CN114409446B

CN114409446B - Low-water solid-state fermentation method of drought-tolerant bacteria and application thereof

Info

Publication number: CN114409446B
Application number: CN202210070787.3A
Authority: CN
Inventors: 徐延平; 李丽艳; 葛振宇; 徐志文
Original assignee: LEADING BIO-AGRICULTURAL CO LTD
Current assignee: LEADING BIO-AGRICULTURAL CO LTD
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-09-16
Anticipated expiration: 2042-01-21
Also published as: CN114409446A

Abstract

The invention provides a low-water solid-state fermentation method of drought-enduring bacteria and application thereof. The low-water solid fermentation method adopts drought-resistant bacteria to carry out solid fermentation under the low-water condition, wherein the drought-resistant bacteria is Bacillus flexus LXBF.1 with the preservation number of CGMCC No. 21705. The method can carry out solid-state fermentation by using drought-resistant bacteria under the low water condition, so that the effective viable count of the drought-resistant bacteria of a solid-state fermentation product obtained by pure culture is obviously improved, the bacterial contamination rate is obviously reduced, the stability of the product is enhanced, and the higher effective viable count and purity are maintained; the mixed bacteria rate of a solid-state fermentation product obtained by non-pure culture is obviously reduced, the loss of effective components is reduced, the directional effect of the product is enhanced, and higher effective viable count and purity are maintained, so that the outstanding problems of complex production process, poor product quality, unstable effect and the like of the existing high-moisture solid-state fermentation are solved; in addition, the solid state fermentation effect of the drought-resistant bacteria under the low water condition can be obviously improved by utilizing the pseudomonas fluorescens CGMCC No. 8820.

Description

Low-water solid-state fermentation method of drought-tolerant bacteria and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a low-water solid-state fermentation method of drought-enduring bacteria and application thereof.

Background

Drought is an adversity stress which is often encountered in the growth process of crops, under the adversity condition, a large number of microorganisms cannot successfully colonize and survive, so that the number of soil bacteria is greatly reduced, and the diversity of community structures is obviously reduced, thereby influencing the growth environment of plant rhizosphere soil, further influencing the absorption of normal moisture and nutrients of plants, and reducing the production capacity of the plants. Meanwhile, crops can cause yield reduction and even death under the condition of long-term drought stress, and great damage is caused to agricultural production. Therefore, the microorganisms in the biofertilizer need to be capable of adapting to adverse environments such as drought and the like so as to better exert the efficacy of the biofertilizer.

Solid state fermentation refers to a biological process of culturing one or more microorganisms in a water-insoluble solid matrix with a certain humidity, in the absence or almost absence of free water. In view of the nature of the biological reaction process, the solid fermentation is a biological reaction process taking a gas phase as a continuous phase, and can simulate a natural growth environment, so that the original growth existing state of microorganisms in the nature can be reduced. Solid state fermentation is one of the technologies for producing required products by using microorganisms, the primary condition of the modern fermentation technology is pure culture, other microorganisms in the nature are not allowed to enter, and the production and application of the solid state fermentation are in a state before stagnation due to the requirement of the modern industry on large-scale intensive production.

However, solid state fermentation has many non-negligible advantages, such as: the raw material cost is low, the raw material is mostly natural substrate or byproduct of industrial production, complex preprocessing procedure is not needed, the source is wide, and the price is low; the water content of the matrix is low, no wastewater treatment is needed, the environmental pollution is less, and the post-treatment processing is convenient; the air existing in the gaps of the solid particles in the fermentation process provides oxygen required by the growth of microorganisms, and continuous ventilation is not needed; simple equipment and technology, greatly reduced volume of the bioreactor, relatively simple process, low energy consumption, simple energy supply equipment, low investment and the like.

