Method for producing organic fertilizer by thermophilic fermentation of citrus fruit rot mixed with compost

Abstract

The present application relates to the technical field of fermentation engineering, and particularly relates to a method for producing organic fertilizer by thermophilic fermentation of mixed compost of rotten citrus fruits and mushroom residue. The method comprises the following steps: (1) crushing and mixing rotten citrus fruits and mushroom residue, adjusting the initial moisture content to 60-65%, and adding lime to obtain mixed materials; (2) adding bacillus velezensis bacterial liquid to the mixed materials obtained in step (1) for composting; (3) when the temperature of the composting pile drops to 35 DEG C, adding coprinus comatus liquid for further composting. The present application uses rotten citrus fruits and mushroom residue as raw materials, and realizes efficient and collaborative resource utilization of the two types of agricultural waste by optimizing the ratio and inoculating exogenous bacterial agents in stages. The method can solve the environmental pollution problem caused by the discarding of rotten fruits in citrus-producing areas from the source, and provides a high-value outlet for the by-products of the mushroom industry.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a method for producing organic fertilizer by thermophilic fermentation of rotten citrus fruit and bacterial residue. Background Technology

[0002] With the continuous expansion of citrus planting areas and the edible fungi industry, the amount of agricultural waste generated is increasing year by year. During harvesting, storage, and transportation, citrus fruits are highly susceptible to rotting due to mechanical damage, pathogen infection, or physiological diseases, resulting in a large amount of rotten fruit each year. If this rotten fruit is not treated promptly, it will not only breed pathogenic fungi such as Penicillium and Green mold, polluting the soil and water sources, but also release unpleasant gases, seriously affecting orchards and the surrounding environment. At the same time, the edible fungi industry generates massive amounts of fungal residue (such as residue from wood-rotting fungi like black fungus and shiitake mushrooms) every year, which is rich in lignocellulose but degrades slowly naturally; indiscriminate dumping also leads to land occupation and environmental pollution. How to efficiently and cleanly utilize these two types of waste has become a critical issue that urgently needs to be addressed for sustainable agricultural development.

[0003] While traditional composting methods can convert organic waste, they have significant shortcomings when dealing with rotten citrus fruit and mushroom residue. Firstly, rotten citrus fruit has high water content and is highly acidic, making it prone to anaerobic fermentation during composting alone, producing foul-smelling gases such as hydrogen sulfide and ammonia, and requiring a maturation period of over 60 days. Mushroom residue, on the other hand, has high lignin and cellulose content and a dense structure, making it difficult for microorganisms to decompose quickly. Traditional composting processes result in slow heating, short high-temperature periods, and incomplete elimination of pathogens. Secondly, conventional composting relies on the natural succession of indigenous microorganisms, leading to low fermentation efficiency, poor compost product stability, and a seed germination index (GI) often below 80%, which can easily cause root burn or disease transmission in crops after application. Furthermore, traditional composting processes often result in insufficient conversion of nutrients (such as total nitrogen, total phosphorus, and total potassium) and inadequate accumulation of nitrate nitrogen, leading to poor fertilizer efficiency and failing to meet the demands of modern agriculture for high-quality bio-organic fertilizers.

[0004] To address these issues, researchers have attempted to improve the results by adding exogenous microbial agents or adjusting composting processes. For example, inoculating common decay-promoting bacteria such as Bacillus subtilis and Bacillus licheniformis at the initial stage of composting can accelerate the decomposition of organic matter to some extent, but its antagonistic effect on pathogenic fungi such as Penicillium spp. carried by rotten citrus fruit is limited, and it is difficult for these fungi to maintain their function throughout the entire composting cycle. Some technologies use a single species for inoculation throughout the entire cycle, but different microorganisms have different requirements for environmental conditions such as temperature and pH, which can easily lead to competition or inactivation of the species. For the degradation of lignocellulose, although some studies have introduced white-rot fungi and brown-rot fungi, these fungi are mostly mesophilic and cannot survive in the high-temperature period of composting (≥50℃). In practical applications, it is often necessary to re-inoculate during the maturation period, but existing technologies lack strategies for precise inoculation during the maturation period, resulting in a still low degradation rate of lignin and cellulose (usually below 30%). In addition, existing composting processes rarely consider generating additional economic value during waste conversion, resulting in low overall economic benefits.

