CN109916840A - A method of measurement soil microbes biomass carbon nitrogen content - Google Patents
A method of measurement soil microbes biomass carbon nitrogen content Download PDFInfo
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- CN109916840A CN109916840A CN201910317938.9A CN201910317938A CN109916840A CN 109916840 A CN109916840 A CN 109916840A CN 201910317938 A CN201910317938 A CN 201910317938A CN 109916840 A CN109916840 A CN 109916840A
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Abstract
The present invention relates to a kind of methods for measuring soil microbes biomass carbon nitrogen content, and described method includes following steps: extracting soil microbial DNA in pedotheque to be measured；Extracted DNA concentration is measured using micro-spectrophotometer and is scaled unit mass soil microbial DNA content；Surveyed soil microbial DNA content multiplied by microbes biomass carbon content transformation ratio and microbial biomass nitrogen content transformation ratio, calculates soil microbes biomass carbon content and soil microbial biomass nitrogen content respectively.Method of the invention only needs a small amount of pedotheque to extract microbial DNA, just it can accurately estimate soil microbes biomass carbon nitrogen content, and chloroform suffocating treatment step is omitted in the method for measuring Soil Microbial Biomass Carbon nitrogen, transformation ratio is accurate and reliable, improve the determination efficiency of soil microbes biomass carbon nitrogen content, and operation is relatively easy, it is highly-safe.
Description
Technical field
The present invention relates to the measuring methods of Soil Microorganism biomass C nitrogen, are specifically combined existing soil
DNA extractive technique and means design a kind of new method for measuring soil microbes biomass carbon nitrogen content.
Background technique
Soil microbial biomass (Microbial Biomass, MB) refers to that those volumes are less than 5 × 10 in soil3μm3
Biological total amount, be the active part of the soil organism, but plant living is not included, such as root system of plant living, some
Big animal body should not also be included.The soil microbial biomass of broad sense includes microbes biomass carbon, nitrogen, p and s, but
It has been generally acknowledged that soil microbial biomass only refers to the microbes biomass carbon content in soil.Edaphon is as the ecosystem
In active a part, exercise important function, on the one hand, edaphon is the important component of soil, regulation life
Object geochemical cycle process, while being also the power of soil organism formation and Nutrient Transformation；On the other hand, due to microorganism
For the sensibility of variation of ecology and environment, edaphon can warn and predict the change of soil ecosystem in a short time
Change, therefore can be using soil microbial biomass as the index of instruction ecosystem function variation.Edaphon has become in recent years
One of the hot issue of domestic and international scientific research, can refer to document: Liu Anrong, Yang Teng, Xu Wei, upper remaining piece is strong, Wang Jinzhou, Liu Hui
Grain husk, Shi Yu, Chu Haiyan, the Qinghai-Tibet Platean He Jinsheng Alpine Grasslands terrestrial life diversity: progress, problem and prospect [J] biology
Diversity, 2018,26 (09): 972-987.
Although soil microbes biomass carbon content only accounts for the 1%-4% of soil organic matter, the micro- life of entire soil is accounted for
The 40%-45% of object dry matter, for soil nutrient, it is a part critically important in carbon base in soil, while the micro- life of soil
The content of object biomass C can largely react Soil Microorganism quantity number, be evaluate soil quality situation weight
Want parameter index.Similar with soil microbes biomass carbon, soil microbial biomass nitrogen is also the important storage of soil nitrogen
Library can generally account for the 2%-7% of soil nitrogen, and equally, it also assists in soil nitrogen cycle, and with nitrogens various forms of in soil
Closely related, for farmland ecosystem, soil microbial biomass nitrogen is agriculture to fertilising, farming and irrigation etc.
Movable response is very violent.In each soil types of Different ecosystems, the variation model of soil microbes biomass carbon nitrogen
It encloses very greatly, even its degree of variation is also very big between the different plant canopy lower parts of the same ecosystem and bare area soil.
Therefore, the method for Accurate Determining soil microbes biomass carbon nitrogen content is all highly paid close attention to all the time.
