NL2030440B1

NL2030440B1 - Application of poly-y-glutamic acid as fertilizer absorption promoter in agricultural planting

Info

Publication number: NL2030440B1
Application number: NL2030440A
Authority: NL
Inventors: Peng Wei; Song Wei; Li Qiang; Yu Binzhou; Zeng Qingzhuo
Original assignee: Wuhan Guanghua Times Biotechnology Co Ltd
Priority date: 2022-01-05
Filing date: 2022-01-05
Publication date: 2023-07-10

Abstract

The present disclosure relates to an application of poly—y— glutamic acid as a fertilizer absorption promoter in agricultural planting. The poly—y—glutamic acid is a high polymer formed by D— glutamic acid (D—Glu) and L—glutamic acid (L—Glu) via d—amino groups and y—carboxyl groups connected in a form of peptide bonds, with a free carboxyl group binding to an d—carbon atom of each repeating unit and an average molecular weight of 10,000— l0,000,000 Da. The poly—v—glutamic acid is used as the fertilizer absorption promoter of nitrogen (N), phosphorus (P), potassium (K) 10 or a combination thereof. The beneficial effects of the present disclosure are that the fertilizer absorption promoter of the poly—y—glutamic acid capable of being degraded by microorganisms in the soil has better environmental protection over TPA.

Description

P981/NLpd

APPLICATION OF POLY-y-GLUTAMIC ACID AS FERTILIZER ABSORPTION

PROMOTER IN AGRICULTURAL PLANTING

TECHNICAL FIELD

The present disclosure relates to an application of poly-y- glutamic acid as a fertilizer absorption promoter in agricultural planting, which relates to the application field of microbial fer- tilizers.

BACKGROUND ART

Chemical fertilizer is a significant change in the history of agricultural development, with the reason that it greatly increas- es the yield of crops and alleviates the food shortage in the globe. The annual consumption of the chemical fertilizer in China reaches more than 40 million tons, accounting for 30% of the total consumption of the chemical fertilizer in the world. However, the utilization rates of nitrogen, phosphorus and potassium are 30%- 35%, 10%-25% and 35%-50%, respectively. According to the statisti- cal data, more than 60 large and medium-sized lakes in China have suffered from eutrophication, with the migration of excessive ni- trogen and phosphorus as the root cause. Therefore, the problem today is not the acquisition but the utilization of fertilizer nu- trition. The loss of a large amount of fertilizer not only in- creases the agricultural cost and causes huge economic losses, but also leads to environmental pollution and the imbalance of the ag- ricultural ecosystem, and brings more and more negative impacts on the environment and sustainable development, such as soil compac- tion and salinization, increased nitrate content in the groundwa- ter, eutrophication of surface water, destruction of atmospheric ozone layer, decline in the quality of crops, greenhouse effect, aggravation of environmental pollution and other negative effects.

Those negative effects seriously affect the human health. Thus, how to reduce the application rate of fertilizer and the environ- mental pollution, and improve the utilization rate of fertilizer without affecting the harvest of crops has been highlighted by re-

searchers. The development of the fertilizer absorption promoter has become an important issue to be solved urgently at present.

Polyamino acid may increase the absorption of nutrients by crops and the yield of crops, and save the application rate of fertilizer. The polyamino acid currently used as the fertilizer absorption promoter is poly-aspartic acid (PAA). The Donlar Compa- ny in America took the lead in the production of thermal polyas- partate (TPA) by thermal polymerization as the chemical synthesis method, which has been industrialized and commercialized and ap- plied to the fertilizer. TPA is the active ingredient in Amisorb which is a fertilizer absorption promoter. TPA has greater affini- ty to nutrients in the fertilizer due to many free carboxyl groups contained, which may accelerate the formation of roots and in- crease the tillering. Amisorb has also been proven to be applied to wheat and corn, soybean and other crops, with the effect of in- creasing the yield, saving the fertilizer usage, and improving the quality of crops. However, the biodegradability of Amisorb is not yet satisfactory.

Poly-y-glutamic acid (y-PGA) is an anionic polyamino acid produced by microbial fermentation in nature. Poly-y-glutamic acid is water-soluble, water-absorbent biodegradable, non-toxic, frost- resistant and environmentally friendly, and has affinity to metal ions and resistance to proteases. Thus, it has broad application prospects in the fields of agriculture, food, medicine, cosmetics, fiber light and chemical industry and the like.

SUMMARY

The technical problems to be solved by the present disclosure is to expand the application range of poly-y-glutamic acid. As a biodegradable fertilizer absorption promoter, poly-y-glutamic acid may save the application rate of fertilizer and reduce the envi- ronmental pollution due to the excessive use of fertilizer, as well as increase the yield and quality of crops.

The present disclosure employs the following technical solu- tions:

The poly-y-glutamic acid is a high polymer formed by D- glutamic acid (D-Glu) and L-glutamic acid (L-Glu) via o-amino groups and y-carboxyl groups connected in a form of peptide bonds, with a free carboxyl group binding to an a-carbon atom of each re- peating unit and an average molecular weight of 10,000-10,000,000

Da.

