CN1833484A - Method of united fixing desert barren sand by utilizing thallose - Google Patents
Method of united fixing desert barren sand by utilizing thallose Download PDFInfo
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- CN1833484A CN1833484A CN 200510055383 CN200510055383A CN1833484A CN 1833484 A CN1833484 A CN 1833484A CN 200510055383 CN200510055383 CN 200510055383 CN 200510055383 A CN200510055383 A CN 200510055383A CN 1833484 A CN1833484 A CN 1833484A
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Abstract
A method for controlling the flowing sand of desert by the combination of alga with bacterium includes proportionally mixing the eremophytic algae powder with Bacillus edaphicus powder, proportionally inoculating the mixture in the flowing sand of desert while sprinkling nutrients conditioning liquid, and covering by ground film.
Description
Technical field
The present invention relates to the fixedly method of desert drift sand of a kind of bacterium united algae, thereby specifically relate to a kind ofly utilize that the algae synergy forms the method that the desert surface crust is checked winds and fixed drifting sand fast in bacillus in the soil and the skinning.
Background technology
Desert earth's surface biological skinning is the product of arid biogeographic zone particular surroundings.By object spectrum and remote sensing technology, can observe desert surface has 40～60% surface to be covered by this special " skin " approximately.But the desert earth's surface biological is green for a long time by the nature dew and the precipitation four seasons, can adapt to the low nutrient environment between subzero 10 ℃ to 60 ℃, select them as fixing the sand pionner, in the large tracts of land drift sand, cultivate, can replenish and improve China's ecosystem of checking winds and fixing drifting sand of " Qiao, filling, grass " combination of employing at present, especially be suitable for the recovery after desertificated area enforcement Important Project is destroyed the face of land.
" biological breadcrust " keeping soil moisture, fixedly playing the part of important role in the content of organic matter of the nitrogen in the atmosphere and increase soil.The high-moisture of skinning can make rainfall be retained in the soil crust surface morely, the growth and the breeding that help unicellular lower eukaryote in the skinning also can and change environment by its life metabolic way influence, play an important role at aspects such as checking winds and fixing drifting sand, prevent the soil erosion, change moisture distributing state.By analysis to the skinning constituent, can learn that one of main component in the skinning is an algae substances, they in case in soil and desert raised growth, just can be to the material of external secretion based on polysaccharide, thus bond soil particle and grains of sand formation skinning.Yet the skinning that Dan Youzao kind tackling quicksand forms is poor growth under field conditions (factors), needs could form for a long time the crust vegetation of high coverage rate.
Summary of the invention
The objective of the invention is to overcome prior art and only use algae kind tackling quicksand, form the slow-paced defective of skinning, thereby the fixedly method of desert drift sand of a kind of simple, bacterium united algae that fixes the sand and be applicable to industrial-scale production that can be quick and lasting is provided.
The objective of the invention is to realize by the following technical solutions:
The invention provides the fixedly method of desert drift sand of a kind of bacterium united algae, it specifically comprises the steps: for desert algae and soil bacillus are united use
1) separation and cultivation bacillus from soil crust:
Selection is mixed from the soil sample and the sterile water at 2～10cm place under arid, semiarid or the desert surface crust, makes the soil dilution after 100 ℃ of high temperature treatment, and this soil dilution is inoculated in the beef-protein medium flat board, is inverted and cultivates; Cultured bacterial classification uses the spore staining method to identify, isolates dyed back observation gemma under oily mirror and is green, the stable on heating soil bacillus that thalline takes on a red color;
The classification called after of this soil bacillus: bacillus sp, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on March 9th, 2005, its preserving number is CGMCC No.1324;
With on the above-mentioned soil bacillus liquid medium within I that from soil crust, separates according to a conventional method through after the liquid culture of continuously fermenting on a large scale, centrifugal, the supernatant that inclines obtains freeze-dried powder after the bacterial sediment thing freeze drying of lower floor, preserves standby in refrigerator; Described liquid nutrient medium I is for formulated in following ratio: add 80～100g corn steep liquor in every premium on currency, and 5g sodium chloride, pH 7.0～7.5;
2) separation and cultivation desert algae from soil crust:
Gather arid, semiarid or desert soil surface skining, remove foreign material such as phytoclasts wherein after, be ground to the sub-sieve that can cross 0.5mm; This culture sample after uviol lamp shines down, is added the sterile distilled water vibration, make homogeneous suspension, will be inoculated in liquid nutrient medium II after it dilution, place illumination box to cultivate; Be transferred on the solid culture medium III after the algae kind chosen, until obtaining purifying, free of contamination desert algae kind;
The desert algae kind access of this purifying is contained in the triangular flask of liquid nutrient medium II, illumination cultivation and gap shaking flasks, the desert algae that obtains is collected by centrifugal or filtration, collects the algae sheet, and pulverizing obtains desert algae dry powder;
Described liquid nutrient medium II prepares as follows: with potassium nitrate 0.8～2.0g, magnesium sulfate 0.05～1.2g, potassium dihydrogen phosphate 0.02～0.05g and citric acid 0.004～0.008g with a small amount of dissolved in distilled water after, add distilled water to 980ml; Behind nitrate of lime 2～4g usefulness 20ml dissolved in distilled water, join in the above-mentioned solution; Add plant growth regulator 0.1～1ppm again;
Described solid culture medium III is the agar of adding 1% in the aforesaid liquid medium ii, after heating is dissolved, and sterilization 30min under 15 pounds of pressure;
3) field inoculation:
With step 1) and 2) from skinning, separate and the desert algae dry powder cultivated and soil bacillus dry powder by 2～3: 1 quality than mixing, form sand-consolidating agent, and by 2.0～4.0g/ rice
2Inoculum concentration be inoculated in the desert drift sand; Spraying liquid nutrition recuperating agent simultaneously, and use plastic mulching, described liquid nutritional conditioner comprises liquid nutrient medium II and acrylic compounds water-loss reducer 0.1～1g/Kg.
The invention reside in the bacillus and the desert algae that extensively exist in the soil crust of arid and semi-arid area are united use, the soil bacillus has the viscosity pod membrane, gelatinous sheath or thick pectic substance outer wall, in growth, can secrete a large amount of mucus, thereby these has the appendicular thalline of viscosity and stick nature liquid by combining the basis that has constituted skinning with the commissure of algae substances.Use this kind method to fix the sand 20～40 days the time, the face of land can form micro-skinning when every square metre of algae coverage rate can reach 50～70%, 50 days.
The present invention compared with prior art, its advantage is:
1, this method is simple, easy to operate, equipment is simple.
2, method provided by the invention is that microorganism and algae are united use, and both interact and promote growth, thereby, to compare with single technology of fixing the sand in the past by the large-scale culture of algae, the desert soil that makes that this kind method can be quicker, lasting forms skinning.
3, this method need not the bacterial classification and the algae kind of being separated are carried out strict kind purifying evaluation, has omitted in the past loaded down with trivial details and unnecessary operating procedure.
4, utilize method provided by the invention to fix the sand the formed soil in back,, be more conducive to its and plant microorganism and some low microtubule fasolculus plant growings that waits, firmer therefrom drift sand for the desert provides abundant carbon source and nitrogenous source.
5, method provided by the invention can be applicable to industrial-scale production.
Embodiment
Embodiment 1,
Selection is mixed with sterile water from the soil sample 10g of 2cm under the soil surface crust of Xinjiang of China Junggar Basin area, puts into to fill 90ml's and have behind the triangular flask vibration 20min of bead triangular flask is placed on and make 10 after boiling water heats 10min
-2, 10
-3, 10
-4, 10
-54 kinds of dilution soil supensions are respectively drawn 0.2ml and are inoculated in the flat board that the bacterium beef-protein medium is housed in each dilution soil supension, culture medium flat plate is upside down in 37 ℃ the incubator to cultivate 2 days.Use spore staining method is identified cultured bacterial classification, and cultured thalline smear is dry fixing, observes under dye liquor dyes dry rearmounted oily mirror, isolates dyed back observation gemma under oily mirror and is green, the stable on heating soil bacillus that thalline takes on a red color; The classification called after of this soil bacillus: bacillus sp, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on March 9th, 2005, its preserving number is CGMCC No.1324.
Use the cheap liquid nutrient medium I-1 that optimizes to carry out large scale fermentation the above-mentioned soil bacillus that from soil crust, separates, consisting of of this liquid nutrient medium I-1 contains corn steep liquor 80g in every premium on currency, sodium chloride 5g, pH 7.0, with this understanding, with the extensive continuous liquid fermented and cultured of the soil bacillus of above-mentioned separation 3 days, centrifugal zymotic fluid, supernatant inclines, the bacterial sediment thing freeze drying of lower floor, obtain the bacillus freeze-dried powder, its yield is every liter of zymotic fluid of 90g/, preserves standby in refrigerator.
