Preparation method of organic garbage carbon source by composite enzyme combined with nitrogen precipitation

Abstract

The application discloses a preparation method of organic garbage carbon source by composite enzyme combined with nitrogen precipitation. Firstly, enzyme pretreatment is performed on organic garbage materials, starch enzyme, cellulase and hemicellulase are used in cooperation to make macromolecular structure of organic matter be broken, so that preliminary release and partial dissolution of carbon source substances are realized; subsequently, sodium tetraphenylborate is added in the pretreatment system to selectively precipitate nitrogen substances in the solution, and centrifugal separation is performed to reduce nitrogen content of the system; on the basis, potassium persulfate and iron substances are added into the obtained suspension liquid to build a Fenton-like reaction system, residual organic macromolecules are further oxidized and degraded, and the structure is optimized, so that availability and stability of the carbon source are improved. In the whole process, each link cooperates, so that the organic garbage can be efficiently converted into high-quality carbon source with high C / N ratio and COD value, and can be used in a sewage treatment system as stable external carbon source.

Description

Technical Field

[0001] This invention belongs to the field of resource utilization technology for perishable waste, specifically relating to a method for preparing organic waste carbon sources through a combination of complex enzymes and nitrogen precipitation. Background Technology

[0002] Excessive nitrogen content in wastewater can easily lead to eutrophication, causing imbalance in aquatic ecosystems and posing potential threats to human health and environmental safety. Currently, the most widely used nitrogen removal technology in urban wastewater treatment systems is biological denitrification, which removes nitrogen from wastewater through nitrification and denitrification. However, in actual operation, the available carbon sources in wastewater are often insufficient to meet the electron donor requirements of the denitrification process, resulting in limited denitrification efficiency. Therefore, it is usually necessary to add external carbon sources to enhance the denitrification reaction. Commonly used external carbon sources in existing projects include methanol, sodium acetate, and glucose. Although these carbon sources can improve the denitrification effect of wastewater treatment systems to some extent, they still have significant limitations: methanol poses safety risks such as flammability and explosiveness; sodium acetate has a low COD equivalent per unit and high addition cost; glucose easily induces the proliferation of miscellaneous bacteria during use, affecting the stability of system operation. Therefore, developing a high-quality, low-cost alternative carbon source with good addition stability is of significant research value and engineering significance for improving the denitrification efficiency and operational safety of wastewater treatment systems.

[0003] Currently, achieving high-value resource utilization of organic waste to achieve harmless and reduced-volume treatment is of great significance for environmental governance and resource recycling. Organic waste is rich in high-value organic matter such as starch, cellulose, hemicellulose, and protein, possessing high carbon source conversion value and development potential. Currently, the main direction for organic waste carbon source conversion is anaerobic fermentation acid production technology. This method, by controlling the hydrolysis and acidification stage of the anaerobic fermentation process and enhancing related treatment effects, can shorten the process and cycle and significantly improve the acid production conversion rate compared to traditional anaerobic digestion processes. To further enhance the effect, chemical pretreatment or wet heat oxidation pretreatment schemes are usually used in conjunction. This method has low operating costs, is simple to operate, and has a relatively high carbon source conversion rate. However, due to the complex mechanism involving multiple synergistic factors in the production process, process control is difficult, and partial nitrogen source release is prone to occur, leading to a low carbon source C / N ratio. In addition to anaerobic fermentation, there are also methods for enzyme-catalyzed carbon source preparation, chemical oxidation carbon source preparation, and acid-catalyzed carbon source preparation. Enzymatic treatment processes offer high conversion rates for starch, cellulose, hemicellulose, and protein in organic waste, significantly reducing reaction time. Under medium-high temperature and high enzyme dosage conditions, carbon source preparation time can be controlled within 10 hours. However, enzyme catalysis alone is insufficient to further improve carbon source conversion rates and cannot separate nitrogen sources, making it difficult to obtain high-quality carbon sources. Chemical oxidation typically uses persulfates as advanced oxidants, achieving rapid carbon source release within approximately 48 hours, resulting in high COD values ​​and good carbon source conversion rates. However, this method releases nitrogen sources along with carbon, leading to increased nitrogen concentration in the final product and higher denitrification costs. Furthermore, the large dosage of persulfates increases production costs, and excessive use can further degrade the carbon source, reducing yield. Acid catalysis is simple to operate and low-cost. By adjusting pH with Lewis acids (such as ferric chloride), it can achieve the removal of substances like proteins through electrostatic precipitation, thus steadily releasing the carbon source. However, for recalcitrant substances (such as cellulose), the conversion rate is low under non-high-pressure heating conditions, resulting in limited final carbon source yield. The main problems with the above-mentioned carbon source preparation methods for organic waste are low carbon source conversion rates and separation of nitrogen-based substances. To address these issues, based on carbon source preparation processes and our own experimental research, we have developed an advanced oxidation process consisting of enzyme-catalyzed pretreatment, sodium tetraphenylborate nitrogen precipitation, and a Fenton-like system. This process achieves efficient conversion of carbon-based substances, thorough precipitation and removal of nitrogen-based substances, and high-efficiency, high-quality carbon source conversion from organic waste. Summary of the Invention

[0004] This invention addresses the problems of low C / N ratio in the carbon source preparation process of organic waste, insufficient carbon source conversion rate, and difficulty in controlling nitrogen source release in existing processes. From the perspective of improving carbon source conversion rate and reducing nitrogen source release, it provides a method for high-quality carbon source conversion of organic waste using a composite enzyme combined with nitrogen precipitation. This method can significantly improve the conversion efficiency of carbon source substances such as starch, cellulose, and hemicellulose in organic waste, increase the COD value of the product, and simultaneously inhibit and reduce total nitrogen (TN) release, significantly improving the C / N ratio of the carbon source. Through process optimization, this method shortens the carbon source preparation time to approximately 5-8 hours, achieving efficient and stable carbon source conversion of organic waste.

[0005] The specific technical solution adopted in this invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation, comprising:

[0007] S1. A composite enzyme composed of amylase, cellulase and hemicellulase is added to organic waste slurry rich in starch, cellulose and hemicellulose as a carbon source catalyst. The enzyme catalysis promotes the degradation and release of macromolecular carbon source substances including starch, cellulose and hemicellulose, and the enzyme-treated solution is obtained.

