CN106124737B - Soil anaerobic ammonia oxidation microbiological structure of community detection method - Google Patents
Soil anaerobic ammonia oxidation microbiological structure of community detection method Download PDFInfo
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Abstract
The invention discloses a kind of soil anaerobic ammonia oxidation microbiological structure of community detection methods, comprising the following steps: 6 containers of setting, each container successively carries out following content respectively: be packed into a reservoir soil to be measured,13C- urea liquid and KNO3Solution, uniform stirring add water and are adjusted to mud state, and closed container；The air in container is replaced with inert gas, anaerobic culture box is then placed in and carries out Anaerobic culturel；After resulting 6 containers cultivate different time respectively, native measurement is taken, according to NO3 ‑- N, NH4 +The changes of contents rate of-N chooses the soil in container corresponding to the highest cultivation stage of rate of change, extracts PLFA therein；It is combined by gas-chromatography-burning-isotopic ratio mass spectrum (IRMS) in the PLFA that method measurement extracts13The abundance D of C13C, to know soil anaerobic ammonia oxidation microbiological structure of community.
Description
Technical field
The invention belongs to structure of soil microbial community detection technique fields, and in particular to a kind of soil anaerobic ammoxidation is micro-
The detection method of biology community structure, it is especially a kind of to measure soil anaerobic ammonia oxidation microbiological structure of community using 13C-PLFA
Method.
Background technique
Anammox acts on (ANAMMOX-Anaerobic ammonium oxidation) and refers under anaerobic,
Microorganism is with NO2 -For electron acceptor, NH4 +For electron donor, by NH4 +、NO2 -It is changed into the biochemical process of nitrogen.1977
Chemically thermodynamics sets out Broda, when calculating its evolutionary relationship with biology, has courageously foretold the presence of Anammox process.The
The bacterial strain of one Anammox named is that physics is utilized from the laboratory enrichment culture bacterial strain of a bioreactor
Method Percoll gradient centrifugation is isolated.Subsequent Vande Graaf et al. confirms the presence of anaerobic ammonium oxidation process.?
The anaerobic ammonium oxidizing bacteria to work during this is very slow to oxygen extreme sensitivity, growth, and in higher bacterial concentration
Under the conditions of just show anaerobic ammoxidation activity, therefore study anaerobism ammonia oxygen with traditional microorganism separation, purification cultivation method
It is extremely difficult to change bacterium.But it with the development of modern technologies, is introduced in Soil Microbes there are many new method.It is analyzing
When anaerobic ammonia oxidizing bacteria group forms in environmental sample, usually using clone, the method for sequencing building 16S rDNA clone library.
This method avoid the requirements to anaerobic ammonia oxidizing bacteria pure culture, but construct clone library heavy workload, costly, Bu Nengshi
The quick analysis of existing anaerobic ammonia oxidizing bacteria structure of community.
Clone and DGGE are the common methods of microorganisms structure of community, but both methods is only capable of embodying group's knot
Similitude and otherness between structure need to combine other technologies if thinking definitely to analyze each content of microorganisms.Lipoid fatty acid
(PLFA) be microbial cell film main component, because microorganism its PLFA of difference composition and configurations, different phosphatide rouge
Fat acid indicates different microorganisms, therefore PLFA is combined with stable isotope labeling and passes through specific Anammox and is trained
During supporting, then each edaphon content that metabolism is participated in anaerobic ammonium oxidation process can be preferably analyzed, after analyzing
Know the structure of community of anaerobic ammonia oxidation microbiological.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of soil anaerobic ammonia oxidation microbiological structure of community detection methods；
The present invention will13The urea of C flag is in conjunction with PLFA and carries out Anaerobic culturel to analyze soil anaerobic ammonia oxidation bacteria structure of community
Method, this method is not only low to experimental condition requirement, stability is good, and sensitivity is high, dosing accuracy is strong, can accurately examine
Measure the structure of community of the microorganism that is active, participating in reaction in anaerobic ammonium oxidation process.