[0005] Therefore, developing a composting process that can synergistically process rotten citrus fruit and edible mushroom residue, achieve rapid heating and efficient composting, effectively inhibit pathogens, significantly improve the degradation rate of lignocellulose, and generate additional benefits during or after composting is of significant practical importance and broad application prospects. This invention is based on this need, proposing a phased inoculation of Bacillus belye and Coprinus comatus liquid, combined with optimized ventilation and proportioning parameters, to achieve efficient resource utilization of rotten citrus fruit and mushroom residue, and also harvest mushroom fruiting bodies, thus achieving both environmental and economic benefits. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing organic fertilizer by thermophilic fermentation of rotten citrus fruit and fungal residue through composting.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for producing organic fertilizer through thermophilic fermentation of rotten citrus fruit and fungal residue, comprising the following steps: (1) Crush and mix the rotten citrus fruit and edible fungus residue, adjust the initial moisture content to 60-65%, add quicklime, and obtain the mixture. (2) Add Bacillus vesiculosus solution to the mixture obtained in step (1) for composting; (3) When the temperature of the compost pile drops to 35°C, add Coprinus comatus liquid to continue composting; the composting cycle is 35 to 45 days.

[0008] Preferably, the mass ratio of the rotten citrus fruit to the edible fungus residue is 6.5~7.5:2.5~3.5.

[0009] Preferably, the amount of quicklime added is 1 to 2% of the mass of the mixture.

[0010] Preferably, in step (2), the concentration of the mixture added is 1-2% by mass, which is 0.8-1.2 × 10⁻⁶. 9 CFU / mL of Bacillus belysiniana bacterial solution (Bacillus belysiniana strain antagonistic to pathogens such as Penicillium spp.) Bacillus from Velez UB201712, abbreviated as BV, accession number CGMCC No.15614, or from CN114921360A).

[0011] Preferably, in step (3), 0.1-0.2% of the biomass of *Coprinus comatus* solution (based on mycelial biomass) of the pile mass is added. Coprinus hirsute JFRL122, abbreviated as CC, is from the seed bank of the Jiangxi Provincial Key Laboratory of Agricultural Microbial Germplasm Discovery and Utilization, or from the literature Huo Guanghua et al., Atlas of Large Fungi in Jiangxi {M}, China, Nanchang, Jiangxi Science and Technology Press, 2020, p. 63.

[0012] Preferably, the edible fungus residue is wood ear fungus residue.

[0013] Preferably, in the composting process, step (2) requires maintaining the compost temperature above 50°C for at least 5 days.

[0014] Preferably, during the composting process, ventilation is carried out at a rate of 0.5~1 L / (min·kg) for 4~6 hours per day.

[0015] This invention provides an organic fertilizer prepared by the method described.

[0016] This invention provides the application of the aforementioned organic fertilizer in promoting strawberry growth.

[0017] Compared with the prior art, the present invention has the following beneficial effects: First, this invention uses whole rotten citrus fruit and edible mushroom residue as raw materials. Through optimized ratios and phased inoculation with exogenous microbial agents, it achieves efficient and synergistic resource utilization of these two types of agricultural waste. This method can solve the environmental pollution problem caused by the disposal of rotten fruit in citrus producing areas at the source, while providing a high-value outlet for by-products of the edible mushroom industry. The prepared bio-organic fertilizer is nutrient-balanced, highly active, and stable, rich in various beneficial microorganisms and trace elements. When applied to the soil, it can significantly improve the soil's physical and chemical properties and enhance crop resistance. Results from strawberry pot experiments show that the treatment group with the added mature compost of this invention significantly outperformed conventional cultivation substrates in terms of growth and yield indicators such as leaf number, leaf length, leaf width, petiole length, number of fruits, and single fruit weight. It also outperformed the treatment group with directly added mushroom residue or uncomposted rotten fruit, fully demonstrating the promoting effect of this organic fertilizer on crop growth.

