Salt-tolerant microbial adsorbent and application thereof

Abstract

The invention discloses a salt-tolerant microbial adsorbent which is prepared by the following steps: performing solid-liquid primary separation on kitchen garbage, removing metal impurities from the separated solid kitchen garbage, and performing mechanical crushing treatment; the water content is adjusted to 75%-85% with deionized water, and then the mixture is put into a reaction kettle to react for 6-10 hours at the temperature of 180-250 DEG C; and drying a product obtained by the reaction to obtain the salt-tolerant microbial adsorbent. According to the method, the kitchen garbage is converted into the carbon-containing substance rich in porous structures in the high-temperature and high-pressure environment, and the problems that salt in the kitchen garbage is difficult to remove, and resource utilization is limited are solved. The adsorbent disclosed by the invention is simple in preparation method and low in cost, meets the requirements of growth and preservation of the salt-tolerant microorganisms for keeping a certain salt content, has a better preservation effect on the salt-tolerant microorganisms than a traditional adsorbent, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] This application relates to the technical field of molecular biology. Specifically, it relates to a method for a salt-tolerant microbial adsorbent and its application. Background Art

[0002] Microbial adsorbents are carrier materials used for immobilizing microorganisms and are widely applied in fields such as sewage treatment, soil remediation, and microbial fertilizers. Traditional microbial adsorbents mainly use materials such as peat and activated carbon. However, these materials have the following problems: Peat resources are limited, mining will damage the ecological environment, and the cost is relatively high; although activated carbon has good adsorption performance, its price is expensive and it is difficult to be applied on a large scale.

[0003] The salt-tolerant characteristics of salt-tolerant microorganisms generally need to be domesticated in a high-salt environment. Growing and metabolizing in a low-salt or salt-free environment for a long time may cause a decline in their salt-tolerant activity or death. Therefore, special attention should be paid to maintaining a certain salt content condition during the cultivation and preservation of salt-tolerant microorganisms. Providing an adsorbent with low cost that can meet the reproduction and preservation of salt-tolerant microorganisms has important market application value.

[0004] Food waste is an important component of urban domestic waste, with characteristics such as high organic matter content, high moisture, and easy rot, and the salt content rate is generally 0.2 - 5%. Traditional food waste treatment methods mainly include landfill, incineration, and anaerobic digestion, etc. However, landfill occupies a large amount of land resources, is prone to generating leachate and odors, and pollutes the soil and groundwater; incineration requires a high energy input and will generate harmful gases such as dioxins, polluting the air environment; although anaerobic digestion can produce biogas, the treatment efficiency is low and the requirements for equipment are high. Therefore, it is necessary to provide an environmentally friendly and efficient food waste treatment method. Summary of the Invention

[0005] The present invention provides a salt-tolerant microbial adsorbent, and its preparation method includes the following steps:

[0006] (1) The food waste is subjected to primary solid-liquid separation. After removing metal impurities from the separated solid food waste, it is subjected to mechanical crushing treatment;

[0007] (2) Use deionized water to adjust the moisture content of the crushed food waste to 75% - 85%. The food waste with adjusted moisture content is placed in a reaction kettle and reacted at 180°C - 250°C for 6 - 10 hours; the product obtained from the reaction is subjected to drying treatment to obtain the salt-tolerant microbial adsorbent.

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

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

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

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

[0012] In step (2), the drying treatment is carried out until the water content is 8 - 10%;

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

[0014] The present invention also provides a preservation method for salt - tolerant microorganisms, which includes mixing the aforementioned salt - tolerant microorganism adsorbent with a salt - tolerant microorganism bacterial solution, placing them in a sealed container, and storing them under cool, dry and room - temperature conditions.

[0015] Preferably, the mass ratio of the salt - tolerant microorganism adsorbent to the salt - tolerant microorganism bacterial solution is 3:1.

[0016] The mixing is to slowly add the salt - tolerant microorganism bacterial solution to the salt - tolerant microorganism adsorbent and continuously stir evenly.

[0017] The sealed container is a sealed bag.

[0018] The beneficial effects of the present invention include:

[0019] The present invention solves the problems that it is difficult to remove salts from kitchen waste and the resource utilization is limited by converting kitchen waste into carbon - rich substances with a porous structure in a high - temperature and high - pressure environment.

