CN1423116A - Dry farm soil microbial phosphorus determination method - Google Patents
Dry farm soil microbial phosphorus determination method Download PDFInfo
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- CN1423116A CN1423116A CN 01138248 CN01138248A CN1423116A CN 1423116 A CN1423116 A CN 1423116A CN 01138248 CN01138248 CN 01138248 CN 01138248 A CN01138248 A CN 01138248A CN 1423116 A CN1423116 A CN 1423116A
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Abstract
The method specifies the temperature of fumigating soil by using chloroform and the technical parameters of the vacuum degree as well as invents the optimal extractant and the ratio of water and soil suitable for different kinds of soils to extract phosphor of the microorganism in soil. Comparing with the prior art, the invention increases the precision and accuracy in detecting phosphor of the microorganism is soil and is applicable to the our country, even to the global. The invention also provides the reliable measurement method for following research: the microorganism conversion of phosphor in soil, the circulation of phosphor in soil and the activation of phosphor in soil.
Description
Technical field
The present invention relates to the assay method of a kind of soil microorganism, more particularly it is a kind of assay method of dry farm soil microbial phosphorus.
Background technology
Soil microorganism is not only controlled the cyclic process of the soil organism and main nutrient (as nitrogen, phosphorus, sulphur), and itself is exactly an important active nutrient storehouse, and the nutrient supply of keeping crop is played an important role.Soil microbial biomass and nutrient content are measured for the function of understanding aspects such as soil microorganism and soil nutrient transformation power and crop deliverability all significant.
The basic skills of measuring soil microbial biomass p is set up respectively by (1982) such as Brookes etc. (1982) and Hedley.
After the Brookes method adopted chloroform vapor stifling 24,0.5 mole of sodium bicarbonate extraction agent (pH8.5), was measured stifling back and is extracted Phos (P than extracting 30 minutes at 1: 20 Tu Shui
i) increment.They suppose that the stifling release rate of soil microbial biomass p is 40%, and the soil fixing rate of microbe-P is consistent with Phos (adding orthophosphate attitude phosphorus) after stifling the release.
The Hedley method adopts liquid chloroform directly to add the method stifling (30 minutes) of soil, extracts 16 hours at 1: 60 Tu Shui ratio with the sodium bicarbonate extraction agent, measures the full phosphorus (P of stifling back extraction
t) increment.They find under the situation of no soil, and the stifling release rate of cultivating microbe-P is far above 40%, the extraction ratio of adding soil also with add Phos and do not have correlativity.Although suggestions such as Hedley have adopted 0.4 as the stifling full phosphorus increment that extracts and the conversion coefficient (K of soil (pH6.2～8.2) microbe-P
p), point out simultaneously and need measure in the recovery of different soils microbe-P.
Above-mentioned two kinds of methods all have some practical problemss, particularly should adopt the sort of method must compare research to acid ground and could determine.The assumed condition of Brookes method not necessarily conforms to actual conditions.Also there are several problems in the Hedley method.The first, the soil sample that adopts air-dry back to cultivate certainly will change the original state of soil microorganism.The second, extraction time is long, for influencing extraction efficiency because of the conversion (one-tenth inorganic states) and the absorption increase of the stifling microbe-P that discharges at the strong soil of phosphorus crystallized ability.The 3rd, adopt 1: 60 Tu Shui than having limited the amount of samples (0.5 gram) of measuring soil, this its representativeness for fresh soil is not enough (crosses 2 mm sieve at least will more than 2 grams).The 4th, full phosphorus measures that not only digestion process is loaded down with trivial details, and dilution ratio further enlarges (3～5 times), thereby strengthens analytical error.These problems make the soil that this method is strong at the phosphorus crystallized ability and microbe-P is very low can not use with error at measurment is relatively large because of stifling increment is too little.
In addition, some scholar also did the improvement Journal of Sex Research to the soil microorganism phosphorus determination method, but the result who does not agree.
Summary of the invention
The objective of the invention is to overcome existing method precision and the poor accuracy of measuring microbe-P in the soil, and narrow application range, process is loaded down with trivial details to wait deficiency, and the method for having invented a kind of microbe-P of accurate mensuration Different Soil.The invention of this method is to carrying out the soil microbial biomass p and the microbial conversion of phosphorus in soil in the China and even the whole world, and researchs such as soil phosphorus activation and circulation provide reliable assay method.
