CN116769671A - Method for enriching ammonia oxidizing microorganisms and ammonia oxidizing microorganism enriching product - Google Patents
Method for enriching ammonia oxidizing microorganisms and ammonia oxidizing microorganism enriching product Download PDFInfo
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Abstract
The application belongs to the technical field of environmental microorganisms, and in particular relates to an enrichment method of ammonia oxidizing microorganisms and an enrichment ammonia oxidizing microorganism product, wherein the method comprises the following steps: and (3) an inhibitor culturing step: adding an inhibitor and ammonia nitrogen into the basic culture medium, inoculating a sample containing the target ammonia oxidizing microorganism to form a culture system, performing incubation culture treatment, and inhibiting the growth of other ammonia oxidizing microorganisms except the target ammonia oxidizing microorganism by using the inhibitor. The scheme of the application utilizes the inhibitor to inhibit the growth of other ammonia oxidizing microorganisms except the required ammonia oxidizing microorganisms, observes the change of the relative abundance of each ammonia oxidizing microorganism, and can utilize the inhibitor to enrich the specific full ammonia oxidizing microorganisms with high abundance; in particular, 1-octyne is firstly proposed as an inhibitor, and the growth of AOB and AOA is effectively inhibited to enrich the complete ammonia oxidizing bacteria.
Description
Technical Field
The application belongs to the technical field of environmental microorganisms, and particularly relates to a method for enriching ammonia oxidizing microorganisms and an ammonia oxidizing microorganism enriched product.
Background
As an essential important element for composing life body nucleic acid and protein, researching the circulating mode and the circulating characteristic of nitrogen has important significance for maintaining ecological balance of nature and removing nitrogen-containing pollutants of sewage plants. The nitration reaction is generally considered to require two steps to complete as the rate limiting step in nitrogen recycle. The first step is to convert ammonia nitrogen into nitrite Nitrogen (NH) by metabolism of ammonia oxidizing bacteria (ammonia-oxidizing bacteria, AOB) or/and ammonia oxidizing archaea (ammonia-oxidizing archaea, AOA) in the presence of dissolved oxygen 3 /NH 4 + →NO 2 - ). The second step is to convert the nitrite produced in the first step into nitrate nitrogen by the action of Nitrite Oxidizing Bacteria (NOB). Since the successful isolation of ammonia oxidizing bacteria at the end of the 19 th century, AOB has been considered by researchers as the only microorganism responsible for ammonia oxidation. However, in 2005, ammonia oxidizing archaea was successfully found to have ammonia oxidizing effect as well, and the conventional knowledge of ammonia oxidizing microorganisms, which are generally referred to as AOB and AOA, was revolutionized. Until 2015, two European research topic groups discovered and enriched a novel nitrifying microorganism, namely complete ammonia oxidizing bacteria (Complete ammonia oxidation Bacteria, CMX), almost simultaneously, and can independently complete the nitrifying reaction which is completed by the cooperation of AOM and NOB in the traditional concept, and the discovery of CMX revolutionarily changes the traditional understanding of the two-step nitrifying theory by researchers, so that the research on the ecological characteristics of the novel nitrifying microorganism becomes a research hotspot in the nitrogen recycling field. Related researches are carried out by collecting environmental samples such as wetland sediments, soil, estuaries, sewage treatment plants and the like, and byThe enrichment culture method of related nitrifying microorganisms and a series of bioinformatics technologies are applied to reveal the various community compositions of the nitrifying microorganisms.
Although the discovery of ammonia oxidizing microorganisms has been a century old today, we have not yet had a deep understanding of it, especially CMX discovery, which further subverts our knowledge of the traditional nitrification concept. However, the high-abundance ammonia oxidizing microorganism strain has great significance for the follow-up continuous promotion of related nitrogen microorganism driving mechanisms and the deep discussion of nitrification. To this end, in order to better promote the ammonia oxidizing microorganisms to be widely applied to the actual process, the application provides a method for enriching ammonia oxidizing microorganisms, which can enrich ammonia oxidizing microorganisms with high abundance from the environment.
Disclosure of Invention
The application aims to provide a method for enriching ammonia oxidizing microorganisms and an ammonia oxidizing microorganism enriching product, which can enrich ammonia oxidizing microorganisms with high abundance from the environment.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect, the present application provides a method of enriching for ammonia oxidizing microorganisms, comprising:
and (3) an inhibitor culturing step: adding an inhibitor and ammonia nitrogen into the basic culture medium, inoculating a sample containing the target ammonia oxidizing microorganism to form a culture system, performing incubation culture treatment, and inhibiting the growth of other ammonia oxidizing microorganisms except the target ammonia oxidizing microorganism by using the inhibitor.
