Normal-temperature preservation method of thiobacillus denitrificans

Abstract

The invention relates to the technical field of environmental microorganisms, in particular to a normal-temperature preservation method of thiobacillus denitrificans. According to the preservation method of thiobacillus denitrificans, mineral materials such as silica fume and coal gangue are selected and matched with a preservation nutrient solution, a specific metal ion Cu < 2 + > is added to serve as a microbial metabolism regulator, and the synergistic effect of the mineral materials and the preservation nutrient solution is utilized, so that thallus metabolism is moderately inhibited through Cu < 2 + > to reduce substrate consumption, and the toxic effect of Cu < 2 + > on thiobacillus denitrificans is relieved through an adsorption material; the microbial inoculum can be effectively preserved at normal temperature for 3 months, is suitable for preserving a large amount of thiobacillus denitrificans for a long time, and has the advantages of high survival rate, quick activity recovery, low preservation energy consumption and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental microbiology, and particularly relates to a normal-temperature preservation method of Thiobacillus denitrificans. BACKGROUND

[0002] Sulfur autotrophic denitrification technology does not need to add carbon source, has low sludge production, low energy consumption, and no secondary pollution, and is particularly one of the best choices for replacing traditional heterotrophic denitrification process in the treatment of low C / N sewage. The core of simultaneous denitrification and desulfurization in sulfur autotrophic denitrification reaction is functional flora, that is, denitrifying desulfurization bacteria (NR-SOB), and therefore it is of great significance to obtain high-efficiency and high-activity functional flora by means of biological reinforcement and the like for the development of sulfur autotrophic denitrification process. Thiobacillus denitrificans (Thiobacillus denitrificans) is a common NR-SOB. Thiobacillus denitrificans

[0003] Thiobacillus denitrificans inoculant faces preservation problems in practical application. Traditional inoculant preservation methods, such as low-temperature refrigeration or freeze-drying, can maintain the activity of the inoculant to some extent, but have problems such as high cost and complex operation, which are not conducive to large-scale application and promotion. Low-temperature refrigeration requires continuous power supply to maintain a low-temperature environment, which increases energy consumption and operating costs; freeze-drying requires special equipment and a complex process, and the freeze-drying process may damage the bacteria, affecting the recovery of the activity of the inoculant. For example, in patent CN 111003813 B, the contents of living cells, early apoptotic cells, late apoptotic cells and dead cells in the biofilm preserved at different temperatures were determined by flow cytometry, and the preservation temperature closest to the activity state of the cells in the pilot operation was determined as the optimum preservation temperature. It was verified by experiments that-20℃ was the optimum preservation temperature, but the removal rates of nitrate nitrogen and total nitrogen nitrate were compared with the initial removal rates, and the activity recovery time was 12-15 days, which prolonged the time of activity recovery. Therefore, it is urgent to develop a method for preserving Thiobacillus denitrificans at normal temperature to avoid damage to Thiobacillus denitrificans caused by low-temperature processes in existing freeze or freeze-drying preservation processes, which has important practical significance and application value.

[0004] In the biological field, mineral carriers are generally used as carriers, adsorbents for preparing microbial fertilizers, or for adsorbing heavy metals for soil remediation, or for microbial immobilization carriers. For example, in patent CN 112011476 A, Thiobacillus denitrificans, oyster powder, volcanic ash and other materials are mixed and embedded with polyvinyl alcohol and sodium alginate to obtain high-strength immobilized microspheres of embedded Thiobacillus denitrificans, so as to reduce the loss of bacterial strains and improve the stability and utilization rate of the bacterial strains. However, there are still limited reports on the use of mineral carriers in microbial normal-temperature preservation methods. SUMMARY

