Biological additive for emission reduction and nitrogen conservation of chicken manure-straw compost and preparation method of biological additive

Abstract

The invention discloses a biological additive for emission reduction and nitrogen conservation of chicken manure-straw compost and a preparation method of the biological additive, and belongs to the field of agricultural waste treatment and resource utilization. The biological additive for emission reduction and nitrogen conservation of chicken manure-straw compost comprises a protein degrading bacterium, two cellulose degrading bacteria and ferrous sulfate, wherein the protein degrading bacterium and the cellulose degrading bacteria are screened from chicken manure. The method comprises the following steps: screening high-temperature-resistant high-efficiency degrading bacteria aiming at macromolecular substances such as protein and cellulose which are difficult to degrade in a chicken manure-straw compost high-temperature period, constructing a synthetic flora on the basis of the high-temperature-resistant protein and cellulose degrading bacteria, compounding the synthetic flora with ferrous sulfate to prepare a compost additive, and researching the physicochemical properties of a compost product at the same time. The emission reduction of ammonia gas and greenhouse gas and the preservation of carbon and nitrogen in the composting process are ensured.

Description

Technical Field

[0001] The invention belongs to the field of agricultural waste treatment and resource utilization, and specifically relates to a biological additive for reducing emission and retaining nitrogen in chicken manure-straw composting and a preparation method thereof. Background Art

[0002] Livestock and poultry manure contains nutrients needed by crops. Aerobic composting is an effective method for treating crop waste and livestock and poultry manure. Composting refers to the process of artificially controlling the appropriate moisture content, C / N and water permeability of materials, relying on naturally existing bacteria, actinomycetes, fungi and other microorganisms in the composting materials or artificial inoculation of exogenous microorganisms, making the compost ferment at high temperature to achieve the effect of killing pathogens, insect eggs, grass seeds and other harmless effects, and at the same time making the compost mineralized and humified to become organic fertilizer. However, the composting process is prone to cause the emission of NH3, N2O, CH4, CO2, etc., resulting in the loss of carbon and nitrogen in the composting materials, thereby reducing the fertilizer efficiency and value of the composting products; it will also cause air pollution, aggravate global warming, promote the formation of haze, and deteriorate air quality. The NH3 and greenhouse gases produced by livestock and poultry manure in the composting process should not be underestimated, and the green and low-carbon resource utilization of agricultural waste is imminent.

[0003] Chicken manure is rich in organic matter, especially macromolecular components such as protein. If it can be fully and efficiently degraded, it can not only be converted into high-quality organic fertilizer, improve soil fertility and promote crop growth, but also improve soil fertility and quality, with significant ecological, economic and social benefits. In the early stage of composting, microorganisms mainly increase the temperature of the pile by degrading substances such as starch, lipids and protein in chicken manure, and enter the high temperature period. The industrialization of crop straw as fertilizer, feed, base material, fuel and industrial raw material has developed rapidly. During the decomposition process of composting, not only a large amount of nutrients such as nitrogen, phosphorus and potassium that can be absorbed and utilized by plants are produced, but also humus can be formed, which is an important organic compound for improving soil fertility. In the process of livestock and poultry manure composting, adding straw can adjust the C / N of the composting process, enhance microbial activity, accelerate the composting process, etc., but there are still many problems in practical application. Since the refractory macromolecular substances in crop straw are mainly cellulose and hemicellulose, of which cellulose accounts for about 33%, the presence of cellulose may lead to asynchronous biotransformation of compost, uneven decomposition, long composting cycle, and ultimately affect the quality of compost products. The composting temperature rise period is mainly used to decompose easily degradable organic matter, and the degradation of cellulose is mainly in the high temperature period and the mature stage. Since the number of mesophiles decreases in the high temperature period, the thermophilic bacteria population gradually dominates. Therefore, it is necessary to exogenously inoculate high-temperature resistant protein and cellulose efficient degrading bacteria to promote the rapid conversion of organic materials, shorten the mature time and improve the quality of compost products. For this reason, a biological additive for emission reduction and nitrogen conservation in chicken manure-straw composting and a preparation method thereof are proposed. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting and a preparation method thereof, thereby solving the problems in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] Biological additives for reducing emissions and retaining nitrogen in chicken manure-straw composting include a protein-degrading bacteria and two cellulose-degrading bacteria screened from chicken manure, as well as ferrous sulfate.

