CN116694502A - Resuscitating method for anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation - Google Patents
Resuscitating method for anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation Download PDFInfo
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 239000010802 sludge Substances 0.000 title claims abstract description 48
- 230000003647 oxidation Effects 0.000 title claims abstract description 45
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 38
- 229910021529 ammonia Inorganic materials 0.000 title claims abstract description 28
- 230000007774 longterm Effects 0.000 title claims abstract description 26
- 238000004321 preservation Methods 0.000 title claims abstract description 24
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 63
- 238000011084 recovery Methods 0.000 claims abstract description 38
- 235000019152 folic acid Nutrition 0.000 claims abstract description 32
- 239000011724 folic acid Substances 0.000 claims abstract description 32
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229960000304 folic acid Drugs 0.000 claims abstract description 31
- 230000000694 effects Effects 0.000 claims abstract description 16
- 239000003102 growth factor Substances 0.000 claims abstract description 8
- 235000015097 nutrients Nutrition 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 22
- 235000013619 trace mineral Nutrition 0.000 claims description 20
- 239000011573 trace mineral Substances 0.000 claims description 20
- 239000000126 substance Substances 0.000 claims description 19
- 239000002351 wastewater Substances 0.000 claims description 19
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 17
- 230000008569 process Effects 0.000 claims description 17
- 210000002966 serum Anatomy 0.000 claims description 14
- 238000005273 aeration Methods 0.000 claims description 9
- 238000011010 flushing procedure Methods 0.000 claims description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 239000001301 oxygen Substances 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 238000005138 cryopreservation Methods 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 230000002950 deficient Effects 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 229920005549 butyl rubber Polymers 0.000 description 3
- 238000010908 decantation Methods 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention discloses a recovery method of anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation, belonging to the technical field of microbial preservation and recovery. According to the invention, the defective growth factor folic acid is added in the recovery process of the anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation, so that the denitrification activity of the anaerobic ammonia oxidation granular sludge and the recovery of the proportion of living cells can be effectively accelerated.
Description
Technical Field
The invention belongs to the technical field of microorganism preservation and recovery, and particularly relates to a recovery method of anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation.
Background
Anaerobic ammoxidation (Anaerobic ammonium oxidation, anammox) technology refers to a novel biological denitrification technology in which anaerobic ammoxidation bacteria (Anaerobic ammonium oxidation bacteria, anAOB) convert ammonia nitrogen into nitrogen gas by taking nitrite nitrogen as an electron acceptor under anaerobic conditions. Compared with the traditional nitrification-denitrification process, the anammox process has the advantages of high volume removal rate, low cost and the like, and is favored in industry.
However, as the multiplication time of AnAOB is as long as half a month or even several months, the anaerobic ammonia oxidation granular sludge (Anammox Granular Sludge, anGS) in the engineering reactor grows slowly, and in addition, a large amount of AnGS is required to be added under various working conditions such as engineering startup, operation breakdown and the like, so that AnGS is often in short supply in engineering, and the storage of AnGS is one of the solutions for providing a 'reservoir' for subsequent engineering application. Prior studies have shown that 4 ℃ cryopreservation is an effective means of long-term preservation of AnGS, during which the AnAOB in AnGS mostly goes into a dormant state. How to quickly recover AnGS is a technical problem to be solved in the current application. .
The existing recovery of AnGS is mostly realized by adding basic water inlet matrix nutrient substances such as ammonia nitrogen, nitrite nitrogen and the like, and the denitrification activity of AnGS is slowly recovered even for several months. AnAOB is a chemolithoautotrophic microorganism, CO 2 The immobilized formation of organic matter is the material basis supporting subsequent metabolic activity. Folic acid is a growth factor necessary for mediating methyl transfer in the AnAOB carbon dioxide fixation pathway (Wood-Ljungdahl pathway), with 4 steps of the fixation pathway involving folic acid and its derivatives. However, genomics studies indicate that AnAOB is a folate synthesis deficient microorganism, which cannot synthesize folic acid itself, and needs to be provided by companion bacteria. After long-term low-temperature preservation, most of microorganisms surviving in AnGS are in a dormant state, the interaction between companion bacteria and AnAOB is weak, and a novel method is provided for promoting the metabolic recovery of AnAOB and accelerating the rapid resumption of AnGS by adding folic acid in the initial stage of resuscitation.