Solid fermentation has not been well developed and utilized for a long time, mainly because not all microorganisms are suitable for solid fermentation, and drought tolerance is one of the important factors for adapting microorganisms to solid fermentation. Therefore, the development of drought-tolerant bacteria to solve the problems that the solid state fermentation of most microorganisms in low-moisture organic materials is difficult, microorganisms are not easy to colonize and survive under drought conditions, so that the microbial fertilizer product cannot fully exert the corresponding efficacy and the like is a technical problem which needs to be solved urgently.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a low-water solid-state fermentation method of drought-enduring bacteria and application thereof, which can realize solid-state fermentation of organic materials under low-water conditions, obviously improve the number of effective viable bacteria in solid-state fermentation products, and obviously improve the agronomic characters and the yield of crops when the crops are planted.

The invention provides a low-water solid fermentation method of drought-enduring bacteria, which adopts the drought-enduring bacteria to carry out solid fermentation under the low-water condition, wherein the drought-enduring bacteria is Bacillus flexus (LXBF.1), and the preservation number is CGMCC No. 21705.

Bacillus flexus (LXBF.1) adopted by the invention is preserved in China general microbiological culture Collection center (CGMCC) at 20.01.22.2021, and the preservation number is CGMCC No. 21705.

The Bacillus flexus LXBF.1 genome contains a series of genes related to plant growth and stress tolerance, including nitrogen fixation (nif U), siderophore synthesis (rhb E, rhb F) and phenazine antibiotic biosynthesis (phz F); at the same time, the gene related to salt tolerance is also provided, and Na is added ⁺ /H ⁺ Genes related to antiporters (mrp B, Nha C and yjb Q), glycine betaine transporter (opuD) and potassium absorption protein (ktrB) show that the strain has strong advantages in growth promoting performance and stress tolerance related functions.

The Bacillus flexus LXBF.1 can grow under the solid fermentation condition of pH5-10, can tolerate 10% NaCl and grow under the drought stress of 20-40% PEG6000, and shows that the strain has strong tolerance to adverse conditions and has the characteristics of high saline-alkali tolerance, drought tolerance and the like.

In the present invention, the low water condition is a condition of lower water content, and the water content is not strictly limited; specifically, the low water condition may be a condition that the water content is 30% or less, for example, a condition that the water content is 20 to 30%.

Furthermore, the low-water solid-state fermentation method of the drought-enduring bacteria can also add pseudomonas fluorescens and/or fermentation products thereof during solid-state fermentation, and the preservation number of the pseudomonas fluorescens is CGMCC No. 8820. Research shows that the composite pseudomonas fluorescens and/or fermentation products thereof can greatly increase the effective viable count of drought-resistant bacteria and obviously reduce the total number of mixed bacteria during solid state fermentation.

The pseudomonas fluorescens adopted by the invention is preserved in the China general microbiological culture Collection center in 2014 at 17.02 and 17, and the preservation number is CGMCC No. 8820.

The addition mode of the pseudomonas fluorescens and/or the fermentation product thereof is not strictly limited, and the addition mode comprises but is not limited to adding pseudomonas fluorescens microbial inoculum, pseudomonas fluorescens fermentation product, fermentation product without pseudomonas fluorescens living body and the like, and the appropriate addition mode can be selected according to actual requirements. In addition, the addition amount of the pseudomonas fluorescens and/or the fermentation product thereof is not strictly limited, and the appropriate addition amount can be selected according to actual requirements.

In one embodiment, the pseudomonas fluorescens fermentation product can be added during solid state fermentation, and the ratio of Bacillus flexus (lxbf) lxbf.1 to the number of pseudomonas fluorescens in the pseudomonas fluorescens fermentation product during solid state fermentation can be 1: (0.4-0.6).

In another embodiment, the solid-state fermentation may be performed using a liquid of an drought-tolerant bacterium, a fermentation product containing no living pseudomonas fluorescens may be added during the solid-state fermentation, and the mass ratio of the liquid of the drought-tolerant bacterium to the fermentation product containing no living pseudomonas fluorescens during the solid-state fermentation may be 1: (0.4-0.6).

