A salt-tolerant microbial adsorbent and its application

Abstract

This invention discloses a salt-tolerant microbial adsorbent. Kitchen waste undergoes initial solid-liquid separation. After removing metal impurities, the separated solid kitchen waste is mechanically crushed. The moisture content is adjusted to 75%-85% with deionized water and then placed in a reaction vessel, reacting at 180℃-250℃ for 6-10 hours. The resulting product is dried to obtain the salt-tolerant microbial adsorbent. This invention solves the problem of difficult salt removal and limited resource utilization in kitchen waste by converting it into a porous, carbonaceous material under high temperature and pressure. The adsorbent preparation method of this invention is simple, low-cost, and meets the requirement of maintaining a certain salt content for the growth and preservation of salt-tolerant microorganisms. Its preservation effect on salt-tolerant microorganisms is superior to traditional adsorbents, making it suitable for large-scale application.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and more specifically, to a method for using a salt-tolerant microbial adsorbent and its application. Background Technology

[0002] Microbial adsorbents are carrier materials used to immobilize microorganisms and are widely used in wastewater treatment, soil remediation, and microbial fertilizers. Traditional microbial adsorbents mainly use materials such as peat moss and activated carbon. However, these materials have the following problems: peat moss resources are limited, and its mining will damage the ecological environment and is costly; although activated carbon has good adsorption performance, it is expensive and difficult to use on a large scale.

[0003] Salt-tolerant microorganisms generally require acclimatization in high-salt environments to develop their salt tolerance. Prolonged growth and metabolism in low-salt or salt-free environments may lead to decreased salt tolerance or even death. Therefore, maintaining a specific salt content is crucial during the cultivation and preservation of salt-tolerant microorganisms. Providing a low-cost adsorbent that meets the requirements for the propagation and preservation of salt-tolerant microorganisms has significant market application value.

[0004] Food waste is a significant component of municipal solid waste, characterized by high organic matter content, high moisture content, and easy biodegradability, with a salt content typically ranging from 0.2% to 5%. Traditional food waste treatment methods mainly include landfill, incineration, and anaerobic digestion. However, landfill occupies a large amount of land resources and easily produces leachate and foul odors, polluting the soil and groundwater; incineration requires high energy input and produces harmful gases such as dioxins, polluting the air environment; while anaerobic digestion can produce biogas, its treatment efficiency is low and it requires sophisticated equipment. Therefore, it is necessary to provide an environmentally friendly and efficient method for food waste treatment. Summary of the Invention

[0005] This invention provides a salt-tolerant microbial adsorbent, the preparation method of which includes the following steps:

[0006] (1) After the kitchen waste undergoes initial solid-liquid separation, the separated solid kitchen waste is removed of metal impurities and then mechanically crushed.

[0007] (2) Adjust the moisture content of the crushed kitchen waste to 75%-85% with deionized water. Place the kitchen waste with the adjusted moisture content in a reaction vessel and react at 180℃-250℃ for 6-10 hours; dry the product obtained from the reaction to obtain the salt-tolerant microbial adsorbent.

[0008] Preferably, the salt-tolerant microorganism is a salt-tolerant Bacillus.

[0009] In step (1), a filter screen with a pore size ≤2cm is used for initial solid-liquid separation;

[0010] In step (1), the crushed particle size is ≤5mm;

[0011] In step (2), the reaction is carried out at 180℃ for 10 hours or at 220℃ for 8 hours;

[0012] In step (2), the product is dried to a moisture content of 8-10%.

[0013] In step (2), the moisture content is adjusted to 75%.

[0014] The present invention also provides a method for preserving salt-tolerant microorganisms, the method comprising mixing the aforementioned salt-tolerant microbial adsorbent with the salt-tolerant microbial liquid and placing it in a sealed container, and storing it in a cool, dry place at room temperature.

[0015] Preferably, the mass ratio of the salt-tolerant microbial adsorbent to the salt-tolerant microbial liquid is 3:1.

[0016] The mixing process involves slowly adding the salt-tolerant microbial culture solution to the salt-tolerant microbial adsorbent while continuously stirring until homogeneous.

[0017] The sealed container is a sealed bag.

[0018] The beneficial effects of this invention include:

[0019] This invention solves the problem of difficult salt removal and limited resource utilization in kitchen waste by converting it into carbonaceous material with a porous structure in a high temperature and high pressure environment.

