CN114574474A - Portable biological crust seed source and preparation method thereof - Google Patents
Portable biological crust seed source and preparation method thereof Download PDFInfo
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- CN114574474A CN114574474A CN202011403458.3A CN202011403458A CN114574474A CN 114574474 A CN114574474 A CN 114574474A CN 202011403458 A CN202011403458 A CN 202011403458A CN 114574474 A CN114574474 A CN 114574474A
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Abstract
The invention discloses a portable biological crust seed source and a preparation method thereof, wherein the portable biological crust seed source comprises biological crust seed source globules prepared from lichen carpolicus (Endocarpon pusillum) F07020, lichen carpolicus (Diplosha chorodii) A07020 and a forming culture matrix. The invention is beneficial to forming the seed source of the lichen collected in a state closer to the nature state, can be applied to the desertification land control engineering in a large scale, and is beneficial to promoting the formation of the artificial biological crust.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and provides a preparation method of a portable biological crust seed source, which can promote the formation of a lichen symbiotic structure.
Background
In desert areas, the rainfall is rare, and the organisms such as cyanobacteria, lichens, mosses, bacteria, fungi and the like in the soil are attached and wound on the ground surface to form a special structure, namely a micro-organism crust. According to the different organisms with different successive stages of crust, the method can be divided into cyanobacteria crust, lichen crust and moss crust. Along with the succession, the stability and the anti-interference ability of biological crust are gradually improved, and the biodiversity also shows a rising trend. Lichen is a reciprocal symbiont composed of lichen type fungi and corresponding algae or cyanobacteria, and is widely distributed in nature. They have a long life cycle and can withstand a harsh living environment. Lichens grown in arid and semiarid regions have special drought adaptability, and lichens crust can reduce wind erosion and water erosion of the ground surface, and is favorable for improving the stability of the ground surface. Meanwhile, lichen contains abundant microbial resources, and plays a role in improving the biological diversity of desert regions. The Wen Jiangchun academia proposes the concept of desert carpet engineering, namely, the micro biological crust is copied by using the modern biotechnology and applied to the ecological restoration in the desert area.
However, lichens grow slowly in nature, and the lichens, which are representative of the lichens in desert regions, grow at a rate of less than 0.8mm per year in diameter, and long-term harvesting of lichens to construct biological crust is obviously not feasible for long-term use. The photinia shiitake symbiotic bacteria and symbiotic algae which are expanded and cultured under the laboratory condition are limited by the difficulty in carrying liquid cultures and the like in the field application process, and are not beneficial to being applied to the improvement of the surface state of desert regions on a large scale.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the phenomena of soil productivity reduction and the like caused by drought, rain loss, wind erosion and water erosion in the desert area and sand storm weather; some sand fixation measures adopted at present have the problems of environmental pollution, high transportation cost and difficult carrying, and the sand fixation biological material applied to the field is environment-friendly, safe and convenient to carry.
The invention provides a portable biological crust seed source, which comprises a biological crust seed source pellet prepared from lichen carpolium symbiotic bacteria (Endocarpon pusillum) F07020, symbiotic algae (Diplophaera choratii) A07020 and a forming culture medium, wherein the preservation number of the lichen carpolium symbiotic bacteria (Endocarpon bacillus) F07020 in the China general microbiological culture Collection center is CGMCCNo.3893, and the preservation number of the symbiotic algae (Diplophaera choratii) A07020 in the China general microbiological culture center is CCCGMCC No.3892.
Further, the mass ratio of the symbiotic bacteria (Endocarpon pusillum) F07020 to the symbiotic algae (Diplosphaera choratii) A07020 is 3: 1.
Further, the forming culture medium comprises a sodium alginate aqueous solution and a calcium nitrate aqueous solution.