[0005] ​In view of this, the present invention proposes a method for the room temperature preservation of *Thiobacillus denitrifyingus* to solve the problems of slow activity recovery, low survival rate, and high energy consumption in existing preservation methods. The preservation method of the present invention uses adsorbent mineral materials such as silica fume and coal gangue, combined with a specific preservation nutrient solution, and adds a specific metal ion, Cu. 2+ As a microbial metabolic regulator, it utilizes the synergistic effect of both Cu and other substances to achieve the desired effect. 2+ Moderately inhibiting bacterial metabolism to reduce substrate consumption, and mitigating Cu through adsorption materials. 2+ It exhibits toxic effects against *Thiobacillus denitrificationus*, enabling effective preservation of the bacterial agent at room temperature for 3 months. It is suitable for large-scale long-term preservation of *Thiobacillus denitrificationus*, and has advantages such as high survival rate, rapid activity recovery, and low energy consumption for preservation.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a method for preserving *Thiobacillus denitrificationus*, comprising the following steps: S1, grind and pulverize the mineral carrier, sieve it to obtain carrier powder for later use; S2, resuspend the bacterial cells of *Thiobacillus denitrificationis* in the preservation nutrient solution, stir evenly to obtain the bacterial solution, and set aside for later use; S3. Mix the carrier powder with the bacterial solution, stir evenly, air dry, and dry to obtain the solid adsorbent.

[0007] Based on the above technical solutions, preferably, in step S1, the mineral carrier includes any one of silica fume, coal gangue, vermiculite powder, zeolite powder, and attapulgite.

[0008] Based on the above technical solutions, preferably, the mineral carrier is silica fume and coal gangue.

[0009] Based on the above technical solution, a further preferred embodiment is that the mass ratio of silica fume to coal gangue is 1:1.

[0010] Based on the above technical solution, preferably, in step S2, the preservation nutrient solution contains Cu. 2+ .

[0011] Based on the above technical solutions, preferably, the Cu 2+ It is selected from any one of CuSO4·5H2O, CuCl2 and Cu(NO3)2·3H2O.

[0012] Based on the above technical solutions, preferably, the Cu 2+ The concentration is 0.0157~0.157 mmol / L. More preferably, the Cu... 2+ The concentration ranges from 0.0785 to 0.1099 mmol / L.

[0013] Denitrifying Thiobacillus against Cu 2+ Cu concentration is very sensitive; a certain concentration of Cu 2+ While it has an inhibitory effect on *Thiobacillus denitrificationis*, slightly higher concentrations can easily damage cell membrane integrity, leading to irreversible loss of enzyme activity, making it difficult to balance "inhibition" and "toxicity." Although using adsorbent mineral materials alone can provide a habitat for microorganisms, it cannot solve the problem of rapid substrate consumption and microbial inactivation due to starvation during preservation. Combining mineral materials with Cu... 2+ This combination avoids the risk of strains becoming inactive due to starvation or undergoing sulfate reduction during preservation caused by rapid substrate consumption, thus ensuring the survival rate of the strains and the rapid recovery of their activity.

[0014] Based on the above technical solutions, preferably, in step S3, the mass-to-volume ratio of the carrier powder to the bacterial solution is 1:(0.8~1.2). More preferably, the mass-to-volume ratio of the carrier powder to the bacterial solution is 1:1.

[0015] Based on the above technical solutions, preferably, in step S3, the moisture content of the dried material is 20-30%, and the air-drying temperature is 20-30°C. More preferably, the moisture content of the dried material is 25%.

[0016] Based on the above technical solutions, preferably, in step S2, the raw materials of the preservation nutrient solution further include NH4Cl, MgCl2·6H2O, KH2PO4, Na2S2O3·5H2O, KNO3, NaHCO3 and FeSO4·7H2O.

[0017] Based on the above technical solutions, preferably, the preservation is carried out at a temperature of 25~30℃. More preferably, the preservation is carried out at a temperature of 28℃.

[0018] Secondly, a denitrifying thiobacillus inoculant is provided, preferably prepared by the preservation method of the denitrifying thiobacillus described above.

