A method for treating excess sludge using white rot fungi

Abstract

This invention discloses a method for treating residual sludge using white-rot fungi, comprising: white-rot fungi cultivation: activating the freeze-dried fungal powder in a sterile environment using a solid culture medium, followed by subculturing in a liquid culture medium to obtain a mycelial suspension; enzyme separation and purification: centrifuging the white-rot fungal culture broth or using other physical methods to separate the mycelia and spores to obtain a crude enzyme solution, or further purifying lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) using ion exchange chromatography; enzyme immobilization: immobilizing the oxidase using calcium alginate encapsulation or related carrier immobilization techniques; sludge pretreatment: without sterilization, adding an equal amount of the immobilized enzyme to the sludge, then placing it in a reactor, and culturing the reactor in a constant-temperature shaker at 28°C and 150 rpm for 4-5 days. According to this invention, the method is efficient, featuring low cost, simple operation, no need for additional chemical inputs, and reduced environmental harm, and has high practical value for the efficient treatment of sludge.

Description

Technical Field

[0001] This invention relates to the technical field of sludge treatment, and in particular to a method for treating excess sludge using white-rot fungi. Background Technology

[0002] Over the past decade, the annual sludge production from domestic wastewater treatment plants has exceeded 60 million tons (based on an 80% moisture content). The cost of sludge treatment and disposal accounts for as much as 60% of the total operating cost of wastewater treatment plants. The sludge treatment process typically involves first thickening and then dewatering, followed by stabilization to reduce the organic matter content in the sludge, before finally landfilling or incinerating. Due to the complex composition of sludge and its low treatment efficiency, sludge disposal often carries the risk of secondary environmental pollution caused by toxic and harmful substances.

[0003] Reducing the moisture content of sludge to 80%~85% is called dewatering. After dewatering, sludge retains solid properties, making it easier for final treatment. The anaerobic digestion efficiency of sludge directly affects dewatering performance. The principle is that anaerobic digestion of sludge is divided into three stages: hydrolysis, acid production, and methanogenesis. Organic matter is mainly decomposed in the hydrolysis stage. After the bonds between organic matter and water are broken, the release of mechanically bound water into free water can be accelerated, thereby improving the dewatering performance of sludge.

[0004] Excessive heavy metal content in sludge is also a problem that needs to be solved in order to improve the efficiency of sludge harmlessness. 70% to 90% of heavy metal elements will be enriched in the remaining sludge through adsorption or precipitation. In the subsequent treatment stage, heavy metals will form complexes with biological ligands in microorganisms, causing the biological ligands to lose their activity, resulting in sludge poisoning and restricting the agricultural use of the remaining sludge. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a more efficient method for treating excess sludge using white-rot fungi. This method is characterized by low cost, simple operation, no need for additional chemical inputs, and reduced environmental harm, making it highly valuable for the efficient treatment of sludge. To achieve the above-mentioned objectives and other advantages of the present invention, a method for treating excess sludge using white-rot fungi is provided, comprising:

[0006] S1. Activate the freeze-dried mycelial powder with a solid culture medium under sterile conditions, and then subculture it with a liquid culture medium to obtain a mycelial suspension.

[0007] S2. The mycelium and spores of the white-rot fungus culture medium were separated by physical methods to obtain crude enzyme solution, and lignin peroxidase, manganese peroxidase and laccase were purified by ion exchange chromatography.

[0008] S3. Immobilization of oxidases using calcium alginate encapsulation or related carrier immobilization techniques;

[0009] S4. Place the immobilized enzyme into the sludge, then into the reactor, and incubate the reactor in a constant temperature shaker at 28℃ and 150rpm for 4-5 days.

[0010] Preferably, in step S1, the white-rot fungi need to be cultured using PDA solid medium. After two generations of subculture, an appropriate amount of inoculum is inoculated into liquid medium using an inoculation loop for amplification to ensure the subsequent experiments can proceed.

[0011] Preferably, the preparation of crude enzyme solution in step S2 includes the following steps:

[0012] S21. After six days of liquid culture of white-rot fungi, the supernatant was obtained by centrifugation to remove mycelia and spores.

[0013] S22. After being stored at 4℃ for 24 hours, a crude enzyme solution for lignin peroxidase and laccase purification was obtained. The crude filtrate was obtained by filtering the culture medium of white-rot fungi using glass wool.