The invention also provides the application of the low-water solid fermentation method in preparing a solid fermentation product under pure culture or non-pure culture conditions.

The invention does not strictly limit the solid fermentation product, and the solid fermentation product prepared under the pure culture condition is pure bacterial powder, an enzyme preparation, a microbial feed or a food-medicine level microecological preparation; the solid fermentation product prepared under the condition of non-pure culture is decomposed compost, a biological organic fertilizer, a bacterial fertilizer or an agricultural microbial preparation.

Further, under pure culture conditions, a fermentation product free from a living body of Pseudomonas fluorescens may be added; under non-pure culture conditions, a Pseudomonas fluorescens fermentation product may be added.

The invention also provides a preparation method of the biological organic fertilizer, which comprises the following steps: adding a drought-resistant microbial inoculum into a low-water fermentation raw material for composting to obtain a bio-organic fertilizer; wherein the drought-resistant microbial inoculum is a fermentation product of Bacillus flexus (LXBF.1) or a microbial inoculum thereof, and the preservation number of the Bacillus flexus (LXBF.1) is CGMCC No. 21705.

Furthermore, pseudomonas fluorescens and/or fermentation products thereof can be added during composting, and the preservation number of the pseudomonas fluorescens is CGMCC No. 8820.

The low-water fermentation raw materials for preparing the bio-organic fertilizer are not strictly limited, and can be conventional low-water fermentation raw materials in the field, such as decomposed materials obtained by composting organic solid wastes such as excrement, straws and kitchen waste, and the water content of the low-water fermentation raw materials is 20-35%.

The drought-resistant microbial inoculum is not strictly limited in form, can be a fermentation product of Bacillus flexus (Bacillus flexus) LXBF.1 or a microbial inoculum thereof, for example, can be a liquid microbial inoculum of the Bacillus flexus (Bacillus flexus) LXBF.1, and the effective viable count of the liquid microbial inoculum is more than or equal to 100 hundred million/mL.

The drought-resistant microbial inoculum is not strictly limited in dosage and can be reasonably added according to actual needs; specifically, the drought-resistant bacterial agent may be used in an amount of 3 to 8%, for example, 5%, by weight to volume ratio, i.e., 3 to 8mL of the liquid bacterial agent of Bacillus flexus (lxbf.1) is added per 1000g of the low water fermentation raw material.

The invention does not strictly limit the composting conditions and can be reasonably set according to the actual situation; specifically, the stacking can be carried out under natural conditions, and the stacking time can be about 25 to 35 days.

The preparation method of the invention can realize the performance of solid state fermentation of organic materials under low water condition by utilizing Bacillus flexus LXBF.1, and the effective viable count in the pile can be increased by more than 25 times after the pile is naturally piled for about 30 days; spores do not germinate under the conditions of low water and low oxygen after being packaged, a higher effective viable count can be maintained within 2 years, and no mixed bacteria are generated; when the strain is applied to the field of low-moisture organic material solid state fermentation, the process method is simple, the production cost is low, fermentation in a low-moisture state can be realized, a higher effective viable count can be obtained, meanwhile, enzymolysis can be realized, protease, amylase and cellulase can be produced, and the problems of high rate of mixed bacteria and the like in the existing high-moisture fermented organic material are solved.

The drought-resistant bacteria curvibacillus flexus can realize solid state fermentation under low water conditions (20% -30%), obviously improve the effective viable count of drought-resistant bacteria, inhibit the generation of other mixed bacteria under high water conditions (50%), and effectively reduce the contamination rate; particularly, after the pseudomonas fluorescens and/or the fermentation product thereof are compounded, the effective viable count of drought-resistant bacteria can be greatly increased, the total number of mixed bacteria is obviously reduced, and the mixed bacteria rate is reduced from 29.95 percent to 4.48 percent.

The invention also provides a biological organic fertilizer prepared by the preparation method.