The present invention adopts following technical scheme for achieving the above object: a kind of dry farm soil microbial phosphorus determination method, and this method in turn includes the following steps:
(1) the fresh soil that will gather is removed visible plant residue (as stem, leaf, root) and soil in the soil sample
Animal (earthworm etc.), sieve (2 millimeters in aperture), thoroughly mixing, adjusting water cut are for full
With about 40～50% of water holding capacity, take by weighing 9 part of 4～10 gram (oven dry basic weight) soil sample;
(2) wherein 3 parts of soil samples add the soil bacteria and the fungi (4: 4～8, weight ratio) of 0.5 milliliter of cultivation
Mixed liquid (phosphorus content is 220～270 micrograms phosphorus/milliliter); 3 parts of soil samples do not add, and exist simultaneously
0.05 under～0.08Mpa vacuum tightness and 20～40 ℃ of conditions, spent the ethanol chloroform stifling 24 hours,
Use 0.025～0.5 mole of extraction agent (Tu Shui is than 1: 10～25) to extract again after removing chloroform; Other is 3 years old
Part soil is directly used in extraction; Measure the extract phosphorus content;
(3) soil microorganism phosphorus content conversion method: soil microbial biomass p=stifling and not fumigating soil extraction
Phosphorus strengthen/add the recovery of the phosphorus of cultivating microorganism.
Described extraction agent and Tu Shui are 0.5mol/LNaHCO3 than optimum value, 1: 20.
The present invention proposes the vacuum tightness and the temperature technical parameter of the soil-fumigating of measuring Different Soil (pH3.3～7.4) microbe-P, optimum extraction agent and Tu Shui ratio, cultivation of soil bacteria and fungi and harvesting method and soil microorganism phosphorus content accurate measuring method.
A kind of dry farm soil microbial phosphorus determination method of the present invention has following advantage:
(1) the standardized vacuum tightness and the temperature parameter of soil-fumigating have improved different batches sample determination knot
The comparability of fruit;
(2) invent optimum extraction agent and Tu Shui ratio, improved microbe-P extraction survey in the Different Soil
Fixed precision;
(3) simple and effective soil bacteria and fungi culture medium and cultivation and harvesting method have been invented;
(4) invented directly to adopt and add the recovery of cultivating microbe-P and survey for dissimilar soil
Decide the method for soil microbial biomass p, improved the little life of soil more greatly with former common method
The accuracy of thing phosphorus measurement result;
(5) this method extraction agent does not have toxic side effect to operating personnel, and simple and safe operation is quick;
(6) wide adaptability is suitable for the microbe-P of the main type dryland soil of China (pH3.3～7.4)
Accurately measure, and even can be used for global dryland soil.
In sum, also do not measure a kind of standard unified method of soil microbial biomass p at present.The method of the microbe-P of a kind of accurate mensuration Different Soil of the present invention provides reliable assay method to soil microbial biomass p and the researchs such as microbial conversion, soil phophorus activation and circulation of phosphorus in soil of carrying out the China and even the whole world.
Description of drawings
Fig. 1 is an assay method schematic flow sheet of the present invention.
Embodiment
Describe the specific embodiment of the present invention in detail below in conjunction with accompanying drawing, reagent configuration, instrument and equipment, operation steps.One, reagent preparation
1.1 go the preparation of ethanol chloroform: in fuming cupboard, will analyze pure chloroform and water and add separating funnel, shake 1 minute, and emit chloroform in beaker, and so wash 3 times by 1: 2 (volume ratio).
1.2 extraction agent: select the soil phophorus extraction agent to be mixed with 0.025～0.5 volumetric molar concentration, extract pH is 4.5～8.5.
1.3 chromogenic reagent preparation:
(1) sulfuric acid solution: get 70 milliliters and analyze pure diluting concentrated sulfuric acids to 0.5 liter;
(2) ammonium molybdate solution: it is soluble in water fixed molten to 0.5 liter to get the pure ammonium molybdate of 20 gram analyses;
(3) ascorbic acid solution: get 1.32 gram ascorbic acid and be dissolved in 75 ml waters and get final product;
(4) antimony tartrate potassium solution: get 0.2743 gram and analyze water-soluble fixed molten 0.1 liter of pure potassium antimony tartrate;
(5) mix chromogenic reagent: reagent (1), (2), (3), (4) were by 10: 3: 6: 1 mixes.
1.4 soil bacteria and fungi culture medium preparation:
(1) bacteria culture media (grams per liter): beef extract 8～12, peptone 2～4, sodium chloride 3～7, fungi
Inhibitor 0.00～0.02, water 1000.
