CN116064283A

CN116064283A - Low-temperature composting microbial inoculum capable of promoting corrosion and preserving nitrogen as well as preparation method and application thereof

Info

Publication number: CN116064283A
Application number: CN202210985083.9A
Authority: CN
Inventors: 王海英; 徐丽萍; 黄祎晨; 张琨; 梅燕; 张芯; 谈重芳
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2023-05-05

Abstract

The invention discloses a corrosion-promoting nitrogen-preserving low-temperature composting composite microbial inoculum and a preparation method and application thereof. The active ingredient of the composite microbial inoculum is providencia alkali (Providencia alcalifaciens) CCTCC No: m2022672, brevibacterium cold-resistant (Brevibacterium frigoritolerans) CCTCC No: m2022673. And mixing the cultured composite microbial inoculum with a carrier to obtain the solid composite microbial inoculum. The solid composite microbial inoculum can be applied to the composting process of agricultural wastes in a low-temperature environment, promotes the conversion of organic matters, reduces nitrogen loss and improves the quality of the compost.

Description

Low-temperature composting microbial inoculum capable of promoting corrosion and preserving nitrogen as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a corrosion-promoting nitrogen-preserving low-temperature composting microbial inoculum and a preparation method and application thereof.

Background

The total amount of crop straws and livestock manure is large, the density is high, a large amount of straws and livestock manure need to be treated every year, and composting fermentation is an important way for returning straws to fields and improving soil. However, in northern areas of China, the winter is long, the air temperature is low, the direct fermentation has the defects of long composting period, large nitrogen loss, serious greenhouse gas emission and the like, and particularly in the alpine environment such as Qinghai-Tibet and the like, the composting fermentation is difficult to start, so that biological macromolecules such as cellulose and the like cannot be effectively degraded, and meanwhile, a large amount of harmful substances are remained.

In the composting process, various microorganisms, organic or inorganic substances are often added into the pile as additives in order to shorten the fermentation period and promote composting. The method is an important way for cultivating straw fermentation bacteria suitable for low temperature in winter.

Disclosure of Invention

The invention aims to solve the technical problems that how to cultivate a microbial inoculum suitable for composting fermentation at low temperature in winter, and the composting fermentation is difficult to start, so that biological macromolecules such as cellulose cannot be effectively degraded, and a large amount of harmful substances remain.

In order to solve the technical problems, the invention firstly provides a composite microbial inoculum.

The active ingredients of the composite microbial inoculum provided by the invention consist of providencia alcaligenes and Brevibacterium cold-resistant bacteria.

The composite microbial inoculum can be a composite microbial inoculum for preparing compost, the providencia is providencia (Providencia alcalifaciens) DW-05, and the preservation number of the providencia is CCTCC NO: m2022672; the cold-resistant bacillus brevis is cold-resistant bacillus brevis (Brevibacterium frigoritolerans) DW-07, and the preservation number of the cold-resistant bacillus brevis in China center for type culture collection is CCTCC NO: m2022673.

In the composite microbial inoculum, the providencia and the cold-resistant Brevibacterium CFU are cultivated until the number of detected viable bacteria reaches 6 multiplied by 10 ⁸ After CFU/mL, the viable bacteria content is 1:1, the compound microbial inoculum is formed by proportioning.

The microbial inoculum can be various dosage forms, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can be added into the above microbial inoculum according to the need.

The composite microbial inoculum comprises a carrier.

The carrier can be one of corncob powder, medical stone, diatomite, biochar, bran and vermiculite.

The invention provides providencia which is providencia (Providencia alcalifaciens) DW-05, and the preservation number of the providencia is CCTCC NO: m2022672.

The invention also provides a cold-resistant bacillus brevis, which is cold-resistant bacillus brevis (Brevibacterium frigoritolians) DW-07, and the preservation number of the cold-resistant bacillus brevis in China center for type culture collection is CCTCC NO: m2022673.

The invention also provides a method for preparing the low-temperature solid composite microbial inoculum.

