CN113736717B - Methane oxidizing bacterium with denitrification function and anoxia resistance and application thereof - Google Patents
Methane oxidizing bacterium with denitrification function and anoxia resistance and application thereof Download PDFInfo
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Abstract
The invention provides a methane-oxidizing bacterium with denitrification function and anoxia resistance and application thereof。The methane oxidizing bacteria can efficiently degrade nitrate under different environmental conditions and simultaneously carry out a methane oxidation process, has stress resistance to an anoxic environment, is simple to culture, has low cost, strong adaptability of bacterial strains and good stability, and has wide application prospects in the field of sewage treatment. The bacterial strain utilizes methane and nitrate as a carbon source and a nitrogen source to form mycoprotein, can be used as feed and the like, realizes the conversion of pollutants into products with higher added values, and has the advantage of comprehensive pollution treatment.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a methane-oxidizing bacterium with a denitrification function and anoxia stress resistance and application thereof.
Background
The atmospheric circulation is affected due to warming of the climate, so that the weather in local areas is unstable, and high-temperature heat waves, heavy rain floods, cold tides and snow disasters frequently occur. In 2020, scientists detected that the north ice ocean ice melted rapidly and that the south pole glacier developed large cracks. Since the industrial revolution, the concentration of methane in the atmosphere has increased rapidly, at a rate of 1.0% to 1.2% per year, and the effect of methane on global climate change has increased day by day. Although the methane content in the atmosphere is far lower than the carbon dioxide content, the greenhouse effect of the methane is 26.5 times that of the carbon dioxide, and the methane has higher global warming potential. Therefore, the enhancement of the management and control of methane emission has great significance for relieving global warming. In addition, the highest methane content in mine gas reaches 90%, and gas explosion is one of main coal mine accidents. Anaerobic sludge and other reactors can also produce methane as biogas. These methane waste gases are urgently needed to be further converted and utilized, and the safety and environmental risks are reduced.
Nitrate is an important discharge nitrogen source causing water eutrophication, can be converted into a harmful substance nitrite under the action of high temperature or biology, causes people to be in oxygen-poor poisoning, and has the risks of carcinogenesis, teratogenesis and mutagenesis. At present, when a sewage treatment plant utilizes microorganisms to treat nitrate, a large amount of carbon sources such as sodium acetate and the like are needed, and the input cost is increased. In addition, the microbial agents are mostly floras, and the nitrate removal activity is easily influenced by the environment and has unstable performance. And the sewage treatment process comprises aerobic condition and anaerobic condition. Therefore, the cultivation of the nitrate removal microbial inoculum which has the advantages of economy in carbon source, strong activity, stable performance and aerobic and anoxic activity is particularly important in the biological sewage treatment process.
The methane-oxidizing bacteria take methane as a unique carbon source and energy source, and can reduce greenhouse gas emission. Methane-oxidizing bacteria are classified into aerobic methane-oxidizing bacteria and anaerobic methane-oxidizing bacteria according to whether oxygen is required. Wherein, the anaerobic methane-oxidizing bacteria have slow growth speed and are difficult to realize pure culture, while the aerobic methane-oxidizing bacteria have fast growth speed and are easy to separate and obtain. CN201010536169.0 discloses a methylobacterium (A) for denitrifying wastewater by using methanolMethylobacterium phyllosphaerae). CN201910598263.X discloses a methane-oxidizing bacterium (Methylobacterium sp) Application in the production of single-cell protein. However, these aerobic methane-oxidizing bacteria cannot survive for a long period of time under anoxic conditions. Therefore, the methane oxidation bacterium which can utilize the greenhouse gas methane and remove the nitrate in the sewage and has stable performance is obtained by separationHas important application value.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a methane-oxidizing bacterium with denitrification function and anoxia stress resistance.
The invention also aims to provide the application of the methane-oxidizing bacteria with denitrification function and anoxia resistance.
In order to achieve the purpose, the invention adopts the technical scheme that:
a methane oxidizing bacterium with denitrification function and anoxia resistance is named methylobacterium (A), (B) and (C)Methylobactersp.) YRD-M2, deposited at 24.9.2021 at the Guangdong province's microorganism culture collection center of Guangdong province institute for scientific microorganisms, No. 59 building, No. 5 building, of Ministry, Zhou, Middu, Guangzhou city, with the collection number GDMCC No. 61952.
The methane oxidizing bacteria with the denitrification function and the anoxic stress resistance can efficiently degrade nitrate under different environments (methane and oxygen), and simultaneously perform the methane oxidation process, the strain has the stress resistance to the anoxic environment, the culture medium components are easy to obtain, the use amount is small, the culture is simple, the cost is low, the adaptability of the strain is strong, the stability is good, the bacteria form mycoprotein by using methane and nitrate, the bacteria can be used as feed, and the like, and have wide application prospects in the field of sewage treatment.
The methane-oxidizing bacteria with denitrification function and anoxia resistance are applied to environmental remediation; preferably comprising the steps of: the methane-oxidizing bacteria with denitrification function and anoxia resistance are cultured in an environment containing methane and nitrate to realize denitrification and methane emission reduction.
The environment is preferably a body of water.
A microbial preparation with denitrification function and anoxia resistance contains culture medium and above methane-oxidizing bacteria.
The culture medium comprises the following components: 0.1-0.3 g/L magnesium sulfate heptahydrate, 0.10-0.18 g/L calcium chloride hexahydrate, 0.5-1.5 g/L potassium nitrate, 45-55 mL/L phosphate buffer solution, 1.5-2.5 mL/L trace element solution and the balance of water; more preferably as follows: 0.2 g/L magnesium sulfate heptahydrate, 0.14 g/L calcium chloride hexahydrate, 1 g/L potassium nitrate, 50 mL/L phosphate buffer solution, 2 mL/L trace element solution and the balance of water.
