Salt-tolerant composite microbial agent and application thereof

Abstract

The application discloses a kind of saline-alkali resistant composite microbial inoculant and application thereof, and is specifically related to the field of microbial soil management.The microbial inoculant includes klebsiella, enterobacter cloacae and humic acid, the ratio of each component is klebsiella:enterobacter cloacae:humic acid=1:0.5~2:1~3, wherein klebsiella and enterobacter cloacae are obtained by fermentation culture.The present application uses a specific pH gradient combined with corresponding increasing sodium chloride concentration subsection fermentation process for klebsiella and enterobacter cloacae, effectively carries out directional domestication to strain, so that it can maintain activity and function in extreme environment of high salt and high alkali.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial soil improvement, in particular to a saline-alkali-tolerant composite microbial agent and application thereof. BACKGROUND

[0002] Soil hardening refers to the phenomenon that the soil surface becomes hard, the soil porosity decreases, the air and water permeability decreases, the tillage performance deteriorates, the effective nutrient content decreases, and thus the normal growth and development of crops are affected, and the yield is reduced, caused by the decrease of soil organic matter content, the poor structure, the soil structure damage, the soil aggregate dispersion under the action of external factors such as irrigation or rainfall, and the cohesion under dry conditions.

[0003] With the development of economy, the wide application of agricultural mechanization, and the rolling of agricultural machinery, the soil suffers from excessive external load, and the original loose soil aggregate structure is destroyed. Long-term shallow plowing makes the soil below the plowing layer more compact, destroys the biological system and soil structure, and deep plowing destroys the soil capillary and enhances the capillary effect of the soil, which sharply reduces the water retention and aeration capacity of the soil. In addition, in order to pursue higher yield, a large amount of chemical fertilizer is applied, which destroys the nutrient structure of the soil, makes the soil structure single, and accelerates the soil degradation. For example, excessive nitrogen fertilizer addition causes rapid decomposition of soil organic matter, and excessive phosphorus fertilizer application causes excessive phosphate and calcium, magnesium and other cations in the soil to form insoluble phosphate precipitates, which destroy the composition and nutrient structure of the soil, and these all aggravate the degree of soil hardening.

[0004] In view of soil hardening, mechanical treatment, inorganic and organic fertilizer treatment, and microbial agent treatment are usually used. Microbial agents are the best treatment for soil hardening at present due to their harmless and efficient advantages, but the current microbial agents are mainly single agents or simple composite bacteria. For example, Chinese patent CN116535269A uses heat-resistant phosphorus-solubilizing bacteria and bacillus subtilis, Chinese patent CN114686238A uses microorganisms such as amylolytic bacillus and bacillus subtilis liquid to prepare microbial agents, but these microbial agents are not resistant to salt and alkali, which limits their further use. Chinese patents CN113186117A and CN114410509A both mention that klebsiella has certain salt and alkali resistance, but the salt and alkali resistance of conventional cultured microorganisms does not meet the expectation in actual application, and single microbial agents have poor effect in treating soil hardening, which further limits their further application.

[0005] Therefore, it is an urgent problem to find a suitable high-salt-alkali-tolerant composite microbial agent for soil hardening treatment. SUMMARY

[0006] To this end, the present application provides a saline-alkali-resistant composite microbial inoculant and application thereof to solve the problems in the prior art.

[0007] To achieve the above-mentioned object, the present application provides the following technical solutions.

[0008] According to one aspect of the present application, a saline-alkali-resistant composite microbial inoculant is provided, which comprises Klebsiella, Enterobacter hormaechei and humic acid, and the ratio of each component is Klebsiella: Enterobacter hormaechei: humic acid = 1:0.5-2:1-3, wherein Klebsiella and Enterobacter hormaechei are obtained by fermentation culture.

[0009] As an example, the ratio of Klebsiella: Enterobacter hormaechei: humic acid is preferably 1:1:2.

[0010] During the granulation process of the saline-alkali-resistant composite microbial inoculant, an auxiliary material is also added, and the addition ratio is Klebsiella: Enterobacter hormaechei: humic acid: auxiliary material = 1:0.5-2:1-3:1-3, and the auxiliary material components are wood ash and vermiculite powder, and the ratio of wood ash: vermiculite powder = 1-2:1.

