A low-temperature composting bacterial agent for promoting decomposition and retaining nitrogen, and its preparation method and application

Abstract

The present invention discloses a composite bacterial agent for promoting decomposition and retaining nitrogen at low temperature composting, and a preparation method and application thereof. The active ingredients of the composite bacterial agent are composed of Providencia alcalifaciens CCTCC No: M 2022672 and Brevibacterium frigoritolerans CCTCC No: M 2022673. The cultured composite bacterial agent is mixed with a carrier to obtain a solid composite bacterial agent. The solid composite bacterial agent can be applied to the composting process of agricultural waste in a low temperature environment to promote the conversion of organic matter and reduce nitrogen loss, thereby improving the quality of compost.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a low-temperature composting bacterial agent that promotes decay and retains nitrogen, and a preparation method and application thereof. Background Art

[0002] The total amount of crop straw and livestock manure is large and the density is high. Every year, a large amount of straw and livestock manure needs to be processed. Composting and fermentation is an important way to return straw to the field and improve the soil. However, in northern my country, the winter is long and the temperature is low. Direct fermentation has the disadvantages of long composting cycle, large nitrogen loss, and serious greenhouse gas emissions. Especially in the high-altitude cold environment such as Qinghai and Tibet, composting and fermentation are difficult to start, resulting in the ineffective degradation of biological macromolecules such as cellulose, and at the same time, a large amount of harmful substances will remain.

[0003] In the composting process, in order to shorten the fermentation cycle and promote compost maturity, various microorganisms, organic or inorganic substances are often added to the compost as additives. Collecting low-temperature resistant bacteria from extreme environments and screening, identifying and culturing them is an important way to cultivate straw fermentation bacteria suitable for low temperatures in winter. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to cultivate a bacterial agent suitable for composting fermentation under low temperature in winter, and the problems that composting fermentation is difficult to start, resulting in the ineffective degradation of biological macromolecules such as cellulose, and at the same time causing a large amount of harmful substances to remain.

[0005] In order to solve the above technical problems, the present invention first provides a composite bacterial agent.

[0006] The composite bacterial agent provided by the present invention comprises active ingredients of Providencia alkaligenes and Brevibacterium frostii.

[0007] The composite bacterial agent can be a composite bacterial agent for preparing compost, the alcalifaciens Providencia is Providencia alcalifaciens DW-05, and its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 2022672; the cold-resistant Brevibacterium is cold-resistant Brevibacterium frigoritolerans DW-07, and its preservation number in the China Center for Type Culture Collection is CCTCC NO: M2022673.

[0008] In the composite bacterial agent, the Providencia alcaligenes and the Brevibacterium frostii were cultured until the number of viable bacteria detected reached 6×10 8 After measuring CFU / mL, a composite bacterial agent was formed according to the 1:1 ratio of live bacteria content.

[0009] The above-mentioned bacterial agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0010] As required, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may be added to the above-mentioned bacterial agents.

[0011] The composite bacterial agent contains a carrier.

[0012] The carrier may be one of corn cob powder, medical stone, diatomaceous earth, biochar, bran, and vermiculite.

[0013] The invention provides a Providencia alcalifaciens, wherein the Providencia alcalifaciens is Providencia alcalifaciens DW-05, and the preservation number of the Providencia alcalifaciens in China Center for Type Culture Collection is CCTCC NO: M 2022672.

[0014] The present invention also provides a cold-resistant Brevibacterium, which is Brevibacterium frigoritolerans DW-07, and its preservation number in China Center for Type Culture Collection is CCTCC NO: M 2022673.

[0015] The invention also provides a method for preparing the low-temperature solid composite bacterial agent.

[0016] In one embodiment, the method includes mixing the above-mentioned composite bacterial agent with a carrier to obtain a solid composite bacterial agent.

[0017] In the above method, the carrier is one of corn cob powder, medical stone, diatomaceous earth, biochar, bran, and vermiculite.

[0018] In the above method, the ratio of the composite bacterial agent to the carrier satisfies: (based on Providencia alcalifaciens DW-05) 6×10 11 CFU Providencia alcalifaciens DW-05: 1 kg carrier.

[0019] The present invention also provides a composite bacterial agent for preparing compost, wherein the bacterial agent contains the above-mentioned Providencia alcaligenes or the above-mentioned Brevibacterium frostii.