[0019] The method for preserving denitrifying thiobacilli at room temperature according to the present invention has the following advantages over the prior art: (1) Synergistic effect: Cu 2+ It forms a synergistic effect of "metabolic regulation-toxicity buffering" with the silica fume-coal gangue composite carrier. 2+ By moderately inhibiting metabolism to reduce substrate consumption, the composite carrier regulates Cu adsorption. 2+ The concentration was optimized to avoid excessive toxicity, and the combination of the two factors—providing a physical barrier and a stable microenvironment to alleviate cell membrane damage—ensured that the activity of the bacterial agent (i.e., nitrate removal rate) remained above 50% after 3 months of preservation, which was significantly better than the activity when the composite carrier was used alone. (2) Material advantage: The materials such as silica ash and coal gangue have the characteristics of large specific surface area, rich pore, good water retention performance and the like, can provide a good habitat for the denitrifying sulfur-oxidizing bacteria, reduce the activity loss of the bacteria in the normal temperature preservation process, and can specifically buffer Cu 2+ concentration, reduce the activity loss of the bacteria in the normal temperature preservation process; (3) Cost and operation: The normal temperature preservation mode does not need low-temperature equipment and complex process, reduces the production cost, simplifies the operation process, and is convenient for large-scale production and practical application promotion; (4) Activity guarantee: The composite carrier of silica ash + coal gangue is adopted, the high specific surface area and pore synergistic effect of the two are combined, and the preservation nutrient solution and the regulator Cu 2+ After preservation for 3 months, the activity of the denitrifying sulfur-oxidizing bacteria solid adsorption bacterial agent is still maintained at more than 50%, the denitrification capacity is stable, and the practical application demand is met. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0022] The denitrifying sulfur-oxidizing bacteria in the present application is a self-screened strain, the preservation number of which is CCTCC NO: M 2023992, and the classification name is Thiobacillus denitrificans SZG-SAD-004.

[0023] Since the growth of the autotrophic bacteria is slow, it is inconvenient to count by coating a plate, therefore, the nitrate removal rate (mg NO3 - -N / (L·h) is used to represent the activity of the denitrifying sulfur-oxidizing bacteria, which is hereby stated.

[0024] The formula of the TD culture medium is as follows: NH4Cl 0.5 g / L, MgCl2·6H2O 0.5 g / L, KH2PO4 2 g / L, Na2S2O3·5H2O 5 g / L, KNO3 2 g / L, NaHCO3 1 g / L, FeSO4·7H2O 0.01 g / L, 1.0 ml trace elements, and pH is adjusted to 7.0-7.2. The trace elements are the same as those of the basic preservation nutrient solution.

[0025] The solid culture medium needs to add agar powder 18-20 g / L, and the liquid culture medium does not need to add.

[0026] The basic preservation nutrient solution consists of: NH4Cl 0.5-1 g / L, MgCl2·6H2O 1 g / L, KH2PO4 4 g / L, Na2S2O3·5H2O 10 g / L, KNO3 4 g / L, NaHCO3 2 g / L, and FeSO4·7H2O 0.01 g / L. Na2S2O3·5H2O can be replaced by other reduced sulfides such as Na2S.

[0027] Example 1: Screening of different mineral carriers Will Thiobacillus denitrificans Strain SZG-SAD-004 was streaked onto TD solid medium plates to obtain single colonies. These colonies were then transferred to 50 mL glass tubes containing 30 mL of TD medium, capped, and incubated statically at 28°C for 6 days until nitrates were completely consumed. The colonies were then transferred to 250 mL blue-necked flasks containing 200 mL of TD medium, capped, and incubated statically at 28°C for 3 days. Finally, the colonies were transferred to 1 L blue-necked flasks containing 1 L of 2×TD medium and incubated statically at 28°C for 3 days. The nitrate removal rate of the fermentation broth was measured to be approximately 300 mg NO3. - -N / (L·h). Collect the bacterial cells after sedimentation and centrifugation of the fermentation broth.

[0028] Mix the bacterial cells with the prepared basic preservation nutrient solution and stir well before use.