In order to solve the above technical problem, the present invention provides a kind of soil anaerobic ammonia oxidation microbiological structure of community detection sides
Method, comprising the following steps:
6 containers are set, and each container successively carries out following step 1)~step 4) respectively:
1), be packed into a reservoir soil to be measured and13C- urea liquid, uniform stirring, to make in soil13C- urea-N
Concentration reach 200mg/kg；
Remarks explanation:13C- urea enter after soil be hydrolyzed under the action of soil urease ammonium and13CO2, it is anaerobism ammonia oxygen
Change provides the inorganic energy, and anaerobic ammonia oxidizing bacteria is that chemosynthetic autotroph is directly utilized13CO2, pass through13C-PLFAs detects soil
The structure of community of Anammox；
2) KNO, is added into the resulting soil of step 1)3Solution, uniform stirring, to make NO in soil3 ?- N concentration reaches
200mg/kg；
3), water (rapidly plus water) is added to be adjusted to mud state, and closed container into the resulting soil of step 2)；
4) air in container, is replaced with inert gas, anaerobic culture box is then placed in and carries out 30 ± 1 DEG C of anaerobism trainings
It supports；
5) resulting 6 containers of step 4), are cultivated into 1~2h, 1d, 3d, 7d, 10d respectively, until NO3 --N、NH4 +- N value
Stable that day；
Content is carried out respectively to the corresponding container of 6 cultivation stages:
Native (such as 5g) is taken, measures soil NO using SKALAR Continuous Flow Analysis instrument3 -- N and NH4 +N content；
For example, concretely: nitrogen cylinder in SKALAR Continuous Flow Analysis instrument being opened nitrogen to 0.2MPa, unclamps flowmeter
45 unit of value, opens heater, and opens 99 DEG C of host of heater, measures soil NO using SKALAR Continuous Flow Analysis instrument3 --
N and NH4 +N content；
Remarks explanation: remaining soil can directly carry out following step in container, carry out following step if not timely,
- 18 DEG C of preservations can also be temporarily put into；
6), according to NO3 -- N, NH4 +The changes of contents rate of-N is chosen corresponding to the highest cultivation stage of rate of change
Soil in container extracts PLFA therein (lipoid fatty acid)；
The judgment rule of the highest cultivation stage of rate of change are as follows: the soil of 6 cultivation stages is (single in identical incubation time
Position incubation time) interior NO3 -- N, NH4 +The highest cultivation stage of the concentration decrease of-N is the highest cultivation stage of rate of change,
If NO3 -- N, NH4 +The rate of change of-N is inconsistent, then takes the mean value of the two rate of change maximum for the highest training of rate of change
The stage of supporting；
Remarks explanation: NO in above-mentioned unit incubation time3 -- N, NH4 +The concentration decrease of-N can be according to conventional utilization
It, can be easily according to the method for Analysis-Mathematics-Differentiate in Origin after Origin matched curve
Detection obtains；
7) PLFA extracted, is measured by gas-chromatography-burning-isotopic ratio mass spectrum (IRMS) combination method (GC-c-IRMS)
In13The abundance D of C13C,13The abundance of C is13The abundance of the lipoid fatty acid of C flag.
Remarks explanation:13The lipoid fatty acid of C flag can be indicated to participate in the microorganism of anaerobic ammonium oxidation process, that is, be known
The abundance of anaerobic ammonia oxidation microbiological, and then analyze and obtain structure of community, it is possible to according to13The abundance of C obtains soil anaerobic
The structure of community of ammonia oxidation microbiological.
Improvement as soil anaerobic ammonia oxidation microbiological structure of community detection method of the invention:
It is 5g/L's that fresh soil and the 1.2mL concentration to be measured of 30g is added in the step 1), in each container13C- urine
Plain solution；
The KNO that 1.2mL concentration is 5g/L is added in the step 2), in each container3Solution.