The trace element liquid comprises the following components: 0.5-1.5 g/L disodium EDTA, 1.5-2.5 g/L ferrous sulfate heptahydrate, 0.7-0.9 g/L zinc sulfate heptahydrate, 0.02-0.04 g/L manganese chloride tetrahydrate, 0.02-0.04 g/L boric acid, 0.15-0.25 g/L cobalt chloride hexahydrate, 0.5-0.7 g/L copper chloride dihydrate, 0.01-0.03 g/L nickel chloride hexahydrate, 0.04-0.06 g/L sodium molybdate dihydrate, and the balance of water; more preferably as follows: 1 g/L of EDTA disodium, 2 g/L of ferrous sulfate heptahydrate, 0.8 g/L of zinc sulfate heptahydrate, 0.03 g/L of manganese chloride tetrahydrate, 0.03 g/L of boric acid, 0.2 g/L of cobalt chloride hexahydrate, 0.6 g/L of copper chloride dihydrate, 0.02 g/L of nickel chloride hexahydrate, 0.05 g/L of sodium molybdate dihydrate and the balance of water.
The phosphate buffer solution comprises the following components: 5.44 g/L KH2PO4、5.68 g/L Na2HPO4And the balance being water.
The water is preferably distilled water or deionized water.
The preparation method of the microbial preparation with denitrification function and anoxia resistance comprises the following steps: inoculating the methane-oxidizing bacteria with the denitrification function and the anoxic stress resistance into a culture medium, and culturing in a methane gas environment in a dark place to obtain the microbial preparation with the denitrification function and the anoxic stress resistance.
The inoculation volume of the methane-oxidizing bacteria with denitrification function and anoxia resistance is preferably 2-5% of the volume of the culture medium; more preferably, it corresponds to 4% of the volume of the medium.
The preferable temperature of the culture is 28-32 ℃; more preferably 30 deg.c.
The culture time is preferably 3-5 days.
The microbial preparation with the denitrification function and the anoxic stress resistance is applied to environmental remediation; preferably comprising the steps of: and (3) placing the microbial preparation with the denitrification function and the anoxia resistance in an environment to be repaired for culture.
The environment is preferably a body of water.
The inoculation amount of the microbial preparation with the denitrification function and the anoxic stress resistance is 1-100: 1000, the calculation is performed.
Compared with the prior art, the invention has the beneficial effects that:
1. the application provides a methane-oxidizing bacteria with denitrogenation function and oxygen deficiency resistance can carry out methane oxidation process simultaneously by high-efficient degradation nitrate under different environmental conditions, and the bacterial strain has the resistance to the oxygen deficiency environment, and the culture medium composition obtains easily, and the use amount is few, cultivates simply, and is with low costs, and the strong adaptability of bacterial strain, stability is good, has extensive application prospect in the sewage treatment field.
2. The microbial preparation provided by the invention takes methane in gas or waste gas as a substrate, can reduce the sewage treatment cost, can reduce the emission of greenhouse gas, and reduces the concentration of combustible and explosive gas methane through methane oxidizing bacteria, thereby reducing the occurrence of accidents caused by methane mis-combustion.
3. The microbial preparation provided by the invention utilizes greenhouse gas methane and nitrate in sewage as a carbon source and a nitrogen source to form mycoprotein through methane-oxidizing bacteria, can be used as feed and the like, realizes conversion of pollutants into products with higher added values, and has the advantage of comprehensive pollution treatment.
4. The strain in the microbial preparation provided by the invention has strong adaptability, high activity and stable performance, has stress resistance to an anoxic environment, can play a role for a long time, and is beneficial to the sustainable development of sewage treatment.
Drawings
FIG. 1 is a colony morphology of methane-oxidizing bacteria YRD-M2.
FIG. 2 is a cell morphology of methane-oxidizing bacterium YRD-M2 under an optical microscope (10X 100).
FIG. 3 is a graph showing the effect of methane utilization and nitrate removal by the methane-oxidizing bacterium YRD-M2 strain; wherein the initial condition is 25% CH475% air and 2mM NO3 -；Methylobactersp, YRD-M2 means inoculation of the culture mediumMethylobactersp. YRD-M2 strain; the sterile control refers to the medium without inoculation of the YRD-M2 strain.
FIG. 4 is a graph showing the effect of methane utilization and nitrate removal by the methane-oxidizing bacterium YRD-M2 strain; wherein the initial condition is 10% CH475% air and 2mM NO3 -；Methylobactersp, YRD-M2 means inoculation of the culture mediumMethylobacterstrain sp. YRD-M2.
FIG. 5 is a graph showing the effect of methane utilization and nitrate removal by the methane-oxidizing bacterium YRD-M2 strain; wherein the initial condition is 25% CH430% air and 2mM NO3 -；Methylobactersp, YRD-M2 means inoculation of the culture mediumMethylobacterstrain sp. YRD-M2.
FIG. 6 is a graph showing the effect of methane utilization and nitrate removal by the methane-oxidizing bacterium YRD-M2 strain; wherein the initial condition is 25% CH475% air and 1mM NO3 -；Methylobactersp, YRD-M2 means inoculation of the culture mediumMethylobacterstrain sp. YRD-M2.
