A Yunnan micrococcus and its application in passivating heavy metals in livestock and poultry manure treatment

Abstract

The present invention relates to a Yunnan micrococcus and its application in passivating heavy metals in livestock and poultry excrement treatment, belonging to the field of heavy metal pollution remediation. The present invention provides Yunnan micrococcus TW001, and a microbial preparation is obtained after strain activation, liquid seed preparation and liquid fermentation. The Yunnan micrococcus TW001 microbial preparation for poultry excrement passivation treatment provided by the present invention is used in soil heavy metal passivation treatment, has the advantages of fast strain growth rate, high fermentation density, strong genetic stability, good compatibility with the environment, and passivation effect on multiple heavy metals such as arsenic, cadmium and mercury, etc., is a good heavy metal biological passivator, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to Yunnan micrococcus and its application, including a microbial preparation of Yunnan micrococcus and its preparation method and application, and in particular to the application of Yunnan micrococcus TW001 in the harmless treatment of poultry feces and a microbial preparation containing Yunnan micrococcus and its preparation method and application, which belongs to the field of heavy metal pollution remediation. Background Art

[0002] With the growth of my country's population, the acceleration of industrialization and the improvement of per capita living standards, a large number of industrial and agricultural production and living pollutants have entered the soil, causing serious pollution to the soil. The mining of non-ferrous metal mines, the discharge of industrial "three wastes", the accumulation of heavy metal wastes, sewage irrigation in agricultural production, and the irrational use of agricultural chemicals can all lead to the direct or indirect entry of harmful heavy metal elements into crop production. According to statistics, the area of ​​arable land in my country with excessive heavy metal content in the soil is about 300 million mu, and 12 million tons of grain are contaminated by heavy metals in the soil every year, causing losses of up to 20 billion yuan each year. Heavy metal pollution destroys soil functions, causes the degradation of arable land, and seriously affects the yield of my country's crops. Some heavy metal ions enter food through the food chain, affecting food safety, and some enter surface water and groundwater, threatening human drinking water safety.

[0003] Heavy metal pollution is not only related to its content, but also to its form. The mobility and bioavailability of heavy metals in different forms are different. According to Tessier's report, heavy metal forms can be divided into exchangeable, carbonate-bound, iron-manganese-oxidation-bound, organic-bound and residual forms. Exchangeable heavy metals are sensitive to environmental changes, easy to migrate and transform, can be absorbed by plants, and have high bioavailability; carbonate-bound, iron-manganese-oxidation-bound and organic-bound forms are not bioavailable under normal circumstances; residual heavy metals are generally stable in nature, in a passivated state in the soil for a long time, and are not easily absorbed by plants. Therefore, in recent years, some researchers have tried to change the form of heavy metals in the environment through some treatment methods, so that they can be transformed from a bioavailable state to a biounavailable state. Bin Dong et al. found that high-solid anaerobic digestion increased the bioavailability of Cu, Zn, Ni and Cr, while reducing the bioavailability of Pb. Baoyi Lv et al. found that earthworm composting reduced the mobility and availability of heavy metals and increased the relative content of unavailable forms of heavy metals. Lina Liu et al. found that composting can reduce 70% of the exchangeable Cd in the soil, fix the Cd in the soil, and can be used to repair soil contaminated by Cd. Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd. reported a strain of Sinensis KY592 that has a passivating effect on heavy metal cadmium and its application. It can reduce the cadmium content in water or soil through the adsorption and passivation of cadmium by the strain, thereby reducing the toxicity of cadmium to corn.

[0004] Micrococcus yunnanensis is a species of Micrococcus. It was originally isolated as an endophyte from the roots of Polyspora axillaris. It has the functions of salt tolerance, nitrogen fixation, and improving plant disease resistance. Patent No. CN201410167816.3 discloses a Yunnan Micrococcus and a microbial agent and their application, which has the function of improving the disease resistance of plants under high salt conditions. Patent CN201610960300.3 discloses a nitrogen-fixing bacterium that produces ACC deaminase and its application in soil ecological restoration. The above patents currently have the following problems: 1. The strain has poor environmental adaptability and is difficult to survive in high temperature environments; 2. The passivation effect on various heavy metals such as arsenic, cadmium and mercury needs to be improved. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a preparation method and application of a Yunnan Micrococcus TW001 microbial preparation for passivation treatment of poultry feces, the strain has fast growth rate, high fermentation density, strong genetic stability, good environmental compatibility, and significant passivation effect on a variety of heavy metals.

[0006] The present invention provides a Yunnan Micrococcus TW001, which is classified and named Micrococcus yunnanensis TW001, separated from farmland soil in Yantai, and preserved in China Center for Type Culture Collection. The preservation date is June 1, 2022, and the preservation number is: CCTCC NO: M 2022768. The 16SrRNA gene sequence of the Yunnan Micrococcus TW001 has a length of 1395bp, as specifically shown in SEQ NO.1.

