CN110218666B - Preparation method of composite bacteria and application of composite bacteria in improving arsenicum sablimatum soil - Google Patents

Preparation method of composite bacteria and application of composite bacteria in improving arsenicum sablimatum soil Download PDF

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CN110218666B
CN110218666B CN201910404456.7A CN201910404456A CN110218666B CN 110218666 B CN110218666 B CN 110218666B CN 201910404456 A CN201910404456 A CN 201910404456A CN 110218666 B CN110218666 B CN 110218666B
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邓琳
王涛
何林燕
杨才千
吴智仁
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Abstract

The invention belongs to the field of agricultural technology, environmental protection and ecological improvement application, and particularly relates to a preparation method of composite bacteria and application of the composite bacteria in improving arsenic sandstone soil, wherein the composite bacteria comprise bacillus P75, rhizobium D10 and bacillus megaterium H3: the compound bacteria or the biological agent prepared from the compound bacteria can be used for improving the fertility of arsenicum soil, promoting the growth of alfalfa, increasing organic matters in arsenicum soil, increasing ammonium nitrogen and nitrate nitrogen in arsenicum soil, increasing available phosphorus and potassium in arsenicum soil, and improving the activity of invertase and urease in arsenicum soil.

Description

Preparation method of composite bacteria and application of composite bacteria in improving arsenic sandstone soil
Technical Field
The invention belongs to the field of agricultural technology, environmental protection and ecological improvement application, and particularly relates to a preparation method of composite bacteria and application of the composite bacteria in improving arsenicum sablimatum soil.
Background
The arsenopyrite is rock interbedded layer composed of thick-layer sandstone, sand shale and argillaceous sandstone in the ages of the ancient and middle ages of the third generation, Jurassic and chalky, and is mainly and intensively distributed in the North Shanxi and the Erdos plateau in the region bordered by Mongolia in Shaanxi. The arsenopyrite belongs to a continental clastic rock system, has low diagenesis, poor cementation degree among sand grains and low structural strength, and has the characteristics of no water, hardness as stone, softness as mud when meeting water and sand formation when meeting wind. The sandstone areas are seriously weathered, plants in the areas distributed with the sandstone areas are difficult to grow, the ecological environment is severe, the water and soil loss conditions are very serious, the local life and production are seriously influenced, and local people consider the harmful toxin like arsenicum, so the sandstone areas are called as 'sandstone'. Meanwhile, the treatment difficulty of the arsenic sandstone is very high, and the arsenic sandstone is called as the world water and soil loss and the earth environment cancer by the experts in China.
As is well known, water and soil loss is becoming a common serious problem in arid or semi-arid regions of the world, and plant or biological measures and other ecological engineering related to vegetation are the most fundamental methods for controlling water and soil loss and achieving a good ecological environment. At present, the measures for controlling water and soil loss in the arsenicum sand region are mainly engineering measures and biological measures, but have limitations and cannot be popularized and applied to large-area construction operation. Cases and researches for improving barren soil by using microorganisms are available at home and abroad. The Liu Li Jun passes a long-term positioning test, treats the aeolian sandy soil in the northeast region by using the microbial soil conditioner, compares the conventional organic fertilizer planting mode with the conventional planting mode, mainly researches the influence of the microbial soil conditioner on the water and fertilizer retention effect of the aeolian sandy soil in the northeast region, and discusses the mechanism of the microbial soil conditioner on the aeolian sandy soil improvement; the Duhuiping and the like research the action and the effect of microorganisms in the matrix improvement process of the mine reclaimed land, the microbial improvement method can effectively improve the plant nutrient condition and promote the growth and development of plants, and simultaneously, the life activity of the microorganisms at the rhizosphere of the plants is utilized to reestablish and recover the soil microbial system of the reclaimed area soil losing the microbial activity, the soil bioactivity is increased, the matrix improvement of the reclaimed land soil is accelerated, the transformation process from natural soil to agricultural soil is accelerated, the raw soil is cured, and the soil fertility is improved. However, the application of the microorganism to the arsenic sandstone treatment is less reported, and the application of the composite microbial inoculum to the improvement of the characteristics of the arsenic sandstone is not seen.
