Electron donor capable of improving reductive dehalogenation efficiency of anaerobic dehalogenation flora

Abstract

The invention belongs to the technical field of bioremediation of organic contaminated sites, and mainly relates to an electron donor capable of improving the reductive dehalogenation efficiency of anaerobic dehalogenation flora, which can be used for improving the reductive dehalogenation efficiency of anaerobic dehalogenation flora. The electron donor comprises lactic acid, butyric acid and hydrogen in a molar ratio of 3: 2: 1 or 2: 2: 2 or 2: 3: 1. During use, lactic acid and butyric acid are added in the form of solution, hydrogen is injected into a polluted area in the form of gas, hydrogen is diffused to a polluted environment as a direct electron donor, flora can be activated more quickly, and then lactic acid and butyric acid gradually release hydrogen after being fermented, so that the activity of hydrogen is improved. The defect that the dehalogenation rate is low under the condition of a single electron donor can be overcome, a complementary electron metabolism path is formed in a flora by means of metabolism of a mixed electron donor based on the complex metabolism path of the dehalogenation flora, the growth and metabolism activity of microorganisms is enhanced, and the efficiency of dehalogenation pseudococcus for obtaining hydrogen for reductive dehalogenation is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioremediation of organic contaminated sites, and mainly relates to an electron donor that can improve the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria, and can be used to improve the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria. Background Art

[0002] Chlorinated olefins are widely used as solvents for dry cleaning and degreasing metal parts. They can easily enter soil and groundwater, causing environmental contamination. As heavy non-aqueous liquids, they migrate downward through soil and groundwater, causing persistent contamination of the soil and aquifers as they flow. Once contaminated, they are difficult to completely dissolve and are only released very slowly into the surrounding environment. For example, trichloroethylene (TCE) has a half-life of approximately six months to a year and a half in soil and as long as eleven months to four and a half years in groundwater. Furthermore, chlorinated olefins can be transported through the groundwater circulation system, contaminating other ecosystems.

[0003] In the existing technology, the methods for chemical degradation of chlorinated olefins mainly include chemical oxidation and degradation-coupled persulfate oxidation. Compared with the traditional chemical method for degrading chlorinated olefins, microbial degradation of chlorinated olefins has the advantages of being green and economical. Under aerobic conditions, chlorinated olefins can serve as substrates for microbial growth, or be degraded through co-metabolic pathways with pollutants such as toluene. In the actual environment, the soil and groundwater environments where chlorinated olefins are located are generally anaerobic environments, so microbial anaerobic reduction is a more suitable degradation method. The main functional microorganisms of microbial anaerobic reduction are organic halogen-respiring bacteria, among which Dehalogenococcus spp. is an obligate anaerobic dehalogenating bacterium that can completely reduce and dehalogenate chlorinated olefins into ethylene. Because the genome of Dehalogenococcus spp. is highly streamlined, it cannot synthesize the electron donor (hydrogen) it needs and needs to obtain it from coexisting microorganisms. In the actual environment, dehalogenating coccidioides often cooperate with acetogens, methanogens, etc. to form anaerobic dehalogenating bacteria groups. This group can use small molecule organic acids as electron donor substrates. Through the metabolism of coexisting microorganisms, hydrogen is produced, which provides electron donors for dehalogenating coccidioides, thereby improving the efficiency of dehalogenation reduction by the dehalogenating bacteria group.

[0004] The electron donor adjuvants used for the reduction and dehalogenation of anaerobic dehalogenating bacteria include ingredients such as lactic acid, pyruvic acid and vegetable oil. For bacteria with complex small molecule organic acid metabolic pathways, a single electron donor adjuvant can specifically improve the efficiency of a certain metabolic pathway of the bacteria, but the overall dehalogenation efficiency is still low, which is difficult to meet the needs of actual remediation. Studies have been conducted to add electron donors such as lactic acid, propionic acid, and formic acid to the dehalogenating bacteria in anaerobic sludge, soil or groundwater to enhance the reduction and dehalogenation efficiency of the bacteria, and some studies have also added ingredients such as surfactants to improve the reduction and dehalogenation efficiency, but this solution increases the cost of actual remediation. Whether a more suitable electron donor can be provided has become the main direction of the inventor's research. Summary of the Invention

[0005] The present invention addresses the problems existing in the above-mentioned technologies and provides an electron donor that can improve the reduction dehalogenation efficiency of anaerobic dehalogenating bacteria, which can be used to improve the reduction dehalogenation efficiency of anaerobic dehalogenating bacteria; the electron donor comprises lactic acid, butyric acid and hydrogen, and the molar ratio of the three is 3:2:1 or 2:2:2 or 2:3:1. When used, lactic acid and butyric acid are directly added to the organic contaminated site, and hydrogen is injected into the organic contaminated site in the form of gas. Hydrogen diffuses into the polluted environment as a direct electron donor, which can activate the bacteria faster. After that, lactic acid and butyric acid gradually release hydrogen through the fermentation process, which can make up for the slow dehalogenation rate under the condition of a single electron donor. Based on the complex metabolic pathway of the dehalogenating bacteria, the mixed electron donor metabolism is used to form a complementary electron metabolic pathway within the bacteria, thereby enhancing the growth and metabolic activity of the microorganisms and improving the efficiency of the dehalogenating coccidioides in obtaining hydrogen for reduction dehalogenation.