More specifically, the effective viable count of Bacillus flexus (LXBF.1) in the bio-organic fertilizer is more than or equal to 10 hundred million/g, and the rate of mixed bacteria before and after stacking has no obvious change; in addition, after the stacked product is packaged, the Bacillus flexus (LXBF.1) hardly germinates, the effective viable count of the bio-organic fertilizer product can be maintained at a high level within 2 years, and no other mixed bacteria are bred.

The invention also provides application of the biological organic fertilizer in crop planting.

The biological organic fertilizer can be well applied to planting crops. The drought-tolerant bacterium solid fermentation product can obviously improve the germination rate and the dry matter accumulation of corn seeds, and can improve the drought resistance of seedlings under the drought condition; when the Bacillus flexus LXBF.1 in the biological organic fertilizer is applied to cucumber planting, the agronomic characters of cucumbers can be obviously improved, the root growth of the cucumbers is promoted, the yield can be increased by 32%, and the Bacillus flexus LXBF.1 has strong colonization and growth promotion capabilities, can obviously improve the agronomic characters and the yield of crops, and has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of measurement of the activity of Bacillus curvatus LXBF.1 amylase;

FIG. 2 shows the results of cellulase activity assay of Bacillus curvatus LXBF.1;

FIG. 3 shows the results of the protease activity assay of Bacillus flexus LXBF.1.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 growth-promoting, stress-resistant genes of the drought-tolerant bacterium Bacillus curvatus LXBF.1

Inoculating Bacillus flexus (Bacillus flexus) LXBF.1 (with the preservation number of CGMCC No.21705) into LB liquid culture solution, culturing at 30 ℃ and 180r/min to logarithmic phase, then centrifuging at 4 ℃ and 4500r/min for 10min, discarding supernatant and collecting thalli, storing by using liquid nitrogen refrigerating fluid, sending to Nuo grass genesis science and technology Limited company for whole genome sequencing to obtain the whole genome sequence of the Bacillus flexus and analyzing related genes; the analysis results are shown in table 1.

TABLE 1 related genes in the Bacillus curvatus LXBF.1 genome

As can be seen from table 1, the campylobacter lxbf.1 genome contains a series of genes associated with plant growth and stress tolerance, including nitrogen fixation (nif U), siderophore synthesis (rhb E, rhb F), phenazine antibiotic biosynthesis (phz F), spermidine biosynthesis (spe a, spe B); meanwhile, the genome also has some genes related to salt tolerance, such as genes related to Na +/H + antiporters (mrp B, Nha C, yjb Q), glycine betaine transporter (opuD) and potassium uptake protein (ktrB).

Example 2 drought-tolerant bacteria Bacillus flexus LXBF.1 stress resistance test

The method comprises the steps of preparing curvularia lunata LXBF.1 seed liquid according to a conventional method, then respectively inoculating the seed liquid into solid fermentation culture media (20 g/L of soybean meal, 300g/L of bran, 5.5g/L of light calcium carbonate, 20g/L of rice hull, 0.5g/L of magnesium sulfate, 0.5g/L of potassium dihydrogen phosphate and 10g/L of cane sugar, and supplementing water to 55% of water) with different pH conditions (pH5, pH7, pH9 and pH10) according to the inoculation amount of 5%, and culturing for 3-5 days at 30 ℃, and determining the effective viable count of the drought-tolerant curvularia lunata in each fermentation system, wherein the results are shown in Table 2.

TABLE 2 stress resistance of drought tolerant bacteria Bacillus curvatus

The results show that: the curvularia Lenzi LXBF.1 can grow under the solid fermentation condition of pH5-10, can tolerate 10% NaCl and can well grow under the drought stress of 20-40% PEG 6000. Therefore, the Bacillus flexus LXBF.1 has strong tolerance to adverse conditions and has the characteristics of high saline-alkali resistance, drought resistance and the like.