Further improvement as soil anaerobic ammonia oxidation microbiological structure of community detection method of the invention:
Extract in the step 6) PLFA method (using the cloth Lay and Dell's method after repair, Bu Lai and Dell's method
It is only applied in food, and can only determine total lipid content, the method after repair is extracted and isolated lipoid fatty acid and comes really
Determine edaphon structure) the following steps are included:
In soil (or freeze-drying soil) addition chloroform: methanol: citric acid sodium citrate buffer solution (0.15M, ph4.0) (1:
2:0.8v/v/v) extract；
By extract (that is, extracting solution) be transferred to after silica gel solid-phase extraction column respectively with chloroform and acetone wash away center rouge and
After glycolipid, phosphatide is eluted with methanol；After phosphatide addition internal standard Nonadecanoic acid methylester (C19:0) after elution, pass through mild alkali
Fatty acid methyl is turned to corresponding fatty acid methyl ester (FAMES) by property methylation method.Mild alkalinity methylation method, that is,
Methanol toluene mixture liquid (methanol: toluene=1:1) and the KOH methanol of 1mL 0.2M that 1mL is added in test tube containing phosphatide are molten
Liquid is put into 37 DEG C of water-bath after heating progress esterification, 2mL water, 0.3mL glacial acetic acid is added, then mixed with n-hexane chloroform
It closes liquid (twice, each 2mL, n-hexane: chloroform=4:1) and extracts lipoid fatty acid methyl esters, and with being dried with nitrogen to obtain fatty acid
Methyl esters.
Remarks illustrate: the relationship of fatty acid methyl ester (FAMES) and PLFA (lipoid fatty acid) is Methanol Decomposition phospholipid fatty
It is sour therefore that fatty acid methyl ester can obtain PLFA's (lipoid fatty acid) easily under the premise of knowing the amount of fatty acid methyl ester
Amount.
Further improvement as soil anaerobic ammonia oxidation microbiological structure of community detection method of the invention:
The step 7) are as follows:
It is separated using fatty acid methyl ester obtained by step 6) through gas-chromatography (GC), specifically: it is first turned on nitrogen gas cylinder
Main valve adjusts pressure to 0.5MPa, opens air generator, hydrogen generator power supply, supply to gas cylinder；Open flow controller
Casing adjusts pressure and flow, and the pressure for adjusting hydrogen and air is 50KPa, nebulizer gas pressure 70KPA, and total gas pressure is
300KPa adjusts tail and is blown to sample introduction after 30ml/min；Ion detector (FID) detection (instrument is fallen by the fire being connected after separation
Device is U.S. Agilent company, has the lipoid fatty acid database of U.S.'s MIDI formula in system), fatty acid is carried out with this
Quantify；Single PLFA's13C/12C is carried out by gas-chromatography-combustion method-stable isotope than mass spectrograph (GC-C-IRMS)
Measurement, wherein it is (all to set to be connected with TraceGCUltra gas-chromatography, GC Combustion III for Delta V isotope ratio matter instrument
Standby is Thermo Finnigan company of Germany)；
The amount of PLFA monomer is calculated according to formula (1):
Mi=dilution × (PFAME×ng Std)/(PISTD×W) (1)
(1) in formula, Mi is the amount (in terms of dry ground) of single lipoid fatty acid (PLFA), and unit is μ g/g；PFAMEFor sample
Peak area；PISTDFor internal standard (methyl ester C19:0,6.2ng/ μ L) peak area；PFAMEAnd PISTDBy by gas chromatograph
It obtains；Ng Std is interior target concentration (6.2ng/ μ L)；Dilution is extension rate；W is to dry native dry weight (in terms of dry ground),
3g。
C is calculated from the amount (Pi, μ g/g) of urea-C with formula (2) in each fatty acid:
Pi=Mi* (δ13Cc-δ13Ccontrol)/(δ13CM-δ13Ccontrol) (2)
Wherein, Mi is the concentration of single PLFA, is calculated by formula (1), δ13CcAnd δ13CcontrolIt is before marking and after label
The δ of single fatty acid13C, δ13CMIt is the δ for adding urea13C is obtained by isotope ratio matter instrument.