The invention also provides a preparation method of the portable biological crust seed source, which comprises the following steps:
1) culturing Shiguojianpinullium F07020 and symbiotic algae Diplochia chorodii A07020, wherein the preservation number of the Shiguojianpinullium F07020 in the China general microbiological culture Collection center is CGMCCNo.3893, and the preservation number of the symbiotic algae (Diplochia chorodii) A07020 in the China general microbiological culture Collection center is CGMCCNo.3893;
2) removing a culture substrate from the culture of the lichen carpolicus F07020 and the symbiotic algae Diplochaera chorodiia 07020 cultured in the step 1), and drying to obtain the dry weight of the lichen carpolicus and the lichen carpolicus in unit volume;
3) mixing, homogenizing and centrifuging the lichen planus and lichen planus to precipitate, and adding the homogenate to the sodium alginate aqueous solution to obtain a mixed solution 1;
4) and adding the mixed solution 1 into a calcium nitrate aqueous solution, and fixing to form bacteria-algae pellets.
Further, the culturing step in the step 1) includes activation culturing and expansion culturing.
Further, the drying temperature in the step 2) is 105 ℃, and the drying time is 2 h.
Further, the concentration of the sodium alginate solution in the step 3) is 1g/150mL, the adding amount of the chlamydomonas stonifera Endocarpop bacillus F07020 in each 5mL of sodium alginate sleeping solution is 0.3g, and the adding amount of the symbiotic algae Diplochia odorati A07020 is 0.1 g; the concentration of the calcium nitrate aqueous solution in the step 4) is 0.5% (mass percentage), and the amount of the mixed solution 1 added into the calcium nitrate aqueous solution each time is 150 μ L.
The invention also provides a set of products for preparing the portable biological crust seed source, which comprises separately packaged lichenococcus shibatae (Endocarpon philumum) F07020, lichenococcus shibatae (Diplophaera chlorophatii) A07020 and a formed culture medium.
The application of the portable biological crust seed source or the preparation method in desert sand stabilization also falls within the protection scope of the invention.
The invention also provides a desert sand fixation method, which comprises the step of using the portable biological crust seed source or the portable biological crust seed source prepared by the preparation method to fix the desert sand.
Compared with other methods, the method has the beneficial technical effects that: the method comprises the steps of carrying out amplification culture on the epiphytic Endocarpon pusillum F07020CGMCC No.3893 and the symbiotic algae Diproshaera chorodiia 07020CGMCC No.3892 special for culturing crusts, collecting culture products in logarithmic phase, and carrying out mass culture according to the ratio of bacteria to algae of 3:1, forming the fungus-algae mixed pellet capable of preserving moisture after mixing. In a sandy soil culture medium, after 1 month of culture, hypha covers the surfaces of the fungus-algae mixture pellets, the lichen carpolium symbiotic bacteria and the symbiotic algae grow well, and a primary fungus-algae contact structure appears. The artificial skin transferred to the field appears a layered structure of symbiotic bacteria and algae. The invention is beneficial to forming the seed source of the lichen collected in a state closer to the nature state, can be applied to the desertification land control engineering in a large scale, and is beneficial to promoting the formation of the artificial biological crust.
Deposit description
The strain name is as follows: lichen carpolite symbiotic bacteria
Latin name: endocarponpuluulium
The strain number is as follows: f07020
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No. 1 Hospital No.3 of Beijing market facing Yang district
The preservation date is as follows: 2010, 05 and 31 days
Registration number of the preservation center: CGMCC No.3893
The strain name is as follows: photinia serrulata symbiotic algae
Latin name: diplosphaera choratii
The strain number is as follows: a07020
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No. 1 Hospital No.3 of Beijing market Chaoyang district
The preservation date is as follows: 2010, 05 and 31 days
Registration number of the preservation center: CGMCC No.3892
Drawings
Figure 1 is the seed form prepared in example 1.
FIG. 2 shows the shape of the mixture of the lichen carpolium symbiotic bacterium Endocarpus F07020CGMCC No.3893 and the lichen carpolium symbiotic alga Diplosaea chlorophatia 07020CGMCC No.3892 cultured for 1 month in example 1.
FIG. 3 shows the microcontact structure of the Shibataea Endocarpa Ilillum F07020CGMCC No.3893 and the Shibataea Diplosphaera Chordatia 07020CGMCC No.3892 cultured for 1 month in example 1.