In one embodiment, the method comprises mixing the composite microbial inoculum with a carrier to obtain a solid composite microbial inoculum.

In the method, the carrier is one of corncob powder, medical stone, diatomite, biochar, bran and vermiculite.

In the method, the ratio of the composite microbial inoculum to the carrier is as follows: (providencia alkali producing providencia)(Providencia alcalifaciens) DW-05 meter) 6×10 ¹¹ CFU Alcaligenes providencia (Providencia alcalifaciens) DW-05:1kg of carrier.

The invention also provides a composite microbial inoculum for preparing compost, which contains the providencia alkali-producing providencia or the cold-resistant Brevibacterium.

The application of the composite microbial inoculum, the providencia and the cold-resistant Brevibacterium in any one of the following:

1) Fermenting and composting under the low-temperature condition;

2) Promoting degradation of organic matters of compost;

3) Accelerating the composting process;

4) Enhancing compost denitrification and/or nitrogen assimilation;

5) Enhancing the conversion of nitrogen in the compost.

The compost is prepared from crop straws and/or livestock and poultry raising excrement as raw materials. The low temperature may be from-15 ℃ to-10 ℃.

Through screening low temperature resistant microorganisms and inoculating the microorganisms to compost materials, the growth and metabolism activities of the microorganisms can be kept in a low temperature environment, and the aim of accelerating fermentation rate and degrading biological macromolecules is fulfilled.

Preservation description

Strain name: alkalogenous providencia

Latin name: providencia alcalifaciens

Strain number: DW-05

Preservation mechanism: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (cctccc)

Address: chinese university of Wuhan

Preservation date: 2022, 05, 19

Accession numbers of the preservation center: cctccc No: m2022672

Strain name: cold-resistant Brevibacterium

Latin name: brevibacterium frigoritolerans

Strain number: DW-07

Preservation mechanism: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (cctccc)

Address: chinese university of Wuhan

Preservation date: 2022, 05, 19

Accession numbers of the preservation center: cctccc No: m2022673

Drawings

FIG. 1 is a schematic diagram showing growth of strains at different temperatures.

FIG. 2 is a morphological image of the strain under a microscope after gram staining.

FIG. 3 is a schematic diagram showing the growth curve of the primary strain at 20 ℃.

FIG. 4 is a graph showing the effects of DW-05 and DW-07 on degrading cellulose, casein and inorganic phosphorus.

FIG. 5 shows the low temperature fermentation composite microbial inoculum obtained.

FIG. 6 is a schematic diagram of the temperature change of low Wen Niufen straw compost.

Fig. 7 is a graph showing the change of total organic carbon in low Wen Niufen straw compost.

Fig. 8 is a schematic diagram of the change of organic loss in low Wen Niufen straw compost.

FIG. 9 is a schematic diagram of ammonia nitrogen change for low Wen Niufen straw compost.

Fig. 10 is a schematic diagram of the change of nitrate nitrogen in low Wen Niufen straw compost.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: screening separation of low temperature resistant bacteria and determination of growth curve

1. Bacterial strain primary screening

Weighing 5g of Qinghai Huang Nazhou wheat field collected soil sample, adding into a triangular flask filled with 50mL of sterile water, and uniformly mixing to obtain 10 ^-1 A soil sample diluent. Placing into a constant temperature shaking table, setting parameters at 20deg.C and 180rpm, shaking for 25min, and standing for 10min to obtain bacterial suspension. Absorbing the upper layer bacterial suspension, and carrying out gradient dilution by using sterile water to prepare 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 The suspension was diluted. Suction 10 ^-3 、10 ^-4 、10 ^-5 100 mu L of each diluted suspension is evenly coated on a sterilized LB solid medium, the medium is placed at 10 ℃ for inversion culture for 3-5d, then colonies growing on a flat plate are observed and recorded, single colonies with different forms are selected and transferred to the sterilized LB solid medium by a flat plate streaking method, and inversion culture is carried out at 10 ℃ for 3-5d, and the culture is repeated for a plurality of times until purified strains are obtained. Purified strains were numbered numerically and inoculated onto LB solid slant medium for storage at 4 ℃. The formula of the LB solid slant culture medium is as follows: 5g of yeast extract, 10g of tryptone, 10g of NaCl, 14-15g of agar, adding double distilled water to 1L, and sterilizing at 121 ℃ for 20min.