Chinese patent CN107488618A discloses a Bacillus megaterium (Bacillus megaterium) H3, which is preserved in the chinese type culture collection center with a preservation date of 2016 years, 10 months and 10 days, and a strain preservation number of CCTCC NO: m2016551; can be used for promoting the growth of crops and reducing the absorption of heavy metals by the crops; chinese patent CN107937302A discloses Sinorhizobium meliloti D10(Sinorhizobium meliloti D10), which is preserved in China center for type culture collection with a preservation date of 2016 years, 10 months and 10 days, and the preservation number of strains is CCTCC NO: m2016554; can be used for promoting the growth of energy plants and improving heavy metal contaminated soil by combining the energy plants; the two microorganisms are separately applied to the arsenic sandstone treatment, and the comprehensive effect is not good.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides a preparation method of composite bacteria and application thereof in improving arsenicum sablimatum soil.
In order to solve the problems, the technical scheme of the invention is as follows:
a composite bacterium comprises bacillus P75, rhizobium D10 and bacillus megaterium H3:
the Bacillus P75 is classified and named as Bacillus halotolerans and is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan, China, the preservation date is 2018, 10 and 17 days, and the strain preservation number is CCTCC NO: m2018690;
the rhizobium D10 is classified and named as Sinorhizobium meliloti and is preserved in China center for type culture Collection, the preservation address is Wuhan university in China, the preservation date is 2016, 10 and 27 days, and the preservation number of the strain is CCTCC NO: m2016554; namely Sinorhizobium meliloti D10 disclosed in Chinese patent CN107937302A
The Bacillus megatherium H3 is classified and named as Bacillus megaterium, and is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan, China, the preservation date is 2016, 10, 27 days, and the strain preservation number is CCTCC NO: m2016551; namely the Bacillus megaterium (Bacillus megaterium H3) disclosed in the Chinese patent CN 107488618A.
Preferably, the ratio of the bacterial amounts of the bacillus P75, the rhizobium D10 and the bacillus megaterium H3 in the composite bacteria is 4-6: 0.5-1.5: 4-6.
A biological agent comprises the compound bacteria.
Preferably, the total number of cells of the compound bacteria in the biological agent is more than 5 hundred million CFU/mL.
The preparation method of the biological agent comprises the following steps:
inoculating bacillus P75 and bacillus megatherium H3 slant strains in an LB solid culture medium for culture; then, selecting a full single colony to be inoculated in an LB liquid culture medium for culture to obtain a P75 bacterial liquid and an H3 bacterial liquid;
inoculating rhizobium D10 slant strains in a TY solid culture medium for culture, and then selecting full and viscous single colonies to be inoculated in a TY liquid culture medium for culture to obtain a D10 bacterial liquid;
and (2) mixing the P75 bacterial liquid, the H3 bacterial liquid and the D10 bacterial liquid according to a volume ratio of 1: 1: 1, mixing to obtain the compound bacterial liquid.
The compound bacteria or biological agent can be used for improving the fertility of the arsenicum sablimatum soil.
The compound bacteria or the biological agent can be used for promoting the growth of the alfalfa.
The compound bacteria or biological agent can be used for increasing organic matters in the arsenopyrite soil, increasing ammonium nitrogen and nitrate nitrogen in the arsenopyrite soil, increasing available phosphorus and available potassium in the arsenopyrite soil, and improving the activity of invertase and urease in the arsenopyrite soil.
Compared with the prior art, the invention has the advantages that,
the composite bacteria can improve the fertility of arsenic sandstone soil, promote the growth of ryegrass and alfalfa, and increase the biomass of plants:
(1) the pH value of the arsenicum sablimatum soil inoculated with the compound bacteria is obviously reduced by 0.45 unit (P is less than 0.05).
(2) The organic matter content of the arsenic sandstone soil inoculated with the compound bacteria is obviously increased by 38.46 percent (P is less than 0.05).
(3) The content of ammonium nitrogen in the arsenopyrite soil inoculated with the compound bacteria is obviously increased by 1.60 times (P is less than 0.05).
(4) The content of nitrate nitrogen in the arsenopyrite soil inoculated with the compound bacteria is obviously increased by 55.60% (P is less than 0.05).
(5) The activity of sucrase in the arsenic sand soil inoculated with the compound bacteria is obviously increased by 22.55 percent (P is less than 0.05).
