Method for synergistically promoting safe and efficient production of farmland through activated sludge-microbial inoculum

Abstract

The invention discloses a method for synergistically promoting safe and efficient production of farmland by activated sludge-microbial inoculum, which comprises the following steps: mixing activated sludge and sawdust, fermenting at 50-60 DEG C for 13-15 days, sieving to remove incompletely decomposed large-particle substances after fermentation to obtain a uniform and fine compost product, mixing the compost product with a soil sample to obtain a culture medium, and culturing the culture medium in the soil sample to obtain the safe and efficient production of the farmland. The method comprises the following steps: planting crops on a culture medium, and inoculating a bacillus marissii bacterial solution into the culture medium in the growth process of the crops. According to the method, Rafs-SNRK2 signal regulation and control channels in plants are induced through the bacillus Yellow Sea, accumulation of heavy metals in the plants is effectively prevented and controlled, crop growth is promoted, use of pesticides and chemical fertilizers is reduced, and the technology is expected to provide a new method for resource utilization of activated sludge and safe and efficient production research of farmlands.

Description

Technical Field

[0001] The invention relates to the technical field of safe agricultural use of activated sludge, and in particular to a method for increasing crop yield and preventing heavy metal accumulation by using Bacillus maris flavi in ​​combination with activated sludge. Background Art

[0002] In agricultural production, in order to ensure high and stable yields of crops, the use of fertilizers and pesticides is increasing, which inevitably leads to a series of environmental problems such as soil compaction, heavy metal pollution, and eutrophication of water bodies, which not only affect the growth environment of crops, but also pose a serious threat to human health and ecological balance. Therefore, it is particularly important to find a method that can reduce the use of fertilizers and pesticides while improving farmland productivity and ensuring food security. Activated sludge is a substance rich in organic matter and microorganisms produced during sewage treatment. It contains a large amount of nutrients required for plant growth, such as nitrogen, phosphorus, potassium, iron, zinc, copper, manganese, and boron, as well as a variety of beneficial microorganisms. Therefore, as a potential fertilizer resource, activated sludge has important application potential in agriculture. However, during the use of activated sludge, a variety of harmful substances such as heavy metals may be introduced, threatening crop growth and human health, and limiting the direct application of activated sludge materials in agricultural production. Therefore, the process development of safe agricultural use of activated sludge and the evaluation of the actual application effect are the prerequisites for its safe application.

[0003] In recent years, a large number of studies have shown that there is a close interactive relationship between microorganisms and plants. For example, microorganisms provide plants with essential nutrients such as nitrogen, phosphorus, and potassium by decomposing organic matter and symbiotic nitrogen fixation (Singh et al., 2004, Trends in Microbiology, 12 (8), 386-393), and regulate plant growth and development by secreting growth hormones, reactive oxygen species, and plant enzymes (Eichmann et al., 2021, The Plant Journal, 105 (2), 518-541). It is worth noting that the technology of using the interaction between microorganisms and plants to regulate the absorption process of heavy metals in plants, thereby preventing the accumulation of heavy metals in plants and ensuring food safety has gradually become the focus of human research. Studies have shown that Pseudomonas aeruginosa, Pseudomonas fluorescens, can not only promote plant growth by producing siderophores, growth hormones, etc., but also can synthesize and secrete a variety of biological chelators to form stable chelates with heavy metal ions in the soil, converting them into a form that is difficult for plants to absorb (Fakhar et al., 2022, Critical Reviews in Environmental Science and Technology, 52 (11), 1868-1914.). Therefore, identifying a strain that can effectively prevent the transport of heavy metals in activated sludge to plants will provide important technical support for the safe agricultural use of activated sludge.

[0004] It is worth pointing out that although the reported microbial control of heavy metal accumulation technology can reduce the plant's uptake of heavy metal ions to a certain extent, it will also have a certain inhibitory effect on the absorption of other key divalent cation nutrients (such as Fe, Mn, Zn, Ca) in plants, thereby affecting plant growth and development, and has certain limitations. Therefore, if nutrient-rich sludge is applied in a coordinated manner, by improving the plant's own endogenous environment, such as plant hormone levels, nutrient status and other biological factors that regulate plant growth and heavy metal accumulation, the effect of microbial control of heavy metal accumulation can be optimized, while taking into account the improvement of farmland soil physical and chemical properties and fertility, which will help develop more efficient and feasible methods for safe and efficient application of activated sludge in farmland. Summary of the invention

[0005] In view of the above-mentioned technical problems and the shortcomings in the art, the present invention provides a method for promoting safe and efficient production in farmland by synergistically using activated sludge and bacterial agents.

