Artificial crust compound biological agent and use method thereof

Abstract

The invention discloses an artificial crust compound biological agent and a use method thereof, and aims to solve the problem of wound ecological restoration in Hexi corridor engineering construction. The compound biological agent is composed of a microalgae and artemisia desertorum gum mixed solution and bacillus powder, the microalgae comprise microcoleus vaginatus, pseudocladosporium javanicum and championia gracilis, the microalgae and the artemisia desertorum gum are mixed according to a specific proportion and then are compatible, and bacillus subtilis or bacillus mucilaginosus can be selected as the bacillus powder. When in use, the bacillus powder is firstly scattered, and then the microalgae artemisia desertorum gum mixed solution is sprayed. The compound biological agent provided by the invention can significantly improve the crust thickness, biomass and compressive strength of the sand soil, reduce the wind erosion rate, increase the contents of nitrogen, phosphorus, potassium and organic matters in the soil, effectively improve the ecology of the sand soil, and provide a new technology for wind erosion prevention and ecological restoration of the engineering slash in the arid region.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an artificial crust compound biological preparation and a use method thereof. Background Art

[0002] In recent years, the Hexi Corridor has carried out a large number of engineering construction projects relying on its regional advantages and abundant solar energy resources. The damage to the ecological environment caused by the slopes and rolling surfaces caused by engineering construction is often difficult to recover. The base types of its engineering wounds are diverse, mostly formed by excavation and accumulation, with problems such as poor stability and easy instability. The construction process destroyed the original soil structure and the original plant community, reduced vegetation coverage and biodiversity, changed the flow of surface water and groundwater, and triggered local microclimate changes, providing a rich source of materials for wind erosion. At the same time, due to the poor site conditions caused by engineering construction, it is difficult for grass and shrubs to be planted on the slopes, which makes the natural recovery process slow, further increasing the risk of soil erosion and posing a certain threat to the normal operation of the projects under construction and completed and the surrounding environment.

[0003] At present, common prevention and control measures include engineering measures, chemical sand fixation agents and forest and grass measures. While these measures have achieved many effects, they also have their own shortcomings. For example, mechanical engineering projects require a large amount of work and have a short maintenance time; chemical sand fixation has problems such as aging of fixatives and the inability to reuse land; forest and grass measures require several years or even decades to truly achieve the effect of windbreak and sand fixation due to scarce precipitation.

[0004] Biological soil crusts (BSC) are complex surface covers formed by cyanobacteria, green algae, lichens, mosses, microorganisms, and other related organisms that are cemented with soil surface particles through mycelium, rhizoids, and secretions. They are an important component of desert ecosystems, accounting for 70% of the bare land area in arid and semi-arid regions around the world, and play an extremely important ecosystem function. As an important component of desert ecosystems, the formation and development of BSCs is one of the main signs of ecosystem health and plays an important role in maintaining the ecological stability and ecological restoration of desert systems. With the implementation of a large number of production and construction projects, BSCs are severely damaged, while the formation of natural BSCs takes several years or even decades. How to accelerate the reconstruction and growth of BSCs through artificial induction of biological soil crusts (IBSCs) is a major demand for ecological restoration and reconstruction of engineering sites, wind erosion and desertification prevention in the Hexi Corridor region.

[0005] There have been some studies on artificial biological crusting technology, but the research content is the combined use of algae and wormwood gum, and the effect is still not ideal, especially for different engineering wounds. The crusting effect is not good. The soil hardness, crusting biomass, and soil enzyme activity are all low, and the crusting time is long, which is not conducive to efficient and rapid repair. Summary of the invention

[0006] The present invention aims at the above-mentioned deficiencies and defects of the prior art and proposes an artificial crust compound biological preparation and a method of using the same.

[0007] The first object of the present invention is to provide an artificial crust compound biological preparation, the compound biological preparation is composed of two parts, one is a microalgae Artemisia sphaerocephala gum mixture, the other is a Bacillus powder, wherein the components of the microalgae Artemisia sphaerocephala gum mixture are: Microcoleus vaginatus , MV), Pseudoclaea javanica ( Scytonema javanicum , SJ), slender phytophora ( Phormidium tenue , PT) Microcoleus ( Microcoleus vaginatus , MV), the fresh weight mixing ratio of the three microalgae was MV:SJ:PT=10:1:5, the concentration of microalgae after mixing was 6.2 μg / mL, and the volume ratio of the mixed algae liquid and 10% Artemisia sphaerocephala gum was 7:3; the spore count of Bacillus powder was ≥30×10 8 CFU / g.

