CN116904342B - Method for culturing anaerobic dehalogenation microbial inoculum and application of anaerobic dehalogenation microbial inoculum in pollution remediation - Google Patents
Method for culturing anaerobic dehalogenation microbial inoculum and application of anaerobic dehalogenation microbial inoculum in pollution remediation Download PDFInfo
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- 238000005695 dehalogenation reaction Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000012258 culturing Methods 0.000 title claims abstract description 13
- 238000005067 remediation Methods 0.000 title abstract description 5
- 239000002068 microbial inoculum Substances 0.000 title description 11
- 241000896321 Dehalogenimonas Species 0.000 claims abstract description 29
- 241000880396 Dehalococcoides Species 0.000 claims abstract description 27
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims abstract description 13
- 239000003673 groundwater Substances 0.000 claims abstract description 11
- 239000002689 soil Substances 0.000 claims abstract description 9
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 claims description 16
- 239000001509 sodium citrate Substances 0.000 claims description 15
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 15
- UBOXGVDOUJQMTN-UHFFFAOYSA-N 1,1,2-trichloroethane Chemical compound ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 claims description 9
- -1 chlorinated hydrocarbon organic compound Chemical class 0.000 claims description 7
- CFXQEHVMCRXUSD-UHFFFAOYSA-N 1,2,3-Trichloropropane Chemical compound ClCC(Cl)CCl CFXQEHVMCRXUSD-UHFFFAOYSA-N 0.000 claims description 6
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 claims description 6
- KFUSEUYYWQURPO-UPHRSURJSA-N cis-1,2-dichloroethene Chemical group Cl\C=C/Cl KFUSEUYYWQURPO-UPHRSURJSA-N 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 2
- KNKRKFALVUDBJE-UHFFFAOYSA-N 1,2-dichloropropane Chemical compound CC(Cl)CCl KNKRKFALVUDBJE-UHFFFAOYSA-N 0.000 claims description 2
- 239000012752 auxiliary agent Substances 0.000 claims description 2
- FNAQSUUGMSOBHW-UHFFFAOYSA-H calcium citrate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FNAQSUUGMSOBHW-UHFFFAOYSA-H 0.000 claims description 2
- 239000001354 calcium citrate Substances 0.000 claims description 2
- 229960004256 calcium citrate Drugs 0.000 claims description 2
- 239000004337 magnesium citrate Substances 0.000 claims description 2
- 229960005336 magnesium citrate Drugs 0.000 claims description 2
- 235000002538 magnesium citrate Nutrition 0.000 claims description 2
- 239000001508 potassium citrate Substances 0.000 claims description 2
- 229960002635 potassium citrate Drugs 0.000 claims description 2
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims description 2
- 235000011082 potassium citrates Nutrition 0.000 claims description 2
- 229960001790 sodium citrate Drugs 0.000 claims description 2
- 235000011083 sodium citrates Nutrition 0.000 claims description 2
- KFUSEUYYWQURPO-OWOJBTEDSA-N trans-1,2-dichloroethene Chemical group Cl\C=C\Cl KFUSEUYYWQURPO-OWOJBTEDSA-N 0.000 claims description 2
- 235000013337 tricalcium citrate Nutrition 0.000 claims description 2
- PLSARIKBYIPYPF-UHFFFAOYSA-H trimagnesium dicitrate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O PLSARIKBYIPYPF-UHFFFAOYSA-H 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 10
- 235000016709 nutrition Nutrition 0.000 abstract description 10
- 230000008439 repair process Effects 0.000 abstract description 9
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 239000003344 environmental pollutant Substances 0.000 abstract description 6
- 231100000719 pollutant Toxicity 0.000 abstract description 6
- 230000000593 degrading effect Effects 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 8
- 238000011529 RT qPCR Methods 0.000 description 7
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 7
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 5
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 5
- 239000005977 Ethylene Substances 0.000 description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 5
- 229940001447 lactate Drugs 0.000 description 5
- 229940076788 pyruvate Drugs 0.000 description 5
- 239000001540 sodium lactate Substances 0.000 description 5
- 229940005581 sodium lactate Drugs 0.000 description 5
- 235000011088 sodium lactate Nutrition 0.000 description 5
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 5
- 235000015112 vegetable and seed oil Nutrition 0.000 description 5
- 239000008158 vegetable oil Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 239000013067 intermediate product Substances 0.000 description 4