[0008] S2. Sodium tetraphenylborate is added to the enzyme-treated solution as a nitrogen removal agent and stirred thoroughly to carry out a nitrogen precipitation reaction. Nitrogen substances, including proteins, ammonia nitrogen and free amino acids, are selectively precipitated. The upper suspension and the lower nitrogen-enriched precipitate are obtained through the first solid-liquid separation operation.

[0009] S3. Add potassium persulfate and iron to the suspension to construct a Fenton-like system. Further degrade and release the suspended substances in the suspension through a Fenton-like advanced oxidation reaction. After removing the iron precipitate through a second solid-liquid separation operation, a high-quality organic waste carbon source product is obtained.

[0010] As a preferred embodiment of the first aspect above, the organic waste slurry is obtained by crushing and homogenizing organic waste with water, and its total solids content is controlled at 5.00% to 7.00%, cellulose content at 25% to 35%, hemicellulose content at 15% to 25%, and pH at 4.0 to 5.5.

[0011] As a preferred embodiment of the first aspect above, the amounts of amylase, cellulase and hemicellulase in the compound enzyme need to be adjusted according to the total solid content a% of the organic waste slurry. The amount of amylase, cellulase and hemicellulase added is (1 / 20000 to 1 / 10000) × a / 6 of the volume of the organic waste slurry, and the activities of the added amylase, cellulase and hemicellulase are not less than 4000 U / mL, 5000 U / mL and 3000 U / mL, respectively.

[0012] Furthermore, in the composite enzyme, the amylase is glucosylamylase, brand name Xiasheng FDY-2223; the cellulase is acidic cellulase, brand name Xiasheng GDG-2012; and the hemicellulase is a mixture of mannanase and xylanase in equal mass ratios, with mannanase brand name Dibai K201675 and xylanase brand name Xiasheng FDG-2221.

[0013] As a preferred embodiment of the first aspect above, the reaction time of the enzyme-catalyzed reaction is 3 to 6 hours, the reaction temperature is 40 to 80°C, and the stirring frequency is 150 to 200 r / min.

[0014] As a preferred embodiment of the first aspect above, the amount of sodium tetraphenylborate added is based on the total nitrogen content in the solution after enzyme treatment, with 1955.54 to 2933.31 mg of sodium tetraphenylborate added per 100 mg of total nitrogen.

[0015] As a preferred embodiment of the first aspect above, the stirring speed of the nitrogen precipitation reaction is 400~800 r / min, and the reaction time is 1~2 h.

[0016] As a preferred embodiment of the first aspect, the first solid-liquid separation operation and / or the second solid-liquid separation operation employ low-speed centrifugation, with a centrifugation speed of 1000~2000 r / min and a duration of 10~20 min.

[0017] As a preferred embodiment of the first aspect above, when constructing the Fenton-like system, the mass of potassium persulfate added to the suspension is 0.83 to 1.00 times the mass of the total residual solids in the suspension, and the amount of iron added to the suspension is 0.01 to 0.02 g / mL of suspension.

[0018] Furthermore, the iron-based substance is composed of iron powder and ferric chloride in equal mass ratios.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) For carbon sources such as starch, cellulose, and hemicellulose in organic waste, this invention employs a coupled process of highly efficient enzyme catalysis and chemical oxidation, which can fully release carbon sources into the liquid phase. The preparation process produces almost no suspended solids, the solution has high clarity, the carbon source conversion rate is significantly improved, and the final product has a high COD value.

[0021] (2) For nitrogen-based substances, this invention uses a combination of chemical reagent precipitation and centrifugal separation to effectively remove inorganic nitrogen and some organic nitrogen from the system, obtaining a high proportion of nitrogen-based solid slag. While improving the potential and value of nitrogen utilization, it significantly increases the C / N ratio of carbon sources.

[0022] (3) The reaction time of each process step of the present invention is significantly shortened compared with the traditional organic waste fermentation carbon source production method, reducing the original 4 to 10 days fermentation cycle to about 5 to 8 hours, realizing rapid carbon source preparation, and greatly improving the carbon source conversion rate and final carbon source quality. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the steps of a method for preparing organic waste carbon source using a complex enzyme combined with nitrogen precipitation;

[0024] Figure 2 This is a typical process diagram of the method of the present invention applied to food waste. Detailed Implementation

[0025] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, a detailed description of specific embodiments is provided below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in the various embodiments of the present invention can be combined accordingly without mutual conflict.

[0026] This invention, based on the perspective of organic waste resource utilization, addresses the problems of unstable fermentation effects, long fermentation cycles, and low carbon source conversion rates and C / N ratios in carbon source preparation, proposing a highly efficient carbon source preparation method. This method uses organic waste with high cellulose and hemicellulose content as raw materials, primarily through a coupling of enzymatic catalysis and chemical oxidation to efficiently convert starch, cellulose, and hemicellulose into soluble carbon source substances, thereby improving the biodegradability of the carbon source. Simultaneously, proteins, free amino acids, and ammonia nitrogen are removed by chemical precipitation, significantly improving the C / N ratio and overall carbon source quality. In the specific process, a selected composite enzyme is first used to preliminarily degrade the large-molecule starch, cellulose, and hemicellulose in the organic waste system, focusing on solving the problem of low degradation rates of cellulose and hemicellulose, causing the three types of carbon source substances to form small-molecule suspensions and soluble reducing sugars, while reducing their residue in the precipitate. Subsequently, sodium tetraphenylborate is added to the system, causing ammonia nitrogen and organic nitrogen substances in the solution to precipitate and accumulate. After medium-low speed centrifugation, an upper turbid liquid and a lower white nitrogen precipitate are obtained. Finally, iron and persulfate were added to the upper turbid liquid to construct a Fenton-like system, which deeply degraded suspended solids and some macromolecular organic matter into soluble carbon source substances, thereby significantly improving carbon source conversion rate and production efficiency.