The poly-y-glutamic acid is used as the fertilizer absorption promoter of nitrogen, phosphorus, potassium or a combination thereof.

The fertilizer absorption promoter of the poly-y-glutamic ac- id is a poly-y-glutamic acid solution by liquid-state fermenta- tion, or a bio-organic fertilizer containing the poly-y-glutamic acid by solid-state fermentation.

The liquid-state fermentation in the patent of ZL03118908.3 is adopted to produce the bio-organic fertilizer containing the poly-y-glutamic acid.

The solid-state fermentation of the bio-organic fertilizer containing the poly-y-glutamic acid comprises using pig manure or cow manure as the main raw material, and adding soybean meal, bran, glutamic acid and citric acid in a certain proportion as the auxiliary material, inoculating with Bacillus subtilis (CCTCC202048), and fermenting by open-air composting. The bio- organic fertilizer obtained contains 10%-12% of the poly-y- glutamic acid.

The application of the poly-y-glutamic acid as the fertilizer absorption promoter in the agricultural planting comprises hydro- ponic farming: diluting a standard nutrient solution (Ca (NO;), * 4H.O 945 mg/L, KNO; 809 mg/L, (NH.)H.PO, 153 mg/L, MgSO, « 7H,0 493 mg/L, HsBO;3 2.86 mg/L, MnSO; *¢ 4H:0 2.13mg/L, ZnSO, + 7H20 0.22 mg/L, CuSO; * 5H:0 0.08mg/L, Fe-EDTA 30 mg/L, (NHs) 6Mo7024 * 4H;0 0.02 mg/L) in a ratio of 1 : 3 to irrigate after a seed germinat- ing 3 d; diluting the standard nutrient solution in a ratio of 1: 1 to irrigate after the seed germinating 7 d; transplanting uni- form seedlings into hydroponic pots {({& 7 cm x 15 cm) when the uni- form seedlings are at a two-leaves-and-one-bud stage (an early growth stage); adding 400 mL of the standard nutrient solution and 4-40 mg of the fertilizer absorption promoter of the poly-y- glutamic acid dissolved in each of the hydroponic pots.

The application of the poly-y-glutamic acid as the fertilizer absorption promoter in the agricultural planting comprises root irrigation: mixing a ternary compound fertilizer in a normal ap- plication rate for different crops with the poly-y-glutamic acid to dissolve in water, performing the root irrigation at a proper growth stage of crops after planting the crops, with an applica- tion rate of the poly-y-glutamic acid being 10-50 g/666 m° (mu).

The application of the poly-y-glutamic acid as the fertilizer absorption promoter in the agricultural planting comprises foliar application: mixing the ternary compound fertilizer in the normal application rate for different crops with the poly-y-glutamic acid to dissolve in water, performing the foliar application at the proper growth stage of the crops after planting the crops, with the application rate of the poly-y-glutamic acid being 10-50 g/666 m?.

The crops planted comprises vegetables, grains, soybeans and economic crops.

The beneficial effects of the present disclosure are as fol- lows: (1) Biodegradability: the poly-y-glutamic acid is a high pol- ymer formed by D-glutamic acid and L-glutamic acid via o-amino groups and y-carboxyl groups connected in the form of peptide bonds, which may be degraded by microorganisms in the soil. Thus, it has better environmental protection over TPA. (2) Applying 10-50 g of the fertilizer absorption promoter of the poly-y-glutamic acid per 666 cm? under the condition of ensur- ing the normal growth of crops may reduce the normal application rate of the chemical fertilizer by 10-30%. Thus, the cost is re- duced. (3) Applying 10-50 g of the fertilizer absorption promoter of the poly-y-glutamic acid per 666 cm’ based on the normal applica- tion rate of the chemical fertilizer for crops may increase the yield of crops by 10-30%. Thus, the economic benefit is increased. (4) The quality of crops is improved, and the content of N, P and K in crops is increased. Thus, the application range of the poly-y-glutamic acid is broadened.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1

Production of the poly-y-glutamic acid by the solid-state fermentation

The poly-y-glutamic acid may be produced by the solid-state 5 fermentation in the present disclosure: using pig manure or cow manure as the main raw material, and adding soybean meal, bran, glutamic acid and citric acid in a certain proportion as the aux- iliary material, inoculating with patented Bacillus subtilis (CCTCC202048), and fermenting by open-air composting, to obtain the bio-organic fertilizer containing the poly-y-glutamic acid.

The solid-state fermentation is performed as follows: (1) Preparation of seed liquid: the strain Bacillus subtilis is activated, and then the activated strain is inoculated into the culture medium of the seed liquid. The culture medium is composed of beef extract 10.0 g, peptone 5.0 g, NaCl 10.0 g and distilled water 1000 ml, and the pH value of the culture medium is 7.2-7.4.