FIG. 7 is a graph showing the results of measurement of the biomass of methane-oxidizing bacteria YRD-M2 using methane and nitrate; wherein, the biomass of the thallus is expressed by the light absorption value of the bacteria culture solution at 600 nm; 2mM N2 mM NO3 -1mM N is 1mM NO3 -(ii) a The sterile control refers to the medium without inoculation of the YRD-M2 strain.
FIG. 8 is a cell morphology diagram of methane-oxidizing bacteria YRD-M2 strain under a scanning electron microscope; FIG. 8 (A) is a morphological diagram of cells of the YRD-M2 strain cultured under anoxic conditions (DO < 22.3. mu.M) for 278 days, wherein DO (dissolved oxygen) is dissolved oxygen; FIG. 8 (B) is a morphological diagram of cells obtained by culturing the YRD-M2 strain under anaerobic conditions for 278 days and then culturing the strain under aerobic conditions (DO of about 220. mu.M).
FIG. 9 is a graph showing methane and nitrate utilization after 278 days of anaerobic culture after the strain YRD-M2 was re-inoculated to aerobic conditions (DO of about 220. mu.M).
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
The aerobic methane-oxidizing bacteria used in the inventionMethylobactersp, YRD-M2 can use methane and nitrate as carbon source and nitrogen source respectively to synthesize mycoprotein with potential application value. The strain has strong applicability, has strong methane oxidation and nitrate utilization functions under different methane concentrations, air amounts and nitrate concentrations, and has stress resistance to anoxic environments. Therefore, when the strain is applied to sewage treatment, the denitrification of sewage and the emission reduction of methane can be synchronously realized.
Example 1
Methane oxidizing bacteriaMethylobacterIsolation and identification of sp, YRD-M2.
1. Enrichment culture of methane-oxidizing bacteria:
and pre-incubation culture is carried out on the soil of the yellow river delta wetland. And (3) taking 10 g of fresh soil into a 125 mL penicillin bottle, and sealing the penicillin bottle by using butyl rubber and an aluminum cover. Approximately 5% methane (6 mL) was added as a substrate and incubated statically in a dark room at 30 ℃. During the period, gas in a bottle of 0.2 mL is taken by a gas sampling needle, the concentration of methane is detected by utilizing high performance gas chromatography (Agilent 7820A, USA), and the methane is supplemented for continuous culture after the methane is exhausted, and the process is repeated for 2 times. And (4) performing methane oxidation enrichment culture by using the pre-incubated soil as an inoculum. 2.5 g of pre-incubated soil was inoculated into 25 mL of nitrate inorganic salt (NMS) medium. NMS culture medium preparation process as follows: 0.2 g of MgSO was weighed respectively with an electronic balance4·7H2O、0.14 g CaCl2·6H2O and 1.0 g KNO3And 50 mL of phosphate buffer (5.44 g/L KH) was taken out with a syringe2PO4、5.68 g/L Na2HPO4) And 2 mL of trace element solution (1.0 g Na/L)2-EDTA、2.0 g FeSO4·7H2O、0.8 g ZnSO4·7H2O、0.03 g MnCl2·4H2O、0.03 g H3BO3、0.2 g CoCl2·6H2O、0.6 g CuCl2·2H2O、0.02 g NiCl2·6H2O and 0.05 g Na2MoO4·2H2O), adding into deionized water, mixing uniformly, fixing the volume to 1L, adding sodium hydroxide to adjust the pH value to 7.0, subpackaging 25 mL of culture medium into 125 mL of penicillin bottles, sealing with a butyl rubber plug, sealing with an aluminum cap, and sterilizing at 121 ℃ for 20 min to obtain the culture medium. Approximately 5% methane (6 mL) was added as a substrate and after resting for 5 days at 30 ℃ in a dark room, an enriched pool of methane-oxidizing bacteria was obtained.
2. Separation and identification of methane-oxidizing bacteria:
methane-oxidizing bacteria were isolated using the Hungate rolling tube technique, and in a 25 mL anaerobic tube, 0.5 mL of the methane-oxidizing bacteria concentrate described above was transferred to 5 mL of 2% agar NMS medium, and approximately 25% methane (5 mL) was added and cultured under static conditions at 30 ℃ in a dark room for 5 days. And selecting a single colony to 5 mL of liquid culture medium, repeating the steps of tube rolling and single colony selection for 3 times to obtain a purified strain named YRD-M2. During the course, 0.2 mL of gas in the tube was sampled by a gas sampling needle, and the methane concentration was measured by high performance gas chromatography (Agilent 7820A, USA) to determine the methane removal effect.