2. Screening of strain for Wen Xingfu

13 strains grown at 10 ℃ after purification are inoculated on a sterilized LB solid medium, the temperature gradient is set to 5 ℃, 10 ℃, 15 ℃ and 20 ℃, the growth condition of the strains is observed after each group of strains are respectively cultured for 1-2 days at 4 different temperatures, the diameters (units: mm) of colonies grown at different temperatures are measured and compared, the growth conditions of the strains are shown in figure 1, the results are summarized in table 1, and the results show that two strains numbered DW-05 and DW-07 grow well at low temperatures of 5 ℃ and 10 ℃.

TABLE 1 growth of cold tolerant bacteria at different temperatures (5 d)

Note that: 1) "+" indicates the extent of colony growth; 2) "-" means no growth

Inoculating DW-05 and DW-07 strain into LB liquid medium, culturing at 20deg.C and 180rpm, and measuring OD every 2 hr _600nm The light absorption value (LB liquid culture medium is taken as blank control) is drawn, and the growth curve of the strain is shown as a typical S-type as shown in figure 3, and accords with the growth rule of microorganisms, so that the microorganisms obtained by screening have good cold resistance. The logarithmic phase span of DW-05 is 4-32h; the logarithmic phase of DW-07 spans 8-26h.

3. Characteristic re-screening of strains

And (3) performing a characteristic screening experiment on bacterial strains DW-05 and DW-07 with low-temperature growth advantages, which are obtained by a Wen Xingfu screen, for degrading cellulose and casein, and for fixing nitrogen and dissolving phosphorus. The strains were inoculated in the center of sterilized cellulose Congo red medium, casein medium, inorganic phosphorus medium and nitrogen-free solid medium, respectively, and the culture medium was inverted and cultured in a biochemical incubator at a constant temperature of 15℃for 1-2d, and the results were observed and recorded. Judging whether the transparent ring has the functions of decomposing casein, degrading cellulose, fixing nitrogen and dissolving phosphorus according to the existence of the transparent ring and the growth condition of the strain.

Wherein, the formula of the cellulose Congo red culture medium comprises: sodium carboxymethylcellulose (CMC-Na) 5.0g, yeast extract 0.5g, casein 0.5g, naNO ₃ 1.0g，Na ₂ HPO ₄ 1.2g，KH ₂ PO ₄ 0.9g，MgSO ₄ 0.5g, 0.5g KCl, 0.2g Congo red and 15.0g agar are added with 1000mL distilled water to be mixed uniformly, the pH is regulated to 6.9-7.1, the sterilization is carried out for 20 minutes at 121 ℃, and the mixture is cooled to about 50 ℃ and poured into a flat plate.

Casein medium: 10.0g of casein, 3.0g of beef extract, 5.0g of NaCl and K ₂ HPO ₄ 2.0g of bromine Bai Lixiang phenol blue 0.05g, agar 15.0g, adding 1000mL of distilled water, uniformly mixing, adjusting pH to 7.3-7.5, sterilizing at 121 ℃ for 20 minutes, and cooling to 50 DEG CThe plate is inverted left and right.

Inorganic phosphorus medium: yeast extract powder 0.5g, (NH) ₄ ) ₂ SO ₄ 0.5g，NaCl 0.3g，KCl 0.3g，MnSO ₄ 0.03g，MgSO ₄ 0.3g，FeSO ₄ ·7H ₂ O 0.03g，Ca ₃ (PO ₄ ) ₂ 5.0g of glucose 10.0g and 15.0g of agar are added with 1000mL of distilled water for uniform mixing, the pH is regulated to 7.0-7.5, sterilization is carried out for 20 minutes at 121 ℃, and the mixture is cooled to about 50 ℃ and poured into a flat plate.

