculum was inoculated into 1 L of enrichment medium with an initial pH of 5.5, which contained 2.0 g / L Na2SO4, 1.5 g / L lactic acid, and 0.8 g / L ethanol (molar ratio of approximately 1:1), and sodium molybdate was added to a final concentration of 0.5 mM to obtain the initial enrichment culture system.

[0020] The initial enrichment culture system was dispensed into three parallel anaerobic reaction bottles, 1 L per bottle. N2 / CO2 (80:20) was bubbled into each bottle to replace the headspace for 15 minutes twice. After sealing, the bottles were incubated at 30°C. Every 48 hours, the pH was adjusted by decreasing it by 0.5 units with 1 M H2SO4, successively decreasing it to 5.0, 4.5, 4.0, and 3.5.

[0021] Before the initial pH adjustment, add 1.5 g / L of rice husk biochar treated with 30% HNO3 oxidation to each reaction flask in one go.

[0022] After 14 days of culture, the dsrB gene copy number was detected by qPCR, and the result was (4.1±0.12)×10⁻⁶. 8 The number of copies / mL (n=3, RSD=2.9%) accounted for (85±2.1)% of the total bacterial abundance; the sulfate removal rate was (88±1.9)%; and the free H2S concentration in the reaction system was maintained at a low level of (1.9±0.2) mg / L, indicating that a highly active and abundant enriched culture of desulfurized spore-forming Campylobacter was successfully obtained, and the results showed good reproducibility.

[0023] Example 2, the second embodiment of the present invention, provides a method for enriching and culturing desulfurized Campylobacter spores using mine sediment as an inoculum source, comprising: 10g of sediment from the same mining area was mixed with 90mL of sterile physiological saline and then heat-shocked in a water bath at 55℃ for 25 minutes. After cooling, it was used as an inoculum.

[0024] The inoculum was evenly inoculated into three groups of 1L enrichment medium with an initial pH of 6.0. The enrichment medium contained 3.0g / L Na2SO4, 2.0g / L lactic acid, and 0.5g / L ethanol (molar ratio of approximately 1:0.3), and 0.8mM sodium molybdate was added.

[0025] Each group underwent gradient acidification at 32℃ under strictly anaerobic conditions (N2 / CO2 80:20, replacement 15min × 2 times, sealed): pH 6.0 for days 1–3, pH 5.0 for days 4–6, pH 4.0 for days 7–9, and pH 3.5 for days 10–15.

[0026] At the start of cultivation, each group was given 2.0 g / L of modified biochar that had been treated with 3% H2O2 solution (v / v) at room temperature for 6 hours.

[0027] After 15 days of culture, the relative abundance of desulfurized Campylobacter was (82±1.8)% (n=3, RSD=2.2%), the sulfate reduction rate was (20.3±0.6) mg / (L·h), the bacterial activity was stable, and there was no significant difference between parallel samples.

[0028] Example 3, the third embodiment of the present invention, provides a method for enriching AMD samples with low copper background, including: Take pH 3.8, Cu 2+ 100 mL of acidic zinc mine wastewater with a concentration of <1 mg / L was used directly as inoculum.

[0029] The samples were evenly inoculated into 1L of pH5.0 enrichment medium (1.2g / L lactic acid, 1.0g / L ethanol, molar ratio ≈1:1.5; 2.5g / L Na2SO4) in three groups, with 0.3mM sodium molybdate added.

[0030] Under anaerobic conditions (N2 / CO2 80:20, sealed after replacement), the pH decreased by 0.4 units every 72 hours, eventually reaching pH 3.2.

[0031] Simultaneously, 1.0 g / L of rice husk biochar treated with 30% HNO3 oxidation was added to each group.

[0032] After 16 days, the abundance of desulfurized Bacillus flexuralis was (76±2.5)% (n=3, RSD=3.3%), proving that even in a low-heavy-metal native environment, the method of the present invention can efficiently enrich the target bacteria, and the results are reproducible.

[0033] Example 4 is the fourth embodiment of the present invention. This embodiment verifies the effect of modified biochar particle size, including: Following the procedure in Example 1, three parallel experiments were set up, using modified biochar treated with HNO3 with particle sizes of 20nm, 50nm, and 100nm respectively (the dosage for each group was 1.5g / L).

[0034] Each group has 3 replicates, for a total of 9 reaction systems.

[0035] Tests were conducted 14 days after culture. 20nm group: Free H2S (2.1±0.3) mg / L, bacterial abundance (83±2.0)%; Group 50nm: Free H2S (1.8±0.2) mg / L, bacterial abundance (87±1.5)%; 100nm group: Free H2S (2.5±0.4) mg / L, bacterial abundance (80±2.2)%; The results showed that the 50nm group had the best performance, confirming that 50nm is the preferred particle size.

[0036] Example 5 This is the fifth embodiment of the present invention, which examines the effect of optimizing the carbon source ratio, including: With other conditions fixed as in Example 1, the molar ratios of lactic acid to ethanol were set to 1:0.2, 1:0.5, 1:1, and 1:2, with 3 replicates in each group.

[0037] The sulfate reduction rate was measured after 14 days of culture. 1:0.2→(18.5±0.7)mg / (L·h); 1:0.5→(20.8±0.8)mg / (L·h); 1:1→(22.6±0.9)mg / (L·h); 1:2 → (15.2±1.0) mg / (L·h), and propionic acid accumulation reached (28±3) mg / L.

