CN115786122A - Phosphorus-dissolving cadmium-reducing microbial agent and preparation method and application thereof - Google Patents

Abstract

The invention discloses a phosphorus-decomposing-cadmium-reducing microbial bacterial agent as well as a preparation method and application thereof. The preparation method comprises the following steps: S1: preparation of microbial fermentation broth: configuring fermentation broth; S2: expanded cultivation of microorganisms: adding phosphorus-reducing cadmium microbial bacterial agent to fermentation broth; S3: preparation of microbial bacterial agent: After the above-mentioned fermentation broth is fermented and cultivated for 18 hours, put in the biomass materials and mix them. After mixing, for the first 8 hours, stir the bacterial agent every 2 hours to make the state of each component in the mixture uniform. After 8 hours, wait for the It is naturally stable for 24 hours, and the production of phosphorus-reducing cadmium microbial bacterial agent is completed; the preparation method of this patented microbial agent is simple, and the microorganisms are immobilized by adsorption. At the same time, it also reduces the absorption of cadmium by plants and increases the content of available phosphorus in the soil.

Description

A kind of phosphorus-decomposing-cadmium-reducing microbial bacterial agent and its preparation method and application

technical field

The invention relates to the field of agricultural microorganism technology and soil restoration technology, in particular to a phosphorus-dissolving-cadmium-reducing microbial bacterial agent and its preparation method and application.

Background technique

Cadmium is a silvery-white heavy metal and a non-essential element for the human body. Cadmium is highly toxic and has a long half-life. Food is closely related to our life, and the production of food crops cannot be separated from the soil. Soil cadmium pollution comes from a wide range of sources, and will eventually accumulate in the human body through the physical chain, causing various harms to people. At present, there are mainly chemical treatment methods, physical treatment methods and biological treatment methods for the remediation of soil cadmium pollution. Chemical treatment can be effective in a short period of time, but it is more disturbing to the environment; physical treatment requires a large cost investment, and biological treatment uses some biomass materials or microorganisms to restore the contaminated site, although it may take a long time in terms of time. However, biological treatment is an environmentally friendly treatment method, which has attracted more and more attention.

In the prior art, most of them only use a single flora for treatment, and each microorganism has a certain suitable growth environment. Using a single flora does not work in some soil environments and does not have universal applicability.

Contents of the invention

The object of the present invention is to propose a phosphorus-dissolving-cadmium-reducing microbial bacterial agent and its preparation method and application in view of the above-mentioned deficiencies of the prior art.

A kind of preparation method of phosphorus solubilizing-cadmium reducing microbial bacterial agent of the present invention, comprises the steps:

S1: preparation of microbial fermentation liquid: configuring fermentation culture liquid;

S2: Expanded cultivation of microorganisms: adding phosphorus-dissolving-cadmium-reducing microbial bacterial agents to the fermentation broth;

S3: Preparation of microbial inoculum: After the above-mentioned fermentation broth is fermented and cultivated for 18 hours, the biomass material will be put in for mixing. After mixing, for the first 8 hours, the inoculum is stirred every 2 hours to make the state of each component in the mixture Uniform, after 8 hours, wait for it to be naturally stable for 24 hours, and complete the production of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent;

The enrichment process of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent is as follows: prepare the bacterial suspension, and the preparation method of the soil bacterial suspension of aerobic flora is as follows: weigh the fresh soil and place it in sterile water for shaking, then take the supernatant after standing The soil bacteria suspension of aerobic flora was prepared from the above liquid; the above-mentioned soil suspension of aerobic flora was added to the medium to enrich the microbial agent, and the enriched product was obtained, and then subcultured twice to obtain The enrichment product of the third generation is a phosphorus-decomposing-cadmium-reducing microbial bacterial agent.

Further, the medium is: glucose 10g/L, (NH ₄ ) ₂ SO ₄ 0.5g/L, NaCl 0.3g/L, MgSO ₄ 0.3g/L, MnSO ₄ 0.03g/L, FeSO ₄ 0.03g /L, K ₂ SO ₄ 0.3g/L, Ca ₃ (PO ₄ ) ₂ 5g/L, pH 7.0-7.5.