FIG. 4 shows the skin of an artificial organism cultured in the field after the same culture in example 1.
FIG. 5 is a layered microstructure of bacteria and algae from the wild-type artificial organism skinning seed source after example 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Description of the main equipment used in the implementation of the invention:
artificial climate box: ningbo Jiangnan instruments Inc., GXZ Intelligent.
Tissue masher: beijing Oulwa Biotechnology Inc., model LB20ES, Waring, USA.
The media formulations used in the following examples are as follows:
optimizing a bacterium slant culture medium: 2g of yeast extract powder, 2g of soybean peptone, 40g of sucrose, 200g/L of potato extract, 20g of agar powder and 1L of distilled water. The preparation method of the potato extract comprises the following steps: cleaning potato, peeling, cutting 200g into small pieces, adding distilled water, boiling for 5-10 min, tearing with glass rod, filtering with eight layers of gauze to obtain potato extract, and adding into optimized bacteria liquid culture medium.
Optimizing the alga slant culture medium: 5g of soybean peptone, 1g of yeast extract powder, 30g of glucose, 20g of agar powder and distilled water to 1L.
Optimizing a bacterium liquid culture medium: 2g of yeast extract powder, 2g of soybean peptone, 40g of sucrose, 200g/L of potato extract and 1L of distilled water.
Optimizing an algae liquid culture medium: 5g of soybean peptone, 1g of yeast extract powder, 30g of glucose and distilled water to 1L.
Sand culture medium: grinding sandy soil, sieving with 60 mesh sieve, sterilizing at 121 deg.C for 30min, subpackaging 50g of sandy soil in each culture dish with diameter of 90mm under aseptic operation, and spraying 5mL of sterile water.
Soybean peptone: beijing Ooboxing Biotechnology Co., Ltd, 01-004; soaking yeast into powder: ANGEL YEAST CO. LTD, FM 888; sucrose: shanghai test, 10021418; glucose: shanghai test, 10010518; agar: BD, 214010.
Example 1 preparation of Portable biological skinning seed sources
1. Activated culture
Inoculating Shiguoba symbiotic bacteria F07020 (which is disclosed in CN201110261963.3 and has the preservation number of CGMCC No.3893 in China general microbiological culture Collection center) on an optimized bacteria slant culture medium, activating for 15 days, and selecting the Shiguoba symbiotic bacteria growing on the culture medium to inoculate into an optimized bacteria liquid culture medium for activation culture under the culture conditions of 22 ℃ and 150rpm for 30 days.
Inoculating the clitocybe diaphorea diproshaera choratiia 07020 (which is disclosed in patent CN201110262013.2 and has the preservation number of CGMCC No.3892 in China general microbiological culture center), activating for 15 days, selecting the clitocybe diaphoresis grown on the culture medium, inoculating the clitocybe diaphoresis algae growing on the culture medium into an optimized algae liquid culture medium, and performing activation culture for 20 days under the conditions of 22 ℃, 150rpm and 12h illumination (illumination intensity 2000Lux)/12h dark culture.
2. Expanding culture
Standing the activated symbiotic bacteria Endocarpop Uliprium F07020 overnight for precipitation, and discarding the supernatant. Uniformly crushing the symbiotic bacteria Endocarpop inum F07020 by using a tissue triturator, setting the rotation speed of the triturator to be 9-10, 15s each time, and three times, adding 10mL of bacterial liquid into 130mL of optimized bacterium liquid culture medium, and carrying out shake culture at 150rpm and 20 ℃ for 30 days.
The activated lichen litsea algae Dipinosphaera chorodiiia 07020 is kept still for overnight precipitation, and the supernatant is discarded. 10mL of the precipitated Pholidota dichotoma 07020 was added to 130mL of the optimized algal liquid medium and cultured at 150rpm, 20 ℃ and 2000lux for 7 days.
