CN110616175A - Ammonia oxidizing archaea and fresh water pond ammonia oxidizing archaea enrichment culture method - Google Patents
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Abstract
The invention provides an enrichment culture method of ammonia oxidizing archaea in a fresh water pond, which comprises the steps of enrichment culture and separation and purification; the ammonia oxidizing archaea is extracted from a fresh water pond, urea in an ammonia oxidizing archaea enrichment culture medium is used as a nitrogen source in the enrichment culture process, the ammonia oxidizing archaea cannot volatilize in the heating process, is stable in state in an aqueous solution, and still can keep an initial concentration value after high-temperature treatment, so that the ammonia oxidizing archaea can be directly sterilized at high temperature, and the probability of mixed bacteria pollution in the culture process is reduced; in the enrichment culture process, calcium carbonate in the enrichment culture medium of the ammonia oxidizing archaea is used as an attachment substrate, so that the relative ratio of the calcium carbonate to the attachment substrate is large, the ammonia oxidizing archaea can be attached and grown, the microbial loss in the culture medium replacement process is reduced, and the pH value of the culture medium can be buffered and stabilized to be about 7.0, so that the stable growth of the ammonia oxidizing archaea is facilitated; in addition, calcium carbonate can also provide an inorganic carbon source, namely carbonate ions, for living bodies to use.
Description
Technical Field
The invention belongs to the technical field of aquaculture environment microorganisms, and particularly relates to an ammonia oxidation archaea and fresh water pond ammonia oxidation archaea enrichment culture method.
Background
The oxidation of ammonia nitrogen has been thought to be performed entirely by ammonia oxidizing bacteria over the past 100 years, but the discovery of ammonia oxidizing archaea has revolutionized this recognition in the last 10 years. Archaea is also called archaea, archaea or pacific and archaeon, is unicellular microorganism, is a domain of another biological classification completely different from bacteria, or a kingdom. They share many similarities with bacteria, none of which have nuclei associated with any other membrane bound organelles, while other features are similar to those of eukaryotes, such as the presence of repetitive sequences and nucleosomes. Since the archaea has strict requirements on the laboratory culture conditions, few archaea strains and cultures are obtained in the world at present. The fresh water pond can provide more than 50% of aquatic products for China every year, and the ammonia oxidizing archaea plays an important role in the transformation of ammonia nitrogen in the pond environment. Therefore, the enrichment and separation of the ammonia oxidizing archaea in the pond environment have important significance.
Publication No. CN 104593304A discloses a method for rapidly enriching and culturing marine ammonia-oxidizing archaea, namely, ammonia-oxidizing archaea is obtained from a marine water body, and tricarboxylic acid cycle metabolites are required to be added in the culture process. Publication No. CN109652568A discloses a method for rapidly enriching Nitrosocosmius ammonia oxidizing archaea from the environment, namely obtaining ammonia oxidizing archaea from a soil habitat, and the culturing process does not need to add any organic substances. This indicates that the life habits of ammonia oxidizing archaea in different habitats are far different. The fresh water pond is completely different from an ecological system of sea and soil, is greatly interfered by human factors, receives a large amount of feed every day in a culture season, has the concentration of organic matters and ammonia nitrogen in water and sediments far higher than that of the sea, is strongly interfered by illumination, and is possibly inapplicable to the culture of the ammonia oxidizing archaea in the habitat of the pond by the existing culture method of the sea and the soil habitat at present.
In addition, ammonium chloride is often used as a nitrogen source of a culture medium in the existing culture method of ammonia oxidizing archaea, and NH is used as ammonium salt in a solution3-N and NH4 +The proportion of N is reduced with the reduction of the pH of the solution and increased with the increase of the pH, and NH with higher concentration3-N and very low concentration of NH3N is not beneficial to the growth of ammonia oxidizing archaea. During the cultivation process, the pH of the culture medium tends to decrease with time due to the continuous proton consumption, which in turn leads to NH in the culture medium solution3Uncertainty of N concentration, unstable growth rate of ammonia oxidizing archaea.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an enrichment culture method for ammonia-oxidizing archaea in a fresh water pond.