Taking the bacterial liquid as a DNA template for PCR amplification. PCR reaction system composition (50 μ L): 10 XExTaq buffer (containing Mg)2+) 5.0 muL, 4.0 muL of dNTPs (2.5 mM), 2.0 muL of primers 27F/1492R (10 muM), 0.25 muL of Ex Taq (5U/muL), 2.0 muL of DNA template and 34.75 muL of sterile ultrapure water. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min, and final extension at 72 ℃ for 10 min after 30 cycles. Primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGYTACCTTGACTT)
The 16S rDNA sequence obtained by sequencing is as follows:
CTGCCCGATCAAGTGGTGAGCGCCCTCCCGAAGGTTAGACTACCCACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTAGGACCGGCTTTTTGGGATTTGCTTACTTTCGCAAGTTCGCTGCCCTCTGTACCGGCCATTGTAGCACGTGTGTAGCCCTACCCATAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCTAGAGTTCCCACCATGATGTGCTGGCAACTAAGGATAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACTCCGCTATCTCTAACAGATTCTCTGGATGTCAAGGGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGCCTGTAAAAAGGCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGTACCTCAGCGTCAGTTTTAATCCAGAGAGTCGCCTTCGCCACTGGTGTTCCTTCAGATCTCTACGCATTTCACCGCTACACCTGAAATTCCACTCTCCTCTATTAAACTCTAGTTGCCCAGTATCAAATGCAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTGACTTAAGCAACCGCCTACGCACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTAAAGGTAATGTCAAGCTGCCGGGTATTGACCGGCAGGTTTTCCTCCCAATTGAAAGTGCTTTACAACCCTCAGGCCTTCTTCACACACGCGGTATTGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCGTCCTCTCAGACCAGCTATAGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCTAACGCAGGCTCATCTCAGAGCGCGAGGCCCGAAGGTCCCCCGCTTTCCCCCGTAGGGCGTATGCGGTATTAGCTTGAGTTTCCCCAAGTTGTCCCCCACTCTGAGGCAGATTCCTACGCGTTACTCACCCGTCCGCCACTCGTCAGCGCCCGAAGG。
3. gram staining:
0.05 mL of bacterial liquid is dripped onto a glass slide, evenly coated, dried and fixed on the flame of an alcohol lamp; dripping 1 drop of ammonium oxalate crystal violet solution, dyeing for 1 min, and washing with water; dripping 1 drop of iodine solution, and washing with water after 1 min; decolorizing with 95% ethanol for 20 s, and washing with water; dripping 1 drop of lycopene to dye for 1 min, and washing with water; after drying, the color and morphology of the cells were observed under an optical microscope.
Bacterial strainsMethylobacterThe colony morphology of sp, YRD-M2 on the wall of the anaerobic tube is shown in FIG. 1, and is round with neat edges, and the colony surface is smooth and yellow and opaque. The strain is in the shape of short rod under an optical microscope (as shown in figure 2), and gram stain is red, and the strain is gram-negative bacteria.
4. Identification of strains
The sequencing result of the 16S rRNA of the screened strain is subjected to Nucleotide BLAST sequence comparison in NCBI database, and the result shows that the strain and the strain are subjected to Nucleotide BLAST sequence comparisonMethylobacter luteus A45 has 98.56% sequence similarity, knotRationalizing the characteristics and morphological characteristics of the thallus, and preliminarily judging the thallus to be the methane-oxidizing bacteriaMethylobacterNamed Methylobacterium (A), (B)Methylobactersp.) YRD-M2, deposited at 24.9.2021 at the Guangdong province's microorganism culture collection center of Guangdong province institute for scientific microorganisms, No. 59 building, No. 5 building, of Ministry, Zhou, Middu, Guangzhou city, with the collection number GDMCC No. 61952.
Example 2
Methane oxidizing bacteriaMethylobactersp. YRD-M2 utilized the effects of methane and nitrate at different methane concentrations, air amounts, and nitrate concentrations.
1) Preparation of NMS medium:
0.2 g of MgSO was weighed respectively with an electronic balance4·7H2O、 0.14 g CaCl2·6H2O and 1.0 g KNO3And 50 mL of phosphate buffer (5.44 g/L KH) was taken out with a syringe2PO4、5.68 g/L Na2HPO4) And 2 mL of trace element solution (1.0 g Na/L)2-EDTA、2.0 g FeSO4·7H2O、0.8 g ZnSO4·7H2O、0.03 g MnCl2·4H2O、0.03 g H3BO3、0.2 g CoCl2·6H2O、0.6 g CuCl2·2H2O、0.02 g NiCl2·6H2O and 0.05 g Na2MoO4·2H2O), adding into deionized water, mixing uniformly, fixing the volume to 1L, adding sodium hydroxide to adjust the pH value to 7.0, subpackaging 25 mL of culture medium into 125 mL of penicillin bottles, sealing with a butyl rubber plug, sealing with an aluminum cap, and sterilizing at 121 ℃ for 20 min to obtain the culture medium.
2) Preparing a methane oxidizing bacterium agent:
in a clean bench, 1 mL of methane-oxidizing bacteria is takenMethylobacterInoculating sp, YRD-M2 bacterial liquid into the above 25 mL culture medium, adding 25 mL methane according to 25% (methane volume/headspace volume), taking out the gas in the 25 mL bottle with the same volume by using a syringe, culturing at 30 deg.C in dark room for 5 days, detecting methane concentration by using high performance gas chromatograph (Agilent 7820A, USA), and using spectrophotometer (Prussian TU 1810)China) detection of the cell Density OD600When the concentration of methane is reduced and the biomass of the bacteria reaches about 0.4, the activation of methane-oxidizing bacteria YRD-M2 is shown, and the preparation of the microbial inoculum is successful.
