CN107189974B - Low-temperature denitrification bacterium for poor nutrition and application thereof - Google Patents
Low-temperature denitrification bacterium for poor nutrition and application thereof Download PDFInfo
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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
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/007—Contaminated open waterways, rivers, lakes or ponds
Abstract
The invention discloses an oligotrophic low-temperature denitrifying bacterium and application thereof, and relates to an oligotrophic low-temperature denitrifying bacterium and application thereof. The invention aims to solve the problem that the existing denitrifying bacteria are not suitable for poor-nutrition low-temperature water bodies. The oligotrophic low-temperature denitrogenation strain is purple bacillus (Janthinobacterium sp.) M-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu No.1 of Beijing Kogyo, Sungyang district, the preservation date of 2017, 07, 03 days and the preservation number of CGMCC No. 14380. The application of the invention refers to the application of the invention in denitrification of poor-nutrition low-temperature water body. Compared with other strains, the oligotrophic low-temperature denitrifying bacteria have smaller inoculum size and are more suitable for treating oligotrophic water bodies. The invention is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to a strain of oligotrophic low-temperature denitrifying bacteria and application thereof.
Background
Nitrogen is a common pollutant in natural water, and excessive accumulation can cause water eutrophication, ecological system unbalance and other serious consequences. The traditional denitrification method mainly comprises physical, chemical and biological methods. Compared with a physical and chemical method, the biological denitrification method has the advantages of low cost, small secondary pollution, good impact resistance and the like.
In northern areas of China, particularly in northeast areas, the growth of traditional denitrifying bacteria is not facilitated due to too low temperature of surface water in winter, and the traditional biological method is greatly difficult due to the impoverishment of C, N and other nutrient substances in the surface water. Therefore, the screening of the high-efficiency low-temperature denitrifying bacteria can be carried out under low-concentration nutrient substances, and the practical application of the low-temperature bacteria is enhanced, so that the method is very necessary for treating low-temperature water bodies in northern areas, particularly micro-polluted water bodies in poor nutrition states such as surface water, underground water and the like.
In recent years, due to the continuous discovery of heterotrophic nitrification aerobic denitrifying bacteria, attention is paid to the advantages of heterotrophic nitrifying aerobic denitrifying bacteria compared with traditional autotrophic bacteria, and particularly, the adaptability to extreme environments is also continuously discovered by researchers. At present, the reports on low-temperature denitrifying bacteria are relatively few, and a few of screened low-temperature bacteria only aim at sewage treatment and the temperature is above 4 ℃. Therefore, screening out the low-temperature denitrification strains which can carry out biological treatment on water bodies such as surface water, underground water and the like at low temperature has important research and practical application values.
Disclosure of Invention
The invention aims to solve the problem that the existing denitrifying bacteria are not suitable for poor-nutrition low-temperature water bodies, and provides a poor-nutrition low-temperature denitrifying bacterium and application thereof.
The oligotrophic low-temperature denitrogenation bacterium is purple bacillus (Janthinobacterium sp.) M-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu No.1 Beijing of Chaozhou, the facing-yang area of Beijing, the preservation date of 2017, 03 days, and the preservation number of CGMCC No. 14380.
The application of the oligotrophic low-temperature denitrifying bacterium is the application in the denitrification of oligotrophic low-temperature water.
The high-efficiency low-temperature resistant denitrifying bacterium Janthinobacterium sp.M-11 screened from the filler in the Chinese-style reactor for treating the water in the Songhua river is identified as gram-negative bacterium and facultative anaerobe, the growth temperature is 2-35 ℃, the optimum temperature is 25-30 ℃, the length of the bacterium is 1-1.5 mu m, the width is 0.5-1 mu m, and the bacterium is short rod-shaped and has no spores. The colony is white, semitransparent, regular and round with the radius of 1-2mm, and is moist and glossy.
DNA extraction of Janthinobacterium sp.M-11 was performed by CTAB method. The 16S rDNA gene is amplified by PCR, and universal primers of the 16S rDNA gene are adopted. And purifying the PCR amplification product and then directly performing bidirectional sequencing. The sequence is input into GenBank, and is compared and analyzed with a database sequence by Blast software, and the result shows that the sequence has higher similarity with the 16SrDNA sequence of Janthinobacterium sp and is purple bacillus. The bacterium, purple bacillus (Janthinobacterium), was a strain based on phylogenetic results analysis of the 16SrDNA gene and physiological and biochemical characteristics. Finally named as purple bacillus (Janthinobacterium sp.) M-11.