Both at home and abroad in research, many methods have been developed and are proposed to measure soil microbial biomass, such as initially
Colony counting method by counting single microbial quantity, converse soil microbial biomass, this method is the most fatal to be lacked
Point is can only to count Bacterial diversity, helpless to not educable edaphon.The substrate proposed later and successively
Induced respiration method, adenosine triphosphate assays, soil chitin analytic approach, phosphoric acid fatty acid analysis method and stifling cultivation and
Fumigation extraction etc., distinct methods respectively have advantage and disadvantage, use condition and use scope.On the whole, Chloroform fumigation extraction method is
Using the soil microbial biomass measuring method more approved, principle is: in vacuum desiccator, being evacuated using vacuum pump
So that the chloroform of drier bottom is boiled under vacuum conditions becomes chloroform vapor, and (generally 24 is small for a period of time for suffocating treatment
When, can be appropriately extended), the microorganism in soil causes edaphon cell rupture to release by chloroform vapor kill, releases a large amount of
Inhereditary material (soil microbial DNA) and cell inclusion；The pedotheque of suffocating treatment and non-suffocating treatment passes through a certain amount of
The K of 0.5mol/L2SO4Solution directly extracts and measures dissolved organic carbon and soluble total nitrogen, and the difference between them is passed through
K2SO4Extracting efficiency (KEC=0.45；KEN=0.54) content of Soil Microorganism biomass C nitrogen can be reacted after correction.But
There is also many problems for the stifling process for killing edaphon of traditional chloroform.First, chloroform vapor is insoluble during stifling
Yu Shui, therefore fumigating effect is easy to be influenced by soil moisture content.Furthermore fumigate incubation will receive the influence of temperature compared with
Greatly, while stifling culture can lose a large amount of inorganic nitrogens, influence the measurement of soil microbial biomass nitrogen；Second, extracting efficiency meeting
It is influenced by P in soil H value, soil agreegate and other factors, general extraction efficiency is possible to not be suitable for soil used
Earth sample；Third, stifling cultivation is inherently time-consuming and laborious, experiment condition requires high and troublesome in poeration.When suffocating treatment, need
Vacuum desiccator air-tightness is gone through, nevertheless, many times still because drier leaks air and causes the failure of an experiment.
The chloroform that experimentation is used is hypertoxic chemicals, and light-exposed easy decomposition, human body, which largely sucks, can cause acute poisoning, long-term to inhale
Enter to will lead to slow poisoning, health be endangered larger.In conclusion being badly in need of developing a kind of soil quickly, accurate, safe
Earth microorganism biological quantity measuring method.
Restriction of the research of edaphon in a very long time by technological means, but in recent years, high pass measures
The development of the modern science and technology such as sequence technology and bioinformatics has greatly pushed the development of soil microbiology.The micro- life of soil
Object DNA extraction process also more depends on DNA extraction kit, but commercial reagents box generally possesses easy to operate, extraction effect
The advantages such as the DNA purity is high that rate is high, obtains, thus by everybody favor.The DNA extraction process of standard helps to obtain high-quality
The soil microbial DNA of amount, and the repeatability tested is high；In addition, there will be a small amount of research has shown that soil microbial DNA content
There is positive correlation between soil microbes biomass carbon nitrogen.Above-mentioned two aspects background provides greatly for realization of the invention
Support.
But how to determine between soil microbial DNA content and soil microbes biomass carbon nitrogen relationship and how bright
All there are no clear for true Microbial nitrogen content transformation ratio.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of sides for measuring soil microbes biomass carbon nitrogen content
Method, described method includes following steps:
Step 1: extracting soil microbial DNA in pedotheque to be measured；
Step 2: measuring extracted DNA concentration using micro-spectrophotometer and be scaled unit mass soil microbial DNA and contain
Amount；
Step 3: surveyed soil microbial DNA content is raw multiplied by microbes biomass carbon content transformation ratio Fc and microorganism respectively
Object amount nitrogen content transformation ratio Fn, calculates soil microbes biomass carbon content and soil microbial biomass nitrogen content.