[0007] The application of the Yunnan Micrococcus TW001 in passivating heavy metals in livestock and poultry manure treatment.

[0008] Meanwhile, the present invention provides a microbial preparation comprising Yunnan Micrococcus TW001.

[0009] The microbial preparation is used to passivate heavy metals in soil.

[0010] The preparation method of the microbial preparation specifically comprises the following steps:

[0011] (1) Strain activation: Aseptically open Yunnan Micrococcus TW001, pick the strain and streak it onto the solid slant culture medium, and culture it in a constant temperature incubator at 30°C for 36 hours. When the slant is full of colonies, it is used to transfer liquid seeds;

[0012] (2) Preparation of liquid seeds: Select the activated strain and inoculate it into a container containing seed culture medium, seal it with eight layers of gauze, and culture it in a shaking incubator at 30°C and 160-200 rpm for 36 hours to grow a large number of bacterial cells to obtain liquid seeds;

[0013] (3) Liquid fermentation: The liquid seeds prepared in step (2) were inoculated into the fermentation medium at a volume ratio of 1-10%, the rotation speed was 300-700 r / min, the dissolved oxygen was controlled at 20%-50%, the pH was 7.0-7.2, the temperature was 30° C., and the aeration culture was carried out for 48 hours to obtain the fermentation liquid of strain TW001.

[0014] Preferably, the solid slant culture medium comprises: 30 g / L tryptone soy broth, 3 g / L yeast extract, 15 g / L agar, dissolved in distilled water to a constant volume, pH 7.0-7.2, and sterilized by high pressure steam at 121° C. for 20 min.

[0015] Preferably, the seed culture medium comprises: 17 g / L trypticase, 3 g / L soy peptone, 2.5 g / L glucose, 0.3 g / L yeast extract, 5 g / L sodium chloride, 2.5 g / L K2HPO4, dissolved in distilled water to a constant volume, pH 7.0-7.2, and sterilized with high-pressure steam at 121°C for 20 min.

[0016] Preferably, the fermentation medium comprises: 10-20 g / L tryptone, 5-10 g / L soy peptone, 1-3 g / L glucose, 1-5 g / L yeast extract, 0.1-0.5 g / L MgSO4, 0.1-1 g / L KH2PO4, 2-5 g / L NaCl, dissolved in distilled water to a constant volume, pH 7.0-7.2, and sterilized with high pressure steam at 121°C for 20 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The Yunnan Micrococcus TW001 and its microbial preparation of the present invention have been screened and repeatedly verified to be high temperature resistant, fast-growing, high fermentation density, strong passivation ability, and obvious passivation effect on heavy metals such as arsenic, cadmium and mercury. Experiments have shown that the microbial preparation prepared by the bacteria has a significant heavy metal passivation effect, is an environmentally friendly preparation with great application prospects, and is a good heavy metal biological passivator;

[0019] 2. The Yunnan Micrococcus TW001 and its microbial preparation of the present invention have strong genetic stability. After multiple continuous passages, the growth rate, fermentation density and passivation ability are not significantly different from those of the original strain, and the genetic stability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the microscopic morphology of Yunnan Micrococcus TW001.

[0021] Figure 2 This is the phylogenetic tree of Micrococcus yunnanensis TW001 based on 16S rRNA sequences. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0023] Example 1 Isolation and screening of strain TW001

[0024] (1) Separation

[0025] 10g of vegetable soil from a long-term use of black soldier fly sand organic fertilizer was selected and dissolved in 90mL of sterile water. The sample was shaken to mix the sample with water thoroughly, and then treated in a boiling water bath for 1 minute after gradient dilution. The sample was then spread on a beef extract peptone medium plate. The plate was placed in a 30°C incubator for 3 days, and the colonies of different forms on the medium were streaked on the beef extract peptone medium plate. The strains obtained were purified and numbered and stored.

[0026] The components of beef extract peptone culture medium include: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, agar 18g / L, pH 7.2, and high-pressure steam sterilization at 121°C for 20min.

[0027] (2) Screening

[0028] The strains obtained in the initial screening were cultured with beef extract peptone liquid culture medium (the above formula does not contain agar) until the bacterial concentration OD600 was 1.0, HgCl2 solution was added to the final concentration of 10-100 mg / L, and the constant temperature shaking culture was continued for 24 hours. The bacterial concentration OD600 was detected by visible spectrophotometry, the bacteria were removed by centrifugation and the supernatant was taken. The Hg2+ concentration in the supernatant was determined by inductively coupled plasma emission spectrometry, and the strains with OD600 values ​​higher than 1.0 and significantly reduced Hg2+ concentrations were selected.