[0027] The method for preparing organic waste carbon source by combining a compound enzyme with nitrogen precipitation provided by this invention has the following basic process:

[0028] S1. A composite enzyme composed of amylase, cellulase and hemicellulase is added to organic waste slurry rich in starch, cellulose and hemicellulose as a carbon source catalyst. The enzyme catalysis promotes the degradation and release of macromolecular carbon source substances including starch, cellulose and hemicellulose, and the enzyme-treated solution is obtained.

[0029] S2. Sodium tetraphenylborate is added to the enzyme-treated solution as a nitrogen removal agent and stirred thoroughly to carry out a nitrogen precipitation reaction. Nitrogen substances, including proteins, ammonia nitrogen and free amino acids, are selectively precipitated. The upper suspension and the lower nitrogen-enriched precipitate are obtained through the first solid-liquid separation operation.

[0030] S3. Add potassium persulfate and iron to the suspension to construct a Fenton-like system. Further degrade and release the suspended substances in the suspension through a Fenton-like advanced oxidation reaction. After removing the iron precipitate through a second solid-liquid separation operation, a high-quality organic waste carbon source product is obtained.

[0031] In the embodiments of the present invention, the specific parameters in the above-mentioned method for preparing organic waste carbon sources can be optimized and adjusted according to actual conditions.

[0032] As a preferred embodiment of the present invention, the organic waste slurry is obtained by adding water to organic waste and crushing and homogenizing it. Its total solids content is controlled at 5.00% to 7.00%, cellulose content at 25% to 35%, hemicellulose content at 15% to 25%, and pH at 4.0 to 5.5.

[0033] As a preferred embodiment of the present invention, the amount of amylase, cellulase and hemicellulase in the composite enzyme needs to be adjusted according to the total solid content a% of the organic waste slurry. The amount of amylase, cellulase and hemicellulase added is (1 / 20000 to 1 / 10000) × a / 6 of the volume of the organic waste slurry, and the activities of the added amylase, cellulase and hemicellulase are not less than 4000U / mL, 5000U / mL and 3000U / mL, respectively.

[0034] Amylase, cellulase, and hemicellulase are all available in a large number of commercially available enzyme reagent products. These products can be screened through testing, as long as they possess good starch, cellulose, and hemicellulose degradation capabilities. In the embodiments of this invention, after experimental optimization, glucosylamylase (brand name: Xiasheng FDY-2223) is recommended as the amylase; acidic cellulase (brand name: Xiasheng GDG-2012) is recommended as the cellulase; and a mixture of mannanase and xylanase in equal mass ratios is recommended as the hemicellulase, with mannanase (brand name: Dibai K201675) and xylanase (brand name: Xiasheng FDG-2221).

[0035] As a preferred embodiment of the present invention, the reaction time of the enzyme-catalyzed reaction is 3-6 hours, the reaction temperature is 40-80°C, and the stirring frequency is 150-200 r / min.

[0036] As a preferred embodiment of the present invention, the amount of sodium tetraphenylborate added is based on the total nitrogen content in the solution after enzyme treatment, with 1955.54 to 2933.31 mg of sodium tetraphenylborate added per 100 mg of total nitrogen.

[0037] As a preferred embodiment of the present invention, the stirring speed of the nitrogen precipitation reaction is 400~800 r / min, and the reaction time is 1~2 h.

[0038] As a preferred embodiment of the present invention, the first solid-liquid separation operation and / or the second solid-liquid separation operation adopts a low-speed centrifugation method, with a centrifugation speed of 1000~2000 r / min and a duration of 10~20 min.

[0039] It should be noted that the iron-containing substances added to the suspension when constructing the Fenton-like system are generally composed of iron powder and ferric chloride. The iron powder and ferric chloride can be added together in S3, or the ferric chloride can be added together with sodium tetraphenylborate in S2 to enhance the nitrogen precipitation effect, and then the iron powder can be added separately in S3. This can also form a Fenton-like system in the solution.

[0040] In a preferred embodiment of the present invention, when constructing the Fenton-like system, the mass of potassium persulfate added to the suspension is 0.83 to 1.00 times the mass of the total residual solids in the suspension, and the amount of iron added to the suspension is 0.01 to 0.02 g / mL of suspension. Preferably, the iron is composed of iron powder and ferric chloride in equal mass ratios.

[0041] It should be noted that the organic waste in this invention is preferably kitchen waste, which is rich in organic matter such as cellulose, hemicellulose, starch, and protein. The method for preparing organic waste carbon sources by combining the complex enzyme with nitrogen precipitation as shown in S1-S3 of this invention, when applied to this type of kitchen waste, can form... Figure 2 The preparation process shown allows for the separation of nitrogen components and sodium tetraphenylborate from the nitrogen-enriched precipitate by adding a strong alkali and heating and condensing. The sodium tetraphenylborate can then be reused. Iron precipitates can also be recycled as Fenton-like catalysts.

[0042] The principles and technical effects of the above technical solutions are illustrated below through several embodiments.

[0043] Example 1

[0044] The composite enzyme used in this invention consists of amylase, cellulase, and hemicellulase. This embodiment conducted a composite enzyme screening experiment, primarily investigating the degradation effects of cellulase and hemicellulase on corresponding substances in organic waste. By screening different enzyme types, reaction temperatures, and pH conditions, the enzyme type and reaction conditions that produce the highest COD value were selected as the components of the subsequent composite enzyme system, thereby achieving a high degradation rate of cellulose and hemicellulose. The specific experimental process is as follows.

[0045] First, organic waste samples with high cellulose and high hemicellulose were prepared. The raw materials selected in the process were as follows: 187.5 g of fresh pork belly, 750.0 g of fresh chicken breast, 6000.0 g of fresh Chinese cabbage, 1500.0 g of edible noodles, 300.0 g of soybean oil, and 75.0 g of refined salt. The preparation process of the organic waste samples is as follows: First, fresh pork, chicken breast, and Chinese cabbage were chopped with a knife and thoroughly mixed, then temporarily stored for later use. Next, 50.0 g of soybean oil was added to a cooking pot and heated until hot. Approximately 1 / 6 of the mixture was placed in the pot and stir-fried until the meat changed color and the cabbage softened. Afterward, approximately 300 mL of deionized water and 12.5 g of refined salt were added to the pot and briefly cooked. After cooking, the meat and vegetable mixture was transferred to a 5 L beaker and stored until all the mixture was cooked. For the noodle processing, edible noodles were added to boiling deionized water and briefly cooked until completely softened. Then, the noodles were transferred to the 5 L beaker containing the meat and vegetable mixture and thoroughly mixed to obtain the organic waste sample. These organic waste samples, after being prepared uniformly, were used for subsequent enzyme screening experiments.