[0020] The preparation method of the adsorbent of the present invention is simple, low - cost, meets the characteristics that the growth and preservation of salt - tolerant microorganisms require a certain salt content, has a better preservation effect on salt - tolerant microorganisms than traditional adsorbents, and is suitable for large - scale popularization and application. Description of the Drawings

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

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

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

[0024] Figure 4 Shows the effect of a temperature of 180 °C on the pores of the prepared salt - tolerant microorganism adsorbent, where A - C are the electron micrographs of the adsorbents prepared by reacting for 6 h, 8 h and 10 h respectively;

[0025] Figure 5Effect of 220°C temperature on the pores of the prepared salt-tolerant microbial adsorbent, where A - C are SEM images of the adsorbents prepared at reaction times of 6 h, 8 h, and 10 h, respectively;

[0026] Figure 6 Effect of 250°C temperature on the pores of the prepared salt-tolerant microbial adsorbent, where A - C are SEM images of the adsorbents prepared at reaction times of 6 h, 8 h, and 10 h, respectively;

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

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

[0029] Figure 9 Effect diagram of preserving salt-tolerant Bacillus spores using the salt-tolerant microbial adsorbent prepared by the present invention and peat. Detailed implementation manners

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

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

[0032] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels. Example 1. Screening of preparation parameters of salt-tolerant microbial adsorbent - Optimization experiment of moisture content of reaction materials. During the preparation process of the adsorbent, the moisture content of the reaction materials plays a crucial role. As the reaction medium, water is required for the decomposition of organic macromolecules in the materials and the polymerization of small molecules into macromolecules with specific structures. In addition, sufficient moisture during the reaction process can promote the uniform mixing of the materials, and the evaporation and removal of water in the later stage of the reaction affect the porosity and specific surface area of the adsorbent. The preparation process of the adsorbent is an energy-consuming process. On the premise of not affecting the performance of the adsorbent, maximizing the yield is the key to reducing the preparation cost. Therefore, it is necessary to select the material ratio with the yield as the main index, and then screen different preparation parameters to obtain the optimal preparation parameters for the adsorbent to meet the use of adsorbed microorganisms.

[0033] Collect kitchen waste from the cafeteria of the Beijing Academy of Agriculture and Forestry Sciences, and conduct primary solid-liquid separation through a filter screen with a pore size of 2 cm. After removing metal impurities from the separated solid kitchen waste, perform mechanical crushing treatment with a crushing particle size ≤ 5 mm. Measure the moisture content of the crushed kitchen waste to be 70%. Set three moisture content gradients of 75%, 85%, and 95%. When the moisture content is insufficient, make up with deionized water. Do 3 replicates for each gradient. Place the kitchen waste with adjusted moisture content in a reaction kettle (model: PPLKH-1000 mL, Xi'an Yibei Instrument and Equipment Co., Ltd., equipment parameters: inner tank temperature ≤ 280 °C, pressure resistance ≤ 3 Mpa, heating and cooling rate ≤ 5 °C / min), and the sample loading amount of the reaction kettle is 80% in all cases. Set the treatments according to reaction temperatures of 180 °C, 220 °C, 250 °C and reaction times of 6, 8, 10 hours respectively. Dry the solid product obtained from the reaction until the moisture content is about 9%, and calculate the adsorbent yield.

[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. It can be seen from the following experimental results that when the reaction temperature is 180 °C and the reaction time is 6 hours, there is no significant difference in the adsorbent yield. As the reaction time extends, when the reaction is 8 hours, the adsorbent yield of the 75% moisture content is significantly higher than that of the 85% and 95% moisture content treatments. When the reaction duration extends to 10 hours, the adsorbent yields of 75% and 85% are both significantly higher than that of the 95% treatment. When the reaction temperature is 220 °C, the product of the 75% moisture content treatment at 6 hours of reaction is significantly higher than the other two treatments. When the reaction is 8 hours, there is no significant difference in the adsorbent yields of the three treatments, but the adsorbent yield of the treatment with lower moisture content is higher. When the reaction is 10 hours, the adsorbent yield of the 75% moisture content is significantly higher than that of the 95% moisture content, and there is no significant difference from the yield of the 85% treatment; when the reaction temperature is 250 °C, at 6 hours of reaction, the adsorbent yield of the 75% moisture content treatment is significantly higher than that of the 95% moisture content treatment, and there is no significant difference from the 85% moisture content treatment. When the reaction is 8 h and 10 h, the adsorbent yields of the 75% and 85% treatments are significantly higher than that of the 95% treatment. By comprehensively evaluating the adsorbent yields of each treatment, it is found that the adsorbent yields of the 75% and 85% moisture content treatments are basically higher than that of the 95% treatment except for the two treatments with shorter reaction times, and there are significant differences in some treatments. On the basis of not affecting the properties of the product, considering maximizing waste utilization and yield, select 75% - 85% as the moisture content parameter for adsorbent preparation, and 75% moisture content is the best reaction material moisture content.

