CN113121284B

CN113121284B - Composite organic bacterial fertilizer and preparation method and application thereof

Info

Publication number: CN113121284B
Application number: CN202110493195.8A
Authority: CN
Inventors: 沈家葆; 李华一
Original assignee: Guangdong Lihao Biological Agriculture Co ltd
Current assignee: Guangdong Lihao Biological Agriculture Co ltd
Priority date: 2021-05-06
Filing date: 2021-05-06
Publication date: 2022-12-30
Anticipated expiration: 2041-05-06
Also published as: CN113121284A

Abstract

The invention belongs to the technical field of compound fertilizers, and particularly relates to a compound organic bacterial fertilizer as well as a preparation method and application thereof. The method for preparing the composite organic bacterial fertilizer comprises the following steps: performing single strain culture to respectively obtain azotobacter bacterial liquid and hydrogenophilic bacterial liquid, performing bacterium group co-culture on the azotobacter bacterial liquid and the hydrogenophilic bacterial liquid in a co-culture medium to obtain primary co-culture bacterial liquid, fermenting the primary co-culture bacterial liquid to obtain co-culture fermentation liquid, infiltrating the co-culture fermentation liquid on a microbial agent carrier, and drying to obtain the composite organic bacterial fertilizer. Based on the protection of the carrier to the microorganism and the reciprocal symbiosis between the azotobacter and the hydrogenophile, the composite organic bacterial fertilizer has the advantages of storage and transportation resistance, capability of being colonized at the root circle of the plant for a long time and suitability for various field environments.

Description

Composite organic bacterial fertilizer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of compound fertilizers, and particularly relates to a compound organic bacterial fertilizer and a preparation method and application thereof.

Background

Since nearly two centuries, with the vigorous development of chemical agriculture, the application amount of chemical fertilizers increases year by year, the soil problem becomes more serious, so that the planting workers must increase the application amount of the chemical fertilizers year by year to ensure or enlarge the original yield, and the soil problem becomes more serious. For the vast majority of the existing farmlands in China, the planting direction of applying a large amount of insecticides, chemical pesticides and hormones is urgently needed to be changed into an ecological friendly type with little or no use of insecticides and chemicals. Under most existing farming conditions, to solve the problem of crop malnutrition, growers often purchase and use commercially available non-standardized agricultural materials, while investing large amounts of organic fertilizers. The measures can increase the types and the quantity of the materials put into production indefinitely, bring adverse effects to an environmental ecosystem and increase the labor cost.

With the increase of population quantity and population density in China, the quantity of agricultural and sideline products to be supplied is increased day by day, so that the requirement of cultivated land on nitrogen fertilizer is increased, and the requirement of cultivated land on nitrogen fertilizer is met in the past mainly by artificial nitrogen fixation, namely industrialized production of nitrogen fertilizer for farming. The long-term application of chemical fertilizers brings a series of problems, such as shortage of available fresh water resources, competition for fertile fields, waste of energy, higher and higher input cost, and reduction of stress resistance of field crops, and the problems are rooted in that long-term chemical agriculture damages soil, causes reduction of soil microbial diversity, and loss of beneficial soil microorganisms.

In recent years, researchers have developed various microbial agents aimed at solving plant nutrition problems and restoring soil microbial diversity. However, the traditional microbial agent has the defects of blind effective active strain proportion, lack of cooperation among strains, narrow adaptation range of a microbial agent carrier to weather, less suitable crop varieties, poor diversity tolerance to field environment, poor capability of effective active bacteria Tian Dingshi and short effective bacteria effect. The microbial fertilizer which can be used by various crops and has high effective active bacteria efficiency, strong colonization ability, lasting drug effect, wide field adaptability and transportation and preservation resistance is urgently needed.

Disclosure of Invention

The invention aims to provide a composite organic bacterial fertilizer with strong colonization ability, storage and transportation resistance and wide application range, and a preparation method and application thereof.

Based on the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a preparation method of a composite organic bacterial fertilizer, which comprises the following steps:

(1) Culturing of individual strain

Respectively inoculating azotobacter and hydrogenophilic bacteria into respective culture solutions to carry out independent culture, and respectively preparing azotobacter bacterial liquid and hydrogenophilic bacterial liquid; the effective viable count of azotobacteria and hydrogenophilia is not less than 1 × 10 ⁹ CFU/mL, and the rate of mixed bacteria is less than 3%;

(2) Co-culture of bacterial groups

Inoculating the azotobacteria liquid and the hydrogenophile liquid prepared in the step (1) into a co-culture medium, and performing mixed culture to prepare a primary co-culture medium liquid; the total number of effective viable bacteria in the primary co-culture bacterial liquid is not less than 1 multiplied by 10 ⁹ CFU/mL, viable count of azotobacteria not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, wherein the rate of mixed bacteria in the primary co-culture bacterial liquid is lower than 3%;

(3) Co-culture bacterial liquid fermentation

Preparing the product of the step (2)Inoculating the obtained primary co-culture bacterial liquid into fermentation liquor, and fermenting to obtain co-culture fermentation liquor; the total number of effective viable bacteria in the co-culture fermentation liquid is not less than 1 × 10 ⁹ CFU/mL, the number of viable bacteria of azotobacteria is not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, the rate of mixed bacteria in the co-culture fermentation liquor is lower than 3%;

(4) And infiltrating the co-culture fermentation liquor into a sterilized microbial agent carrier, and drying to obtain the compound organic bacterial fertilizer.