[0046] After cooking, take small amounts of organic waste sample from a 5 L beaker and add them repeatedly to a 1 L blender, simultaneously adding deionized water at a ratio of 1:1 to 2:1. Blend at a speed of 10,000–30,000 rpm for 1–2 minutes. During blending, ensure no large solid particles appear in the slurry. After blending, transfer the slurry to a mixing tank. The slurry height is measured to be 47.81 cm, the inner diameter of the tank is 64.25 cm, and the total volume is approximately 15.50 L. Then, dispense the slurry into plastic bottles, stirring for approximately 10 seconds each time to ensure even mixing and form an organic waste slurry sample. After dispensing, store the organic waste slurry sample in a -20°C freezer. After removing the organic waste slurry sample, thaw it in a 40°C water bath for 3–5 hours until completely thawed. After thawing, the three groups of organic waste slurry samples were weighed and found to be 628.48 g, 604.28 g, and 663.07 g, respectively, and diluted approximately three times with deionized water. Subsequently, the three groups of organic waste slurry samples were transferred to beakers for recording their masses, with masses of 1885.46 g, 1818.88 g, and 1989.20 g, respectively, and dried in a 65℃ oven until constant weight. The dried organic waste slurry samples weighed 115.17 g, 111.44 g, and 126.40 g, respectively, and the average TS content of the organic waste slurry samples was determined to be 6.20%. The dried samples were then ground into powder in a mortar and stored at low temperature. A portion of the powder was sent to the Vibrant testing platform for analysis of six main components. The results showed that the starch content was 24.05%, the cellulose content was 29.96%, the hemicellulose content was 18.82%, the crude protein content was 22.25%, the crude fat content was 13.25%, and the lignin content was 7.92%.

[0047] In the enzyme screening experiment, two bottles of organic waste slurry samples were first thawed by heating them in a 40℃ water bath for 3–5 hours. After thawing, the organic waste slurry samples were transferred to a large beaker, the mass was recorded, and the samples were diluted with deionized water to approximately three times their original volume, with the diluted mass also recorded. The experiment was conducted in groups of different temperatures, and different pH conditions were adjusted by slowly adding 1 mol / L hydrochloric acid to the organic waste slurry samples. For example, when the pH decreased from 5.3 to 4.3, due to the presence of buffer substances in the system, the amount of hydrochloric acid needed to be increased appropriately; approximately 5–8 mL of hydrochloric acid was added to 2 L of diluted solution to complete the adjustment. After preparing the organic waste slurry samples at different pH values, three parallel samples were further set up for each enzyme. The enzymes used included: 9 commercial cellulases, 3 commercial hemicellulases, 3 mannanases, 5 xylanases, and 15 mannanases and xylanases in a 1:1 mixture. A total of 105 enzyme-treated samples and 3 blank control samples were included in the single pH group, with two pH conditions in the experimental design, totaling 216 samples. For each sample, 10 mL of diluted slurry was placed in a 15 mL centrifuge tube, and the mass was recorded. The enzyme addition was controlled at 1 / 10000. The tubes were then placed in a water bath at the corresponding temperatures (5 temperature gradients: 40℃, 50℃, 60℃, 70℃, and 80℃) and reacted at 180 r / min for 3 hours. The mass and dilution factor of the slurry before and after dilution at each temperature condition are as follows:

[0048] (1) 40℃: The mass before dilution was 617.48 g, and the mass after dilution was 1865.86 g, with a dilution factor of 3.02;

[0049] (2) 50℃: Mass before dilution 526.71 g, mass after dilution 1575.36 g, dilution factor 2.99;

[0050] (3) 60℃: Mass before dilution 560.83 g, mass after dilution 1683.42 g, dilution factor 3.00;

[0051] (4) 70℃: Mass before dilution 550.51 g, mass after dilution 1656.86 g, dilution ratio 3.01;

[0052] (5) 80℃: The mass before dilution was 427.55 g, and the mass after dilution was 1285.27 g, with a dilution ratio of 3.01.

[0053] After the reaction was complete, the centrifuge tubes were centrifuged at 4000 r / min for 10 minutes. The supernatant was then transferred to a new 15 mL centrifuge tube and filtered using a 0.45 μm microporous membrane. The samples were subsequently diluted, and total nitrogen (TN) was determined using the national standard method. Chemical oxygen demand (COD) was determined by diluting the samples 80-fold using the Hach reagent method.

[0054] Based on the experimental results above, the optimal enzyme combination of cellulase and hemicellulase, selected under the reaction conditions of pH = 4.3 and temperature T = 70℃, was as follows: the cellulase used was acidic cellulase, brand name Xiasheng GDG-2012; the hemicellulase consisted of an equal mass ratio of mannanase and xylanase, brand name Dibai K201675 and brand name Xiasheng FDG-2221. Under these reaction conditions and with the optimal enzyme combination, the COD conjugate value was 43040 mg / L, and the COD increase reached its maximum of 24760 mg / L.

[0055] Based on the above optimal reaction conditions and the optimal enzyme combination of cellulase and hemicellulase, the selection of high-temperature amylase was further optimized on the basis of cellulase and hemicellulase. Finally, it was found that when glucosylamylase (brand name Xiasheng FDY-2223) is combined with the above optimal enzyme combination of cellulase and hemicellulase, it has a good degradation effect on starchy substances in organic waste, and can achieve a COD increase of nearly 30,000 mg / L within 1 hour.

[0056] Therefore, the optimal enzyme combination obtained by screening the composite enzyme of amylase + cellulase + hemicellulase under the reaction conditions of pH = 4.3 and temperature T = 70℃ is as follows: the amylase used is glucosylamylase, brand name Xiasheng FDY-2223; the cellulase used is acidic cellulase, brand name Xiasheng GDG-2012; and the hemicellulase used is a mixture of mannanase and xylanase in equal mass ratio, with mannanase brand name Dibai K201675 and xylanase brand name Xiasheng FDG-2221.