[0020] Use of the composite bacterial agent, the alkaline-producing Providencia or the cold-resistant Brevibacterium in any of the following:

[0021] 1) Fermentation and composting under low temperature conditions;

[0022] 2) Promote the degradation of compost organic matter;

[0023] 3) Accelerate the composting process;

[0024] 4) Enhance denitrification and / or nitrogen assimilation in compost;

[0025] 5) Enhance the conversion of nitrogen in compost.

[0026] The compost is made from crop straw and / or livestock and poultry excrement. The low temperature can be from -15°C to +10°C.

[0027] By screening low-temperature-resistant microorganisms and inoculating them into compost materials, the growth and metabolic activities of microorganisms can be maintained in a low-temperature environment, thereby accelerating the fermentation rate and degrading biomacromolecules faster.

[0028] Collection Instructions

[0029] Bacteria name: Providencia alcaligenes

[0030] Latin name: Providencia alcalifaciens

[0031] Strain ID: DW-05

[0032] Collection institution: China Center for Type Culture Collection

[0033] Abbreviation of depository institution: CCTCC

[0034] Address: Wuhan University, Wuhan, China

[0035] Date of deposit: May 19, 2022

[0036] CCTCC No: M 2022672

[0037] Bacterial species name: Brevibacterium frostii

[0038] Latin name: Brevibacterium frigoritolerans

[0039] Strain ID: DW-07

[0040] Collection institution: China Center for Type Culture Collection

[0041] Abbreviation of depository institution: CCTCC

[0042] Address: Wuhan University, Wuhan, China

[0043] Date of deposit: May 19, 2022

[0044] CCTCC No: M 2022673 BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the growth of strains at different temperatures.

[0046] Figure 2 This is the morphology of the strain under a microscope after Gram staining.

[0047] Figure 3 Schematic diagram of the growth curve of the initial screening strains at 20°C.

[0048] Figure 4 Schematic diagram of the effects of DW-05 and DW-07 in degrading cellulose, casein and inorganic phosphorus.

[0049] Figure 5 The invention relates to a low-temperature fermentation composite bacterial agent prepared.

[0050] Figure 6 This is a schematic diagram of temperature changes in low-temperature cow dung and straw composting.

[0051] Figure 7 Schematic diagram of the changes in total organic carbon during low-temperature cow dung straw composting.

[0052] Figure 8 Schematic diagram of the change in organic matter loss during low-temperature cow dung and straw composting.

[0053] Fig. 9 This is a schematic diagram of the changes in ammonia nitrogen during low-temperature cow dung straw composting.

[0054] Fig.10 This is a schematic diagram of the changes in nitrate nitrogen during low-temperature cow dung straw composting. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] Example 1: Screening and Isolation of Refractory Bacteria and Determination of Growth Curves

[0058] 1. Preliminary screening of bacterial strains

[0059] Weigh 5 g of soil sample collected from wheat fields in Huangnan Prefecture, Qinghai, add it into a triangular flask containing 50 mL of sterile water, and mix well to obtain 10 -1 Soil sample dilution. Place in a constant temperature shaking incubator, set the parameters to 20°C, 180 rpm, and shake for 25 min, then take out and let stand for 10 min to obtain bacterial suspension. Take the upper bacterial suspension and dilute it with sterile water to prepare 10 -2 , 10 -3 , 10 -4 , 10 -5 Dilute the suspension. Pipette 10 -3 , 10 -4 , 10 -5 100 μL of each diluted suspension was evenly spread on the sterilized LB solid medium, and the medium was placed in an inverted culture at 10°C for 3-5 days. Then the colonies grown on the plate were observed and recorded, and single colonies of different morphologies were selected and transferred to the sterilized LB solid medium by the plate streak method. The culture was inverted at 10°C for 3-5 days, and repeated many times until the purified strains were obtained. After the purified strains were digitally numbered, they were inoculated on the LB solid slant medium and stored at 4°C. The formula of LB solid slant medium is: 5g yeast extract, 10g tryptone, 10g NaCl, 14-15g agar, add double distilled water to 1L, and sterilize at 121°C for 20min.