[0018] Secondly, this invention introduces *Bacillus belye* during the initial stage of composting heating. This bacterium, through competitive exclusion and the secretion of antimicrobial metabolites, effectively inhibits the proliferation of various pathogenic fungi carried by rotten citrus fruit, thus reshaping the microbial community structure of the compost. Compared to the control group without this bacterium, the treated compost heated up more rapidly, entered the high-temperature period earlier, lasted longer, and had a higher peak temperature, thereby more effectively killing pathogens. Precise addition of *Coprinus comatus* solution during the maturation period further significantly improved the maturity of the compost. After treatment with this process, the seed germination index of the compost significantly increased, the pH approached neutral, the electrical conductivity decreased, and nutrient indicators such as organic carbon, total nitrogen, and nitrate nitrogen were optimized. The degradation rates of lignin and cellulose were significantly improved, and recalcitrant organic matter was efficiently converted into stable organic matter.

[0019] Third, this invention not only improves compost quality but also provides additional economic benefits. The *Coprinus comatus* (chicken leg mushroom) used is a safe and edible fungus. Unlike the poisonous *Coprinus indica*, this fungus produces a large number of edible fruiting bodies during or after composting, characterized by tender flesh, high protein, and low fat. Therefore, this technical solution not only produces high-quality bio-organic fertilizer but also harvests chicken leg mushrooms, achieving a dual output of "fertilizer + edible fungus," significantly improving overall economic benefits. The contrast with the treatment group directly applying uncomposted rotten citrus fruit resulted in stunted crop growth or even no fruit set, further validating the crucial role of this invention's composting process in eliminating the toxicity of rotten fruit and enhancing fertilizer efficiency. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1 1. Raw material pretreatment: Crush the rotten citrus fruit (from Xinyu tangerines that have been preserved and rotten after harvest) into a residue, retaining the juice components; and naturally sun-dry the edible fungus residue (from the Jiangxi Provincial Key Laboratory of Agricultural Microbial Germplasm Discovery and Utilization [residue after harvesting edible fungi of the Auricularia auricula type (crisp Auricularia auricula, hairy Auricularia auricula, short-haired Auricularia auricula, wrinkled Auricularia auricula) after harvesting) and crush it into particles of 1cm.

[0022] 2. Composting fermentation system: Mix citrus rotten fruit residue and edible fungus residue at a mass ratio of 7:3, adjust the moisture content to 65%, add 1.5% quicklime to adjust the initial pH to 6.5, mix well and pour into a composting container.

[0023] 3. Specifications of composting device: The main body of the composting reactor is a cuboid with holes at the bottom for air inlet and liquid outlet. A temperature measuring port is provided in the middle of the device, and the top is covered with a perforated plastic plate to ensure air circulation.

[0024] 4. Initial stage of composting: Introduce 1.5% of the compost mass at a concentration of 1×10⁻⁶. 9 cfumL -1 Bacillus vesiculosus culture ( Bacillus velezensis UB201712).

[0025] 5. Ventilation treatment: Forced ventilation of the pile is carried out for 5 hours daily at a ventilation rate of 0.75 L / (min·kg) to ensure oxygen supply to the pile and promote the activity of aerobic microorganisms.

[0026] 6. Inoculation Stage: When the compost enters the maturation stage (and the temperature drops below 35℃), add 0.2% (by mass, based on mycelial biomass) of *Coprinus comatus* solution to the compost. Coprinus hirsute JFRL122), with a total composting cycle of 40 days.