[0038] The results show that the 1:1 group has the best performance, and the RSD of parallel samples within each group is less than 5%, indicating that the data is stable and reliable.

[0039] Comparative Example 1 To compare the effect of heat shock treatment, the same process as the modified Example 1 was used, but the 60°C heat shock pretreatment step was omitted, and the original wastewater was directly used as inoculum, with 3 parallel trials.

[0040] After 14 days of cultivation, the abundance of desulfurized spores of Campylobacter was only (12±1.8)%, the sulfate removal rate was (18±2.1)%, and the proportion of contaminating bacteria (Acidithiobacillus) exceeded 80%, proving that the mild heat shock treatment of 55–60℃ used in this invention is a key step in selectively activating target spores, inhibiting non-spore contaminating bacteria, and achieving efficient enrichment.

[0041] Comparative Example 2 To compare the effect of copper ions, the same procedure as the modified Example 1 (i.e., including 60°C heat shock pretreatment) was used, but CuSO4 was added to a final concentration of 0.2 mM, and three replicates were set up.

[0042] The abundance of desulfurized spores was (62±3.5)%, lower than that in Example 1 (p<0.01), indicating that in real copper-containing AMD, even with the presence of background copper, the excessive addition of exogenous copper ions still produces toxic inhibition, verifying the superiority of the copper-free strategy of the present invention.

[0043] Comparative Example 3 To verify the necessity of modified biochar, the revised process of Example 1 was used, but without adding any modified biochar, with 3 parallel runs.

[0044] From day 7 onwards, free H2S increased to (28±2.8) mg / L, and on day 14, the abundance of desulfurized spores Campylobacter was only (55±3.1)%, and the sulfate removal rate stagnated at (60±3.5)%, proving that modified biochar-mediated in-situ sulfide passivation and electron transfer are crucial for maintaining high activity enrichment.

[0045] In summary, this invention utilizes nanoscale modified biochar, which has undergone surface oxidation treatment with nitric acid or hydrogen peroxide and washed to neutrality, as an electron mediator, sulfide adsorption / passivation carrier, and microenvironment buffer platform. Combined with mild heat shock treatment at 55–60℃ to selectively activate spores, an optimized lactic acid-ethanol mixed carbon source, sodium molybdate as a selective inhibitor, and a precise gradient acidification and acclimatization strategy, it effectively overcomes the challenges of inhibited growth, community imbalance, and low reducing activity of *Campylobacter desulfurization* under strong acid conditions. This not only avoids the non-specific killing and toxic inhibition caused by traditional high-temperature heat shock and copper ion addition, but also achieves in-situ synergistic passivation of toxic H2S through modified biochar, improving the enrichment efficiency, abundance, and sulfate reduction activity of the target bacteria. This provides a stable and reliable bacterial resource and technical pathway for low-cost, high-efficiency bioremediation of acidic wastewater from sulfur-containing mines.

[0046] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines, characterized in that: include: Take the original water sample of acidic wastewater from sulfur-containing mines or the acidic sediment suspension after heat shock treatment at 55℃ to 60℃ for 20 to 30 minutes as inoculum; The inoculum was inoculated into an enrichment medium with an initial pH of 5.0 to 6.0, wherein the enrichment medium used lactic acid and ethanol as a mixed carbon source and sodium sulfate or potassium sulfate as a sulfur source, wherein the final concentration of sulfate was 1.0 g / L to 5.0 g / L, the total organic carbon concentration of lactic acid and ethanol was 1.0 g / L to 3.0 g / L, and the COD / SO4 ratio was [not specified]. 2- The molar ratio was 2.0 to 3.0, and sodium molybdate was added to obtain the initial enrichment culture system; Under strictly anaerobic conditions, modified biochar was added once to the initial enrichment culture system at a dosage of 0.5 g / L to 2.0 g / L; Gradient acidification and acclimatization culture were carried out. When the culture medium became significantly darker or black metal sulfide precipitates appeared, the bacterial culture was transferred to a freshly prepared culture system with the pH adjusted to the target value and cultured again. This process was repeated until a highly active and abundant culture of desulfurized spore-forming Campylobacter was obtained.

2. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: The molar ratio of lactic acid to ethanol is from 1:0.2 to 1:

2.

3. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: The final concentration of sodium molybdate in the enrichment medium is 0.1 mmol / L to 1.0 mmol / L.

4. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: During the gradient acidification acclimatization culture process, the pH of the culture system is reduced every 24 to 72 hours, with each reduction being 0.3 to 0.6 pH units, until the final pH drops to 3.0 to 4.

0.

5. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: The strict anaerobic conditions are achieved by introducing high-purity nitrogen or argon into the headspace of the reaction system and replacing it with a mixed gas of 80% N2:20% CO2 by volume for 10 to 15 minutes, repeated 1 to 3 times, so that the dissolved oxygen concentration in the system is reduced to below 0.1 mg / L and then sealed.

6. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: The modified biochar has a particle size of 20 nanometers to 100 nanometers.

7. The method for enriching and culturing Campylobacter desulfurization spores in acidic wastewater from sulfur-containing mines as described in claim 1, characterized in that: The modified biochar is biochar that has undergone surface oxidation treatment with nitric acid or hydrogen peroxide and is repeatedly washed with deionized water until the washing solution is neutral.

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