[0008] Preferably, the Bacillus is Bacillus subtilis.

[0009] Preferably, the Bacillus is Bacillus subtilis.

[0010] The second object of the present invention is to provide a method for using the artificial crust compound biological agent, which comprises the following steps: firstly, sprinkling the Bacillus powder on the surface of the sand, and adding an amount of 20 g / m 2 Then, spray the mixture of microalgae and Artemisia sphaerocephala glue on the sand at a rate of 2 L / m 2 .

[0011] The present invention provides an artificial crust compound biological preparation and a method of use, wherein an appropriate amount of Artemisia sphaerocephala gum is added to the artificial algae liquid and combined with Bacillus to spray the sandy soil. Microcoleus, Pseudobranchium javanicum, and Sclerophyllum gracilis are suitable for neutral and alkaline environments, and have a high survival rate in sandy soil environments. When inoculated at a ratio of 10:1:5, the desert algae crust formed has the largest compressive strength and the highest stability; Artemisia sphaerocephala gum is an environmentally friendly sand fixation agent, and the present invention uses a 0.1% concentration of the compound biological preparation to further reduce the spraying cost and increase the crusting rate; Bacillus can secrete a large amount of extracellular polysaccharides, induce calcium carbonate precipitation, increase the stability of sandy soil aggregates, and thus improve the anti-erosion ability of sandy soil. BS can also improve the quality of sandy soil and promote the growth of sand-dwelling plants. The addition of the bacterial agent effectively promotes the growth of algae. When the composite biological agent provided by the present invention is used, the crust thickness at 60 days is more than 50% higher than that of the prior art, which can significantly increase the cementation of sand particles, promote the increase of crust biomass, and effectively promote the growth of sandy soil biological crust. At the same time, it can significantly increase the content of nitrogen, phosphorus, potassium and organic matter in the sandy soil, and has the effect of improving the ecology of the sandy soil. It provides a new artificial biological crust technology for preventing and controlling wind erosion in engineering sites in arid areas and improving sandy soil nutrients. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The biological crust after being treated for 30 days in Example 1; Figure 2 The changes in crust thickness, biomass, compressive strength and wind erosion rate of each treatment in Example 1; Figure 3 The soil moisture content, pH and soil hardness two years after the algae solution was inoculated in Example 2; Figure 4 The growth of biological crust after 2 years of algae liquid inoculation in Example 2; Figure 5 The soil enzyme activity status after 3 months and 2 years of algae liquid inoculation in Example 2; Figure 6 This is the soil nutrient status 2 years after inoculation with algae liquid. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with specific implementation examples. The following implementation examples are helpful for those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form.

[0014] The algae species used in this experiment were Microcoleus vaginatus (MV), Scytonema javanicum (SJ) and Phormidium tenue (PT), all of which were purchased from the freshwater algae species bank of the Institute of Hydrobiology, Chinese Academy of Sciences. MV and PT were cultured in BG11 liquid medium, and SJ was cultured in BG110 liquid medium. The culture conditions were: 25℃, 2000 Lx light, and 8 h / d light time. After expansion culture, the three microalgae were mixed in proportion (MV:SJ:PT=10:1:5, fresh weight ratio) for use, and the mixed microalgae was Cy.

[0015] Bacillus powder and Artemisia sphaerocephala glue ( Artemisia sphaerocephala Krasch . gum, ASKG) was obtained through commercial channels, of which the effective bacterial count of Bacillus mucilaginosus (BM) was > 10 billion / g, and the effective bacterial count of Bacillus subtilis (BS) was > 30 billion / g.

[0016] The outdoor test was conducted in Mengjiazhuang, Gulang County (103°36′37″E, 37°26′55″N) from July 2021 to September 2023. The experimental site is located in the eastern section of the Hexi Corridor, the northern foot of Wushaoling, and the southern edge of the Tengger Desert. It has a temperate continental climate and an altitude of 1,980 meters. The average annual temperature in the area is 5.9°C, and the extreme maximum temperature is 33°C. The average annual rainfall is 360.5 mm, and the annual evaporation is 2,300 mm.