Disclosure of Invention
The invention aims to solve the problem of slow recovery of denitrification activity after long-term low-temperature preservation of anaerobic ammonium oxidation granular sludge (AnGS) in the prior art, and provides a recovery method of the anaerobic ammonium oxidation granular sludge after long-term low-temperature preservation. According to the method, the restriction growth factor folic acid of anammox bacteria (AnAOB) is added into a resuscitating system, and the anammox activity and the recovery of the proportion of living cells can be obviously improved in the early stage of resuscitating after the addition.
In order to achieve the above purpose, the specific technical scheme adopted by the invention is as follows:
the invention provides a recovery method of anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation, which comprises the following specific steps:
s1, placing anaerobic ammonia oxidation granular sludge preserved for a long time in a room temperature environment, flushing with phosphate buffer solution after the anaerobic ammonia oxidation granular sludge is recovered to room temperature, removing a residual buffer on the surface of the anaerobic ammonia oxidation granular sludge, and transferring to a resuscitating container;
s2, adding simulated wastewater containing nutrient substances and growth factor folic acid into the recovery container to form a recovery system; aeration deoxidizing treatment is carried out on the resuscitation system, and a resuscitation container is sealed after the resuscitation system reaches an anoxic state;
s3, placing the sealed resuscitating container in a constant-temperature shake culture environment for resuscitating culture; the simulated wastewater containing the nutrient substances and folic acid are replaced at regular time in the resuscitating culture process, so that the anaerobic ammoxidation granular sludge is ensured to obtain sufficient nutrient substances and growth factors in the resuscitating culture process; and after the denitrification activity of the anaerobic ammoxidation granular sludge and the proportion of living cells are restored to the specified level, finishing resuscitating.
The long-term cryopreservation of anaerobic ammonium oxidation granular sludge described in this example refers to anaerobic ammonium oxidation granular sludge that is preserved in a low-temperature environment for more than 3 months.
Preferably, the concentration of the anaerobic ammonium oxidation granular sludge is 1.2 to 1.5gVSS/L.
Preferably, the phosphate buffer has a concentration of 0.1M and a pH of 7.5. The number of times of flushing the phosphate buffer solution is 3-5 times.
Preferably, the volume ratio of the anaerobic ammonia oxidation granular sludge to the simulated wastewater containing the nutrient substances in the recovery system is 1 (40-50).
Preferably, the folic acid concentration in the resuscitation system is 0.1-1 mu M.
Preferably, the resuscitation container is an anaerobic serum bottle.
Preferably, the aeration deoxidization treatment is carried out by introducing gas containing 95% Ar and 5% CO 2 The aeration deoxidization treatment time is 10-15 min.
Preferably, the anoxic state is a resuscitation system with dissolved oxygen concentration < 0.2mg/L.
Preferably, the components of the simulated wastewater containing nutrients include: (NH) 4 ) 2 SO 4 0.24g/L、NaNO 2 0.25g/L、NaHCO 3 0.8g/L、KHCO 3 0.24g/L、KH 2 PO 4 0.0175g/L、CaCl 2 0.0175g/L、MgSO 4 ·7H 2 O0.3 g/L, trace element I concentrated solution 1ml/L and trace element II concentrated solution 1ml/L.
Further, the components of the trace element I concentrated solution comprise EDTA 5g/L and FeSO 4 5g/L。
Further, the components of the trace element II concentrated solution comprise ZnSO 4 ·7H 2 O 0.43g/L、CoCl 2 ·6H 2 O 0.24g/L、MnCl 2 ·4H 2 O 0.99g/L、CuSO 4 ·5H 2 O 0.25g/L、NaMoO 4 ·2H 2 O0.22g/L、NiCl 2 ·6H 2 O 0.19g/L、NaSeO 4 ·10H 2 O0.21 g/L and H 3 BO 4 0.014g/L。
Preferably, the resuscitating culture process is carried out under a light-shielding condition for 8-12 days. The constant temperature shake culture environment conditions are as follows: the culture temperature is 30 ℃, and the shaking speed is 150rpm/min.