In order to achieve the above purpose, the solution of the invention is as follows:
an ammonia oxidation archaea enrichment culture method for a fresh water pond comprises the following steps:
(1) inoculating a sample in an ammonia oxidizing archaea enrichment culture medium, and carrying out enrichment culture in a first stage at 36 +/-1 ℃;
(2) when the urea in the archaea ammonia oxidation enrichment culture medium in the step (1) is exhausted, putting 100 +/-10 mu L of archaea ammonia oxidation enrichment culture into a fresh archaea ammonia oxidation enrichment culture medium, and respectively adding 50 +/-1 mg/L of penicillin and streptomycin to carry out enrichment culture of a second stage;
(3) when the urea in the ammonia oxidizing archaea enrichment culture medium in the step (2) is exhausted, pouring out the supernatant, retaining the calcium carbonate at the bottom, then supplementing a fresh sterile ammonia oxidizing archaea enrichment culture medium to the inside, respectively adding 50 +/-1 mg/L of penicillin and streptomycin, and standing and culturing at 36 +/-1 ℃;
(4) and (5) repeating the operation step (3) for 5-6 times, extracting the total DNA of the microorganism in the ammonia oxidizing archaea enrichment culture medium, carrying out PCR amplification on 16S rDNA, carrying out deep sequencing on the PCR amplified product, and identifying the microbial diversity in the sample.
Wherein the enrichment medium of ammonia oxidizing archaea is as follows: each L of enrichment medium contained 0.2. + -. 0.01mmol of CO (NH)2)2、1±0.1mmol KH2PO4、1±0.1mmol KCl、0.2±0.01mmol MgSO4、10±1mmol NaCl、40±1mmol CaCO31 plus or minus 0.1mL of trace elements and 1 plus or minus 0.1mL of selenium-tungsten solution;
the trace elements are: each L of the solution contains 0.2 plus or minus 0.01mmol of MnSO4、1±0.1mmol H3BO3、0.5±0.01mmol ZnCl2、0.3±0.01mmol Na2MoO4·2H2O、0.1±0.01mmol CuCl2、0.1±0.01mmol NiCl2、0.5±0.01mmol CoCl2、3.5±0.01mmol FeSO4·7H2O and 2.5mL 37% HCl;
the selenium tungsten solution is as follows: each L of the solution contained 12.5. + -. 0.01mmol NaOH, 10. + -. 1. mu. mol Na2SeO3And 10. + -. 1. mu. mol of Na2WO4。
Preferably, in step (1), the sample is a freshwater pond sediment.
Preferably, the specific surface area of calcium carbonate in the ammonia oxidizing archaea enrichment medium is 1 +/-0.1 m2(iii) a particle diameter of 0.5 to 3 μm/g.
Preferably, in step (4), the primers for PCR amplification of 16S rDNA are:
338F：ACTCCTACGGGAGGCAGCAG；
806R：GGACTACHVGGGTWTCTAAT。
an ammonia oxidizing archaea obtained by the enrichment culture method of the ammonia oxidizing archaea.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the ammonia oxidation archaea is extracted from a fresh water pond, urea in an ammonia oxidation archaea enrichment culture medium is used as a nitrogen source in the enrichment culture process, the ammonia oxidation archaea cannot volatilize in the heating process, is stable in state in a water solution, and still can keep an initial concentration value after high-temperature treatment, so that the ammonia oxidation archaea can be directly sterilized at high temperature, the probability of mixed bacteria pollution in the culture process is reduced, and the operation is simple, convenient and practical.
Secondly, calcium carbonate in the enrichment culture medium of the ammonia oxidation archaea is used as an attachment substrate in the enrichment culture process, the surface area is relatively large, the ammonia oxidation archaea can be attached and grow, the microbial loss in the culture medium replacement process is reduced, the pH value of the culture medium can be buffered and stabilized to be about 7.0, and therefore the ammonia oxidation archaea can be stably grown; in addition, calcium carbonate can also provide carbonate ions in an inorganic carbon source for living bodies to use.
Thirdly, in the enrichment culture process, penicillin and streptomycin can inhibit and kill most of mixed bacteria in the culture medium, but the growth of the ammonia-oxidizing archaea is not inhibited, so that the purity of the ammonia-oxidizing archaea reaches more than 85 percent.