Gather the thick soil surface crust of the area 0.5cm of the Xinjiang of China Junggar Basin with the sampler of washing and sterilizing.The sample of gathering is placed in the culture dish and ground reagent bottle of cleaning sterilization, gets the sample in the bottle, carefully choose residual foreign material such as phytoclasts, be placed in the mortar and break into pieces gently, until all can be by the sub-sieve of 0.5mm.Take by weighing this culture sample 20g and add sterile distilled water 180ml in the 250ml triangular flask in shining about 2min under the efficient uviol lamp of 60W, being placed on then, vibration 20min makes homogeneous suspension, is diluted to 10 in proportion then
-2, 10
-3, 10
-4, 10
-54 grades also are inoculated in liquid nutrient medium II-1 with it.
Condition of culture is in the illumination box of 4 40 watts fluorescent tube, the photoperiod: light/dark=16h/8h, and temperature is cultivated down for about 25 ℃, and cultivating fate is about 10 days.The algae kind of growing in the plating medium is chosen with aseptic glass bar or pincet, on aseptic slide, clean with liquid nutrient medium II-1, it is inoculated on the new flat board that solid culture medium III-1 is housed again, obtains to be transferred to the inclined-plane preservation after the pollution-free algae kind.The pollution-free algae kind of growing in the inclined-plane is carried out liquid culture in enormous quantities, and inoculum concentration is that algae liquid optical density is controlled at about 0.1A, and periodicity of illumination is the same, and cultivation cycle is 6 days, the cell number after instrumentation is cultivated, 70,000 cells/ml as a result.The desert algae that obtains after the cultivation is applied on the tiffany by centrifugal or filter to collect, collect the algae sheet after, pulverize with cracker, obtain desert algae dry powder.Cultivate and all under aseptic condition, carry out.
Described liquid nutrient medium II-1 prepares as follows: with potassium nitrate 1g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 0.03g and citric acid 0.005g with a small amount of dissolved in distilled water after, add distilled water to 980ml; Behind nitrate of lime 2.5g usefulness 20ml dissolved in distilled water, join in the above-mentioned solution; Add plant growth regulator triacontanol 0.1ppm again.
Described solid culture medium III-1 is the agar of adding 1% in aforesaid liquid medium ii-1, after heating is dissolved, and sterilization 30min under 15 pounds of pressure.
After the above-mentioned desert algae dry powder that separates and cultivate and soil bacillus dry powder mixed with 2: 1 mass ratio, press 2.0g/ rice from skinning
2Amount be inoculated in the desert drift sand; Press 0.5-1 kilogram/rice simultaneously
2Use portable sprinkler rig spray to irrigate the liquid nutritional conditioner, and use plastic mulching, pricking on the mulch film has the cavity to be convenient to circulation of air, contains aforesaid liquid medium ii-1 in this liquid nutritional conditioner, also contains Sodium Polyacrylate-polyacrylamide water-loss reducer 0.1g/Kg simultaneously.
Under this kind condition of culture, occur the algal grown sign in the mulch film after 5～7 days, throw off mulch film after forming micro-skinning when the algae coverage rate reaches 50%, 50 day left and right sides during 40 days left and right sides, let alone freely to grow.
Embodiment 2,
Selection is mixed with sterile water from the soil sample 10g of 6cm under the soil surface crust of Xinjiang of China Junggar Basin area, puts into to fill 90ml's and have behind the triangular flask vibration 20min of bead triangular flask is placed on and make 10 after boiling water heats 10min
-2, 10
-3, 10
-4, 10
-54 kinds of dilution soil supensions are respectively drawn 0.2ml and are inoculated in the flat board that the bacterium beef-protein medium is housed in each dilution soil supension, culture medium flat plate is upside down in 37 ℃ the incubator to cultivate 3 days.Use spore staining method is identified cultured bacterial classification, and cultured thalline smear is dry fixing, observes under dye liquor dyes dry rearmounted oily mirror, isolates dyed back observation gemma under oily mirror and is green, the stable on heating soil bacillus that thalline takes on a red color.
Use the cheap liquid nutrient medium I-2 that optimizes to carry out large scale fermentation the above-mentioned soil bacillus that from soil crust, separates, consisting of of this liquid nutrient medium I-2 contains corn steep liquor 90g in every premium on currency, sodium chloride 5g, pH 7.5, with this understanding, with the extensive continuous liquid fermented and cultured of the soil bacillus of above-mentioned separation 3 days, centrifugal zymotic fluid, supernatant inclines, the bacterial sediment thing freeze drying of lower floor, obtain the bacillus freeze-dried powder, its yield is every liter of zymotic fluid of 120g/, preserves standby in refrigerator.