(6) The urease activity of the arsenicum sablimatum soil inoculated with the compound bacteria is obviously increased by 1.36 times (P is less than 0.05).
(7) The content of available phosphorus in the arsenic sandstone soil inoculated with the compound bacteria is obviously increased by 76.32 percent (P is less than 0.05).
(8) The content of the quick-acting potassium in the arsenicum sablimatum soil inoculated with the compound bacteria is obviously increased by 41.38 percent (P is less than 0.05).
(9) The biomass of the alfalfa inoculated with the compound bacteria treatment is remarkably increased by 44.52% (P < 0.05).
Detailed Description
Example 1: activation of strains P75, D10 and H3 and preparation of composite bacteria
Slant strains of P75(CCTCC NO: M2018690) and H3(CCTCC NO: M2016551) were inoculated in LB solid medium (tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, distilled water 1000mL, agar 20g, pH 7.0) and cultured at 30 ℃ for 1 day. Then, a single full colony is selected and inoculated in an LB liquid culture medium (tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, distilled water 1000mL, pH 7.0), shaking culture is carried out at 30 ℃ and 160rpm for 32H, so as to ensure the formation of spores, and P75 bacterial liquid and H3 bacterial liquid are obtained.
D10(CCTCC NO: M2016554) slant strain was inoculated on TY solid medium (peptone 5.0g, yeast extract 3.0g, anhydrous CaCl)20.647g, 1L of distilled water, pH 7.0), and cultured at 30 ℃ for 1 day. Then, a single colony of the full and viscous liquid is selected and inoculated on TY liquid culture medium (peptone 5.0g, yeast extract 3.0g, anhydrous CaCl)20.647g of distilled water, 1L of distilled water, pH 7.0), 30 ℃, and shaking culture at 160rpm for 18h to obtain D10 bacterial liquid;
and finally, mixing the components in a volume ratio of 1: 1: 1, mixing the bacterial liquid P75, D10 and H3 to obtain a composite bacterial liquid. The ratio of the bacterial amounts of the bacillus P75, the rhizobium D10 and the bacillus megaterium H3 in the composite bacterial liquid is 4-6: 0.5-1.5: 4-6.
Example 2: influence of composite microbial inoculum on pH of arsenicum sablimatum soil
The bacterial liquid P75, the bacterial liquid D10, the bacterial liquid H3 and the compound bacterial liquid in example 1 were respectively inoculated in arsenic sandstone soil according to the inoculum size of 5%, and alfalfa was used as a test plant for scientific management for one month. Mixing air-dried, ground and sieved arsenopyrite soil and deionized water according to the proportion of 1: 2.5, and placing at 200 r.min-1Shaking the mixture on a shaking bed for 2 hours at room temperature, centrifuging the mixture to precipitate insoluble substances in the soil, and measuring the pH value of the supernatant by a pH meter to obtain the pH value of the soil. As shown in Table 1, the pH of the soil in the inoculated group was reduced by 0.43-0.45 units and the pH of the soil in the sandstone treated with complex bacteria was reduced by 0.45 units (P) compared to the non-inoculated control group<0.05)。
TABLE 1 influence of composite microbial inoculum on pH of arsenicum sand soil
Treatment of pH of soil
CK 8.21±0.05
P75 7.78±0.12
D10 7.77±0.06
H3 7.76±0.05
Complex microbial inoculum 7.76±0.03*
Example 3: function of composite microbial inoculum for improving content of organic matters in arsenicum sand soil
The bacterial liquid P75, the bacterial liquid D10, the bacterial liquid H3 and the compound bacterial liquid in example 1 were respectively inoculated in arsenic sandstone soil according to the inoculum size of 5%, and alfalfa was used as a test plant for scientific management for one month. And measuring the content of organic matters in the soil by adopting a potassium dichromate volumetric method-dilution heat method. As shown in Table 2, compared with the non-inoculated control group, the content of organic matters in the inoculated sandstone soil is increased by 6-38.46%, and the content of organic matters in the compounded sandstone soil is significantly increased by 38.46% (P < 0.05).