In the above method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the target ammonia oxidizing microorganism is a complete ammonia oxidizing bacteria.
In the above method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the inhibitor is 1-octyne. The inventor of the application discovers that 1-octyne has good inhibition effect on ammonia oxidizing microorganisms AOB and AOA, and complete ammonia oxidizing bacteria CMX has anti-octyne capability, and the 1-octyne can be used as an inhibitor to realize high-efficiency inhibition on the AOB and the AOA, thereby providing favorable conditions for enrichment of CMX. More preferably, 1-octyne is added at a concentration of 50-400mg/L (e.g., 80mg/L, 100mg/L, 125mg/L, 150mg/L, 180mg/L, 200mg/L, 250mg/L, 300mg/L, 350mg/L, etc.).
In the above method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, in the inhibitor culturing step, ammonia nitrogen (NH 4 + The addition amount of the-N) is 5-10mg/L, namely 5-10mg of ammonia nitrogen is added into each liter of culture medium; the ammonia nitrogen is applied at a constant rate of 0.1-0.25mg/L per hour, such as 0.1136-0.2272mg/L per hour.
In the method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the basic culture medium comprises the following formula: 50mg/L KH 2 PO 4 ，75mg/L KCl，50mg/L MgSO 4 ·7H 2 O,584mg/LNaCl,1mL TES,1mL SWS; the TES consists of the following components: 34.4mg/LMnSO 4 ·H 2 O，50mg/L H 3 BO 3 ，70mg/L ZnCl 2 ，72.6mg/L Na 2 MoO 4 ·2H 2 O，20mg/L CuCl 2 ·H 2 O，24mg/L NiCl 2 ·6H 2 O，80mg/L CoCl 2 ·6H 2 O，1g/LFeSO 4 ·7H 2 O; the SWS consists of the following components: 0.5g/L NaOH,3mg/LNa 2 SeO 3 ·5H 2 O，4mg/L Na 2 WO 4 ·2H 2 O。
In the above method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the temperature of the incubation and culture in the inhibitor culturing step is 15-35 ℃ (e.g., 18 ℃,20 ℃, 25 ℃, 30 ℃, 32 ℃, etc.) for 70-100 days (e.g., 75 days, 80 days, 90 days, 95 days, etc.).
As a preferred embodiment, the concentration DO of dissolved oxygen in the reaction system should be maintained at 0.5mg/L to 2mg/L in the above-mentioned method for enriching ammonia oxidizing microorganisms; preferably, the reactor is closed, and the oxygen content in the reactor is controlled to be about 5%.
In the method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the inhibitor culturing step further includes a dilution switching step:
firstly, inoculating a culture obtained in the inhibitor culturing step into a first culture medium, incubating and culturing until ammonia nitrogen is exhausted, and then carrying out continuous dilution culture by using a dilution gradient method until a culture with only target ammonia oxidizing microorganisms is obtained;
next, the culture in which only the target ammonia oxidizing microorganism is present is inoculated into a second medium for expansion culture.
In the method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, in the dilution and transfer step, the first medium and the second medium are media in which ammonia nitrogen is added to the basic medium, and the addition amount of ammonia nitrogen in the first medium is 0.007mmol/L; the addition amount of ammonia nitrogen in the second culture medium is 0.5mmol/L.
In the method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, in the dilution switching step, the dilution factor is 10 -4 For example, the ratio of the amount of culture obtained in the inhibitor culturing step to the amount of the basal medium containing ammonia nitrogen is 0.1mL:1L, and the ratio of the inoculum to the culture medium used in the subsequent serial dilution culture and expansion culture by a dilution gradient method is 0.1mL:1L.
In the method for enriching ammonia oxidizing microorganisms, as a preferred embodiment, the culturing temperature in the dilution transfer step is 15-35℃such as 18℃at 20℃at 25℃at 30℃at 32 ℃.
In a second aspect, the application provides an ammonia-oxidizing enriched microbial product prepared by the method described above.
The technical principle of the method for enriching ammonia oxidizing microorganisms disclosed by the application is as follows: on the basis of inhibiting the growth of other ammonia oxidizing microorganisms except the target ammonia oxidizing microorganisms by using the inhibitor, the other ammonia oxidizing microorganisms are further eliminated by adopting dilution transfer, so that the aim of enriching the target ammonia oxidizing microorganisms is fulfilled. Firstly, selecting a sample containing target pollutants from the natural world, and inhibiting the growth of other ammonia oxidizing microorganisms except the target ammonia oxidizing microorganisms by using an inhibitor; and then, continuously purifying the enriched target ammonia oxidizing bacteria culture by adopting dilution transfer so as to realize the purpose of enriching specific ammonia oxidizing microorganisms.