[0007] Furthermore, the protein-degrading bacteria belong to Burkholderia gladioli; the two strains of cellulose-degrading bacteria belong to Bacillus stercoris and Bacillus velezensis, respectively.

[0008] The preparation method of the above-mentioned biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting comprises the following steps:

[0009] S1, screening of protein-degrading and cellulose-degrading bacteria from chicken manure;

[0010] S2, the screened protein-degrading bacteria and cellulose-degrading bacteria were subjected to in-dish antagonism tests and 16S rRNA sequencing analysis to eliminate animal and plant pathogenic microorganisms, and strains without antagonistic effects were selected to construct synthetic bacterial communities;

[0011] S3, compounding the synthetic bacterial flora with ferrous sulfate to obtain a biological additive.

[0012] Further, the steps of screening out protein-degrading bacteria are:

[0013] Carrying out enrichment culture on chicken manure to obtain enrichment solution, and applying it on a plate for culture;

[0014] After the culture is completed, the strains are separated and purified, and then the protein hydrolysis test is carried out to preliminarily screen the strains with protein degradation ability;

[0015] After the initial screening strains were activated, they were inoculated and cultured, and then the protein degradation ability of the strains at 30°C was determined using the Coomassie Brilliant Blue method, and the four strains with the best protein degradation ability were obtained;

[0016] The strains with better protein degradation ability at room temperature were continued to be inoculated and cultured, and then the protein degradation ability of the strains at 50°C was determined using the Coomassie Brilliant Blue method to obtain the two best protein-degrading bacteria.

[0017] Furthermore, the steps of screening cellulose degrading bacteria are:

[0018] Carrying out enrichment culture on chicken manure to obtain enrichment solution, and applying it on a plate for culture;

[0019] After the culture is completed, the strains are separated and purified, and the bacterial solution of the purified strains is placed in an 80°C water bath for 15 minutes to screen out the high-temperature resistant strains;

[0020] The selected thermostable strains were inoculated on Congo red sodium cellulose agar medium to preliminarily screen out cellulose-degrading bacteria;

[0021] The cellulose-degrading bacteria preliminarily screened were inoculated and cultured, and the CMCase activity and FPA enzyme activity of the strains were determined by the DNS method, and the six strains with the highest cellulase activity were obtained.

[0022] Furthermore, the steps of the in-dish antagonism test include:

[0023] The screened protein-degrading bacteria and cellulose-degrading bacteria are activated, spread on a solid culture medium for culture, and then cultured in an incubator;

[0024] Use a pipette to suck up the antagonistic bacterial suspension, apply it on a solid culture medium and culture it; after the puncher is sterilized and cooled, punch holes in the antagonistic bacteria to make round bacterial blocks; turn the taken colony agar block upside down on a plate coated with indicator bacteria and culture it in an incubator;

[0025] After the indicator bacteria grow out, observe whether there is an inhibition zone around the antagonistic bacteria agar block on the indicator bacteria plate. If there is, it indicates that there is antagonism between the strains. The larger the diameter of the inhibition zone, the greater the antagonism between the strains.

[0026] Further, the synthetic bacterial community includes: one strain of protein-degrading bacteria and two strains of cellulose-degrading bacteria;

[0027] The protein-degrading bacteria belong to Burkholderia gladioli; the two cellulose-degrading bacteria belong to Bacillus stercoris and Bacillus velezensis, respectively.

[0028] The above-mentioned chicken manure-straw composting biological additive for reducing emission and retaining nitrogen is used in degrading chicken manure-straw composting.

[0029] Furthermore, the application process is: spraying the biological additive on the straw powder, then fully mixing the straw powder and chicken manure, and turning the pile.

[0030] Furthermore, the mass ratio of the chicken manure to the straw powder is 5:1; and the biological additive accounts for 2% of the total mass of the chicken manure-straw compost.