According to the invention, azotobacter and hydrogenophile are taken as microorganism combinations and soaked in a carrier to prepare the composite organic bacterial fertilizer, the composite organic bacterial fertilizer is based on the reciprocal symbiosis of azotobacter and hydrogenophile, H2 generated by azotobacter nitrogen fixation can be utilized by hydrogenophile, the concentration of a product in the azotobacter reaction is reduced, and the azotobacter reaction is accelerated; meanwhile, the hydrogenophile decomposed macromolecular carbon chain organic matter can be used as an energy and material source of the azotobacter, so that the azotobacter reaction is promoted, the benign interaction of the azotobacter and the hydrogenophile is beneficial to enhancing the nitrogen content of soil and plants, and the growth promoting effect on the plants is obvious.

In addition, carriers suitable for loading azotobacter and hydrogenophilous bacteria are screened out, the effective strains are reasonably matched and have proper concentration, the effective active bacteria can be protected when changing the living environment, and substances and energy are supplied, so that the composite organic bacterial fertilizer can adapt to most kinds of field climates, even field environments with poor environmental conditions, such as environments with over-high or over-low pH value, over-high or over-low temperature, drought, waterlogging, high permeability and the like.

In addition, the invention further solves the problem that active bacteria in the existing microbial agent are difficult to colonize in a rhizosphere environment, besides the protective effect of a carrier on bacteria is beneficial to colonizing of the microorganisms in a plant rhizosphere environment, the plant rhizosphere environment is mainly gram-negative bacteria, and is different from the traditional agricultural microbial agent which is mostly gram-positive bacteria.

The invention also further solves the problem that the prior agricultural microbial agent is difficult to transport and store, the prior microbial agent usually adopts water as a carrier of effective active bacteria and also adopts fine sand, bran, wheat bran and carbon dust, however, the concentration of the effective active bacteria of the microbial agent is greatly reduced along with the transportation and the storage; the preferable carrier adopted by the microbial agent can ensure the activity and concentration of effective active bacteria in the processes of long-distance transportation and long-term storage.

Furthermore, the concentration of the hydrogenophilic bacteria in the composite organic bacterial fertilizer is 1 multiplied by 10 ⁵ ～1×10 ¹¹ CFU/g; the concentration of azotobacteria is 1 × 10 ⁵ ～1×10 ¹¹ CFU/g。

Further, the Azotobacter is at least one of Rhodobacter aquaticus (mangrove marinum), rhodopseudomonas palustris (Rhodopseudomonas palustris), rhodobacter sphaeroides (Rhodobacter sphaeroides), rhodospirillum palustris (Magnetospirillum belliums), azospirillum brasilense (Azospirillum brasilense), azospirillum melitensis (Azospirillum melini), azospirillum oryzae (Azospirillum oryzae), and Azotobacter vinelandii.

Further, the hydrogenophile is at least one of a hydrogenophile atypical (hydrogenophoga atypica), a hydrogenophile xanthium (hydrogenophoga flava), a hydrogenophile lisophorbide leinshi (hydrogengenophaga laconensis), and a hydrogenophile pa tienii (hydrogenophohaga pallronii).

Further, the microbial agent carrier is at least one of ammonium bicarbonate, urea, ammonium sulfate, calcium magnesium phosphate fertilizer, potassium sulfate, lime, calcium carbonate, charcoal, plant ash, green manure, compost, biogas fertilizer, petroleum distillate, waste fertilizer, coconut husk, kaolin, river sand and wood dust.

Further, the azotobacter is Azospirillum brasiliensis (Azospirillum brasilense); the hydrogenophile is hydrogenophile pa (Hydrogenophaga pallronii) or hydrogenophile lainsm (Hydrogenophaga laconesensis); the microbial agent carrier is charcoal.

Further, the azotobacter is Azospirillum melinii (Azospirillum melinis) or Azospirillum irakense (Azospirillum irakense); the hydrogenophile is atypical hydrogenophile spore (Hydrogenophaga typica); the microbial agent carrier is charcoal.

Further, the azotobacter is Azospirillum irakense (Azospirillum irakense); the hydrogenophile is hydrogenophile yellow spore bacillus (Hydrogenophaga flava); the microbial agent carrier is charcoal.

The invention preferably selects the combination of the five different azotobacter, hydrogenophile and carrier, the selected combination has more excellent growth promoting effect on plants, can still keep higher effective activity and concentration of active bacteria in the storage and transportation process, can colonize the root circle of plants for a long time, and is suitable for various field environments.

In a second aspect, the invention provides a composite organic bacterial fertilizer prepared by the method.

In a third aspect, the composite organic bacterial fertilizer provided by the invention is applied to nitrogen fixation of soybean and rejuvenation of peanut, wheat, oat and corn.

