AU2020101766A4 - Manufacturing method and application of biological fertilizer prepared by kelp residue and bacillus - Google Patents

Abstract

The present invention provides a method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus, and belongs to the technical field of biological fertilizer manufacturing. The method includes the following steps: 1) mixing kelp residue with water, followed with microwave treatment, and centrifuging a resulting mixture to obtain a first filter residue and a first supernatant; centrifuging the first supernatant to obtain a second supernatant and a second filter residue, and freeze-drying the second supernatant to obtain a degradation product of the kelp residue; and 2) dissolving the degradation product of the kelp residue obtained in step 1) in water, inoculating Bacillus, and fermenting to obtain a biological fertilizer. The present invention manufactures a biological fertilizer prepared by kelp residue and Bacillus. The biological fertilizer can promote plant growth, and prevent and control plant diseases. 1/2 FIG1 FIG2 LIW+C NTC FIG3

Description

1/2

FIG1

FIG2

LIW+C

NTC

FIG3

MANUFACTURING METHOD AND APPLICATION OF BIOLOGICAL FERTILIZER PREPARED BY KELP RESIDUE AND BACILLUS TECHNICAL FIELD The present invention relates to the technical field of biological fertilizer manufacturing, and in particular to a method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus and application thereof. BACKGROUND China is a traditional agricultural country, and agriculture plays a very important role in national production. Modem agricultural technology promotes the rapid development of agricultural production, and the promotion of chemical fertilizers makes the agricultural production obtain enormous economic benefits. In recent years, environmental pollution, soil hardening, soil degradation, ecological deterioration, and other problems caused by extensive use of chemical fertilizers have become increasingly serious. These problems damage the environment, affect the soil fertility and reduce the quality of agricultural products. In order to realize the sustainable development of the agriculture, and achieve the purposes of high yield, high quality, ecology, and safety, development of an economic and efficient natural fertilizer is of great significance. SUMMARY In view of this, an objective of the present invention is to provide a method manufacturing a biological fertilizer prepared by kelp residue and Bacillus and application thereof. To achieve the above objective, the present invention provides the following technical solutions: The present invention provides a method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus, including the following steps: 1) mixing kelp residue with water, followed with microwave treatment, and centrifuging a resulting mixture to obtain a first filter residue and a first supernatant; centrifuging the first supernatant to obtain a second supernatant and a second filter residue, and freeze-drying the second supernatant to obtain a degradation product of the kelp residue; and 2) dissolving the degradation product of the kelp residue obtained in step 1) in water, inoculating Bacillus, and fermenting to obtain a biological fertilizer. Preferably, the Bacillus in step 2) includes Bacillus subtilis and/or Bacillus amyloliquefaciens. Preferably, a ratio of a mass of the kelp residue to a volume of the water in step 1) is (1-5) g:(10-50) ml. Preferably, the microwave treatment in step 1) is conducted at 150-200°C with a power of 800-1,200 W for10-30 min.

Preferably, the centrifugation in step 1) is conducted for 15-25 min at 9,000-11,000 rpm. Preferably, the Bacillus is inoculated in the form of Bacillus suspension, and the Bacillus suspension has an OD 60 0 value of 0.1-0.5. Preferably, a ratio of a mass of the degradation product of the kelp residue to a volume of the Bacillus suspension is (1-5) g:(1-5) ml. Preferably, the fermentation in step 2) is conducted for 90-150 h at 150-200 rpm at 25-30°C. The present invention further provides application of a biological fertilizer manufactured by the manufacturing method according to the above technical solution in promoting plant growth. The present invention further provides application of a biological fertilizer manufactured by the manufacturing method according to the above technical solution in the prevention and control of plant diseases. The present invention provides a method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus. The method includes the following steps: 1) mixing kelp residue with water, followed with microwave treatment, and centrifuging a resulting mixture to obtain a first filter residue and a first supernatant; centrifuging the first supernatant to obtain a second supernatant and a second filter residue, and freeze-drying the second supernatant to obtain a degradation product of the kelp residue; and 2) dissolving the degradation product of the kelp residue obtained in step 1) in water, inoculating Bacillus, and fermenting to obtain a biological fertilizer. The present invention manufactures the biological fertilizer prepared by kelp residue and Bacillus. The biological fertilizer can promote plant growth, and prevent and control plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 shows the growth promoting result of a biological fertilizer in Example 1, where LJW+C is a biological fertilizer manufactured with Enteromorphaand Bacillus amyloliquefaciens, and NTC is a control; FIG 2 shows the growth promoting result of a biological fertilizer in Example 1, where LJW+T is a biological fertilizer manufactured with Enteromorphaand Bacillus subtilis, and NTC is a control; FIG. 3 shows the result of controling plant diseases by a biological fertilizer in Example 2, where LJW+C is a biological fertilizer manufactured with Enteromorpha and Bacillus amyloliquefaciens, and NTC is a control; FIG. 4 shows the result of controlling plant diseases by a biological fertilizer in example 2, where LJW+T is a biological fertilizer manufactured with Enteromorphaand Bacillus subtilis, and NTC is a control. DETAILED DESCRIPTION