[0031] Beneficial effects of the present invention:

[0032] In view of the large ammonia and greenhouse gas emissions and high carbon and nitrogen nutrient losses in the chicken manure-straw composting process, the present invention separates and screens high-temperature resistant protein and cellulose efficient degrading bacteria, and then constructs a synthetic bacterial community, and compounding it with ferrous sulfate, to develop a biological additive for reducing emissions and retaining nitrogen in the composting process, providing new ideas and new products for the coordinated control of ammonia and carbon emissions in the agricultural waste composting process and improving the quality of compost products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 is a phylogenetic tree of 16S rRNA sequences of isolated strains of the present invention;

[0035] Figure 2 It is a characteristic diagram of the temperature change of the composting process of the present invention;

[0036] Figure 3 It is a characteristic diagram of moisture content change in each treatment during the composting process of the present invention;

[0037] Figure 4 It is a characteristic diagram of pH and EC changes in each treatment during the composting process of the present invention;

[0038] Figure 5 The present invention composting process each processing NH4 + -N and NO 3- -N content change characteristic diagram;

[0039] Figure 6 It is a characteristic diagram of TN and TC changes in each treatment during the composting process of the present invention;

[0040] Figure 7 It is a graph of NH3 emission flux and cumulative emission in each treatment during the composting process of the present invention;

[0041] Figure 8 It is a graph of N2O emission flux and cumulative emission in each treatment during the composting process of the present invention;

[0042] Fig. 9 is a graph showing nitrogen loss in each treatment during the composting process of the present invention;

[0043] Fig.10 It is a graph of CH4 emission flux and cumulative emission in each treatment during the composting process of the present invention;

[0044] Fig.11It is a graph of CO2 emission flux and cumulative emission in each treatment during the composting process of the present invention;

[0045] Fig.12 It is a graph of CO2 emission equivalents for each treatment in the composting process of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] The preparation method of a biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting comprises the following steps:

[0049] S1, screening thermostable protein-degrading bacteria and thermostable cellulose-degrading bacteria from chicken manure;

[0050] (1) Isolation and screening of thermostable protein-degrading bacteria;

[0051] Enrichment culture: Take 10 grams of fresh chicken manure and place it in 100 mL of distilled water. Incubate in a constant temperature shaking incubator at 30°C and 180 r / min. -1 , shake and culture for 1 hour, mix evenly, take 10 ml of the mixture under sterile conditions, add it to the newly prepared 100 ml liquid culture medium, shake and culture continuously at 30°C for 24 hours, and repeat the enrichment 3 times.

[0052] Spreading culture: Under sterile conditions, use a 10mL sterile pipette to draw 9mL of sterile water into a sterilized screw-capped glass tube, then add 1mL of enrichment solution to perform concentration gradient dilution to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 In a sterile operating room, use 0.1mL 10 -1 The bacterial solution of 6 dilution concentrations was added dropwise to the solid plate culture medium prepared in advance and spread with a triangular glass rod. Similarly, 6 dilution concentrations of bacterial solution were spread on the solid plate culture medium, with 2-3 plates spread for each gradient.

[0053] Isolation and purification: After the culture is completed, take out the plate from the constant temperature incubator, select the plate with the appropriate dilution multiple, ensure that the number of colonies on the plate is between 30-300, select colonies with different morphological characteristics on the protein decomposition medium for streak separation, and select bacteria with transparent circles for single colony streak culture, and repeat the streak three times until pure culture or single colony.

[0054] Initial screening: A protein hydrolysis test was conducted, and a "+" dot was made on the casein hydrolysis medium. The diameter of the colorless transparent circle around the colony reflected the strength of the bacteria's protein hydrolysis ability. A preliminary screening of 48 strains with protein degradation ability was conducted.

