AU2020103175A4

AU2020103175A4 - An application of silkworm excrement in the preparation of a pesticide-degrading bacteria preparation

Info

Publication number: AU2020103175A4
Application number: AU2020103175A
Authority: AU
Inventors: Jie Liu; Hongxia YANG; Guohua Zhong
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2021-01-14
Anticipated expiration: 2028-11-02

Abstract

The invention discloses an application of silkworm excrement in the preparation of a pesticide-degrading bacteria preparation, and also provides a pesticide-degrading bacteria preparation and its preparation method and its application. The pesticide-degrading bacteria preparation contains the following components: (1) Strains that can degrade pesticides (such as the HZ-2 strain of Bacillus pumilus). The strain depository is the China Type Culture Collection, and the preservation number is It is CCTCC NO: M 2013380; As for the BSF1 strain of Bacillus subtilis, of which the strain depository is the China Type Culture Collection, and the deposit number is CCTCC NO: AB 2014103; (2) Microbial preparation carrier: the aforementioned silkworm excrement. The pesticide-degrading bacteria preparation of the present invention uses silkworm excrement as the microbial preparation carrier, which can greatly improve the utilization rate of silk industrial waste, it has low production cost, and it can provide a stable carrier for the microorganisms in the preparation, in order to ensure the persistence of the preparation. The pesticide degrading bacteria preparation of the invention has simple operation and good degradation effect, and it has good promotion and application value. -1/3 LP 5/11/2018 HV cur det mode mag [B WD HPN 10 pm 5:35:31 PM 5.00 kV 25p TL SE 5 000 x 3.8mm 41.4 pm SCAU(Verios 460) Figure 1. F -r 5/11/2018 HV cur det mode mag ED WD HPIN - 5pm 4:40:32 PM |5.00 kV 25 -pA' TLD SE 9 70 x 5.1 mm 20.8 pm SCAU(Verios 460) Figure 2.

Description

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LP

/11/2018 HV cur det mode mag [B WD HPN 10 pm :35:31 PM 5.00 kV 25p TL SE 5 000 x 3.8mm 41.4 pm SCAU(Verios 460)

Figure 1.

F -r

/11/2018 HV cur det mode mag ED WD HPIN - 5pm 4:40:32 PM |5.00 kV 25-pA' TLD SE 9 70 x 5.1 mm 20.8 pm SCAU(Verios 460)

Figure 2.

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

An application of silkworm excrement in the preparation of a pesticide-degrading bacteria

preparation

The invention is described in the following statement:-

An application of silkworm excrement in the preparation of a pesticide-degrading

bacteria preparation

TECHNICAL FIELD

The invention relates to the technical field of microbial agent and pesticide

degradation. More specifically, the invention relates to a pesticide degrading bacteria

preparation and a preparation method and its application.

BACKGROUND

China is a big agricultural country, and the production and consumption of pesticides

account for one tenth of the world total. In modern agricultural production, the government

and agricultural scientists strongly advocate the adoption of Integrated Pest Management

(IPM), in order to change the old mode of destroying ecological environment in exchange

for economic development, and developing agricultural production and economy steadily

while preventing and controlling pests and diseases sustainably. However, the new

varieties of pesticides and the increasing output of pesticides have revealed an unavoidable

fact. Chemicals still occupy an unshakable and important position in the control of crop

diseases, insect pests and weeds. Studies have shown that most of the pesticides applied to

farmland will remain in the soil environment medium. Although pesticides themselves

have different degrees of natural degradation characteristics, long-term large-scale use will

inevitably lead to environmental pollution; In addition, some pesticides have a long

degradation cycle due to their own characteristics, and some kinds of their degradation products are more toxic. Therefore, how to reduce or eliminate the accumulation of pesticides in the environment, so as to prevent the occurrence of ecological security problems, has become an urgent problem to be solved in many countries and regions.