The invention adopts the method for treating the Songhua river waterThe high-efficiency low-temperature resistant denitrifying bacterium Janthinobacter sp.M-11 screened by the filler in the Chinese reactor can react with NH at the temperature of 2 DEG C 4 + -N,NO 2 - -N and NO 3 - And respectively carrying out denitrification treatment on the eutrophic water body with N as the unique nitrogen source. The initial N concentration is about 5mg/L, and the inoculation concentration is 4X 10 6 cfu/ml, initial OD 600 About 0.03, the oligotrophic low-temperature denitrifying bacteria Janthinobacterium sp.M-11 can grow at 2 ℃, the ammonia nitrogen removal rate reaches over 90 percent in 48 hours, and no nitrite nitrogen and nitrate nitrogen are accumulated; nitrite and nitrate are removed at 2 ℃, the removal rate reaches more than 80%, and nitrite accumulation has no inhibiting effect on the bacteria, which shows that the oligotrophic low-temperature denitrifying bacteria Janthinobacterium sp.M-11 has smaller inoculation amount compared with other strains, and is more suitable for the treatment of oligotrophic low-temperature water.
Drawings
FIG. 1 is a graph of the concentration of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and bacteria in the culture medium as a function of time; wherein a is ammonia nitrogen concentration, b is total nitrogen concentration, c is nitrite nitrogen concentration, d is nitrate nitrogen concentration, and e is bacteria concentration;
FIG. 2 is a graph of nitrate nitrogen and nitrite in a denitrification medium with sodium nitrite as the sole nitrogen source as a function of time; wherein f is nitrate and g is nitrite;
FIG. 3 is a graph showing the change with time of nitrate nitrogen and nitrite in a denitrification medium using sodium nitrate as the sole nitrogen source; wherein f is nitrate and g is nitrite;
FIG. 4 shows the ammonia nitrogen concentration in the nitrification medium at various pH values.
Detailed Description
The first embodiment is as follows: the oligotrophic low-temperature denitrifier is purple bacillus (Janthinobacterium sp.) M-11, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation address of No. 3 Xilu No.1 Beijing of Chaozhou, facing the sunny region, the preservation date of 2017, 07 and 03 days, and has the preservation number of CGMCC No. 14380.
The high-efficiency low-temperature resistant denitrifying bacterium Janthinobacterium sp.M-11 screened from the filler in the Chinese-style reactor for treating the Songhua river water is identified as gram-negative bacteria and facultative anaerobe, the growth temperature is 2-35 ℃, the optimal temperature is 25-30 ℃, the length of the strain is 1-1.5 mu m, the width is 0.5-1 mu m, and the strain is in a short rod shape and has no spores. The bacterial colony is in a white semitransparent regular round shape with the radius of 1-2mm, and is moist and glossy.
DNA extraction of Janthinobacterium sp.M-11 was performed by CTAB method. The 16S rDNA gene is amplified by PCR, and universal primers of the 16S rDNA gene are adopted. And purifying the PCR amplification product and then directly performing bidirectional sequencing. The sequence is input into GenBank, and Blast software is used for comparing and analyzing with a database sequence, and the result shows that the sequence has higher similarity with the 16SrDNA sequence of Janthinobacterium sp and is purple bacillus. The bacterium, purple bacillus (Janthinobacterium), was a strain based on phylogenetic results analysis of the 16SrDNA gene and physiological and biochemical characteristics. Finally named as purple bacillus (Janthinobacterium sp.) M-11.
The embodiment adopts the high-efficiency low-temperature resistant denitrifying bacterium Janthinobacter sp.M-11 screened by the filler in the Chinese-style reactor for treating the water of the Songhua river, and the strain can treat the water with NH at the temperature of 2 DEG C 4 + -N,NO 2 - -N and NO 3 - And respectively carrying out denitrification treatment on eutrophic water bodies with N as a unique nitrogen source.
The second embodiment is as follows: the application of the oligotrophic low-temperature denitrifying bacteria in the embodiment refers to the application of the oligotrophic low-temperature denitrifying bacteria in denitrification of oligotrophic low-temperature water bodies.