[0006] The method of the present invention is a method for improving plant growth and preventing heavy metal accumulation in plants by using activated sludge and bacterial agents in a coordinated manner, which can solve the problem of heavy metal pollution in the application process of activated sludge. The application of activated sludge improves soil fertility, promotes plant absorption of key nutrients such as Ca, Mg, Fe, and Zn, and improves plant stress resistance. At the same time, Bacillus marisflavi is inoculated in coordination to increase the expression level of Raf-like protein kinase (APK) in plants, activate the Rafs-SnRK2 signal regulation pathway, and significantly inhibit the absorption of heavy metals such as Cd, Pb, Ni, and Cr in sludge by plants. The method of the present invention provides a green and economical improvement strategy for the safe agricultural use of activated sludge, and is an effective means to achieve safe and efficient production of farmland.

[0007] A method for promoting safe and efficient production in farmland by using activated sludge and bacterial agents in a coordinated manner, comprising:

[0008] The activated sludge and sawdust are mixed, fermented at 50-60° C. for 13-15 days (e.g., 14 days), and after the fermentation is completed, the incompletely decomposed large particles are removed by sieving to obtain a uniform and fine compost product, the compost product is mixed with a soil sample to obtain a cultivation matrix, crops are planted on the cultivation matrix, and during the growth of the crops, a bacterial solution of Bacillus marisflavi is inoculated into the cultivation matrix.

[0009] The present invention found that the application of activated sludge combined with Bacillus subtilis can increase the expression level of Raf-like protein kinase in plants, significantly inhibit the plant's uptake of heavy metals such as Cd, Cr, and Pb, and increase the plant's chlorophyll content and the content of key nutrients such as Ca, Mg, Fe, and Zn.

[0010] Compared with the group without activated sludge, the addition of activated sludge significantly improved the photosynthetic characteristics of the plants, the SPAD (Soil and Plant Analyzer Development) value increased by 95.6%, and the nutrient contents of Ca, Mg, Fe, and Zn increased by 106.7%, 42.8%, 166.7%, and 91.7%. Compared with the treatment of adding activated sludge alone, the activated sludge synergized with Bacillus marisflavi, on the basis of improving plant nutrients and photosynthesis, further reduced the accumulation of Cd, Hg, Pb, and Ni per plant in pakchoi by 49.4%, 45.9%, 18.7%, and 44.4%, respectively.

[0011] Therefore, the method of the present invention is feasible for optimizing the application effect of activated sludge in farmland soil.

[0012] In addition, the present invention further discovered that the Raf-like kinase target protein receptor antagonist AA1 inhibited the effect of activated sludge-Bacillus subtilis synergistically controlling heavy metals, indicating that the Rafs-SNRK2 signaling regulation pathway activated by Bacillus subtilis plays a key role in the above-mentioned mediation process.

[0013] In some embodiments, in the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland, the activated sludge may be municipal sludge and may have a water content of 50 wt% to 60 wt%.

[0014] In some embodiments, in the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland, the activated sludge and sawdust can be mixed in a volume ratio of 2.5 to 3.5:1 (eg, 3:1).

[0015] In some embodiments, the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland is performed by passing the activated sludge through a 10-mesh sieve after fermentation is completed.

[0016] In some embodiments, in the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland, the mass ratio of the compost product to the soil sample can be 2 to 8:100, for example, 4:100.

[0017] In some embodiments, in the method for promoting safe and efficient production of farmland by activated sludge-microbial agent synergistically, the soil sample can be a mixture of perlite and soil particles with a particle size of less than 100 mesh, which can make the soil sample have better air permeability. Furthermore, the volume ratio of soil particles to perlite can be 1:1.

[0018] In some embodiments, in the method of promoting safe and efficient production in farmland by synergistic activation of activated sludge and bacterial agents, the Bacillus marisflavi may be a strain with the German Collection of Microorganisms (DSMZ) accession number of DSM16204.

[0019] In some embodiments, in the method of promoting safe and efficient production in farmland by synergistic use of activated sludge and bacterial agents, the Bacillus marisflavi bacterial solution can be inoculated to a depth of 0.4 to 0.6 cm in the soil surface layer at the root of the crop.