Compared with the prior art, the application has the following technical advantages:
(1) Inhibiting the growth of other ammonia oxidizing microorganisms except the required ammonia oxidizing microorganisms by using the inhibitor, observing the change of the relative abundance of each ammonia oxidizing microorganism, and verifying the feasibility of enriching the specific complete ammonia oxidizing microorganism by using the inhibitor; especially, a scheme of using 1-octyne as an inhibitor to efficiently inhibit the growth of AOB and AOA and enrich CMX is proposed for the first time;
(2) On the basis of inhibiting other ammonia oxidizing microorganisms except the required ammonia oxidizing microorganisms by using an inhibitor, further enriching the specific ammonia oxidizing microorganisms by using dilution switching, and observing the change of the relative abundance of the specific ammonia oxidizing microorganisms after the dilution switching is performed to culture the ammonia oxidizing microorganisms with high relative abundance;
(3) The idea of enriching specific ammonia oxidizing microorganisms by combining an inhibitor method and a dilution switching method is provided, and the feasibility of the method is verified, so that a high-abundance ammonia oxidizing microorganism product is obtained, the ammonia nitrogen affinity and the oxygen affinity are higher, the method can be used for further removing ammonia nitrogen from low ammonia nitrogen wastewater, and the method has good scientific research and engineering application prospects.
Drawings
FIG. 1 shows the relative abundance of amoA genes for AOB (a in FIG. 1), CMX (b in FIG. 1), AOA (c in FIG. 1) determined by RT-qPCR analysis (base-10 log transformation) in reactors of first and second stage cultures at different 1-octyne concentrations; it should be noted that in panel b of FIG. 1, the 200mg/L and 400mg/L curves substantially overlap, which is difficult to distinguish under the current mapping conditions, and also indicates that the inhibitor concentration of 200mg/L is sufficient to inhibit the growth of other ammonia oxidizing microorganisms than the target.
FIGS. 2-4 show the community structure and relative abundance of all microorganisms likely to be involved in ammoxidation in the inhibitor culture phase based on 16S rRN high throughput sequencing. Wherein FIG. 2 shows a phylogenetic tree of reference sequences and AOB, AOA and NOB representative sequences retrieved from the reactors of the first stage and second stage cultures; FIG. 3 shows a heat map of relative abundance after logarithmic transformation of 10 OTU base-10 belonging to CMX retrieved from the microcosm; figure 4 shows OTU abundance composition extracted in microcosm at different 1-octyne concentrations in the first and second stages.
FIGS. 5-7 show community structure and relative abundance of target ammonia oxidizing microorganisms based on CMX amoA genes during the inhibitor cultivation stage. FIG. 5 shows a phylogenetic tree of reference sequences and CMX representative sequences retrieved from the reactor of the first stage culture; FIG. 6 is a heat map (Heatm) showing the relative abundance of 13 OTUbase-10 logarithmic transformations belonging to CMX retrieved from the microcosm; FIG. 7 shows the CMX amoA gene composition extracted from microcosm at different 1-octyne concentrations in the first stage.
FIG. 8 shows the community structure and relative abundance of target ammonia-oxidizing microorganisms determined based on 16S rRNA and CMX amoA genes after dilution culture; specifically, phylogenetic trees of reference sequences and sequences of AOB, AOA, nitrospira 16S rRNA (a in FIG. 8) and CMX (b in FIG. 8) retrieved from M5Y3.
FIG. 9 shows the distribution of each ammonia-oxidizing microorganism determined by Fluorescence In Situ Hybridization (FISH) technique after the end of dilution transfer culture; wherein a shows that all detected bacteria appear blue (probe EUB 338); b shows that CMX is detected in M5Y3 (probe Ntspa476 is red); c shows that AOB is not detected in M5Y3 (probe NSO190 is green); d is the overall observation image.
Detailed Description
The following examples are given to illustrate the present application in further detail with reference to the accompanying drawings, and the scope of the present application includes but is not limited to the following examples.
The examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions that are routine procedures described in the literature in this field.
The reagents and starting materials used in the examples were the same as those commercially available except as otherwise indicated.