[0027] 7. Monitoring and Control of the Composting Process: During composting, the pile temperature should rapidly rise to above 50℃ and be maintained at a high temperature (≥50℃) for 5 days to kill pathogens and accelerate the decomposition of organic matter. Forced ventilation and turning of the pile should be used to regulate the temperature, preventing excessive moisture from creating an anaerobic environment and excessively high temperatures from reducing microbial activity. During composting, the pH value will initially rise and then fall, eventually stabilizing. If the pH value is too high or too low, it can be adjusted by adding appropriate amounts of acidic or alkaline substances.

[0028] Example 2 1. Raw material pretreatment: Crush rotten citrus fruit into a pulpy state, retaining the juice components; dry the edible fungus residue naturally in the sun and then crush it into particles of 2 cm.

[0029] 2. Composting fermentation system: Mix citrus rotten fruit residue and edible fungus residue in a mass ratio of 6.5:3.5, adjust the moisture content to 60%, add 1.5% quicklime to adjust the initial pH to 7, mix well and pour into a composting container.

[0030] 3. Specifications of composting device: The main body of the composting reactor is a cuboid with holes at the bottom for air inlet and liquid outlet. A temperature measuring port is provided in the middle of the device, and the top is covered with a perforated plastic plate to ensure air circulation.

[0031] 4. Initial stage of composting: Introduce 2% of the compost mass at a concentration of 1×10⁻⁶. 9 cfumL -1 Bacillus vesiculosus culture ( Bacillus velezensis UB201712).

[0032] 5. Ventilation treatment: Forced ventilation of the pile is carried out for 5 hours daily at a ventilation rate of 0.75 L / (min·kg) to ensure oxygen supply to the pile and promote the activity of aerobic microorganisms.

[0033] 6. Inoculation Stage: When the compost enters the maturation stage (and the temperature drops below 35℃), add 0.1% (by mass, based on mycelial biomass) of *Coprinus comatus* solution to the compost. Coprinus hirsute JFRL122), with a total composting cycle of 40 days.

[0034] 7. Monitoring and Control of the Composting Process: During composting, the pile temperature should rapidly rise to above 50℃ and be maintained at a high temperature (≥50℃) for 5 days to kill pathogens and accelerate the decomposition of organic matter. Forced ventilation and turning of the pile should be used to regulate the temperature, preventing excessive moisture from creating an anaerobic environment and excessively high temperatures from reducing microbial activity. During composting, the pH value will initially rise and then fall, eventually stabilizing. If the pH value is too high or too low, it can be adjusted by adding appropriate amounts of acidic or alkaline substances.

[0035] Example 3 1. Raw material pretreatment: Crush rotten citrus fruit into a pulpy state, retaining the juice components; dry the edible fungus residue naturally in the sun and then crush it into particles of 1.5 cm.

[0036] 2. Composting fermentation system: Mix citrus rotten fruit residue and edible fungus residue at a mass ratio of 7.5:2.5, adjust the moisture content to 63%, add 2% quicklime to adjust the initial pH to above 6, mix well and pour into composting container.

[0037] 3. Specifications of composting device: The main body of the composting reactor is a cuboid with holes at the bottom for air inlet and liquid outlet. A temperature measuring port is provided in the middle of the device, and the top is covered with a perforated plastic plate to ensure air circulation.

[0038] 4. Initial stage of composting: Introduce 1% of the compost mass at a concentration of 1×10⁻⁶. 9 cfumL -1 Bacillus vesiculosus culture ( Bacillus velezensis UB201712).

[0039] 5. Ventilation treatment: Forced ventilation of the pile is carried out for 5 hours daily at a ventilation rate of 0.75 L / (min·kg) to ensure oxygen supply to the pile and promote the activity of aerobic microorganisms.

[0040] 6. Inoculation Stage: When the compost enters the maturation stage (and the temperature drops below 35℃), add 0.2% (by mass, based on mycelial biomass) of *Coprinus comatus* solution to the compost. Coprinus hirsute JFRL122), with a total composting cycle of 40 days.