[0020] In some embodiments, in the method for promoting safe and efficient production of farmland by synergistic action of activated sludge and bacterial agent, the cell concentration of the bacterial solution of Bacillus marisflavi can be 1×10 7 ~1×10 8 CFU mL -1 .

[0021] In some embodiments, in the method of promoting safe and efficient production in farmland by synergistic use of activated sludge and bacterial agents, the volume of the Bacillus marisflavi bacterial solution inoculated each time may be 20 to 30 mL / 2 crops.

[0022] In some embodiments, the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland may include inoculating the Bacillus marisflavi bacterial solution once a week for four consecutive weeks.

[0023] In some embodiments, in the method of promoting safe and efficient production in farmland by synergistic use of activated sludge and bacterial agents, the water content of the cultivation matrix is ​​maintained at 60wt% to 70wt%.

[0024] In some embodiments, the method of promoting safe and efficient production of farmland by synergistically using activated sludge and bacterial agents can control the photoperiod to 12h / 20-26°C (further 20-25°C) / d (day) and the light intensity to 50-60μmolphotons (photons) m -2 s -1 .

[0025] In some embodiments, in the method of using activated sludge and bacterial agents to synergistically promote safe and efficient production in farmland, the crop is Chinese cabbage.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention successfully realizes the application of activated sludge in promoting the safe and efficient production of crops such as pakchoi through the pretreatment of wastewater activated sludge and the preparation of compost cultivation medium, turning waste into treasure. When Bacillus spp. is not applied, the addition of activated sludge increases the nutrients Ca, Mg, Fe and Zn in pakchoi by 106.7%, 42.8%, 166.7% and 91.7% respectively.

[0028] 2. Compared with the treatment of adding activated sludge alone, the application of activated sludge synergistically with Bacillus subtilis can reduce the single-plant accumulation of heavy metals Cd, Hg, Pb, and Ni in plants by 49.4%, 45.9%, 18.7%, and 44.4%, respectively. This shows that the application of Bacillus subtilis can further prevent the accumulation of heavy metals in plants on the basis of ensuring the resource utilization of activated sludge.

[0029] In summary, the present invention can regulate the Rafs-SNRK2 signal regulation pathway by applying Bacillus subtilis to activated sludge, activate the plant stress signal response, and thus block the transfer of trace heavy metals in activated sludge to plants. This technology is expected to provide a new method for achieving the safe application of activated sludge in agricultural production processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figures are actual photos of the growth of pakchoi in a control group without activated sludge and experimental groups with different amounts of activated sludge after 8 weeks of operation with and without inoculation with Bacillus marisflavi bacterial solution in a specific implementation manner. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0032] The test plants were Suzhou green pakchoy, purchased from Qingxian Xingye Co., Ltd. (Qingxian, Hebei, China). The test soil was collected from the surface soil (0-20 cm) of wasteland in Sandun Town, Xihu District, Hangzhou City, Zhejiang Province, China, and was naturally air-dried for later use.

[0033] Soil sample preparation: After the test soil was coarsely ground, it was sieved with a 10-mesh (1.5 mm) sieve to remove gravel larger than 2 mm. It was further sieved with a 0.15 mm (100-mesh) sieve to obtain soil particles, which were mixed with perlite in a 1:1 volume ratio and used as soil samples for subsequent tests. The experimental pot had a hole diameter of 12.5 cm and a height of 11 cm. 300 g of soil sample was weighed for each pot.

[0034] Wastewater sludge pretreatment and compost cultivation medium preparation: First, the municipal sludge is dehydrated and pretreated to reduce its water content to 50wt%~60wt%, and sawdust is selected as the compost auxiliary material. The sludge and auxiliary materials are mixed in a volume ratio of 3:1 and loaded into a fermentation tank, and fermented at 50~60℃ for 2 weeks. After the fermentation is completed, the composted sludge is sieved, and a 10-mesh sieve is used to remove large particles that are not completely decomposed to obtain a uniform and fine compost product. The compost product is mixed with the above-mentioned soil sample (300g) in a ratio of 0, 2%, 4%, and 8% to ensure that the activated sludge and the soil are fully mixed, and it is placed in a pot as a cultivation medium for use, and is recorded as a control group, a 2% sludge experimental group, a 4% sludge experimental group, and an 8% sludge experimental group, respectively.