[0055] Secondary screening: After the primary screening strains are activated, they are inoculated into seed liquid culture medium and placed at 30°C and 180r / min -1 The culture was shaken on a shaker for 24 h, and then the seed solution was transferred to the basic fermentation medium at a 2% inoculation rate and incubated at 30 °C and 180 r / min. -1 The culture was shaken in a shaker under the conditions for 48 hours. 2 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 15000 r·min-1 for 3 minutes. 0.1 mL of supernatant was taken into a test tube, and the protein degradation ability of the strain was determined by the Coomassie Brilliant Blue method. The strains with better protein degradation ability (G15, G13, G2, G9) were obtained, as shown in Table 1:

[0056] Table 1 Protein degradation rate of each isolated strain

[0057]

[0058] Screening of thermotolerant strains: The protein-efficient degrading bacteria (G15, G13, G2, and G9) screened under normal temperature culture were inoculated on solid culture medium and cultured at 50°C for 36 h. The growth of the strains was observed, as shown in Table 2. The four strains were inoculated into seed liquid culture medium and placed at 50°C, 180 r / min -1 The culture was shaken for 24 hours, and then the seed solution was transferred to the basic fermentation medium at a 2% inoculation rate. -1 The culture was shaken on a shaker for 48 h under the same conditions, and 2 mL of the bacterial solution was placed in a centrifuge tube and centrifuged at 15000 r / min. -1 The mixture was centrifuged at a speed of 3000 nm for 3 min, 0.1 mL of the supernatant was taken into a test tube, and the ability of the strain to degrade protein under high temperature environment was determined by the Coomassie Brilliant Blue method. As shown in Table 3, thermostable protein-degrading bacteria (G13 and G15) were obtained.

[0059] Table 2 Growth status of isolated protein-degrading bacteria under 50°C culture conditions

[0060]

[0061] Note: +++ indicates good growth; + indicates average growth; - indicates no growth.

[0062] Table 3 Protein degradation rate of isolated protein-efficient degrading bacteria under 50°C culture conditions

[0063]

[0064] (2) Isolation and screening of thermostable and efficient cellulose-degrading bacteria

[0065] Enrichment culture: Take 10 grams of fresh chicken manure and place it in 100 mL of distilled water. -1 The culture was shaken in a constant temperature shaking incubator for 1 h under the conditions. After mixing evenly, 10 ml of the bacterial solution was taken under sterile conditions and transferred to the newly prepared 100 ml liquid culture medium. The culture was shaken continuously at 30 ° C for 24 h. The enrichment was repeated 3 times.

[0066] Spreading culture: Under sterile conditions, use a 10mL sterile pipette to draw 9mL of sterile water into a sterilized screw-capped glass tube, then add 1mL of enrichment solution to perform concentration gradient dilution to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 In the clean bench, use 0.1mL 10 -1 The bacterial solution of 100 μg / ml was inoculated into the solid plate medium prepared in advance and spread with a triangular glass rod. The bacterial solution of 6 dilution gradients was spread on the solid plate medium in sequence, and 2-3 plates were spread for each gradient.

[0067] Isolation and purification: After the culture is completed, take out the plate from the constant temperature incubator, select the plate with the appropriate dilution multiple, ensure that the number of colonies on the plate is between 30-300, select colonies with different morphological characteristics on the sodium carboxymethyl cellulose selective medium for streak separation, and repeat the streak three times until pure culture or single colony is obtained.

[0068] Screening of thermotolerant strains: The bacterial solution of the purified strains was placed in an 80°C water bath for 15 min, and 110 thermotolerant strains were preliminarily screened out.

[0069] Initial screening: Inoculate the screened single colonies onto Congo red sodium cellulose agar medium, “+” four-point inoculation, observe the colony diameter and transparent circle diameter every 12 hours, calculate D / d, and preliminarily screen out 30 strains of cellulose-degrading bacteria, and preserve the strains in glycerol tubes.

[0070] Secondary screening: After the primary screening strains were activated, they were inoculated into the cellulase-producing bacteria seed culture medium and placed at 50°C and 180 r / min. -1 The culture was shaken on a shaker for 2 days. Then the seed solution was transferred to the cellulase-producing bacteria fermentation medium at a 2% inoculation rate and incubated at 50°C and 180 r / min. -1 The culture was shaken on a shaker for 4 days. Finally, 2 mL of bacterial solution was placed in a centrifuge tube and centrifuged at 9000 r / min. -1 The mixture was centrifuged at a speed of 10 min, and 0.1 mL of the supernatant was taken into a test tube. The CMCase activity and FPAase activity of the strains were determined by the DNS method, and strains with higher cellulase activity (M4, KZ3, MX5, GX9, MX2, and MX7) were obtained, as shown in Table 4.