PLFA is total in each soil13C is enriched with ratio (pt) i.e.13The abundance D of C13C is calculated with following formula (3):
pt=∑ Pi/ ∑ Mi (3)
Wherein Mi is the content (μ g/kg) of C in single fatty acid.
Further improvement as soil anaerobic ammonia oxidation microbiological structure of community detection method of the invention: the step
1) in, container is the glass scre-cap culture bottle of 100mL, is jumped a queue with soft rubber plug and realizes sealing.The glass scre-cap culture bottle is for example
Are as follows: base diameter about 4.5cm, bore about 1.3cm , bottle height about 9.5cm.
Further improvement as soil anaerobic ammonia oxidation microbiological structure of community detection method of the invention: the step
3) in, every 30g soil adds 5~7ml of water (preferably 6mL).
The present invention specifies the detection method of soil anaerobic ammonia oxidation microbiological structure of community, while can also be used as bottom
The reference that anaerobic ammonia oxidation microbiological structure of community detects in the environment such as mud, sewage sludge reactor, wetland.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is vegetable garden soil NO3 --N,NH4 +- N tendency chart；
Fig. 2 is vegetable garden soil13C-PLFA map；
Fig. 3 is inorganic fertilizer processing rice soil NO3 --N,NH4 +- N tendency chart；
Fig. 4 is inorganic fertilizer processing rice soil13C-PLFA map；
Fig. 5 is organic-inorganic with applying processing rice soil NO3 --N,NH4 +- N tendency chart；
Fig. 6 is organic-inorganic with applying processing rice soil13C-PLFA map；
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The feasibility and application effect of the method for the present invention will be further illustrated in following Examples.
The detection method of soil anaerobic ammonia oxidation microbiological structure of community in embodiment 1, vegetable garden soil successively carries out following step
It is rapid:
Prepare 6 100mL culture bottles, each culture bottle carries out following step 1)~step 4) respectively:
1) the fresh soil to be measured of 30g, is added in each culture bottle, drawing the fresh 1.2mL concentration matched is 5g/L's13C- urine
Plain solution uniformly spills in the soil of each culture bottle and stirs and evenly mixs rapidly, makes in soil13The concentration of C- urea-N reaches about
200mg/kg；
2) KNO that 1.2mL concentration is 5g/L, is drawn3Solution is uniformly spilt in the resulting culture bottle soil of step 1), and is stirred
Mixing mixing makes NO3 ?- N concentration about 200mg/kg；
3), water 6ml is added to be adjusted to mud state rapidly and adds rubber septum 100mL culture bottle；
4), with syringe, into culture bottle, filling with inert gas He drives air away, is put into anaerobic culture box and carries out 30 DEG C of anaerobism
Culture.
5) resulting 6 containers of step 4), are cultivated into 1~2h, 1d, 3d, 7d, 10d respectively, until NO3 --N,NH4 +- N value
Stable that day (about 14 days)；
Content is carried out respectively to the corresponding container of 6 cultivation stages:
Destructiveness takes soil, and each culture bottle respectively takes 5g native, in 150ml shake flask, adds 50ml 1mol/L KCl leaching liquor
At 20 DEG C, 180r/s shakes 1 hour, and machine measures in filtering, and nitrogen cylinder in SKALAR Continuous Flow Analysis instrument is opened nitrogen extremely
0.2Mpa is unclamped 45 unit of flow meter value, opens heater, and open 99 DEG C of host of heater, continuously flowed using SKALAR
Analysis-e/or determining NO3 -- N, NH4 +- N, remaining soil (25g) can be put into -18 DEG C of refrigerators and save (also i.e. in each culture bottle
Subsequent step can directly be carried out).
Measure NO3 -- N, NH4 +- N concentration results are as shown in Figure 1, the result is fitted according to conventional using Origin
After curve, operation, NO are carried out according to the method for Analysis-Mathematics-Differentiate in Origin3 -- N, NH4 +-
N concentration fall off rate it is most fast a few days ago for culture after the 7th day and the 14th day.