Microbial remediation of contaminated soil is a new method as well as a new way to

solve the problem of pesticide residue enrichment. The natural microbial resources are

extremely rich. Microbial remediation has great potential for remediation of pesticide

pollution in ecological environment because of its advantages of less secondary pollution,

simple operation and low cost, which is worthy of further research and development.

However, the technical difficulty of microbial remediation of pesticide contaminated soil

lies in the development of microbial agents. How to concentrate and fix the active

microorganisms in the preparation to ensure the activity of microorganisms, while being

friendly to the environment and harmless to people and animals, is the breakthrough and

key point of developing pesticide degrading bacteria preparation. Because the influence of

various additives in traditional pesticide formulations on microbial organisms is not clear,

the preparation of traditional pesticide formulations is not completely suitable for the

development of microbial formulations.

China is the birthplace of the world silk industry, with abundant resources and

remarkable advantages. Feces discharged from silkworm rearing, leftover mulberry and

bedding in silkworm seats are collectively called silkworm excrement, which is a multi

component substance rich in crude protein and carbohydrate. The dried silkworm

excrement contains 15.4% of crude protein, 3.9% of crude fat, 36.2% of nitrogen-free

extract, 19.6% of crude fiber, trace elements such as copper, iron and zinc, and it is rich in

chlorophyll, pectin, flavonoids, alkaloids and adrenocorticoid hormones. At present, the research on the reuse of silkworm excrement mainly focuses on the extraction of various effective components, such as chlorophyll and pectin. Because silkworm excrement also contains a certain amount of plant growth hormone indoleacetic acid analogues, it has become a hot spot in the industry to use silkworm excrement as raw material to develop nutrient-rich organic fertilizer technology. It is not difficult to find that silkworm excrement is a resource with low cost, environmental protection and great development value, but its application in pesticide degrading bacteria preparation has not been reported.

SUMMARY

The technical problem to be solved by the invention is to overcome the technical

shortage of the existing pesticide degrading bacteria preparation, and provide an

application of silkworm excrement in the preparation for it.

Another object of the invention is to provide a pesticide degrading bacteria preparation.

Another object of the invention is to provide a preparation method of the pesticide

degrading bacteria preparation.

Further, the invention is also to provide the application of the pesticide degrading

bacteria preparation.

The objects of the invention is realized by the following technical scheme:

The invention provides the application of silkworm excrement in the preparation of

pesticide degrading bacteria preparation. The invention makes silkworm excrement into

carrier required for microbial preparation, which can not only realize energy reuse, but also can reduce the risk of biological agent affecting environment.

The invention also provides a pesticide degrading bacteria preparation, which

comprises the following components:

The strain described in component (1) can be a conventional strain that can degrade

pesticides. Preferably, the present invention provides two most preferred technical

solutions. The strains are respectively the HZ-2 strain of Bacillus pumilus, of which the

strain depository is the China Type Culture Collection, and the strain preservation number

is CCTCC NO: M 2013380, and BSF01 strain of Bacillus subtilis. The strain depository is

the China Type Culture Collection, and the deposit number is CCTCC NO: AB 2014103.

The silkworm excrement of component (2) is the silk industrial waste silkworm

excrement or the silkworm excrement obtained by other ways.

The pesticide degradation bacteria agent of the invention can achieve a very good

effect by using common silkworm excrement. Preferably, the silkworm excrement is

physically crushed to a particle size of 20-200 mesh or after chemical modification. The

modified silkworm excrement has better water absorption and sustained-release property.

Si. Firing with the muffle furnace filled with nitrogen and anoxic carbonization for 5

h at 700 °C. After cooling, taking out the silkworm excrement, which is mixed and grind

through a 100-mesh sieve. After cooling, taking it out, then which is mixed, ground, and

screened by 100-mesh sieve.