The present invention provides a method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus, including the following steps: 1) mixing kelp residue with water, followed with microwave treatment, and centrifuging a resulting mixture to obtain a first filter residue and a first supernatant; centrifuging the first supernatant to obtain a second supernatant and a second filter residue, and freeze-drying the second supernatant to obtain a degradation product of the kelp residue; and 2) dissolving the degradation product of the kelp residue obtained in step 1) in water, inoculating Bacillus, and fermenting to obtain a biological fertilizer. In the present invention, the kelp residue is mixed with water, followed with microwave treatment, and a resulting mixture is centrifuged to obtain a first filter residue and a first supernatant; the first supernatant is centrifuged to obtain a second supernatant and a second filter residue, and the second supernatant is freeze-dried to obtain a degradation product of the kelp residue. In the present invention, the kelp residue is preferably the residue left after extracting iodine and sodium alginate from kelp. In the present invention, a ratio of a mass of the kelp residue to a volume of the water is preferably (1-5) g:(10-50) ml, and more preferably (2-4) g:(20-40) ml. In the present invention, the microwave treatment includes the following conditions: a temperature of the microwave treatment is preferably 150-200°C, and more preferably 160-180°C; a power of the microwave treatment is 800-1,200 W, and more preferably 900-1,100 W; the microwave treatment preferably lasts for 10-30 min, and more preferably 15-25 min. In the present invention, the kelp residue is degraded by microwave treatment. In the present invention, the microwave-treated mixture is centrifuged at 50-70°C. The centrifugation includes the following conditions: the centrifugation is preferably conducted for -25 min at 9,000-11,000 rpm, and more preferably for 20 min at 10,000 rpm. In the present invention, the centrifugation removes solid matters from the kelp residue. No particular limitation is imposed on freeze-drying time of the second supernatant in the present invention, and conventional freeze-drying conditions may be used. In the present invention, the degradation product of the kelp residue obtained is preferably stored at -20°C. In the present invention, the degradation product of the kelp residue obtained is dissolved in water, inoculated with Bacillus, and fermented to obtain a biological fertilizer. In the present invention, the Bacillus preferably includes Bacillus subtilis and/or Bacillus amyloliquefaciens; when the Bacillus includes Bacillus subtilis and Bacillus amyloliquefaciens, the mixing is carried out in an arbitrary ratio. In the present invention, the Bacillus subtilis is preferably Bacillus subtilis Tbp55, with an accession number of CGMCC No. 2853; the Bacillus amyloliquefaciens is preferably Bacillus amyloliquefaciens Cas02, with an accession number of CGMCC No. 15514. In the present invention, the Bacillus ferments the degradation product of the kelp residue. In the present invention, a ratio of a mass of the degradation product of the kelp residue to a volume of the water is preferably (1-5) g:(0.1-2) L, and more preferably (2-3) g:(0.8-1.5) L. In the present invention, the Bacillus is preferably inoculated in the form of Bacillus suspension, and the Bacillus suspension preferably has an OD 600 value of 0.1-0.5, and more preferably 0.3. In the present invention, preparation of the Bacillus suspension preferably includes the following steps: inoculating the into a Luria-Bertani (LB) liquid medium for culture, diluting a resulting culture medium, and measuring an OD 600 