Preferably, in the above method, microbes biomass carbon content transformation ratio Fc is 37.20 in step 3, and microorganism is raw
Object amount nitrogen content transformation ratio Fn is 4.02.
Preferably, in the above method, the concrete operations of step 1 are as follows: soil microbial DNA extracts kit is used, from one
Determine to extract soil microbial DNA in the pedotheque to be measured of quality；
Preferably, in the above method, the soil microbial DNA extracts kit is Power Soil DNA Isolation
Kit kit.
Preferably, in the above method, the concrete operations of step 2 are as follows: measure extracted DNA's using micro-spectrophotometer
Concentration, and surveyed DNA concentration is scaled to the content of DNA in unit mass soil；
Preferably, in the above method, the micro-spectrophotometer is 2000 UV-Vis of NanoDropTM
spectrophotometer。
Preferably, in the above method, the quality of the pedotheque to be measured of the certain mass is 0.2 ~ 0.5g.
Preferably, in the above method, extracted DNA concentration is measured using micro-spectrophotometer and is scaled unit mass
The method of soil microbial DNA content are as follows: 2000 the surveyed DNA concentration of UV-Vis spectrophotometer of NanoDropTM
Unit be ng/ μ l, by this unit conversion be unit quality soil in contained DNA amount, unit be μ g/g, specific method be by
DNA concentration is multiplied by the DNA volume finally extracted again divided by the quality of soil, as unit mass soil microbial DNA content.
The present invention also provides a kind of calculating microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen to contain
The method for measuring transformation ratio Fn, described method includes following steps:
Step 1: collecting microorganism
It collects the pedotheque of terrestrial ecosystems and is subdivided into different microhabitats；
Step 2: establishing the regression equation of DNA content Yu soil microbes biomass carbon and biomass nitrogen
1) it extracts microorganism: extracting soil microbial DNA in microhabitat；
2) it measures soil microbial DNA concentration: measuring soil microbial DNA concentration with micro-spectrophotometer, and by surveyed DNA
Concentration unit ng/ μ l is scaled μ g/g, with DNA concentration multiplied by the DNA volume finally extracted, then divided by the quality of soil, obtains
Unit mass soil microbial DNA content；
3) it establishes regression equation: calculating the average value of unit mass soil microbial DNA content in microhabitat, then with traditional chlorine
The average value of imitative fumigation extraction measurement soil microbes biomass carbon nitrogen content, establishes unit mass edaphon respectively
The regression equation of the average value of the average value and soil microbes biomass carbon nitrogen content of DNA content is built using least square method
The regression equation of vertical soil microbial DNA content and microbes biomass carbon nitrogen content, when unit mass soil microbial DNA contains
When amount is significantly positively correlated with actual measurement soil microbes biomass carbon nitrogen content, using the regression slope of equation as edaphon
DNA content is converted into microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio Fn；
Step 3: verifying the accuracy and reliability of transformation ratio
In the data of all pedotheques, fitting DNA concentration is calculated microbes biomass carbon nitrogen multiplied by transformation ratio and contains
Data are measured, microbes biomass carbon nitrogen content is calculated by following formula:
Microbes biomass carbon content=Fc × unit mass soil microbial DNA content
Microbial biomass nitrogen content=Fn × unit mass soil microbial DNA content
Utilize standard main shaft regression fit microbes biomass carbon nitrogen content measured data and microbes biomass carbon nitrogen content meter
The relationship for the evidence that counts, when soil microbes biomass carbon nitrogen content measured data and microbes biomass carbon nitrogen content calculate data
It is significant to be positively correlated, it is determined that microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio Fn is closed
Lattice.
Preferably, in the above method, the terrestrial ecosystems are 3-8, and the quantity of microhabitat is 15-30, preferably
, terrestrial ecosystems are a variety of in desert, farmland, meadow, grassy marshland and forest.
Beneficial effects of the present invention
Method of the invention only needs a small amount of pedotheque to extract microbial DNA, just can accurately estimate soil microbial biomass
Carbon nitrogen content.