[0036] Table 1. Analysis of adsorbent yields of different treatments

[0037]

[0038]

[0039] Example 2. Screening of preparation parameters of salt-tolerant microbial adsorbent - Optimization experiment of reaction temperature and reaction time

[0040] Collect kitchen waste from the cafeteria of Beijing Academy of Agriculture and Forestry Sciences, and conduct primary solid-liquid separation through a filter screen with a pore size of 2 cm. After removing metal impurities from the separated solid kitchen waste, perform mechanical crushing treatment with a crushing particle size ≤ 5 mm. Measure the moisture content of the crushed kitchen waste and adjust its moisture content to 75%. When the moisture content is insufficient, make up the difference with deionized water. Place the kitchen waste with adjusted moisture content in a reaction kettle (model: PPLKH-1000 mL, Xi'an Yibel Instrument and Equipment Co., Ltd., equipment parameters: inner tank temperature ≤ 280 °C, pressure resistance ≤ 3 Mpa, heating and cooling rate ≤ 5 °C / min), and set a total of 9 treatments according to reaction temperatures of 180 °C, 220 °C, 250 °C and reaction times of 6, 8, 10 hours. Dry the solid product obtained from the reaction until the moisture content is about 9% to obtain the salt-tolerant microbial adsorbent.

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

[0042] Under the condition of a moisture content of 75%, the effect of different temperatures on the yield of the prepared salt-tolerant microbial adsorbent is as Figure 1 shown. Among them, the adsorbent yield (%) = [adsorbent mass * (1 - adsorbent moisture content)] * 100 / kitchen waste * (1 - kitchen moisture content).

[0043] From Figure 1 it can be seen that under the condition of 180 °C, the yield is the highest at 47.8% when reacting for 8 hours, and the lowest at 6 hours. Under the condition of 220 °C, the yield is the highest at 45.6% when reacting for 8 hours, and the lowest at 10 hours. Under the condition of 250 °C, the yield is the highest at 43.9% when reacting for 6 hours, and the lowest at 10 hours. The experimental results show that at lower temperatures, extending the reaction time will increase the adsorbent yield, but at higher temperatures, extending the reaction time will decrease the adsorbent yield.

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

[0045] Under the condition of a moisture content of 75%, the effect of different temperatures on the pH value of the prepared salt-tolerant microbial adsorbent is as Figure 2 shown.

[0046] From Figure 2It can be seen that temperature has little effect on the pH value of the salt-tolerant microbial adsorbent. The pH values of all treated adsorbents are between 4.0 and 4.7. The pH of natural peat 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 the EC of the salt-tolerant microbial adsorbent

[0048] During the preservation of the strain, 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 the strain, especially salt-tolerant microorganisms. Appropriate ionic concentration helps to maintain the osmotic pressure balance of the strain cells and prevent the cells from bursting or losing water due to excessive or insufficient water.

[0049] Under the condition of 75% moisture content, the effect of different temperatures on the EC of the prepared salt-tolerant microbial adsorbent is as Figure 3 shown.

[0050] From Figure 3 it can be seen that temperature has a greater effect on the EC value of the salt-tolerant microbial adsorbent. Specifically, at 180 °C, the EC value of the adsorbent is the highest after reacting for 6 hours, reaching 7.98 ms / cm. At 220 °C, the EC value of the adsorbent decreases with the extension of the reaction time; at 250 °C, the EC value of the adsorbent first increases and then decreases with the extension of the reaction time.

[0051] (4) Effect of temperature on the pores of the salt-tolerant microbial adsorbent

[0052] The surface of the microbial adsorbent has more pores and rough particles, which have better adsorption performance.