[0014] S23. Concentrate the filtrate using ultrafiltration, increase the concentration by 20 times, and then dilute it 1:1 with deionized water to obtain a crude enzyme solution for purifying manganese peroxidase.

[0015] Preferably, after preparing the crude enzyme solution, the enzyme is purified, including the following steps:

[0016] S24. Ion exchange chromatography was performed using diethylaminoethyl-dextran at a temperature of 4°C.

[0017] S25. The lignin peroxidase was purified by elution with NaCl gradient solution, and the manganese peroxidase was used to elute the protein with sodium tartrate.

[0018] S26. Concentration was performed using ultrafiltration, and laccase was eluted using a gradient elution with an acetate-sodium acetate buffer.

[0019] Preferably, the crude enzyme solution is stored at 4°C for later use after preparation.

[0020] Preferably, the purified enzyme should be stored at -20°C to maintain its activity for one year.

[0021] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses white-rot fungi to treat sludge, which can effectively destroy the cell walls of the sludge, degrade most recalcitrant organic matter, release internal and adsorbed water, and improve dewatering efficiency. Using white-rot fungi to passivate heavy metals in the sludge can reduce secondary pollution to the environment from subsequent sludge treatment. Using calcium alginate to immobilize the white-rot fungi can maintain the catalytic activity of enzymes and improve treatment efficiency. Attached Figure Description

[0022] Figure 1 The UV254 variation trend of sludge after treatment with white-rot fungi is shown in the method for treating residual sludge using white-rot fungi according to the present invention.

[0023] Figure 2 The changes in polysaccharide and protein content after sludge treatment by white-rot fungi according to the method of treating residual sludge with white-rot fungi according to the present invention are described in terms of the method of treating residual sludge with white-rot fungi. EPS-TB represents tightly bound extracellular polymeric polymer, EPS-LB represents loosely bound extracellular polymeric polymer, and EPS-SB represents dissolved extracellular polymeric polymer.

[0024] Figure 3 To illustrate the drying performance of sludge treated with white-rot fungi according to the method of treating residual sludge using white-rot fungi of the present invention, KB represents blank sludge and HF represents sludge treated with white-rot fungi. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] White-rot fungi possess a strong ability to degrade various natural and chemical substances, primarily relying on the secretion of their own enzymes. These enzymes constitute the enzymatic degradation system of white-rot fungi, exhibiting a broad spectrum of substrate degradation capabilities. Representative enzymes include lignin peroxidase, manganese peroxidase, and laccase. Sludge adsorbs a large amount of recalcitrant organic matter. White-rot fungi can disrupt sludge cell walls by secreting extracellular enzymes, causing the sludge colloid to lose stability. Simultaneously, they degrade recalcitrant organic matter, releasing internal and adsorbed water from the sludge, reducing its specific resistance, and thus improving sludge dewatering performance. The extracellular enzymes of white-rot fungi also participate in the complexation and transformation of metals, converting heavy metals into less toxic elemental forms, thereby completing the detoxification of sludge. Encapsulating white-rot fungi with calcium alginate can maintain their enzymatic catalytic activity. Adding them to excess sludge can improve the anaerobic digestion and dewatering performance of the excess sludge and passivate heavy metals.

[0027] The white-rot fungi used in the following examples can be Proteobacterium chrysosporum, Trametes versicolor, Pleurotus ostreatus, etc., and the other materials and instruments are commercially available.

[0028] White-rot fungi grow in the following manner:

[0029] The purchased lyophilized white-rot fungus powder was mixed with sterile water and inoculated onto PDA solid medium. The mixture was then incubated at 28°C for 5-6 days. After 5-6 days, the strains showing good growth were selected and subcultured using liquid medium to obtain a white-rot fungus suspension. This step was to obtain a stable and active strain. The PDA solid medium consisted of 200g potato, 20g glucose, 20g agar, and 1000mL water. The liquid medium did not contain agar.

[0030] Example 1

[0031] A method for treating residual sludge using white-rot fungi includes the following steps:

[0032] White-rot fungi were encapsulated with calcium alginate and added to sludge. The mixture was placed in a constant-temperature shaker at 130 rpm and 28°C for 5 days. The capillary time (CST) was used to represent the dewatering performance of the sludge. The results are shown in Table 1.