Nitrogen-free solid medium: caCO (CaCO) ₃ 5.0g，，MgSO ₄ 0.2g，KH ₂ PO ₄ 0.2g，CaSO ₄ 0.1g of NaCl 0.2g, mannitol 10.0g and agar 15.0g, adding 1000mL of distilled water, mixing, adjusting pH to 7.0, sterilizing at 121 ℃ for 20 minutes, cooling to about 50 ℃, and pouring into a plate.

As shown in FIG. 3, DW-05 showed transparent degradation circles on all three media, indicating that it had the effects of decomposing casein, degrading cellulose, fixing nitrogen and dissolving phosphorus. DW-07 showed transparent degradation circles on the Congo red medium and the casein medium, which indicates that the DW-07 has the functions of decomposing casein and degrading cellulose.

4. Carrying out 16S rRNA molecular biological identification on the strain DW-05 and the strain DW-07, sequencing and splicing to obtain a nucleotide sequence of a 16S rRNA gene of the strain DW-05, such as a nucleotide sequence 1 in a sequence table, carrying out comparison analysis on the nucleotide sequence of the 16S rRNA gene of the strain DW-07, such as a nucleotide sequence of a sequence 2 in the sequence table, and selecting a sequence with higher homology to construct a phylogenetic tree.

The results show that the reference species for DW-05 is providencia (providencia califas.) with homology up to 99%. The reference species of DW-07 was Brevibacterium cold-resistant (Brevibacterium frigoritolerans), with homology up to 99%.

After DW-05 and DW-07 strains are dyed by a gram staining method, the strains are respectively observed under a microscope, and as shown in figure 2, the gram staining results of the DW-07 strain are positive, and the gram staining result of the DW-05 strain is negative.

Preservation of DW-05, DW-07 Strain

The providencia (Providencia alcalifaciens) DW-05 provided by the invention is preserved in China center for type culture collection (CCTCC for short) in year 2022, wherein the address is the university of Wuhan, post code is 430072, and the preservation number is CCTCC No. M2022672. Providencia (Providencia alcalifaciens) DW-05 is abbreviated as providencia alkali DW-05.

The cold-resistant bacillus brevis (Brevibacterium frigoritolerans) DW-07 provided by the invention is preserved in China center for type culture collection (CCTCC for short) in the year 2022, the address is the university of Wuhan, the mail code is 430072, and the preservation number is CCTCC No. M2022673. The cold-resistant bacillus brevis (Brevibacterium frigoritolerans) DW-07 is called cold-resistant bacillus brevis DW-07 for short.

Example 2 preparation method of Low temperature microbial agent

1. Seed liquid preparation

Picking single colony of the providencia DW-05 from the flat plate, inoculating the single colony into an LB liquid culture medium, and shaking and culturing at 20 ℃ and 180rpm for 24 hours to obtain seed liquid of the providencia DW-05. Picking single colony of the cold-resistant bacillus brevis DW-07 from the flat plate, inoculating the single colony into an LB liquid culture medium, and shake-flask culturing at 20 ℃ and 180rpm for 24 hours to obtain seed liquid of the cold-resistant bacillus brevis DW-07.

The formula of the LB liquid medium is as follows: yeast extract 5g, tryptone 10g, naCl 10g, double distilled water to 1L, and sterilizing at 121 ℃ for 20min.