[0011] Further, the fermentation culture method of Klebsiella comprises: first, controlling the reaction conditions to preliminarily culture Klebsiella to obtain a primary seed liquid; then, performing secondary culture on the primary seed liquid to obtain a secondary seed liquid; finally, performing segmented fermentation on the secondary seed liquid under different pH gradients to obtain a Klebsiella fermentation liquid; wherein the pH gradient is 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and 9.5-10.0, and the culture time of each stage is 5-10 h.

[0012] Further, the conditions for the preliminary culture are that the culture is performed in a 25-40℃ incubator with shaking for 18-30 h, and the rotation speed is controlled at 150-200 rpm.

[0013] Further, the conditions for the secondary culture are that the culture is performed at a temperature of 25-40℃, a rotation speed of 150-200 rpm, a pH of 6.5-7.5, and a dissolved oxygen of 20-50% for 18-30 h.

[0014] Further, the sodium chloride concentration corresponding to the pH gradient is 8-15 g / L, 15-30 g / L, 30-50 g / L, 50-80 g / L, and 80-100 g / L.

[0015] Further, the fermentation culture method of the Enterobacter hormaechei comprises the following steps: firstly, controlling the reaction conditions to preliminarily culture the Enterobacter hormaechei to obtain a first-stage seed liquid; secondly, carrying out secondary culture on the first-stage seed liquid to obtain a second-stage seed liquid; and finally, carrying out segmented fermentation on the second-stage seed liquid under different pH gradients to obtain the Enterobacter hormaechei fermentation liquid; wherein the pH gradient is 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, 9.5-10.0, and the culture time of each stage is 5-10 h.

[0016] Further, the culture conditions of the preliminary culture are as follows: the temperature is 25-40℃, the culture is carried out in a shaking incubator, and the culture time is 18-30 h, with the control of the rotation speed at 150-200 rpm.

[0017] Further, the culture conditions of the secondary culture are as follows: the temperature is 25-40℃, the rotation speed is 150-200 rpm, the dissolved oxygen is 20-50%, and the culture time is 18-30 h.

[0018] Further, the sodium chloride concentration corresponding to the pH gradient is 8-15 g / L, 15-30 g / L, 30-50 g / L, 50-80 g / L, or 80-100 g / L.

[0019] The second-stage seed liquid is inoculated into the fermentation tank according to the inoculation amount with the integral number of 10-20%.

[0020] According to another aspect of the present application, a preparation method of the salt-tolerant and alkali-tolerant composite microbial agent is provided, and the method comprises the following steps:

[0021] In step one, the Klebsiella fermentation liquid and the Enterobacter hormaechei fermentation liquid are respectively centrifuged, the solid content of the bacterial liquid is adjusted to 50-80%, and 0.5-15% of the dry protective agent is added to the bacterial liquid to obtain the Klebsiella bacterial slurry and the Enterobacter hormaechei bacterial slurry, respectively.

[0022] In step two, the Klebsiella bacterial slurry, the Enterobacter hormaechei bacterial slurry, humic acid and auxiliary materials in step one are mixed and granulated according to the proportion to obtain the solid granules.

[0023] In step three, the solid granules in step two are vacuum dried to obtain the composite microbial agent.

[0024] As an example, the dry protective agent is preferably one or more of galactose, trehalose, dimethyl sulfoxide and gelatin.

[0025] As an example, the dry temperature is preferably 30-45℃, and the dry time is 5-15 h.

[0026] The present application has the following advantages:

[0027] The present application effectively carries out directional domestication on the strains by adopting the segmented fermentation process of specific pH gradient combined with corresponding increasing sodium chloride concentration on Klebsiella and Enterobacter hormaechei, so that the strains can still maintain activity and function in the extreme environment of high salt and high alkali. This far exceeds the tolerance limit of conventional microbial culture.