3) The utilization effect of the methane oxidizing bacteria agent on nitrate and methane under different environments is as follows:
to simulate wastewater containing different concentrations of nitrate, 1mM and 2mM KNO were prepared3NMS culture medium (KNO)3The amounts of addition of (b) were modified to 0.10 g and 0.23 g, respectively, and the other components and the preparation process were the same as in 1). Depending on the process of wastewater treatment, the reactor may develop different conditions of methane concentration and air content, and the concentration of nitrate in the wastewater may also vary. Therefore, the conditions of different methane concentrations, air contents and nitrate concentrations are simulated, and the methane consumption and nitrate removal effects and the thallus biomass forming conditions of the methane-oxidizing bacteria YRD-M2 under different conditions are verified. Respectively setting up experimental groups, including: 25% methane +75% air + 2mM nitrate; ② 10 percent of methane, 15 percent of argon, 75 percent of air and 2mM nitrate; ③ 25% of methane, 30% of air, 45% of argon and 2mM of nitrate; 25% methane +75% air + 1mM nitrate, wherein the methane content is v/v (methane volume/headspace volume) and the air content is v/v (air volume/headspace volume). In a clean bench, 1 mL of bacterial suspension of methane-oxidizing bacteria YRD-M2 was inoculated as an experimental group into the culture medium of the above different treatments, while 3 replicates of each treatment were used as a control group without addition of methane-oxidizing bacteria YRD-M2, and the culture was incubated at 30 ℃ for 5 days in a dark room. The methane removal effect was determined by taking 0.2 mL of gas in a bottle with a gas needle using destructive sampling, and measuring the methane concentration using high performance gas chromatography (Agilent 7820A, USA). 1 mL of the bacterial solution was taken out by a sterile syringe, filtered and sterilized through a 0.22 μm filter, and the nitrate removal effect was determined by measuring the nitrate concentration using an ion chromatograph (Dionex ICS-600, USA). Meanwhile, 1 mL of the bacterial liquid is taken by a sterile syringe, and the absorbance of the bacterial liquid is detected by a spectrophotometer (Puseout TU1810, China) at a wavelength of 600 nm, namely the bacterial density OD600。
As can be seen from FIG. 3, under the conditions of about 25% methane, 75% air and 2mM nitrate, that is, the system has high concentration of methane, air and nitrate, the strain YRD-M2 can degrade 5.47 mM methane and 1.42 mM nitrate within 5 days, the cell density can reach 0.40 (FIG. 7), and the strain has the effect of strongly reducing the contents of methane and nitrate and simultaneously forms more cells with application value. As can be seen from FIG. 4, under the conditions of about 10% methane, 75% air and 2mM nitrate, i.e., the system has lower concentration methane, high content air and nitrate, the methane-oxidizing bacteria YRD-M2 can degrade 3.83 mM methane and 1.06 mM nitrate within 5 days, the thallus density can reach 0.40 (FIG. 7), the effect of strongly reducing the contents of methane and nitrate is still achieved, and more thallus with application value is formed.
As can be seen from FIG. 5, under the conditions of about 25% methane, 30% air and 2mM nitrate, i.e., high concentration methane and nitrate and lower content air exist in the system, the methane-oxidizing bacteria YRD-M2 can degrade 4.34 mM methane and 1.05 mM nitrate within 5 days, the thallus density can reach 0.24 (FIG. 7), the effect of strongly reducing the contents of methane and nitrate is still achieved, and a certain amount of thallus with application value is formed. As can be seen from FIG. 6, under the conditions of about 25% methane, 75% air and 1mM nitrate, i.e., high concentration methane and air, and lower concentration nitrate, the methane-oxidizing bacterium YRD-M2 strain can degrade 5.00 mM methane and 0.91 mM nitrate within 5 days, the thallus density can reach 0.30 (FIG. 7), the effect of strongly reducing the contents of methane and nitrate is still shown, and a large number of thallus with application value is formed.
Example 3
Methane oxidizing bacteriaMethylobacterMethane and nitrate utilization effects after long-term anaerobic culture of sp, YRD-M2.
1) Preparation of NMS medium under anoxic conditions:
0.2 g of MgSO was weighed respectively with an electronic balance4·7H2O、 0.14 g CaCl2·6H2O and 0.23 g KNO3And 50 mL of phosphate buffer (5.44 g/L KH) was taken out with a syringe2PO4、5.68 g/L Na2HPO4) And2 mL of trace element liquid (1.0 g Na/L)2-EDTA、2.0 g FeSO4·7H2O、0.8 g ZnSO4·7H2O、0.03 g MnCl2·4H2O、0.03 g H3BO3、0.2 g CoCl2·6H2O、0.6 g CuCl2·2H2O、0.02 g NiCl2·6H2O and 0.05 g Na2MoO4·2H2O), adding into deionized water, mixing uniformly, fixing the volume to 1L, adding sodium hydroxide to adjust the pH value to 7.0, subpackaging 25 mL of culture medium into 125 mL of penicillin bottles, sealing with a butyl rubber plug, and sealing with an aluminum cap. Extracting with intelligent anaerobic preparation instrument (Beijing Elastu technology Co., Ltd.) for 1 min, charging high purity nitrogen gas for 30 s, and repeating the above steps for 10 times to form anoxic environment. Thereafter, sterilization was carried out at 121 ℃ for 20 min. The initial dissolved oxygen concentration in the vial was determined by destructive sampling using a microelectrode (Unisense, Denmark) and found to be 2.13. mu.M.
2) Culturing methane-oxidizing bacteria under an anoxic condition and observing the shapes of the bacteria:
in a clean bench, 1 mL of methane-oxidizing bacteria is takenMethylobacterInoculating sp, YRD-M2 bacterial liquid into 25 mL culture medium in the penicillin bottle, adding 25 mL methane according to 25% (methane volume/headspace volume), taking out the gas in the 25 mL bottle with the same volume by using a syringe, and statically culturing for 278 days at 30 ℃ in a dark room. At the end of the incubation, the dissolved oxygen concentration in the vial was determined by destructive sampling using a microelectrode (Unisense, Denmark) and found to be 22.3. mu.M. Then, scanning electron microscopy was performed: taking 2 mL of bacterial liquid, centrifuging for 3 min at 5000 r/min, and removing supernatant; adding 2 mL of 0.1 mol/L PBS buffer solution, centrifuging for 3 min at 5000 r/min after resuspension, and repeatedly washing for 3 times; then adding 1 mL of 2.5% glutaraldehyde phosphate buffer (pH 7.2), and fixing at 4 ℃ overnight; the next day, washing with 0.15% glutaraldehyde phosphate buffer; then, carrying out gradient dehydration by sequentially using 30%, 50%, 70%, 90% and absolute ethyl alcohol, and centrifuging for 3 min at 5000 r/min after 15 min of dehydration each time; adding 2 mL of tert-butyl alcohol to replace ethanol, centrifuging for 3 min at 5000 r/min, discarding most of supernatant, leaving a little supernatant for resuspending the thallus, sucking 0.1 mL of bacterial liquid, and drippingDrying on a clean cover glass sheet on a superclean workbench; after plating gold, the shape of the cells was observed under a scanning electron microscope.