Chloroform suffocating treatment step is omitted in the method that the present invention measures Soil Microbial Biomass Carbon nitrogen, and transformation ratio accurately may be used
It leans on, improves the determination efficiency of soil microbes biomass carbon nitrogen content, and operation is relatively easy, it is highly-safe.
The present invention also provides a kind of reproducible, accuracy is high, and universality is good, and simple and easy Microbial nitrogen contains
The method for measuring transformation ratio, and microbes biomass carbon nitrogen content transformation ratio can be accurately determined using this method, it applies
The transformation ratio being capable of Accurate Determining soil microbes biomass carbon content and nitrogen content.
Detailed description of the invention
Fig. 1 is soil microbial DNA content and microbes biomass carbon nitrogen content correlativity figure, wherein CmicWith
NmicSoil microbes biomass carbon and biomass nitrogen are represented, * * * represents the conspicuousness of statistics as P < 0.001, pictorial representation 5
25 microhabitats of a ecosystem.
Fig. 2 is that microbes biomass carbon nitrogen content measured data is related to microbes biomass carbon nitrogen content calculating data
Relational graph, wherein SMA represents the recurrence of standard main shaft, and slope represents regression slope, * * * represents the conspicuousness that counts as P <
0.001。
English meaning difference in Fig. 1 and Fig. 2 is as follows:
Calculated value calculated value, observed value observation, Desert desert, the farmland Cropland,
The meadow Meadow, Forest forest, Steppe grassy marshland, Bare land bare area, under Under canopy is preced with, Corn high
Density corn high density, density in Corn medium density corn, Corn low density corn low-density,
Density in Soybeans medium density soybean, Soybeans low density soybean low-density, Weed weeds,
The level land Flat, Fertilize fertilising ground, Sunny slopes tailo, the hillside fields Slopes, Ungrazed wetlands taboo is herded wet
Ground, Grazed wetlands herd wetland.
Specific embodiment
It is the preferred embodiment of the present invention below, it is noted that for those skilled in the art,
Following embodiments do not have any limitation to the present invention, and without departing from the principle of the present invention, can also make
Several improvements and modifications, these modifications and embellishments are also considered to be within the scope of the present invention.
Embodiment 1
Step 1: collecting microorganism
Collect desert, farmland, meadow, grassy marshland and forest 5 main terrestrial lands ecosystems 138 pedotheques, these soil
Earth sample is further subdivided into 25 different microhabitats according to the difference in locating habitat, wherein specific terrestrial ecosystems and micro-
Habitat are as follows:
Desert: under hat, bare area；
Farmland: 900 plants/m of corn high density2, 100 plants/m of density in corn2, 1 plant/m of corn low-density2, soybean high density 900
Strain/m2, 100 plants/m of density in soybean2, 1 plant/m of soybean low-density2, weeds samples；
Meadow；
Grassy marshland: level land, fertilising ground, tailo, hillside fields, taboo herd wetland, herd wetland；
Forest: 2245 meters of height above sea level soil depth 0-20cm, 20-40cm, 40-60cm, 2416 meters of height above sea level of soil depth 0-
20cm, 20-40cm, 40-60cm, 2587 meters of height above sea level soil depth 0-20cm, 20-40cm, 40-60cm.
Step 2: establishing the regression equation of DNA content Yu soil microbes biomass carbon and biomass nitrogen
1) microorganism is extracted
Soil microbial DNA in 25 microhabitats is extracted, the kit for extracting soil microbial DNA selects Power Soil
DNA Isolation Kit (MOBIO Laboratories Inc., Carlsbad, CA, USA) kit, accurately weighs
0.4g pedotheque, according to Power Soil DNA Isolation Kit (MOBIO Laboratories Inc.,
Carlsbad, CA, USA) kit specification, soil DNA is extracted in draught cupboard.
2) soil microbial DNA content is measured
Soil microbial DNA concentration is measured, the instrument of measurement soil microbial DNA concentration is micro-spectrophotometer
2000 UV-Vis spectrophotometer of NanoDropTM (Thermo Fisher Scientific, USA),
The surveyed DNA concentration unit of 2000 UV-Vis spectrophotometer of NanoDropTM is ng/ μ l, is by this unit conversion
Unit mass soil microbial DNA content, unit are μ g/g, and method is by DNA concentration multiplied by the DNA volume finally extracted,
Again divided by soil quality, unit mass soil microbial DNA content is obtained.