2. Preparation of fermentation broth

Adding the seed solution of the providencia alkali-producing providencia DW-05 in the step 1 into a sterilized LB liquid culture medium according to the proportion of 1%, shaking and culturing for 24 hours at 20 ℃ and 180rpm, and collecting fermentation liquor to obtain the providencia alkali-producing providencia DW-05 fermentation liquor. The content of Alcaligenes providencia DW-05 in the fermentation liquid of Alcaligenes providencia DW-05 is not lower than 6×

10 ⁸ CFU/mL。

Adding the seed liquid of the cold-resistant Brevibacterium DW-07 in the step 1 into a sterilized LB liquid culture medium according to a proportion of 1%, shaking and culturing for 24 hours at 20 ℃ and 180rpm, and collecting fermentation liquid to obtain the cold-resistant Brevibacterium DW-07 fermentation liquid. The content of the cold-resistant Brevibacterium DW-07 in the fermentation liquid is not less than 6 multiplied by 10 ⁸ CFU/mL。

3. Preparation of solid microbial inoculum

3.1 preparation of compost fermentation composite microbial inoculum

Uniformly mixing the alkali-producing providencia DW-05 fermentation liquor, the cold-resistant bacillus brevis DW-07 fermentation liquor and the sterilized corncob powder in the step 2, airing in a shade, and enabling the water content to be lower than 30%, thus obtaining the compost fermentation composite microbial inoculum. The content of Alcaligenes providencia DW-05 in the compost fermentation composite microbial inoculum is about 6 multiplied by 10 ⁸ CFU/g, the content of the cold-resistant Brevibacterium DW-07 is about 6 multiplied by 10 ⁸ CFU/g。

3.2 preparation of single agent of providencia alkali-producing Provisions by composting fermentation

The preparation method of the single agent of the providencia alkali-producing providencia is different from that of the step 3.1 only in that the 3.1 cold-resistant short bacillus DW-07 fermentation liquor is replaced by the providencia alkali-producing providencia DW-05 fermentation liquor, and other operations are the same. The specific method comprises the following steps: uniformly mixing the fermentation liquor of the alkali-producing providencia DW-05 in the step 2 with the sterilized corncob powder, airing in a shade, and enabling the water content to be lower than 30%, thus obtaining the providencia-producing single microbial agent for composting fermentation. The content of the Alcaligenes providencia DW-05 in the compost fermentation Alcaligenes providencia single microbial inoculum is about 1 multiplied by 10 ⁹ CFU/g。

3.3 preparation of Cold-resistant Brevibacterium single microbial inoculum by composting fermentation

The preparation method of the compost fermentation cold-resistant bacillus brevis single microbial inoculum is different from that of the step 3.1 only in that the Alcaligenes providencia DW-05 fermentation liquor of the step 3.1 is replaced by the cold-resistant bacillus brevis DW-07 fermentation liquor, and other operations are the same. The specific method comprises the following steps: and (3) uniformly mixing the cold-resistant Brevibacterium DW-07 fermentation liquor obtained in the step (2) with the sterilized corncob powder, and airing in a shade to ensure that the water content is lower than 30%, thus obtaining the compost fermentation cold-resistant Brevibacterium single microbial inoculum. The content of the cold-resistant Brevibacterium DW-07 in the compost fermentation cold-resistant Brevibacterium single microbial inoculum is about 1 multiplied by 10 ⁹ CFU/g。

Example 3 Carrier optimization of compost fermentation Complex microbial inoculant

The effective viable bacteria rate was measured to verify the affinity of the liquid bacterial agent to the carrier.

Uniformly mixing the Alcaligenes providencia DW-05 fermentation liquor, the cold-resistant Brevibacterium DW-07 fermentation liquor and the sterilized carrier in the step 2, airing in a shade, and enabling the water content to be lower than 30%, thus obtaining the compost fermentation composite microbial inoculum. In the composite microbial inoculum for compost fermentation, the content of providencia-producing providencia DW-05 is 6 multiplied by 10 ⁸ CFU/g, content of cold-resistant Brevibacterium DW-07 is 6×10 ⁸ CFU/g. The carriers are corncob powder, biochar or vermiculite respectively, and the bacteria carrying capacity of each carrier, namely the effective bacterial rate after compounding, is measured, and the experimental method is as follows:

1g of the immobilized microbial inoculum sample was weighed and placed in a conical flask containing 100mL of PBS buffer, and was subjected to gradient dilution after stirring for 2h on a constant temperature magnetic stirrer. The dilution is coated on LB solid culture medium by adopting a coating plate method, and the colony number on the culture medium is counted after culturing for 48 hours at 20 ℃ to calculate the number of viable bacteria released by the carrier. Meanwhile, the colony number of the fermentation broth is counted as the number of inoculated bacteria, and the average value is obtained by experimental determination for 3 times. Viable bacteria ratio (%) =number of viable bacteria released/number of bacteria inoculated (CFU) ×100%. The results are shown in Table 2, and the number of microorganisms contained in the three microbial agents is that corncob powder is more than vermiculite is more than biochar. The effective viable bacteria rate in the corncob powder is highest and reaches 66.88%, which shows that the corncob powder has the strongest bacteria carrying capacity. The microbial inoculum taking the biochar as the carrier has the lowest effective viable count, on one hand, the water absorption performance is poor, on the other hand, the biochar is a substance obtained after the anaerobic high-temperature conversion of biomass, and different biomass sources and carbonization process parameters can have great influence on the microstructure of the biochar, so that the bacteria carrying capacity of the biochar shows remarkable difference.

TABLE 2 effective viable count in different Carrier microbial Agents

The viable bacteria remained after the composting fermentation composite microbial inoculum taking corncob powder as a carrier is stored for 3 months at the normal temperature of 25 ℃ is 9 multiplied by 10 ⁷ CFU/g。

The finally obtained low-temperature compost fermentation composite microbial agent is in a yellow brown powder shape and has spicy and irritated microorganism smell as shown in figure 3.

Example 4 application of Low temperature microbial agent in agricultural waste treatment

The compost raw material of the embodiment is fresh cow dung pulled from a cow farm to a composting site and mixed straw picked from local farmers, and the mass ratio of the raw material is 1:1:1, sunflower stalk, chrysanthemum stalk and wheat stalk form mixed stalk. According to the measured C/N ratio of the materials, the wet weight ratio of the fresh cow dung to the straw is determined to be 10:1. 10 parts by mass (wet weight) of fresh cow dung and 1 part by mass (wet weight) of the mixed straw were mixed to obtain a compost fermentation raw material, and the following compost fermentation was carried out for 84 days in a scientific and scientific exhibition garden of Qianzhen Zhenzi Ziyuan agriculture in Zhongxiang county, qinghai province, west Ning City, 24 months 2021.

4.1 composting fermentation by using composting fermentation composite microbial inoculum

And (3) performing composting fermentation by using the composting fermentation composite microbial inoculum of the step 3.1 in the example 2. And (3) uniformly mixing the compost fermentation raw material with the compost fermentation composite microbial inoculum of the step 3.1 of the embodiment 2 to obtain a pile material. The content of providencia DW-05 in the bulk material is 6×10 ⁵ CFU/g, content of cold-resistant Brevibacterium DW-07 is 6×10 ⁵ CFU/g, designated as Pile2 group (P2).

Composting the stacked materials in a stack manner, wherein the volume of each stack is 18m ³ (length 4m, width 3m, height 1.5 m), the water content was adjusted to 65%. And the surface of the pile is covered with a plastic film, so that the water and heat loss is reduced. The top and the periphery of the pile body are provided with inserted electrodes, the electrodes penetrate into the 40cm position of the pile body, and the wires are connected with a digital thermometer.

During the experiment, the ambient temperature and the stack temperature were measured once each of 10 a.m. and 4 a.m. each day, and the average value was taken as the current day temperature. Mechanical and manual turning is used when the temperature reaches the top point and starts to continuously drop. Determination of Total Organic Carbon (TOC), organic Matter (OM), ammonia nitrogen and nitrate nitrogen content of compost during fermentation

4.2 Natural fermentation without microbial inoculum

The difference from 4.1 is that the composting fermentation composite microbial inoculum of the step 3.1 of the example 2 is replaced by the sterilized corncob powder of the example 2 with the same quality, and the other operations are the same and are defined as a Pile1 natural fermentation group (P1).