3) Preparation of culture medium under aerobic condition and verification of strain activity:
the preparation process is the same as 1) NMS culture medium preparation in example 2. Then, 1 mL of methane-oxidizing bacteria cultured for 278 days were collected on a clean benchMethylobacterThe sp, YRD-M2 bacterial solution was inoculated into the above 25 mL culture medium, 25 mL of methane was added in an amount of 25% (volume of methane/headspace volume), and previously, the same volume of gas in a 25 mL flask was taken out by a syringe and cultured in a dark room at 30 ℃ for 9 days. During the course of the measurement, the gas in a 0.2 mL bottle was sampled by a gas needle, and the methane concentration was measured by high performance gas chromatography (Agilent 7820A, USA) to determine the methane removal effect. 1 mL of the bacterial solution was taken out by a sterile syringe, filtered and sterilized through a 0.22 μm filter, and the nitrate removal effect was determined by measuring the nitrate concentration using an ion chromatograph (Dionex ICS-600, USA). In addition, the methane-oxidizing bacteria cultured for 9 daysMethylobactersp. YRD-M2 was observed for cell morphology under a scanning electron microscope in the same manner as described in 2) above.
As can be seen from (a) of fig. 8,Methylobacterafter being cultured for 278 days by sp, YRD-M2, the thalli are gathered and have uneven sizes, a small amount of damaged thalli cells can be seen, and most of intact thalli cells can be seen even though the anaerobic environment has the stress effect on the strain, which indicates that the strain has stress resistance to the anaerobic environment. As can be seen from (B) in FIG. 8, cultured for a long period of time in the absence of oxygenMethylobacterThe sp, YRD-M2 can still grow rapidly after being transferred to the aerobic environment again, the thallus forms are uniform, the distribution is uniform, the thallus density is high, further showing thatMethylobactersp. YRD-M2 is viable under hypoxic conditions.
As can be seen from FIG. 9, the culture was carried out for a long period of time in the absence of oxygenMethylobacterAfter sp, YRD-M2 was transferred to aerobic environment again, it still had the ability to oxidize methane and utilize nitrate, and could utilize 5.22 mM methane and 1.46 mM nitrate in 7 days, and had higher methane oxidation and nitrate removal rate, and the function was the same as that of the strain not cultured in oxygen deficiency. The results show that it is possible to determine,Methylobacterthe sp, YRD-M2 still has vitality under anoxic condition, can rapidly recover normal growth after contacting aerobic condition, recover methane oxidation and nitrate removal functions, and has strong anoxic stress resistance.
Review the methane-oxidizing bacteriaMethylobactersp, YRD-M2 have strong methane and nitrate removal function under the condition of different methane concentrations and oxygen contents, can synchronously perform denitrification and methane oxidation processes under aerobic conditions, and has stress resistance to anoxic environment, easily obtained culture medium components, small use amount, simple culture, low cost, strong adaptability of strains and good stability, so that the microbial inoculum can be widely applied to sewage treatment. Thus, methane-oxidizing bacteriaMethylobactersp, YRD-M2 can synchronously realize denitrification and methane oxidation, has anoxia resistance and can realize the purpose of the invention.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> institute for ecological environment and soil of academy of sciences of Guangdong province
<120> methane-oxidizing bacterium with denitrification function and anoxia resistance and application thereof
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<170> SIPOSequenceListing 1.0
<210> 1
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<213> methane-oxidizing bacterium YRD-M2(Methylobacter sp. YRD-M2)
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ctgcccgatc aagtggtgag cgccctcccg aaggttagac tacccacttc ttttgcaacc 60
cactcccatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcgacatt 120
ctgattcgcg attactagcg attccgactt catggagtcg agttgcagac tccaatccgg 180
actaggaccg gctttttggg atttgcttac tttcgcaagt tcgctgccct ctgtaccggc 240
cattgtagca cgtgtgtagc cctacccata agggccatga tgacttgacg tcgtccccac 300
cttcctccgg tttatcaccg gcagtctccc tagagttccc accatgatgt gctggcaact 360
aaggataagg gttgcgctcg ttacgggact taacccaaca tctcacgaca cgagctgacg 420
acagccatgc agcacctgtc tcagagttcc cgaaggcact ccgctatctc taacagattc 480
tctggatgtc aagggtaggt aaggttcttc gcgttgcatc gaattaaacc acatgctcca 540
ccgcttgtgc gggcccccgt caattcattt gagttttaac cttgcggccg tactccccag 600
gcggtcaact taatgcgtta gctgcgccac taagcctgta aaaaggccca acggctagtt 660
gacatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctacc cacgctttcg 720
tacctcagcg tcagttttaa tccagagagt cgccttcgcc actggtgttc cttcagatct 780
ctacgcattt caccgctaca cctgaaattc cactctcctc tattaaactc tagttgccca 840
gtatcaaatg cagttcccag gttaagcccg gggctttcac atctgactta agcaaccgcc 900
tacgcacgct ttacgcccag taattccgat taacgcttgc accctccgta ttaccgcggc 960
tgctggcacg gagttagccg gtgcttcttc taaaggtaat gtcaagctgc cgggtattga 1020
ccggcaggtt ttcctcccaa ttgaaagtgc tttacaaccc tcaggccttc ttcacacacg 1080
cggtattgct ggatcaggct tgcgcccatt gtccaatatt ccccactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggctgatcgt cctctcagac cagctataga 1200
tcgtcgcctt ggtaggcctt taccccacca actagctaat ctaacgcagg ctcatctcag 1260
agcgcgaggc ccgaaggtcc cccgctttcc cccgtagggc gtatgcggta ttagcttgag 1320
tttccccaag ttgtccccca ctctgaggca gattcctacg cgttactcac ccgtccgcca 1380
ctcgtcagcg cccgaagg 1398
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer 27F
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agagtttgat cctggctcag 20
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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ggytaccttg ttacgactt 19
Claims (10)
1. A methane oxidizing bacterium with denitrification function and anoxia resistance is characterized in that: the name is Methylobacterium (A), (B)Methylobactersp.) YRD-M2, deposited at 24.9.2021 at the Guangdong province's microorganism culture collection center of Guangdong province institute for scientific microorganisms, No. 59 building, No. 5 building, of Ministry, Zhou, Middu, Guangzhou city, with the collection number GDMCC No. 61952.