[0020] The adsorbent preparation method of the present invention is simple and low in cost, and meets the requirement of maintaining a certain salt content for the growth and preservation of salt-tolerant microorganisms. The preservation effect on salt-tolerant microorganisms is better than that of traditional adsorbents, and it is suitable for large-scale promotion and application. Attached Figure Description

[0021] Figure 1 The effect of different temperatures on the yield of the prepared salt-tolerant microbial adsorbent;

[0022] Figure 2 The effect of different temperatures on the pH value of the prepared salt-tolerant microbial adsorbent;

[0023] Figure 3 The effect of different temperatures on the prepared salt-tolerant microbial adsorbent EC;

[0024] Figure 4 The effect of temperature 180℃ on the pore size of the prepared salt-tolerant microbial adsorbent is shown, where A and C are electron micrographs of the adsorbent prepared after 6 h, 8 h and 10 h of reaction, respectively.

[0025] Figure 5The effect of 220℃ temperature on the pore size of the prepared salt-tolerant microbial adsorbent is shown, where A and C are electron micrographs of the adsorbent prepared after 6h, 8h and 10h of reaction, respectively.

[0026] Figure 6 The effect of 250℃ temperature on the pore size of the prepared salt-tolerant microbial adsorbent is shown, where A and C are electron micrographs of the adsorbent prepared after 6h, 8h and 10h of reaction, respectively.

[0027] Figure 7 The effect of different temperatures on the humic acid content of the prepared salt-tolerant microbial adsorbent;

[0028] Figure 8 The effect of different temperatures on the total salt content of the prepared salt-tolerant microbial adsorbent;

[0029] Figure 9 The diagram shows the effect of using the salt-tolerant microbial adsorbent prepared by this invention and peat moss to preserve salt-tolerant Bacillus. Detailed Implementation

[0030] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1: Screening of Salt-Tolerant Microbial Adsorbent Preparation Parameters - Optimization of Reactant Moisture Content Experiment. The moisture content of the reactants plays a crucial role in the properties of the adsorbent. Water serves as the reaction medium; the decomposition of organic macromolecules in the material requires water, and the polymerization of small molecules into macromolecules with specific structures also requires water molecules. Furthermore, sufficient moisture during the reaction promotes uniform mixing of the materials. The evaporation and removal of water in the later stages of the reaction affect the porosity and specific surface area of ​​the adsorbent. The adsorbent preparation process is energy-intensive. Maximizing the yield without affecting the adsorbent performance is key to reducing preparation costs. Therefore, it is necessary to select material ratios with yield as the primary indicator, and then screen different preparation parameters to obtain the optimal adsorbent preparation parameters for the adsorbent microorganisms.

[0033] Kitchen waste was collected from the canteen of the Beijing Academy of Agricultural and Forestry Sciences. It underwent initial solid-liquid separation using a 2cm pore size filter. After removing metal impurities, the separated solid kitchen waste was mechanically crushed to a particle size ≤5mm. The moisture content of the crushed kitchen waste was determined to be 70%. Three moisture content gradients (75%, 85%, and 95%) were established. Deionized water was used to supplement the moisture content if necessary. Each gradient was repeated three times. The kitchen waste with adjusted moisture content was placed in a reaction vessel (model: PPLKH-1000mL, Xi'an Yibei Instrument Equipment Co., Ltd., equipment parameters: inner tank temperature ≤280℃, pressure resistance ≤3Mpa, heating and cooling rate ≤5℃ / min). The sample loading volume in the reaction vessel was 80%. Reaction temperatures were set at 180℃, 220℃, and 250℃, and reaction times were set at 6, 8, and 10 hours. The resulting solid product was dried to a moisture content of approximately 9%, and the adsorbent yield was calculated.

[0034] Adsorbent yield (%) = [Adsorbent mass * (1 - Adsorbent moisture content)] * 100 / Kitchen waste * (1 - Kitchen waste moisture content)

[0035] The experimental results are shown in Table 1 below. The results show that when the reaction temperature is 180℃ and the reaction time is 6 hours, the adsorbent yield does not differ significantly. However, with the extension of time, after 8 hours of reaction, the adsorbent yield with 75% water content is significantly higher than that with 85% and 95% water content. When the reaction time is extended to 10 hours, the adsorbent yields with 75% and 85% water content are significantly higher than those with 95% water content. When the reaction temperature was 220℃, the product yield of the 75% moisture content treatment after 6 hours of reaction was significantly higher than that of the other two treatments. After 8 hours of reaction, there was no significant difference in adsorbent yield among the three treatments, but the adsorbent yield of the treatment with lower moisture content was higher. After 10 hours of reaction, the adsorbent yield of the 75% moisture content treatment was significantly higher than that of the 95% moisture content treatment, but there was no significant difference in yield compared to the 85% moisture content treatment. When the reaction temperature was 250℃, after 6 hours of reaction, the adsorbent yield of the 75% moisture content treatment was significantly higher than that of the 95% moisture content treatment, but there was no significant difference compared to the 85% moisture content treatment. After 8 and 10 hours of reaction, the adsorbent yield of the 75% and 85% moisture content treatments was significantly higher than that of the 95% moisture content treatment. A comprehensive evaluation of the adsorbent yield of each treatment revealed that, except for two treatments with shorter reaction times, the adsorbent yields of the 75% and 85% moisture content treatments were generally higher than those of the 95% moisture content treatment, with some treatments showing significant differences. Based on maximizing waste utilization and yield without affecting the product properties, 75%-85% was selected as the moisture content parameter for adsorbent preparation, with 75% moisture content being the optimal moisture content of the reactants.