Example 3 enzyme assay of drought-tolerant bacterium Bacillus flexus LXBF.1

1) And (3) amylase activity determination:

inoculating Bacillus flexus LXBF.1 on an amylase selective culture medium solid plate (10 g of peptone, 10g of soluble starch, 5g of yeast extract powder, 10g of sodium chloride, 20g of agar powder, constant volume of distilled water to 1000mL, pH7.2-7.5), culturing at constant temperature of 30 ℃ for 2-3 days, and covering a layer of iodine solution (5mol/L I) on the surface of the solid plate ₂ And 5mol/L KI), observing and measuring the diameter of the transparent circle, and if the transparent circle appears, indicating that the amylase has the capability of producing amylase.

2) And (3) cellulase activity determination:

the method comprises the steps of inoculating curvularia Lemansonii LXBF.1 on a cellulase biopsy detection culture medium plate (10 g/L of sodium carboxymethylcellulose, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of potassium dihydrogen phosphate, 1g/L of disodium hydrogen phosphate, 5g/L of yeast extract, 10g/L of peptone, 20g/L of agar powder and distilled water to reach a constant volume of 1000mL and a pH value of 7.2-7.4), culturing at a constant temperature of 30 ℃ for 2 days, dropwise adding 0.1% Congo red dye solution on the surface of a culture medium, soaking for 15min, then eluting for 20min by using a proper amount of 1mol/L NaCl solution, and observing whether a transparent ring appears.

3) Protease activity assay:

inoculating Bacillus flexus LXBF.1 on a solid plate of a screening culture medium (10 g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 10g of skimmed milk powder or 10mL of skimmed milk, 20g of agar powder, constant volume of distilled water to 1000mL, pH7.2-7.5), culturing at constant temperature of 30 ℃ for 2-3 days, observing whether a transparent ring (black background) exists, and if the transparent ring appears, indicating that the bacillus flexus LXBF.1 has the capability of producing protease activity.

The measurement results are shown in FIGS. 1 to 3.

The results show that: the Bacillus flexus LXBF.1 has the activities of amylase, protease and cellulase at the same time, and has good application potential when being applied to the decomposition of organic materials.

EXAMPLE 4 growth characteristics of drought-tolerant bacteria on different aqueous materials

Adopts a solid fermentation culture medium (20 g/L of soybean meal and 300g/L of bran)5.5g/L of light calcium carbonate, 20g/L of rice hull, 0.5g/L of magnesium sulfate, 0.5g/L of monopotassium phosphate and 10g/L of cane sugar, respectively adjusting the water content to be 20%, 30% and 50%, and then performing high-temperature steam sterilization treatment (121 ℃, 50 min). The test strains are Bacillus subtilis of commercial Jinan Luya Biotechnology Co., Ltd, Bacillus amyloliquefaciens of Shandong Xin Zhuo Yun chemical Co., Ltd and Bacillus flexus of drought-enduring bacteria. Respectively preparing fermentation liquor of bacillus subtilis, bacillus amyloliquefaciens and drought-enduring bacteria bacillus flexus by adopting LB liquid culture medium, and diluting to ensure that the effective bacteria number reaches 10 ⁸ And (3) respectively inoculating the CFU/ml into cow dung pure culture materials with different water contents according to the inoculation amount of 1%, culturing for 7 days at the temperature of 30 ℃, and counting the effective viable count and the contamination condition of the initial and cultured 7 days according to the following formulas:

the bacterial contamination ratio [ (% total colony-effective viable bacteria)/total colony ] × 100

The results are shown in tables 3 and 4.