[0035] The test strain was Bacillus flavus (German Collection of Microorganisms (DSMZ), with the accession number DSM16204). -1 Peptone, 3 g L -1 Beef extract and 5g·L -1The cells were cultured in a Nutrient Agar (NA) liquid medium containing NaCl and pH 7.2 at a constant temperature of 150 rpm and 35°C for 2 days, then centrifuged at 3000×g for 10 min and washed twice with sterile 0.9 wt% saline. The concentration of 1×10 8 CFU mL -1 of bacterial suspension.

[0036] Sowing Chinese cabbage: dig a small hole every 3 to 5 cm on the surface of the cultivation medium, with a depth of about 0.5 to 1 cm. Put 3 to 4 Chinese cabbage seeds in each hole, and then cover the seeds with fine soil. There are about 10 seeds in total. After sowing, gently compact the soil covering the seeds to ensure that the seeds are in close contact with the soil. Use a spray bottle to evenly spray a thin layer of water on the soil surface to ensure that the seeds get enough water. On the 7th day, inoculate 30 mL of 10 8 CFU mL -1 The bacterial suspension was inoculated in the experimental group once a week according to the above operation, and the control group was inoculated with sterile water once a week according to the above operation, for a total of 4 weeks. After 4 weeks, the inoculation of bacterial suspension was stopped and cultivation was continued for a total of 8 weeks. This experiment was carried out in an artificial climate chamber with the following conditions: temperature 20-25°C, relative humidity 60%-70%, photoperiod 12h / d, light intensity 50-60μmol photons m -2 s -1 During the plant growth period, 50 mL of deionized water was added to the bottom of the tray every 2 days to maintain the soil's appropriate water holding capacity.

[0037] Sample harvesting and index determination: After 8 weeks, the plants were harvested, separated from the soil, and the soil adhering to the root surface was washed with tap water. The plant tissues were divided into glass weighing bottles, placed in an oven to kill green, and then dried at 80°C to constant weight. After weighing the dried samples, they were digested with high-grade pure HNO3 at 180°C until the solution was clear. After the digestion solution was fixed to 20mL, it was filtered through a 0.22μm filter membrane, and the Cd, Pb, Hg and Ni contents in the digestion solution were determined using a microwave plasma atomic emission spectrometer MP-AES (4210, Agilent Technologies, USA). The Cd, Pb, Hg, and Ni contents and the decrease in the body of a single Chinese cabbage were calculated.

[0038] Table 1 shows the effect of adding sludge on the nutrient content of Chinese cabbage in the above examples. The Chinese cabbage treated for 8 weeks was harvested, dried in an electric constant temperature drying oven at 65°C until the weight was constant, weighed with an electronic balance, and the dry weight (DW) of the plant was recorded. The Chinese cabbage plants were digested with nitric acid, and the obtained digestion solution was subjected to metal ion detection by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the application of sludge can significantly increase the content of Ca, Mg, Fe, and Zn nutrient ions in Chinese cabbage. Compared with the group without sludge addition, the Ca, Mg, Fe, and Zn in the Chinese cabbage treated with 8% composting sludge increased by 106.7%, 42.8%, 166.7%, and 91.7%, respectively.

[0039] Table 1

[0040]

[0041] Table 2 shows the effect of inoculating Bacillus yellow sea on the biomass and SPAD value of plants without sludge treatment (control group) and plants under sludge treatment in the above examples. The pakchoy treated for 8 weeks was harvested, and the soil and other impurities on the leaves were removed. The surface moisture of the plant samples was wiped clean with absorbent paper, placed in a container of known weight, and the fresh weight (FW) was weighed with an electronic balance (accuracy 0.0001g). Using a SPAD-502 chlorophyll meter, the plant leaves were clamped in the clamp of the instrument, and multiple leaves of the same leaf position on the plant were clamped separately while avoiding the main veins to ensure that the leaves were in good contact with the light source. The results show that sludge addition promotes the growth of pakchoy, increases its chlorophyll content, and improves photosynthetic activity.

[0042] Table 2

[0043]

[0044] Table 3 shows the effects of the above-mentioned embodiments on the accumulation of heavy metals in plants without sludge treatment (control group) and plants under sludge treatment. The pakchoi treated for 8 weeks were harvested, dried to constant weight, and the dried samples were digested with nitric acid, and metal ion detection was performed using an inductively coupled plasma mass spectrometer (ICP-MS). While the addition of sludge improves plant nutrients, it also increases the enrichment of heavy metal ions (Cd, Pb, Hg, Ni) in the plant body. However, the inoculation of Bacillus treatment significantly reduced the above-mentioned heavy metal content, indicating that the inoculation of Bacillus blocked the migration process of heavy metals in the sludge from the growth medium to the plant body.