TABLE 2 influence of composite microbial inoculum on organic matter content of arsenicum sand soil
Treatment of Organic matter content g/kg
CK 14.51±2.07
P75 15.62±1.30
D10 15.38±1.55
H3 18.63±0.97
Complex microbial inoculum 20.10±2.18*
Example 4: function of composite microbial inoculum for improving nitrogen, phosphorus and potassium contents of arsenic sandstone soil
The bacterial liquid P75, the bacterial liquid D10, the bacterial liquid H3 and the compound bacterial liquid in example 1 were respectively inoculated in arsenic sandstone soil according to the inoculum size of 5%, and alfalfa was used as a test plant for scientific management for one month. By using 2 mol. L-1KCl leaching-indophenol blue colorimetric method for measuring content of ammonium nitrogen in soil, and ultraviolet spectrophotometry method for measuring content of nitrate nitrogen in soil, wherein 0.5mol/LNaHCO3The method is used for measuring the content of the available phosphorus in the soil, and the flame photometry is used for measuring the content of the available potassium in the soil. As can be seen from Table 3, compared with the control group without inoculation, the content of ammonium nitrogen in the arsenopyrite soil treated by the inoculation is increased by-10% -160%, the content of nitrate nitrogen is increased by 16.28% -55.60%, the content of available phosphorus is increased by 18.10% -76.32%, and the content of available potassium is increased by 24.56% -72.04%, wherein the content of ammonium nitrogen in the arsenopyrite soil treated by the composite bacteria is obviously increased by 1.60 times, and the content of nitrate nitrogen is obviously increased55.60%, the content of available phosphorus is increased by 76.32%, and the content of available potassium is increased by 41.38% (P)<0.05)。
TABLE 3 influence of composite bacteria on NPK content in arsenopyrite soil
Figure GDA0003507848320000051
Example 5: function of compound microbial inoculum for improving activity of arsenicum sablimatum soil enzyme
The bacterial liquid P75, the bacterial liquid D10, the bacterial liquid H3 and the compound bacterial liquid in example 1 were respectively inoculated in arsenic sandstone soil according to the inoculum size of 5%, and alfalfa was used as a test plant for scientific management for one month. The soil sucrase activity is measured by a 3, 5-dinitrosalicylic acid (DNS) colorimetric method, and the urease activity is measured by a sodium phenolate-sodium hypochlorite colorimetric method. As can be seen from Table 4, compared with the control group without inoculation, the sucrase activity of the arsenopyrite soil treated by the inoculation is increased by 22.55-36.77%, and the urease activity is increased by 104-148%, wherein the sucrase activity of the arsenopyrite soil treated by the compound bacteria is obviously increased by 22.55%, and the urease activity is obviously increased by 1.38 times (P < 0.05).
TABLE 4 influence of composite microbial inoculum on enzyme activity of arsenicum sand soil
Treatment of Sucrase activity mg/g Urease activity mg/g
CK 5.63±0.82 0.21±0.02
P75 7.70±0.66 0.49±0.03
D10 7.29±0.75 0.43±0.02
H3 7.30±0.08 0.52±0.01
Complex microbial inoculum 6.90±0.42* 0.50±0.01*
Example 6: function of composite microbial inoculum in arsenic sandstone soil for promoting growth of alfalfa
The soil is obtained from the soil in inner Mongolia arsenicum sablimatum area. The experiment adopts a pot experiment, 300g of soil is filled in each hole tray, and 30g of deionized water is added into the hole trays, so that the whole soil environment is kept at a certain drought degree. Respectively carrying out surface disinfection on ryegrass and alfalfa seeds to be tested by using 5% sodium hypochlorite solution for 20min, placing the seeds on sterile gauze, placing the sterile gauze in a sterile incubator at 30 ℃, keeping the humidity at 60% -80% for accelerating germination, and picking the seeds with the consistent sizes and exposed white and placing the seeds on a water culture bowl for culture. When the seed buds grow to about 2cm, alfalfa seedlings with uniform growth vigor and size are selected to be placed in a hole tray, 20ml of the P75 bacterial liquid, the D10 bacterial liquid, the H3 bacterial liquid and the compound bacterial liquid in the embodiment 1 are inoculated, three treatments are repeated, 20ml of bacterial liquid is inoculated again every half month for strengthening treatment, and scientific management is carried out for one month. And measuring the dry weight of the alfalfa one month later to judge the growth promoting effect of the compound microbial inoculum on the alfalfa.