Fourthly, the enrichment culture process of the invention adopts a gradient dilution method, which not only can shorten the enrichment and screening period, but also can improve the abundance of the ammonia-oxidizing archaea, and after the enrichment culture, the method can be used for controlling the ammonia nitrogen concentration of the aquaculture water body and has great application value.
Drawings
FIG. 1 is a graph showing the results of the ammonia oxidizing archaea of the present invention at the species level.
FIG. 2 is a diagram showing the results of the genus-level ammonia oxidizing archaea of the present invention.
FIG. 3 is a graph showing the results of the ammonia oxidizing archaea of the present invention at the phylum level.
Detailed Description
The invention provides an enrichment culture method for ammonia oxidizing archaea in a fresh water pond.
The enrichment culture method of ammonia oxidizing archaea in the fresh water pond comprises the following steps:
(1) preparing an ammonia oxidizing archaea enrichment culture medium, weighing 100mL of the culture medium in a blue-covered bottle (with the volume of 100mL), sterilizing at 121 ℃, continuing for 15min, cooling, inoculating 0.5g of pond sediments, and standing and culturing for 1 month in an incubator at 36 +/-1 ℃;
(2) and detecting water quality indexes including the concentrations of nitrite and nitrate in the ammonia oxidizing archaea enrichment culture medium every 7 days during the culture period. Through calculation, when the urea in the archaea ammonia oxidizing bacteria enrichment culture medium in the step (1) is exhausted, shaking and uniformly mixing the culture medium, putting 100 +/-10 mu L of archaea ammonia oxidizing bacteria enrichment culture into a fresh archaea ammonia oxidizing bacteria enrichment culture medium, and respectively adding 50 +/-1 mg/L of penicillin and streptomycin to carry out enrichment culture of a second stage;
(3) when the urea in the ammonia oxidizing archaea enrichment culture medium in the step (2) is exhausted, pouring out the supernatant, retaining the calcium carbonate at the bottom, then supplementing a fresh sterile ammonia oxidizing archaea enrichment culture medium to the inside, respectively adding 50 +/-1 mg/L of penicillin and streptomycin, and continuously standing and culturing at 36 +/-1 ℃;
(4) and repeating the operation step (3) for 5-6 times, extracting the total DNA of the microorganisms in the ammonia oxidizing archaea enrichment medium, and performing PCR amplification on the 16S rDNA by using primers (338F: ACTCCTACGGGAGGCAGCAG; 806R: GGACTACHVGGGTWTCTAAT) to identify the microbial diversity in the sample. The abundance of the ammonia oxidizing archaea in each mL of culture medium (calcium carbonate suspension) can reach 10 within about 4 months7Above, the purity (i.e. abundance) of the aminoxyarchaea strain can reach more than 85%, and the archaea can account for more than 93% of the total microorganism number.
Wherein in the step (1), the enrichment medium of ammonia oxidizing archaea is: each L of enrichment medium contained 0.2. + -. 0.01mmol of CO (NH)2)2、1±0.1mmol KH2PO4、1±0.1mmol KCl、0.2±0.01mmol MgSO4、10±1mmol NaCl、40±1mmol CaCO31 plus or minus 0.1mL of trace elements and 1 plus or minus 0.1mL of selenium-tungsten solution.
The trace elements are: each L of the solution contains 0.2 plus or minus 0.01mmol of MnSO4、1±0.1mmol H3BO3、0.5±0.01mmol ZnCl2、0.3±0.01mmol Na2MoO4·2H2O、0.1±0.01mmol CuCl2、0.1±0.01mmol NiCl2、0.5±0.01mmol CoCl2、3.5±0.01mmol FeSO4·7H2O and 2.5mL 37% HCl.
The selenium tungsten solution is as follows: each L of the solution contained 12.5. + -. 0.01mmol NaOH, 10. + -. 1. mu. mol Na2SeO3And 10. + -. 1. mu. mol of Na2WO4。
In the step (2), ammonia oxidizing archaea, ammonia oxidizing bacteria and nitrite oxidizing bacteria exist simultaneously in the early enrichment culture process, and the ammonia oxidizing bacteria can convert most of nitrogen (CO (NH) in urea2)2-N) to nitrite Nitrogen (NO)2 --N), nitrite oxidizing bacteria will convert most of the NO2 -Conversion of-N to nitro-Nitrogen (NO)3 --N). Determination of NO2 --N and NO3 -N concentration, according to the law of conservation of the elements, by NO2 --N and NO3 -The sum of-N is reasonably feasible to judge the consumption of nitrogen in urea.