In a fourth aspect, the composite organic bacterial fertilizer provided by the invention is applied to improving soil acidity and alkalinity and enriching soil microorganism species.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, azotobacter and hydrogenophile are taken as microbial agents and soaked in a carrier to prepare the solid composite organic bacterial fertilizer, and the composite organic bacterial fertilizer has the advantages of storage and transportation resistance, capability of long-term colonization in a plant root ring and suitability for various field environments based on the protection of the carrier on microorganisms and the reciprocal symbiosis between the azotobacter and the hydrogenophile.

Drawings

FIG. 1 is a streaked plate of Azospirillum brasilense;

FIG. 2 is a streak plate of atypical hydrogenophiles;

FIG. 3 is a microscopic examination of Azospirillum brasilense;

FIG. 4 is a microscopic examination of atypical hydrogenophiles;

FIG. 5 is a microscopic examination of the primary co-culture broth;

FIG. 6 is a graph of isolation of a soil rhizosphere plate at the beginning of the test in example 4;

FIG. 7 is a graph of isolation of a soil rhizobacteria plate 2 months after the test in example 4;

FIG. 8 is a cut view of peanut stalks from example 4.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It should be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

The embodiment provides a preparation method of a composite organic bacterial fertilizer, which comprises the following steps:

(1) Culturing of individual strain

Obtaining single strains of azotobacter and hydrogenophile through a commercial way, adding the single strains into corresponding culture solution according to the proportion of 1.

Respectively dropping the cultured single strain culture solution on a glass slide, performing microscopic examination under oil microscope to test the concentration and the rate of undesired bacteria, wherein the microscopic examination images of azotobacter bacterial solution and hydrogenophile bacterial solution are respectively shown in fig. 3 and 4, and the effective viable count of azotobacter bacterial solution and hydrogenophile bacterial solution is not less than 1 × 10 ⁹ CFU/mL, and the rate of mixed bacteria is less than 3%.

The azotobacter culture solution comprises the following components: KH (natural Kill) ₂ PO ₄ 0.2g、K ₂ HPO ₄ 0.8g、MgSO ₄ ·7H ₂ O 0.2g、CaSO ₄ ·2H ₂ O 0.1g、FeCl ₃ 0.0005g、Na ₂ MoO ₄ ·2H ₂ 0.0005g of O, 0.5g of yeast extract, 20.0g of mannitol, 15.0g of agar and 1.0L of distilled water; the pH of the broth was adjusted to 7.2 with HCl and NaOH.

The formula of the hydrogenophilic bacteria culture solution is as follows: 10.0g of peptone, 3.0g of beef extract, 5.0g of NaCl, 5.0g of sodium chloride and 1.0L of distilled water; the pH of the broth was adjusted to 7.0 with HCl and NaOH.

The Azotobacter used in this embodiment is Azospirillum brasilense, and may be at least one of Rhodobacter sphaeroides (mangrove marine), rhodopseudomonas palustris (Rhodopseudomonas palustris), rhodobacter sphaeroides (Rhodobacter sphaeroides), rhodospirillum palustris (magnotrichum belliums), azospirillum melitensis (Azospirillum melini), azospirillum irillum (Azospirillum melini), azospirillum oryzae (Azospirillum oryzae), and Azotobacter vinelandii.

The hydrogenophile used in this example is atypical hydrogenophile (Hydrogenophaga typica), and may be at least one of hydrogenophile chrysosporium (Hydrogenophaga flava), hydrogenophile leprecheckii (Hydrogenophaga laconensis), and hydrogenophile pa li (Hydrogenophaga pallronii).

(2) Co-culture of bacterial groups

Adding azotobacteria bacterial liquid and hydrogenophilic bacteria bacterial liquid into co-culture liquid according to the proportion of 0.5 to 1000, culturing the inoculated co-culture liquid in a constant-temperature oscillator, adjusting the culture temperature to 28 ℃, the rotation speed to 150rpm/min, and co-culturing for 24-48 h to obtain primary co-culture bacterial liquid, and performing microscopic examination on the primary co-culture bacterial liquid, wherein the total number of effective viable bacteria in the primary co-culture bacterial liquid is not less than 1 x 10 as shown in figure 5 ⁹ CFU/mL, viable count of azotobacteria not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, and the rate of mixed bacteria in the primary co-culture bacterial liquid is lower than 3%.

The formula of the co-culture medium is as follows: 5.0g of peptone and beef extract1.5g of extract, 10.0g of sucrose, 0.15g of yeast extract and NaCl ₂ .5g、KH ₂ PO ₄ 0.2g、K ₂ HPO ₄ 0.8g, 0.1g of magnesium sulfate heptahydrate, 0.05g of calcium sulfate dihydrate, 0.0005g of ferric chloride, 0.0005g of sodium tungstate, 0.0005g of sodium molybdate, 0.0005g of manganese sulfate and 1.0L of water.

(3) Co-culture bacterial liquid fermentation

Adding the primary co-culture bacterial liquid into a fermentation tank containing fermentation liquor, wherein the weight ratio of the primary co-culture bacterial liquid to the fermentation liquor is 1; controlling the fermentation temperature to be 28 ℃, continuously introducing sterile air into the fermentation tank in the fermentation process, wherein the introduction flow rate of the sterile air is 0.5-5L/min, fermenting for 24-48 h to obtain co-culture fermentation liquor, and the total number of effective viable bacteria in the co-culture fermentation liquor is not less than 1 multiplied by 10 ⁹ CFU/mL, the number of viable bacteria of azotobacteria is not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, and the rate of mixed bacteria in the co-culture fermentation liquid is lower than 3%.