3. Preparation of Photinia serrulata Endocarpon pusillum F07020 and Photinia serrulata Diplosphaera choratiia 07020
Collecting the symbiotic bacteria Endocarpop inum F07020 cultured to logarithmic phase, standing overnight for precipitation, and discarding the supernatant. Crushing the symbiotic bacteria Endocarpon pusillum F07020 by using a sterile tissue triturator, setting the rotating speed to be 9-10, crushing for 15s each time, and placing the obtained homogenates of the symbiotic bacteria Endocarpon pusillum in a sterile container for later use. And (3) uniformly mixing the homogenate, taking 5-10mL, centrifuging at 3000rpm for 5min, then removing the supernatant, resuspending once with sterile water, centrifuging at 3000rpm for 5min, then removing the supernatant, drying the precipitate at 105 ℃ for 2h, and drying to constant weight to obtain the dry weight of the symbiotic bacteria Endocarpon pusilllum F07020 in unit volume (mL).
Collecting culture solution of Diaplosphaera rhodochrous Hayata 07020 cultured to logarithmic phase, standing overnight for precipitation, discarding supernatant, and transferring into sterile container for use. Mixing the algae solution uniformly to obtain stonecrop symbiotic algae solution, taking out 5-10mL, centrifuging at 3000rpm for 5min, then discarding the supernatant, resuspending once with sterile water, centrifuging at 3000rpm for 5min, then discarding the supernatant, drying the precipitate at 105 ℃ for 2h, and drying to constant weight to obtain the dry weight of the stonecrop symbiotic algae Diprosphera 07020 in unit volume (mL).
According to the dry weight of the chlamydomonas stonoticus Endocarpon bacillus F07020 and the chlamydomonas stonoticus Dipinosa Chodotii in unit volume (mL), the homogenates of the chlamydomonas stonoticus and the volume of the chlamydomonas stonoticus liquid corresponding to the dry weight required in the experiment can be obtained.
4. Preparation of bacteria and algae pellet
Preparing sodium alginate aqueous solution (1g/150mL) and 0.5% (mass percent) calcium nitrate aqueous solution, sterilizing at 121 ℃ for 30min, and cooling for later use.
Mixing homogenate corresponding to 0.3g of Shiguo chlamydomonas Endocarpus F07020 with algae liquid corresponding to 0.1g of Shiguo chlamydomonas Diprosphera odorati A07020, centrifuging at 3000rpm for 5min to discard culture medium supernatant, re-suspending precipitate with sterile water, centrifuging to discard supernatant, and repeating twice. Adding 5mL of sterile sodium alginate aqueous solution, properly adjusting the adding amount according to the amount of the bacteria-algae mixture, uniformly stirring and mixing by using a sterile glass rod, absorbing 150 mu L of the mixture into the sterile calcium nitrate aqueous solution for fixing to form bacteria-algae pellets, wherein the bacteria-algae pellets are portable biological crust seed sources.
The sodium alginate aqueous solution and the calcium nitrate aqueous solution can form a formed culture medium.
Example 2 verification of the application of Portable Biodermic seed sources
The pellet of the fungus algae prepared in example 1 was clamped to a sandy soil medium using sterile forceps and inoculated at a density of 9 at intervals on a 90mm sterile plate. Placing a culture dish in an artificial climate box for culture, culturing at 18 ℃, 16h illumination (illumination intensity 2000lux)/8h in darkness, observing the shape and the internal structure of the bacteria-algae mixture pellet by using an environment scanning electron microscope after culturing for 1 month, as shown in figures 2 and 3, and as can be seen from figures 2 and 3, the growth state of bacteria-algae is good, and a microscopic bacteria-algae contact structure is finally generated due to the fact that sufficient contact of the lichen shiitake symbiotic bacteria and the lichen shiitake symbiotic algae can be ensured.
After 3 months of culture in an artificial climate box, the plate culture, namely the bacteria-algae mixture pellet and the soil matrix are transferred to a field test field (as shown in figure 4), so that the field test field is subjected to dual culture in an indoor suitable environment and a field drought and windy environment, the bacteria-algae pellet is taken back after 2 months of growth in a natural state, formed crusts are observed, as shown in figure 5, the bacteria-algae layered structure appears after 3 months of indoor culture and 2 months of field culture after transferring the artificial biology crust.