- 229940054269 sodium pyruvate Drugs 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 241000722234 Pseudococcus Species 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- FUWDYYPNMYKOBA-UPHRSURJSA-N (Z)-1,3,4,5,6,6,7,7,8,8,8-undecachlorooct-1-ene Chemical compound ClC(C(Cl)(Cl)Cl)(C(C(C(C(Cl)\C=C/Cl)Cl)Cl)(Cl)Cl)Cl FUWDYYPNMYKOBA-UPHRSURJSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical group ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229950011008 tetrachloroethylene Drugs 0.000 description 2
- MWSOSEYIOCGKDW-UHFFFAOYSA-N 1,1,2-tris(chloranyl)ethene Chemical compound ClC=C(Cl)Cl.ClC=C(Cl)Cl MWSOSEYIOCGKDW-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- KRGNPJFAKZHQPS-UHFFFAOYSA-N chloroethene;ethene Chemical group C=C.ClC=C KRGNPJFAKZHQPS-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- Processing Of Solid Wastes (AREA)
Abstract
The invention discloses a method for culturing anaerobic dehalogenation bacteria and application thereof in pollution remediation, wherein the anaerobic dehalogenation bacteria simultaneously comprise Dehalogenimonas and Dehalococcoides, and the anaerobic Dehalogenimonas and the Dehalococcoides are characterized in that a culture medium contains citrate. In the method, the citrate can be used as a nutritional aid in a polluted site for a long time to continuously play a role without frequent addition, so that the repair engineering cost is greatly saved, dehalogenimonas and Dehalococcoides are balanced to grow and play a role in degradation, and the aim of degrading chlorohydrocarbon pollutants in soil or groundwater for a long time and high efficiency is fulfilled.
Description
Technical Field
The invention belongs to the technical field of biodegradation treatment of polluted sites, and mainly relates to a method for culturing an anaerobic dehalogenation microbial inoculum and application thereof in pollution repair, in particular to a method for culturing the anaerobic dehalogenation microbial inoculum by citrate and application thereof in pollution repair.
Background
Chlorinated hydrocarbons, including chlorinated alkanes and chlorinated alkenes, are excellent cleaning agents and organic solvents, are widely applied to industries such as machinery, chemical industry, electronics, cleaning and the like, are easy to enter soil and groundwater environments through leakage, emission and other ways in the production and use processes, become common volatile organic pollutants in domestic pollution sites, and seriously threaten human health and ecological environmental safety. The chlorinated hydrocarbon pollutant has the advantages of high toxicity, low viscosity and low polarity, is easy to form a large-scale pollution plume after entering an aquifer, and has strict risk control value and difficult natural degradation, thus being an important characteristic of chlorinated hydrocarbon pollution sites.
Most chlorinated hydrocarbons are difficult to aerobically biodegrade, but can be anaerobically biodegraded with high efficiency. Because the soil and groundwater environment are usually anaerobic conditions, the in-situ anaerobic biological repair technology is the most economical and green repair technology for solving the pollution of chlorinated hydrocarbon in soil and groundwater.
Dehalogenimonas and pseudococcus dehalogenccoides are the primary obligate anaerobic dehalogenation respiratory strains that degrade chlorinated hydrocarbon contaminants. When the two bacteria exist at the same time, the method can efficiently and anaerobically dehalogenate and degrade chlorinated hydrocarbon pollutants, and the degradable chlorinated hydrocarbon pollutants have a wider range.
Common nutritional aids for anaerobic dehalogenation bioremediation are lactate, pyruvate and vegetable oil. These nutritional aids are decomposed by anaerobic bacteria, providing a carbon source and electron donor hydrogen for anaerobic dehalogenation bacteria. Pyruvate allows balanced growth of Dehalogenimonas and dehalococcoides but is substantially completely decomposed by anaerobic bacteria within 7 days. Lactate and vegetable oil continue to decompose for a longer period of time, but do not allow for the balanced growth of Dehalogenimonas and dehalococcoides, the number of Dehalogenimonas will be greatly reduced or even eliminated. Dehalogenimonas is missing and will greatly affect the variety and efficiency of anaerobically degradable chlorinated hydrocarbons, such as dehalococcoides cannot degrade 1, 2-trichloroethane, 1, 2-tetrachloroethane and 1,2, 3-trichloropropane. The nutrition auxiliary agent which can not only play a long-acting role, but also enable Dehalogenimonas and dehalococcoides to grow uniformly is found, and is valuable for restoring chlorinated hydrocarbon pollution of soil and underground water.