[0057] All enzyme reagents in the above optimal enzyme combination are commercially available. Enzymes with the brand name Xiasheng can be purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and enzymes with the brand name Dibai can be purchased from Shanghai Dibai Biotechnology Co., Ltd.

[0058] Subsequent embodiments will conduct orthogonal experiments based on this optimal enzyme combination and reaction conditions to further explore the synergistic relationship between enzymes and optimize the enzyme dosage.

[0059] Example 2

[0060] This embodiment serves as a verification and condition optimization experiment for the effect of the composite enzyme, aiming to verify the synergistic degradation and release effect of three highly efficient enzymes on carbon sources (starch, cellulose, and hemicellulose) in organic waste. Based on the enhancement effect of each single enzyme in the screening experiment conducted in Example 1, this embodiment sets the target benefit of the composite enzyme system at a COD increase of 30,000 mg / L to achieve the initial release of carbon sources in organic waste, providing experimental basis for subsequent optimization of process parameters and composite enzyme dosage.

[0061] First, one bottle of the organic waste slurry sample prepared in Example 1 was placed in a 40°C water bath and heated for 3–5 hours to thaw. After thawing, the slurry was transferred to a large beaker, and its mass was recorded as 307.27 g. It was then diluted with deionized water to approximately three times its original volume, and the diluted mass was recorded as 926.97 g, corresponding to a total solids (TS) content of 6.13%. After stirring and mixing, the pH of the slurry was measured to be 5.49. The pH of the slurry system was adjusted to 4.34 by slowly adding 1 mol / L hydrochloric acid while stirring. Subsequently, the corresponding enzymes and complex enzyme systems were added according to the amounts shown in Table 1. This embodiment sets dosage gradients for amylase (Xiasheng FDY-2223), cellulase (Xiasheng GDG-2012), and hemicellulase (a mixture of mannanase Dibai K201675 and xylanase Xiasheng FDG-2221 in equal mass ratios). Specifically: three gradients are set for cellulase dosage: 0, 1 / 20000, and 1 / 10000; three gradients are set for hemicellulase dosage: 0, 1 / 20000, and 1 / 10000; and three gradients are set for amylase dosage: 0, 1 / 20000, and 1 / 10000. The enzyme dosages mentioned above are the ratios of the added enzyme solution volume to the organic waste slurry volume. The activities of the amylase solution, cellulase solution, and hemicellulase solution are prepared to be 4000 U / mL, 5000 U / mL, and 3000 U / mL, respectively. The volume of each slurry sample is uniformly controlled to 50 mL, and a total of 16 samples are prepared. After enzyme addition, the sample was placed in a 70℃ constant temperature water bath and heated at 180 r / min for 3 hours for reaction. After the reaction, the centrifuge tube was removed and centrifuged at 4000 r / min for 10 minutes. The supernatant was then filtered through a 0.45 μm microporous membrane. The filtered supernatant was used to determine total nitrogen (TN, national standard method) and chemical oxygen demand (COD, Hach reagent method). The specific experimental results are shown in Table 1.

[0062] Based on the results of the above-mentioned compound enzyme verification experiment, an orthogonal experimental analysis was conducted to analyze the relationship between enzyme addition and COD release. The results showed that when the enzyme addition increased from 0 to 1 / 10000, the addition of cellulase, hemicellulase, and amylase were all significantly correlated with COD release (sig. ≤ 0.05). When the enzyme addition increased from 1 / 20000 to 1 / 10000, the cellulase addition was still significantly correlated with COD release (sig. = 0.049), while amylase (sig. = 0.862) and hemicellulase (sig. = 0.132) did not show significant correlations. Parameter estimation analysis showed that cellulase and hemicellulase showed a significant increasing trend in COD release when the enzyme addition increased from 0 to 1 / 10000; while amylase did not show a significant increase in the range of 1 / 20000 to 1 / 10000, exhibiting an excess effect. In summary, the combined addition of the three types of enzymes can significantly increase the COD release from carbon sources in organic waste. Within the range of enzyme addition from 1 / 20,000 to 1 / 10,000, cellulase and hemicellulase showed the best COD-enhancing effects, with cellulase being the most significant, while the enhancement effect of amylase became less pronounced at higher addition levels.

[0063] Based on this, in subsequent applications of compound enzymes, when the activities of amylase solution, cellulase solution, and hemicellulase solution are prepared to be 4000 U / mL, 5000 U / mL, and 3000 U / mL, respectively, the recommended enzyme solution addition amounts are calculated based on the ratio of the added enzyme solution volume to the organic waste slurry volume: 1 / 10000 for cellulase, 1 / 10000 for hemicellulase, and 1 / 20000 for amylase, in order to achieve the best COD release effect.

[0064] Table 1. Orthogonal Experiment Data

[0065]

[0066] Example 3

[0067] This embodiment serves as an experiment to optimize the dosage and application effect of the compound enzyme, aiming to evaluate the application effect of the optimized compound enzyme system in the preparation of carbon sources from organic waste. First, enzyme screening experiments were conducted to determine the optimal reaction effects of each enzyme under different reaction temperatures and pH conditions. Subsequently, orthogonal experimental analysis was used to clarify the significance and interactions of each individual enzyme in the compound enzyme system, and the enzyme dosage was further optimized. This embodiment focuses on evaluating the COD and TN release from organic waste under compound enzyme treatment, as well as the degradation of starch, cellulose, and hemicellulose—three major carbon source molecules. The specific experimental steps of this embodiment are as follows:

[0068] First, one bottle of the organic waste slurry sample prepared in Example 1 was placed in a 40°C water bath and heated for 3–5 hours to thaw. After thawing, the slurry sample was transferred to a large beaker, and the mass of the slurry sample was recorded as 316.69 g. It was then diluted with deionized water to approximately three times its volume, and the mass after dilution was recorded as 953.81 g, corresponding to a total solids (TS) content of 6.26%. After stirring and mixing, the pH value of the slurry sample was measured to be 5.35. The pH of the system was adjusted to 4.33 by slowly adding 1 mol / L hydrochloric acid while stirring. Subsequently, the slurry samples were divided into two categories: 200 mL solutions of three groups of compound enzymes (referred to as compound enzyme 1, compound enzyme 2, and compound enzyme 3) were used to add the compound enzyme system, that is, 1 / 20000 ratio of amylase, 1 / 10000 ratio of cellulase, and 1 / 10000 ratio of hemicellulase were added simultaneously, and each group was set up in parallel experiments; the other three groups of 50 mL solutions were used for control and single enzyme verification, namely: (1) blank control, (2) amylase group: only 1 / 20000 ratio of amylase was added, (3) cellulase + hemicellulase group: only 1 / 10000 ratio of cellulase and 1 / 10000 ratio of hemicellulase were added. The above enzyme dosage ratio is the ratio of the volume of added enzyme solution to the volume of organic waste slurry, wherein the activities of amylase solution, cellulase solution and hemicellulase solution were prepared to be 4000 U / mL, 5000 U / mL and 3000 U / mL, respectively. The three enzymes were selected according to the optimal enzyme combination obtained in Example 1: Xiasheng FDY-2223 for amylase, Xiasheng GDG-2012 for cellulase, and a mixture of mannanase Dibai K201675 and xylanase Xiasheng FDG-2221 in equal mass ratios for hemicellulase. After enzyme addition, the slurry sample was placed in a 70℃ water bath and reacted at 180 r / min for 3 hours. After the reaction, the slurry sample was centrifuged at 4000 r / min for 10 minutes. After centrifugation, the supernatant was filtered through a 0.45 μm microporous membrane, and the filtrate was used to determine COD and TN. The precipitate was dried in a 65℃ oven to constant weight, and the dried mass was recorded. The dried precipitate was ground into powder in a mortar and pestle, stored at low temperature, and sent for starch, cellulose, and hemicellulose content analysis. The final experimental results of the three groups of compound enzymes, blank control, and single enzyme verification are shown in Tables 2 and 3.

[0069] Based on the experimental results of the above-mentioned compound enzyme application, the compound enzyme treatment significantly increased the COD value, with an average increase of 33920 mg / L, initially achieving the design target. Simultaneously, compared with the control group, the total nitrogen (TN) content did not increase significantly after treatment, indicating that the compound enzyme treatment enhanced carbon source release without leading to a large release of nitrogen. The average C / N ratio of the system after compound enzyme treatment was 89.19, showing a significant improvement compared to treatments using cellulase + hemicellulase alone or amylase alone. Analysis of the solid dry weight reduction ratio showed that the solid weight reduction rate of the compound enzyme treatment was significantly higher than that of the single enzyme treatment or the cellulase + hemicellulase combination, and a large amount of suspended matter remained in the supernatant after centrifugation, indicating that the carbon source in the system still had high availability and optimization potential after enzyme pretreatment. Further analysis from the perspective of material composition showed that the degradation rates of starch, cellulose, and hemicellulose by the compound enzyme were generally higher than those of treatments using cellulase + hemicellulase alone or amylase alone, demonstrating a synergistic enhancing effect on the three types of carbon sources and significantly improving the carbon source conversion rate. The degradation rates of starchy substances reached 91.25%, hemicellulose substances reached 95.71%, and cellulose substances reached 74.46%. These results indicate that the composite enzyme can efficiently promote the release and transformation of major carbon sources in organic waste, providing a reliable technical foundation for the subsequent preparation of high-quality carbon sources.

[0070] Table 2 Results of the compound enzyme application test (COD and TN determination)

[0071]

[0072] Table 3. Changes in the content and degradation rate of the three types of carbon source substances during the preparation process.

[0073]

[0074] Example 4

[0075] This embodiment presents a complete carbon source preparation process experiment, encompassing compound enzyme pretreatment, sodium tetraphenylborate nitrogen precipitation, and the deep degradation of suspended solids and macromolecular organic matter by a Fenton-like system constructed from iron and persulfate. The feasibility and overall benefits of the entire carbon source preparation process are verified through the combined application of these process steps. The specific experimental steps of this embodiment are as follows.

[0076] First, take one bottle of the organic waste slurry sample prepared in Example 1 and place it at 40°C. Thawing was performed by heating in a water bath for 3–5 hours. The dissolved slurry was then placed in a large beaker, and its mass was recorded as 298.03 g. It was then diluted with deionized water to approximately three times its original volume, and its mass was recorded as 912.97 g, with a corresponding total slurry (TS) value of 6.13%. After stirring and mixing, the pH was measured to be 5.40. 1 mol / L hydrochloric acid was added dropwise with stirring until the system pH reached 4.34. Six conical flask experiments were then conducted, with approximately 100 mL of diluted slurry added to each group. Three groups served as the compound enzyme addition group, and three groups served as the blank control group. The compound enzyme group received 1 / 20000 ratio of amylase, 1 / 10000 ratio of cellulase, and 1 / 10000 ratio of hemicellulase. The blank control group received no other added substances, and all other treatments were the same. The enzyme dosage ratios mentioned above refer to the ratio of the added enzyme solution volume to the organic waste slurry volume. The amylase, cellulase, and hemicellulase solutions were prepared with activities of 4000 U / mL, 5000 U / mL, and 3000 U / mL, respectively. The specific selection of the three enzymes followed the optimal enzyme combination optimized in Example 1: amylase was Xiasheng FDY-2223, cellulase was Xiasheng GDG-2012, and hemicellulase was a mixture of mannanase Dibai K201675 and xylanase Xiasheng FDG-2221 in equal mass ratios. After enzyme addition, the slurry samples with added enzymes were placed at 70°C. The enzyme-catalyzed reaction was carried out at a constant temperature of 180 rpm for 3 hours in a water bath. After the enzyme-catalyzed reaction was completed, 5 mL of the slurry from each group was thoroughly mixed and stored for subsequent determination. Then, 20 mL of 61.10 g / L sodium tetraphenylborate solution was added to each group's sample after the enzyme-catalyzed reaction. After thorough mixing, a magnetic stir bar was added, and the sample was placed on a magnetic stirrer and stirred at 600 rpm for 1 hour to ensure complete reaction. The reacted solution was then centrifuged at 1000 rpm for 20 minutes to remove nitrogen precipitates. A portion of the separated solution was transferred to an Erlenmeyer flask, thoroughly mixed, and 5 mL of the solution was stored for subsequent determination. After centrifugation, a significant amount of suspended matter remained in the solution. Therefore, 0.834 g of potassium persulfate and an iron mixture (composed of 0.500 g of iron powder and 1.000 g of ferric chloride) were added to each group. After addition, the sample was placed on a magnetic stirrer and stirred at 600 rpm for 1 hour to carry out a nitrogen precipitation reaction, ensuring complete precipitation of nitrogenous substances in the solution. After the nitrogen precipitation reaction is complete, the reaction solution is centrifuged at a medium-low speed, specifically at 1000 r / min for 20 min, to fully precipitate and remove the iron ion precipitate, yielding a clear carbon source solution. All samples were taken using a 0.45... Microporous membrane filtration was performed, and the COD and TN of the solution were measured after treatment. The results are shown in Table 4. According to the final results of this experiment, enzyme treatment can increase COD by 24400 mg / L. After nitrogen precipitation, the COD value of the prepared carbon source reaches 75808 mg / L, which is 54421 mg / L higher than the blank group, achieving a high carbon source conversion rate. In addition, no other nitrogen source is released except for the dissolved nitrogen source in the process. Nitrogen precipitation effectively removes nitrogen ions in the solution and enhances the nitrogen precipitation enrichment effect, achieving extremely low TN in the final solution, averaging 75.09 mg / L, and the C / N ratio can reach as high as 992.92, which has excellent carbon source utilization value.