[0028] The present application is compounded with humic acid after directional domestication of Klebsiella and Enterobacter hormaechei, which on the one hand increases the salt and alkali tolerance of the strains, and the two strains synergistically act on improving soil structure, and on the other hand, after being compounded with humic acid, humic acid not only acts as a carrier and adhesive, but also can improve soil structure, improve water and fertilizer retention capacity, stimulate microbial activity, and can synergistically promote the formation and stability of soil aggregates with the bacterial body. The combination of the three can more effectively decompose organic matter in the soil, promote mineral dissolution, secrete extracellular polysaccharides and other substances to bind soil particles, thereby significantly improving soil porosity, aeration and water permeability, tillage performance and nutrient availability, and fundamentally alleviating the problem of soil hardening, and the effect is better than that of single bacterial agent or simple compound bacteria.

[0029] The compound microbial inoculant of the present application helps to break the hardening layer caused by long-term improper land management by improving soil structure and microbial activity, and creates a more favorable environment for root growth.

[0030] The outstanding salt and alkali tolerance of the compound microbial inoculant of the present application makes it particularly suitable for saline-alkali soil, secondary salinization and soil hardened by long-term use of chemical fertilizers, greatly expanding the application range of microbial inoculants.

[0031] The compound microbial inoculant of the present application itself has the characteristics of harmless, efficient and environment-friendly, and meets the requirements of green agriculture and sustainable management of soil. Improving hardening by biological means reduces the over-reliance on mechanical deep plowing and chemical modifiers.

[0032] The compound microbial inoculant of the present application has remarkable effect on treating soil hardening caused by factors such as calcium sulfate accumulation and organic matter decline, and is particularly suitable for saline-alkali hardened soil which is difficult to be effectively treated by the prior art, solves the long-standing technical bottleneck in this field, and has broad application prospect and important practical value. DETAILED DESCRIPTION

[0033] The following specific embodiments illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosed content. Obviously, the described embodiments are part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0034] Klebsiella: China General Microbiological Culture Collection Center CGMCC 1.839;

[0035] Enterobacter hormaechei: China General Microbiological Culture Collection Center CGMCC 1.10608;

[0036] Humic acid: Guangdong Baisi Chemical Technology Co., Ltd., CAS: 1415-93-6;

[0037] Wood ash: Shuyang Linzhi Garden Art Co., Ltd., batch number: 03215;

[0038] Vermiculite powder: Hebei Hongyao Mineral Product Processing Co., Ltd., batch number: 200;

[0039] Galactose: Shanghai Maikelin Biotechnology Co., Ltd., CAS: 59-23-4;

[0040] Dimethyl sulfoxide: Shanghai Maikelin Biotechnology Co., Ltd., CAS: 67-68-5;

[0041] Gelatin: Komiyu Chemical Reagent Co., Ltd., CAS: 9000-70-8;

[0042] Trehalose: Komiyu Chemical Reagent Co., Ltd., CAS: 6138-23-4.

[0043] Example 1

[0044] The present embodiment provides a saline-alkali-resistant composite microbial agent:

[0045] 1. The fermentation method of Klebsiella is as follows:

[0046] 1) Klebsiella was inoculated into the seed culture medium, and was cultured in a 30℃ incubator for 24h to obtain a first-stage seed liquid, and the rotation speed was controlled at 180rpm;

[0047] 2) The seed liquid obtained in step 1) was inoculated into a first-stage seed tank at a volume fraction of 10%, and the culture conditions of the first-stage seed tank were temperature 30℃, rotation speed 180rpm, pH 7.0, and dissolved oxygen 40%, and was cultured for 24h to obtain a second-stage seed liquid;

[0048] 3) The second-stage seed liquid obtained in step 2) was inoculated into a fermentation tank at a volume fraction of 15%, and the culture conditions of the fermentation tank were temperature 30℃, rotation speed 180rpm, and was subjected to segmented fermentation under different pH gradients to obtain a fermentation liquid; the pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0, and the culture time of each stage was 5h, 6h, 6h, 8h, and 10h, respectively;

[0049] Corresponding to different pH gradients, the concentration of sodium chloride is 10 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L. The Klebsiella liquid inoculum is obtained.