Preferably, the simulated wastewater containing nutrients and folic acid are replaced every 2 days during the resuscitating culture process.
Compared with the prior art, the invention has the following beneficial effects:
the invention adds the restriction growth factor folic acid of AnAOB in the AnGS recovery process after long-term low-temperature preservation, and accelerates the recovery of the metabolic activity of AnAOB. Compared with the recovery process of only adding nutrient substances at present, the recovery method provided by the invention can better promote the denitrification activity of the anaerobic ammonia oxidation granular sludge and the recovery of the proportion of living cells.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.
Example 1
The embodiment provides a recovery method for anaerobic ammonia oxidation granular sludge with added folic acid concentration of 0.1 mu M after long-term low-temperature preservation, which comprises the following specific steps:
(1) Taking out anaerobic ammonia oxidation granular sludge (AnGS) preserved at the low temperature of 4 ℃ for 150 days in a laboratory, and allowing AnGS to return to room temperature after being placed in a room temperature environment for 12 hours; then, phosphate buffer solution (0.1M, pH=7.5) is used for flushing for 3-5 times, then buffer and the like in the anaerobic ammonia oxidation granular sludge preservation process are removed, natural sedimentation and decantation are carried out to finish the flushing, and 10g of flushed AnGS is transferred to an anaerobic serum bottle;
(2) 500mL of simulated wastewater containing nutrient substances is added into an anaerobic serum bottle, and then 0.1 mu M folic acid is added to form a resuscitation system; introducing a resuscitation system containing 95% Ar and 5% CO 2 The mixed gas of (2) is subjected to aeration deoxidization treatment for 10min, and then oxygen is removed; after the concentration of dissolved oxygen in the resuscitating system is less than 0.2mg/L, plugging the bottle mouth of the anaerobic serum bottle by a butyl rubber plug, and sealing by an aluminum cover;
the components of the simulated wastewater containing the nutrient substances comprise: (NH) 4 ) 2 SO 4 0.24g/L、NaNO 2 0.25g/L、NaHCO 3 0.8g/L、KHCO 3 0.24g/L、KH 2 PO 4 0.0175g/L、CaCl 2 0.0175g/L、MgSO 4 ·7H 2 O0.3 g/L, trace element I concentrated solution 1ml/L and trace element II concentrated solution 1ml/L; the components of the trace element I concentrated solution comprise EDTA 5g/L and FeSO 4 5g/L; the components of the trace element II concentrated solution comprise ZnSO 4 ·7H 2 O 0.43g/L、CoCl 2 ·6H 2 O 0.24g/L、MnCl 2 ·4H 2 O0.99g/L、CuSO 4 ·5H 2 O 0.25g/L、NaMoO 4 ·2H 2 O 0.22g/L、NiCl 2 ·6H 2 O 0.19g/L、NaSeO 4 ·10H 2 O0.21 g/L and H 3 BO 4 0.014g/L。
(3) The sealed anaerobic serum bottle was transferred to a constant temperature shaking incubator, and light-protected cultivation was performed at 30℃and 150rpm/min for 8 days. The simulated wastewater containing nutrient substances and folic acid are replaced every 2 days in the resuscitating culture process.
In this example, anGS recovered after treatment with 0.1. Mu.M folic acid was obtained by the steps (1) to (3) described above.