[0017] All technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Example

[0018] The treatment groups of the sand surface experiment are shown in Table 1. Each plate (Φ = 120 mm) was filled with 300 g of sterilized sand with a bulk density of 1.54 g / cm 3 The spraying volume of algae liquid is 2 L / m 2 The amount of microbial agent added in treatments 3 and 4 was 20 g / m 2 In treatment 5, 10 g / m 2 . The experimental method was to spray the inoculum solution directly on the sand for treatments CK, 1, and 2; and to sprinkle the bacterial powder on the sand first, and then spray the inoculum solution for treatments 3, 4, and 5. During the experiment, water was added to the original weight every 3 days, and the soil was maintained for 2 months. The culture conditions were: 25℃, 2000-3000 Lx of light, and 8 h / d of light duration.

[0019] Table 1 Treatment groups of sand surface experiment serial number Inoculation solution CK water 1 Microalgae (Cy) 2 Cy+0.1% ASKG 3 Cy+0.1% ASKG+BM 4 Cy+0.1% ASKG+BS 5 Cy+0.1% ASKG+BM+BS After 1 month and 2 months of sand surface tending, various indicators were measured. The crust thickness was measured using a vernier caliper (mm, N=6), the crust compressive strength was measured using a soil hardness tester (kg.cm-2, N=8), the biomass was measured using a spectrophotometer, and the wind erosion rate was measured using a fan (N=3), with a wind speed of 7-10 m / s.

[0020] Wind erosion rate (Re) = (m0 - m1) / (s·t) m0: initial sand weight; m1: sand weight after erosion; s: sand tray area; t: 30 min The soil nutrient determination methods are as follows: Potassium dichromate volumetric method was used to determine soil organic matter content; sulfuric acid-accelerator digestion and Kjeldahl method were used to determine soil total nitrogen content; NaOH alkali fusion and molybdenum antimony anti-spectrophotometry were used to determine soil total phosphorus content; and flame photometer method was used to determine soil total potassium content.

[0021] like Figure 1 As shown, obvious biological crust formed on the sand surface one month after the algae liquid was sprayed and cultivated.

[0022] Changes in crust thickness, biomass, compressive strength, and wind erosion rate Figure 2 As shown: The thickness of the crust after sandy soil cultivation is as follows Figure 2 As shown in A, CK had no obvious crust at 32 days, and a physical crust with a thickness of 0.31 mm appeared after 60 days. The crust thickness of treatments 1-5 was 1.29-2.28 mm (32 days), and reached 1.58-3.58 mm after 60 days. The crust thickness of treatment 5 was the highest (60 days, P < 0.05), 53.6% higher than that of treatment 2, indicating that the simultaneous addition of BM and BS can significantly increase the cementation of sand particles and make the crust thicker.

[0023] The changes in biomass Figure 2 As shown in B, the growth of crust biomass under different treatments gradually became obvious after 60 days. After 60 days, the biomass of treatments 1 and 2 was 7 to 12 times that of CK, and treatments 3 to 5 was 16 to 20 times that of CK and higher than treatments 1 and 2 (P < 0.05), indicating that the addition of the inoculant accelerated the growth of algae. After 60 days, the biomass of treatment 3 reached 0.35 μg chl-a.cm-2, which was higher than treatment 2 but the difference was not significant, indicating that ASKG had no significant promoting effect on the growth of microalgae.

[0024] Compressive strength Figure 2As shown in C, treatment 2 had the lowest compressive strength at the initial stage (32 days), which was 26.0% lower than the control, but the compressive strength increased significantly after 60 days, reaching 0.42 kg.cm-2, indicating that the addition of ASKG can improve soil hardness in a short period of time. The compressive strength of the treatments with added microbial agents (treatments 3-5) was higher (32 days), which was 40% higher than CK; There are significant differences in wind erosion rates under different treatments ( Figure 2 D). The wind erosion rates of treatments 3 and 4 were significantly lower than those of other treatments and CK after 32 days (P < 0.05), and were only 0.59 gm-2.min-1 and 0.45 gm-2.min-1 after 60 days, indicating that Cy+BM / BS has the best anti-wind erosion effect. The wind erosion rate of treatment 1 decreased rapidly after 1 month, and the wind erosion rate was 1.11 gm-2.min-1 (decreased by 57.6%) after 60 days, which was 36.9% lower than that of CK. The wind erosion rates of treatments 2 and 5 were not significantly different from those of CK. The wind erosion rate of the treatment with simultaneous addition of BM and BS (treatment 5) was higher, indicating that although the simultaneous addition of the two increased the cementation of sand particles, the cementation strength was not enhanced, and the two may not have a synergistic effect on the growth of crust.