Example 1
1. Inoculating activated sludge:
the method comprises the steps of taking activated sludge in an aeration tank from a sewage treatment plant as an experimental sample, wherein the sewage treatment plant adopts a mode of combining A2O and MBR processes to treat domestic sewage, the Hydraulic Retention Time (HRT) is 12 hours, the Sludge Retention Time (SRT) is 30 days, the concentration of ammonia nitrogen in the inlet water is about 29.8mg/L, the pH is about 7.3, and the dissolved oxygen is about 5mg/L. Prior to inoculation, the activated sludge mixed liquor suspension (MLSS) concentration was about 8447mg/L. The activated sludge is investigated for the presence of a certain abundance of the desired ammonia oxidizing microorganisms, namely complete ammonia oxidizing bacteria.
2. Inhibitor culture experiments:
(1) The first stage:
(1.1) the inoculated activated sludge was placed in six 300mL reactors (i.e., 300mL neutral brown blue cap flask) containing 50mL activated sludge and 90mL ultrapure water and a certain amount of 1-octyne. In this step, 1-octyne was used as an inhibitor of other ammonia oxidizing microorganisms than the desired ammonia oxidizing microorganism at different concentrations, and M1 (0 mg/L), M2 (20 mg/L), M3 (50 mg/L), M4 (100 mg/L), M5 (200 mg/L), and M6 (400 mg/L) were used as six groups, respectively.
(1.2) the reactor was sealed with a butyl rubber stopper, the top gas was completely replaced with pure nitrogen, and then a fixed volume of pure oxygen was injected using a syringe, thereby establishing a relatively stable reactor environment and oxygen content, the oxygen content in the top gas being set to 5%. Each reactor is shaken sufficiently to allow the gas to equilibrate rapidly.
(1.3) 10mL of the concentrated solution of the medium was uniformly injected into each reactor by a syringe pump over 44 hours so that the concentration of ammonia nitrogen in the medium was 7mg/L (0.5 mmol NH) 4 Cl), when the injection was completed, the liquid medium formulation in the reactor was (per liter): 0.5mmol NH 4 Cl，50mg KH 2 PO 4 ，75mg KCl，50mg MgSO 4 ·7H 2 O,584mg NaCl,1mL TES,1mL SWS; each liter of TES consists of the following components: 34.4mg MnSO 4 ·H 2 O，50mg H 3 BO 3 ，70mg ZnCl 2 ，72.6mg Na 2 MoO 4 ·2H 2 O，20mg CuCl 2 ·H 2 O，24mg NiCl 2 ·6H 2 O，80mg CoCl 2 ·6H 2 O，1g FeSO 4 ·7H 2 O; SWS per liter consists of the following components: 0.5g NaOH,3mg Na 2 SeO 3 ·5H 2 O，4mg Na 2 WO 4 ·2H 2 O; the above-mentioned culture medium formulation was (a) designed to inject the above-mentioned amounts of components within 44 hours, regardless of the consumption of nutrients during injection and during subsequent precipitation, (b) a culture medium concentrate was slowly injected within a prescribed period of time to ensure a low ammonia nitrogen concentration environment for the normal growth and consumption of ammonia oxidizing microorganisms, and (c) the culture medium used subsequently in this example was based on this culture medium formulation and was only changed in the designed ammonia nitrogen concentration. The step is mainly an incubation and culture step.
(1.4) after the injection of step (1.3) was completed, the medium was precipitated for 4 hours.
(1.5) draining and replenishing: 110mL of supernatant was withdrawn from each reactor by syringe, 100mL of fresh ultrapure water and a certain amount of 1-octyne were supplemented to eliminate the influence of 1-octyne residue and degradation, and the octyne concentrations in the six groups of reactors were maintained as M1 (0 mg/L), M2 (20 mg/L), M3 (50 mg/L), M4 (100 mg/L), M5 (200 mg/L), M6 (400 mg/L), respectively.
(1.6) aeration: the headspace gas of each reactor was replaced to maintain the designed oxygen concentration at 5%. This step is the same as step (1.2).
(1.7) in 44 hours, using a syringe pump to evenly inject 10mL of culture medium concentrate into each reactor, ensuring that the culture medium formulation after injection is the same as step (1.3).
The cycle is followed according to steps (1.4) -step (1.7). The operation in this experiment belongs to the sequencing batch reactor operation and goes through four stages in sequence called a cycle: aeration is the first stage, see step (1.2), step (1.6); injecting the culture medium into the culture medium for the second stage, see step (1.7); the precipitation medium is the third stage, see step (1.4); the water discharge and water replenishment is the fourth stage, see step (1.5). At the end of each cycle, 110mL of supernatant was drained and fresh ultrapure water 100mL and a set amount of octyne was replenished to eliminate the effects of 1-octyne residue and degradation by having an octyne density less than the concentration of water and insoluble reset 1-octyne.