The temperature of the compost is shown in figure 6, the temperature of the compost can characterize the degradation condition of organic matters to a certain extent, and the composting process approximately comprises a low-temperature stage, a high-temperature stage and a cooling stage. At the low temperature stage, the microbial activity is lower, and the composting temperature is greatly influenced by the environmental temperature. With the enhancement of the metabolic activity of microorganisms in the compost, the decomposition speed of organic matters is increased, a large amount of heat is generated, the temperature of the compost is promoted to rise, the faster the temperature of the compost rises, the higher the temperature is, the stronger the activity of the microorganisms in the compost is, and the more severe the decomposition of the organic matters is. After reaching 50 ℃, i.e. at the high temperature stage, the microbial activity reaches a peak value, and the high temperature of the compost can inhibit the microbial activity, so that only some heat-resistant microorganisms are active. Along with the decrease of the microbial activity, the temperature of the compost also begins to decrease, and the compost enters a cooling and decomposing stage, at this time, the organic matters which are easy to degrade are basically consumed, the residual cellulose, lignin and the like which are difficult to degrade are difficult to utilize, and at this time, mesophilic actinomycetes and fungi can take the dominant position, so that the organic matters in the compost are basically converted into stable humus.

The average temperature of the composting environment is between 13 ℃ below zero and 6 ℃ below zero within 0 to 63 days, and the composting temperature shows a rising and falling trend. After the first Pile turning in 18 days, the temperature of the PIle2 experimental group (P2) is raised to 54.3 ℃ in the next day (20 days) and reaches the high temperature period (50 ℃), and the temperature of the PIle1 natural fermentation group (P1) is 43.1 ℃. The temperature of the natural fermentation group (P1) of the PIle1 is raised to 54.2 ℃ only after 22 days, and the time for the experimental group of the PIle2 to reach the high temperature period is obviously earlier than that of the blank group. The temperature of the pilot 2 experimental group reached 61.4℃at 24 days and the pilot 1 reached 60.1℃at 29 days. And the Pile body temperature of the PIle2 experimental group is kept above 55 ℃ for more than 10 days, and meets the requirements of national standards (NY/T1168-2006). Subsequently, starting at 28 days, the pilot 2 group started to cool earlier than the pilot 1 group and continued for up to 64 days. Then, as the air temperature rises, the ambient temperature begins to remain above 0 ℃, and the stack temperature of both groups begins to rise slowly. After the microbial inoculum is added, microorganisms are quickly adapted to a low-temperature environment in the compost, organic matters are metabolized and decomposed, the organic matters which are easy to degrade at a high temperature are quickly brought into a high-temperature period, the organic matters are consumed completely, and the pile body is more quickly brought into a stable decomposition stage.

Samples were taken at different stages to determine the Total Organic Carbon (TOC) change (as shown in fig. 7), the Organic Matter (OM) loss change (as shown in fig. 8), the ammonia nitrogen (as shown in fig. 9) and the nitrate nitrogen (as shown in fig. 10) changes of the compost to evaluate the effect of the low-temperature microbial inoculum on the degradation of the organic matter and the conversion of nitrogen in the compost.

And (5) measuring the water content and TOC by adopting a drying weight loss method. Weighing a certain amount of fresh sample, and placing the fresh sample into a container with weight of w ₀ In the crucible of (2), the total weight is denoted as w ₁ The method comprises the steps of carrying out a first treatment on the surface of the The crucible and sample were then placed in an oven and dried at 105℃for 24 hours, cooled and weighed, denoted w ₂ The method comprises the steps of carrying out a first treatment on the surface of the Then directly placing the crucible and the dried sample into a muffle furnace to burn for 6 hours at 550 ℃, taking out the crucible when the temperature of the muffle furnace is reduced to about 250 ℃, placing the crucible into a dryer, weighing after cooling, and marking as w ₃ . Finally, the moisture content, ash, OM, TOC, OM losses of the compost samples were calculated according to the formulas 4.1, 4.2, 4.3, 4.4 and 4.5, respectively.