[0050] 2. The fermentation method of Enterobacter hormaechei is as follows:

[0051] 1) Enterobacter hormaechei is inoculated into a seed culture medium, and is cultured in a 28°C incubator for 28 h to obtain a first-stage seed liquid, and the rotation speed is controlled at 160 rpm;

[0052] 2) The seed liquid obtained in step 1) is inoculated into a first-stage seed tank at a volume fraction of 15%, and the culture conditions of the first-stage seed tank are temperature 28°C, rotation speed 160 rpm, pH 7.2, and dissolved oxygen 35%, and the first-stage seed liquid is cultured for 28 h to obtain a second-stage seed liquid;

[0053] 3) The second-stage seed liquid obtained in step 2) is inoculated into a fermentation tank at a volume fraction of 15%, and the culture conditions of the fermentation tank are temperature 28°C and rotation speed 160 rpm, and the fermentation is carried out under different pH gradients to obtain a fermentation liquid; the pH gradient is 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and 9.5-10.0, and the culture time of each stage is 5 h, 6 h, 6 h, 8 h, and 10 h, respectively; corresponding to different pH gradients, the concentration of sodium chloride is 10 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L, and the Enterobacter hormaechei liquid inoculum is obtained.

[0054] 3. The preparation method of the composite microorganism is as follows:

[0055] 1) Klebsiella fermentation liquid and Enterobacter hormaechei are centrifuged respectively, the solid content of the adjusted bacterial liquid is 60%, and 10% dry protective agent is added to the adjusted bacterial liquid to obtain bacterial slurry, and the dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0056] 2) The Klebsiella bacterial slurry, Enterobacter hormaechei bacterial slurry, humic acid, and auxiliary materials obtained in step 1) are mixed and granulated according to a mass ratio of 1:1:2:2 to obtain solid particles, and the auxiliary material components are wood ash and vermiculite powder, and the ratio of wood ash to vermiculite powder is 2:1;

[0057] 3) The solid particles obtained in step 2) are vacuum dried at a drying temperature of 30°C for 10 h to obtain the composite microorganism.

[0058] Example 2

[0059] The present embodiment provides a saline-alkali-resistant composite microbial inoculum:

[0060] 1. The fermentation method of Klebsiella is as follows:

[0061] 1) Klebsiella pneumoniae was inoculated into seed culture medium and cultured in an incubator at 30°C with shaking for 24 hours to obtain primary seed liquid, with the rotation speed controlled at 180 rpm;

[0062] 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 25℃, rotation speed 200rpm, pH 7.5, dissolved oxygen 50%, and culture for 30h to obtain the secondary seed solution.

[0063] 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 10%. The fermentation conditions were 25°C and 200 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively.

[0064] For different pH gradients, sodium chloride concentrations of 8 g / L, 15 g / L, 30 g / L, 50 g / L, and 80 g / L were used to obtain Klebsiella liquid inoculum.

[0065] 2. The fermentation method for Enterobacter hooligans is as follows:

[0066] 1) Inoculate Enterobacter holmie into seed culture medium and culture in an incubator at 25°C with shaking for 30 hours to obtain primary seed culture, and control the rotation speed at 200 rpm;

[0067] 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 25℃, rotation speed 200rpm, pH 7.5, dissolved oxygen 50%, and culture for 30h to obtain the secondary seed solution.

[0068] 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 10%. The fermentation conditions were 25°C and 200 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively. The sodium chloride concentrations corresponding to the different pH gradients were 8g / L, 15g / L, 30g / L, 50g / L, and 80g / L, respectively, to obtain the liquid inoculum of Enterobacter holmieae.

[0069] 3. The preparation method of the compound microorganism is as follows:

[0070] 1) Centrifuge Klebsiella and Enterobacter hormaechei separately, adjust the solid content of the bacterial solution to 60%, add 10% dry protective agent to the adjusted bacterial solution to obtain the bacterial slurry, and the dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0071] 2) Mix the Klebsiella bacterial slurry, Enterobacter hormaechei bacterial slurry, humic acid, and auxiliary materials obtained in step 1) according to a mass ratio of 1:0.8:1.5:1.5 to granulate, to obtain solid particles, and the auxiliary material composition is wood ash and vermiculite powder, wood ash: vermiculite powder = 1:1;

[0072] 3) Vacuum dry the solid particles obtained in step 2) at a drying temperature of 45°C for 5h to obtain the composite microorganism.