[0060] 2. Rescreening of strains for thermophilicity

[0061] The 13 strains grown at 10°C after purification were inoculated on sterilized LB solid medium. The temperature gradient was set to 5°C, 10°C, 15°C, and 20°C. Each group of bacteria was cultured at 4 different temperatures for 1-2 days and then the growth of the strains was observed. The diameters of the colonies grown at different temperatures (unit: mm) were measured and compared. The growth of the strains was shown in Figure 2. Figure 1 The results are summarized in Table 1, which show that the two strains numbered DW-05 and DW-07 grew well at low temperatures of 5°C and 10°C.

[0062] Table 1 Growth of cold-resistant bacteria at different temperatures (5d)

[0063]

[0064]

[0065] Note: 1) "+" indicates the degree of colony growth; 2) "-" indicates no growth

[0066] The DW-05 and DW-07 strains were inoculated into LB liquid medium and cultured at 20°C and 180 rpm. The OD was measured every 2 h.600 nm The absorbance value at LB liquid medium was used as blank control, and the growth curve was drawn, as shown in Figure 3 As shown, the growth curve of the strain presents a typical S-type, which is in line with the law of microbial growth, indicating that the screened microorganisms have good cold resistance. The logarithmic phase span of DW-05 is 4-32h; the logarithmic phase span of DW-07 is 8-26h.

[0067] 3. Rescreening of strain characteristics

[0068] The strains DW-05 and DW-07, which were screened for low-temperature growth, were screened for their ability to degrade cellulose and casein, as well as their ability to fix nitrogen and dissolve 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. The medium was inverted in a 15°C constant temperature biochemical incubator and cultured for 1-2 days. The results were observed and recorded. The presence or absence of transparent circles and the growth of the strains were used to determine whether they had the ability to decompose casein, degrade cellulose, and fix nitrogen and dissolve phosphorus.

[0069] Among them, the formula of cellulose Congo red medium: sodium carboxymethyl cellulose (CMC-Na) 5.0g, yeast extract powder 0.5g, casein 0.5g, NaNO 3 1.0g, Na 2 HPO 4 1.2g, KH 2 PO 4 0.9 g, MgSO 4 0.5g, 0.5g KCl, 0.2g Congo red, 15.0g agar, add 1000mL distilled water and mix well, adjust pH 6.9-7.1, sterilize at 121℃ for 20 minutes, cool to about 50℃ and pour into plates.

[0070] Casein medium: casein 10.0 g, beef extract 3.0 g, NaCl 5.0 g, K 2 HPO 4 2.0g, 0.05g of bromovanol blue, 15.0g of agar, add 1000mL of distilled water and mix well, adjust the pH to 7.3-7.5, sterilize at 121℃ for 20 minutes, cool to about 50℃ and pour into plates.

[0071] Inorganic phosphorus medium: yeast extract powder 0.5g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, KCl 0.3g, MnSO 4 0.03 g, MgSO 4 0.3 g, FeSO4 7H 2 O 0.03g, Ca 3 (PO 4 ) 2 5.0g, glucose 10.0g, agar 15.0g, add 1000mL distilled water and mix well, adjust pH 7.0-7.5, sterilize at 121℃ for 20 minutes, cool to about 50℃ and pour into plates.

[0072] Nitrogen-free solid medium: CaCO 3 5.0 g, MgSO 4 0.2g, KH 2 PO 4 0.2 g, CaSO 4 0.1g, 0.2g NaCl, 10.0g mannitol, 15.0g agar, add 1000mL distilled water and mix well, adjust pH to 7.0, sterilize at 121℃ for 20 minutes, cool to about 50℃ and pour into plates.

[0073] The results are as follows Figure 4 As shown in the figure, DW-05 showed transparent degradation circles on all three culture media, indicating that it has the functions of decomposing casein, degrading cellulose, fixing nitrogen and dissolving phosphorus. DW-07 showed transparent degradation circles on both cellulose Congo red culture medium and casein culture medium, indicating that it has the functions of decomposing casein and degrading cellulose.

[0074] 4. The strains DW-05 and DW-07 were subjected to 16S rRNA molecular biological identification. The nucleotide sequence of the 16S rRNA gene of strain DW-05 was obtained by sequencing and splicing, such as the nucleotide sequence of sequence 1 in the sequence list, and the nucleotide sequence of the 16S rRNA gene of DW-07 was obtained by sequencing and splicing. Comparison analysis was performed in the NCBI database, and sequences with higher homology were selected to construct a phylogenetic tree.