Thus, the first stage was incubated at 25℃for 3 months (45 cycles).
(2) And a second stage: the stage mainly searches the regrowth condition of all ammonia oxidizing microorganisms after the inhibitor 1-octyne is not added, and judges whether other ammonia oxidation except the target ammonia oxidizing microorganisms are completely inhibited. Specifically, after the first stage test was completed, the addition of the inhibitor to all the reactors was stopped, and the inhibitor concentration was reset to 0mg/L, and the operation was continued under the same conditions for 2 months. Each reactor is shaken sufficiently to allow the gas to equilibrate rapidly.
3. Dilution transfer culture experiment:
although most of the ammonia oxidizing microorganisms other than the target ammonia oxidizing microorganisms are eliminated by the inhibition culture, it is still difficult to achieve the effect of only the target ammonia oxidizing microorganisms, and therefore it is necessary to further enrich the target ammonia oxidizing microorganisms by the dilution transfer culture.
(1) After the inhibitor culturing process (first stage) is completed, 0.1mL of a sample (namely M5 sample) with relatively high concentration of the target ammonia oxidizing microorganism and relatively low concentration of other ammonia oxidizing microorganisms is selected and inoculated into 1L of primary ammonia consumption enrichment medium (dilution factor 10) -4 ) This system is designated M5Y1'; wherein, the formula of the primary ammonia consumption enrichment medium is as follows (per liter): 7mgNH 4 + -N(0.5mmol NH 4 Cl)，50mg KH 2 PO 4 ，75mg KCl，50mg MgSO 4 ·7H 2 O,584mg NaCl,1mL TES,1mL SWS; each liter of TES consists of the following components: 34.4mg MnSO 4 ·H 2 O，50mg H 3 BO 3 ，70mg ZnCl 2 ，72.6mg Na 2 MoO 4 ·2H 2 O，20mg CuCl 2 ·H 2 O，24mg NiCl 2 ·6H 2 O，80mg CoCl 2 ·6H 2 O，1g FeSO 4 ·7H 2 O; SWS per liter consists of the following components: 0.5g NaOH,3mg Na 2 SeO 3 ·5H 2 O，4mg Na 2 WO 4 ·2H 2 O; incubating and culturing the M5Y1 'system in a constant temperature shaking table at 30deg.C and 120 r/min for one period (namely culturing until ammonia nitrogen in the culture medium is reduced to 0), and observing the growth condition of ammonia oxidizing microorganism in the M5Y1' system by PCR techniqueThe inventors found that not only the whole ammonia oxidizing bacteria but also other ammonia oxidizing microorganisms AOA and AOB other than the whole ammonia oxidizing bacteria were grown in the culture, and therefore, the inventors adjusted the culture medium in time and selected dilution transfer culture.
(2) The mixed solution (i.e., M5 sample containing microbial growth to form a colony) as an inoculum was suspended, and 0.1mL of the mixed solution was inoculated into 1L (dilution ratio 10) by a pipette -4 ) The first medium (which has the same formulation as the primary ammonia-depleted enrichment medium in step (1), the only difference being that it contains 0.007mM NH 4 Cl), named M5Y1 system; the M5Y1 system was incubated at 30℃in a 120rpm constant temperature shaker for one cycle (i.e., until the ammonia nitrogen in the medium was substantially reduced to 0). During the period, the growth of the target ammonia oxidizing microorganisms in the M5Y1 system was observed by PCR technology, and it was found that other ammonia oxidizing microorganisms than the complete ammonia oxidizing bacteria were also present in the culture. Continuing to dilute, transfer and culture once: 0.1mL of the culture was inoculated into 1L (dilution 10) by a pipette -4 ) In the first medium, the system takes over the previous designation, the M5Y1 system; the M5Y1 system is incubated and cultured for one period (namely, until ammonia nitrogen in the culture medium is basically reduced to 0) in a constant temperature shaking table at 30 ℃ and 120 revolutions per minute, and the PCR technology shows that only the target ammonia oxidizing microorganism exists in the M5Y1 system.
(3) When it is found that only the target ammonia oxidizing microorganism is present in the M5Y1 system obtained in the step (2), the medium formulation is adjusted to increase the medium ammonium concentration to 0.5mM NH 4 Cl, otherwise the same as the primary ammonia-depleted enrichment medium in step (1) (i.e., the second medium) to further increase its propagation rate.