[0006] The main principle of this invention is that dehalogenating bacteria have a complex nutrient interaction network. For example, Desulfovibrio and Clostridium can ferment lactic acid to produce hydrogen, which is then used by Dehalogenococcus, while methanogens can compete with Dehalogenococcus for hydrogen. Based on this interaction network between microorganisms, different electron components are used to form complementary electron metabolic pathways, constructing a multi-level transfer network, and enhancing the efficiency of Dehalogenococcus in acquiring electrons for reductive dehalogenation.

[0007] After research, the inventors discovered that, compared to using a single component (lactic acid, butyric acid, or hydrogen) or a two-component electron donor, the present invention utilizes a three-component mixed substrate electron donor consisting of a small molecule organic acid and hydrogen. This allows for the formation of complementary electron metabolic pathways, building a multi-level transfer network and enhancing the efficiency of Dehalogenobacteria in acquiring hydrogen for reductive dehalogenation. Compared to the 20-plus days required to degrade TCE using a single electron donor, the electron donor component provided in this application can completely convert TCE into the non-toxic product ethylene in as little as 14 days.

[0008] The specific technical solutions of the present invention are as follows: An electron donor capable of improving the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria. The electron donor comprises lactic acid, butyric acid and hydrogen, and the molar ratio of the three is 3:2:1 or 2:2:2 or 2:3:1.

[0009] The above-mentioned anaerobic dehalogenating bacteria group at least includes Dehalogenating Coccidioides ( Dehalococcoides mccartyi )、Desulfovibrio spp.( Desulfovibrio desulfuricans ), Clostridium ( Clostridium sp. ) and methanogens ( Methanobacterium formicicum ), the above are all commonly used strains in this field (as shown by Ningbo Mingzhou Biotechnology Co., Ltd.), and the inventors will not elaborate on them.

[0010] The concentration range of the above bacteria is: Dehalogenobacterium (2.1×10 7 -2.3×10 7 copy / mL), Desulfovibrio spp. (6.8×10 6 -7.4×10 6 copy / mL), Clostridium (8.4×10 5 -1.4×10 6 copy / mL) and methanogens (1.6×10 5 -6.2×10 5 copy / mL); the total concentration of bacteria in pollutants is generally controlled to be 2.9×10 7 -3.2×10 7 copy / mL.

[0011] When the pollutants in the contaminated site are chlorinated olefins, especially trichloroethylene, when the trichloroethylene content does not exceed 500 μmol / L and the corresponding chloride ion concentration does not exceed 1500 μmol / L, the corresponding bacterial population concentration controlled in this application is 2.9×10 7 -3.2×10 7 The total concentration of the mixed electron donor is 5-15 mmol / L, and more preferably the total concentration of the mixed electron donor is 10 mmol / L.

[0012] When it is used in the actual contaminated site remediation process, the electron donor concentration can also be appropriately adjusted according to the actual concentration of the pollutant and the amount of bacterial agent added. Generally, it is sufficient to control the bacterial population and the electron donor to be excessive. In addition to chlorinated olefins, the pollutants can also be other halogenated olefins or other halogen-containing pollutants. The dosage is referenced to chlorinated olefins. Preferably, the bacterial population and the electron donor are both excessive.

[0013] More preferably, the lactic acid can be replaced by one or both of sodium lactate and potassium lactate; the butyric acid can be replaced by one or both of sodium butyrate and potassium butyrate.

[0014] After providing the above-mentioned electron donor component, the inventor verified through experiments that it is used as an electron donor substrate for anaerobic dehalogenating bacteria. Lactic acid and butyric acid can be directly added to the culture medium, and hydrogen is injected into the culture medium in the form of gas. At this time, hydrogen diffuses into the contaminated environment as a direct electron donor, which can activate the bacteria faster. After that, lactic acid and butyric acid gradually release hydrogen through the fermentation process, which can make up for the slow dehalogenation rate under the condition of a single electron donor. Based on the complex metabolic pathway of the dehalogenating bacteria, mixed electron donor metabolism is used to form a complementary electron metabolic pathway within the bacteria, thereby enhancing the growth and metabolic activity of microorganisms and improving the efficiency of dehalogenating coccidioides in obtaining hydrogen for reduction dehalogenation. In actual contaminated sites, lactic acid and butyric acid can be directly added to the soil and mixed evenly, and hydrogen is introduced into the soil at the same time, so that the total concentration of electron donors per unit volume of soil eventually reaches the above-mentioned standard, more preferably in excess.