[0073] Example 3

[0074] The present embodiment provides a saline-alkali-tolerant composite microbial agent:

[0075] 1. The fermentation method of Klebsiella is as follows:

[0076] 1) Inoculate Klebsiella into the seed culture medium, shake culture in a 30°C incubator for 24h to obtain the first-stage seed solution, and control the rotation speed at 180rpm;

[0077] 2) Inoculate the seed solution obtained in step 1) into the first-stage seed tank at an inoculation amount of 20% by volume, and the culture conditions of the first-stage seed tank are temperature 40°C, rotation speed 150rpm, pH 6.5, and dissolved oxygen 20%, and culture for 18h to obtain the second-stage seed solution;

[0078] 3) Inoculate the second-stage seed solution obtained in step 2) into the fermentation tank at an inoculation amount of 20% by volume, and the culture conditions of the fermentation tank are temperature 40°C, rotation speed 150rpm, and segmented fermentation is carried out at different pH gradients to obtain the fermentation liquor; the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0, and the culture time of each stage is 5h, 6h, 6h, 8h, and 10h, respectively;

[0079] Corresponding to different pH gradients, the concentration of sodium chloride is 15g / L, 30g / L, 50g / L, 80g / L, and 100g / L; to obtain the Klebsiella liquid bacterial agent.

[0080] 2. The fermentation method of Enterobacter hormaechei is as follows:

[0081] 1) Inoculate Enterobacter hormaechei into the seed culture medium, shake culture in a 40°C incubator for 18h to obtain the first-stage seed solution, and control the rotation speed at 150rpm;

[0082] 2) The seed liquid obtained in step 1) is inoculated into a primary seed tank at a volume fraction of 20%, and the culture conditions of the primary seed tank are temperature 40°C, rotation speed 150 rpm, pH 6.5, and dissolved oxygen 20%, and the culture is carried out for 18 h to obtain a secondary seed liquid;

[0083] 3) The secondary seed liquid obtained in step 2) is inoculated into a fermentation tank at a volume fraction of 20%, and the culture conditions of the fermentation tank are temperature 40°C, rotation speed 150 rpm, and the culture is carried out at different pH gradients to obtain a fermentation liquid; the pH gradients are 7.2-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and 9.5-10.0, and the culture time of each stage is 5 h, 6 h, 6 h, 8 h, and 10 h, respectively; and the sodium chloride concentrations corresponding to the different pH gradients are 15 g / L, 30 g / L, 50 g / L, 80 g / L, and 100 g / L, respectively, to obtain Enterobacter hormaechei liquid inoculum.

[0084] 3. The preparation method of the composite microorganism is as follows:

[0085] 1) The Enterobacter cloacae fermentation liquid and the Enterobacter hormaechei are centrifuged respectively, and the solid content of the adjusted bacterial liquid is 60%, and 10% dry protective agent is added to the adjusted bacterial liquid to obtain bacterial slurry, and the dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0086] 2) The Enterobacter cloacae bacterial slurry, the Enterobacter hormaechei bacterial slurry, humic acid, and auxiliary materials obtained in step 1) are mixed and granulated according to a mass ratio of 1:1.5:3:3 to obtain solid particles, and the auxiliary material components are wood ash and vermiculite powder, and the ratio of wood ash to vermiculite powder is 1.5:1;

[0087] 3) The solid particles obtained in step 2) are vacuum dried at a drying temperature of 30°C for 15 h to obtain the composite microorganism.

[0088] Comparative Example 1

[0089] This comparative example provides a saline-alkali-resistant composite microbial inoculum:

[0090] 1. The fermentation method of Klebsiella is as follows:

[0091] 1) Klebsiella is inoculated into a seed culture medium, and the culture is carried out in a 30°C incubator for 24 h to obtain a primary seed liquid, and the rotation speed is controlled at 180 rpm;

[0092] 2) The seed liquid obtained in step 1) is inoculated into a primary seed tank at a volume fraction of 10%, and the culture conditions of the primary seed tank are temperature 30°C, rotation speed 180 rpm, pH 7.0, and dissolved oxygen 40%, and the culture is carried out for 24 h to obtain a secondary seed liquid;

[0093] 3) The secondary seed liquid obtained in step 2) is inoculated into a fermenter at an inoculation amount of 15% by volume, and the culture conditions of the fermenter are temperature 30°C, rotation speed 180 rpm, pH 7.2, and sodium chloride concentration 10 g / L, and the culture is carried out for 35 h to obtain the liquid bacterial agent of Klebsiella.