[0041] 7. Monitoring and Control of the Composting Process: During composting, the pile temperature should rapidly rise to above 50℃ and be maintained at a high temperature (≥50℃) for 5 days to kill pathogens and accelerate the decomposition of organic matter. Forced ventilation and turning of the pile should be used to regulate the temperature, preventing excessive moisture from creating an anaerobic environment and excessively high temperatures from reducing microbial activity. During composting, the pH value will initially rise and then fall, eventually stabilizing. If the pH value is too high or too low, it can be adjusted by adding appropriate amounts of acidic or alkaline substances.

[0042] Comparative Example 1 The difference from Example 1 is that Bacillus berleis is not added, but the rest of the steps are the same as in Example 1.

[0043] Comparative Example 2 The difference from Example 1 is that no Coprinus comatus liquid was added, but the rest of the steps are the same as in Example 1.

[0044] Comparative Example 3 The difference from Example 1 is that Bacillus vesiculosus and Coprinus comatus were not added, but the rest of the steps were the same as in Example 1.

[0045] Experimental Example 1 Relevant indicators were measured for the well-rotted compost of Example 1 and Comparative Examples 1-3. The germination index was measured using Jinyan No. 4 cucumber seeds. The results are shown in Tables 1, 2 and 3.

[0046] This experiment used cucumber seeds to determine the seed germination index. When the GI was greater than 80%, it indicated that the compost was fully decomposed and had no toxic effect on plant growth. The pH, EC (electrical conductivity), total organic carbon, total nitrogen, total phosphorus, total potassium, and nitrate nitrogen in the compost were measured at different stages to assess the nutrient composition and degree of decomposition. The degradation rates of lignin and cellulose in the compost were determined using the Panthen washing method and the acetyl bromide method to assess the degree of decomposition of recalcitrant organic matter during the composting process.

[0047] Table 1. Peak Temperature, EC Value, and Seed Germination Indicators

[0048] Table 2 Total Organic Carbon, Total Nitrogen, Total Phosphorus, and Total Potassium

[0049] Table 3 Degradation rates of nitrate nitrogen, lignin, and cellulose

[0050] This invention introduces *Bacillus belye* bacterial solution during the initial stage of composting heating. This bacterium effectively inhibits the proliferation of putrefactive bacteria and pathogenic fungi such as *Penicillium italicum*, *Penicillium fingernail*, and *Colletotrichum discoidus* through competitive exclusion and the secretion of antimicrobial metabolites, thus reshaping the microbial community structure in the composting system. Compared to Comparative Example 3, the treatment group with added *Bacillus belye* reached the high-temperature period 48 hours earlier, the duration of the high-temperature period was extended by 2 days, and the peak temperature increased by 1.1°C; simultaneously, it accelerated the decomposition of organic matter such as lignocellulose and pectin, promoting the mineralization and humification of organic matter.

[0051] This invention precisely adds *Coprinus comatus* liquid during the composting stage (when the compost temperature drops to 35℃), significantly improving compost maturity and product quality. Compared to Comparative Example 2 without *Coprinus comatus*, the seed germination index (GI) increased to 126.83% (Comparative Example 2: 122.25%); after composting, the pH stabilized at 7.0-7.5, and the electrical conductivity decreased to 0.72 mS·cm. -1 (0.84 mS·cm for Comparative Example 2) -1 The total organic carbon content was 27.36% (compared to 26.79% in Comparative Example 2), the total nitrogen content was 2.40% (compared to 2.34% in Comparative Example 2), the nitrate nitrogen content reached 0.802% (compared to 0.796% in Comparative Example 2), the lignin degradation rate reached 24.1% (compared to 21.6% in Comparative Example 2), and the cellulose degradation rate reached 41.2% (compared to 35.5% in Comparative Example 2). The recalcitrant organic matter was efficiently converted into stable organic matter.