[0075] The results showed that the reference species of DW-05 was Providencia alcalifaciens, with a homology of 99%, and the reference species of DW-07 was Brevibacterium frigoritolerans, with a homology of 99%.

[0076] After being stained with Gram stain, strains DW-05 and DW-07 were observed under a microscope. Figure 2 As shown, the Gram staining results of strain DW-07 were all positive, and the Gram staining results of strain DW-05 were negative.

[0077] 5. Storage of DW-05 and DW-07 strains

[0078] The Providencia alcalifaciens DW-05 provided by the present invention has been deposited in the China Center for Type Culture Collection (CCTCC for short; address: Wuhan University, Wuhan, China; zip code: 430072) on May 19, 2022, with a deposit number of CCTCC No. M2022672. Providencia alcalifaciens DW-05 is referred to as Providencia alcalifaciens DW-05 for short.

[0079] The Brevibacterium frigoritolerans DW-07 provided by the present invention has been deposited in the China Center for Type Culture Collection (CCTCC for short; address: Wuhan University, Wuhan, China; zip code: 430072) on May 19, 2022, with a deposit number of CCTCC No.M2022673. Brevibacterium frigoritolerans DW-07 is referred to as Brevibacterium frigoritolerans DW-07 for short.

[0080] Example 2: Preparation method of low-temperature bacterial agent

[0081] 1. Seed Solution Preparation

[0082] A single colony of Providencia alcaligenes DW-05 was picked from the plate, inoculated into LB liquid culture medium, and cultured at 20°C, 180rpm for 24h to obtain a seed solution of Providencia alcaligenes DW-05. A single colony of Brevibacterium frostii DW-07 was picked from the plate, inoculated into LB liquid culture medium, and cultured at 20°C, 180rpm for 24h to obtain a seed solution of Brevibacterium frostii DW-07.

[0083] The formula of LB liquid culture medium is: 5 g yeast extract, 10 g tryptone, 10 g NaCl, add double distilled water to 1 L, and sterilize at 121°C for 20 min.

[0084] 2. Preparation of Fermentation Broth

[0085] The Providencia alcaligenes DW-05 seed liquid of step 1 was added to the sterilized LB liquid medium at a ratio of 1%, and the mixture was shaken at 20°C and 180 rpm for 24 hours, and the fermentation liquid was collected to obtain the Providencia alcaligenes DW-05 fermentation liquid. The content of Providencia alcaligenes DW-05 in the Providencia alcaligenes DW-05 fermentation liquid was not less than 6×10 8 CFU / mL.

[0086] The seed liquid of Brevibacterium refractory DW-07 in step 1 was added to the sterilized LB liquid medium at a ratio of 1%, and the mixture was shaken at 20°C and 180 rpm for 24 hours. The fermentation liquid was collected to obtain the fermentation liquid of Brevibacterium refractory DW-07. The content of Brevibacterium refractory DW-07 in the fermentation liquid of Brevibacterium refractory DW-07 was not less than 6×10 8 CFU / mL.

[0087] 3. Preparation of solid bacterial agent

[0088] 3.1 Preparation of composite bacterial agent for composting fermentation

[0089] The Providencia alcaligenes DW-05 fermentation liquid, the Brevibacterium frost-tolerant DW-07 fermentation liquid and the sterilized corn cob powder in step 2 are uniformly mixed and air-dried in a cool place to make the moisture content less than 30%, thereby obtaining a composting fermentation composite bacterial agent. The content of Providencia alcaligenes DW-05 in the composting fermentation composite bacterial agent is about 6×10 8 CFU / g, the content of Brevibacterium frostii DW-07 is about 6×10 8 CFU / g.

[0090] 3.2 Preparation of Providencia alkali-producing single bacterial agent in compost fermentation

[0091] The preparation method of the Providencia alkali-producing single bacterial agent for compost fermentation is different from step 3.1 only in that the fermentation liquid of cold-resistant Brevibacterium DW-07 in 3.1 is replaced with the fermentation liquid of Providencia alkali-producing DW-05, and the other operations are the same. The specific method is as follows: the fermentation liquid of Providencia alkali-producing DW-05 in step 2 and the sterilized corn cob powder are evenly mixed, and dried in a cool place to make the moisture content less than 30%, so as to obtain the Providencia alkali-producing single bacterial agent for compost fermentation. The content of Providencia alkali-producing DW-05 in the Providencia alkali-producing single bacterial agent for compost fermentation is about 1×10 9 CFU / g.