Disclosure of Invention
The first object of the invention is to provide citrate as a nutritional aid for culturing anaerobic dehalogenation agents comprising Dehalogenimonas and Dehalococcoides, so that the dehalogenation agents grow uniformly and provide an efficient biological agent for repairing chlorinated hydrocarbon contaminated sites.
In one aspect, the invention relates to a method of culturing an anaerobic dehalogenation agent comprising both Dehalogenimonas and pseudococcus Dehalococcoides, wherein the medium comprises citrate. The concentration of citrate in the medium is not particularly limited, but is preferably 0.1 to 10mM in view of facilitating the balanced growth of Dehalogenimonas and Dehalococcoides.
In a preferred embodiment of the invention, the medium further comprises an N source and a P source.
In a preferred embodiment of the present invention, the citrate includes one or two or more of sodium citrate, potassium citrate, calcium citrate and magnesium citrate.
In a preferred embodiment of the invention, the ratio of the number of Dehalogenimonas to Dehalococcoides after 15 days of culture by the method is 1 to 5:1-5.
The invention also aims to provide the application of the citrate as a nutrition aid in restoring chlorinated hydrocarbon organic matter polluted soil or underground water, which can continuously play a role for a long time, so that Dehalogenimonas and pseudococcus Dehalococcoides are balanced in growth, and finally the effect of degrading chlorinated hydrocarbon pollutant in soil or underground water for a long time is achieved.
Preferably, the chlorinated hydrocarbon organic compound is one or two or more of tetrachloroethylene, trichloroethylene, 1-dichloroethylene, trans-1, 2-dichloroethylene, cis-1, 2-dichloroethylene, vinyl chloride, 1, 2-dichloroethane, 1, 2-trichloroethane, 1, 2-tetrachloroethane, 1, 2-dichloropropane and 1,2, 3-trichloropropane.
Compared with common lactate, pyruvate, vegetable oil and other nutritional aids, the invention has the following advantages:
(1) Nutritional aids such as lactate, pyruvate, and vegetable oil are decomposed by anaerobic bacteria to provide a carbon source and electron donor hydrogen for anaerobic dehalogenation bacteria. Pyruvate allows balanced growth of Dehalogenimonas and dehalococcoides but is substantially completely decomposed by anaerobic bacteria within 7 days. Lactate and vegetable oil continue to decompose for a longer period of time, but do not allow for the balanced growth of Dehalogenimonas and dehalococcoides, the number of Dehalogenimonas will be greatly reduced or even eliminated. The citrate has six carbon atoms, can continuously play a role for a long time, is decomposed into formate, acetate and hydrogen by anaerobic bacteria, provides electron donors and carbon sources for anaerobic dehalogenation bacteria, and can lead Dehalogenimonas and Dehalococcoides to grow uniformly.
(2) The citrate can be used as a nutritional aid in a polluted site for a long time to continuously play a role without frequent addition, so that the repair engineering cost is greatly saved, dehalogenimonas and Dehalococcoides are enabled to grow uniformly, and the degradation effect is exerted, so that the aim of degrading chlorinated hydrocarbon pollutants in soil or groundwater in a lasting and efficient manner is achieved.
Under the support of the project of the national key research and development plan of "site groundwater halogenated hydrocarbon pollution repair material and technology", the technology of the invention has the advantages that the sodium citrate is used as a nutrition aid in the anaerobic dehalogenation biological repair pilot scale of the actual polluted site, a good repair effect is obtained, and the high-concentration chlorinated hydrocarbon reaches the IV standard of groundwater in four months through anaerobic dehalogenation degradation, and the removal rate is more than 99%.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.