The formula of the fermentation liquid for fermentation of the primary co-culture bacterial liquid is as follows: 15g/L of glucose, 10g/L of fish meal and 1g/L of yeast extract. The pH value of the fermentation liquor is adjusted to 5.0-6.0 by using citric acid and calcium carbonate.

(4) Soaking the co-culture fermentation broth in a microbial agent carrier subjected to ozone sterilization and ultraviolet irradiation sterilization, then placing the carrier soaked with the bacterial liquid in a cool and ventilated drying place, and quickly drying in the shade to obtain the composite organic bacterial fertilizer, wherein the concentration of hydrogenophiles in the composite organic bacterial fertilizer is 1 x 10 ⁵ ～1×10 ¹¹ CFU/g; the concentration of azotobacteria is 1 × 10 ⁵ ～1×10 ¹¹ CFU/g。

In the operation process, the co-culture fermentation liquor is required to completely infiltrate the carrier, and the air flow in a shade drying place is fast, the humidity is low and the temperature is low. Before drying in the shade, the shade drying place should be cleaned and disinfected.

The microbial agent carrier used in this example is charcoal, and may be at least one of ammonium bicarbonate, urea, ammonium sulfate, calcium magnesium phosphate, potassium sulfate, lime, calcium carbonate, plant ash, green manure, compost, biogas manure, petroleum distillate, waste fertilizer, coconut husk, kaolin, river sand, and wood dust.

Example 2

In this example, the performance of the composite organic bacterial manure prepared in example 1 is tested in the following three aspects.

1. Detection of nitrogen fixation capability of composite organic bacterial fertilizer

Mixing the co-culture fermentation liquid obtained in the step (3) in the example 1 and the azotobacteria liquid obtained in the step (1) with LB agar to prepare a slant culture medium, adding 5mL of the slant culture medium into a test tube with the specification of 15mm × 150mm, simultaneously using an equal amount of clear water as a reference, sealing the test tube with a soft rubber plug for culturing, standing for 24h until a lawn grows, extracting 5mL of air, injecting 5mL of acetylene, culturing for 48h, then absorbing 100 μ L of gas in the test tube, and measuring the ethylene production by using a gas chromatograph.

The method is characterized in that nmol of acetylene produced by each mg of bacterial liquid per hour is used for evaluating the nitrogen fixation capacity of each bacterial agent, the higher the ethylene production amount is, the stronger the nitrogen fixation capacity of the bacterial agents is, the test results are shown in table 1, the average value of the nitrogen fixation capacity of the composite organic bacterial fertilizer is 1.81 times of that of the nitrogen-fixing bacteria, and the nitrogen fixation capacity of the composite organic bacterial fertilizer is obviously higher than that of the nitrogen-fixing bacteria.

TABLE 1 mean value of nitrogen fixation capacity of composite organic bacterial fertilizer and nitrogen fixation bacteria

Test group of species of strains	Mean nitrogen fixation (nmol)
		Azotobacteria and hydrogenophilic bacteria	6195
Azotobacteria	3422
		Control group	0

2. Determination of hydrogen production capacity of composite organic bacterial fertilizer

Mixing the co-culture fermentation broth obtained in the step (3) in the example 1 and the hydrogenophilic bacteria liquid obtained in the step (1) with LB agar to prepare a slant culture medium, adding 5mL of the slant culture medium into a test tube with the specification of 15mm × 150mm, simultaneously using an equal amount of clear water as a control, sealing and culturing by using a soft rubber plug, standing for 24h, extracting 5mL of gas after bacterial lawn grows out, adding 5mL of hydrogen, extracting 100 mu L of gas in the test tube after culturing for 48h, and measuring the hydrogen absorption rate by using a gas chromatograph.

The detection result of the hydrogen absorption rate is shown in table 2, and it can be seen that the composite organic bacterial fertilizer has good hydrogen production capacity compared with the hydrogenophiles.

TABLE 2 different inoculants to H ₂ Absorption rate value

Test group of species of strains	H2 absorption (%)
		Azotobacteria and hydrogenophilic bacteria	-9.58
Hydrogen-philic bacterium	39.09

3. Hemolytic reaction of composite organic bacterial fertilizer

The co-culture fermentation broth obtained in the step (3) in the example 1 is inoculated on a blood agar plate and cultured for 24 hours at 37 ℃, and no hemolytic cycle is observed to be generated, which indicates that the composite organic bacterial fertilizer prepared by the invention can be used as an agricultural microbial fertilizer.

The blood agar plate formulation in the experiment was as follows: 10g of peptone, 10g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of distilled water, wherein the pH value of a blood agar plate is 7.5, 60mL of sterile defibrinated sheep blood is added when the blood agar plate is sterilized under high pressure and cooled to 60 ℃, the plate is fully rotated and poured, and the thickness of the blood agar layer is 5mm.