The low-temperature denitrification bacterium Janthinobacterium sp.M-11 with poor nutrition can grow at 2 ℃, the removal rate of ammonia nitrogen reaches more than 90% in 48 hours, and no nitrite nitrogen and nitrate nitrogen are accumulated; nitrite and nitrate are removed at 2 ℃, the removal rate reaches more than 80%, and nitrite accumulation has no inhibiting effect on the bacteria, which shows that the oligotrophic low-temperature denitrifying bacteria Janthinobacterium sp.M-11 has smaller inoculation amount compared with other strains, and is more suitable for the treatment of oligotrophic low-temperature water.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: low-temperature water for poor nutritionThe dosage of the low-temperature denitrogenation bacteria in the body is 4 multiplied by 10 6 cfu/mL. The rest is the same as the second embodiment.
The fourth concrete implementation mode is as follows: the screening method of the oligotrophic low-temperature denitrifier Janthinobacterium sp.M-11 in the embodiment comprises the following steps: and taking the filler in the reactor which runs stably, shaking and centrifuging to obtain supernatant. Separation was carried out by dilution plate coating, starting with NH at 2 ℃ 4 + N concentration 5mg/L, NH of the isolated strain in culture medium 4 + N removal experiments for NH 4 + The strain with the highest N removal rate is the required low-temperature denitrification strain. The specific strain screening step is to obtain 10g of filler from a reactor which operates stably. Placing the mixture into a 50mL centrifuge tube, adding 30mL sterile water, and shaking uniformly to uniformly disperse the bacteria into the sterile water. Then, 1mL of the suspension was transferred to a test tube containing 9mL of sterile water to give a concentration of 10 1 Gradient bacterial suspension, repeating the steps to obtain the strain concentration of 10 2 、10 3 …10 7 A gradient of bacterial suspension. And then, adopting a plate coating method, taking 100 mu L of bacterial suspension from each gradient, putting the bacterial suspension into a plate culture medium, carrying out low-temperature culture at 2 ℃, respectively picking typical colonies with different forms and clear colonies, carrying out streak separation on the plate culture medium, and repeating for 3-4 times until a single colony with consistent colony characteristics appears. All the above operations are carried out under sterile conditions.
The components of the plate culture medium are as follows (g/L): sodium acetate 1, ammonium chloride 1, disodium hydrogen phosphate 0.2, sodium dihydrogen phosphate 0.2, agar 18 and 2mL of trace element solution. Microelement solution composition (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
Respectively adding 20 strains obtained by primary screening into an enrichment medium for enrichment culture, wherein the enrichment medium comprises (g/L) ammonium chloride 0.4, sodium acetate 1, disodium hydrogen phosphate 0.2, potassium chloride 0.1 and 2mL trace element solution; microelement solution composition (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
Centrifuging the enriched bacterial liquid, washing with PBS solution for three times, and respectively adding into a nitrification culture medium, wherein the components of the nitrification culture medium are (g/L): 0.02 part of ammonium chloride, 0.09 part of sodium acetate, 0.02 part of disodium hydrogen phosphate, 0.01 part of potassium chloride and 1mL of trace element solution; microelement solution composition (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
And culturing the inoculated nitrification culture medium at the temperature of 2 ℃, measuring the ammonia nitrogen removal efficiency, and identifying the strain with the highest ammonia nitrogen removal efficiency. The main biological characteristics of the bacterial strain are gram-negative bacteria, the bacterial strain is in a short rod shape, and the bacterial colony is in a regular milky semitransparent circular shape. The DNA extraction was performed by CTAB method. The 16S rDNA gene is amplified by PCR, and universal primers of the 16S rDNA gene are adopted. And purifying the PCR amplification product and then directly performing bidirectional sequencing. The sequence is input into GenBank, and Blast software is used for comparing and analyzing with a database sequence, and the result shows that the sequence has higher similarity with the 16SrDNA sequence of Janthinobacterium sp and is purple bacillus. The bacterium, purple bacillus (Janthinobacterium), was a strain based on phylogenetic results analysis of the 16SrDNA gene and physiological and biochemical characteristics. Finally, it was named as purple bacterium (Janthinobacterium sp.) M-11.