[0092] 3.3 Preparation of cold-resistant Brevibacterium monoculture agent for compost fermentation

[0093] The preparation method of the compost fermentation cold-resistant Brevibacterium single bacterial agent is different from step 3.1 only in that the alkaline-producing Providencia DW-05 fermentation liquid in 3.1 is replaced with cold-resistant Brevibacterium DW-07 fermentation liquid, and the other operations are the same. The specific method is as follows: the cold-resistant Brevibacterium DW-07 fermentation liquid in step 2 and the sterilized corn cob powder are evenly mixed, and dried in a cool place to make the moisture content less than 30%, thereby obtaining the compost fermentation cold-resistant Brevibacterium single bacterial agent. The content of cold-resistant Brevibacterium DW-07 in the compost fermentation cold-resistant Brevibacterium single bacterial agent is about 1×10 9 CFU / g.

[0094] Example 3: Optimization of carriers for compost fermentation composite bacterial agents

[0095] The effective viable bacterial rate was measured to verify the affinity between the liquid bacterial agent and the carrier.

[0096] The Providencia alcaligenes DW-05 fermentation liquid, the Brevibacterium frost-tolerant DW-07 fermentation liquid and the sterilized carrier in step 2 of Example 2 were uniformly mixed and air-dried in a cool place to make the moisture content less than 30%, thereby obtaining a compost fermentation composite bacterial agent. The content of Providencia alcaligenes DW-05 in the composite bacterial agent for compost fermentation was 6×10 8 CFU / g, and the content of Brevibacterium frostii DW-07 was 6×10 8 CFU / g. The carriers were corncob powder, biochar or vermiculite, and the bacterial carrying capacity of each carrier, that is, the effective viable bacterial rate after compounding, was determined. The experimental method is as follows:

[0097] Weigh 1g of the immobilized bacterial sample and place it in a conical flask containing 100mL PBS buffer. Stir it on a constant temperature magnetic stirrer for 2h and then perform gradient dilution. The dilution was spread on LB solid culture medium by the coating plate method. After culturing at 20℃ for 48h, the number of colonies on the culture medium was counted to calculate the number of live bacteria released by the carrier. At the same time, the number of colonies in the fermentation liquid was counted as the number of inoculated bacteria, and the average value was taken after 3 experimental measurements. Live bacteria rate (%) = number of released live bacteria / number of inoculated bacteria (CFU) × 100%. The results are shown in Table 2. The relationship between the number of microorganisms contained in the three bacterial agents is corn cob powder > vermiculite > biochar. The effective live bacteria rate in corn cob powder is the highest, reaching 66.88%, indicating that it has the strongest bacterial carrying capacity. The effective live bacteria rate of the inoculant with biochar as the carrier is the lowest. On the one hand, this is because of its poor water absorption performance. On the other hand, biochar is a substance obtained after anaerobic and high-temperature conversion of biomass. Different biomass sources and carbonization process parameters will have a huge impact on the microstructure of biochar, and also make its bacterial carrying capacity show significant differences.

[0098] Table 2 Effective live bacteria rate in different carrier bacterial agents

[0099]

[0100] The number of live bacteria remaining in the compost fermentation composite bacterial agent with corn cob powder as the carrier after being stored at 25℃ for 3 months was 9×10 7 CFU / g.

[0101] The low-temperature composting fermentation composite bacterial agent finally obtained is Figure 5 As shown, it is a yellow-brown powder with a pungent and irritating microbial odor.

[0102] Example 4: Application of low-temperature bacterial agent in agricultural waste treatment

[0103] The composting raw materials in this embodiment are fresh cow dung pulled from the dairy farm to the composting site, and mixed straw collected from local farmers, which is composed of sunflower straw, chrysanthemum straw and wheat straw in a mass ratio of 1:1:1. The wet weight ratio of fresh cow dung and straw was determined to be 10:1 based on the measured C / N ratio of the material. 10 parts by mass (wet weight) of fresh cow dung and 1 part by mass (wet weight) of the above-mentioned mixed straw were mixed to obtain a composting fermentation raw material, and the following composting fermentation was carried out for 84 days starting on December 24, 2021 at Qianziyuan Agricultural Science and Technology Expo Park in Tianjiazhai Town, Huangzhong County, Xining City, Qinghai Province.