Example 2
The embodiment provides a recovery method for anaerobic ammonia oxidation granular sludge with added folic acid concentration of 1 mu M after long-term low-temperature preservation, which comprises the following specific steps:
(1) Taking out anaerobic ammonia oxidation granular sludge (AnGS) preserved at the low temperature of 4 ℃ for 150 days in a laboratory, and allowing AnGS to return to room temperature after being placed in a room temperature environment for 12 hours; then, phosphate buffer solution (0.1M, pH=7.5) is used for flushing for 3-5 times, then buffer and the like in the anaerobic ammonia oxidation granular sludge preservation process are removed, natural sedimentation and decantation are carried out to finish the flushing, and 10g of flushed AnGS is transferred to an anaerobic serum bottle;
(2) 500mL of simulated wastewater containing nutrient substances is added into an anaerobic serum bottle, and 1 mu M folic acid is added to form a resuscitation system; introducing a resuscitation system containing 95% Ar and 5% CO 2 The mixed gas of (2) is subjected to aeration deoxidization treatment for 10min, and then oxygen is removed; after the concentration of dissolved oxygen in the resuscitating system is less than 0.2mg/L, plugging the bottle mouth of the anaerobic serum bottle by a butyl rubber plug, and sealing by an aluminum cover;
the components of the simulated wastewater containing the nutrient substances comprise: (NH) 4 ) 2 SO 4 0.24g/L、NaNO 2 0.25g/L、NaHCO 3 0.8g/L、KHCO 3 0.24g/L、KH 2 PO 4 0.0175g/L、CaCl 2 0.0175g/L、MgSO 4 ·7H 2 O0.3 g/L, trace element I concentrated solution 1ml/L and trace element II concentrated solution 1ml/L; the components of the trace element I concentrated solution comprise EDTA 5g/L and FeSO 4 5g/L; the components of the trace element II concentrated solution comprise ZnSO 4 ·7H 2 O 0.43g/L、CoCl 2 ·6H 2 O 0.24g/L、MnCl 2 ·4H 2 O0.99g/L、CuSO 4 ·5H 2 O 0.25g/L、NaMoO 4 ·2H 2 O 0.22g/L、NiCl 2 ·6H 2 O 0.19g/L、NaSeO 4 ·10H 2 O0.21 g/L and H 3 BO 4 0.014g/L。
(3) The sealed anaerobic serum bottle was transferred to a constant temperature shaking incubator, and light-protected cultivation was performed at 30℃and 150rpm/min for 8 days. The simulated wastewater containing nutrient substances and folic acid are replaced every 2 days in the resuscitating culture process.
In this example, anGS recovered after treatment with 1. Mu.M folic acid was obtained by the steps (1) to (3) described above.
Comparative example 1
The comparative example provides a recovery method of anaerobic ammonia oxidation granular sludge without folic acid after long-term low-temperature preservation, which comprises the following specific steps:
(1) Taking out anaerobic ammonia oxidation granular sludge (AnGS) preserved at the low temperature of 4 ℃ for 150 days in a laboratory, and allowing AnGS to return to room temperature after being placed in a room temperature environment for 12 hours; then, phosphate buffer solution (0.1M, pH=7.5) is used for flushing for 3-5 times, then buffer and the like in the anaerobic ammonia oxidation granular sludge preservation process are removed, natural sedimentation and decantation are carried out to finish the flushing, and 10g of flushed AnGS is transferred to an anaerobic serum bottle;
(2) 500mL of simulated wastewater containing nutrient substances is added into an anaerobic serum bottle, and distilled water with the volume equal to that of the folic acid in the embodiment 1 is added to form a recovery system; introducing a resuscitation system containing 95% Ar and 5% CO 2 The mixed gas of (2) is subjected to aeration deoxidization treatment for 10min, and then oxygen is removed; after the concentration of dissolved oxygen in the resuscitating system is less than 0.2mg/L, plugging the bottle mouth of the anaerobic serum bottle by a butyl rubber plug, and sealing by an aluminum cover;
the components of the simulated wastewater containing the nutrient substances comprise: (NH) 4 ) 2 SO 4 0.24g/L、NaNO 2 0.25g/L、NaHCO 3 0.8g/L、KHCO 3 0.24g/L、KH 2 PO 4 0.0175g/L、CaCl 2 0.0175g/L、MgSO 4 ·7H 2 O0.3 g/L, trace element I concentrated solution 1ml/L and trace element II concentrated solution 1ml/L; the components of the trace element I concentrated solution comprise EDTA 5g/L and FeSO 4 5g/L; the components of the trace element II concentrated solution comprise ZnSO 4 ·7H 2 O 0.43g/L、CoCl 2 ·6H 2 O 0.24g/L、MnCl 2 ·4H 2 O0.99g/L、CuSO 4 ·5H 2 O 0.25g/L、NaMoO 4 ·2H 2 O 0.22g/L、NiCl 2 ·6H 2 O 0.19g/L、NaSeO 4 ·10H 2 O0.21 g/L and H 3 BO 4 0.014g/L。
(3) The sealed anaerobic serum bottle was transferred to a constant temperature shaking incubator, and light-protected cultivation was performed at 30℃and 150rpm/min for 8 days. The simulated wastewater containing nutrient substances and folic acid are replaced every 2 days in the resuscitating culture process.