TABLE 3 pure culture characteristics of different aqueous materials

TABLE 4 pure culture characteristics of different aqueous materials

The results show that the drought-enduring bacillus flexus can be subjected to solid state fermentation under the conditions of low water content of 20% and 30%, the effective viable count is obviously improved, and the effective viable count of the bacillus subtilis and the bacillus amyloliquefaciens is not obviously changed, which indicates that spores are not germinated and still in a dormant state; under 50% high water condition, 3 strains of bacillus can germinate and grow, but the existence of drought-resistant bacteria can obviously reduce the bacterial contamination rate (15.82%), and the bacterial contamination rates of bacillus amyloliquefaciens and bacillus subtilis are 73.32% and 72.58% respectively. The strain shows that the drought-enduring bacillus curvatus can realize solid state fermentation under low water conditions (20-30 percent), obviously improve the effective viable count of the drought-enduring bacteria, inhibit the generation of other mixed bacteria under high water conditions (50 percent) and effectively reduce the contamination rate.

EXAMPLE 5 non-pure culture characteristics of drought-resistant bacteria on Low Water organic materials

A solid fermentation culture medium (20 g/L of soybean meal, 300g/L of bran, 5.5g/L of light calcium carbonate, 20g/L of rice hull, 0.5g/L of magnesium sulfate, 0.5g/L of monopotassium phosphate and 10g/L of cane sugar) is adopted, the water content is adjusted to be about 30%, and no sterilization treatment is carried out. The test treatments are respectively as follows:

treatment 1: 1% drought-resistant bacterium Bacillus flexus solution (10) ⁸ CFU/ml)；

And (3) treatment 2: 1% drought-resistant bacterium Bacillus flexus liquid (10) ⁸ CFU/ml) + 0.5% Pseudomonas fluorescens liquid fermentation product (10) ⁸ CFU/ml)。

After inoculation for 15 days at 30 ℃ for each treatment, the total number of colonies (bacteria and fungi) at the beginning and 15 days of culture was counted and the rate of undesired bacteria was calculated as follows:

percent infectious microbe ═ [ (total colony-effective viable count)/total colony ] × 100

The results are shown in Table 5.

TABLE 5 non-pure culture characteristics of Low Water materials

The results show that the drought-tolerant bacteria curvibacillus flexus of the invention has little influence on the total number of the mixed bacteria when being subjected to solid fermentation under the condition of 30 percent low water, and after the liquid fermentation product of the pseudomonas fluorescens is compounded, the effective viable count of the drought-tolerant bacteria can be greatly improved, the total number of the mixed bacteria can be obviously reduced, and the mixed bacteria rate is reduced from 29.95 percent to 4.48 percent, which indicates that the drought-tolerant bacteria curvibacillus flexus is compounded with the liquid fermentation product of the pseudomonas fluorescens in the process of solid fermentation of low water organic materials, so that the bacteria number of the drought-tolerant bacteria can be effectively improved, and the generation of the mixed bacteria can be inhibited.

Example 6 preparation of Bacillus curvatus LXBF.1 microbial Agents

1) Culture medium

The liquid culture mediums of the curvibacillus LXBF.1 seed tank and the fermentation tank are as follows:

5.5g/L glucose, 3.5g/L maltose, 3g/L peptone, 4g/L ammonium sulfate, 0.3g/L magnesium sulfate, 0.3g/L potassium dihydrogen phosphate, 0.3g/L dipotassium hydrogen phosphate, 5g/L corn meal, 10g/L soybean meal, 3g/L light calcium carbonate, and the pH value is 7.2.

2) Culturing

Inoculating Bacillus flexus LXBF.1 preserved in a glycerol tube at the temperature of-80 ℃ on an LB agar culture medium, and performing activated culture at the temperature of 28 ℃ for 2-3 days to obtain a solid seed culture.

Inoculating the solid seed culture of the curvatus LXBF.1 into a liquid NB culture medium, and carrying out shake culture for 24-48h at the temperature of 30 ℃ and the rotating speed of 180rpm to obtain a liquid primary fermentation seed.

Respectively inoculating the liquid primary fermentation seeds into a liquid seed fermentation tank according to the inoculation amount of 5%, and performing fermentation culture for 24-48h under the conditions of the temperature of 30 ℃, the rotation speed of 180rpm and the ventilation amount of 2% to obtain liquid secondary fermentation seeds.