[0077] Table 4. COD and TN measurements at each stage of the carbon source preparation process.

[0078]

[0079] Example 5

[0080] This embodiment serves as an experiment to explore the effects of different organic waste treatment conditions on carbon source preparation. It aims to evaluate the influence of key parameters during the entire carbon source preparation process on the product COD, TN, and the degradation of carbon source macromolecules, including the type of food waste and the range of total solids (TS) content in the slurry. The specific experimental steps are as follows.

[0081] First, two samples of homemade kitchen waste slurry (i.e., the organic waste slurry samples prepared in Example 1) were taken, and the TS value of the samples was measured to be 15.49%. Subsequently, the two sets of homemade kitchen waste slurry samples were diluted by 3.098 times and 2.210 times, respectively, and the TS was controlled at approximately 5.00% and 7.00%, respectively. The pH values ​​after dilution were 5.42 and 5.37, respectively. Then, the pH values ​​of both sets of samples were adjusted to 4.29 by adding dilute hydrochloric acid. Three parallel samples were set for each TS value.

[0082] Secondly, two types of food waste slurry samples were taken from a certain factory before and after the three-phase separation process of food waste to biogas production. Following the procedure in Example 1, they were frozen in a -20℃ freezer and then thawed in a 40℃ water bath for 3-5 hours. After thawing, deionized water was added for dilution, and the samples were dried before measuring the total sulfide (TS) content. The final TS values ​​were 17.68% and 12.35%, respectively. The samples with the two TS values ​​were then diluted to approximately 5.00% and 7.00%, respectively. Specifically, the former was diluted 3.536 times and 2.520 times, while the latter was diluted 2.470 times and 1.760 times. The pH values ​​of the diluted former were 3.66 and 3.67, respectively, while the pH values ​​of the diluted latter were 3.55 and 3.56, respectively. Three parallel samples were set up for each TS value of the food waste slurry sample.

[0083] The above-mentioned self-made food waste slurry samples and food waste slurry samples were subjected to enzyme treatment. During the enzyme treatment process, all three types of slurry samples were treated using the same method:

[0084] Based on the optimized addition amounts obtained in the aforementioned embodiments, with a slurry TS of approximately 6.00% as a baseline, it is necessary to add 1 / 10000 ratio of cellulase, 1 / 10000 ratio of hemicellulase, and 1 / 20000 ratio of amylase. Therefore, in this embodiment, the enzyme addition amounts for slurries with TS contents of 5.00% and 7.00% need to be adjusted appropriately according to the TS content. Specifically, for slurries with TS of 5.00% and 7.00%, the enzyme addition amounts are adjusted according to dilution: when TS=5.00%, the amounts are 1 / 12000 ratio of cellulase, 1 / 12000 ratio of hemicellulase, and 1 / 24000 ratio of amylase; when TS=7.00%, the amounts are 1 / 8571 ratio of cellulase, 1 / 8571 ratio of hemicellulase, and 1 / 17143 ratio of amylase. The enzyme dosage ratios mentioned above refer to the ratio of the added enzyme solution volume to the organic waste slurry volume. The amylase, cellulase, and hemicellulase solutions were prepared with activities of 4000 U / mL, 5000 U / mL, and 3000 U / mL, respectively. The specific selection of the three enzymes followed the optimal enzyme combination optimized in Example 1: Xiasheng FDY-2223 for amylase, Xiasheng GDG-2012 for cellulase, and a mixture of equal mass ratios of mannanase Dibai K201675 and xylanase Xiasheng FDG-2221 for hemicellulase. After enzyme addition, each group of slurry samples with added enzymes was placed in a 70℃ water bath and stirred at 180 r / min for 3 hours to complete the enzyme-catalyzed reaction. After the enzyme-catalyzed reaction, 5 mL of the enzyme-catalyzed sample from each group was thoroughly mixed and stored for subsequent measurements; simultaneously, another 5 mL of the supernatant was taken for COD and TN determination. Subsequently, 20 mL of 61.10 g / L sodium tetraphenylborate solution was added to every 100 mL of the sample after the enzyme-catalyzed reaction, along with 0.500 g of anhydrous ferric chloride to assist in nitrogen precipitation. After reacting for 1 hour with stirring (600 r / min), the precipitate was removed by low-speed centrifugation (1000 r / min for 20 min). 5 mL of the supernatant was used for the determination of the physicochemical properties of the carbon source, and the remaining supernatant was used for the subsequent advanced oxidation stage reaction. The nitrogen precipitate could be recovered and reused as a precipitating agent. In the advanced oxidation stage, 0.500 g of iron powder and potassium persulfate (0.695 g when TS=5.00%, 0.973 g when TS=7.00%) were added to the aforementioned supernatant. The mixture was stirred at 600 r / min for 1 hour at room temperature until fully reacted. After the reaction was completed, solid-liquid separation was performed by low-speed centrifugation (1500 r / min for 15 min), and 5 mL of the supernatant was used for the determination of the physicochemical properties of the carbon source.