value of 0.1-0.5. No particular limitation is imposed on the LB liquid medium in the present invention, and conventional one may be used. In the present invention, the culture is preferably conducted for 20-30 h at 25-30°C. In the present invention, the fermentation includes the following conditions: the fermentation preferably lasts for 90-150 h, and more preferably 110-130 h; the fermentation is preferably conducted at 25-30°C; the fermentation is preferably conducted at 150-200 rpm, and more preferably for at 160-180 rpm. In the present invention, the biological fertilizer is a liquid fertilizer; the liquid fertilizer has a laminarin content of 1-3 g/L; a Bacillus count is 1 x 1010-101 cells/L. The present invention further provides application of a biological fertilizer manufactured by the manufacturing method according to the above technical solution in promoting plant growth. The present invention further provides application of a biological fertilizer manufactured by the manufacturing method according to the above technical solution in the prevention and control of plant diseases. In the present invention, the plant preferably includes pepper, tobacco, eggplant, or cucumber. In the present invention, the plant disease preferably includes bacterial wilt of pepper. In the present invention, a method for applying the biological fertilizer is preferably to pour roots with 15-25 ml, and more preferably 20 ml. The technical solution of the present invention will be described in detail below in conjunction with examples, but should not be construed as limiting the scope of the invention. Example 1 1) Degradation of kelp residue: Two grams of kelp residue was mixed with 20 mL of water, and degraded in a microwave reactor (CEM Mars 6) for 20 min at 180°C and a microwave power of 1,000 W. When the reaction was over and the system was cooled to 60°C, an extract was centrifuged, and a second extract was centrifuged again to obtain a final extract. The final extract obtained was freeze-dried under vacuum to obtain a degradation product of the kelp residue, which was stored in a refrigerator at -20°C. Herein, the kelp residue was the residue left after extracting iodine and sodium alginate from kelp. 2) Microbial fermentation: Bacillus subtilis Tbp55 was cultured in LB medium for 24 h and diluted to an OD 600 value of 0.3 to obtain a Bacillus subtilis Tp55 suspension. Five grams of degradation product of the kelp residue was dissolved in 1 L of water, and then 1 ml of Bacillus subtilis Tbp55 suspension was inoculated thereinto, and shake-cultured for 120 h at 175 rpm and 28°C to manufacture a kelp residue/Bacillussubtilis Thp55 functional liquid fertilizer. Alternatively Microbial fermentation: Bacillus amyloliquefaciens Cas02 was cultured in LB medium for 24 h and diluted to an OD 60 0 value of 0.3 to obtain a Bacillus amyloliquefaciens Cas02 suspension. Five grams of degradation product of kelp residue was dissolved in L of water, and then 1 ml of Bacillus amyloliquefaciens Cas02 bacterial suspension was inoculated thereinto, and shake-cultured for 120 h at 175 rpm and 28°C to manufacture a kelp residue/Bacillus amyloliquefaciens Cas02 functional suspension fertilizer. Example 2 Pepper seedlings with consistent growth vigor were transplanted into sterilized matrices. When the seedlings grew to two true leaves, 20 ml of the liquid fertilizer manufactured in Example 1 was slowly poured into the rhizosphere of each pepper. A control group was watered with an equal volume of water. The seedlings were watered every five days, three consecutive times. The growth promoting effect of the liquid fertilizer on peppers was measured seven days after the third watering. Results are shown in Table 1 and FIGS. 1 and 2.