S2. Treating the silkworm excrement with 0.1 mol-L-1 of phosphoric acid solution for

hours, which is repeated for 3 times to remove the ash. After the complete filtration,

rinsing the material with deionized water to neutrality, then putting it in the desiccator for drying at 60 °C. It is recorded it as carbonized silkworm excrement (MCS) and stored.

Preferably, the pesticide degrading bacteria preparation also contains auxiliary

materials, which are industrial waste glucose or molasses; According to the mass

percentage, the concentration of the auxiliary material is 1%-95%.

The present invention uses waste molasses or glucose as the auxiliary material of the

pesticide degrading bacterial agent, which provides a stable and targeted carbon source for

the microorganisms in the bacterial agent to ensure the efficacy of the formulation. At the

same time, it greatly improves the utilization rate of industrial waste.

The invention also provides a preparation method of the pesticide degrading bacteria

preparation, which comprises the following steps:

Si. Activating the pesticide-degrading bacteria, which is inoculated in liquid LB

medium, and cultivated to the logarithmic phase with shaking;

S2. Centrifuging the strain liquid obtained from SI, removing the supernatant, and

adding sterile water to prepare the strain liquid;

S3. Putting the strain obtained from S2 into the silkworm excrement. Respectively

performing sealing, shaking and biofilm culturing by shaking, centrifuging, vacuum drying

at room temperature to nearly dryness, and finally sub-packaging and sealing.

Preferably, the strain liquid obtained by S2 is put into silkworm excrement, and adding

the auxiliary material industrial waste glucose or molasses.

After activation, the strain Si is activated and degraded by the conventional method.

Preferably, the culture method provided by the invention is as follows: according to

-'7

the properties of different kinds of degradation bacteria, the method of freezing

preservation can be adopted, the strains of pesticide degradation bacteria are activated, the

strains are selected by the inoculation needle for inoculation in the inclined medium, and it

is cultured in the biochemical incubator of 28 °C. After the strain grows on and fully covers

the slope, it is moved to the 4 °C refrigerator for preservation. The bacteria can be preserved

in liquid paraffin. The bacteria can be cultured in the medium to obtain robust bacteria or

spores. Using the sterile straw to suck the sterilized liquid paraffin and injecting it into the

slope where the bacteria have grown. The dosage should be 1cm above the top of the slope

to isolate the bacteria from the air. The test tube should be kept upright and stored at 4 °C.

Selecting suitable culture medium for strains. Preparing LB medium according to the

following formula: sodium chloride: 10 g/L; peptone: 10 g/L; yeast extract: 5 g/L;

Preparing PDA medium according to the following formula: potato: 200 g/L; glucose: 20

g /L. Preparing Chappey's medium according to the following formula: sodium nitrate: 2

g/L; potassium chloride: 0.5 g/L; magnesium sulfate: 0.5 g/L; dipotassium hydrogen

phosphate: 1.0 g/L; iron sulfate: 0.01 g/ L; sucrose: 30.0 g/L. In the liquid culture medium

with pH 7.0, 1% wet bacteria are inoculated and cultured in the gas bath oscillator at

constant temperature for 1 ~ 7 days.

The amount of sterile water added in S2 is 10 ~ 20ml.

The amount of strain liquid, silkworm excrement and auxiliary materials mentioned

in S3 is determined according to the following proportion: in each Ig silkworm excrement,

the inoculation amount of 1107 ~ 110 cfu / ml is added into S2 to obtain 1 ml ~ 5ml of

strain liquid; According to the mass percentage content, adding 1% ~ 95% waste molasses

or glucose.

The method of shaking film formation described in S3 is as follows: the bacteria are

cultured in the air bath thermostatic oscillator for 24-48 hours, so that the bacteria are

biofilm cultured in the silkworm excrement.

The invention provides an application of the pesticide degrading bacteria preparation

in degrading pesticides.

Preferably, when the strain in the pesticide degrading bacteria preparation is Bacillus

pumilus HZ-2 strain, the pesticide is mesotrione; And when that strain in the pesticide

degrade bacteria preparation is Bacillus subtilis BSFO1 strain, the pesticide is beta

cypermethrin.