(2) fungi culture medium (grams per liter): sodium nitrate 0.5～0.8, potassium dihydrogen phosphate 0.2～0.5, magnesium sulphate 0.1～0.2,
Iron sulfate 0.005～0.007, yeast extract 0.15～0.18 and sucrose 9～11, streptomycin sulfate
0.06～0.12, tetracycline hydrochlorinate compound 0.003～0.006, water 1000.Two, instrument and equipment
Vacuum dryer, water pump vacuum extractor (preparation), reciprocal shaker, high-pressure sterilizing pot, shaking table, soil sieve, Plastic Drum (the band screw top is salable), pipettor, charger, spectrophotometer, tygon extraction flask, volumetric flask, beaker, triangular flask.Three, operation steps 3.1 sample pre-treatments:
The fresh soil of gathering should be handled immediately or be kept in 4 ℃ of refrigerator, removes visible plant residue (as stem, Ye Hegen) and soil animal (earthworm etc.) in the soil sample before the mensuration, sieve (2 millimeters in aperture), thoroughly mixing.Processing procedure should avoid destroying soil texture as far as possible, and water cut is too high should be indoor suitably air-dry, and is moistening loose but do not lump and be advisable (be 40～50% saturation moisture capacity) with feel.This sample promptly can be used for the soil The real time measure.Carry out in the big Plastic Drum that other research (as culture experiment) can place soil sealing and cultivated 7～15 days, water should be arranged keeping humidity in the bucket, in put the carbon dioxide that a cuvette sodium hydroxide solution absorbs the soil respiration generation, cultivation temperature is 25 ℃.Soil through preceding cultivation should be analyzed immediately.Keep if desired, should be positioned in 4 ℃ the constant temperature oven, need to cultivate at least under these conditions 24 hours before using next time.These processes reach the interference of plant residue tissue to measuring for eliminating the influence of soil moisture restriction to microorganism.3.2 soil bacteria and fungal culture and mixing suspension preparation:
The cultivation of soil bacteria and fungi, results and matching method have innovation.In every bottle of inoculum, inoculate 10
-40.5～1 milliliter of soil suspending liquid was cultivated 5 days under 25 ℃ of dark conditions.The results bacterium is adopted centrifugal method (15 minutes, 1500～4000 rev/mins), uses 0.85% sodium chloride solution repeated washing 3～7 times again.In every bottle of fungus culture medium, inoculate 10
-10.5～1 milliliter of soil suspending liquid was cultivated 5 days down at 25 ℃.Adopt filter method to collect hypha,hyphae, with 0.85% sodium chloride solution washing 3～7 times.Washed bacterium and fungi add an amount of 0.85% sodium chloride solution respectively and make suspending liquid, place 4 ℃ of refrigerators to preserve.Fungi is suitably broken up before using.Bacterium and fungi suspending liquid are made mixing suspension by 4: 4～8 (dry weight ratios), make it substantially near bacterium in the soil and fungi ratio.Measure bacterium and fungi mixing suspension content of tatal phosphorus, make the mixed liquor phosphorus content at 220～270 micrograms phosphorus/milliliter.3.3 soil-fumigating and extraction:
Soil through pre-treatment takes by weighing 3 parts of 4～10 gram (oven dry basic weight) soil samples, in beaker, bottom set portion has in the vacuum dryer of low amounts of water and chloroform, and put into and fill 1～3 in 25 milliliters of beakers that go ethanol chloroform and explosion-proof pearl,-0.04～-make the violent boiling of chloroform 3～5 minutes under 0.08Mpa vacuum tightness and 20～40 ℃ of temperature, 25 ℃ stifling 24 hours down.Change soil sample over to clean vacuum dryer, vacuumize 3～5 times, remove chloroform in the soil.Change soil sample over to extraction flask then, add 0.025～0.5 mole of extraction agent, concussion is filtered.Simultaneously, other gets 3 part of 4～10 gram soil sample directly extraction in extraction flask.3.4 add the microbe-P determination of recovery rates:
Take by weighing 3 part of 4～10 gram soil, add 0.5 milliliter of bacterium and fungi mixing suspension, mixing, stifling as stated above the extraction.3.5 extract phosphorus is measured:
Extract phosphorus is measured and should in time be carried out.In 25 milliliters of volumetric flasks, add an amount of (1～5ml) extract.While preparation standard phosphorus working curve, before adding mixed developer, extract and working curve thereof neutralized with hydrochloric acid, placed 1～4 hour and constantly shook to get rid of carbon dioxide.Adding distil water adds 4 milliliters and mixes chromogenic reagent to about 20 milliliters under 25～30 ℃ of temperature, constant volume, and mixing is used spectrophotometric determination.3.6 soil microorganism phosphorus content conversion method:
Soil microbial biomass p=stifling phosphorus with not fumigating soil extraction strengthens/adds the recovery of the phosphorus of cultivating microorganism.