Example 3

This example will explore the soil improvement effect of the compound organic bacterial manure of example 1 through the following two aspects.

1. Regulating and controlling effect of composite organic bacterial fertilizer on soil pH

Taking far red soil of Guangdong Qing dynasty, humus soil of Changbai mountain and bottom soil of a fish pond as test soil, sampling 10kg of each soil, adding 100mL of co-culture fermentation liquor obtained in the step (3) in the embodiment 1, mixing and stirring uniformly, putting into a constant-temperature constant-humidity incubator for culture, and sampling and detecting the pH value of the soil on the 1 st day, the 7 th day and the 28 th day of culture respectively.

The sampling detection method comprises the following specific steps: respectively putting 100g of a test soil sample into a beaker, adding distilled water to a constant volume of 1L, uniformly mixing by using a vortex stirrer, and standing for 10min; and (3) taking 100mL of the supernatant, pouring the supernatant into a triangular flask, measuring the pH of the supernatant by using a pH meter, and repeating the operation process for three times to obtain an average value.

The results of the pH measurements of the three samples are shown in table 3, and it can be seen that the pH value of the alkaline bottom soil of the fishpond gradually becomes neutral after being treated by the compound organic bacterial manure; after the acidic Guangdong Qingyuan red soil and Changbai mountain humus soil are treated by the compound organic bacterial manure, the pH value is increased to some extent; the results show that the composite organic bacterial fertilizer has the effect of improving the pH value of soil.

TABLE 3 pH value of different soil samples treated by composite organic bacterial manure

Test soil sample	Day 1 pH of the soil sample	Day 7 pH of the soil sample	pH value of soil sample at day 28
				Guangdong Qingyuan red soil	4.67	4.81	5.31
Changbai mountain humus soil	5.21	5.28	5.56
				Bottom soil for fishpond	9.21	9.01	8.28

2. Influence of composite organic bacterial fertilizer on nitrogen content of soil

Taking far red soil of Guangdong Qing dynasty, humus soil of Changbai mountain and bottom soil of a fish pond as test soil, sampling 10kg of each soil, adding 100mL of co-culture fermentation liquor obtained in the step (3) in the embodiment 1, mixing and stirring uniformly, putting into a constant-temperature constant-humidity incubator for culture, and sampling and detecting the nitrogen content in the soil on the 1 st day, the 7 th day and the 28 th day of culture respectively.

The sampling detection method comprises the following specific steps: respectively sampling 100g of test soil, putting the test soil into a beaker, adding distilled water to a constant volume of 1L, uniformly mixing the test soil and the distilled water by using a vortex stirrer, and standing the test soil for 10min; and (3) taking 100mL of the supernatant, pouring the supernatant into a triangular flask, measuring the nitrogen content of the supernatant by using a Kjeldahl azotometer, and repeating the operation process for three times to obtain an average value.

The results of measuring the nitrogen content of the three samples are shown in table 4, and it can be seen that the nitrogen content of the Guangdong Qingyuan red soil and Changbai mountain humus soil treated by the composite organic bacterial fertilizer gradually increases, which indicates that the composite organic bacterial fertilizer of the invention is beneficial to improving the nitrogen content of the soil.

TABLE 4 Nitrogen content of different soil samples after treatment with composite organic bacterial manure

Test soil sample	Nitrogen content of soil sample on day 1	Soil nitrogen content at day 7	Nitrogen content of soil at 28 th day
				Guangdong Qingyuan red soil	＜0.1％	0.04％	0.19％
Changbai mountain humus soil	0.80％	0.88％	1.16％
				Bottom soil for fishpond	＜0.1％	＜0.1％	＜0.1％

Example 4

This example will analyze the effect of the compound organic bacterial manure on plants in four ways as follows.

1. Colonization test of microorganism in composite organic bacterial manure in plant rhizosphere environment

The crops are potted in an incubator, and soil for potting is humus and is from Changbai mountain in Jilin province. The experimental culture pot is 21cm × 21cm, and the physical and chemical properties of the soil are as follows: the average particle size in dry state was 223 μm, the particle size in wet state was 66.5 μm, and the particle volume was 4.64 × 105cm ³ The density of the dry humus soil is about 2.47g/cm ³ . The mass fraction of the organic matters is 29.13 percent, the solid content is 89.50 percent, and the planted crop is peanut.

The method comprises the steps of soaking seeds of the Luyu peanuts in a microbial agent prepared by the co-culture fermentation liquid obtained in the step (3) of the embodiment 1 diluted by 25 times, planting the seeds into culture bowls, planting 4 strains in each pot, and spraying the microbial agent diluted by 100 times on the pages and the roots every 2 weeks.

The plate isolation map of the rhizosphere bacteria in the soil at the beginning of the test is shown in fig. 6, the plate isolation map of the rhizosphere bacteria in the soil after 2 months of the test is shown in fig. 7, the bacteria in the rhizosphere environment of the soil after 2 months of the test are isolated, the contents of the azotobacter and the hydrogenophile are estimated by adopting a plate counting method, and the results are shown in table 5.