Thus, the following detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 sodium citrate for use in the extended cultivation of anaerobic dehalogenating bacteria
(1) 1000ml of liquid medium preparation:
1000ml of deionized water, 1g of NaCl and 0.5g of MgCl 2 ·6H 2 O、0.2g KH 2 PO 4 、0.3gNH 4 Cl、0.3g KCl、0.015g CaCl 2 ·2H 2 O、30mM NaHCO 3 、1.5mg FeCl 2 ·4H 2 O、0.1mg MnCl 2 ·4H 2 O、0.2mM Na 2 S, anaerobic treatment, ph=7.2-7.4, sterilization at 121 ℃ for 20min.
Subsequently, 5mM sodium citrate and 0.5mM tetrachloroethylene were added.
(2) Inoculating culture
The enriched and domesticated anaerobic dehalogenation bacteria solution containing Dehalogenimonas and Dehalococcoides strain is added into the liquid culture medium, and the mixture is kept stand in a dark place and cultured at a constant temperature of 30 ℃. The tetrachloroethylene degradation was detected every 3 days. After culturing for about 15 days, the intermediate products such as tetrachloroethylene, trichloroethylene, cis-1, 2-dichloroethylene, and vinyl chloride are all degraded into ethylene, and the culturing is terminated.
Sodium citrate is used for vinyl chloride detection data (unit: mM) during the cultivation of anaerobic dehalogenation microbial inoculum
| Time (d) | PCE | TCE | cis-DCE | VC | Ethylene |
| 0 | 0.5 | 0 | 0 | 0 | 0 |
| 3 | 0.45 | 0.02 | 0.01 | 0.01 | 0.01 |
| 6 | 0.2 | 0.015 | 0.01 | 0.15 | 0.12 |
| 9 | 0.05 | 0.01 | 0.01 | 0.18 | 0.25 |
| 12 | 0.02 | 0.01 | 0.00 | 0.09 | 0.37 |
| 15 | 0.00 | 0.00 | 0.00 | 0.00 | 0.49 |
(3) Detection of
Through qPCR detection, the Dehalogenimonas and the Dehalococcoides are balanced in number, and the anaerobic dehalogenides are obtained in 1000ml of enlarged culture, wherein the number of the Dehalogenimonas and the Dehalococcoides are different in countless orders. About 4.5mM sodium citrate remains in the bacterial solution, i.e., only 10% of the sodium citrate is consumed for 15 days.
qPCR detection result after sodium citrate is used for culturing anaerobic dehalogenation microbial inoculum for 15d
| Genus of bacteria | CT value | Copies/mL |
| Dehalococcoides | 14.5 | 2.61E+07 |
| Dehalogenimonas | 15.78 | 1.22E+07 |
Comparative example 1: sodium lactate for expanding culture of anaerobic dehalogenation microbial inoculum
The 5mM sodium citrate of example 1 was replaced with 5mM sodium lactate, with the other conditions unchanged.
Experimental results: after culturing for about 21 days, the intermediate products such as tetrachloroethylene, trichloroethylene, cis-1, 2-dichloroethylene, vinyl chloride and the like are all degraded into ethylene. No Dehalogenimonas was detected by qPCR, the Dehalogenimonas strain was predominantly the dehalococides strain. About 4mM sodium lactate remained in the bacterial liquid, namely 20% sodium lactate was consumed for 21 days.
qPCR detection result after 21d culture of anaerobic dehalogenation microbial inoculum by sodium lactate
Comparative example 2: sodium pyruvate for enlarged culture of anaerobic dehalogenation bacteria agent
The 5mM sodium citrate of example 1 was replaced with 5mM sodium pyruvate, with the other conditions unchanged.
Experimental results: after culturing for about 15 days, the intermediate products such as tetrachloroethylene, trichloroethylene, cis-1, 2-dichloroethylene, vinyl chloride and the like are all degraded into ethylene. The amounts of Dehalogenimonas and Dehalococcoides strains were balanced and varied in countless orders of magnitude as measured by qPCR. No sodium pyruvate was detected on day 9, indicating that sodium pyruvate was consumed.