[0071] Table 4 Degradation ability of isolated cellulose degrading bacteria

[0072]

[0073]

[0074] In summary, the thermostable protein-degrading bacteria and thermostable cellulose-degrading bacteria are as follows:

[0075] Thermotolerant protein-degrading bacteria: G13, G15;

[0076] High temperature resistant cellulose degrading bacteria: M4, KZ3, MX5, GX9, MX2, MX7.

[0077] S2, the selected thermostable protein-degrading bacteria and thermostable cellulose-degrading bacteria were subjected to in-dish antagonism tests and 16S rRNA sequencing analysis to eliminate animal and plant pathogenic microorganisms, and strains without antagonism were selected to construct synthetic bacterial communities;

[0078] (1) In vitro antagonism test

[0079] After the degradation performance of the strains was determined, strains with high degradation ability were screened out, activated, and cultured on solid culture media, placed in a 30°C incubator, and cultured for 2 days.

[0080] Use a pipette to draw 100 μL of the antagonistic bacterial suspension, apply it on the solid culture medium and culture it. After the puncher is sterilized and cooled, punch holes in the antagonistic bacteria to make round bacterial blocks. Place the taken colony agar block upside down on the plate coated with indicator bacteria and culture it in a 30℃ incubator for 2 days.

[0081] After the indicator bacteria grow out, observe whether there is an inhibition zone around the antagonistic bacteria agar block on the indicator bacteria plate, as shown in Table 5. If there is, it indicates that there is antagonism between the strains. The larger the diameter of the inhibition zone, the greater the antagonism between the strains.

[0082] Table 5 Results of in-dish antagonism test of high temperature resistant and efficient degradation strains

[0083]

[0084]

[0085] Note: √ indicates no antagonistic effect; × indicates antagonistic effect.

[0086] (2) 16S rRNA sequencing analysis of strains

[0087] like Figure 1 As shown, a bacterial whole genome extraction kit was used to extract the DNA of the strain, and the 16SrRNA gene was amplified using the DNA as a template, and the forward primer was 27F:

[0088] 5'-AGAGTTTGATCMTGGCTCAG-3'; reverse primer is 1492R: 5'-TACCTTGTTACGACTT-3'. PCR reaction system: DNA polymerase (Taq enzyme) 0.1μL, upstream primer (10μmol·L -1 )0.4μL, downstream primer (10μmol·L -1 )0.4μL, 10×PCR buffer2μL, dNTP (2.5mmol·L -1 )1.6μL, DNA template amount (50-100ng·μL -1 )0.1μL, dd H2O 15.4μL. The PCR amplification program was: 95℃ preheating for 10min; 95℃30s, 55℃30s, 72℃45s, 32 cycles; 72℃ final extension for 7min. After gel excision and purification, the PCR product was sequenced and analyzed. After sequencing, the sequencing results were compared and analyzed in the NCBI database. The 16S rRNA gene sequences of strains similar to the sample were found in the library for homology comparison.

[0089] Based on the results of 16S rRNA sequence comparison, potential animal and plant pathogenic microorganisms were eliminated; further, efficient strains without antagonistic effects were selected to construct synthetic bacterial communities.

[0090] These eight strains were subjected to in-dish antagonism test (Table 5), 16S rRNA sequencing analysis ( Figure 1 ), and combined with their degradation effects to construct synthetic bacterial consortia (M4, MX5, G15), among which strain M4 belongs to Bacillus stercoris; strain MX5 belongs to Bacillus velezensis; strain G15 belongs to Burkholderia gladioli.

[0091] (3) Pure culture test was carried out by simulating the temperature of composting heating period (37°C) and high temperature period (55°C): the constructed synthetic bacterial community was inoculated into solid culture medium and placed in a constant temperature biochemical incubator at 37°C and 55°C for 48 hours to simulate the growth environment of composting heating period and high temperature period. The growth of synthetic bacterial community was recorded every 12 hours, and the protein degradation ability and cellulase activity of synthetic bacterial community under two temperature culture conditions were measured, as shown in Table 6 and Table 7, respectively.