Table 5 test the content of azotobacteria and hydrogenophila isolated from the rhizosphere soil of plants for 2 months

Species class	Content (CFU/g)
		Azospirillum brasilense	8.5×10 ⁶
Atypical hydrogenophila	4.9×10 ⁵

2. Influence of composite organic bacterial manure on plant tissues

A stem plant tissue section experiment is carried out on peanuts planted for 1 month in a culture bowl under an incubator environment, and the specific test method is as follows.

The incubator environment is: the light is 14 hours, the dark is 10 hours, the light intensity is 16000Lux, the temperature is 28 ℃, and the humidity is 75%.

The planting management scheme is as follows: the experimental group used the co-culture fermentation broth obtained in step (3) of example 1 to soak the seeds of the peanut in the co-culture fermentation broth diluted by 25 times at 28 ℃ for 1 hour, after germination accelerating, the seeds were planted in a culture bowl, and the co-culture fermentation broth diluted by 100 times was irrigated once every two weeks while the leaves were sprayed. The control group used the same management mode, but the inoculum was replaced with distilled water.

The stem tissue of the experimental group is thicker than that of the control group, the fibrous root is more vigorous, the aerial root is more developed, the leaf is thicker, and the wax layer is thicker. As shown in FIG. 8, when the cut peanut stems are planted for 1 month, the stem phloem tissue is thickened, the xylem is developed, and the air permeability is increased.

3. Influence of composite organic bacterial manure on soluble sugar of corn

(1) Establishing a standard curve of absorbance-sugar content

The specific steps for establishing the standard curve of the absorbance-sugar content are as follows: taking 6 test tubes with 20mL scales, numbering 1-6 in sequence, adding 100 mu g/mL sucrose solutions with the sugar contents of 0 mu g/mL, 10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL and 50 mu g/mL into the 6 test tubes respectively, supplementing the solution to 2mL with water, adding 0.5mL anthrone ethyl acetate reagent and 5mL concentrated sulfuric acid into the test tubes in sequence, fully oscillating, immediately putting the test tubes into a boiling water bath, accurately preserving the temperature for 1min tube by tube, taking out, naturally cooling to room temperature for comparison, measuring the absorbance at the wavelength of 630nm, drawing a standard curve by taking the absorbance as a vertical coordinate and the sugar content as a horizontal coordinate, and calculating a standard linear equation.

(2) Extraction and detection of corn sugar content

The method comprises the following steps of soaking corn in the microbial inoculum prepared in the embodiment 1 to obtain an experimental group, soaking corn in an equivalent amount of sterile culture solution to obtain a control group, planting the corn in the same soil and culturing in the same environment, irrigating the experimental group and the control group with the microbial inoculum and the sterile culture solution every 7 days after planting, and taking the corn seedlings on the same day to extract and detect the sugar content after the corn seedlings grow to three leaves, wherein the specific method comprises the following steps:

and (3) placing the overground parts of the corns in the three-leaf stage of the test group and the control group in a drying box for drying for 24h, crushing, weighing 0.10-0.30 g of crushed samples, placing the crushed samples in a 20mL graduated test tube, adding 5-10 mL of distilled water, sealing a plastic film, extracting in boiling water for 30min for 2 times, filtering the extracting solution, placing the extracting solution in a 25mL volumetric flask, repeatedly washing the test tube and residues, and fixing the volume to the graduation to obtain a sample solution.

Sucking 0.5mL of sample liquid into a 20mL graduated test tube, adding 1.5mL of distilled water, adding an anthrone ethyl acetate reagent and a concentrated sulfuric acid solution in sequence according to the steps of making a standard curve, developing, measuring absorbance, and calculating the sugar content of the test sample by a standard linear equation.

The results are shown in table 6, compared with the control group, the corn treated by the compound organic bacterial manure of the invention has higher soluble sugar content, which indicates that the compound organic bacterial manure of the invention can promote the growth of the corn.

TABLE 6 soluble sugar content of corn treated with different bacterial manure

Test group	Soluble sugar content of corn (mg/g)
		Test group	10.9
Control group	7.2

4. Influence of composite organic bacterial manure on nitrogen content of soybean seeds

After diluting 50 times the co-culture broth obtained in step (3) of example 1, the soybean seeds were soaked for 20min to serve as a test group; the soybean seeds soaked in the sterile fermentation liquor diluted by the same times for 20min are used as a control group. The sampling place of the soybean seeds is a clear city district of Qingyuan, guangdong province; the growing environment is a plant incubator. The soybean seed protein content was measured with a portable soy protein determinator (FOSS infra 1241).

The protein content of the soybean seeds treated by different methods is shown in table 7, and the protein content of the soybean seeds treated by the composite organic bacterial fertilizer is higher than that of a control group, so that the composite organic bacterial fertilizer has the effect of increasing the nitrogen content of the soybean seeds.