Specifically, 0.1mL of the M5Y1 system culture obtained in the step (2) is inoculated to 1L of a second culture medium, and is named as an M5Y2 system, the M5Y2 system is incubated and cultured for one period in a constant temperature shaking table at 30 ℃ and 120 revolutions per minute (the consumption of ammonia nitrogen is observed periodically until the complete consumption of ammonia nitrogen is observed, and the culture time is about two weeks), and the PCR technology is observed to find that only target ammonia oxidizing microorganisms, namely complete ammonia oxidizing microorganisms, still exist in the M5Y2 system.
(4) In order to further enrich the completely ammonia oxidizing microorganisms, the inventors continued the dilution transfer once, in which no more ammonia oxidizing microorganisms other than completely ammonia oxidizing microorganisms have been observed in the product obtained by the incubation culture of step (3).
Specifically, 0.1mL of the M5Y2 system culture obtained in the step (3) was added to 1L of the second medium by a pipette (dilution factor 10) -4 ) Named M5Y3 system; and (3) continuously culturing the M5Y3 system for one period at 30 ℃ and 120rpm (namely, basically reducing ammonia nitrogen in a culture medium to 0), so as to obtain the M5Y3 with higher enrichment degree in the study.
4. And (3) detection:
the application adopts quantitative PCR (q-PCR), high throughput sequencing and Fluorescence In Situ Hybridization (FISH) technology as a means for measuring the enrichment effect of the complete ammonia oxidizing bacteria.
(1) q-PCR technique: biological samples enriched for complete ammoxidation bacteria during enrichment were collected and genomic DNA from each sample was extracted using Fast DNA kit (MP Biomedical, illkirch, france) according to the manufacturer's instructions. The gene Copy number of each species was determined using all specific amoA gene primers on Commox, AOA and AOB, and the ratio of amoA gene Copy number in Commox to all amoA gene Copy number was calculated. The base sequences of the q-PCR primers used in this example are shown in Table 1.
Table 1 shows the base sequences of the q-PCR primers used in the present application
(2) FISH technology: the slides were placed in a solution of 70% ethanol and 1% hcl for several hours, rinsed with deionized water, and sterilized at 121 degrees for 20min. 0.6g of gelatin is weighed and dissolved in 250mL of deionized water, the gelatin is heated (60 ℃) and stirred for dissolution, and after the gelatin is completely dissolved, 0.6g of methyl alum is added for use. After the prepared glass slide is cooled at room temperature, the glass slide is immersed in gelatin liquid up and down for several times, dispersed and opened on a frame, and naturally dried in air. After 1mL of the sample was centrifuged at 8000rpm in a 2mL centrifuge tube to remove the supernatant, 1mL of PBS was added, centrifuged at 8000rpm (repeated once), 3 volumes (about 1.5 mL) of 4% Paraformaldehyde (PFA) was added, and the mixture was fixed at 4℃for 4 to 12 hours. The fixative was removed by centrifugation at 8000rpm and washed 2 times with PBS, and the samples were suspended in an equal volume of PBS (200. Mu.L) and 100% ethanol solution (200. Mu.L). Taking 10 mu L of a fixed sample, coating the sample on a glass slide embedded with gelatin, naturally airing, and dehydrating with 50%,80% and 96% ethanol at room temperature in sequence (3 minutes each time, and naturally airing the ethanol coated on the sample of the glass slide). Mixing 18 μl of hybridization buffer with 2 μl of probe (9:1), coating 15 μl on the slide with the sample fixed, placing the slide horizontally in a hybridization wet box (the slide lower layer is coated with paper and soaked with water), covering with a cover to maintain humidity, wrapping with aluminum foil paper to prevent light, and hybridizing at 46 deg.C for 1.5 hr. After hybridization, the slide glass is put into preheated cleaning solution at 48 ℃ for soaking for 10-15min, taken out, washed by distilled water in ice bath and air-dried in air.