[0029] Preparation of the mineral carriers for adsorption: Vermiculite powder, silica fume, zeolite powder, attapulgite clay, and coal gangue were ground and pulverized, sieved through a 60-mesh sieve, and placed at 110℃ for 3 hours. After removal, they were cooled to room temperature for later use. 100g of mineral carrier powder was added to 100mL of a mixture of bacterial cells and preservation nutrient solution, stirred for 30min, and dried in cold air at 25℃ for 4 hours to obtain a solid bacterial agent with a water content of approximately 30%. This agent was stored at 28℃ for 3 months, and samples were taken monthly to test the nitrate removal rate to assess the preservation effect. The nitrate removal rate represents, to some extent, the survival rate of *Thiobacillus denitrifyingus*. The control group consisted of bacteria without any added mineral carrier, with all other conditions being the same. The nitrate removal rates of *Thiobacillus denitrifyingus* adsorbed by different mineral carriers after 3 months of storage are shown in Table 1.

[0030] Table 1. Nitrate removal rate of different mineral carriers adsorbing denitrifying thiobacilli after 3 months of preservation.

[0031] As shown in Table 1, the mineral carriers silica fume, attapulgite, and coal gangue adsorbed and preserved Thiobacillus denitrification bacteria well. The nitrate removal rates remained high after 2 months of preservation, at 41.80%, 36.11%, and 43.92% respectively, and there was still a certain nitrate removal capacity after 3 months of preservation.

[0032] Example 2: Screening using mineral carrier combinations The embodiment is basically the same as the method of Example 1, except that the silica ash, attapulgite and coal gangue are combined in pairs with a mass ratio of 1:1 to prepare the mineral carrier adsorbed solid bacterial agent for preservation effect observation, and the results are shown in Table 2.

[0033] Table 2 Nitrate removal rate of mineral carrier combination adsorbed denitrifying sulfur bacteria preserved for 3 months

[0034] As shown in Table 2, the combination of mineral carrier silica ash and coal gangue adsorbed denitrifying sulfur bacteria has the best preservation effect at room temperature, and the nitrate removal rate is 161.23 mg N / (L·h) after 2 months of preservation, with a removal rate of 53.74% of the initial value. After 3 months, the removal rate still maintains 40.41% of the initial value.

[0035] Example 3: Screening of different water contents On the basis of Example 2, silica ash and coal gangue adsorbed denitrifying sulfur bacteria were selected, and the water content of the adsorbed bacterial powder was controlled at 10%, 15%, 20%, 25%, 30% and 35%, respectively. The remaining conditions were the same as those of Example 2, and the preservation observation results are shown in Table 3.

[0036] Table 3 Nitrate removal rate of silica ash + coal gangue adsorbed denitrifying sulfur bacteria with different water contents preserved for 3 months

[0037] As shown in Table 3, when the water content is 20% to 30%, the preservation effect of silica ash + coal gangue adsorbed denitrifying sulfur bacteria at room temperature is better.

[0038] Example 4

[0039] On the basis of Example 3, silica ash and coal gangue adsorbed denitrifying sulfur bacteria were selected, and the water content of the adsorbed bacterial powder was controlled at 30%. Pure water and basic preservation nutrient solution were used to treat the bacterial agent, respectively. The remaining conditions were the same as those of Example 3, and the preservation observation results are shown in Table 4.

[0040] Table 4 Effect of basic preservation nutrient solution on nitrate removal rate

[0041] According to the test results in Table 4, the basic preservation nutrient solution plays an important role in the preservation of denitrifying sulfur bacteria adsorbed bacterial powder at room temperature, provides nutrients for denitrifying sulfur bacteria, and prolongs the preservation time.

[0042] Example 5

[0043] On the basis of Example 3, the silicon ash and coal gangue were selected to adsorb Thiobacillus denitrificans, and the water content of the adsorbed bacteria powder was controlled at 30%, and 0, 2.11, 10.55, 14.77, and 21.11 mg / L of CuCl2 solution, i.e., Cu 2+ The concentration of Cu was 0, 0.0157, 0.0785, 0.1099, and 0.1570 mmol / L, respectively, and the remaining conditions were the same as in Example 3. The preservation observation results are shown in Table 5.