The invention has the following beneficial effects:

The invention provides a brand-new application for industrial waste silkworm

excrement, which is applied to preparing pesticide degrading bacteria preparation. In the

preparation process of the pesticide degrading bacteria preparation, silkworm excrement is

used as the carrier of the microbial preparation. It not only realizes the energy reuse, low

carbon environmental protection, low cost, rich resources and easy operation, but also

reduces the risk of biological agents affecting the environment, without secondary pollution,

and provides new carrier materials for the development of microbial preparations.

The pesticide degradation bacteria preparation provided by the invention can reduce

the pesticide residues by more than 70%. It has a high degradation effect, and successfully

resolves the contradiction between the use of herbicides and the elimination of

environmental enrichment of pesticides in the process of planting agricultural products,

which is of great significance to ensure the safety of crops, soil, water, non-target organisms and ecological environment.

The invention further provides a new application of industrial waste glucose or

molasses. The industrial waste glucose or molasses is added as an auxiliary material of the

pesticide degrading bacteria preparation, which greatly improves the utilization rate of

industrial waste, provides a stable carbon source for microorganisms in the preparation,

and can ensure the persistence of the preparation. The microbial inoculum still has a high

degradation rate after being applied for 14 days, which keeps the target pesticide residue

in the field at a low level.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the surface morphology of silkworm excrement before bacteria fixation

under environmental electron microscope.

Fig. 2 shows the surface morphology of silkworm excrement after scanning and

immobilizing bacteria under environmental electron microscope.

Fig. 3 shows the degradation of mesotrione by degrading bacteria preparation in LB

medium.

Fig. 4 shows the degradation of beta-cypermethrinby degrading bacteria preparation

in LB medium.

Fig. 5 is the comparison of the field degradation of mesotrione by bacteria preparation

and bacteria solution.

Fig. 6 is the comparison of the field degradation of beta-cypermethrin by degrading bacteria preparation and bacteria solution.

DESCRIPTION OF THE INVENTION

The present invention is further described in combination with the drawings and

specific embodiments, but the embodiment does not define the invention in any form.

Unless otherwise specified, the reagents and equipment used in the invention are

conventional reagents and equipment in the technical field.

Example 1 Preparation process of carbonized silkworm excrement (MCS)

The carbonized silkworm excrement MCS is prepared by chemical modification of

silkworm excrement. The carbonized silkworm excrement after chemical modification has

sufficient voids and better water absorption and slow release.

The chemical modification method comprises the following steps:

screened by 100-mesh sieve.

rinsing the material with deionized water to neutrality, then putting it in the desiccator for

drying at 60 °C. It is recorded it as carbonized silkworm excrement (MCS) and stored.

Carbonized silkworm excrement is shown in Fig. 1

Example 2 Preparation of pesticide degrading bacteria preparation

1. Materials

(1) The silkworm excrement used is the waste of silk industry, which is crushed by

machine to the size of 60 mesh.

(2) The strain adopted is HZ-2 strain of Bacillus pumilus, of which the depository is

China typical culture preservation center, and the preservation number is CCTCC No: M

2013380.

2. The preparation steps are as follows:

The strain HZ-2 is activated on the culture dish and inoculated on the slope of the test

tube.

Si. Inoculating the single colony on the slope into medium shake flask of LB liquid,

shaking culture to logarithmic phase to obtain the strain liquid;

S2. Centrifuging the strain liquid obtained from SI at 4800 R / min for 10 min,

removing the supernatant, adding 10-20 ml sterile water, the liquid formulation of strain

liquid is preliminarily prepared;

S3. In every ig of silkworm excrement, 2 ml of strain liquid obtained in S2 is

inoculated according to the inoculation amount of 1109 cfu/ml. Then adding 5% waste

molasses according to the mass percentage. Carrying out sealing, the strain is cultured in

the air bath thermostatic oscillator for 24 hours, so that the bacteria are biofilm cultured in

the silkworm excrement. Then respectively performing centrifuging, vacuum drying at

room temperature to nearly dryness, and finally sub-packaging and sealing.