As can be seen from Table 5, compared with the control without inoculation, the biomass of the inoculated alfalfa is increased by 18.58-44.52%, and the biomass of the alfalfa after the compound bacteria treatment is significantly increased by 44.52%.
TABLE 5 growth promoting effect of the complex microbial inoculum on alfalfa
Treatment of Alfalfa dry weight g/pot
CK 0.103±0.005
P75 0.130±0.011
D10 0.122±0.020
H3 0.146±0.012
Complex microbial inoculum 0.149±0.017*
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all equivalent substitutions or substitutions made on the above-mentioned embodiments are included in the scope of the present invention.

Claims (9)

1. The composite bacterium is characterized by consisting of bacillus P75, rhizobium D10 and bacillus megaterium H3:
the Bacillus P75 is classified and named as Bacillus halotolerans and is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan, China, the preservation date is 2018, 10 and 17 days, and the strain preservation number is CCTCC NO: m2018690;
the rhizobium D10 is classified and named as Sinorhizobium meliloti and is preserved in China center for type culture Collection, the preservation address is Wuhan university in China, the preservation date is 2016, 10 and 27 days, and the preservation number of the strain is CCTCC NO: m2016554;
the Bacillus megaterium H3 is classified and named as Bacillus megaterium and is preserved in China center for type culture Collection, the preservation address is Wuhan university in Wuhan, China, the preservation date is 2016, 10, 27 days, and the strain preservation number is CCTCC NO: m2016551.
2. The complex bacterium according to claim 1, wherein the ratio of the bacterial amounts of Bacillus sp 75, Rhizobium D10, and Bacillus megaterium H3 in the complex bacterium is 4-6: 0.5-1.5: 4-6.
3. A biological agent comprising the complex bacterium according to claim 1 or 2.
4. The biological agent according to claim 3, wherein the total number of cells of the complex bacteria in the biological agent is 5 hundred million CFU/mL or more.
5. The method for preparing a biological agent according to claim 3, comprising the steps of:
inoculating bacillus P75 and bacillus megatherium H3 slant strains in an LB solid culture medium for culture; then, selecting a full single colony to be inoculated in an LB liquid culture medium for culture to obtain a P75 bacterial liquid and an H3 bacterial liquid;
inoculating rhizobium D10 slant strains in a TY solid culture medium for culture, and then selecting full and viscous single colonies to be inoculated in a TY liquid culture medium for culture to obtain a D10 bacterial liquid;
and (2) mixing the P75 bacterial liquid, the H3 bacterial liquid and the D10 bacterial liquid according to a volume ratio of 1: 1: 1, mixing to obtain the compound bacterial liquid.
6. The use of the compound bacteria of claim 1 or 2 in improving the fertility of arsenicum soil or promoting the growth of alfalfa.
7. The compound bacteria of claim 1 or 2, which can be used for increasing organic matters in arsenopyrite soil, increasing ammonium nitrogen in arsenopyrite soil, increasing nitrate nitrogen in arsenopyrite soil, increasing available phosphorus in arsenopyrite soil, increasing available potassium in arsenopyrite soil, increasing invertase in arsenopyrite soil or increasing urease activity in arsenopyrite soil.
8. The application of the biological agent as claimed in claim 3 or 4 in improving fertility of arsenic sandstone soil or promoting growth of alfalfa.
9. The application of the biological agent of claim 3 or 4 in increasing organic matters in arsenopyrite soil, increasing ammonium nitrogen in arsenopyrite soil, increasing nitrate nitrogen in arsenopyrite soil, increasing available phosphorus in arsenopyrite soil, increasing available potassium in arsenopyrite soil, increasing invertase in arsenopyrite soil or increasing urease activity in arsenopyrite soil.
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CA2845408A1 (en) * 2012-11-30 2014-05-30 Manas Ranjan Banerjee Phosphate solubilizing rhizobacteria bacillus firmus as biofertilizer to increase canola yield
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CA2845408A1 (en) * 2012-11-30 2014-05-30 Manas Ranjan Banerjee Phosphate solubilizing rhizobacteria bacillus firmus as biofertilizer to increase canola yield
CN105861420A (en) * 2016-05-20 2016-08-17 广州赛莱拉干细胞科技股份有限公司 Culture medium for inducing inner ear stem cells to be differentiated into hair cells and method of culture medium

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