Water content (%) = (w) ₁ -w ₂ )/(w ₁ -w ₀ )×100％ (4.1)

Dry matter (%) = (w) ₂ -w ₀ )/(w ₁ -w ₀ )×100％

Ash (%) = (w) ₃ -w ₀ )/(w ₂ -w ₀ )×100％ (4.2)

OM(％)＝(w ₂ -w ₃ )/(w ₂ -w ₀ )×100％ (4.3)

TOC(％)＝OM(％)/1.8 (4.4)

OM loss (%) = [1- (initial ash× (100-corresponding composting time ash))/(corresponding composting time ash× (100-initial ash)) ] ×100% (4.5)

The method for measuring the change of ammoniacal nitrogen nitrate nitrogen of the compost is as follows

Ammoniacal Nitrogen (NH) ₄ ⁺ -N), nitrate nitrogen (NO ₃ ^- -N) determination of extraction-separation Using Potassium chloride solutionSpectrophotometry (according to HJ634-2012, GB/T32737-2016).

Compared with the blank group P2, the treatment group P1 added with the low-temperature microbial inoculum has obvious difference in total organic carbon, shows lower concentration, combines with the loss change of organic matters, and concludes that the low-temperature microbial inoculum can promote the degradation of the organic matters of the compost and accelerate the composting process.

The ammonia nitrogen content of the P2 experimental group drops more severely than that of the blank group in the high-temperature maintaining stage (27-43 d), and the nitrate nitrogen peak value appears at the moment; the ammonia nitrogen content of P2 in the later stage of composting is higher than that of a blank group, and the nitrate nitrogen is obviously reduced, which indicates that the denitrification effect and the nitrogen assimilation effect of the composting added with the low-temperature bacteria are stronger. The result shows that the addition of the low-temperature microbial inoculum can compost to enhance the nitrification in the high-temperature period and the conversion of nitrogen.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. The composite microbial inoculum is characterized in that the active ingredients of the composite microbial inoculum consist of providencia and Brevibacterium cold-resistant bacteria.

2. The composite microbial agent according to claim 1, wherein: the composite microbial inoculum is used for preparing compost, the providencia is providencia (Providencia alcalifaciens) DW-05, and the preservation number of the providencia is CCTCC NO: m2022672; the cold-resistant bacillus brevis is cold-resistant bacillus brevis (Brevibacterium frigoritolerans) DW-07, and the preservation number of the cold-resistant bacillus brevis in China center for type culture collection is CCTCC NO: m2022673.

3. The composite microbial agent according to claim 1 or 2, wherein: in the composite microbial inoculum, the CFU ratio of the providencia and the cold-resistant Brevibacterium is 1:1.

4. the composite microbial agent of claim 1, 2 or 3, wherein: the composite microbial inoculum comprises a carrier.

5. The composite microbial inoculant of claim 4, wherein: the carrier is corncob powder.

6. Providencia is providencia (Providencia alcalifaciens) DW-05, and the preservation number of the providencia is CCTCC NO: m2022672.

7. The cold-resistant bacillus brevis is characterized in that the cold-resistant bacillus brevis is cold-resistant bacillus brevis (Brevibacterium frigoritolerans) DW-07, and the preservation number of the cold-resistant bacillus brevis in China center for type culture collection is CCTCC NO: m2022673.

8. The composite microbial inoculum for preparing compost is characterized in that: the microbial inoculum contains providencia stuartii according to claim 6 or Brevibacterium cold-resistant bacteria according to claim 7.

9. Use of the complex bacterial agent of any one of claims 1-5, providencia of claim 6, brevibacterium cold-resistant of claim 7 or bacterial agent of claim 8 in any one of the following:

1) Fermenting and composting under the low-temperature condition;

2) Promoting degradation of organic matters of compost;

3) Accelerating the composting process;

4) Enhancing compost denitrification and/or nitrogen assimilation;

5) Enhancing the conversion of nitrogen in the compost.