[0094] 2. The fermentation method of Enterobacter hormaechei is as follows:

[0095] 1) Enterobacter hormaechei is inoculated into a seed culture medium, and the culture is carried out in a 28°C incubator for 28 h to obtain a primary seed liquid, and the rotation speed is controlled at 160 rpm;

[0096] 2) The seed liquid obtained in step 1) is inoculated into a primary seed tank at an inoculation amount of 15% by volume, and the culture conditions of the primary seed tank are temperature 28°C, rotation speed 160 rpm, pH 7.2, and dissolved oxygen 35%, and the culture is carried out for 28 h to obtain a secondary seed liquid;

[0097] 3) The secondary seed liquid obtained in step 2) is inoculated into a fermenter at an inoculation amount of 15% by volume, and the culture conditions of the fermenter are temperature 28°C, rotation speed 160 rpm, pH 7.2, and sodium chloride concentration 10 g / L, and the culture is carried out for 35 h to obtain the liquid bacterial agent of Klebsiella.

[0098] The other steps are the same as in Example 1.

[0099] Comparative Example 2

[0100] The microbial agent in the present comparative example only contains Klebsiella, and does not contain Enterobacter hormaechei and humic acid:

[0101] The difference between the present comparative example and Example 1 is that the present comparative example does not include the fermentation of Enterobacter hormaechei.

[0102] The preparation method of the composite microorganism in the present comparative example is as follows:

[0103] 1) The fermentation liquid of Klebsiella is centrifuged, and the solid content of the bacterial liquid is adjusted to 60%, and 10% of dry protective agent is added to the adjusted bacterial liquid to obtain bacterial slurry. The dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0104] 2) The Klebsiella bacterial slurry obtained in step 1) and the auxiliary material are mixed and granulated at a mass ratio of 4:2 to obtain solid particles. The auxiliary material comprises wood ash and vermiculite powder, and the mass ratio of wood ash to vermiculite powder is 2:1.

[0105] 3) The solid particles obtained in step 2) are vacuum dried at a drying temperature of 30°C for 10 h to obtain the composite microorganism.

[0106] Comparative Example 3

[0107] The microbial agent in the present comparative example contains only Enterobacter cloacae, and does not contain Klebsiella and humic acid:

[0108] Different from Example 1, the present comparative example does not include fermentation of Klebsiella.

[0109] The preparation method of the composite microorganism in the present comparative example is as follows:

[0110] 1) Centrifuge the Enterobacter cloacae fermentation broth, adjust the solid content of the bacterial solution to 60%, and add 10% dry protective agent to the adjusted bacterial solution to obtain a bacterial slurry. The dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0111] 2) Mix the Enterobacter cloacae bacterial slurry obtained in step 1) and the auxiliary material according to a mass ratio of 4:2 to granulate to obtain solid particles. The auxiliary material components are wood ash and vermiculite powder, and the ratio of wood ash to vermiculite powder is 2:1.

[0112] 3) Vacuum dry the solid particles obtained in step 2) at a drying temperature of 30°C for 10 hours to obtain the composite microorganism.

[0113] Comparative Example 4

[0114] The microbial agent in the present comparative example contains Klebsiella and Enterobacter cloacae, and does not contain humic acid:

[0115] The fermentation method of Klebsiella and Enterobacter cloacae in the present comparative example is the same as that in Example 1.