3) regression equation is established
The average value of soil microbial DNA content in 25 microhabitats is calculated, is then surveyed respectively with tradition Chloroform fumigation extraction method
Determine the average value of soil microbes biomass carbon nitrogen content, establishes soil microbial DNA content and edaphon biology respectively
The regression equation of amount carbon nitrogen content establishes soil microbial DNA content and microbes biomass carbon using least square method respectively
The regression equation of nitrogen content, the result is shown in Figure 1.The result shows that soil microbial DNA content contains with soil microbes biomass carbon nitrogen
Amount is significant positive correlation, and the degree of fitting of equation is high, R2Respectively 0.94 and 0.92, the regression slope of equation is respectively as soil
Microbial DNA content is converted into microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio
Fn is calculated transformation ratio and is respectively as follows: Fc=37.20, Fn=4.02.
Step 3: verifying the accuracy and reliability of transformation ratio
In the data of all 138 pedotheques, soil microbial DNA content is fitted multiplied by microbes biomass carbon nitrogen content
Transformation ratio, is calculated microbes biomass carbon nitrogen content, and soil microbes biomass carbon nitrogen content passes through following formula meter
It calculates:
Soil microbes biomass carbon content=37.20 × unit mass soil microbial DNA content
Soil microbial biomass nitrogen content=4.02 × unit mass soil microbial DNA content
Utilize standard main shaft regression fit microbes biomass carbon nitrogen content measured data and microbes biomass carbon nitrogen content meter
The relationship for the evidence that counts, as a result as shown in Fig. 2, as a result, it has been found that soil microbes biomass carbon nitrogen content measured data and the micro- life of soil
Object biomass C nitrogen content calculates data and is significantly positively correlated, R2Respectively 0.91 and 0.88, the results showed that DNA content respectively multiplied by
The two transformation ratios can accurately react actual measurement soil microbes biomass carbon nitrogen content.
Content provided by embodiment of the present invention is described in detail above, herein to the principle of the present invention and reality
The mode of applying is expounded and illustrates, described above to be merely used to help understand method and its core concept of the invention；Meanwhile
For those of ordinary skill in the art, according to the thought of the present invention, has change in specific embodiments and applications
Become place, in conclusion the contents of this specification are not to be construed as limiting the invention.
Claims (10)
1. a kind of method for measuring soil microbes biomass carbon nitrogen content, described method includes following steps:
Step 1: extracting soil microbial DNA in pedotheque to be measured；
Step 2: measuring extracted DNA concentration using micro-spectrophotometer and be scaled unit mass soil microbial DNA and contain
Amount；
Step 3: surveyed soil microbial DNA content is raw multiplied by microbes biomass carbon content transformation ratio Fc and microorganism respectively
Object amount nitrogen content transformation ratio Fn, calculates soil microbes biomass carbon content and soil microbial biomass nitrogen content.
2. according to the method described in claim 1, it is characterized in that microbes biomass carbon content transformation ratio Fc is in step 3
37.20, microbial biomass nitrogen content transformation ratio Fn are 4.02.
3. according to the method described in claim 1, it is characterized in that the concrete operations of step 1 are as follows: mentioned using soil microbial DNA
Kit is taken, extracts soil microbial DNA from the pedotheque to be measured of certain mass.
4. according to the method described in claim 3, it is characterized in that the soil microbial DNA extracts kit is Power
Soil DNA Isolation Kit kit.
5. according to the method described in claim 1, it is characterized in that the concrete operations of step 2 are as follows: utilize micro-spectrophotometer
The concentration of extracted DNA is measured, and surveyed DNA concentration is scaled to the content of DNA in unit mass soil.