The functionality of the oligotrophic low-temperature denitrifier Janthinobacterium sp.M-11 in the denitrification of the oligotrophic low-temperature water body is verified as follows:
Culturing the low-temperature denitrifying bacterium Janthinobacterium sp.M-11 in an enrichment medium to logarithmic growth phase, inoculating in a nitrification medium at 2 ℃, and performing OD (origin-destination) inoculation 600 Aerobic culture was carried out at a rotation speed of about 0.03 and 150rpm, and ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and bacterial concentration in the culture medium were measured as a function of time, and the results are shown in FIG. 1.
The detection result in figure 1 shows that the bacteria can grow at 2 ℃, the removal rate reaches over 90% in 48 hours, and no nitrite nitrogen and nitrate nitrogen are accumulated in the process of reducing ammonia nitrogen, which indicates that the bacteria can directly convert ammonia nitrogen into gases such as nitrogen and the like.
Culturing the low-temperature denitrifying bacterium Janthinobacterium sp.M-11 in an enrichment medium to logarithmic growth phase, respectively inoculating in a denitrification medium with sodium nitrite and sodium nitrate as unique nitrogen sources, and inoculating to OD 600 Aerobic culture was performed at a rotation speed of 150rpm at approximately 0.03, and the nitrate nitrogen and nitrite in the culture medium were measured as a function of time, respectively, and the results are shown in FIGS. 2 and 3.
The results in fig. 2 and 3 show that the bacteria can remove nitrite and nitrate at 2 ℃, the removal rate reaches more than 80%, and the accumulation of nitrite has no inhibition effect on the bacteria, which indicates that the bacteria has certain tolerance to nitrite.
The denitrification culture medium comprises the following components in percentage by weight (g/L): 0.03 percent of sodium nitrate or 0.025 percent of sodium nitrite, 0.09 percent of sodium acetate, 0.02 percent of disodium hydrogen phosphate, 0.01 percent of potassium chloride and 1mL of trace element solution. Microelement solution composition (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
Test 3 growth of Janthinobacterium sp.M-11 at a suitable pH
Culturing the low-temperature denitrifying bacterium Janthinobacterium sp.M-11 in an enrichment medium to logarithmic phase, respectively inoculating into a nitrification medium with pH of 5-10 at 2 deg.C, and inoculating to OD 600 Approximately 0.03, aerobic culture was carried out at 150rpm, and the ammonia nitrogen concentration in the nitrified medium was measured after 24 hours. The measured value is the ammonia nitrogen concentration of the supernatant after the bacteria are centrifuged, and the result is shown in figure 4.
The results in fig. 4 show that the cold-resistant bacteria showed the highest removal rate of ammonia nitrogen (76.33%) at pH 7, the acidic condition could significantly inhibit the low-temperature denitrification effect of the bacteria, and the alkaline condition could inhibit the low-temperature denitrification effect of the bacteria to some extent, but the bacteria still expressed a certain denitrification capability under the alkaline condition compared to the acidic condition.
The enrichment medium in tests 1-3 comprises (g/L) ammonium chloride 0.4, sodium acetate 1, disodium hydrogen phosphate 0.2, potassium chloride 0.1 and 2mL trace element solution; microelement solution ingredient (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
The components of the nitrification culture medium are (g/L): 0.02 part of ammonium chloride, 0.09 part of sodium acetate, 0.02 part of disodium hydrogen phosphate, 0.01 part of potassium chloride and 1mL of trace element solution; microelement solution composition (g/L): 0.5 part of manganese chloride, 0.5 part of ferrous sulfate, 1 part of magnesium sulfate, 1 part of calcium chloride, 0.5 part of zinc sulfate and 0.2 part of cobalt chloride.
The experiments show that the oligotrophic low-temperature denitrifying bacteria Janthinobacterium sp.M-11 have smaller inoculation amount than other strains, and are more suitable for treating oligotrophic low-temperature water.
Sequence listing
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<120> a strain of oligotrophic low-temperature denitrogenation bacteria and application thereof
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Claims (3)
1. A strain of low-temperature denitrification bacterium with poor nutrition is characterized in that the strain is purple bacillus (purple bacillus)Janthinobacteriumsp.) M-11, wherein the culture is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 of West Lu No.1 of the morning-Yang district of Beijing, the preservation date is 2017, 07 and 03 days, and the preservation number is CGMCC number 14380.
2. The use of the strain M-11 according to claim 1 for denitrification of low temperature water with poor nutrient content.
3. Use according to claim 2, characterized in that the strain M-11 is dosed in an amount of 4X 10 6 cfu/mL。
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