Inoculating the liquid fermentation secondary seeds into a fermentation tank filled with a liquid fermentation culture medium according to the inoculation amount of 10%, fermenting for 24-48h at 30 ℃, 150rpm and the ventilation amount of 2%, stopping fermentation, wherein the bacterial count in the fermentation liquor of the Bacillus flexus LXBF.1 is 1.0 multiplied by 10 ¹⁰ cfu/mL。

Example 7 application to solid fermentation of Low Water organic materials

Collecting a later-stage sample (with the water content of 20-30%) of a normal cow dung compost decomposing system, uniformly spraying a bacillus curvatus microbial agent (100 hundred million/mL) prepared in example 6 on one part of the later-stage sample according to the weight volume ratio of 5 per mill, uniformly stirring, and naturally stacking for 30 days to prepare a biological bacterial fertilizer; and sterilizing the other part by high-temperature steam to obtain a pure culture material with the water content of 20-30%, uniformly spraying the curvularia lunata microbial inoculum (100 hundred million/mL) prepared in the example 6 according to the weight volume ratio of 5 per thousand, uniformly stirring, and naturally stacking for 30 days to obtain the pure culture (27.6 hundred million/g) of the cow dung organic material.

Respectively taking pile samples piled for 0 day and 30 days to detect the effective viable count and the quantity of mixed bacteria of the bacillus curvatus in the pile; and stacking the samples, normally packaging the products, naturally storing the products in a storehouse, and detecting the number of the campylobacter bacteria and the growth condition of the mixed bacteria every half year.

The detection result shows that: the effective viable count of the curvularia lunata in the pile is increased to 12.9 hundred million/mL after being naturally piled for 30 days from 0.5 hundred million/mL, which is increased by more than 25 times compared with the initial count, the effective viable count of the curvularia lunata is more than or equal to 10 hundred million/g, and the rate of mixed bacteria before and after piling is not obviously changed; after the product is stacked and packaged, the bent bacillus hardly germinates, the effective viable count of the product can be maintained at a higher level within 2 years, and no other mixed bacteria are bred.

Example 8 drought stress test with drought-tolerant bacteria

A drought stress test was performed using the pure culture of organic material (27.6 hundred million/g) prepared in example 7. The air-dried soil was sub-packed in plastic pots, each containing 1.5kg of soil. The test treatments are respectively as follows: a treatment was a blank control, 80g of the drought-resistant bacteria solid pure culture prepared in example 7 was added to B treatment, and 80g of the inactivated drought-resistant bacteria solid pure culture prepared in example 7 was added to C treatment (autoclaving at 121 ℃ C. for 40min), each treatment being repeated 3 times. Selecting corn seeds which are plump, uniform and consistent, sowing the seeds, watering the seeds in 5 seeds per pot according to the watering amount of 50% of the field water capacity, carrying out drought stress treatment on the corn seedlings after the corn seedlings emerge for 20 days, namely stopping watering the corn seedlings until the control seedlings wither, investigating the germination rate, the dry matter quantity and the chlorophyll content of each treatment, and the results are shown in table 6.

TABLE 6 Effect of drought stress on maize seedling growth vigor

The results show that the germination rate of the control treatment (namely, the treatment A) is 70 percent, while the germination rate of the treatment B added with the drought-enduring bacterium solid fermentation product is improved to 86.7 percent, the germination rate is improved by 23.86 percent, and the germination rate of the treatment C after inactivation is not obviously changed (73.3 percent); after drought stress, the dry matter and chlorophyll content of the corn treated by the B in the seedling stage are both higher than those of the control A, the dry matter reaches 0.231 g/plant, the chlorophyll content (SPAD) is 54.8, and the change of the inactivated C treatment on the dry matter and the chlorophyll content is not obvious. Therefore, the drought-tolerant bacterium solid fermentation product can obviously improve the germination rate and the dry matter accumulation of the corn seeds and improve the drought resistance of seedlings under the drought condition.