2. The use of methanotrophs having denitrification and anoxic stress resistance as claimed in claim 1, comprising the steps of: the methane-oxidizing bacteria with denitrification function and anoxia resistance of claim 1 is cultured in an environment containing methane and nitrate to realize denitrification and methane emission reduction.
3. The use of methanotrophs having denitrification and hypoxia stress tolerance as claimed in claim 2, wherein: the environment is a water body.
4. A microbial preparation with denitrification function and anoxia resistance is characterized in that: comprises a culture medium and the methane-oxidizing bacteria with denitrification function and anoxia resistance of claim 1.
5. The microbial preparation with denitrification function and anoxia resistance according to claim 4, wherein: the composition of the culture medium is as follows: 0.1-0.3 g/L magnesium sulfate heptahydrate, 0.10-0.18 g/L calcium chloride hexahydrate, 0.5-1.5 g/L potassium nitrate, 45-55 mL/L phosphate buffer solution, 1.5-2.5 mL/L trace element solution and the balance of water;
the phosphate buffer solution comprises the following components: 5.44 g/L KH2PO4、5.68 g/L Na2HPO4The balance being water;
the trace element liquid comprises the following components: 0.5-1.5 g/L disodium EDTA, 1.5-2.5 g/L ferrous sulfate heptahydrate, 0.7-0.9 g/L zinc sulfate heptahydrate, 0.02-0.04 g/L manganese chloride tetrahydrate, 0.02-0.04 g/L boric acid, 0.15-0.25 g/L cobalt chloride hexahydrate, 0.5-0.7 g/L copper chloride dihydrate, 0.01-0.03 g/L nickel chloride hexahydrate, 0.04-0.06 g/L sodium molybdate dihydrate, and the balance of water.
6. The microbial preparation with denitrification function and anoxia resistance according to claim 5, wherein:
the composition of the culture medium is as follows: 0.2 g/L magnesium sulfate heptahydrate, 0.14 g/L calcium chloride hexahydrate, 1 g/L potassium nitrate, 50 mL/L phosphate buffer solution, 2 mL/L trace element solution and the balance of water;
the trace element liquid comprises the following components: 1 g/L of EDTA disodium, 2 g/L of ferrous sulfate heptahydrate, 0.8 g/L of zinc sulfate heptahydrate, 0.03 g/L of manganese chloride tetrahydrate, 0.03 g/L of boric acid, 0.2 g/L of cobalt chloride hexahydrate, 0.6 g/L of copper chloride dihydrate, 0.02 g/L of nickel chloride hexahydrate, 0.05 g/L of sodium molybdate dihydrate and the balance of water.
7. The method for preparing the microbial preparation with denitrification function and anoxia resistance of any one of claims 4 to 6, comprising the steps of: the culture medium is inoculated with the methane-oxidizing bacteria with denitrification function and anoxia resistance as described in claim 1, and is cultured in the absence of methane gas in the environment, so as to obtain the microbial preparation with denitrification function and anoxia resistance.
8. The method for preparing a microbial preparation having denitrification function and anoxia resistance according to claim 7, wherein:
the inoculation volume of the methane-oxidizing bacteria is 2-5% of the volume of the culture medium;
the culture temperature is 28-32 ℃;
the culture time is 3-5 days.
9. The use of the microbial preparation with denitrification and anoxia resistance of any one of claims 4 to 6 in environmental remediation.