[0029] Through extensive screening and repeated verification, a strain of Yunnan Micrococcus TW001 was obtained that can passivate heavy metal mercury. Experiments have shown that the microbial preparation prepared from this bacterium has a significant heavy metal passivation effect and is an environmentally friendly preparation with great application prospects.

[0030] Example 2 Identification of Yunnan Micrococcus TW001 strain

[0031] After culturing Yunnan Micrococcus TW001 on beef extract peptone medium at 30°C for 2 days, the colonies were yellow, round and opaque, with a smooth, moist and raised surface, regular edges and no pigment. Figure 1As shown, the bacteria were observed to be spherical, without spores, unable to move, and Gram-positive by microscopic examination. As shown in Table 1, compared with the existing Yunnan Micrococcus YIM65004T, the strain TW001 did not grow at 4°C and 6.5% sodium chloride, but grew at 45°C, and could tolerate temperatures above 60°C or even 100°C.

[0032] Table 1 Physiological and biochemical characteristics of strain TW001

[0033]

[0034]

[0035] (2) Molecular identification of Yunnan Micrococcus TW001 strain

[0036] The length of the 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing is 1395bp. BLAST comparison on GenBank showed that the strain had a base similarity of 99.85% with Micrococcus yunnanensis (NR116578). Combined with morphological and physiological and biochemical indicators, the strain was identified as Micrococcus yunnanensis. The 16S rRNA sequence information is shown in SEQ NO.1. The phylogenetic tree based on the 16S rRNA sequence is shown in Figure 2 shown.

[0037] SEQ NO.1 Sequence Listing

[0038]

[0039] (3) Genetic stability

[0040] Yunnan Micrococcus TW001 was subcultured 50 times on solid slant medium by streak method. The bacterial morphology, growth rate and heavy metal passivation ability were determined. There was no significant difference with the original strain and the genetic stability was good.

[0041] (4) Preservation of bacterial strains

[0042] The Yunnan Micrococcus TW001 described in this invention has been deposited in the China Center for Type Culture Collection on June 1, 2022, with the deposit number: CCTCC NO:M 2022768. The address of the deposit unit is: China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0043] Example 3 Preparation method of microbial preparation

[0044] (1) Strain activation

[0045] Pick the bacterial strain and streak it onto the solid slant culture medium, 30 g / L tryptone soy broth, 3 g / L yeast extract, 15 g / L agar, dissolve it in distilled water to make up the volume, pH 7.2, sterilize it with high-pressure steam at 121℃ for 20 min, and culture it in a constant temperature incubator at 30℃ for 36 hours. Wait until a large number of bacterial cells grow on the slant for transfer of liquid seeds.

[0046] (2) Liquid seed preparation

[0047] The activated strain was selected and inoculated into a medium containing seed culture medium (17 g / L trypticase, 3 g / L soy peptone, 2.5 g / L glucose, 0.3 g / L yeast extract powder, 5 g / L sodium chloride, 2.5 g / L K2HPO4, dissolved in distilled water to make up the volume, pH 7.2, sterilized by high-pressure steam at 121°C for 20 min), sealed with gauze, and cultured in a shaker at 30°C and 180 r / min for 36 hours to grow a large number of bacteria and obtain liquid seeds.

[0048] (3) Liquid fermentation

[0049] The liquid seeds prepared in step (2) were inoculated into a fermentation medium (tryptone 10 g / L, soy peptone 5 g / L, glucose 2.5 g / L, yeast extract 5 g / L, MgSO4 0.5 g / L, KH2PO4 1 g / L, NaCl 5 g / L, pH 7.2, and sterilized by high-pressure steam at 121°C for 20 min) at a volume ratio of 5%, the dissolved oxygen was controlled at 30%, the pH was 7.2, the temperature was 30°C, and the culture was aerated for 48 hours.

[0050] Example 4 Experiment on the passivation of heavy metals by Yunnan Micrococcus TW001 strain in chicken manure treatment

[0051] 2000 kg of fresh chicken manure was added with appropriate amount of sawdust and mixed well. The water content was adjusted to about 60%. The fermentation liquid of strain TW001 was inoculated with 0.5% inoculum and mixed well. The compost was fermented for 10 days. The compost was turned twice on the 2nd and 4th days. The chicken manure samples before and after fermentation were taken. The chemical forms of heavy metals arsenic, cadmium and mercury were analyzed by Tessier continuous extraction method. The exchangeable state, carbonate-bound state, iron-manganese oxide-bound state, organic-bound state and residual state were obtained respectively. The chemical forms of heavy metals arsenic, cadmium and mercury were analyzed by ICPE-9000 spectrometer (Shimadz u, Japan) was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). Heavy metal standards were provided by Beijing Wanjia Shouhua Biotechnology Co., Ltd., the National Center for Standard Materials. The results are shown in Table 2. In the test group with strain TW001 added, the exchangeable contents of heavy metals arsenic, cadmium and mercury in chicken manure after composting were significantly lower than those in the control group, and most of them were converted into bound and residual states, especially the residual state content was significantly higher than that in the control group, indicating that strain TW001 had a significant passivation effect on heavy metals in chicken manure.