[0104] 4.1 Composting using compound bacterial agents

[0105] Composting was carried out using the composting fermentation composite bacterial agent of step 3.1 of Example 2. The composting fermentation raw materials were mixed evenly with the composting fermentation composite bacterial agent of step 3.1 of Example 2 to obtain a pile material. In the pile material, the content of Providencia alcaligenes DW-05 was 6×10 5 CFU / g, and the content of Brevibacterium frostii DW-07 was 6×10 5 CFU / g, designated as Pile2 experimental group (P2).

[0106] The pile materials are composted in piles, with a volume of 18m 3 (4m long, 3m wide, 1.5m high), adjust the moisture content to 65%. Cover the surface of the pile with plastic film to reduce the loss of moisture and heat. Set up inserted electrodes on the top and around the pile, 40cm deep into the pile, and connect the wires to the digital thermometer.

[0107] During the experiment, the ambient temperature and the temperature of the compost were measured at 10 a.m. and 4 p.m. every day, and the average value was taken as the temperature of the day. Mechanical and manual compost turning was used when the temperature reached its peak and began to drop continuously. During the fermentation process, the total organic carbon (TOC), organic matter (OM), ammonia nitrogen content and nitrate nitrogen content of the compost were measured.

[0108] 4.2 Natural fermentation without adding bacteria

[0109] The only difference from 4.1 is that the compost fermentation composite bacterial agent in step 3.1 of Example 2 is replaced with the sterilized corn cob powder of Example 2 of equal mass, and the other operations are the same, which is designated as Pile1 natural fermentation group (P1).

[0110] The temperature change of the pile is as follows Figure 6The temperature of compost can characterize the degradation of organic matter to a certain extent. The composting process generally includes low temperature stage, high temperature stage and cooling stage. In the low temperature stage, the microbial activity is low and the compost temperature is greatly affected by the ambient temperature. As the metabolic activity of microorganisms inside the compost increases, the decomposition rate of organic matter is accelerated, and a large amount of heat is generated, which will cause the compost temperature to rise. The faster the compost temperature rises and the higher the temperature, the stronger the internal microbial activity and the more intense the decomposition of organic matter. After reaching 50°C, that is, the high temperature stage, the microbial activity reaches its peak. The high temperature of the compost will inhibit the microbial activity. At this time, only some heat-resistant microorganisms are relatively active. As the microbial activity decreases, the compost temperature also begins to decrease and enters the cooling and mature stage. At this time, the easily degradable organic matter has been basically consumed, and the remaining difficult-to-degrade cellulose, lignin, etc. are difficult for most bacteria to use. At this time, mesophilic actinomycetes and fungi will occupy a dominant position, and the organic matter in the compost is basically transformed into stable humus.

[0111] From 0 to 63 days, the average temperature of the composting environment was -13-6℃, and the composting temperature showed a trend of first rising and then falling. Among them, after the first composting on the 18th day, the Pile2 experimental group (P2) rose to 54.3℃ on the second day (20 days), reaching the high temperature period (50℃), while the Pile1 natural fermentation group (P1) was still at 43.1℃. The Pile1 natural fermentation group (P1) did not rise to 54.2℃ until the 22nd day, and the Pile2 experimental group reached the high temperature period significantly earlier than the blank group. The temperature of the Pile2 experimental group reached a peak of 61.4℃ on the 24th day, and the Pile1 reached a peak of 60.1℃ on the 29th day. The temperature of the Pile2 experimental group remained above 55℃ for more than 10 days, meeting the requirements of the national standard (NY / T 1168-2006). Then from the 28th day, the Pile2 experimental group began to cool down earlier than the Pile1 group, and it lasted until the 64th day. Later, as the temperature rose, the ambient temperature began to remain above 0°C, and the temperatures of the two groups began to rise slowly. This shows that after adding the bacterial agent, the microorganisms in the compost quickly adapted to the low temperature environment, metabolized and decomposed organic matter, and quickly entered the high temperature period. The organic matter that is easily degradable at high temperature was consumed, and the pile entered the stable decomposition stage faster.

[0112] Samples were taken at different stages to determine the changes in total organic carbon (TOC) in the compost (e.g. Figure 7 ), changes in organic matter (OM) loss (as shown in Figure 8 As shown), ammonia nitrogen (as Fig. 9 as shown) and nitrate nitrogen (as Fig.10 The changes in the content of nitrogen (as shown) were used to evaluate the effects of low-temperature bacterial agents on the degradation of compost organic matter and nitrogen transformation.