Further, the fermented culture medium is configured as beef extract: 3g, peptone: 10g, sodium chloride: 5g, pH: 7.4-7.6.

Further, the total inoculation amount in the fermentation broth is 2%.

Further, the biomass material includes rice husk powder, and the solid-to-liquid ratio of the biomass material to the mixed fermentation broth is 1g:2-10mL.

A phosphorus-solubilizing-cadmium-reducing microbial bacterial agent prepared by the above-mentioned preparation method.

The application of the above-mentioned phosphorus-dissolving-cadmium-reducing microbial bacterial agent is used to reduce the content of available cadmium in the soil and increase the content of available phosphorus in the soil.

The phosphorus-solubilizing-cadmium-reducing microbial bacterial agent of the present invention can reduce the content of available cadmium in the soil, thereby achieving the effect of reducing crop absorption, increasing the content of available phosphorus in the soil, and further changing other physical and chemical factors in the soil. Specifically, the phosphorus-solubilizing-cadmium-reducing microbial bacterial agent can convert phosphorus in the soil that is difficult to be absorbed by plants into available phosphorus that is easily absorbed by plants. Available phosphorus mainly exists in the form of phosphate, and cadmium can react with heavy metals to form precipitation. xM ^m+ +yPO ₄ ^3- →My(PO ₄ )x.

Compared with the prior art, this bacterial agent has the following beneficial effects: the preparation method of the patented bacterial agent is simple, and the microorganisms are immobilized by adsorption, which saves the complicated production process of other microorganism immobilization methods; this patent uses cheap and easy The obtained biomass material is used as a carrier to repair soil cadmium pollution by using microorganisms. Compared with other treatment methods, the input cost is small; compared with other chemical treatment methods, the selected materials are less disturbing to the environment and are effective in reducing soil pollution. While reducing cadmium, it also reduces the absorption of cadmium by plants, and increases the content of available phosphorus in the soil, which has the value of popularization and application.

Description of drawings

The photo of the nitrogen-fixing microbial bacterial agent prepared in Fig. 1;

Figure 2 is a diagram of the change of available cadmium in the soil system (the standards a, b, c, and d in the figure represent whether there is a significant difference);

Figure 3 is the soil cadmium form analysis diagram (Cd(F1)-acid extractable cadmium Cd(F2)-reducible cadmium Cd(F3)-oxidizable cadmium Cd(F4)-residual cadmium, CK is the blank group , DP is bacterial agent group);

Figure 4 is a partial physical and chemical display diagram in the system;

Fig. 5 is the monitoring diagram of cadmium content and plant growth status in the plant body;

Fig. 6 is a graph showing the measurement results of cadmium content in plants (field test, P<0.01, ** means significant difference).

Detailed ways

The following are specific embodiments of the present invention and in conjunction with the accompanying drawings, the technical solutions of the present invention are further described, but the present invention is not limited to these embodiments.

Preparation of Phosphorus Solubilizing-Cadmium-Reducing Microbial Inoculant

First prepare the bacterial suspension. The preparation method of the aerobic flora soil bacterial suspension is as follows: Weigh 10g of fresh soil and place it in a triangular conical flask filled with 90mL sterile water, vibrate at 150r/min for 2 minutes, then take Soil bacteria suspension (aerobic) was obtained from the clear liquid; 1 mL of the above-mentioned aerobic soil suspension was added to the phosphorus-dissolving medium (glucose 10 g/L, (NH ₄ ) ₂ SO ₄ 0.5 g/L, NaCl 0.3 g/L L, MgSO ₄ 0.3g/L, MnSO ₄ 0.03g/L, FeSO ₄ 0.03g/L, K ₂ SO ₄ 0.3g/L, Ca ₃ (PO ₄ ) ₂ 5g/L, pH7.0-7.5), Enrichment of the phosphorus-dissolving functional flora was carried out to obtain a phosphorus-dissolving enrichment product, and then subcultured and transferred twice to obtain the enriched product (P) of the third generation. It was then stored in 20% glycerol and stored in a -80°C refrigerator for subsequent studies.