In this comparative example, anGS recovered after treatment without folic acid was obtained by the above steps (1) to (3).
Finally, the comparative results of example 1, example 2 and comparative example 1 are as follows:
from the recovery of the anags denitrification activity: at the beginning of resuscitation, the specific activities of AnGS denitrification in examples 1 and 2 and comparative example 1 were 160.7.+ -. 15.1, 158.2.+ -. 8.1 and 159.3.+ -. 2.7mg-N/g-VSS-d, respectively, and after 8 days of resuscitation, the specific activities of denitrification in three groups were 267.2.+ -. 18.6, 305.2.+ -. 9.2 and 238.3.+ -. 12.7mg-N/g-VSS-d, respectively, with examples 1 and 2 being 12% and 28% higher than comparative example 1, respectively. The results show that the recovery of the denitrification activity of AnGS can be obviously promoted by adding 0.1 mu M folic acid and 1 mu M folic acid in the resuscitation process.
From the recovery of the proportion of viable cells in AnGS: at the beginning of resuscitation, the ratios of living cells in AnGS of examples 1, 2 and comparative example 1 were 59.2.+ -. 3.6%, 60.0.+ -. 3.5% and 63.0.+ -. 4.7%, respectively, and after 8 days of resuscitation, the ratios of the three groups of living cells were 73.2.+ -. 1.4%, 75.5.+ -. 2.2% and 68.5.+ -. 3.8%, respectively, and the ratios of living cells in examples 1 and 2 were 4.7% and 7.0%, respectively, which indicates that the recovery of the ratios of living cells in AnGS was promoted by the addition of 0.1 and 1. Mu.M folic acid during resuscitation.
In theory, the denitrification activity and the ratio of living cells in the recovery process of the anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation can be increased along with the increase of the culture time. The invention does not limit the level of denitrification activity and living cell proportion to be achieved, namely, the completion of resuscitation. In practical applications, the denitrification activity and the proportion level of living cells of the anaerobic ammonium oxidation granular sludge can be determined according to the requirements of subsequent use.
In conclusion, folic acid is added in the recovery process after the AnGS is preserved at a low temperature for a long time, so that the denitrification activity of the AnGS and the recovery of the proportion of living cells can be effectively improved. Provides a solution for the slow denitrification activity of AnGS and the slow recovery of living cells after the existing long-term low-temperature preservation.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (9)
1. The recovery method of the anaerobic ammonia oxidation granular sludge after long-term low-temperature preservation is characterized by comprising the following specific steps:
s1, placing anaerobic ammonia oxidation granular sludge preserved for a long time in a room temperature environment, flushing with phosphate buffer solution after the anaerobic ammonia oxidation granular sludge is recovered to room temperature, removing a residual buffer on the surface of the anaerobic ammonia oxidation granular sludge, and transferring to a resuscitating container;
s2, adding simulated wastewater containing nutrient substances and growth factor folic acid into the recovery container to form a recovery system; aeration deoxidizing treatment is carried out on the resuscitation system, and a resuscitation container is sealed after the resuscitation system reaches an anoxic state;
s3, placing the sealed resuscitating container in a constant-temperature shake culture environment for resuscitating culture; the simulated wastewater containing the nutrient substances and folic acid are replaced at regular time in the resuscitating culture process, so that the anaerobic ammoxidation granular sludge is ensured to obtain sufficient nutrient substances and growth factors in the resuscitating culture process; and after the denitrification activity of the anaerobic ammoxidation granular sludge and the proportion of living cells are restored to the specified level, finishing resuscitating.
2. The method for resuscitating a granular anaerobic ammonium oxidation sludge after long-term low-temperature storage according to claim 1, wherein the concentration of the granular anaerobic ammonium oxidation sludge is 1.2-1.5 gVSS/L.