[0033] Table 1. Changes in sludge CST at different pH values

[0034]

[0035] As shown in Table 1, the CST of the sludge after adding white rot fungi was lower than that before adding fungi. The treatment effect was best at pH=4, which indicates that white rot fungi can break down sludge flocs to a certain extent and improve the dewatering performance of sludge. However, the breaking effect is affected by pH value, so the suitable pH value of this invention is 4.

[0036] Example 2

[0037] A method for treating residual sludge using white-rot fungi includes the following steps:

[0038] White-rot fungi were encapsulated with calcium alginate and added to the sludge. The mixture was placed in a constant-temperature shaker at 130 r / min and 28℃ for 5 days. The passivation rate of heavy metals in the remaining sludge was determined, represented by the contents of Zn, Cu, and Pb. The results are shown in Table 2.

[0039] Table 2. Heavy metal passivation rate (%) of different sludge samples after addition of white-rot fungi

[0040] 1 2 3 4 5 6 Zn 2.00 32.95 6.49 5.16 -4.99 3.16 Cu 5.65 31.45 1.94 3.89 36.04 1.41 Pb 3.17 45.35 1.36 1..81 22.22 3.4

[0041] As shown in Table 2, white-rot fungi exhibit significant passivation rates for all three metals. This indicates that the number of valence states changed after the oxidases bind to the heavy metals increases. The oxidases convert the exchangeable and reducible states of the heavy metals into residual states, thus completing the heavy metal remediation of the sludge and reducing biotoxicity.

[0042] Example 3

[0043] A method for treating residual sludge using white-rot fungi includes the following steps:

[0044] White rot fungi were encapsulated with calcium alginate and added to sludge. The mixture was placed in a constant temperature shaker at 130 r / min and 28℃ for 5 days. During this period, the sludge particle size change was measured using a laser particle size analyzer. The results are shown in Table 3.

[0045] Table 3. Changes in sludge particle size before and after treatment (μm)

[0046] d(0.1) d(0.5) d(0.9) / μm blank 14.021 44.563 220.016 Adding bacteria 10.421 32.294 79.256

[0047] As shown in Table 2, the sludge particle size was significantly reduced after the addition of white rot fungi. This indicates that white rot fungi play a significant role in breaking down sludge flocs, turning larger sludge flocs into smaller particles. This is beneficial for releasing the organic matter and water adsorbed on the sludge, which can improve the hydrolysis efficiency of anaerobic digestion and provide favorable conditions for subsequent dewatering.

[0048] Example 4

[0049] A method for treating residual sludge using white-rot fungi includes the following steps:

[0050] White-rot fungi were encapsulated with calcium alginate and added to anaerobic digested sludge. The mixture was placed in a constant-temperature shaker at 130 r / min and 28℃ for 5 days. During this period, the removal rates of SCOD and TCOD in the sludge were measured. The specific results are shown in Table 4.

[0051] Table 4. SCOD and TCOD removal rates (%) of anaerobic digestion sludge

[0052] blank 1 2 3 4 5 6 SCOD 3 44 52 46 51 71 42 TCOD 0 14 12 13 14 15 11

[0053] As shown in Table 4, after the addition of white rot fungi, both SCOD and TCOD showed significant degradation effects on the sludge, especially SCOD, with a maximum removal rate of 71%. This indicates that the organic matter in the sludge was gradually degraded under the action of white rot fungi, proving that white rot fungi can cooperate with anaerobic digestion to degrade most organic matter during the hydrolysis stage, thereby improving the overall anaerobic fermentation efficiency.

[0054] Example 5

[0055] A method for treating residual sludge using white-rot fungi includes the following steps:

[0056] White-rot fungi were encapsulated with calcium alginate and added to gravity-concentrated sludge. The mixture was placed in a constant-temperature shaker at 130 r / min and 28℃ for 5 days. During this period, the changes in sludge COD were measured, and the specific results are shown in Table 5.

[0057] Table 5. SCOD and TCOD removal rates (%) of gravity thickened sludge

[0058] blank 1 2 SCOD 9.86 33.33 20.83 TCOD 2.61 7.42 5.06

[0059] Example 6

[0060] A method for treating residual sludge using white-rot fungi includes the following steps:

[0061] White-rot fungi were encapsulated with calcium alginate and added to sludge. The mixture was placed in a constant-temperature shaker at 130 rpm and 28°C for 5 days. During this period, the changes in UV254 of the sludge were measured. Specific results are as follows: Figure 1 As shown.