[0044] Table 5 Influence of Cu 2+ concentration on preservation

[0045] According to the test results in Table 5, the addition of 0.0785-0.1099 mmol / L of Cu 2+ in the preservation nutrient solution has the best preservation effect, and the removal rate (i.e., survival rate) is 55.74% and 50.90% after 3 months of preservation. When the Cu 2+ addition amount is 0.0157 mmol / L, the survival rate is 48.63%, which is not significantly different from the survival rate of 49.48% when 0 mmol / L is added. This concentration is at the lower limit of the control range, and the inhibition intensity of the bacteria metabolism is insufficient: the bacteria maintain a relatively active metabolic state during preservation at room temperature, and quickly consume key substrates such as KNO3 (nitrogen source) and Na2S2O3·5H2O (sulfur source) in the preservation nutrient solution.

[0046] Comparative Example 1 This comparative example is basically the same as Example 1, except that after the bacteria are mixed with the prepared basic preservation nutrient solution, no mineral carrier is added, and 0, 2.11, 10.55, 14.77, and 21.11 mg / L of CuCl2 solution, i.e., Cu 2+ with a concentration of 0, 0.0157, 0.0785, 0.1099, and 0.1570 mmol / L, respectively, is directly added, and the remaining conditions are the same as in Example 1. The results are shown in Table 6.

[0047] Table 6 Influence of directly added Cu 2+ on the strain

[0048] As can be seen from Table 6, in the case of no carrier adsorption, directly adding Cu 2+ to the preservation nutrient solution causes the Thiobacillus denitrificans inoculant to completely lose activity after 3 months of preservation, and cannot maintain activity for a long time.

[0049] In summary, the denitrifying sulfur-oxidizing bacteria is added into the preservation nutrient solution, is adsorbed by using the composite carrier combined by the mineral carrier silica ash and coal gangue, and 0.0785~0.1099 mmol / L Cu 2+ The regulator has a good preservation effect at 28 DEG C under the condition that the moisture content is maintained at 20~30%, and the survival rate is still above 50% after 3 months of preservation.

[0050] The above merely describes preferred embodiments of the present application, but should not be used to restrict the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preserving *Thiobacillus denitrificationus*, characterized in that, Includes the following steps: S1, grind and pulverize the mineral carrier, sieve it to obtain carrier powder for later use; S2, resuspend the bacterial cells of Thiobacillus denitrification in the preservation nutrient solution, stir evenly to obtain the bacterial solution, and set aside for later use; S3. Mix the carrier powder with the bacterial solution, stir evenly, air dry, and dry to obtain a solid adsorbent for preservation.

2. The method for preserving denitrifying thiobacilli as described in claim 1, characterized in that: In step S1, the mineral carrier comprises any one or more of silica fume, coal gangue, vermiculite powder, zeolite powder, and attapulgite.

3. The method for preserving denitrifying thiobacilli as described in claim 2, characterized in that: The mineral carriers are silica fume and coal gangue.

4. The method for preserving denitrifying thiobacilli as described in claim 1, characterized in that: In step S2, the preservation nutrient solution contains Cu 2+ .

5. The method for preserving denitrifying thiobacilli as described in claim 3, characterized in that: The Cu 2+ The concentration is 0.0157~0.157 mmol / L.

6. The method for preserving denitrifying thiobacilli as described in claim 1, characterized in that: In step S3, the mass-to-volume ratio of the carrier powder to the bacterial solution is 1:(0.8~1.2).

7. The method for preserving denitrifying thiobacilli as described in claim 1, characterized in that: In step S3, the moisture content of the dried material is 20-30%, and the air-drying temperature is 20-30°C.

8. The method for preserving denitrifying thiobacilli as described in claim 1, characterized in that: In step S2, the raw materials of the preservation nutrient solution include NH4Cl, MgCl2·6H2O, KH2PO4, Na2S2O3·5H2O, KNO3, NaHCO3 and FeSO4·7H2O.

9. The method for preserving *Thiobacillus denitrificationus* as described in claim 1, characterized in that: The preservation method is to preserve the food at a temperature of 25~30℃.

10. A denitrifying thiobacillus agent, characterized in that, It is prepared by the preservation method of denitrifying thiobacillus according to any one of claims 1 to 9.

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