Prepare beef extract-peptone medium: weigh 3g of beef extract, 10g of peptone, and 5g of sodium chloride, and adjust to 7.4-7.6 with sodium hydroxide solution.

The activated strain was expanded overnight, and the culture was stopped when the OD ₆₀₀ in the medium system reached 1.0-1.2. Centrifuge in a refrigerated centrifuge (setting centrifugation conditions: 8000r/min, 5min), discard the supernatant, and then resuspend on a vortex apparatus with sterilized deionized water to collect the bacteria.

Mix the above bacteria with sterilized rice husk powder, shake on a shaker (setting condition: 180r/min, 24h), make the rice husk powder adsorb and fix the microorganisms, and complete the preparation of microbial inoculum. Referring to accompanying drawing 1, it is the phosphorus-dissolving-cadmium-reducing microbial bacterial agent prepared by the technical scheme of this embodiment.

Potted Plant Restoration Test of Phosphorus Solubilizing and Cadmium Reducing Microbial Agents

Indoor pot experiments were carried out (the top diameter of the flower pot was 23cm, the height was 13cm, and the bottom diameter was 13cm), and each pot was filled with 2kg of aged cadmium-contaminated soil. Phosphorus solubilizing microbial inoculum was added according to the addition amount of 1%. Monitor the form of cadmium, available phosphorus and other physical and chemical factors in the system.

①Monitoring of available cadmium in soil system

Monitor the available cadmium in the system. The higher the availability of cadmium, the stronger the activity of cadmium and the easier it is absorbed and utilized by plants. The experimental results are shown in Figure 2. The experimental results showed that the addition of phosphorus solubilizing bacteria significantly decreased the available cadmium content in the soil, and the decreasing trend tended to be stable after 25 days.

②Cadmium speciation monitoring in the system

Further monitoring of cadmium species in the soil system is used to explain the reasons for the decline in cadmium bioavailability. Residual cadmium. The bioavailability of cadmium decreases successively, and the activity becomes weaker and weaker. The experimental results show that the content of acid-extractable and reducible cadmium in the system is significantly reduced. On the contrary, the content of oxidizable cadmium and residue cadmium in the system is significantly increased. This explains the decrease of soil available cadmium content in the system, and the decrease of cadmium bioavailability is due to the transformation of cadmium form in the system from an unstable state to a stable state in the system. The experimental results are shown in Figure 3.

③ Some physical and chemical monitoring in the system

The data results in Fig. 4 show that after adding the microbial agent, the pH value in the soil has significantly increased compared with the blank treatment group; although the organic matter content has no significant difference, it has increased on the mean value; the ferrous iron in the system The content of ferrous ions has increased significantly, and the content of ferrous ions can reflect the redox environment in the system. When the content of ferrous ions is higher, the system tends to be more reducing environment. Previous studies have shown that when the soil tends to be more alkaline, the activity of cadmium in the soil will decrease, and the content of organic matter will increase, which will combine with heavy metal cadmium, and the content of oxidizable cadmium will increase. The hair tends to reduce the environment, and the content of cadmium in the precipitated state such as CdS in the system will be higher. In addition, it is worth noting that after adding the phosphate-dissolving bacteria agent, the content of available phosphorus in the soil system increased significantly in the early stage, and there was a significant difference, which indicated that the microbial phosphate-dissolving bacteria agent increased the available phosphorus content in the soil to a certain extent. The above explains that the decrease of cadmium content in the system is due to the reaction of cadmium and phosphate to form cadmium phosphate precipitation, and also explains that the improvement of plant growth is related to the improvement of soil nutrients.

④ Correlation analysis between cadmium form and soil physicochemistry

Table 1 Spearman correlation analysis between cadmium speciation and soil physicochemistry

^** p<0.01, ^* p<0.05.

(Positive value represents positive correlation, negative value represents negative correlation, * represents whether there is a significant difference)

Experimental data show that acid extractable cadmium, reducible cadmium, oxidizable cadmium, and residue cadmium are significantly negatively correlated; the bioavailability of cadmium is significantly negatively correlated with the environment, and the bioavailability of cadmium is significantly negatively correlated with the content of organic matter. Negative correlation, the bioavailability of cadmium did not change significantly with T, H ₂ O and other indicators.