[0025] This example uses sterilized fine sand with extremely low nutrients. The soil nutrient status of different treatments is shown in Table 2. The soil organic matter content increased significantly under different treatments. After 32 days of cultivation, the organic matter content of different treatments increased by 27.0 (treatment 2) to 194.6% (treatment 4) compared with CK; after 60 days, the organic matter content was 5.05 (treatment 3) to 7.78 times that of the control (treatment 5). The total nitrogen content of the soil increased significantly after 60 days, increasing by 45.2 (treatment 1) to 134.9% (treatment 3) compared with the control, and the total nitrogen content of the sandy loam in the Cy+BM treatment was the highest. Under different treatments, the total nitrogen and total potassium contents of the soil were not much different, and there was no significant increase compared with the control. Cy+ASKG+BM / BS has a good ability to improve soil nutrients.

[0026] Table 2 Soil nutrients under different treatments From the above experimental results, it can be seen that the compound biological preparation (treatment 3 and treatment 4) provided by the present invention has the most significant effect on promoting biological crusting and soil remediation. Example

[0027] The test site is 50×50 m in size, with no related buildings or hills around it. To avoid wild animals and other human interference, the test site is enclosed by a wire fence, and the height of the fence is 1.5 m above the ground. The disturbance and damage to the ground surface during construction mainly manifests itself in three forms, namely, excavation and filling, dumping and rolling. To simulate production and construction activities, we set up three basic characteristic plots, namely: excavation slope, filling slope, and mechanical rolling disturbance surface (construction road). Referring to the construction characteristics of general construction projects, this study selected the characteristics of each plot as follows: excavation plot: excavation depth of 2.0 m, slope ratio of 1:1.3; filling plot: filling height of 2.0 m, slope ratio of 1:1.5; mechanical rolling plot: mechanical rolling disturbance surface width of 6.0 m, length of 30 m, surface fill of 50 cm, and manual tamping to simulate rolling. After the construction was completed, there were 5 types of wound surfaces (S), namely, the sunny slope of the excavated slope (Ⅰ), the shady slope of the excavated slope (Ⅱ), the sunny slope of the fill slope (Ⅲ), the shady slope of the fill slope (Ⅳ), and the mechanical rolling disturbance surface (Ⅴ). Each slope surface was arranged with 12 experimental plots of 2×1 m, totaling 60 plots. The soil type in this area is light brown calcareous soil. The soil water content (SWC) before treatment was 0.03-0.12% (110℃, drying for 6 h), the soil hardness was 0.7-1.9 kg.cm-2, and the pH value was 8.40 (soil-water extraction method, water-soil ratio was 1:2.5).

[0028] Four treatments (T) were set up for each wound surface, with three replicates for each treatment, which were randomly distributed in 60 2m×1m plots on the slope. Treatment 1 was no inoculation (CK), treatment 2 was Cy (MV:SJ:PT=10:1:5, mixed to Cy, biomass 63μg / L), and Cy was supplemented with 0.1% ASKP and Bacillus subtilis (BS; spore number ≥ 30×10 8 CFU / g), the volume ratio of algae solution to 0.1% ASKP was 7:3, and BS was added to the soil surface at an addition amount of 20 g / m 2 The liquid inoculum volume for each treatment was 2 L / m 2. After inoculation of algae liquid and ASKP, the same as in Example 1 was used, with bacterial powder being spread first and then inoculation liquid being sprayed. The treatments were maintained for 3 to 4 weeks, and all treatments were watered appropriately daily (except on rainy days). The crust and subcutaneous soil were collected after 3 m and 2 years, respectively, for the determination of relevant parameters. A measuring rod was set in the middle of each plot, with a length of 50 cm. The measuring rod was driven vertically into the ground with an exposed height of 20 cm. The wind erosion rate was measured at the end of the test.