[0045] Table 3

[0046]

[0047] Table 4 shows the effect of inoculation with Bacillus flavus on the expression level of APK in plants without sludge treatment (control group) and plants under sludge treatment in the above examples. Young and tender tissues of pakchoi treated with bacteria for one week were taken, and total RNA was extracted using RNAiso Plus (TaKaRa, Otsu, Japan), and the first-strand cDNA was synthesized using PrimeScript RT Kit (TaKaRa, Otsu, Japan), and qRT-PCR analysis was performed using SYBR Green RT-PCR Kit (Vazyme, Nanjing, China). Quantitative PCR analysis used a StepOne Plus instrument (ABI, CA, USA), and the relative transcription abundance was calculated using the comparative cycle threshold method, and the results were normalized using Actin2 as the internal reference gene. The results showed that inoculation with Bacillus flavus induced the expression level of Raf-like protein kinase (APK) in the plant.

[0048] Table 4

[0049]

[0050] According to the method of the above embodiment, after the plants of the 8% sludge experimental group were treated with Bacillus flavus, 100 μM of APK target protein receptor antagonist (AA1) (F0544-0152) was sprayed on the leaves of the cabbage, and the changes in the heavy metal content of the cabbage were measured. The results showed that spraying the receptor inhibitor weakened the control effect of the inoculation on heavy metals, proving that the control of heavy metal accumulation by inoculation with Bacillus flavus was achieved by inducing the expression of Raf-like protein kinase (APK) and the activation of its downstream signaling pathways.

[0051] Table 5

[0052]

[0053] In summary, the present invention provides a method for promoting safe and efficient production of farmland by synergistically using activated sludge and Bacillus marisflavi, including: mixing composted activated sludge into the cultivation matrix of crops such as pakchoi, and inoculating Bacillus marisflavi bacterial liquid into the cultivation matrix during the growth of the crops. The present invention induces the Rafs-SNRK2 signal regulation pathway in the plant body by Bacillus marisflavi, effectively blocks the accumulation of heavy metals in the plant, promotes crop growth, and reduces the use of pesticides and fertilizers. This technology is expected to provide a new method for realizing the resource utilization of activated sludge and the research on safe and efficient production of farmland.

[0054] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for promoting safe and efficient production in farmland by using activated sludge and bacterial agents, characterized in that: include: The activated sludge and sawdust are mixed and fermented at 50-60°C for 13-15 days. After the fermentation is completed, the large particles that are not completely decomposed are sieved to obtain a uniform and fine compost product. The compost product is mixed with a soil sample to obtain a cultivation matrix, and crops are planted on the cultivation matrix. During the growth of the crops, a solution of Bacillus marisflavi is inoculated into the cultivation matrix.

2. The method according to claim 1, characterized in that The activated sludge is municipal sludge with a water content of 50wt% to 60wt%; The activated sludge and sawdust are mixed in a volume ratio of 2.5 to 3.5:1; After fermentation is completed, pass through a 10-mesh sieve; The mass ratio of compost product to soil sample is 2 to 8:100; The soil sample was a mixture of perlite and soil particles with a particle size less than 100 mesh; the volume ratio of soil particles to perlite was 1:

1.

3. The method according to claim 1, characterized in that The yellow sea bacillus (Bacillusmarisflavi) is a strain with the preservation number of DSM 16204 of the German Microbiological Culture Collection Center.

4. The method according to claim 1, characterized in that: The Bacillus marisflavi bacterial solution is inoculated to a depth of 0.4 to 0.6 cm on the soil surface at the root of the crop.

5. The method according to claim 1, characterized in that The cell concentration of the Bacillus marisflavi bacterial solution is 1×10 7 ~1×10 8 CFU mL -1 .

6. The method according to claim 1, characterized in that The volume of the Bacillus maris flavi bacterial solution inoculated each time is 20-30 mL / 2 plants.

7. The method according to claim 1, characterized in that The bacterial liquid of Bacillus marisflavi was inoculated once a week for 4 consecutive weeks.

8. The method according to claim 1, characterized in that The water content of the cultivation substrate is maintained at 60wt% to 70wt%.

9. The method according to claim 1, characterized in that: During the crop growth period, the photoperiod was controlled at 12 h / 20-26 °C / d and the light intensity was 50-60 μmol photons m -2 s -1 .

10. The method according to claim 1, characterized in that The crop is Chinese cabbage.

Images