[0116] The preparation method of the composite microorganism in the present comparative example is as follows:

[0117] 1) Centrifuge the Klebsiella fermentation broth and the Enterobacter cloacae, respectively, adjust the solid content of the bacterial solution to 60%, and add 10% dry protective agent to the adjusted bacterial solution to obtain a bacterial slurry. The dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1;

[0118] 2) Mix the Klebsiella bacterial slurry, Enterobacter cloacae bacterial slurry, and auxiliary material according to a mass ratio of 2:2:2 to granulate to obtain solid particles. The auxiliary material components are wood ash and vermiculite powder, and the ratio of wood ash to vermiculite powder is 2:1.

[0119] 3) Vacuum dry the solid particles obtained in step 2) at a drying temperature of 30°C for 10 hours to obtain the composite microorganism.

[0120] Comparative Example 5

[0121] The microbial agent in the present comparative example contains Enterobacter cloacae and humic acid, and does not contain Klebsiella:

[0122] Unlike Example 1, this comparative example does not include fermentation of Klebsiella.

[0123] The preparation method of the composite microorganism in this comparative example is as follows:

[0124] 1) Centrifuge the Enterobacter hormaechei fermentation broth, adjust the solid content of the bacterial solution to 60%, and add 10% dry protective agent to the adjusted bacterial solution to obtain the bacterial slurry. The dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1.

[0125] 2) Mix the Enterobacter hormaechei bacterial slurry obtained in step 1), humic acid, and auxiliary materials according to a mass ratio of 2:2:2 to granulate, to obtain solid particles. The auxiliary material components are wood ash and vermiculite powder, and the wood ash: vermiculite powder = 2:1.

[0126] 3) Vacuum dry the solid particles obtained in step 2) at a drying temperature of 30°C for 10h, to obtain the composite microorganism.

[0127] Comparative Example 6

[0128] The microbial agent in this comparative example contains Klebsiella and humic acid, but does not contain Enterobacter hormaechei:

[0129] Unlike Example 1, this comparative example does not include fermentation of Enterobacter hormaechei.

[0130] The preparation method of the composite microorganism in this comparative example is as follows:

[0131] 1) Centrifuge the Enterobacter hormaechei fermentation broth, adjust the solid content of the bacterial solution to 60%, and add 10% dry protective agent to the adjusted bacterial solution to obtain the bacterial slurry. The dry protective agent is galactose: dimethyl sulfoxide: gelatin = 1:1:1.

[0132] 2) Mix the Enterobacter hormaechei bacterial slurry obtained in step 1), humic acid, and auxiliary materials according to a mass ratio of 2:2:2 to granulate, to obtain solid particles. The auxiliary material components are wood ash and vermiculite powder, and the wood ash: vermiculite powder = 2:1.

[0133] 3) Vacuum dry the solid particles obtained in step 2) at a drying temperature of 30°C for 10h, to obtain the composite microorganism.

[0134] Comparative Example 7

[0135] This comparative example provides a saline-alkali tolerant composite microbial agent:

[0136] Replace Enterobacter hormaechei and Klebsiella with thermotolerant phosphorus solubilizing bacteria and Bacillus subtilis.

[0137] Experimental Example 1

[0138] The composite microbial inoculants prepared from Examples 1, 2, 3, Comparative Examples 1-7 and humic acid are respectively applied to the soil with different degrees of salinization and different degrees of soil compaction, and the changes of the degrees of soil compaction and salinization after one month are shown in Tables 1-3.

[0139] Table 1 Influence of microbial inoculants on the degree of soil compaction of light salinization soil

[0140]

[0141] Table 2 Influence of microbial inoculants on the degree of soil compaction of moderate salinization soil

[0142]

[0143] Table 3 Influence of microbial inoculants on the degree of soil compaction of heavy salinization soil

[0144]

[0145] From Tables 1-3, it can be seen that the composite microbial inoculants prepared by the present application have good effects on soil compaction of different degrees of salinization, but the strains not domesticated by the specific pH gradient of the present application have poor effects on soil compaction of salinization, and single strains have poor effects on soil compaction of salinization, and humic acid not only acts as a carrier and adhesive, but also can improve soil structure, improve water and fertilizer retention capacity, stimulate microbial activity, and promote the formation and stability of soil aggregates in cooperation with the bacterial bodies.