Example 4 Immobilization of Silkworm Feces on Degradation Bacteria HZ-2

One of the degradation bacteria preparations obtained in Examples 2-3 of the present

invention was randomly selected to be placed in a scanning electron microscope, and was

scanned and observed by an environmental electron microscope to obtain its solid bacteria

form. The results are shown in Figures 1-2.

Fig. 1 shows the morphology of silkworm excrement before and after immobilization;

Fig. 2 shows the morphology of silkworm excrement and bacteria after immobilization.

The results showed that there are obvious gaps on the surface of silkworm excrement

before immobilization, with damages and fragments (as shown in Figure 1), which should

be the mechanical damage to silkworm excrement caused by carbonization and high

temperature firing.

After immobilization, the surface morphology of silkworm excrement changed

obviously, and a large number of bacteria adhered to the gaps of silkworm excrement (as

shown in Figure 2), which is widely distributed and clearly visible. This indicates that the

degradation bacteria could be rapidly enriched and immobilized in the carrier silkworm

excrement.

Example 5 The degradation effect of the degradation bacterial preparation of the

present invention on mesotrione

In this example, mesotrione is commercially available.

The degradation bacteria preparation prepared in Example 2 is inoculated into the

initial concentration of 50 mg/L of mesotrione LB liquid culture medium according to the volume fraction of 1%, and the pesticide residues are detected by HPLC after being cultured for 2, 3 and 5 days respectively. The results are shown in Figure 3.

The results show that the degradation rate of mesotrione is over 95% after 3 days of

culture.

The residual amounts are 1.05 mg/L and the degradation rate is 97.9% after 5 days of

culture.

The experimental results show that the degradation bacteria preparation of the

invention has outstanding degradation ability on the herbicide mesotrione, it can rapidly

reduce the residual amount of mesotrione in a short time.

Example 6 Degradation effect of the degradation bacteria preparation of the invention

on beta-cypermethrin

In this example, beta-cypermethrin is commercially available.

The degradation bacteria preparation prepared in Example 3 is inoculated into the

mesotrione LB liquid culture medium with the initial concentration of 50 mg/L according

to the volume fraction of 1%, and the pesticide residues are detected by high performance

liquid chromatography after being cultured for 2 days, 3 days and 5 days respectively. The

results are shown in Figure 3.

The results show that the degradation rate of beta-cypermethrin is over 85% after 3

days of culture.

The residual amounts were 6.34 mg/L and the degradation rate was 87.3% after 5 days

of culture.

Experimental results show that the degradation bacteria preparation of the invention

has outstanding degradation ability on beta-cypermethrin, and it can rapidly reduce the

residual amount of beta-cypermethrin in a short time.

Example 7 Field experiment on degradation of mesotrione by the degradation bacteria

preparation of the present invention

In this example, mesotrione is commercially available.

Corn fields without herbicide application are selected and divided into plots, each plot

with an area of not less than 20 m2 and three repetitions. The 40% mesotrione suspension

is prepared into 100 mg/L of aqueous solution, which is uniformly sprayed in the field

according to the dosage of 50 L/667 M 2 .

Two hours later, the degradation bacteria preparation prepared in Example 2 and the

pure bacteria solution are applied respectively, and a blank control CK is set up. According

to the five-point sampling method, five time points are selected for sampling analysis.

Calculating the degradation rate, and the field degradation effect of the degradation bacteria

preparation on mesotrione is evaluated. The results are shown in Fig. 5.