[0053] Under the condition of 75% moisture content, the effect of different temperatures on the pores of the prepared salt-tolerant microbial adsorbent is as Figures 4 - 6 shown.

[0054] From Figure 4 it can be seen that when the kitchen waste reacts at 180 °C for 6 hours, the adsorbent begins to show carbonization characteristics, the surface becomes relatively rough, but still retains many structural characteristics of the original kitchen waste. As the reaction time is extended to 8 hours, the hydrothermal degree further deepens, the organic matter in the kitchen waste continues to decompose and transform, the surface of the adsorbent becomes rougher, and the pore structure gradually increases. 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 5It can be seen that for food waste reacting at 220°C for 6 hours, compared with 180°C, the surface of the adsorbent is rougher, the pore structure is more developed, and the particles exhibit smaller sizes and more irregular shapes. After 8 hours of reaction, the morphology of the adsorbent becomes more complex. More wrinkles and protrusions appear on its surface, and these structures help increase the specific surface area and improve the adsorption performance of the adsorbent. At the same time, the agglomeration phenomenon between particles may be more obvious, forming larger blocky structures. After 10 hours of reaction, the dense structure of the adsorbent begins to increase.

[0056] From Figure 6 It can be seen that for food waste reacting at 250°C for 6 hours, the morphology of the adsorbent shows highly carbonized characteristics, the surface is extremely rough, and the pore structure is very rich and complex. The particle size decreases, and the specific surface area reaches a relatively large value. As the reaction time extends to 8 - 10 hours, the reaction continues, the carbon layer structure on the surface of the adsorbent becomes more perfect, the pore distribution becomes more uniform, and due to the high temperature and long reaction time, the phenomenon of pore structure collapse begins to occur.

[0057] (5) Influence of temperature on the humic acid content of salt - tolerant microbial adsorbent

[0058] The humic acid content in the strain preservation agent has important effects on the vitality, stability, activity after resuscitation, and community structure and function of the strains. Humic acid can provide a nutrient source for microorganisms, regulate environmental conditions (buffer pH, adjust redox potential), serve as a physical barrier for microbial cells, and have antioxidant effects.

[0059] Under the condition of a moisture content of 75%, the influence of different temperatures on the humic acid content of the prepared salt - tolerant microbial adsorbent is as Figure 7 shown.

[0060] From Figure 7 It can be seen that at a reaction temperature of 180°C, as the reaction time increases, the humic acid content gradually increases. When the reaction temperature rises to 220°C, the change in the humic acid content of the adsorbent with the reaction time is very small. However, as the reaction temperature increases to 250°C, due to excessive reaction with the increase in temperature, the humic acid content in the adsorbent decreases compared with the lower - temperature conditions.

[0061] (6) Influence of temperature on the salt content of salt - tolerant microbial adsorbent

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

[0063] Under the condition of a moisture content of 75%, the influence of different temperatures on the total salt content of the prepared salt - tolerant microbial adsorbent is as Figure 8 shown.

[0064] FromFigure 8 It can be seen that at a reaction temperature of 180°C, as the reaction time increases, the total salt content gradually increases. When the reaction temperature rises to 220°C, the total salt content of the adsorbent decreases compared to the adsorbent produced at 180°C, and the salt content is the largest under the condition of 8 hours of reaction. As the reaction temperature rises to 250°C, the total salt content in the adsorbent further decreases compared to the adsorbent produced at a lower temperature. Finally, the total salt content of the adsorbent can basically be maintained at 0.5 - 3.0%.

[0065] Based on the above data results, without any desalination pretreatment of food waste and without adding a catalyst, the salt-tolerant microbial adsorbent obtained by reacting at 180°C for 10 hours or 220°C for 8 hours is relatively more economical and of better quality. Especially 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 adsorbent: 1 kg of food waste was actually collected from the cafeteria of the Beijing Academy of Agriculture and Forestry Sciences. The solid-liquid initial separation of food waste was carried out through a filter screen (pore size 2 cm), and after removing metal impurities, mechanical crushing treatment was carried out with a crushing particle size ≤ 5 mm. The water content of the crushed food waste was measured to be 73%, and its water content was adjusted to 75% with deionized water. The food waste with adjusted water content was placed in 2 reaction kettles, and the reaction temperatures were set at 180°C and 220°C respectively, and the reaction times were 10 and 8 hours respectively. The solid products obtained from the reaction were dried to a water content of 8% to obtain two groups of salt-tolerant microbial adsorbents (adsorbent 1 and adsorbent 2).