[0146] Experimental Example 2

[0147] Examples 1, Comparative Examples 2-7 and humic acid are respectively applied to the salinization soil for planting corn, and the changes of the degrees of soil compaction, salinization and corn yield after four months are as follows:

[0148] Table 4 Influence of microbial inoculants on corn planted in salinization soil

[0149]

[0150] From Table 4, it can be seen that the composite microbial inoculants prepared by the present application can further improve the yield of crops after repairing soil compaction.

[0151] Experimental Example 3

[0152] The composite microbial inoculants obtained in Example 1 are preserved at room temperature, and the changes of the effective viable bacterial number are shown in Table 5.

[0153] Table 5

[0154]

[0155] As shown in Table 5, the microbial agent of the present application has good stability and is still effective after 24 months.

[0156] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A salt- and alkali-resistant compound microbial agent, characterized in that, The microbial agent is composed of Klebsiella pneumoniae, Enterobacter holmium, and humic acid, with the mass ratio of each component being Klebsiella pneumoniae: Enterobacter holmium: Humic acid = 1:0.5~2:1~3, wherein Klebsiella pneumoniae and Enterobacter holmium are obtained by fermentation culture. The preservation unit and preservation number of the Klebsiella pneumoniae are: China General Microbiological Culture Collection Center CGMCC1.839; The depository and accession number of the *Enterobacter holmieae* are: China General Microbiological Culture Collection Center (CGMCC1.10608). The fermentation culture method of Klebsiella includes: firstly, controlling the reaction conditions to conduct a preliminary culture of Klebsiella to obtain a primary seed culture; then, conducting a secondary culture of the primary seed culture to obtain a secondary seed culture; finally, conducting a staged fermentation of the secondary seed culture under different pH gradients to obtain Klebsiella fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours; The fermentation culture method of *Enterobacter holmieae* includes: firstly, controlling the reaction conditions to perform a preliminary culture of *Enterobacter holmieae* to obtain a primary seed culture; then, performing a secondary culture of the primary seed culture to obtain a secondary seed culture; finally, performing a staged fermentation of the secondary seed culture under different pH gradients to obtain *Enterobacter holmieae* fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours; The fermentation culture method for *Enterobacter holmieae* has a pH gradient corresponding to sodium chloride concentrations of 8-15 g / L, 15-30 g / L, 30-50 g / L, 50-80 g / L, and 80-100 g / L. The fermentation culture method for Klebsiella pneumoniae uses a pH gradient corresponding to sodium chloride concentrations of 8-15 g / L, 15-30 g / L, 30-50 g / L, 50-80 g / L, and 80-100 g / L.

2. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method for Klebsiella pneumoniae, the preliminary culture conditions are: shaking culture in an incubator at a temperature of 25~40℃ for 18~30h, and controlling the rotation speed at 150~200rpm.

3. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method for Klebsiella pneumoniae includes secondary culture conditions of 25-40℃, 150-200 rpm, pH 6.5-7.5, dissolved oxygen 20-50%, and culture time of 18-30 h.

4. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method for the aforementioned Enterobacter holmie involves preliminary culture under the following conditions: a shaking culture at a temperature of 25-40℃ for 18-30 hours, with the rotation speed controlled at 150-200 rpm.

5. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method for *Enterobacter holmieae* has the following secondary culture conditions: temperature 25-40℃, rotation speed 150-200 rpm, dissolved oxygen 20-50%, and culture time 18-30 h.

6. A method for preparing composite microbial agent particles, characterized in that... The preparation method comprises a salt- and alkali-resistant composite microbial agent as described in any one of claims 1-5, wherein the preparation method includes: Step 1: Centrifuge the Klebsiella fermentation broth and the Enterobacter hopnea fermentation broth separately, adjust the solid content of the bacterial broth to 50-80%, and add 0.5-15% desiccant to the bacterial broth to obtain Klebsiella bacterial slurry and Enterobacter hopnea bacterial slurry, respectively. Step 2: Mix the Klebsiella pneumoniae slurry, Enterobacter holmium slurry, humic acid and excipients from Step 1 in a certain proportion and granulate to obtain solid particles; Step 3: Vacuum dry the solid particles from Step 2 to obtain composite microbial agent particles.

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