(3) High throughput sequencing: the DNA extract is rapidly sent to Shanghai Meijia biomedical science and technology limited company, high-throughput sequencing is carried out through an Illumina Miseq platform, sequencing data are returned through fastq files, and the phylogenetic and community structures of three ammonia oxidizing microorganisms are visually analyzed. The RStudio software is used for carrying out classification unit OTU clustering and phylogenetic analysis on the original sequencing data and carrying out visual processing on the data, and main analysis steps comprise quality control operation, redundancy removal operation, clustering operation, flattening operation and species annotation. For phylogenetic analysis of ammonia oxidizing microorganisms, the representative sequences of genes in the database were aligned with the representative sequences of each OTU, respectively, using MEGA6.0 software. The comparison result is imported into MEGA6.0, and a phylogenetic tree is manufactured by using Neighbor-training, wherein the Bootstrap value is 1000, so that the accuracy and the reliability of the result are improved. Through the operation, phylogenetic tree of ammonia oxidizing microorganisms can be obtained to better understand the evolutionary process and community structure of ammonia oxidizing microorganisms.
5. Results of inhibitor culture study:
for the first and second stage of test runs, samples were collected every 10 days, and sample detection was performed using PCR techniques, with the detection results shown in fig. 1.
Inhibition of AOB by 1-octyne is evident (see a in FIG. 1). The AOB amoA genes in the M1 and M2 groups were significantly higher than those in the other groups, and their inhibition was more pronounced with increasing inhibitor 1-octyne concentration. On day 90, the inhibition effect was greatest in the M5 group and the M6 group.
For CMX, CMX starts to increase at the beginning of culture and then its abundance remains at a steady level (see b in fig. 1). The abundance levels were not greatly different and fluctuated across a range between the different octyne concentration treatment groups, except for the M5 and M6 groups. However, for high concentrations of 1-octyne, it still inhibits the growth of CMX.
The inhibition of AOA by 1-octyne is beyond expectations and should be more resistant than AOB (see c in fig. 1). Within 30 days, the inhibition effect of 1-octyne on AOA is not obviously different. However, there was a significant difference in AOA amoA gene between groups, eventually stabilizing with increasing culture time on day 50. The low concentration of 1-octyne showed no significant inhibition similar to group M1, but the high concentration of 1-octyne showed comparable inhibition as group M5 or group M6.
From the second phase, see the trend from day 90 to day 150 in fig. 1, it is clear that, contrary to the first phase, no significant improvement in CMX could be obtained without the addition of 1-Xin Guishi, but after stopping the addition of 1-octyne, AOB and AOA grew rapidly to a level comparable to CMX. Demonstrating that efficient inhibition of AOB and AOA by 1-octyne provides an advantage for CMX enrichment.
The results of the phylogenetic analysis of the first and second stages under long-term inhibitor culture conditions are shown in FIGS. 2-4. The AOB colony consisted mainly of Nitrosospira lineage cluster (nitrosaminium) Nitrosomonas europaea cluster (nitrosaminium European), N.marian cluster, N.oligophacluster and N.com cluster. On the OTUs level, OTU1935 is classified as n.oligophas, OTU290 as n.marian, OTU189 and OTU344 as n.communications.
In the AOA community, OTU3 belongs to the Nitrosopurlus class as the most abundant AOA class, OTU1462 belongs to the Nitrososphaera class, and OTU7 belongs to the Nitrosocosmus class.
From the Nitrospira community, OTU12 belongs to Nitrospira lineage I and OTU1 belongs to Nitrospira lineage II. In both the M5 and M6 groups, each cluster of AOB and the main cluster of AOA were very severely inhibited. In contrast, nitrospira lineage II did not vary much in the M5 and M6 groups.
Based on CMX amoA gene sequencing, phylogenetic analysis showed that OTUs are broadly divided into 3 classes: nitrospira nitrifican, nitrospira nitrosa, and a New cluster (see FIGS. 5-7). OTU1, OTU2, OTU3 and OTU5 have less effect in the M5 group and M6 group. The relative abundance of Nitrospira nitrosa from group M1 to group M6 is 96.99%, 98.46%, 97.64%, 95.56%, 98.95%, 99.85%, respectively, all above 95%. There is sufficient reason to conclude that Nitrospira nitrosa has sufficient octyne resistance in the flora.
6. Study results of dilution transfer culture:
the results of high throughput sequencing transfer of the M5Y3 sample obtained from the dilution transfer culture experiment are shown in FIG. 8. OTU1 belongs to branch Nitrospira lineage II, its abundance reaches 70.27%, OTU2 belongs to Nitrospira lineage I, and its abundance reaches 15.77%. Further by sequencing CMX amoA, OTU1, OTU2, OTU3 were all scored into the Nitrospira nitrosa branch, which was 100% in ratio.