6) the cloth Lay of soil repair in 7 days and 14 days and Dell's method are extracted after the culture for, taking -18 DEG C of refrigerators to save
PLFA.The chloroform of addition 15.2ml in 3.0g (freeze-drying soil sample): methanol: citric acid sodium citrate buffer solution (0.15M,
Ph4.0) (1:2:0.8v/v/v) is extracted and (250rpm, room temperature, is extracted 2h), and 7.6ml is added in extracting resulting residue
Chloroform: methanol: citric acid sodium citrate buffer solution (0.15M, ph4.0) (1:2:0.8v/v/v) carries out extraction again and (mentions
Taking technique parameter is same as above)；It extracts resulting extracting solution for 2 times to merge, as extract.
Extract is turned after being dried with nitrogen, after being then dried with nitrogen with methanol dissolution (volumetric usage is 1ml*2 times), uses chlorine
Imitative (volumetric usage is 0.5ml*3 times) dissolution moves to after silica gel solid-phase extraction column (the silica gel 500mg of built-in C18 model/specification)
After washing away center rouge and glycolipid with chloroform (10ml, flow velocity 0.3m/min) and acetone (10ml, flow velocity 0.3m/min) respectively,
Phosphatide is eluted with methanol (8ml, flow velocity 0.3m/min)；Collect phosphatide eluent.
Phosphatide (that is, phosphatide eluent) after elution added after being dried with nitrogen 200 μ l internal standard Nonadecanoic acid methylester (19:
0,6.2ng/ μ L) after be dried with nitrogen, thus formed the test tube containing phosphatide.By mild alkaline methylation method by fatty acid methyl
Base turns to corresponding fatty acid methyl ester (FAMES).Mild alkalinity methylation method in the above-mentioned test tube containing phosphatide that is, be added
The KOH/methanol solution of methanol toluene mixture liquid (methanol: toluene=1:1) and 1mL 0.2M of 1mL, is put into 37 DEG C of water-bath
Heating carry out esterification after, be added 2mL water, 0.3mL glacial acetic acid, then with n-hexane chloroform mixed liquor (twice, each 2mL, just oneself
Alkane: chloroform=4:1) extraction lipoid fatty acid methyl esters, and with being dried with nitrogen to obtain fatty acid methyl vinegar.
7) it, is separated using fatty acid methyl ester obtained by step 6) through gas-chromatography (GC), is first turned on the main valve of nitrogen gas cylinder,
Pressure is adjusted to 0.5MPa, air generator, hydrogen generator power supply is opened, is supplied to gas cylinder；Flow controller casing is opened,
Pressure and flow are adjusted, the pressure for adjusting hydrogen and air is 50KPa, nebulizer gas pressure 70KPA, total gas pressure 300KPa, is adjusted
Section tail is blown to sample introduction after 30ml/min, and by the fire that is connected falling into ion detector (FID) detection after separation, (instrument is the U.S.
Agilent company has the lipoid fatty acid database of U.S.'s MIDI formula in system), quantifying for fatty acid is carried out with this.It is single
A PLFA's13C/12C is measured by gas-chromatography-combustion method-stable isotope than mass spectrograph (GC-C-IRMS), wherein
Delta V isotope ratio matter instrument is connected with TraceGCUltra gas-chromatography, (all devices are Germany to GC Combustion III
Thermo Finnigan company)；
The amount of PLFA monomer is calculated according to formula (1):
Mi=dilution* (PFAME*ng Std)/(PISTD*W) (1)
(1) in formula, Mi is the amount (in terms of dry ground) of single lipoid fatty acid (PLFA), and unit is μ g/g；PFAMEFor sample
Peak area；PISTDFor internal standard (methyl ester C19:0,6.2ng/ μ L) peak area；PFAMEAnd PISTDBy by gas chromatograph
It obtains；Ng Std is interior target concentration (6.2ng/ μ L)；Dilution is extension rate；W is to dry native dry weight (in terms of dry ground),
3g。
C is calculated from the amount (Pi, μ g/g) of urea-C with formula (2) in each fatty acid:
Pi=Mi* (δ13Cc-δ13Ccontrol)*100/(δ13CM-δ13Ccontrol) (2)
Wherein, Mi is the concentration of single PLFA, is calculated by formula (1), δ13CcAnd δ13CcontrolIt is before marking and after label
The δ of single fatty acid13C, δ13CMIt is the δ for adding urea13C is obtained by isotope ratio matter instrument.