Table 1 The growth promoting effect of peppers

Treatment Plant height (mm) Leaf number (mm) Leaf length (mm) Leaf width (mm)

NTC 88.80±11.67 8.40±1.06 70.13±8.44 37.30±4.81

LJW+C 116.53±22.87 10.13±1.13 79.80±9.66 41.13±8.41

LJW+T 88.93±16.02 9.07±1.10 70.53±12.52 38.00±5.10 Annotations: NTC - Water blank; LJW - Degradation product of the kelp residue; T - Bacillus subtilis; C - Bacillus amyloliquefaciens As is apparent from Table 1 and FIGS. 1 and 2, the kelp residue/Bacillus functional fertilizer has a significant growth promoting effect on peppers. Example 3 After the pepper was transplanted, 20 ml of the liquid fertilizer manufactured in Example 1 was slowly poured into the rhizosphere of each pepper, and a control group was watered with an equal volume of Hoagland's nutrient solution. The seedlings were watered every five days, three consecutive times. The seedlings were watered every 5 day, three consecutive times. Twenty milliliters of Ralstonia solanacearum was inoculated around the rhizosphere of each pepper (an objective of adding the bacterium was to cause bacterial wilt of pepper) seven days after the third watering, and inoculated in the form of bacterial suspension (OD6 0 0 = 0.3), 20 ml per plant, and the onset was counted five days after inoculation. Results are shown in Table 2 and FIGS. 3 and 4. According to Kempe's (1983) classification standard of bacterial wilt, the disease was classified into five grades: grades 0, 1, 2, 3, and 4. Grade 0: no symptom of bacterial wilt; Grade 1: wilting symptoms in 1-25% of leaves; Grade 2: wilting symptoms in 26-50% of leaves; Grade 3: wilting symptoms in 51-75% of leaves; and Grade 4: wilting symptoms in 76-100% of leaves. Disease index and biocontrol effect were calculated according to the following formulas: disease index = 100 x Y (number of diseased plants x disease grade)/(total number of plants x highest disease grade); relative control effect (%) = (control disease index - treated disease index)/control disease index x 100.

Table 2 Statistics on the incidence of bacterial wilt of pepper

Treatment NTC LJW+T LJW+C

Incidence (%) 80.00 33.33 20.00

Disease index 72.59 25.93 15.56

Relative control effect (%) ---- 64.29 78.57

Annotations: NTC - Water blank; LJW - Degradation product of the kelp residue; T - Bacillus subtilis; C - Bacillus amyloliquefaciens As is apparent from Table 2 and FIGS. 3-4, results show that the kelp residue/Bacillus functional fertilizer has a significant control effect on bacterial wilt of pepper. It can be concluded from the above examples that the present invention manufactures the biological fertilizer prepared by kelp residue and Bacillus, and the biological fertilizer can promote plant growth and prevent and control plant diseases. The foregoing descriptions is merely preferred examples of the present invention; it should be noted that various modifications and variations can be made by those skilled in the art without departing from the principles of the present invention and are within the scope of the invention.

Claims (5)

1. CLAIMS 1. A method for manufacturing a biological fertilizer prepared by kelp residue and Bacillus, comprising the following steps: 1) mixing kelp residue with water, followed with microwave treatment, and centrifuging a resulting mixture to obtain a first filter residue and a first supernatant; centrifuging the first supernatant to obtain a second supernatant and a second filter residue, and freeze-drying the second supernatant to obtain a degradation product of the kelp residue; and 2) dissolving the degradation product of the kelp residue obtained in step 1) in water, inoculating Bacillus, and fermenting to obtain a biological fertilizer.
2. 2. The manufacturing method according to claim 1, wherein the Bacillus in step 2) comprises Bacillus subtilis and/or Bacillus amyloliquefaciens.
3. 3. The manufacturing method according to claim 1, wherein a ratio of a mass of the kelp residue to a volume of the water in step 1) is (1-5) g:(10-50) ml; wherein the microwave treatment in step 1) is conducted at 150-200°C with a power of 800-1,200 W for10-30 min; wherein the centrifugation in step 1) is conducted for 15-25 min at 9,000-11,000 rpm.
4. 4. The manufacturing method according to claim 1, wherein the Bacillus is inoculated in the form of Bacillus suspension, and the Bacillus suspension has an OD6 0 0 value of 0.1-0.5; wherein a ratio of a mass of the degradation product of the kelp residue to a volume of the Bacillus suspension is (1-5) g:(1-5) ml.
5. 5. The manufacturing method according to claim 1, wherein the fermentation in step 2) is conducted for 90-150 h at 150-200 rpm at 25-30°C.

   Tobacco Research Institute of Chinese Academy of Agricultural Sciences

   Patent Attorneys for the Applicant/Nominated Person

   SPRUSON&FERGUSON