The culture M5Y3 obtained after the end of the dilution transfer culture was examined by in situ fluorescence hybridization experiments. FIG. 9 shows the distribution of each ammonia-oxidizing microorganism determined by Fluorescence In Situ Hybridization (FISH) technique after the end of dilution transfer culture; wherein a shows that all detected bacteria are hybridized blue (probe EUB 338); b shows that CMX was detected in M5Y3 samples (probe Ntspa476 was red); c shows a photograph of whether AOB is detected in the M5Y3 sample (probe NSO190 is green), and the AOB is not found in the sample, which proves that the M5Y3 sample is all the strain to be enriched; d is the overall observation image.
Finally, it is further noted that in this disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
While the disclosure has been disclosed by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.
Claims (10)
1. A method of enriching for ammonia oxidizing microorganisms, comprising:
and (3) an inhibitor culturing step: adding an inhibitor and ammonia nitrogen into the basic culture medium, inoculating a sample containing the target ammonia oxidizing microorganism to form a culture system, performing incubation culture treatment, and inhibiting the growth of other ammonia oxidizing microorganisms except the target ammonia oxidizing microorganism by using the inhibitor.
2. The method of enriching ammonia oxidizing microorganisms according to claim 1, wherein the target ammonia oxidizing microorganism is a complete ammonia oxidizing bacteria.
3. The method of enriching an ammonia oxidizing microorganism according to claim 1 or 2, wherein the inhibitor is 1-octyne; preferably, 1-octyne is added at a concentration of 50-400mg/L.
4. A method for enriching an ammonia oxidizing microorganism according to any one of claims 1-3, wherein in the inhibitor culturing step, the ammonia nitrogen is added in an amount of 5-10mg/L in the culture system; the ammonia nitrogen is applied at a constant speed, and the application speed is 0.1-0.25mg/L per hour.
5. The method for enriching ammonia oxidizing microorganisms according to any one of claims 1 to 4, wherein the basal medium is formulated as follows: 50mg/L KH 2 PO 4 ，75mg/L KCl，50mg/LMgSO 4 ·7H 2 O,584mg/L NaCl,1mL TES,1mL SWS; the TES consists of the following components: 34.4mg/L MnSO 4 ·H 2 O，50mg/L H 3 BO 3 ，70mg/L ZnCl 2 ，72.6mg/LNa 2 MoO 4 ·2H 2 O，20mg/L CuCl 2 ·H 2 O，24mg/L NiCl 2 ·6H 2 O，80mg/LCoCl 2 ·6H 2 O，1g/L FeSO 4 ·7H 2 O; the SWS consists of the following components: 0.5g/L NaOH,3mg/L Na 2 SeO 3 ·5H 2 O，4mg/L Na 2 WO 4 ·2H 2 O；
Preferably, in the inhibitor culturing step, the medium is replaced every 48 hours.
6. The method for enriching ammonia oxidizing microorganisms according to any one of claims 1 to 5, wherein the temperature of the incubation culture treatment is 15 to 35 ℃ for 70 to 100 days in the inhibitor culture step.
7. The method for enriching an ammonia oxidizing microorganism according to any one of claims 1-6, wherein the dissolved oxygen concentration DO in the reaction system is maintained at 0.5mg/L to 2mg/L; preferably, the reactor is closed, and the oxygen content in the reactor is controlled to be about 5%.
8. The method of enriching ammonia oxidizing microorganisms of any one of claims 1-7, wherein the inhibitor culturing step is followed by a dilution switching step:
firstly, inoculating a culture obtained in the inhibitor culturing step into a first culture medium, incubating and culturing until ammonia nitrogen is exhausted, and then carrying out continuous dilution culture by using a dilution gradient method until a culture with only target ammonia oxidizing microorganisms is obtained;
secondly, inoculating the culture with only the target ammonia oxidizing microorganism into a second culture medium for expansion culture;
preferably, in the dilution transfer step, the culture temperature is 15-35 ℃.
9. The method for enriching an ammonia oxidizing microorganism according to claim 8, wherein in the dilution switching step, the first medium and the second medium are a medium in which ammonia nitrogen is added to the basal medium, and an addition amount of ammonia nitrogen in the first medium is 0.007mmol/L; the addition amount of ammonia nitrogen in the second culture medium is 0.5mmol/L;
preferably, in the dilution switching step, the ratio of the amount of the culture obtained in the inhibitor culturing step to the amount of the basal medium containing ammonia nitrogen is 0.1mL:1L, and the ratio of the inoculum to the culture medium used in the subsequent serial dilution culture and expansion culture by a dilution gradient method is 0.1mL:1L.
10. An enriched ammonia oxidizing microorganism product prepared by the method of any one of claims 1-9.
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