[0015] The above-mentioned chlorinated olefin organic compounds include one or two of tetrachloroethylene, trichloroethylene, 1,1-dichloroethylene, trans-1,2-dichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride. The anaerobic dehalogenating bacteria and corresponding electron donor combination targeted by the present invention can treat systems where the chloride ion concentration of pollutants is less than 1500 μmol / L. If the pollution concentration of individual sites is higher than this range, existing technologies such as vapor phase extraction can be used to reduce its concentration to this range, and then the bacterial community reduction dehalogenation remediation can be carried out.

[0016] In summary, compared to using a single component (lactic acid, butyric acid, or hydrogen) or a two-component electron donor, the present invention utilizes a three-component mixed substrate electron donor consisting of a small molecule organic acid and hydrogen. This can form complementary electron metabolic pathways, construct a multi-level transfer network, and enhance the efficiency of Dehalogenobacteria in acquiring hydrogen for reductive dehalogenation. Compared to the degradation cycle of more than 20 days required to degrade TCE using a single electron donor component, the electron donor component provided in this application can completely convert TCE into the non-toxic product ethylene in as little as 14 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the reduction dehalogenation rate diagram of the single-component electron donor system in Example 3; Figure 2 This is the reduction dehalogenation rate diagram of the two-component electron donor system in Example 3; Figure 3 This is the reductive dehalogenation rate diagram of the three-component electron donor system in Example 3; Figure 4 This is a graph showing the reduction dehalogenation rate of the three-component electron donor system in Example 4; In each figure, PCE is tetrachloroethylene, TCE is trichloroethylene, DCE is cis-1,2-dichloroethylene, VC is vinyl chloride, and ETH is ethylene. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments, which may enable those skilled in the art to more fully understand the present invention, but does not limit the present invention in any way. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0019] Example 1 Preparation of liquid culture medium Liquid culture medium composition for verifying the function of electron donor components: 1000 mL deionized water, 1.0 g NaCl, 0.5 g MgCl2·6H2O, 0.2 g KH2PO4, 0.3 g NH4Cl, 0.3 g KCl, 0.015 g CaCl2·2H2O, 1 mL Se / W stock solution (6 mg / L Na2SeO3·5H2O, 8 mg / LNa2WO4·2H2O, 500 mg / L NaOH), 1 mL trace element stock solution (1.5 g / L FeCl2·4H2O, 190 mg / L CoCl2·6H2O, 100 mg / L MnCl2·4H2O, 70 mg / L ZnCl2, 6 mg / L H3BO3, 36 mg / LNa2MoO4·2H2O, 24 mg / L NiCl2·6H2O, 2 mg / L CuCl2·2H2O), 2.292 g Tris ethanesulfonic acid, boiled under nitrogen to remove oxygen, pH about 7.2, and then sterilized at 121 ℃ for 20 min.

[0020] Example 2 Obtaining anaerobic dehalogenating bacteria The anaerobic dehalogenating bacteria group used includes at least Dehalogenating Coccidioides ( Dehalococcoides mccartyi )、Desulfovibrio spp.( Desulfovibrio desulfuricans ), Clostridium ( Clostridium sp. ) and methanogens ( Methanobacterium formicicum ).

[0021] The concentration range of each bacteria is: Dehalogenobacterium (2.1×10 7 -2.3×10 7 copy / mL), Desulfovibrio spp. (6.8×10 6 -7.4×10 6 copy / mL), Clostridium (8.4×10 5 -1.4×10 6 copy / mL) and methanogens (1.6×10 5-6.2×10 5 copy / mL). They can be purchased directly from the market, such as Ningbo Mingzhou Biotechnology Co., Ltd. When used, it is only necessary to control the concentration of each bacteria in the mixed flora to reach the above minimum limit and meet the requirement that the total concentration of the flora in the pollutant is 2.9×10 7 -3.2×10 7 copy / mL is sufficient.