Under the situation of basic operation with embodiment 1, desert algae liquid nutrient medium II-2 consists of: potassium nitrate 1.5g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 0.04g, citric acid 0.006g, nitrate of lime 3g, plant growth regulator triacontanol 0.5ppm.Described solid culture medium III-2 is the agar of adding 1% in aforesaid liquid medium ii-2, after heating is dissolved, and sterilization 30min under 15 pounds of pressure.Frustule number after fermentation back instrumentation is cultivated with this understanding, the result is 300,000 cells/ml.
After the above-mentioned desert algae dry powder that separates and cultivate and soil bacillus dry powder mixed with 3: 1 mass ratio, press 3.0g/ rice from skinning
2Amount be inoculated in the desert drift sand; Press 0.5-1 kilogram/rice simultaneously
2Use portable sprinkler rig spray to irrigate the liquid nutritional conditioner, and use plastic mulching, pricking on the mulch film has the cavity to be convenient to circulation of air, contains aforesaid liquid medium ii-2 in this liquid nutritional conditioner, also contains Sodium Polyacrylate-polyacrylamide water-loss reducer 0.5g/Kg simultaneously.
Under this kind condition of culture, occur the algal grown sign in the mulch film after 3～5 days, throw off mulch film after forming micro-skinning when the algae coverage rate reaches 70%, 50 day left and right sides during 30 days left and right sides, let alone freely to grow.
Embodiment 3,
Selection is mixed with sterile water from the soil sample 10g of 10cm under the soil surface crust of Xinjiang of China Junggar Basin area, puts into to fill 90ml's and have behind the triangular flask vibration 20min of bead triangular flask is placed on and make 10 after boiling water heats 10min
-2, 10
-3, 10
-4, 10
-54 kinds of dilution soil supensions are respectively drawn 0.2ml and are inoculated in the flat board that the bacterium beef-protein medium is housed in each dilution soil supension, culture medium flat plate is upside down in 37 ℃ the incubator to cultivate 3 days.Use spore staining method is identified cultured bacterial classification, and cultured thalline smear is dry fixing, observes under dye liquor dyes dry rearmounted oily mirror, isolates dyed back observation gemma under oily mirror and is green, the stable on heating soil bacillus that thalline takes on a red color.
Use the cheap liquid nutrient medium I-3 that optimizes to carry out large scale fermentation the above-mentioned soil bacillus that from soil crust, separates, consisting of of this liquid nutrient medium I-3 contains corn steep liquor 100g in every premium on currency, sodium chloride 5g, pH 7.5, with this understanding, with the extensive continuous liquid fermented and cultured of the soil bacillus of above-mentioned separation 3 days, centrifugal zymotic fluid, supernatant inclines, the bacterial sediment thing freeze drying of lower floor, obtain the bacillus freeze-dried powder, its yield is every liter of zymotic fluid of 80g/, preserves standby in refrigerator.
Under the situation of basic operation with embodiment 1, desert algae liquid nutrient medium II-3 consists of: potassium nitrate 2.0g, magnesium sulfate 1.2g, potassium dihydrogen phosphate 0.05g, citric acid 0.008g, nitrate of lime 4g, plant growth regulator triacontanol 1ppm.Described solid culture medium III-3 is the agar of adding 1% in aforesaid liquid medium ii-3, after heating is dissolved, and sterilization 30min under 15 pounds of pressure.Cell number after fermentation back instrumentation is cultivated with this understanding, 10,000 cells/ml as a result, this is because the plant growth regulator of high concentration suppresses the growth of algae substances.
After the above-mentioned desert algae dry powder that separates and cultivate and soil bacillus dry powder mixed with 3: 1 mass ratio, press 4.0g/ rice from skinning
2Amount be inoculated in the desert drift sand; Press 0.5-1 kilogram/rice simultaneously
2Use portable sprinkler rig spray to irrigate the liquid nutritional conditioner, and use plastic mulching, pricking on the mulch film has the cavity to be convenient to circulation of air, contains aforesaid liquid medium ii-3 in this liquid nutritional conditioner, also contains Sodium Polyacrylate-polyacrylamide water-loss reducer 1fg/Kg simultaneously.