PLFA is total in each soil13C is enriched with ratio (pt) i.e.13Abundance (the D of C13C it) is calculated with following formula (3):
pt=∑ Pi/ ∑ Mi*100 (3)
Specific experiment is as follows:
Experiment: Zhejiang Province Yuhang barrow head village is picked up from for examination soil；
The data obtained is by taking the 15:0iso of 14d as an example:
PFAME=6.651, PISTD=18.105, ng Std=6.2, W=3, so Mi=0.759205；
δ13Cc=1.279, δ13Ccontrol=1.074, δ13CM=99, so Pi=0.208；
∑ Pi=1.170, ∑ Mi=66.242, pt=1.766.
Because data volume is larger, do not repeat one by one.
20 kinds of lipoid fatty acids are measured altogether, are computed and are obtained each lipoid fatty acid13C is enriched with ratio (pt) as shown in Fig. 2,
Abscissa is the various lipoid fatty acids measured, and ordinate, that is, various lipoid fatty acids account for the percentage of total phospholipids fatty acid.From
Fig. 2 can be seen that, 14d vegetable garden soil soil anaerobic ammonia oxidation microbiological structure of community diversity after culture.
The lipoid fatty acid for characterizing bacterium as the result is shown of the different microorganisms according to indicated by lipoid fatty acid, 14d contains
Amount accounts for about 93.9 ± 0.7%, and fungi accounts for about 0.49 ± 0.02%, and actinomyces account for about 5.5 ± 0.2%.
Illustrate: soil conclusion is same as above substantially after culture 7d.
Experiment 2, the detection of inorganic fertilizer processing rice soil soil anaerobic ammonia oxidation microbiological structure of community
Jintan City of Jiangsu Province town Zhi Qian village Jian Chun, pure NPK processing (100% local dosage, N are picked up from for examination soil
300kg·hm-2, P2O5 120kg·hm-2, K2O 100kg·hm-2).Take the technological parameter and technique in same embodiment 1
Step.
Measure NO3 -- N, NH4 +- N concentration results are as shown in figure 3, through operation, NO3 -- N, NH4 +- N concentration after incubation 7 days
And 14 days concentration fall off rates are most fast.
The data obtained is by taking the 15:0iso of 14d as an example:
PFAME=31.656, PISTD=28.249, ng Std=6.2, W=3, so Mi=2.3159；
δ13Cc=1.245, δ13Ccontrol=1.074, δ13CM=99, so Pi=0.174；
∑ Pi=2.985, ∑ Mi=102.258, pt=2.920.
Because data volume is larger, do not repeat one by one.
20 kinds of lipoid fatty acids are measured altogether, are computed and are obtained each lipoid fatty acid13C is enriched with ratio (pt) as shown in figure 4,
Abscissa is the various lipoid fatty acids measured, and ordinate, that is, various lipoid fatty acids account for the percentage of total phospholipids fatty acid.From
Fig. 4 can be seen that, cultivate the soil anaerobic ammonia oxidation microbiological structure of community diversity of the inorganic processing rice soil of 14d.
The different microorganisms according to indicated by lipoid fatty acid, the phospholipid fatty acid content for characterizing bacterium account for about 94.0%,
And fungi accounts for about 0.60%, actinomyces account for about 5.4%.
Illustrate: soil conclusion is same as above substantially after culture 7d.
Embodiment 3, organic fertilizer inorganic fertilizer, which are matched, applies processing rice soil soil anaerobic ammonia oxidation microbiological structure of community diversity.