[0036] Table 1. Adsorbent Yield Analysis under Different Treatments

[0037]

[0038]

[0039] Example 2: Screening of preparation parameters for salt-tolerant microbial adsorbents - Optimization experiment of reaction temperature and reaction time

[0040] Kitchen waste was collected from the canteen of the Beijing Academy of Agricultural and Forestry Sciences. It underwent initial solid-liquid separation using a 2cm pore size filter. After removing metal impurities, the separated solid kitchen waste was mechanically crushed to a particle size ≤5mm. The moisture content of the crushed kitchen waste was measured and adjusted to 75%. If the moisture content was insufficient, deionized water was added. The kitchen waste with adjusted moisture content was placed in a reaction vessel (model: PPLKH-1000mL, Xi'an Yibei Instrument Equipment Co., Ltd., equipment parameters: inner tank temperature ≤280℃, pressure resistance ≤3Mpa, heating and cooling rate ≤5℃ / min). Nine treatments were set up with reaction temperatures of 180℃, 220℃, and 250℃, and reaction times of 6, 8, and 10 hours. The resulting solid product was dried to a moisture content of approximately 9%, yielding a salt-tolerant microbial adsorbent.

[0041] (1) Effect of temperature on the yield of salt-tolerant microbial adsorbents

[0042] The effect of different temperatures on the yield of the prepared salt-tolerant microbial adsorbent at a moisture content of 75% is as follows: Figure 1 As shown. Wherein, the adsorbent yield (%) = [adsorbent mass * (1 - adsorbent moisture content)] * 100 / kitchen waste * (1 - kitchen waste moisture content).

[0043] from Figure 1 The results show that at 180℃, the highest yield (47.8%) was achieved after 8 hours of reaction, while the lowest yield was achieved after 6 hours. At 220℃, the highest yield (45.6%) was achieved after 8 hours of reaction, while the lowest yield was achieved after 10 hours. At 250℃, the highest yield (43.9%) was achieved after 6 hours of reaction, while the lowest yield was achieved after 10 hours. The experimental results indicate that at lower temperatures, extending the reaction time increases the adsorbent yield, but at higher temperatures, extending the reaction time decreases the adsorbent yield.

[0044] (2) Effect of temperature on pH of salt-tolerant microbial adsorbents

[0045] The effect of different temperatures on the pH value of the prepared salt-tolerant microbial adsorbent at a moisture content of 75% is as follows: Figure 2 As shown.

[0046] from Figure 2It can be seen that temperature has little effect on the pH value of salt-tolerant microbial adsorbents. The pH values ​​of all treated adsorbents are between 4.0 and 4.7, while the pH of natural peat moss is usually between 4.0 and 6.6, which is suitable for the reproduction and preservation of salt-tolerant microorganisms.

[0047] (3) Effect of temperature on salt-tolerant microbial adsorbent EC

[0048] During the preservation of microbial strains, the EC value of the preservation matrix can characterize information such as dissolved salts and ionic strength in the adsorbent. These ions are crucial for maintaining the physiological activity and metabolic processes of microorganisms, especially salt-tolerant microorganisms. Appropriate ion concentrations help maintain the osmotic pressure balance of microbial cells, preventing cell rupture or dehydration due to excessive or insufficient water.

[0049] The effect of different temperatures on the prepared salt-tolerant microbial adsorbent EC at a moisture content of 75% is as follows: Figure 3 As shown.

[0050] from Figure 3 As can be seen, temperature has a significant impact on the EC value of salt-tolerant microbial adsorbents. Specifically, at 180℃, the adsorbent exhibits the highest EC value, reaching 7.98 ms / cm, after 6 hours of reaction. At 220℃, the EC value decreases with increasing reaction time; at 250℃, the EC value initially increases and then decreases with increasing reaction time.