[0068] (2) Preparation of salt-tolerant microbial liquid: The microbial strain used in this example is salt-tolerant Bacillus, which was screened and isolated from the saline-alkali soil in Weihai, Shandong and preserved in this experiment. 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 (salt content 2%) at a volume ratio of 1%, and cultured in a shaker at 32°C and 180 r / min for 24 h to obtain a seed liquid. Subsequently, each seed liquid was inoculated into 500 mL of liquid medium (salt content 2%) at a volume ratio of 1%, and cultured in a shaker at 32°C and 180 r / min. When the sporulation rate reached 80%, the fermentation was stopped, and the fermentation broth was counted by plate counting to ensure that the viable bacteria count reached 5.0×10 8 CFU / mL or more for standby.

[0069] (3) Adsorbent preservation of salt-tolerant microbial agents: Mix the two groups of dried salt-tolerant microbial adsorbents with the salt-tolerant microbial liquid above. The mass ratio of the salt-tolerant microbial adsorbent to the microbial liquid is 3:1. Slowly add the microbial liquid to the salt-tolerant microbial adsorbent, stir evenly, and place it in a sealed bag. Store it in a cool, dry place at room temperature. At the same time, use commercially available peat as a control experiment and measure the number of salt-tolerant microorganisms in it every month. The selected medium for measurement is LB medium with a salt content of 2%.

[0070] (4) Evaluation of the results of strain preservation

[0071] As Figure 9 shown, the two groups of salt-tolerant microbial adsorbents prepared from kitchen waste were used to preserve salt-tolerant Bacillus for 5 months, and the measured effective viable count was significantly higher than that of the traditional peat preservation method. Moreover, the activity retention rate of using the two groups of salt-tolerant microbial adsorbents to preserve salt-tolerant Bacillus was better, and there was a significant difference compared with using commercially available peat to adsorb and preserve salt-tolerant microorganisms. It shows that the adsorbent prepared from kitchen waste in the present invention can completely replace and is significantly superior to peat as the adsorption carrier of salt-tolerant microbial agents.

Claims

1. A salt-tolerant microbial adsorbent, characterized in that, The preparation method of the adsorbent comprises the following steps: (1) The kitchen waste is subjected to primary solid-liquid separation. After the separated solid kitchen waste is removed of metal impurities, it is subjected to mechanical crushing treatment; (2) The water content of the crushed kitchen waste is adjusted to 75%-85% with deionized water. The kitchen waste with the adjusted water content is placed in a reaction kettle and reacted at 180°C-250°C for 6-10 hours; the product obtained from the reaction is subjected to drying treatment to obtain the salt-tolerant microbial adsorbent.

2. The adsorbent according to claim 1, characterized in that, The salt-tolerant microorganism is Bacillus halodurans.

3. The adsorbent according to claim 1, wherein In the step (1), a filter screen with a pore diameter ≤ 2 cm is used for primary solid-liquid separation; and / or, in the step (1), the crushing particle size ≤ 5 mm.

4. The adsorbent according to claim 1, characterized in that, In the step (2), the reaction is carried out at a temperature of 180°C for 10 h or 220°C for 8 h.

5. The adsorbent according to claim 1, characterized in that, In the step (2), the drying treatment is carried out until the water content is 8-10%.

6. The adsorbent according to claim 1, wherein In the step (2), the water content is adjusted to 75%.

7. A preservation method for salt-tolerant microorganisms, characterized in that, The preservation method comprises mixing the salt-tolerant microbial adsorbent according to any one of claims 1-6 with the salt-tolerant microbial liquid and placing the mixture in a sealed container, and storing it in a cool, dry place at room temperature.

8. The adsorbent according to claim 7, wherein The mass ratio of the salt-tolerant microbial adsorbent to the salt-tolerant microbial liquid is 3:

1.

9. The adsorbent according to claim 7, wherein The mixing is to slowly add the salt-tolerant microbial liquid to the salt-tolerant microbial adsorbent and continuously stir evenly.

10. The adsorbent according to claim 7, characterized in that, The sealed container is a sealed bag.

Images