3. The method for resuscitating after long-term cryopreservation of anaerobic ammonium oxidation granular sludge according to claim 1, wherein the concentration of phosphate buffer is 0.1m and the ph is 7.5; the phosphate buffer solution is washed 3-5 times.
4. The method for resuscitating the anaerobic ammonium oxidation granular sludge after long-term low-temperature preservation according to claim 1, wherein the volume ratio of the anaerobic ammonium oxidation granular sludge to the simulated wastewater containing the nutrient substances in the resuscitating system is 1 (40-50); the concentration of the folic acid in the recovery system is 0.1-1 mu M.
5. The method for resuscitating granular sludge after long-term cryopreservation of anaerobic ammonia oxidation according to claim 1, wherein the resuscitating container is an anaerobic serum bottle.
6. The method for resuscitating granular sludge after long-term cold storage of anaerobic ammonium oxidation according to claim 1, wherein the aeration deoxidization treatment is performed by introducing gas containing 95% Ar and 5% CO 2 Is a mixed gas of (a) and (b); the aeration deoxidization treatment time is 10-15 min.
7. The method for resuscitating granular sludge after long-term cryopreservation of anaerobic ammonium oxidation according to claim 1, wherein the anoxic state is a concentration of dissolved oxygen in the resuscitating system of < 0.2mg/L.
8. A method for resuscitating a long-term cryopreservation of anaerobic ammonium oxidation granular sludge according to claim 1, wherein the composition of the nutrient-containing simulated wastewater comprises: (NH) 4 ) 2 SO 4 0.24g/L、NaNO 2 0.25g/L、NaHCO 3 0.8g/L、KHCO 3 0.24 g/L、KH 2 PO 4 0.0175 g/L、CaCl 2 0.0175 g/L、MgSO 4 ·7H 2 O0.3 g/L, trace element I concentrated solution 1ml/L and trace element II concentrated solution 1ml/L; the components of the trace element I concentrated solution comprise EDTA 5g/L and FeSO 4 5g/L; the components of the trace element II concentrated solution comprise ZnSO 4 ·7H 2 O 0.43g/L、CoCl 2 ·6H 2 O 0.24g/L、MnCl 2 ·4H 2 O 0.99g/L、CuSO 4 ·5H 2 O 0.25g/L、NaMoO 4 ·2H 2 O 0.22g/L、NiCl 2 ·6H 2 O 0.19g/L、NaSeO 4 ·10H 2 O0.21 g/L and H 3 BO 4 0.014g/L。
9. The recovery method after long-term low-temperature preservation of anaerobic ammonium oxidation granular sludge according to claim 1, wherein the recovery culture process is carried out under a light-proof condition for 8-12 days; the constant temperature shake culture environment conditions are as follows: the culture temperature is 30 ℃, and the shaking speed is 150 rpm/min; preferably, the simulated wastewater containing nutrients and folic acid are replaced every 2 days during the resuscitating culture process.
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| CN102627379A (en) * | 2012-03-27 | 2012-08-08 | 杭州师范大学 | Method for storing and exciting anaerobic ammonium oxidation sludge |
| CN103043788A (en) * | 2013-01-04 | 2013-04-17 | 哈尔滨工业大学 | Cultivation method of anammox granular sludge |
| CN110129203A (en) * | 2019-04-23 | 2019-08-16 | 杭州师范大学 | A kind of preservation and restoration methods of Anammox strain |
| CN116730486A (en) * | 2023-05-26 | 2023-09-12 | 浙江沃乐科技有限公司 | Anaerobic ammonia oxidizing bacteria proliferation agent |
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| CN102627379A (en) * | 2012-03-27 | 2012-08-08 | 杭州师范大学 | Method for storing and exciting anaerobic ammonium oxidation sludge |
| CN103043788A (en) * | 2013-01-04 | 2013-04-17 | 哈尔滨工业大学 | Cultivation method of anammox granular sludge |
| CN110129203A (en) * | 2019-04-23 | 2019-08-16 | 杭州师范大学 | A kind of preservation and restoration methods of Anammox strain |
| CN116730486A (en) * | 2023-05-26 | 2023-09-12 | 浙江沃乐科技有限公司 | Anaerobic ammonia oxidizing bacteria proliferation agent |
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