[0113] The moisture content and TOC were determined by the drying loss method. A certain amount of fresh sample was weighed and placed in a 0 The total weight of the crucible is w 1 Then put the crucible and the sample into an oven and dry them at 105℃ for 24h. After cooling, weigh them and record them as w 2 ; Then directly put the crucible and the dried sample into a muffle furnace and burn it at 550℃ for 6h. When the temperature of the muffle furnace drops to about 250℃, take out the crucible and put it into a desiccator. After cooling, weigh it and record it as w 3 Finally, the moisture content, ash, OM, TOC, and OM loss of the compost samples were calculated according to equations 4.1, 4.2, 4.3, 4.4, and 4.5, respectively.

[0114] Moisture content (%) = (w 1 -w 2 ) / (w 1 -w 0 )×100% (4.1)

[0115] Dry matter (%) = (w 2 -w 0 ) / (w 1 -w 0 )×100%

[0116] Ash content (%) = (w 3 -w 0 ) / (w 2 -w 0 )×100% (4.2)

[0117] OM(%)=(w 2 -w 3 ) / (w 2 -w 0 )×100% (4.3)

[0118] TOC(%)=OM(%) / 1.8 (4.4)

[0119] OM loss (%) = [1-(initial ash × (100-corresponding composting time ash)) / (corresponding composting time ash × (100-initial ash))] × 100% (4.5)

[0120] The method for determining the changes in ammonia nitrogen and nitrate nitrogen in compost is as follows

[0121] Ammonia nitrogen (NH 4 + -N), nitrate nitrogen (NO 3 --N) was determined using potassium chloride solution extraction-spectrophotometry (in accordance with HJ634-2012, GB / T 32737-2016).

[0122] There was a significant difference in total organic carbon between the treatment group P1 with the addition of low-temperature bacteria and the blank group P2, showing a lower concentration. Combined with the changes in organic matter loss, it was concluded that the addition of low-temperature bacteria can promote the degradation of compost organic matter and accelerate the compost maturity process.

[0123] The ammonia nitrogen content of the P2 experimental group decreased more dramatically than that of the blank group during the high temperature maintenance stage (27-43 days), and the peak of nitrate nitrogen appeared at this time; the ammonia nitrogen content of P2 was higher than that of the blank group in the late composting period, and the nitrate nitrogen was significantly reduced, indicating that the denitrification and nitrogen assimilation of the compost with the addition of low-temperature bacteria at this time were stronger. The results show that the addition of low-temperature bacteria can enhance the nitrification and nitrogen conversion of composting during the high temperature period.

[0124] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Compound bacterial agent, It is characterized in that The active ingredients of the composite bacterial agent are composed of Providencia alcalifaciens and Brevibacterium frigoritolerans. The composite bacterial agent is a composite bacterial agent used for preparing compost. The Providencia alcalifaciens is Providencia alcalifaciens (Providencia alcalifaciens) DW-05, and its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 2022672; the Brevibacterium frigoritolerans is Brevibacterium frigoritolerans (Brevibacterium frigoritolerans) DW-07, and its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 2022673.

2. The composite bacterial agent according to claim 1, Features: In the composite bacterial agent, the CFU ratio of the alkaline-producing Providencia and the cold-resistant Brevibacterium is 1:

1.

3. The composite bacterial agent according to claim 1 or 2, Features: The composite bacterial agent contains a carrier.

4. The composite bacterial agent according to claim 3, Features: The carrier is corn cob powder.

5. Providencia alcaligenes, It is characterized in that The Providencia alcalifaciens is Providencia alcalifaciens DW-05, and its preservation number in the China Center for Type Culture Collection is CCTCCNO: M 2022672.

6. Use of the composite bacterial agent according to any one of claims 1 to 4 in any of the following: 1) Fermentation and composting under low temperature conditions; 2) Promote the degradation of compost organic matter; 3) Accelerate the composting process; 4) Enhance denitrification and / or nitrogen assimilation in compost; 5) Enhance the conversion of nitrogen in compost.

7. Use of the Providencia alcaligenes according to claim 5 in any of the following: 1) Fermentation and composting under low temperature conditions; 2) Promote the degradation of compost organic matter; 3) Accelerate the composting process.

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