Jintan City of Jiangsu Province town Zhi Qian village Jian Chun, pig manure 6000kghm are picked up from for examination soil-2+ 50%NPK processing；
Wherein, pure NPK (urea, calcium superphosphate, potassium sulfate) handles fertilizer amount are as follows: N 300kghm-2, P2O5 120kg·hm-2,
K2O 100kg·hm-2；Pig manure nutrient content are as follows: organic matter 45.4%, N 2.3%, P2O52.9%, K2O1.2% contains
Water 29.1%.Take the technological parameter and processing step in same embodiment 1.
Measure NO3 -- N, NH4 +- N concentration results are as shown in figure 5, through operation, NO3 -- N, NH4 +- N concentration after incubation 7 days
And 14 days concentration fall off rates are most fast.
The data obtained is by taking the 15:0iso of 14d as an example:
PFAME=16.273, PISTD=24.569, ng Std=6.2, W=3, so Mi=1.3688；
δ13Cc=1.242, δ13Ccontrol=1.074, δ13CM=99, so Pi=0.171；
∑ Pi=1.734, ∑ Mi=62.648, pt=2.768.
Because data volume is larger, do not repeat one by one.
20 kinds of lipoid fatty acids are measured altogether, are computed and are obtained each lipoid fatty acid13C is enriched with ratio (pt) as shown in fig. 6,
Abscissa is the various lipoid fatty acids measured, and ordinate, that is, various lipoid fatty acids account for the percentage of total phospholipids fatty acid.From
Fig. 6 can be seen that, cultivate the soil anaerobic ammonia oxidation microbiological structure of community diversity of 14d organic process rice soil.
The different microorganisms according to indicated by lipoid fatty acid, the phospholipid fatty acid content for characterizing bacterium account for about 92.1%,
And fungi accounts for about 0.6%, actinomyces account for about 7.3%.
Illustrate: soil conclusion is same as above substantially after culture 7d.
The high DGGE combination qPCR of confirmatory experiment, the detection accuracy generally acknowledged using the existing industry is to embodiment 1, implementation
Example 2, soil described in embodiment 3 are detected, and the summary of acquired results is as described in Table 1:
Table 1
Comparative example 1-1, will make in soil in embodiment 113The concentration of C- urea-N is changed to 150mg/kg by 200mg/kg,
By NO3 ?- N concentration is changed to 150mg/kg by 200mg/kg, remaining is equal to embodiment 1.
The results show that bacterial content is decreased significantly (only 80% or so), fungi variation less, on unwrapping wire bacterial content has
It rises, differs larger with the data of confirmatory experiment, cannot correctly show actual result.
Comparative example 1-2, will make in soil in embodiment 113The concentration of C- urea-N is changed to 250mg/kg by 200mg/kg,
By NO3 ?- N concentration is changed to 250mg/kg by 200mg/kg, remaining is equal to embodiment 1.
The results show that bacterial content rises, fungi and unwrapping wire bacterial content reduce (actinomyces are only 3.5% or so), with
The data difference of confirmatory experiment is larger, cannot correctly show actual result.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (5)
1. soil anaerobic ammonia oxidation microbiological structure of community detection method, it is characterized in that the following steps are included:
6 containers are set, and each container successively carries out following step 1)~step 4) respectively:
1) it is 5g/L's that fresh soil and the 1.2mL concentration to be measured of 30g, is added in each container13C- urea liquid, is uniformly stirred
It mixes, to make in soil13The concentration of C- urea-N reaches 200mg/kg；
2) KNO that 1.2mL concentration is 5g/L, is added into the soil of the resulting each container of step 1)3Solution, uniform stirring, from
And make NO in soil3 ?- N concentration reaches 200mg/kg；
3) mud state, and closed container, are added with water into the resulting soil of step 2)；
4) air in container, is replaced with inert gas, anaerobic culture box is then placed in and carries out 30 ± 1 DEG C of anaerobism cultures；
5) resulting 6 containers of step 4), are cultivated into 1~2h, 1d, 3d, 7d, 10d respectively, until NO3 --N、NH4 +- N value is stablized
That day；
Content is carried out respectively to the corresponding container of 6 cultivation stages:
Soil is taken, measures soil NO using SKALAR Continuous Flow Analysis instrument3 -- N and NH4 +N content；
6), according to NO3 -- N, NH4 +The changes of contents rate of-N is chosen in container corresponding to the highest cultivation stage of rate of change
Soil, extract PLFA therein, PLFA is lipoid fatty acid；
The judgment rule of the highest cultivation stage of rate of change are as follows: the soil of 6 cultivation stages NO in identical incubation time3 --
N, NH4 +The highest cultivation stage of the concentration decrease of-N is the highest cultivation stage of rate of change, if NO3 -- N, NH4 +The change of-N
Change rate is inconsistent, then takes the mean value of the two rate of change maximum for the highest cultivation stage of rate of change；
7) it, is combined by gas-chromatography-burning-isotopic ratio mass spectrum (IRMS) in the PLFA that method measurement extracts13The abundance D of C13C,13The abundance of C is13The abundance of the lipoid fatty acid of C flag.