TABLE 7 Soybean seed protein content after treatment with different bacterial manure

Test group	Soybean seed protein content
		Test group	45.2％
Control group	41.6％

Example 5

In this embodiment, the influence of different groups of composite organic bacterial fertilizers on the growth rate of corn, wheat, oat, rape and peanut is analyzed through a field test, so as to screen out a better microbial inoculum combination, and the specific test method is as follows:

the test field is located in the Guangdong Qingyuan Xinxin area, and the test crops comprise corn, wheat, oat, rape and peanut. The specific operation is as follows: crop seeds with uniform size, glossiness and similar shape are taken and soaked in a test mixed bacterial sample diluted by 50 times, and each crop is provided with 5 plants in one group. The planting site was irrigated with 100-fold diluted mixed bacterial samples of the test every 2 weeks within 1 month after sowing. And spraying leaves of the crops by using the tested mixed bacterial sample diluted by 100 times every 2 weeks in the second February after sowing. Collecting the overground part of the whole plant 2 months after planting, drying to constant weight, measuring the dry weight growth rate of the overground part, and taking the average value of the growth rates of the crops of corn, wheat, oat, rape and peanut species as the final growth rate. The control group was treated with the corresponding clear water.

The tested strains were as follows:

test hydrogenophiles: a total of 5 species of the species Hydrogenphaga (Hydrogenophaga typica), hydrogenphaga flava (Hydrogenophaga flava), hydrogenphaga leinshi (Hydrogenophaga laconensis), and Hydrogenphaga palustris (Hydrogenophaga pallronii).

The tested azotobacter: rhodobacter hydrophila (mangrove marinum), rhodopseudomonas palustris (Rhodopseudomonas palustris), rhodobacter sphaeroides (Rhodobacter sphaeroides), magnetitum fus (Magnetospirillum belliums), azospirillum brasilense (Azospirillum brasilense), azospirillum melissa (Azospirillum melinis), azospirillum illucens (Azospirillum melinis), azospirillum iraillucens (Azospirillum irakense), azospirillum oryzae (Azospira oryzae), azotobacter vinelandii (Azotobacter vinelandii), 9 in total.

The combination of the tested hydrogenophiles and azotobacteria has 36 groups of experimental groups, the increase rate of the experimental groups is shown in table 8 by taking the dry weight increase rate on the ground of the control group as 100%, and it can be seen that when the hydrogenophiles are hydrogenophiles pa stutzeri and azospirillum brasilense, the dry weight increase rate on the ground of the plant can reach 144.88%, and then the combination of atypical hydrogenophiles and agaricus, the combination of atypical hydrogenophiles and azospirillum melitensis, the combination of hydrogenophiles xanthium and agaricus, and the combination of hydrogenophiles laishii and azospirillum brazianum, the dry weight increase rate on the ground of the plant of the five combinations is over 140% and is obviously higher than that of the other combinations.

TABLE 8 Effect of different groups of microbial Agents on the aboveground Dry weight growth Rate of crops

Example 6

In this embodiment, the influence of different carriers on the survival condition of microorganisms is explored to screen out the optimal carrier of the optimal microbial agent, and the specific test method is as follows:

the microbial agents obtained by the optimal combination of the microbial agents selected in example 5, i.e., hydrogenophilus pahnsonii and azospirillum brasilense, were tested for survival on various carriers according to the method described in example 1: lime, charcoal, plant ash, biogas residue, coconut chaff, sawdust and river sand are respectively used as carriers, 7 kinds of composite biological bacterial fertilizers are prepared according to the method in the embodiment 1, after the composite biological bacterial fertilizers are placed in a shade place for 6 months, 1g of the composite biological bacterial fertilizers are added into 100mL of sterile water for dilution, 50 mu L of the diluted composite biological bacterial fertilizers are coated on a flat plate, and the residual rate of active bacteria and the effective active bacteria ratio are measured. The higher the residual rate of the active bacteria is, the smaller the influence of the selected carrier on the activity of the strain is; the effective active bacteria refer to hydrogenophiles and azotobacteria which still keep activity after preservation or storage, and can be further propagated and rejuvenated, and the effective active bacteria ratio is relative to infectious microbes and refers to the ratio of the number of the hydrogenophiles and the azotobacteria in the total number of the active bacteria; the higher the residual rate of the active bacteria and the higher the effective active bacteria ratio, the carrier is relatively more suitable for hydrogenophiles and azotobacter.

The residual rates of the active bacteria loaded with different carriers are shown in table 9, and it can be seen that the residual rates of the active bacteria loaded with different carriers are, from high to low: biogas residue, charcoal, coconut chaff, sawdust, plant ash, river sand and lime; the effective active bacteria ratio is from high to low: plant ash, wood chips, charcoal, coconut husk, river sand, biogas residue and lime. Because the residual rate of the active bacteria of the plant ash and the wood dust is relatively lower than that of the charcoal, the residual rate of the active bacteria and the effective active bacteria ratio are comprehensively considered, and the optimally selected microbial agent carrier is the charcoal.

TABLE 9 residual ratio of active bacteria for different vectors

Example 7

The composite organic bacterial fertilizer prepared by taking Azospirillum brasilense and atypical hydrogenophilus as microbial agents and soaking on carrier charcoal in the embodiment 1 is canned in a shade at 15 ℃, opened after 90 days, and the number of active bacteria measured by a coating plate is 6.8 multiplied by 10 ⁷ (ii) a The composite organic bacterial fertilizer preserved for 3 months under the condition has strong rejuvenation potential, and the composite organic bacterial fertilizer in the embodiment 1 can also be added into cooling rice water according to the mass ratio of 2.5 percent and stored and transported in 15 ℃ for cold storage, which is more beneficial to storage, transportation and rejuvenation of the composite organic bacterial fertilizer.