[0092] Table 6 Degradation of protein by synthetic bacterial consortia at 37°C and 55°C

[0093]

[0094] Table 7 Cellulase activity of synthetic bacterial consortia under 37°C and 55°C culture conditions

[0095]

[0096] From the results in Tables 6 and 7, it can be seen that the protein degradation rate of the synthetic bacterial consortium at 37°C was >70%, among which M4 had the best degradation effect; the protein degradation rate decreased at 55°C, but was >55%. Compared with 37°C, the CMCase activity and FPAase activity of the two strains G15 and MX5 showed higher enzyme activity at 55°C, indicating that the synthetic bacterial consortium can further efficiently degrade cellulose under high temperature conditions.

[0097] S3, compounding the above synthetic bacteria with ferrous sulfate to obtain a biological additive.

[0098] The synthetic bacterial consortium includes: M4, MX5 and G15, wherein strain M4 belongs to Bacillus stercoris; strain MX5 belongs to Bacillus velezensis; strain G15 belongs to Burkholderia gladioli.

[0099] The dosage of ferrous sulfate is 1% of the mass of the chicken manure-straw compost raw material.

[0100] Example 2

[0101] In this example, a chicken manure-straw composting test was conducted on the biological additive prepared in the example to illustrate the superiority of the biological additive.

[0102] (1) The test was set up as follows: The test device was a 25L composting fermentation barrel. A pad was placed under the barrel to facilitate the seepage of the leachate inside the pile. A water outlet was installed under the barrel to increase the permeability of the bottom of the pile and to allow the leachate to flow out. The compost raw material came from fresh chicken manure from a chicken farm. Wheat straw powder was added to adjust the moisture content of the compost material. The mass ratio was chicken manure: straw powder = 5:1. Four test treatments were set up for composting, as shown in Table 8. The inoculation amount of the microbial liquid was 2% of the total mass of the pile, and it was evenly added to the pile in an inoculated manner. That is, the bacterial liquid was evenly sprayed on the wheat straw powder in the form of spraying, and the straw powder and chicken manure were fully mixed. The amount of ferrous sulfate was 1% of the total mass of the chicken manure-straw pile, and it was evenly mixed with the pile after addition. The turning frequency was set to once every 48 hours, and the turning frequency was determined later according to temperature changes.

[0103] Table 8 Treatment settings for the small-scale chicken manure-straw composting test

[0104]

[0105] (2) Temperature measurement: Measure and record the pile temperature and room temperature at 9:00 a.m. and 6:00 p.m. every day.

[0106] (3) Gas sample collection: Gas samples are collected every day during the warming stage and high temperature stage, and every 2-3 days during the cooling stage to measure the concentrations of NH3 and greenhouse gases (N2O, CH4, CO2).

[0107] (4) Compost material sample collection: The sampling date was determined according to the temperature change of the compost body. Compost material samples were collected at the initial stage (day 0), the heating stage (i.e., the temperature was lower than 55°C), the high temperature stage (i.e., the temperature was higher than 55°C), the cooling stage (i.e., the temperature was lower than 55°C), and the mature stage (i.e., the gas emission tended to be stable). The moisture content, pH, EC, and NH4 + -N, NO 3- -N, TC, TN, GI index and other properties.

[0108] Test results:

[0109] like Figure 2 As shown in the figure, the treatment inoculated with synthetic flora (T2 treatment) and the treatment combined with synthetic flora and FeSO4 (T3 treatment) entered the high temperature period (>55℃) on the third day of composting, one day earlier than the uninoculated control (CK treatment); the pile temperature of T2 and T3 treatments reached above 62℃, of which the temperature of T3 treatment reached as high as 65℃, and the high temperature time lasted for 5 days. On the fourth day of composting, compared with the control treatment (59℃), the temperature of T2 and T3 treatments increased by 3℃ and 6℃ respectively.

[0110] like Figure 3As shown in the figure, the initial moisture content of each treatment was about 50%, and the moisture content of the compost showed a downward trend during the composting process. After the composting was completed, the moisture content of each treatment was 20-30%, which met the conditions for microbial nutritional metabolism.

[0111] like Figure 4 As shown in (a), the pH of each treatment during the composting process showed a trend of decline-increase-stabilization. The pH of the treatments with added FeSO4 (T1, T3) was significantly lower than that of other treatments (p < 0.05). The lower pH effectively reduced NH3 emissions. Figure 4 As shown in (b), the EC values ​​of the compost products of each treatment were all lower than 4 mS·cm -1 , reaching the compost maturity requirements.