[0062] Depend on Figure 1 It can be seen that the UV254 content of the sludge treated by the two white rot fungi showed an overall decreasing trend, and the final degradation rates reached 66.42% and 53.29%, respectively. This indicates that the white rot fungi gradually adsorbed and degraded the humic substances and aromatic compounds containing C=C double bonds and C=O double bonds in the sludge.

[0063] Example 7

[0064] A method for treating residual sludge using white-rot fungi includes the following steps:

[0065] White-rot fungi were encapsulated with calcium alginate and added to sludge. The mixture was placed in a constant-temperature shaker at 130 rpm and 28°C for 5 days. The sludge was then subjected to hot water hydrolysis to determine the polysaccharide and protein content. Specific results are shown below. Figure 2 As shown.

[0066] according to Figure 2 It can be seen that, compared with the blank, the polysaccharide and protein content in the sludge with added white rot fungi is reduced. This indicates that the white rot fungi metabolize some of the polysaccharides and proteins during the growth process, and the hyphae can grow into the sludge flocs, which helps to break down the dense sludge flocs, degrade polysaccharides, and release the internal proteins in the sludge flocs.

[0067] Example 8

[0068] A method for treating residual sludge using white-rot fungi includes the following steps:

[0069] White-rot fungi were encapsulated with calcium alginate and added to sludge. The mixture was placed in a constant-temperature shaker at 130 rpm and 28°C for 5 days. The sludge drying performance was analyzed using TG-DSC. Specific results are shown below. Figure 3 As shown.

[0070] according to Figure 3 It can be seen that, compared with the blank, the sludge treated with white rot fungi first reaches constant temperature in a shorter time and then completes incineration at a lower temperature. From the treatment results, the treatment time of sludge treated with white rot fungi is shortened by about 20%, and the energy consumption at the end of the reaction is reduced by 43.5%. This shows that white rot fungi can make sludge treatment more efficient and consume less energy.

[0071] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention, and applications, modifications and variations thereof will be apparent to those skilled in the art.

[0072] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for treating residual sludge using white-rot fungi, characterized in that, Includes the following steps: S1. After subculturing the white-rot fungus on PDA solid medium for two generations, it was inoculated into liquid medium using an inoculation loop for amplification culture. The supernatant was then centrifuged to obtain crude enzyme solution. S2. The crude enzyme solution is purified by ion exchange chromatography, and the components containing lignin peroxidase, manganese peroxidase and laccase are collected stepwise by gradient elution. S3. Immobilization of oxidases using calcium alginate encapsulation immobilization technology; S4. Place the immobilized enzyme into the sludge, then into the reactor, and incubate the reactor in a constant temperature shaker at 28℃ and 150rpm for 4-5 days.

2. The method for treating residual sludge using white-rot fungi as described in claim 1, characterized in that, Step S2, the preparation of the crude enzyme solution, includes the following steps: S21. After six days of liquid culture of white-rot fungi, the supernatant was obtained by centrifugation to remove mycelia and spores. S22. After being stored at 4℃ for 24 hours, a crude enzyme solution for lignin peroxidase and laccase purification was obtained. The crude filtrate was obtained by filtering the culture medium of white-rot fungi using glass wool. S23. Concentrate the filtrate using ultrafiltration, increase the concentration by 20 times, and then dilute it 1:1 with deionized water to obtain a crude enzyme solution for purifying manganese peroxidase.

3. The method for treating residual sludge using white-rot fungi as described in claim 2, characterized in that, After preparing the crude enzyme solution, the enzyme is purified, including the following steps: S24. Ion exchange chromatography was performed using diethylaminoethyl-dextran at a temperature of 4°C. S25. The lignin peroxidase was purified by elution with NaCl gradient solution, and the manganese peroxidase was used to elute the protein with sodium tartrate. S26. Concentration was performed using ultrafiltration, and laccase was eluted using a gradient elution with an acetate-sodium acetate buffer.

4. The method for treating residual sludge using white-rot fungi as described in claim 2, characterized in that, After the crude enzyme solution is prepared, it should be stored at 4°C for later use.

5. The method for treating residual sludge using white-rot fungi as described in claim 3, characterized in that, The purified enzyme needs to be stored at -20℃ to maintain its activity for one year.

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