[0022] Example 3 Verification of the function of anaerobic dehalogenating bacteria Trichloroethylene was added to the liquid culture medium to a concentration of 450 μmol / L (corresponding to a chloride ion concentration of 1350 μmol / L); electron donors (single-component system, two-component system, and three-component system) were added to the liquid culture medium to a total concentration of 10 mmol / L. The anaerobic dehalogenating bacteria in Example 2 were then added to the liquid culture medium to a total concentration of 2.9×10 7 -3.2×10 7 The cells were incubated at 30°C in the dark. The concentrations of trichloroethylene and its products were measured daily for the first four days, and then every two or four days thereafter. The results are as follows: (1) Single-component electron donor system Experimental results: After 22 days of culture, trichloroethylene, cis-1,2-dichloroethylene, vinyl chloride and other intermediate products of the lactic acid group were all degraded into ethylene, followed by the hydrogen group (complete degradation in 26 days), but the butyric acid group could not be completely degraded; the results are as follows Figure 1 shown.

[0023] (2) Two-component electron donor culture system Experimental results: With the electron donor equipped at a molar ratio of 1:1, after 18 days of cultivation, trichloroethylene and intermediate products in the lactic acid-butyric acid system were completely dehalogenated to ethylene. The butyric acid-hydrogen system was completely dehalogenated to ethylene after 22 days. The dehalogenation rate of the lactic acid-hydrogen system was the slowest and could not be completely degraded. The results are as follows Figure 2 shown.

[0024] (3) Three-component electron donor culture system Experimental results: When lactic acid, butyric acid and hydrogen are used as mixed electron donors, when the ratio of the three is 3:2:1 and 2:2:2, TCE and its intermediates can be reduced and dehalogenated to ethylene within 14 days, and the dehalogenation rate is significantly better than that of the single-component and two-component electron donor systems; when the ratio of the three is 2:3:1, TCE and its intermediates can be reduced and dehalogenated to ethylene within 18 days, which is slightly faster than the two-component system. Although the dehalogenation rate of other three-component systems is slower, its dehalogenation rate is still higher than that of the single-component butyric acid system and the two-component lactic acid-hydrogen system, and TCE and its intermediates of all systems are completely dehalogenated within 30 days. The results are as follows Figure 3 As shown, the dehalogenation efficiency is highest when the three-component electron donor culture system with the above ratio is adopted.

[0025] Example 4: Simulation of actual remediation site verification The soil from the actual contaminated site was used as the matrix. Testing confirmed that the soil contained some tetrachloroethylene and trichloroethylene. A simulation experiment was conducted with a water-soil mass ratio of 1:1. The molar ratio of tetrachloroethylene to trichloroethylene was artificially adjusted to 1:2, and the total chloride ion concentration of the pollutants was 1500 μmol / L. The dehalogenating bacteria from Example 2 were added to the above-mentioned mixed system to degrade tetrachloroethylene and trichloroethylene in the soil, resulting in a total bacterial concentration of 2.9×10 7 -3.2×10 7 copy / mL.

[0026] A three-component electron donor was added with a molar ratio of lactic acid, butyric acid, and hydrogen of 3:2:1, 2:2:2, and 2:3:1, respectively, to make the total concentration of the electron donor reach 10 mmol / L. Reductive dehalogenation experiments were carried out, and the results are as follows: When lactic acid, butyric acid and hydrogen are used as mixed electron donors, when the ratio of the three is 3:2:1 and 2:2:2, PCE, TCE and their intermediates can be completely reduced and dehalogenated to ethylene within 18 days; when the ratio of the three is 2:3:1, PCE, TCE and their intermediates can be completely dehalogenated to ethylene within 24 days. Figure 4 shown.

[0027] It can be seen that the present invention uses a three-component mixed substrate electron donor of a mixed small molecule organic acid and hydrogen to form a complementary electron metabolic pathway, construct a multi-level transfer network, and enhance the efficiency of Dehalogenococcus to obtain hydrogen for reductive dehalogenation.

[0028] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An electron donor that can improve the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria, characterized in that: The electron donor comprises lactic acid, butyric acid and hydrogen, and the molar ratio of the three is 3:2:1 or 2:2:2 or 2:3:

1.

2. The electron donor capable of improving the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria according to claim 1, characterized in that: The lactic acid is replaced by one or both of sodium lactate and potassium lactate; the butyric acid is replaced by one or both of sodium butyrate and potassium butyrate.

3. The electron donor capable of improving the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria according to claim 1, characterized in that: The anaerobic dehalogenating bacteria group includes at least Dehalogenococcus, Desulfovibrio, Clostridium and methanogens.

4. The electron donor capable of improving the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria according to claim 1, characterized in that: When the pollutant is calculated as trichloroethylene and the concentration does not exceed 500 μmol / L, the bacterial concentration is 2.9×10 7 -3.2×10 7 copy / mL, and the total concentration of the mixed electron donor is 5-15 mmol / L.

5. The electron donor capable of improving the reduction and dehalogenation efficiency of anaerobic dehalogenating bacteria according to claim 4, characterized in that: The total concentration of the electron donor was 10 mmol / L.

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