[0085] The experimental results of this embodiment are shown in Table 5. The results analysis shows that the TS value significantly affects the carbon source output of the self-made kitchen waste in Example 1. When TS=5.00%, the COD increased by 18640 mg / L compared to the blank group; when TS=7.00%, the COD increased by 33066 mg / L; compared to the enzyme treatment of the sample with TS=6.13%, the COD increased by 24400 mg / L, showing a certain linear relationship, indicating that TS has a strong positive correlation with COD output. For the kitchen waste before and after the three-phase separation process of a certain plant's kitchen waste to biogas production, the enzyme treatment effect was not significant, possibly because the proportion of large carbon molecules in the three-phase separation stage is low, limiting the amount of carbon source that can be enzymatically hydrolyzed. In the nitrogen precipitation stage, the TN of various types of kitchen waste slurry can be controlled within the range of approximately 100 mg / L, indicating good denitrification effect; the COD of the kitchen waste before three-phase separation showed a significant increase in this stage, with an increase of 214 mg / L when TS=5.00%. In the advanced oxidation stage, the COD of the self-made kitchen waste was further improved: COD increased by 37,576 mg / L when TS=5.00% and by 70,682 mg / L when TS=7.00%, significantly increasing the carbon source output value, and the C / N ratio remained above 300; the COD increase of the sample before three-phase separation was limited; after three-phase separation, the COD of the sample increased significantly in the advanced oxidation stage, by 10,795 mg / L when TS=5.00% and by 19,435 mg / L when TS=7.00%, and finally the C / N ratio increased to above 800.

[0086] The above results indicate that TS value and organic waste type have a significant impact on carbon source output and carbon source optimization effect. Self-made kitchen waste shows higher COD release and C / N improvement effect in the whole process of carbon source preparation, while the enzyme treatment output of three-phase separated kitchen waste is relatively low due to the limited available carbon source.

[0087] Table 5. Exploration of the effects of different TS and organic waste types on the entire process of carbon source preparation.

[0088]

[0089] This invention includes, but is not limited to, the present embodiment. It should be noted that, for those skilled in the art, other methods can be used to make substitutions without departing from the technical principles of this invention. All modifications and equivalent substitutions made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing a composite enzyme combined nitrogen precipitation organic waste carbon source, characterized in that, include: S1. A composite enzyme composed of amylase, cellulase, and hemicellulase is added as a carbon source catalyst to organic waste slurry rich in starch, cellulose, and hemicellulose. The enzyme catalytic reaction promotes the degradation and release of macromolecular carbon source substances, including starch, cellulose, and hemicellulose, to obtain an enzyme-treated solution. The amount of amylase, cellulase, and hemicellulase in the composite enzyme needs to be adjusted according to the total solid content a% of the organic waste slurry. The amount of amylase, cellulase, and hemicellulase added is (1 / 20000~1 / 10000)×a / 6 of the volume of the organic waste slurry, and the activities of the added amylase, cellulase, and hemicellulase are not less than 4000U / mL, 5000U / mL, and 3000U / mL, respectively. S2. Sodium tetraphenylborate is added to the enzyme-treated solution as a nitrogen removal agent and stirred thoroughly to carry out a nitrogen precipitation reaction. Nitrogen substances, including proteins, ammonia nitrogen and free amino acids, are selectively precipitated. The upper suspension and the lower nitrogen-enriched precipitate are obtained through the first solid-liquid separation operation. S3. Add potassium persulfate and iron to the suspension to construct a Fenton-like system. Further degrade and release the suspended substances in the suspension through a Fenton-like advanced oxidation reaction. After removing the iron precipitate through a second solid-liquid separation operation, a high-quality organic waste carbon source product is obtained. The first solid-liquid separation operation and the second solid-liquid separation operation adopt low-speed centrifugation method, with a centrifugation speed of 1000~2000 r / min and a duration of 10~20 min.

2. The method for preparing organic waste carbon source by combining complex enzymes with nitrogen precipitation as described in claim 1, characterized in that, The organic waste slurry is obtained by adding water to organic waste and crushing and homogenizing it. Its total solids content is controlled at 5.00% to 7.00%, cellulose content at 25% to 35%, hemicellulose content at 15% to 25%, and pH at 4.0 to 5.

5.

3. The method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation as described in claim 1, characterized in that, In the complex enzyme, the amylase is glucosylamylase, brand name Xiasheng FDY-2223; the cellulase is acidic cellulase, brand name Xiasheng GDG-2012; and the hemicellulase is a mixture of mannanase and xylanase in equal mass ratios, with mannanase brand name Dibai K201675 and xylanase brand name Xiasheng FDG-2221.

4. The method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation as described in claim 1, characterized in that, The enzyme-catalyzed reaction takes 3–6 hours, is carried out at a temperature of 40–80°C, and is stirred at a frequency of 150–200 r / min.

5. The method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation as described in claim 1, characterized in that, The dosage of sodium tetraphenylborate is based on the total nitrogen content in the solution after enzyme treatment, with 1955.54 to 2933.31 mg of sodium tetraphenylborate added per 100 mg of total nitrogen.

6. The method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation as described in claim 1, characterized in that, The stirring speed for the nitrogen precipitation reaction is 400~800 r / min, and the reaction time is 1~2 h.

7. The method for preparing organic waste carbon source by combining a complex enzyme with nitrogen precipitation as described in claim 1, characterized in that, When constructing the Fenton-like system, the mass of potassium persulfate added to the suspension is 0.83 to 1.00 times the mass of the total residual solids in the suspension, and the amount of iron added to the suspension is 0.01 to 0.02 g / mL of suspension.

8. The method for preparing organic waste carbon source by combining complex enzymes with nitrogen precipitation as described in claim 7, characterized in that, The iron-based substance is composed of iron powder and ferric chloride in equal mass ratios.

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