[0051] (4) Effect of temperature on the pore size of salt-tolerant microbial adsorbents

[0052] Microbial adsorbents with more surface pores and more coarse particles have better adsorption performance.

[0053] The effect of different temperatures on the pore size of the prepared salt-tolerant microbial adsorbent at a moisture content of 75% is as follows: Figure 4-6 As shown.

[0054] from Figure 4 As can be seen, after 6 hours of reaction at 180℃, the adsorbent begins to exhibit carbonization characteristics, and its surface becomes relatively rough, but it still retains many of the original structural features of the kitchen waste. With the reaction time extended to 8 hours, the hydrothermal intensity further increases, and the organic matter in the kitchen waste continues to decompose and transform, making the surface of the adsorbent even rougher and gradually increasing the pore structure. When the reaction reaches 10 hours, the surface roughness of the adsorbent increases, and the surface particles become finer and more uniform.

[0055] from Figure 5As can be seen, after 6 hours of reaction at 220℃, the adsorbent surface is rougher, the pore structure is more developed, and the particles exhibit smaller size and more irregular shape compared to 180℃. After 8 hours of reaction, the morphology of the adsorbent becomes more complex. More wrinkles and protrusions appear on its surface, which help increase the specific surface area and improve the adsorption performance. Simultaneously, particle aggregation may be more pronounced, forming larger blocky structures. After 10 hours of reaction, the density of the adsorbent structure begins to increase.

[0056] from Figure 6 As can be seen, after reacting kitchen waste at 250℃ for 6 hours, the adsorbent exhibits highly carbonized characteristics, with an extremely rough surface and a very rich and complex pore structure. The particle size decreases, and the specific surface area reaches a large value. As the reaction time is extended to 8-10 hours, the reaction continues, the carbon layer structure on the adsorbent surface becomes more complete, and the pore distribution becomes more uniform. Due to the high temperature and long reaction time, the pore structure begins to collapse.

[0057] (5) Effect of temperature on humic acid content of salt-tolerant microbial adsorbents

[0058] The humic acid content in microbial strain preservatives has a significant impact on the viability, stability, post-resuscitation activity, community structure, and function of the strains. Humic acid can provide nutrients for microorganisms, regulate environmental conditions (buffer pH, regulate redox potential), act as a physical barrier for microbial cells, and has antioxidant effects.

[0059] The effect of different temperatures on the humic acid content of the prepared salt-tolerant microbial adsorbent at a moisture content of 75% is as follows: Figure 7 As shown.

[0060] from Figure 7 As can be seen, at a reaction temperature of 180℃, the humic acid content gradually increases with increasing reaction time. When the reaction temperature rises to 220℃, the humic acid content of the adsorbent changes very little with reaction time. However, as the reaction temperature increases to 250℃, due to excessive reaction, the humic acid content in the adsorbent decreases somewhat compared to lower temperature conditions.

[0061] (6) Effect of temperature on the salt content of salt-tolerant microbial adsorbents

[0062] The salt content of microbial adsorbents can provide a certain acclimatization environment for salt-tolerant microorganisms, ensuring that the cells of salt-tolerant microorganisms are protected from the harm of low osmotic pressure.

[0063] The effect of different temperatures on the total salt content of the prepared salt-tolerant microbial adsorbent at a moisture content of 75% is as follows: Figure 8 As shown.

[0064] from Figure 8 As can be seen, at a reaction temperature of 180℃, the total salt content gradually increases with increasing reaction time. When the reaction temperature rises to 220℃, the total salt content of the adsorbent decreases compared to the adsorbent produced at 180℃, and the salt content is highest after 8 hours of reaction. Furthermore, as the reaction temperature increases to 250℃, the total salt content in the adsorbent decreases further compared to the adsorbent produced at lower temperatures. Ultimately, the total salt content of the adsorbent can be maintained at approximately 0.5-3.0%.

[0065] Based on the above data, the salt-tolerant microbial adsorbent obtained by reacting kitchen waste at 180℃ for 10 hours or at 220℃ for 8 hours without any pre-treatment for desalination or the addition of a catalyst is relatively more economical and of higher quality. In particular, its salt content is above 1.5% and below 3.0%, which can meet the growth requirements of highly salt-tolerant microorganisms.