Need to prove that for this area alternative technique personnel under the situation that does not need creative work, can also make some improvement and modification to assay method of the present invention, this belongs to protection scope of the present invention equally.
Claims (2)
1, a kind of dry farm soil microbial phosphorus determination method is characterized in that this method comprises the steps:
(1) soil pH 3.3～7.4 is removed visible plant residue and soil animal in the soil sample, sieved, mixing, adjusting water cut take by weighing soil sample;
(2) soil bacteria and the fungi with 0.5 milliliter of cultivation of 3 parts of soil samples addings mixes liquid; Bacteria culture media is (grams per liter): beef extract 8～12, peptone 2～4, sodium chloride 3～7, fungistat 0.00～0.02, water 1000; Fungi culture medium (grams per liter): sodium nitrate 0.5～0.8, potassium dihydrogen phosphate 0.2～0.5, magnesium sulphate 0.1～0.2, iron sulfate 0.005～0.007, yeast extract 0.15～0.18 and sucrose 9～11, streptomycin sulfate 0.06～0.12, tetracycline hydrochlorinate compound 0.003～0.006, water 1000; Soil sample does not add, simultaneously-0.05～-0.08Mpa vacuum tightness and 20～40 ℃ of conditions under, spent the ethanol chloroform stifling 24 hours, remove behind the chloroform again with 0.025～0.5 mole of extraction agent extraction; Soil sample is directly extracted with extraction agent; Measure phosphorus content in the extract;
(3) soil microorganism phosphorus content conversion method: soil microbial biomass p=stifling phosphorus with not fumigating soil extraction strengthens/adds the recovery of the phosphorus of cultivating microorganism.
2, a kind of dry farm soil microbial phosphorus determination method according to claim 1 is characterized in that described extraction agent and Tu Shui than being 0.5mol/LNaHCO
3, 1: 20.
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| CN 01138248 CN1423116A (en) | 2001-12-05 | 2001-12-05 | Dry farm soil microbial phosphorus determination method |
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| CN 01138248 CN1423116A (en) | 2001-12-05 | 2001-12-05 | Dry farm soil microbial phosphorus determination method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419175B (en) * | 2007-10-24 | 2010-11-10 | 中国科学院沈阳应用生态研究所 | Method for detecting organic matter in soil |
| CN102156185A (en) * | 2010-02-11 | 2011-08-17 | 日本有限公司Dgc综合研究所 | Crop culture degree measuring method for soil and judgment method for origin place camouflage |
| WO2017029592A1 (en) * | 2015-08-14 | 2017-02-23 | Indian Council Of Agricultural Research | Device and method for measurement of soil health parameters and fertilizer recommendation |
| CN106846295A (en) * | 2016-12-19 | 2017-06-13 | 河北民族师范学院 | The assay method and device of the soil organism |
| CN109507182A (en) * | 2018-12-04 | 2019-03-22 | 罗永学 | A kind of soil acidity or alkalinity is unbalance testing equipment and its application method |
| CN112540166A (en) * | 2020-12-11 | 2021-03-23 | 黑龙江省森林工程与环境研究所 | Method for rapidly determining microbial biomass nitrogen in forest soil by using carbon nitrogen analyzer |
-
2001
- 2001-12-05 CN CN 01138248 patent/CN1423116A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419175B (en) * | 2007-10-24 | 2010-11-10 | 中国科学院沈阳应用生态研究所 | Method for detecting organic matter in soil |
| CN102156185A (en) * | 2010-02-11 | 2011-08-17 | 日本有限公司Dgc综合研究所 | Crop culture degree measuring method for soil and judgment method for origin place camouflage |
| WO2017029592A1 (en) * | 2015-08-14 | 2017-02-23 | Indian Council Of Agricultural Research | Device and method for measurement of soil health parameters and fertilizer recommendation |
| CN106846295A (en) * | 2016-12-19 | 2017-06-13 | 河北民族师范学院 | The assay method and device of the soil organism |
| CN106846295B (en) * | 2016-12-19 | 2020-05-19 | 河北民族师范学院 | Method and device for measuring soil organic matter |
| CN109507182A (en) * | 2018-12-04 | 2019-03-22 | 罗永学 | A kind of soil acidity or alkalinity is unbalance testing equipment and its application method |
| CN109507182B (en) * | 2018-12-04 | 2021-07-30 | 中山市中能检测中心有限公司 | Soil pH value imbalance detection equipment and use method thereof |
| CN112540166A (en) * | 2020-12-11 | 2021-03-23 | 黑龙江省森林工程与环境研究所 | Method for rapidly determining microbial biomass nitrogen in forest soil by using carbon nitrogen analyzer |
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