[0029] SWC was measured by weighing method, and soil pH was measured by acidometer (Leci DHS-3C). The thickness and compressive strength of the crust were measured by electronic vernier caliper and soil hardness meter (TYD-1, Hangzhou Green Expo), respectively, and the compressive strength was expressed by soil hardness. Five points were evenly selected in each treatment plot, and the thickness of the biological crust was measured three times at random points. The biomass of the crust was expressed by the content of Chla, and Chl a was extracted with 95% ethanol. The amount of soil wind erosion was measured by the rod method.

[0030] The methods for determining soil enzyme activity and soil nutrients are as follows. Soil urease was determined using the sodium benzoate-sodium hypochlorite colorimetric method, soil alkaline phosphatase was determined using the disodium phenyl phosphate colorimetric method, and soil catalase activity was determined using the potassium permanganate titration method. Soil organic matter was determined using the potassium dichromate volumetric method. Soil total nitrogen was digested using the Kjeldahl method and determined using the AA3 continuous flow analyzer. Soil available potassium was measured using the ammonium acetate extraction-flame photometer method. Available phosphorus was measured using the sodium bicarbonate extraction-molybdenum antimony colorimetric method.

[0031] Figure 3 The soil water content, pH and soil hardness 2 years after algae liquid inoculation. The soil hardness of the Cy group was significantly higher than that of the CK, especially the wound surface II, where the soil hardness was 242.77% higher than that of the CK. The algae liquid had a significant effect on soil hardening. Figure 4 This is the growth of biological crusts 2 years after algae inoculation. It can be seen from the figure that the biological crusts of each wound surface 2 years after algae inoculation are significantly higher than those of CK, and the thickness of the crusts in each group is more than 20% higher than that of the CK group; Figure 5 The activity of soil enzymes 3 months and 2 years after algae liquid inoculation. It can be seen from the figure that the activity of soil alkaline phosphatase increased significantly after algae liquid treatment, and the alkaline phosphatase activity in wound surface treatment III was 93.63% higher than that in CK group. Figure 6 This is the soil nutrient status 2 years after inoculation with algae liquid. It can be seen that in each wound treatment group, the contents of organic matter, available phosphorus and quick-acting potassium in Cy are higher than those in CK, indicating that inoculation with algae liquid can effectively increase soil nutrients and improve the soil ecological environment.

Claims

1. An artificial crust compound biological preparation, characterized in that: The composite biological preparation consists of a microalgae wormwood gum mixed solution and a bacillus powder, wherein: the microalgae wormwood gum mixed solution comprises: Microsphaeria sheathii, Pseudobranchium javanica, and Pseudosphaeria gracilis, the fresh weight mixing ratio of the three microalgae is 10:1:5, the microalgae concentration after mixing is 6.2 μg / mL, and the mixing volume ratio of the mixed algae liquid to 10% wormwood gum is 7:3; the spore number of the bacillus powder is ≥30×10 8 CFU / g.

2. The artificial skin compound biological preparation according to claim 1, characterized in that: The bacillus is Bacillus subtilis.

3. The artificial skin compound biological preparation according to claim 1, characterized in that: The bacillus is Bacillus mucilaginosus.

4. A method for using the artificial skin compound biological preparation according to claim 1, characterized in that: The following steps are involved: (1) Sprinkle Bacillus powder evenly on the surface of the sand, adding 20 g / m 2 ; (2) Spray the mixture of microalgae and Artemisia sphaerocephala gel on the sand at a rate of 2 L / m 2 .

5. The method for using the artificial skin compound biological preparation according to claim 4, characterized in that: The bacillus powder is bacillus subtilis or bacillus mucilaginosus.

6. The method for using the artificial skin compound biological preparation according to claim 4, characterized in that: The microalgae in the microalgae artemisia gum mixed liquid are Microsciaena sheathiola, Pseudoclase javanica and Pseudoclase javanica, and the fresh weight mixing ratio of the three microalgae is 10:1:

5.

7. The method for using the artificial skin compound biological preparation according to claim 4, characterized in that: The concentration of Artemisia sphaerocephala gum in the microalgae Artemisia sphaerocephala gum mixed solution is 0.1%.

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