①CO(NH2)2-N→NO2 --N→NO3 --N
In step (3), the added penicillin and streptomycin can inhibit and kill ammonia oxidizing bacteria and nitrite oxidizing bacteria, and the nitrogen in the added urea is finally converted into NO under the action of ammonia oxidizing archaea2 -And N, judging whether the urea is exhausted or not according to the element conservation law through the generated nitrous nitrogen amount.
②CO(NH2)2-N→NO2 --N
In the step (3), the calcium carbonate is heavy calcium carbonate with the specific surface area of 1 +/-0.1 m2About/g, the grain diameter is 0.5-3 μm, and calcium carbonate is used as an attachment carrier of the ammonia oxidizing archaea, has larger attachment surface area, not only allows the ammonia oxidizing archaea to attach and grow, but also reduces the loss of microorganisms in the process of replacing the culture medium.
In addition, in the enrichment culture process, proton generation is accompanied, the solution tends to be weakly acidic, but in the presence of calcium carbonate, bicarbonate ions dissociated from the calcium carbonate ions are combined with part of protons, so that the culture medium is always kept near 7.0, and stable growth of the ammonia oxidizing archaea is facilitated.
The present invention will be further described with reference to the following examples.
Example (b):
the sediment used in this example was obtained from a fresh water pond at the ecological engineering research center of the institute of aquatic science, china, and after sampling, was stored at 4 ℃ and quickly transported back to the laboratory.
Specifically, the method for enriching and culturing ammonia-oxidizing archaea in the fresh water pond comprises the following steps:
(1) preparing an ammonia oxidizing archaea enrichment culture medium: each L of enrichment medium contained 0.2mmol of CO (NH)2)2、1mmol KH2PO4、1mmol KCl、0.2mmol MgSO4、10mmol NaCl、40mmol CaCO31mL of trace element and 1mL of selenium tungsten solution.
Composition of trace elements: each L of the solution contains 0.2mmol of MnSO4、1mmol H3BO3、0.5mmol ZnCl2、0.3mmol Na2MoO4·2H2O、0.1mmol CuCl2、0.1mmol NiCl2、0.5mmol CoCl2、3.5mmol FeSO4·7H2O and 2.5mL 37% HCl.
The selenium tungsten solution comprises the following components: each L of the solution contained 12.5mmol of NaOH and 10. mu. mol of Na2SeO3、10μmol Na2WO4。
(2) Weighing 100mL of the culture medium in a blue-cap bottle (volume 100mL), sterilizing at 121 ℃ for 15min, cooling, inoculating 0.5g of sediment, and standing and culturing in an incubator at 36 ℃ for 1 month;
(3) and detecting water quality indexes including the concentrations of nitrite and nitrate in the ammonia oxidizing archaea enrichment culture medium every 7 days during the culture period. Through calculation, when the urea in the archaea ammonia oxidizing bacteria enrichment culture medium in the step (2) is exhausted, shaking and uniformly mixing the culture medium, putting 100 mu L of archaea ammonia oxidizing bacteria enrichment culture into a fresh archaea ammonia oxidizing bacteria enrichment culture medium, and respectively adding 50mg/L of penicillin and streptomycin to carry out enrichment culture of a second stage;
(4) when the urea in the ammonia oxidizing archaea enrichment culture medium in the step (3) is exhausted, pouring out the supernatant, retaining the calcium carbonate at the bottom, then supplementing a fresh sterile ammonia oxidizing archaea enrichment culture medium to the inside, respectively adding 50mg/L of penicillin and streptomycin, and standing and culturing continuously at 36 ℃;
(5) and repeating the operation step (4) for 5-6 times, extracting the total DNA of the microorganism in the enrichment medium of the ammonia oxidizing archaea, and carrying out PCR amplification on the 16S rDNA by using primers (338F: ACTCCTACGGGAGGCAGCAG; 806R: GGACTACHVGGGTWTCTAAT). Sending the PCR product to Mergiz biological medicine science and technology Limited company, then carrying out deep sequencing on the PCR product, and identifying the microbial diversity in the enrichment culture (namely the sediment).