Under this kind condition of culture, occur the algal grown sign in the mulch film after 3～5 days, throw off mulch film after forming micro-skinning when the algae coverage rate reaches 60%, 50 day left and right sides during 35 days left and right sides, let alone freely to grow.
As can be seen from the above-described embodiment, the present invention unites use with the bacillus and the desert algae that extensively exist in the soil crust of arid and semi-arid area, both interact and promote growth, thereby, compare with single technology of being fixed the sand by the large-scale culture of algae in the past, the desert soil that makes that this kind method can be quicker, lasting forms skinning, uses this kind method to fix the sand 20～40 days the time, the face of land can form micro-skinning when every square metre of algae coverage rate can reach 70～90%, 50 days.And, utilize method provided by the invention to fix the sand the formed soil in back, for the desert provides abundant carbon source and nitrogenous source, be more conducive to its and plant microorganism and some low microtubule fasolculus plant growings that waits, firmer therefrom drift sand.
Claims (5)
1, the fixing method of desert drift sand of a kind of bacterium united algae, it is for to unite use with desert algae and soil bacillus.
2, the fixing method of desert drift sand of bacterium united algae as claimed in claim 1, it is characterized in that: the classification called after of described soil bacillus: bacillus sp, be preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on March 9th, 2005, its preserving number is CGMCC No.1324.
3, the fixing method of desert drift sand of bacterium united algae as claimed in claim 1 is characterized in that: described soil bacillus is to separate from soil crust and cultivate by following step:
Selection is mixed from the soil sample and the sterile water at 2～10cm place under arid, semiarid or the desert surface crust, makes the soil dilution after 100 ℃ of high temperature treatment, and this soil dilution is inoculated in the bacterium beef-protein medium flat board, is inverted and cultivates; Cultured bacterial classification uses the spore staining method to identify, isolates dyed back observation gemma under oily mirror and is green, the stable on heating soil bacillus that thalline takes on a red color;
With on the above-mentioned soil bacillus liquid medium within I that from soil crust, separates according to a conventional method through after the liquid culture of continuously fermenting on a large scale, centrifugal, the supernatant that inclines obtains freeze-dried powder after the bacterial sediment thing freeze drying of lower floor, preserves standby in refrigerator;
Described liquid nutrient medium I is for formulated in following ratio: add 80～100g corn steep liquor in every premium on currency, 5g sodium chloride, pH7.0～7.5.
4, the fixing method of desert drift sand of bacterium united algae as claimed in claim 1 is characterized in that: described desert algae is to separate from soil crust and cultivate by following step:
Gather arid, semiarid or desert soil surface skining, remove foreign material such as phytoclasts wherein after, be ground to the sub-sieve that can cross 0.5mm; This culture sample after uviol lamp shines down, is added the sterile distilled water vibration, make homogeneous suspension, will be inoculated in liquid nutrient medium II after it dilution, place illumination box to cultivate; Be transferred on the solid culture medium III after the algae kind chosen, until obtaining purifying, free of contamination desert algae kind;
The desert algae kind access of this purifying is contained in the triangular flask of liquid nutrient medium II, illumination cultivation and gap shaking flasks, the desert algae that obtains is collected by centrifugal or filtration, collects the algae sheet, and pulverizing obtains desert algae dry powder;
Described liquid nutrient medium II prepares as follows: with potassium nitrate 0.8～2.0g, magnesium sulfate 0.05～1.2g, potassium dihydrogen phosphate 0.02～0.05g and citric acid 0.004～0.008g with a small amount of dissolved in distilled water after, add distilled water to 980ml; Behind nitrate of lime 2～4g usefulness 20ml dissolved in distilled water, join in the above-mentioned solution; Add plant growth regulator 0.1～1ppm again;
Described solid culture medium III is the agar of adding 1% in the aforesaid liquid medium ii, after heating is dissolved, and sterilization 30min under 15 pounds of pressure.
5, the fixing method of desert drift sand of bacterium united algae as claimed in claim 1, it is characterized in that: described desert algae and soil bacillus are united uses when inoculating in the open air, desert algae dry powder that will separate from skinning and cultivate and soil bacillus dry powder are by 2～3: 1 quality compares mixing, form sand-consolidating agent, and by 2.0～4.0g/ rice
2Inoculum concentration be inoculated in the desert drift sand; Spraying liquid nutrition recuperating agent simultaneously, and use plastic mulching, described liquid nutritional conditioner comprises liquid nutrient medium II and acrylic compounds water-loss reducer 0.1～1g/Kg.
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