The results show that after 14 days of treatment, the degradation rate of the degrading

bacteria preparation reaches 76.1%, and the degradation rate is significantly higher than

that of the pure bacterial liquid treatment, which indicates that the degrading bacteria

preparation prepared by the method of the present invention, with silkworm excrement as

an effective biological carrier, could significantly improve the degradation effect of the

degrading bacteria and had a high degradation effect.

Example 8 Field experiment on degradation of beta-cypermethrin by the degradation bacteria preparation of the present invention

In this example, beta-cypermethrin is commercially available.

Cabbage fields without pesticides are selected and divided into plots, each plot with

an area of not less than 20 m2 and 3 repetitions. The 4.5% beta-cypermethrin

microemulsion is uniformly sprayed in the field according to the dosage of 13.5 g/ha and

L/667 M2 .

Two hours later, the preparation of degrading bacteria prepared in Example 3 and the

pure bacterial solution are applied respectively, and a blank control CK is set up. According

to the five-point sampling method, five time points are selected for sampling analysis, the

degradation rate is calculated, and the field degradation effect of the preparation of

degrading bacteria on beta-cypermethrin is evaluated. The results are shown in Fig. 6.

The results show that after 15 days of treatment, the degradation rate of the degrading

bacteria preparation reaches 71.3%, and the degradation rate is significantly higher than

degrading bacteria with a high degradation effect.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The application of silkworm excrement in the preparation of a pesticide-degrading

bacteria preparation.

2. The invention relates to a pesticide degrading bacterial preparation, which is

characterized in that it comprises the following components: (1) Strains that can degrade

pesticides (such as the HZ-2 strain of Bacilluspumilus); (2) Microbial preparation carrier.

It is the silkworm excrement of claim 1.

3. The pesticide degrading bacteria preparation according to claim 2, which is

characterized in that: the strain is the HZ-2 strain of Bacilluspumilus, the strain depository

is the China Center for Type Culture Collection, and the strain deposit number is CCTCC

NO: M 2013380; The BSF01 strain of Bacillus subtilis is deposited by the China Center

for Type Culture Collection and the deposit number is CCTCC NO: AB 2014103.

4. The pesticide degrading bacteria preparation according to claim 1 or 2, which is

characterize in that that silkworm excrement is silkworm excrement of silk industry waste.

5. The pesticide degrading bacteria preparation according to claim 4, which is

characterized in that the silkworm excrement is physically crushed to a particle size of 20

200 meshes or chemically modified.

The chemical modification method is as follows:

Sl. Firing with the muffle furnace filled with nitrogen and anoxic carbonization for 5

through a 100-mesh sieve. After cooling, taking it out, then which is mixed, ground, and screened by 100-mesh sieve.

S2. Treating the silkworm excrement with 0.1 mol-L- of phosphoric acid solution for

6. The pesticide degrading bacteria preparation according to claim 2, it is

characterized in that it also contains auxiliary materials, which are industrial waste glucose

or molasses; According to the mass percentage, the concentration of the auxiliary material

is 1%-95%.

7. The preparation method of the pesticide degrading bacteria preparation according

to claim 2, which is characterized by the following steps:

medium, and cultivated to the logarithmic phase with shaking;

adding sterile water to prepare the strain liquid;

at room temperature to nearly dryness, and finally sub-packaging and sealing.

8. The preparation method of pesticide degrading bacteria preparation according to

claim 7, which is characterized in that the strain liquid obtained in step S2 is inoculated

into silkworm excrement, and adding auxiliary industrial waste glucose or molasses.

9. The application of the pesticide degrading bacteria preparation according to claim

2 in degrading pesticides.

10. The application according to claim 9, characterized in that, when the strain in the

pesticide-degrading bacteria preparation is the HZ-2 strain of Bacillus pumilus, the

pesticide is mesotrione; When the strain of the pesticide-degrading bacteria is the BSF1

strain of Bacillus subtilis, the pesticide is beta-cypermethrin.

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Figure 1.

Figure 2.

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Figure 3.

Figure 4.

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Figure 5.

Figure 6.