6. according to the method described in claim 5, it is characterized in that the micro-spectrophotometer is NanoDropTM 2000
UV-Vis spectrophotometer。
7. according to the method described in claim 3, it is characterized in that the quality of the pedotheque to be measured of the certain mass is 0.2
~0.5g。
8. according to the method described in claim 5, it is characterized in that measuring extracted DNA concentration simultaneously using micro-spectrophotometer
The method for being scaled unit mass soil microbial DNA content are as follows: 2000 UV-Vis of NanoDropTM
The surveyed DNA concentration unit of spectrophotometer is ng/ μ l, is contained DNA in unit quality soil by this unit conversion
Amount, unit are μ g/g, and specific method is multiplied by the DNA volume finally extracted by DNA concentration again divided by the quality of soil, as
Unit mass soil microbial DNA content.
9. a kind of side for calculating microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio Fn
Method, described method includes following steps:
Step 1: collecting microorganism
It collects the pedotheque of terrestrial ecosystems and is subdivided into different microhabitats；
Step 2: establishing the regression equation of DNA content Yu soil microbes biomass carbon and biomass nitrogen
1) it extracts microorganism: extracting soil microbial DNA in microhabitat；
2) it measures soil microbial DNA concentration: measuring soil microbial DNA concentration with micro-spectrophotometer, and by surveyed DNA
Concentration unit ng/ μ l is scaled μ g/g, with DNA concentration multiplied by the DNA volume finally extracted, then divided by the quality of soil, obtains
Unit mass soil microbial DNA content；
3) it establishes regression equation: calculating the average value of unit mass soil microbial DNA content in microhabitat, then with traditional chlorine
The average value of imitative fumigation extraction measurement soil microbes biomass carbon nitrogen content, establishes unit mass edaphon respectively
The regression equation of the average value of the average value and soil microbes biomass carbon nitrogen content of DNA content is built using least square method
The regression equation of vertical soil microbial DNA content and microbes biomass carbon nitrogen content, when unit mass soil microbial DNA contains
When amount is significantly positively correlated with actual measurement soil microbes biomass carbon nitrogen content, using the regression slope of equation as edaphon
DNA content is converted into microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio Fn；
Step 3: verifying the accuracy and reliability of transformation ratio
In the data of all pedotheques, fitting DNA concentration is calculated microbes biomass carbon nitrogen multiplied by transformation ratio and contains
Data are measured, microbes biomass carbon nitrogen content is calculated by following formula:
Microbes biomass carbon content=Fc × unit mass soil microbial DNA content
Microbial biomass nitrogen content=Fn × unit mass soil microbial DNA content
Utilize standard main shaft regression fit microbes biomass carbon nitrogen content measured data and microbes biomass carbon nitrogen content meter
The relationship for the evidence that counts, when soil microbes biomass carbon nitrogen content measured data and microbes biomass carbon nitrogen content calculate data
It is significant to be positively correlated, it is determined that microbes biomass carbon content transformation ratio Fc and microbial biomass nitrogen content transformation ratio Fn is closed
Lattice.
10. according to the method described in claim 9, the quantity of microhabitat is it is characterized in that terrestrial ecosystems are 3-8
20-30, terrestrial ecosystems are a variety of in desert, farmland, meadow, grassy marshland and forest.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112379078A (en) * | 2020-10-22 | 2021-02-19 | 广东省科学院生态环境与土壤研究所 | Method for measuring carbon isotope content of soil microorganisms |
-
2019
- 2019-04-19 CN CN201910317938.9A patent/CN109916840A/en active Pending
Non-Patent Citations (3)
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| HAIYANG GONG ET.AL: "Soil microbial DNA concentration is a powerful indicator for estimating soil microbial biomass C and N across arid and semi-arid regions in northern China", 《APPLIED SOILECOLOGY》 * |
| SAKI YOKOYAMA ET.AL: "The high correlation between DNA and chloroform-labile N in various types of soil", 《APPLIED SOIL ECOLOGY》 * |
| 陶澍: "《应用数理统计方法》", 31 August 1994, 中国环境科学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112379078A (en) * | 2020-10-22 | 2021-02-19 | 广东省科学院生态环境与土壤研究所 | Method for measuring carbon isotope content of soil microorganisms |
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