10. The use of the microbial preparation with denitrification and anoxia resistance of claim 9 for environmental remediation, wherein: and (3) placing the microbial preparation with the denitrification function and the anoxia resistance in an environment to be repaired for culture.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6982161B1 (en) * | 2003-10-15 | 2006-01-03 | Markus Donald Herrema | Process for the utilization of ruminant animal methane emissions |
| CN101627109A (en) * | 2006-12-01 | 2010-01-13 | 格沃股份有限公司 | Engineered microorganisms for producing n-butanol and related methods |
| CN112574916A (en) * | 2020-12-16 | 2021-03-30 | 南京理工大学 | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment |
| WO2021117981A1 (en) * | 2019-12-13 | 2021-06-17 | 한국건설기술연구원 | Method of removing high concentration of ammonia from reject water in sewage treatment process by using methane- and methanol-dependent symbiotic bacteria and biogas |
| AU2019374529A1 (en) * | 2018-11-09 | 2021-06-17 | String Bio Private Limited | A growth media composition and improved methods of producing biomass and value added product |
| CN113308410A (en) * | 2021-07-08 | 2021-08-27 | 山东绿邦生物科技有限公司 | Denitrifying bacterium for efficient denitrification and application thereof |
| CN113795570A (en) * | 2019-05-03 | 2021-12-14 | 奥博拉有限公司 | Bioreactor apparatus and method |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100250830B1 (en) * | 1997-12-09 | 2000-04-01 | 성재갑 | Method for producing optically active hydroxycarboxylic acids by autodegradation of polyhydroxyalkanoates |
| CN101165172B (en) * | 2007-10-15 | 2010-11-10 | 广西大学 | Recombination methyl nourishment bacillus and application thereof |
| CN101993839B (en) * | 2010-07-23 | 2012-06-27 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN102071161B (en) * | 2010-08-23 | 2012-08-01 | 重庆理工大学 | Chryseobacterium sp. capable of using methane and application thereof |
| CN102465103B (en) * | 2010-11-04 | 2013-04-10 | 中国石油化工股份有限公司 | Aerobic denitrification methylobacterium phyllosphaerae and application thereof |
| WO2014047103A2 (en) * | 2012-09-18 | 2014-03-27 | The Translational Genomics Research Institute | Isolated genes and transgenic organisms for producing biofuels |
| DK3202900T3 (en) * | 2013-02-04 | 2019-04-08 | Dsm Ip Assets Bv | CARBOHYDRATE DEGRADING POLYPEPTIDE AND APPLICATIONS THEREOF |
| CN103224896B (en) * | 2013-03-13 | 2014-10-15 | 重庆理工大学 | Facultative methanotroph capable of degrading chlorohydrocarbons, and its applications |
| CN103833186B (en) * | 2014-03-10 | 2015-08-19 | 北京工业大学 | The method of Anammox coupling anaerobic methane oxidation lignin-sludge Digestive system |
| CN103898011B (en) * | 2014-03-11 | 2016-01-20 | 江南大学 | A kind of method of methylotrophic bacteria and fermentative production pyrroloquinoline quinone thereof |
| US10793823B2 (en) * | 2014-04-24 | 2020-10-06 | Rapid Micro Biosystems, Inc. | Microbiological growth media and methods of using the same |
| CN103979732B (en) * | 2014-05-09 | 2015-10-07 | 中国科学院生态环境研究中心 | The film biological treating equipment of methane and sulfur-bearing nitrogenous effluent associated treatment and method thereof |
| CN104611193A (en) * | 2015-02-11 | 2015-05-13 | 重庆大学 | Device and method for quickly enriching Nx-DAMO bacteria |
| CN105385620B (en) * | 2015-11-19 | 2018-11-13 | 中国科学技术大学 | A method of separation denitrification anaerobic methane oxidation flora middle ancient times bacterium |
| CN109706202B (en) * | 2018-12-13 | 2021-06-29 | 广东省科学院生态环境与土壤研究所 | Method for extracting chitin by acidating and decalcifying ferrous oxide thiobacillus |
| CN110002608B (en) * | 2019-04-17 | 2021-07-27 | 哈尔滨工业大学 | Method for synchronously realizing sewage denitrification and methane desulfurization |
| CN111484138A (en) * | 2020-05-06 | 2020-08-04 | 太原理工大学 | Control method for oxidation coupling denitrification process for synchronously producing methane and aerobic methane |
| CN112479507B (en) * | 2020-12-11 | 2022-01-28 | 中国科学院生态环境研究中心 | Method for enrichment culture of anaerobic methane-oxidizing bacteria by using coupled biological-membrane-electrochemical waste gas and wastewater co-treatment device |
-
2021
- 2021-11-03 CN CN202111291004.6A patent/CN113736717B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6982161B1 (en) * | 2003-10-15 | 2006-01-03 | Markus Donald Herrema | Process for the utilization of ruminant animal methane emissions |
| CN101627109A (en) * | 2006-12-01 | 2010-01-13 | 格沃股份有限公司 | Engineered microorganisms for producing n-butanol and related methods |
| AU2019374529A1 (en) * | 2018-11-09 | 2021-06-17 | String Bio Private Limited | A growth media composition and improved methods of producing biomass and value added product |
| CN113795570A (en) * | 2019-05-03 | 2021-12-14 | 奥博拉有限公司 | Bioreactor apparatus and method |
| WO2021117981A1 (en) * | 2019-12-13 | 2021-06-17 | 한국건설기술연구원 | Method of removing high concentration of ammonia from reject water in sewage treatment process by using methane- and methanol-dependent symbiotic bacteria and biogas |
| CN112574916A (en) * | 2020-12-16 | 2021-03-30 | 南京理工大学 | N-methylpyrrolidone degrading bacterium and application thereof in wastewater treatment |
| CN113308410A (en) * | 2021-07-08 | 2021-08-27 | 山东绿邦生物科技有限公司 | Denitrifying bacterium for efficient denitrification and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| "低氧生境中好氧甲烷氧化菌的缺氧耐受机理及种群结构研究进展";马若潺等;《应用生态学报》;20070412(第06期);第2047-2054页 * |
| "湿地甲烷代谢微生物产甲烷菌和甲烷氧化菌的研究进展";李思琦等;《海洋环境科学》;20200526(第03期);第488-496页 * |
| "自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展";赵子闻等;《微生物学通报》;20200521;第47卷(第11期);第3709-3718页 * |
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