[0052] Table 2 Changes in heavy metal content in chicken manure after composting with Yunnan Micrococcus TW001 strain

[0053]

[0054] Example 5 Field test effect of Yunnan Micrococcus TW001 microbial preparation on passivation of heavy metals in soil

[0055] A common apple orchard was selected and divided into two plots of equal size. One plot was the control group, and ordinary chicken manure fermented organic fertilizer was applied; the second plot was the treatment group, and the microbial preparation of Yunnan Micrococcus TW001 strain was added to the above ordinary chicken manure fermented organic fertilizer to a content of 200 million / g, and then applied. Both groups applied fertilizer in October of the first year, with an application rate of 2 tons per mu, and the fertilization method was a combination of strip ditch application and whole garden broadcasting. Soil samples were collected in September of the second year, and the chemical forms of heavy metal arsenic, cadmium and mercury were analyzed by Tessier continuous extraction method, and determined by inductively coupled plasma emission spectrometry (ICP-OES) using ICPE-9000 spectrometer (Shimadzu, Japan). The heavy metal standards were provided by Beijing Wanjia Shouhua Biotechnology Co., Ltd., National Standard Material Center. The results are shown in Table 3. The treatment group with Yunnan Micrococcus TW001 was found to have a significantly higher passivation rate of heavy metal arsenic, cadmium and mercury in the plots where the fermentation liquid of Yunnan Micrococcus TW001 strain was applied than that in the control group where the bacterial liquid was not applied.

[0056] Table 3 Effect of Yunnan Micrococcus TW001 strain microbial preparation on passivation of heavy metals in soil

[0057]

[0058] The specific embodiments of the present invention are described in detail above, but are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A Yunnan Micrococcus TW001, classified and named Micrococcus yunnanensis TW001, isolated from farmland soil in Yantai, and deposited in the China Center for Type Culture Collection. The preservation date is June 1, 2022, and the preservation number is: CCTCCNO: M 2022768. The 16S rRNA gene sequence of the Yunnan Micrococcus TW001 is 1395bp in length, and the gene sequence is shown in SEQ NO.

1.

2. Use of the Yunnan Micrococcus TW001 described in claim 1 to passivate any one of arsenic, cadmium and mercury in chicken manure treatment.

3. A microbial preparation comprising the Yunnan Micrococcus TW001 according to claim 1.

4. Use of the microbial preparation according to claim 3 to passivate any one of arsenic, cadmium and mercury after treating chicken manure in soil.

5. The method for preparing the microbial preparation according to claim 3, characterized in that: The specific steps include: (1) Strain activation: Aseptically open Yunnan Micrococcus TW001, pick the strain and streak it onto the solid slant culture medium, and culture it in a constant temperature incubator at 30°C for 36 hours. When the slant is full of colonies, it is used to transfer liquid seeds; (2) Preparation of liquid seeds: Select the activated strain and inoculate it into a container containing seed culture medium, seal it with eight layers of gauze, and culture it in a shaking incubator at 30°C and 160-200 rpm for 36 hours to grow a large number of bacterial cells to obtain liquid seeds; (3) Liquid fermentation: The liquid seeds prepared in step (2) were inoculated into the fermentation medium at a volume ratio of 1-10%, the rotation speed was 300-700 r / min, the dissolved oxygen was controlled at 20%-50%, the pH was 7.0-7.2, the temperature was 30° C., and the aeration culture was carried out for 48 hours to obtain the fermentation liquid of strain TW001; The solid slant medium: 30 g / L tryptone soy broth, 3 g / L yeast extract, 15 g / L agar, dissolved in distilled water to make up the volume, the pH value was adjusted to 7.0-7.2, and sterilized at 121° C. high pressure steam for 20 min; The seed culture medium: 17 g / L trypticase, 3 g / L soy peptone, 2.5 g / L glucose, 0.3 g / L yeast extract, 5 g / L sodium chloride, 2.5 g / L K2HPO4, dissolved in distilled water to a constant volume, pH 7.0-7.2, sterilized with high pressure steam at 121°C for 20 min; The fermentation medium comprises: 10-20 g / L tryptone, 5-10 g / L soy peptone, 1-3 g / L glucose, 1-5 g / L yeast extract, 0.1-0.5 g / L MgSO4, 0.1-1 g / L KH2PO4, 2-5 g / L NaCl, dissolved in distilled water to a constant volume, pH 7.0-7.2, and sterilized with high pressure steam at 121° C. for 20 min.

Images