[0052] Experimental Example 2 The experiment was conducted in gallon pots with a diameter of 16 cm. Four treatment groups were set up, with five replicates for each treatment. The substrate composition of the different treatments was different: CK was conventional cultivation substrate soil (peat soil: leaf mold: perlite = 3:1:1), and the T1, T2 and T3 treatment groups were respectively composed of conventional cultivation substrate soil with 20% volume fraction of well-rotted compost (prepared by the method in Example 1), 20% volume fraction of crushed edible fungi residue, and 20% volume fraction of rotten citrus fruit residue.

[0053] Select healthy strawberry seedlings with well-developed root systems and similar sizes. During the strawberry planting period, follow standard planting procedures, water regularly, and provide ample sunlight. Approximately 70 days after transplanting, uniformly measure the growth indicators of the strawberry seedlings (Red Cream Strawberry). Table 4 shows the number of leaves, leaf length, leaf width, petiole length, number of fruits, and single fruit weight.

[0054] Table 4 Growth indicators of strawberry seedlings under different substrate ratios

[0055] Analysis of the experimental results in Table 4 shows that different substrate ratios have a significant impact on the growth and yield indicators of strawberry seedlings. Compared with the conventional cultivation substrate (CK), the T1 treatment group, which added 20% of the composted manure of this invention, performed best in all measured indicators, indicating that the organic fertilizer of this invention can effectively promote the vegetative and reproductive growth of strawberries and increase fruit yield. The T2 treatment group, which added 20% of crushed fungal residue, had lower photosynthetic-related indicators (number of leaves, leaf length, leaf width, and petiole length) and yield indicators (number of fruits and single fruit weight) than the T1 treatment group. The number of leaves, leaf length, and number of fruits were slightly better than CK, but the leaf width and petiole length, especially the single fruit weight, were lower than CK, indicating that the effect of directly returning fungal residue to the field is limited. The T3 treatment group, which added 20% of citrus rotten fruit residue, had all indicators lower than CK, and even had no fruits, indicating that directly applying uncomposted citrus rotten fruit to the soil will have a significant toxic or inhibitory effect on strawberries. In summary, the organic fertilizer prepared by this invention effectively transforms harmful substances in rotten citrus fruit and fungal residue through thermophilic fermentation and improves nutrient availability, thus significantly promoting strawberry growth.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing organic fertilizer through thermophilic fermentation of rotten citrus fruit and fungal residue, characterized in that, Includes the following steps: (1) Crush and mix the rotten citrus fruit and edible fungus residue, adjust the initial moisture content to 60-65%, add quicklime, and obtain the mixture. (2) Add Bacillus vesiculosus solution to the mixture obtained in step (1) for composting; (3) When the temperature of the compost pile drops to 35°C, add Coprinus comatus liquid to continue composting; the composting cycle is 35-45 days.

2. The method according to claim 1, characterized in that, The mass ratio of the rotten citrus fruit to the edible mushroom residue is 6.5~7.5:2.5~3.

5.

3. The method according to claim 1, characterized in that, The amount of quicklime added is 1 to 2% of the mass of the mixture.

4. The method according to claim 1, characterized in that, Step (2) Add 1-2% by mass of the mixture to a concentration of 0.8-1.2 × 10⁻⁶. 9 Bacillus berberis bacterial suspension at cfu / mL.

5. The method according to claim 1, characterized in that, Step (3) Add 0.1~0.2% of the biomass of Coprinus comatus solution.

6. The method according to claim 1, characterized in that, The edible fungus residue is wood ear fungus residue.

7. The method according to claim 1, characterized in that, During the composting process, step (2) requires maintaining the temperature of the compost pile above 50°C for at least 5 days.

8. The method according to claim 1, characterized in that, During the composting process, ventilation is carried out at a rate of 0.5~1 L / (min·kg) for 4~6 hours per day.

9. The organic fertilizer prepared by the method according to any one of claims 1 to 8.

10. The application of the organic fertilizer according to claim 9 in promoting strawberry growth.

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