⑤Monitoring of cadmium content in plants and plant growth status

The cadmium content in plants was monitored by wet digestion method, the plant height was measured by ruler, and the fresh weight of plants was measured by balance. The experimental results are shown in Figure 5. The experimental results showed that the cadmium content in the plants of the phosphate-dissolving bacteria treatment group decreased significantly compared with the blank treatment group, and the decline rate reached 36.24%. It is worth noting that when the cadmium content in the plants decreased, the plants The height, fresh weight, etc. were significantly increased, which indicated that the addition of phosphate solubilizing bacteria alleviated the toxic effect of heavy metal cadmium on plants.

⑥Discussion on the application of field experiments

The microbial agent prepared above was added to cadmium-contaminated soil in the field, and lettuce was selected as the research object. Determination of cadmium content in plants. The measurement results are shown in Figure 6. The data showed that the addition of phosphorus-solubilizing microbial agents can produce results that are basically consistent with those in the laboratory, and can significantly reduce the cadmium content in plants, with a reduction rate of 32.16%. It shows that the microbial agent also has a certain application development prospect.

What is not involved above is applicable to the prior art.

Although some specific embodiments of the present invention have been described in detail by examples, those skilled in the art should understand that the above examples are only for illustration, rather than for limiting the scope of the present invention. Various modifications or additions or similar substitutions can be made to the described specific embodiments without departing from the direction of the present invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above implementations based on the technical essence of the present invention shall be included in the protection scope of the present invention.

Claims (7)

1. a preparation method of phosphorus-reducing cadmium microbial bacterial agent, is characterized in that: comprise the steps: S1: preparation of microbial fermentation liquid: configuring fermentation culture liquid; S2: Expanded cultivation of microorganisms: adding phosphorus-dissolving-cadmium-reducing microbial bacterial agents to the fermentation broth; S3: Preparation of microbial inoculum: After the above-mentioned fermentation broth is fermented and cultivated for 18 hours, the biomass material will be put in for mixing. After mixing, for the first 8 hours, the inoculum is stirred every 2 hours to make the state of each component in the mixture Uniform, after 8 hours, wait for it to be naturally stable for 24 hours, and complete the production of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent; The enrichment process of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent is as follows: prepare the bacterial suspension, and the preparation method of the soil bacterial suspension of aerobic flora is as follows: weigh the fresh soil and place it in sterile water for shaking, then take the supernatant after standing The soil bacteria suspension of aerobic flora was prepared from the above liquid; the above-mentioned soil suspension of aerobic flora was added to the medium to enrich the microbial agent, and the enriched product was obtained, and then subcultured twice to obtain The enrichment product of the third generation is a phosphorus-decomposing-cadmium-reducing microbial bacterial agent. 2. The preparation method according to claim 1, characterized in that: the medium is: glucose 10g/L, (NH ₄ ) ₂ SO ₄ 0.5g/L, NaCl 0.3g/L, MgSO ₄ 0.3g/L L, MnSO ₄ 0.03g/L, FeSO ₄ 0.03g/L, K ₂ SO ₄ 0.3g/L, Ca ₃ (PO ₄ ) ₂ 5g/L, pH 7.0-7.5. 3. The preparation method according to claim 1, characterized in that: the configured fermentation culture liquid is beef extract: 3g, peptone: 10g, sodium chloride: 5g, pH: 7.4-7.6. 4. The preparation method according to claim 1, characterized in that: the total inoculum size in the fermentation broth is 2%. 5. The preparation method according to claim 1, characterized in that: the biomass material comprises rice husk powder, and the solid-to-liquid ratio of the biomass material and the mixed fermentation broth is 1g:2-10mL. 6. A phosphorus-dissolving-cadmium-reducing microbial bacterial agent prepared by the preparation method described in any one of claims 1-5. 7. The application of the phosphorus-dissolving-cadmium-reducing microbial bacterial agent as claimed in claim 6, characterized in that: it is used to reduce the content of available cadmium in the soil and increase the content of available phosphorus in the soil.

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