[0112] like Figure 5 As shown, after the composting was completed, the treatments inoculated with synthetic bacteria (T2, T3) had NH4 + -N and NO 3- -N content was significantly higher than that of the other two treatments. Figure 5 As shown in (a), T2 and T3 treat NH4 + -N content increased by 16.71% and 32.31% compared with the control; Figure 5 As shown in (b), NO 3- -N content increased by 46.97% and 49.70%.

[0113] like Figure 6 As shown in (a), after the composting was completed, the TN content of the treatments inoculated with synthetic flora (T2 and T3) was significantly higher than that of the other two treatments. The TN content of the T2 and T3 treatments increased by 36.55% and 38.90% relative to the control, which means that the addition of synthetic flora has a nitrogen-retaining effect. Figure 6 As shown in (b), the TC content of T2 and T3 treatments increased by 14.43% and 11.78% relative to the control.

[0114] like Figure 7 As shown in (a) of Figure 2, the inoculation of synthetic bacterial flora treatments had a significant reduction effect on NH3 emissions during composting (p < 0.05). Figure 7 As shown in (b), compared with the control, the cumulative NH3 emissions of T2 and T3 treatments decreased by 24.73% and 44.15%, respectively, among which the combination of synthetic bacteria and FeSO4 (T3 treatment) had the most significant emission reduction effect.

[0115] like Figure 8 As shown in (a) in the figure, each treatment during the composting process had a significant effect on reducing N2O emissions (p < 0.05). Figure 8As shown in (b), compared with the control, the cumulative N2O emissions of the T1, T2 and T3 treatments were reduced by 34.82%, 38.58% and 49.13%, respectively, among which the combination of synthetic bacteria and FeSO4 (T3 treatment) had the most significant emission reduction effect.

[0116] like Fig. 9 As shown in the figure, the nitrogen loss of composting is mainly caused by NH3 and N2O emissions, among which NH3 volatilization is the main loss pathway. T1, T2, and T3 treatments reduced nitrogen losses by 18.14%, 25.08%, and 44.28% respectively compared with the control. Among them, the nitrogen loss of the combination of gregarious bacteria and FeSO4 (T3 treatment) was significantly lower than that of the other three treatments (p < 0.05), which further shows that the combination of gregarious bacteria and FeSO4 has the best nitrogen retention effect and can increase the nitrogen nutrient content of compost products.

[0117] like Fig.10 As shown in (a), all treatments had significant CH4 emission reduction effects compared with the control during composting (p < 0.05); Fig.10 As shown in (b), the cumulative CH4 emissions of treatments T1, T2, and T3 were reduced by 33.82%, 60.16%, and 63.03%, respectively. The CH4 emission reduction effect of the treatments inoculated with synthetic bacteria (T2 and T3) was more significant.

[0118] like Fig.11 As shown in (a), each treatment during the composting process had a significant effect on reducing CO2 emissions compared to the control (p < 0.05); Fig.11 As shown in (b), the cumulative CO2 emissions of treatments T1, T2, and T3 were reduced by 9.83%, 16.12%, and 39.19%, respectively. Among them, the treatment (T3) using a combination of syngeneic bacteria and FeSO4 had the most obvious emission reduction effect.

[0119] like Fig.12 As shown, during the composting process, each treatment significantly reduced CO2 emission equivalent compared with the control (p < 0.05). The CO2 emission equivalent of treatments T1, T2, and T3 decreased by 10.79%, 17.08%, and 39.63%, respectively. Among them, the treatment (T3) with the combined use of gregarious bacteria and FeSO4 had the most obvious emission reduction effect.

[0120] The GI index of mung bean and wheat seeds of the compost products treated with FeSO4 addition (T1), inoculation with synthetic flora (T2), and combined use of synthetic flora and FeSO4 (T3) all exceeded 70%, meeting the GI requirements of the agricultural industry standard "Organic Fertilizer" (NY 525-2021) (>70%), so the compost products have no toxic inhibitory effect on plants (Table 9).