In addition, the composite organic bacterial fertilizer prepared in the example 1 is canned and placed on an oscillator, the frequency is set to be 5rpm/min to simulate the bumpy environment in transportation, the tank is opened after 2d, and the number of active bacteria is measured to be 2.4 multiplied by 10 through plate coating ⁸ (ii) a The compound organic bacterial fertilizer has certain tolerance to transportation.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The preparation method of the composite organic bacterial fertilizer is characterized by comprising the following steps:

(1) Culturing of individual strain

Respectively inoculating azotobacter and hydrogenophilic bacteria into respective culture solutions to carry out independent culture, and respectively preparing azotobacter bacterial liquid and hydrogenophilic bacterial liquid; the effective viable count of the azotobacteria bacterial liquid and the hydrogenophilic bacteria bacterial liquid is not less than 1 multiplied by 10 ⁹ CFU/mL, and the rate of mixed bacteria is less than 3%;

(2) Co-culture of bacterial groups

Inoculating the azotobacteria liquid and the hydrogenophile liquid prepared in the step (1) into a co-culture medium, and performing mixed culture to prepare a primary co-culture medium liquid; the total number of effective viable bacteria in the primary co-culture bacterial liquid is not less than 1 multiplied by 10 ⁹ CFU/mL, the number of viable bacteria of azotobacteria is not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, wherein the rate of mixed bacteria in the primary co-culture bacterial liquid is lower than 3%;

(3) Co-culture bacterial liquid fermentation

Inoculating the primary co-culture bacterial liquid prepared in the step (2) into fermentation liquor, and fermenting to prepare co-culture fermentation liquor; the total number of effective viable bacteria in the co-culture fermentation liquid is not less than 1 × 10 ⁹ CFU/mL, the number of viable bacteria of azotobacteria is not less than 1 × 10 ⁶ CFU/mL, viable count of hydrogenophilic bacteria not less than 1 × 10 ⁶ CFU/mL, the rate of mixed bacteria in the co-culture fermentation liquor is lower than 3%;

(4) Infiltrating the co-culture fermentation liquor into a sterilized microbial agent carrier, and drying to obtain a compound organic bacterial fertilizer;

when the hydrogenophile is atypical hydrogenophila oxyphylla (Hydrogenophaga oxyphylla), the Azotobacter is selected from one of Rhodopseudomonas palustris (Rhodopseudomonas palustris), azospirillum brasilense (Azospirillum brasilense), azospirillum melini mellis (Azospirillum melinis), azospirillum iraillum iraense (Azospirillum irakense), azospirillum oryzae (Azospira oryzae), azospirillum vinelandii (Azotobacter vinelandii);

when the hydrogenophile is hydrogenophile flavivirus (hydrogengenophaga flava), the Azotobacter is selected from one of Rhodopseudomonas palustris (Rhodopseudomonas palustris), azospirillum brasilense (Azospirillum brasilense), azospirillum melini (Azospirillum melinis), azospirillum iraillum iraense (Azospirillum irakense), azospirillum oryzae (Azospira oryzae), azospirillum vineladii;

when the hydrogenophilic bacteria is hydrogenophilic spore bacteria (Hydrogenophaga laconensis), the Azotobacter is one of Azospirillum brasilense, azospirillum melitensis, azospirillum iranthitum and Azotobacter vinelandii;

when the hydrogenophile is hydrogenophile (hydrogengenophaga pallronii), the azotobacter is selected from one of Azospirillum brasilense (Azospirillum brasilense), azospirillum melinii (Azospirillum melinis) and Azospirillum iractense (Azospirillum irakense);

the microbial agent carrier is charcoal.

2. The method for preparing the composite organic bacterial fertilizer as claimed in claim 1, wherein the concentration of hydrogenophiles in the composite organic bacterial fertilizer is 1 x 10 ⁵ ～1×10 ¹¹ CFU/g; the concentration of azotobacteria is 1 × 10 ⁵ ～1×10 ¹¹ CFU/g。

3. The method for preparing the composite organic bacterial fertilizer as claimed in claim 1, wherein the azotobacter is Azospirillum brasiliensis (Azospirillum brasiliense); the hydrogenophile is hydrogenophile pa (hydrogengenophaga pallronii) or hydrogenophile lainsm (hydrogengenophaga laconesensis); the microbial agent carrier is charcoal.

4. The preparation method of the compound organic bacterial fertilizer as claimed in claim 1, wherein the azotobacter is Azospirillum melinis or Azospirillum irakense; the hydrogenophile is atypical hydrogenophile spore (Hydrogenophaga typica); the microbial agent carrier is charcoal.

5. The composite organic bacterial fertilizer prepared by the preparation method of the composite organic bacterial fertilizer of any one of claims 1 to 4.

6. The application of the composite organic bacterial fertilizer in nitrogen fixation of soybean, rejuvenation of peanut, wheat, oat and corn.

7. The use of the compound organic bacterial fertilizer of claim 5 for improving soil acidity and alkalinity and enriching soil microbial species.