2. soil anaerobic ammonia oxidation microbiological structure of community detection method according to claim 1, it is characterized in that:
The method of PLFA is extracted in the step 6) the following steps are included:
Soil be added volume ratio be 1:2:0.8 chloroform: methanol: citric acid sodium citrate buffer solution (0.15M, ph4.0) into
Row extracts；
Extract is transferred to after washing away center rouge and glycolipid with chloroform and acetone respectively after silica gel solid-phase extraction column, it will with methanol
Phosphatide elution；After phosphatide addition internal standard Nonadecanoic acid methylester after elution, by mild alkalinity methylation method by fatty acid methyl
Base turns to corresponding FAMES, and FAMES is fatty acid methyl ester.
3. according to soil anaerobic ammonia oxidation microbiological structure of community detection method according to claim 2, it is characterized in that:
The step 7) are as follows:
It is separated using fatty acid methyl ester obtained by step 6) through gas-chromatography, ion detector inspection is fallen by the fire being connected after separation
It surveys, quantifying for fatty acid is carried out with this；Single PLFA's13C/12C compares mass spectrum by gas-chromatography-combustion method-stable isotope
Instrument is measured, and wherein Delta V isotope ratio matter instrument is connected with TraceGCUltra gas-chromatography, GC Combustion III；
The amount of PLFA monomer is calculated according to formula (1):
Mi=dilution × (PFAME×ng Std)/(PISTD×W) (1)
(1) in formula, Mi is the amount of single lipoid fatty acid PLFA, and unit is μ g/g；PFAMEFor sample peak area；PISTDFor internal standard
Peak area；PFAMEAnd PISTDBy being obtained by gas chromatograph；Ng Std is interior target concentration；Dilution is extension rate；W is
Dry native dry weight；
C is calculated from the amount Pi of urea-C with formula (2) in each fatty acid:
Pi=Mi* (δ13Cc-δ13Ccontrol)/(δ13CM-δ13Ccontrol) (2)
Wherein, Mi is the concentration of single PLFA, is calculated by formula (1), δ13CcAnd δ13CcontrolIt is single after marking preceding and label
The δ of fatty acid13C, δ13CMIt is the δ for adding urea13C is obtained by isotope ratio matter instrument；
PLFA is total in each soil13C is enriched with ratio ptI.e.13The abundance D of C13C is calculated with following formula (3):
pt=∑ Pi/ ∑ Mi (3)
Wherein Mi is the content of C in single fatty acid.
4. according to soil anaerobic ammonia oxidation microbiological structure of community detection method according to claim 1, it is characterized in that: institute
It states in step 1), container is the glass scre-cap culture bottle of 100mL, is jumped a queue with soft rubber plug and realizes sealing.
5. according to soil anaerobic ammonia oxidation microbiological structure of community detection method according to claim 1, it is characterized in that: institute
It states in step 3), every 30g soil adds 5~7ml of water.
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