The microbial diversity composition of the enriched culture diversity at the species, genus and phylum level is shown in FIGS. 1 to 3, which are cultured by the above method and subjected to high throughput sequencing. As shown in FIG. 1, the abundance of the ammonia oxidizing archaea Unsilaged _ g _ Candiadat us _ Nitrosotenuus can reach 86.09% at the seed level. As shown in fig. 2, at the genus level, the abundance of Candidatus Nitrosotenuis can reach 86.09%. As shown in FIG. 3, the abundance of ammonia-oxidizing archaea in the phylum chiasmata (Thaumarchaeota) was 93.11%.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (5)
1. An ammonia oxidation archaea enrichment culture method for a fresh water pond is characterized by comprising the following steps: which comprises the following steps:
(1) inoculating a sample in an ammonia oxidizing archaea enrichment culture medium, and carrying out enrichment culture in a first stage at 36 +/-1 ℃;
(2) when the urea in the archaea ammonia oxidizing bacteria enrichment culture medium in the step (1) is exhausted, putting 100 +/-10 mu L of archaea oxidizing bacteria enrichment culture into a fresh archaea oxidizing bacteria enrichment culture medium, and respectively adding 50 +/-1 mg/L of penicillin and streptomycin to carry out enrichment culture of a second stage;
(3) when the urea in the ammonia oxidation archaea enrichment culture medium in the step (2) is exhausted, pouring out the supernatant, retaining the calcium carbonate at the bottom, then supplementing a fresh ammonia oxidation archaea enrichment culture medium, respectively adding 50 +/-1 mg/L of penicillin and streptomycin, and standing and culturing at 36 +/-1 ℃;
(4) repeating the step (3) for 5-6 times, extracting the total DNA of the microorganisms in the ammonia oxidizing archaea enrichment culture medium, performing PCR amplification on the 16S rDNA, and identifying the microbial diversity in the sample;
the ammonia oxidizing archaea enrichment medium comprises: each L of enrichment medium contains 0.2 + -0.01 mmol CO (NH)2)2、1±0.1mmol KH2PO4、1±0.1mmol KCl、0.2±0.01mmol MgSO4、10±1mmol NaCl、40±1mmol CaCO31 plus or minus 0.1mL of trace elements and 1 plus or minus 0.1mL of selenium-tungsten solution;
the trace elements are: each L of the solution contains 0.2 +/-0.01 mmol of MnSO4、1±0.1mmolH3BO3、0.5±0.01mmol ZnCl2、0.3±0.01mmolNa2MoO4·2H2O、0.1±0.01mmolCuCl2、0.1±0.01mmol NiCl2、0.5±0.01mmol CoCl2、3.5±0.01mmol FeSO4·7H2O and 2.5mL 37% HCl;
the selenium tungsten solution is as follows: each L of the solution contained 12.5. + -. 0.01mmol NaOH, 10. + -. 1. mu. mol Na2SeO3And 10. + -. 1. mu. mol of Na2WO4。
2. The method for enriching and culturing ammonia-oxidizing archaea in a freshwater pond according to claim 1, wherein the method comprises the following steps: in the step (1), the sample is sediment of a fresh water pond.
3. The method for enriching and culturing ammonia-oxidizing archaea in a freshwater pond according to claim 1, wherein the method comprises the following steps: the specific surface area of calcium carbonate in the ammonia oxidizing archaea enrichment medium is 1 +/-0.1 m2(iii) a particle diameter of 0.5 to 3 μm/g.
4. The method for enriching and culturing ammonia-oxidizing archaea in a freshwater pond according to claim 1, wherein the method comprises the following steps: in the step (4), the primers for PCR amplification of 16S rDNA are as follows:
338F：ACTCCTACGGGAGGCAGCAG；
806R：GGACTACHVGGGTWTCTAAT。
5. an ammonia oxidizing archaea characterized by: the method for enriching and culturing ammonia-oxidizing archaea according to any one of claims 1 to 4.
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| CN113736703B (en) * | 2021-09-14 | 2023-08-18 | 华南理工大学 | Culture medium and application thereof in separation and purification culture of ammonia oxidizing bacteria |
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