[0121] Table 9 Germination index of mung bean and wheat seeds in each treatment compost product

[0122]

[0123] Note: Different letters indicate significant differences among treatments (p<0.05).

[0124] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0125] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting, characterized in that: It includes screening out a protein-degrading bacteria and two cellulose-degrading bacteria, as well as ferrous sulfate, from chicken manure.

2. The biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 1, characterized in that: The protein-degrading bacteria belong to Burkholderia gladioli; the two cellulose-degrading bacteria belong to Bacillus stercoris and Bacillus velezensis, respectively.

3. The method for preparing the biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 1 or 2, characterized in that: The following steps are involved: S1, screening of protein-degrading and cellulose-degrading bacteria from chicken manure; S2, the screened protein-degrading bacteria and cellulose-degrading bacteria were subjected to in-dish antagonism tests and 16S rRNA sequencing analysis to eliminate animal and plant pathogenic microorganisms, and strains without antagonistic effects were selected to construct synthetic bacterial communities; S3, compounding the synthetic bacterial flora with ferrous sulfate to obtain a biological additive.

4. The method for preparing a biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 3, characterized in that: The steps for screening out protein-degrading bacteria are: Carrying out enrichment culture on chicken manure to obtain enrichment solution, and applying it on a plate for culture; After the culture is completed, the strains are separated and purified, and then the protein hydrolysis test is carried out to preliminarily screen the strains with protein degradation ability; After the initial screening strains were activated, they were inoculated and cultured, and then the protein degradation ability of the strains at 30°C was determined using the Coomassie Brilliant Blue method, and the four strains with the best protein degradation ability were obtained; The strains with better protein degradation ability at room temperature were continued to be inoculated and cultured, and then the protein degradation ability of the strains at 50°C was determined using the Coomassie Brilliant Blue method to obtain the two best protein-degrading bacteria.

5. The method for preparing a biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 3, characterized in that: The steps for screening cellulose degrading bacteria are: Carrying out enrichment culture on chicken manure to obtain enrichment solution, and applying it on a plate for culture; After the culture is completed, the strains are separated and purified, and the bacterial solution of the purified strains is placed in an 80°C water bath for 15 minutes to screen out the high-temperature resistant strains; The selected thermostable strains were inoculated on Congo red sodium cellulose agar medium to preliminarily screen out cellulose-degrading bacteria; The cellulose-degrading bacteria preliminarily screened were inoculated and cultured, and the CMCase activity and FPA enzyme activity of the strains were determined by the DNS method, and the six strains with the highest cellulase activity were obtained.

6. The biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 3, characterized in that: The steps of the in-dish antagonism assay include: The screened protein-degrading bacteria and cellulose-degrading bacteria are activated, spread on a solid culture medium for culture, and then cultured in an incubator; Use a pipette to suck up the antagonistic bacterial suspension, apply it on a solid culture medium and culture it; after the puncher is sterilized and cooled, punch holes in the antagonistic bacteria to make round bacterial blocks; turn the taken colony agar block upside down on a plate coated with indicator bacteria and culture it in an incubator; After the indicator bacteria grow out, observe whether there is an inhibition zone around the antagonistic bacteria agar block on the indicator bacteria plate. If there is, it indicates that there is antagonism between the strains. The larger the diameter of the inhibition zone, the greater the antagonism between the strains.

7. The method for preparing a biological additive for reducing emissions and retaining nitrogen in chicken manure-straw composting according to claim 3, characterized in that: The synthetic bacterial community includes: one strain of protein-degrading bacteria and two strains of cellulose-degrading bacteria; The protein-degrading bacteria belong to Burkholderia gladioli; the two cellulose-degrading bacteria belong to Bacillus stercoris and Bacillus velezensis, respectively.

8. Use of the biological additive for reducing emission and retaining nitrogen in chicken manure-straw compost according to claim 1 or 2 in degrading chicken manure-straw compost.

9. The use according to claim 8, characterized in that: The application process is: spraying the biological additive on the straw powder, then fully mixing the straw powder and chicken manure, and turning the pile.

10. The use according to claim 9, characterized in that: The mass ratio of the chicken manure to the straw powder is 5:1; the biological additive accounts for 2% of the total mass of the chicken manure-straw compost.

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