Example 2 application of sodium citrate in the remediation of trichloroethylene contaminated groundwater
The polluted underground water adopted in the test is collected in underground water of a polluted site in Jiangsu province, and the concentration of trichloroethylene in the underground water is detected to be 130mg/L, and the concentration of cis-1, 2-dichloroethylene is 48.47mg/L, and the concentration of chloroethylene is 12.50mg/L. 100ml of collected underground water is added into a 160ml anaerobic culture flask, 10mM sodium citrate, 100mg/L sodium dihydrogen phosphate and 500mg/L ammonium chloride are added, dissolved oxygen in the water is removed, and then an anaerobic dehalogenation microbial inoculum (comprising dehalogenimas and Dehalococcoides strains) is inoculated according to an inoculum size of 5 percent, and the mixture is subjected to a light-shielding standing experiment at a constant temperature of 30 ℃. Every 5 days, 100 μl of gas phase sample is taken, and the trichloroethylene degradation is detected.
After about 30 days of the experiment, the intermediate products such as trichloroethylene, cis-1, 2-dichloroethylene, chloroethylene and the like are completely degraded into ethylene.
The qPCR detection shows that the Dehalogenimonas and the Dehalococcoides are balanced in number and innumerable in order of magnitude.
Detection data (unit: mg/L) of sodium citrate applied to trichloroethylene polluted groundwater remediation
| Time (d) | Trichloroethylene (trichloroethylene) | Cis-1, 2-dichloroethylene | Vinyl chloride | Ethylene |
| 0 | 130.00 | 48.47 | 12.50 | 0.00 |
| 5 | 99.86 | 62.50 | 14.19 | 0.56 |
| 10 | 59.13 | 42.53 | 30.30 | 7.28 |
| 15 | 3.94 | 28.02 | 41.80 | 16.24 |
| 20 | 2.63 | 7.76 | 36.31 | 24.88 |
| 25 | 1.31 | 1.94 | 23.75 | 35.81 |
| 30 | 0.00 | 0.00 | 0.00 | 46.29 |
qPCR detection result of sodium citrate applied to trichloroethylene polluted groundwater restoration
Claims (6)
1. A method of culturing an anaerobic dehalogenation agent comprising both dehalogenimons and Dehalococcoides, wherein the medium comprises citrate.
2. The method of claim 1, wherein the medium further comprises an N source and a P source.
3. The method of claim 1, wherein the citrate comprises one or two or more of sodium citrate, potassium citrate, calcium citrate, and magnesium citrate.
4. The method of claim 1, wherein the ratio of the number of Dehalogenimonas Dehalococcoides to the number of Dehalococcoides is from 1 to 5 after 15 days of culture: 1-5.
5. A method for restoring chlorinated hydrocarbon organic-contaminated soil or groundwater, which comprises adding Dehalogenimonas and Dehalococcoides and adding citrate as a culture auxiliary agent.
6. The method for repairing according to claim 5, wherein the chlorinated hydrocarbon organic compound is one or two or more of tetrachloroethylene, trichloroethylene, 1-dichloroethylene, trans-1, 2-dichloroethylene, cis-1, 2-dichloroethylene, vinyl chloride, 1, 2-dichloroethane, 1, 2-trichloroethane, 1, 2-tetrachloroethane, 1, 2-dichloropropane and 1,2, 3-trichloropropane.
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| JP2002355055A (en) * | 2000-07-24 | 2002-12-10 | Kurita Water Ind Ltd | Nucleic acid, nucleic acid for detecting ethylene chloride-decomposing bacterium, probe, method of detecting ethylene chloride-decomposing bacterium and method of decomposing ethylene chloride or ethane chloride |
| JP2012086191A (en) * | 2010-10-21 | 2012-05-10 | Kurita Water Ind Ltd | Method of cleaning soil and ground water |
| CN113151100A (en) * | 2021-05-10 | 2021-07-23 | 中山大学 | Anaerobic dehalogenation microbial inoculum and amplification production method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002355055A (en) * | 2000-07-24 | 2002-12-10 | Kurita Water Ind Ltd | Nucleic acid, nucleic acid for detecting ethylene chloride-decomposing bacterium, probe, method of detecting ethylene chloride-decomposing bacterium and method of decomposing ethylene chloride or ethane chloride |
| JP2012086191A (en) * | 2010-10-21 | 2012-05-10 | Kurita Water Ind Ltd | Method of cleaning soil and ground water |
| CN113151100A (en) * | 2021-05-10 | 2021-07-23 | 中山大学 | Anaerobic dehalogenation microbial inoculum and amplification production method thereof |
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