In the invention, the chlamydomonas lycocarpi Endocarpon pusillum F07020 and the chlamydomonas chalcopyara chlorophatiia 07020 are mixed according to the ratio of 3: the artificial biological crust seed source prepared according to the proportion of 1 has stable shape in the culture process and good growth state of bacteria and algae. Can promote the stonecrop symbiotic bacteria to fully contact with the stonecrop symbiotic algae, and finally generates a microscopic bacteria-algae contact structure. After indoor culture for 3 months and switching to field culture for 2 months, artificial organisms are skinned to generate bacteria-algae layered structures. The bacteria-algae mixture pellets constructed according to a certain proportion are used as artificial biological crust seed sources, can relatively fix bacteria and algae and create close contact opportunities, are beneficial to formation of a bacteria-algae symbiotic form, promote formation of stone fruit peel crust, and can be used for preparing a large number of artificial biological crust seed sources for desert regions in a short time.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A portable biological crust seed source is characterized by comprising biological crust seed source pellets made of lichen carpolicus (Endocarpon pusillum) F07020, lichen carpolicus (Diplopharmala choratii) A07020 and a forming culture medium, wherein the preservation number of the lichen carpolicus (Endocarpon pusillum) F07020 in the China general microbiological culture Collection center is CGMCC No.3893, and the preservation number of the lichen carpolicus (Dipcophaera choratii) A07020 in the China general microbiological culture collection center is CGMCC No. 3892.
2. The portable biological seed source of claim 1, wherein the mass ratio of the lichen fomentarius (endogropon pusillum) F07020 to lichen commensal (Diplophaera odorati) A07020 is 3: 1.
3. The portable biological seed source of claim 1, wherein the shaped culture medium comprises an aqueous solution of sodium alginate and an aqueous solution of calcium nitrate.
4. A preparation method of a portable biological crust seed source is characterized by comprising the following steps:
1) culturing Shibataea chinensis symbiotic Endocarpon pusillum F07020 and Shibataea chinensis symbiotic Diplochia A07020, wherein the preservation number of the Shibataea chinensis symbiotic F07020 in China general microbiological culture Collection center is CGMCCNo.3893, and the preservation number of the Shibataea chinensis symbiotic A07020 in China general microbiological culture Collection center is CGMCCNo.3893;
2) removing culture medium from the culture of the clitocybe Endocarpon pusillum F07020 and clitocybe diplosaera choratii A07020 cultured in the step 1), and drying to obtain the dry weight of the clitocybe and the clitocybe in unit volume;
3) mixing, homogenizing, centrifuging and precipitating the lichen parvum and lichen parvum to obtain a mixed solution 1;
4) and adding the mixed solution 1 into a calcium nitrate aqueous solution, and fixing to form bacteria-algae pellets.
5. The method according to claim 4, wherein the culturing step in step 1) comprises activation culturing and expansion culturing.
6. The method according to claim 4, wherein the drying temperature in step 2) is 105 ℃ and the drying time is 2 hours.
7. The process according to claim 4, wherein the concentration of the aqueous solution of sodium alginate in step 3) is 1g/150mL, and the amount of the Synechocystis Endocarpon pusillum F07020 added and the amount of the Synechocystis diplosaera choratii A07020 added are 0.1g per 5mL of the aqueous solution of sodium alginate; in the step 4), the mass percentage concentration of the calcium nitrate aqueous solution is 0.5%, and the amount of the mixed solution 1 added into the calcium nitrate aqueous solution each time is 150 μ L.
8. A set of products for preparing a portable biological seed source comprises a lichen carpolicus (Endocarpon pusillum) F07020, a lichen carpolicus (Diplophora chlorophatis) A07020 and a shaped culture medium.
9. Use of the portable biogenetic seed source of any of claims 1 to 3 or the process of manufacture of any of claims 4 to 7 or the kit of claim 8 in desert sand stabilization.
10. The desert sand-fixing method is characterized by comprising the step of carrying out desert sand-fixing by using the portable biological crust seed source of any one of claims 1 to 3 or the portable biological crust seed source prepared by the preparation method of any one of claims 4 to 7.
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