Example 9 field application test

The test site is selected from test fields for planting cucumbers in Chongli gathering in Hebei province.

Test area: before transplanting the cucumbers, 20 kg/mu of the biological bacterial manure prepared in the embodiment 7 is broadcast; control zone: application of Compound fertilizers (N: P) ₂ O ₅ :K ₂ O26: 12:8)20 kg/mu, and the agronomic characters and economic characters of the cucumbers are respectively investigated in the seedling stage and the harvest stage, and the results are shown in tables 7 and 8.

TABLE 7 influence on agronomic traits of cucumber in seedling stage

TABLE 8 influence on economic traits of cucumber

The results show that: the biological bacterial fertilizer prepared in the embodiment 7 is applied to the cucumber, so that the agronomic characters of the cucumber can be obviously improved, the root growth of the cucumber is promoted, and the yield can be increased by 32%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A low-water solid fermentation method of drought-enduring bacteria is characterized in that the drought-enduring bacteria are adopted to carry out solid fermentation under the low-water condition, wherein the drought-enduring bacteria are Bacillus flexus (LXBF.1) with the preservation number of CGMCC No. 21705; and adding pseudomonas fluorescens and/or a fermentation product thereof during solid state fermentation, wherein the preservation number of the pseudomonas fluorescens is CGMCC No. 8820.

2. The low water solid state fermentation process of claim 1, wherein the low water conditions are a water content of 20-35%.

3. The low water solid state fermentation process of claim 1, wherein the pseudomonas fluorescens fermentation product is added during solid state fermentation, and the ratio of Bacillus flexus (Bacillus flexus) lxbf.1 to pseudomonas fluorescens in the pseudomonas fluorescens fermentation product during solid state fermentation is 1: (0.4-0.6).

4. The low water solid-state fermentation method according to claim 1, wherein the solid-state fermentation is performed using a drought-resistant bacterium solution, and a fermentation product containing no living pseudomonas fluorescens is added during the solid-state fermentation, and the mass ratio of the drought-resistant bacterium solution to the fermentation product containing no living pseudomonas fluorescens during the solid-state fermentation is 1: (0.4-0.6).

5. Use of the low water solid state fermentation process of any one of claims 1 to 4 in the preparation of a solid state fermentation product under pure or non-pure culture conditions; wherein, under pure culture conditions, a fermentation product without the living body of the pseudomonas fluorescens is added; adding a pseudomonas fluorescens fermentation product under non-pure culture conditions.

6. A preparation method of a biological organic fertilizer is characterized by comprising the following steps: adding a drought-resistant microbial inoculum into a low-water organic fermentation raw material for composting to obtain a bio-organic fertilizer; wherein the drought-resistant microbial inoculum is a fermentation product of Bacillus flexus (LXBF.1) or a microbial inoculum thereof, and the preservation number of the Bacillus flexus (LXBF.1) is CGMCC No. 21705; adding pseudomonas fluorescens and/or fermentation products thereof during composting, wherein the preservation number of the pseudomonas fluorescens is CGMCC No. 8820.

7. The preparation method according to claim 6, wherein the low-water organic fermentation raw material is decomposed material obtained by composting organic solid waste, and the water content of the low-water organic fermentation raw material is 20-35%.

8. The method of claim 7, wherein the organic solid waste is at least one selected from the group consisting of manure, straw, and kitchen waste.

9. The preparation method according to claim 6, wherein the drought-enduring microbial agent is a Bacillus flexus (LXBF.1) liquid microbial agent, and the effective viable count of the liquid microbial agent is more than or equal to 100 hundred million/mL.

10. The method according to claim 6, wherein the amount of the drought-resistant microbial inoculum is 3 to 8% o in composting.

11. A bio-organic fertilizer, characterized in that, it is prepared by the preparation method of any one of claims 6 to 10.

12. Use of the bio-organic fertilizer of claim 11 for growing crops.