[0066] Example 3: Verification of the Preservation Effect of Salt-Tolerant Microbial Adsorbent Strains

[0067] (1) Preparation of salt-tolerant microbial adsorbents: 1 kg of kitchen waste was collected from the canteen of the Beijing Academy of Agricultural and Forestry Sciences. The waste was initially separated into solid and liquid components using a filter (2 cm pore size) to remove metal impurities, and then mechanically crushed to a particle size ≤ 5 mm. The moisture content of the crushed kitchen waste was determined to be 73%, and was adjusted to 75% using deionized water. The adjusted moisture content kitchen waste was placed in two reaction vessels, with reaction temperatures set at 180℃ and 220℃, and reaction times of 10 and 8 hours, respectively. The resulting solid products were dried to a moisture content of 8%, yielding two groups of salt-tolerant microbial adsorbents (adsorbent 1 and adsorbent 2).

[0068] (2) Preparation of Salt-Tolerant Microbial Culture: The microbial strain used in this example is salt-tolerant Bacillus, which was isolated and preserved from saline-alkali soil in Weihai, Shandong Province. This strain can grow normally in a medium with a salt content of 5%. The activated salt-tolerant Bacillus was inoculated into 100 mL of liquid medium (2% salt content) at a volume ratio of 1% and cultured on a shaker at 32℃ and 180 r / min for 24 h to obtain the seed culture. Subsequently, various seed cultures were inoculated into 500 mL of liquid medium (2% salt content) at a volume ratio of 1% and cultured on a shaker at 32℃ and 180 r / min. Fermentation was stopped when the sporulation rate reached 80%. The fermentation broth was plate-counted to ensure a viable count of 5.0 × 10⁻⁶. 8 For use with concentrations of CFU / mL or higher.

[0069] (3) Preservation of Salt-Tolerant Microbial Agents with Adsorbents: The two groups of dried salt-tolerant microbial adsorbents were mixed with salt-tolerant microbial inoculum, with a mass ratio of 3:1. The microbial inoculum was slowly added to the salt-tolerant microbial adsorbents and stirred evenly. The mixture was then placed in a sealed bag and stored in a cool, dry place at room temperature. Simultaneously, a control experiment was conducted using commercially available peat moss to determine the number of salt-tolerant microorganisms monthly. LB medium with a salt content of 2% was used for the determination.

[0070] (4) Evaluation of strain preservation results

[0071] like Figure 9 As shown, two groups of salt-tolerant microbial adsorbents prepared from kitchen waste preserved salt-tolerant Bacillus for 5 months, and the measured number of effective viable bacteria was significantly higher than that of the traditional peat preservation method. Furthermore, the activity retention rate of salt-tolerant Bacillus preserved using the two groups of salt-tolerant microbial adsorbents was better, showing a significant difference compared to the use of commercially available peat for adsorption and preservation. This indicates that the adsorbents prepared from kitchen waste in this invention can completely replace, and are significantly superior to, peat as an adsorption carrier for salt-tolerant microbial agents.

Claims

1. A method for preserving salt-tolerant microorganisms, characterized in that, The preservation method includes mixing the salt-tolerant microbial adsorbent with the salt-tolerant microbial liquid, placing it in a sealed container, and storing it in a cool, dry place at room temperature. The preparation method of the adsorbent includes the following steps: (1) After the kitchen waste undergoes initial solid-liquid separation, the separated solid kitchen waste is mechanically crushed after removing metal impurities; (2) Adjust the moisture content of the crushed kitchen waste to 75%-85% with deionized water, place the kitchen waste with the adjusted moisture content in a reaction vessel, and react at 180℃-250℃ for 6-10 hours; dry the product obtained from the reaction to obtain the salt-tolerant microbial adsorbent.

2. The preservation method according to claim 1, characterized in that, The salt-tolerant microorganism is a salt-tolerant Bacillus.

3. The preservation method according to claim 1, characterized in that, In step (1), a filter screen with a pore size ≤ 2 cm is used for initial solid-liquid separation; and / or, in step (1), the particle size of the crushed particles is ≤ 5 mm.

4. The preservation method according to claim 1, characterized in that, In step (2), the reaction is carried out at 180℃ for 10 hours or at 220℃ for 8 hours.

5. The preservation method according to claim 1, characterized in that, In step (2), the product is dried to a moisture content of 8-10%.

6. The preservation method according to claim 1, characterized in that, In step (2), the moisture content is adjusted to 75%.

7. The preservation method according to claim 1, characterized in that, The mass ratio of the salt-tolerant microbial adsorbent to the salt-tolerant microbial liquid is 3:

1.

8. The preservation method according to claim 1, characterized in that, The mixing process involves slowly adding the salt-tolerant microbial culture solution to the salt-tolerant microbial adsorbent while continuously stirring until homogeneous.

9. The preservation method according to claim 1, characterized in that, The sealed container is a sealed bag.

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