The seed is cultured at 30 °C for 4.5 h, and the strain growing to the mid-log phase (OD600nm = 1.4) is used, to obtain the seed lig- uid. (2) Preparation of the culture medium for amplified culture in the solid-state fermentation: The culture medium is composed of the following components in weight percentage (w/w):

Formulation 1: It is composed of fresh pig manure 62.5%, soy- bean meal 25.0%, wheat bran 5.0%, glutamic acid 5.0%, and citric acid 2.5%. Water is added to the culture medium so that the mois- ture content of the culture medium is 60%, and the pH value is ad- justed to be 9.0.

Formulation 2: It is composed of fresh cow dung 56.32%, soy- bean meal 23.35%, wheat bran 16.08%, and glutamic acid 4.25%. Wa- ter is added to the culture medium so that the moisture content of the culture medium is 60%, and the pH value is adjusted to be 9.0. (3) The seed liquid obtained in the step (1) is inoculated into the culture medium prepared in the step (2) for the solid- state fermentation. (4) The solid-state fermentation is performed under the fol- lowing conditions: The charge amount of the fermenter is 10-30%, the inoculation amount is 8%, and the initial pH value is 9.0. The culturing temperature is at 40 °C, and the fermentation is per- formed in a ventilation condition for 48 h, to obtain the ferment- ed product containing 5-64 ( 6% in the formulation 1 and 5% in the formulation 2) of the poly-y-glutamic acid. (5) The fermented product obtained in the step (4) is dried at 80 °C to make the moisture content less than 5%, to obtain the bio-organic fertilizer containing 10-12% of the poly-y-glutamic acid by the solid-state fermentation.

Example 2

The accumulation of nutrients in the stems and leaves of Chi- nese cabbage cultured in the nutrient pots applied with the poly- v—glutamic acid {with the average molecular weight 200 kDa)

Experimental steps: The plump and uniform seeds of Chinese cabbage (with the variety of Brassica campestris L., Shanghaiging) are selected to germinate. The standard nutrient solution (see more details for the formulation in the Summary) is diluted in the ratio of 1 : 3 to irrigate after germinating 3 d, and diluted in the ratio of 1 : 1 to irrigate after germinating 7 d. The uniform seedlings are transplanted into the black and plastic hydroponic pots (7 cm x 15 cm) when the uniform seedlings are at the two- leaves-and-one-bud stage (the early growth stage), dividing into four groups. And 400 mL of the standard nutrient solution is added into each of the black and plastic hydroponic pots in one of the four groups, and the standard nutrient solution diluted in two- fold, fourfold and eightfold is sequentially added into the re- maining three groups. Each of the four groups is divided into five subgroups added with the poly-y-glutamic acid, and the final con- centration of the poly-y-glutamic acid in each subgroup is 0, 50 mg/kg and 100 mg/kg, respectively. 4 seedlings are planted in each of the black and plastic hydroponic pots, and the experiment is repeated 10 times in random arrangement. The standard nutrient so- lution is changed once a week, and the poly-y-glutamic acid (with the average molecular weight of 200 kDa) in corresponding concen- tration is added. The position of the black and plastic hydroponic pots is changed frequently. The stems and leaves of the seedlings at the stage of 5-7 leaves are harvested to dry and weigh. The to- tal nitrogen is measured by the Semi-Micro-Kjeldahl Nitrogen meth-

od after digestion, and phosphorus and potassium are determined using the VISTA-MPX ICP-OES spectrometer manufactured by the VARI-

AN company in America.

The experiment results are as follows:

Table 1 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) in different concentrations on the accumulation of potassium in the stems and leaves of Chinese cabbage (%) (The weight ratio of potassium in the stems and leaves of

Chinese cabbage) ‘Dilution ratio of standard nutrient eightfold fourfold twofold stock solu- solution tion

Addition amount of poly-y- glutamic acid 'o 85.05 5.1 5.51 &.2 50 mg/kg 5.28 5.23 5.6 6.34 100 mg/kg ~~ 5.4 6.15 5.78 &.85 © Table 2 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) in different concentrations on the accumulation of phosphorus in the stems and leaves of Chinese cabbage (3%) (The weight ratio of phosphorus in the stems and leaves of

Addition amount of poly-y- glutamic acid 0 12983 1.45 1.583 1.65 mg/kg 1.637 1.71% 1.84 2.031 100 mg/kg 1.516 1.831 1.688 1.59 © Table 3 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) in different concentrations on the accumulation of nitrogen in the stems and leaves of Chinese cabbage (3%) (The weight ratio of nitrogen in the stems and leaves of Chi- nese cabbage) ‘Dilution ratio of standard nutrient eightfold fourfold twofold stock solu- solution tion

Addition amount of poly-y- glutamic acid 0 4.66 4.78 5.32 6 50 mg/kg 5.7 5.32 6.67 7.79 "100 mg/kg 4.33 6.12 7.44 6.05 ~~ The experiment results indicate that the poly-y-glutamic acid added in the standard nutrient solution is beneficial to the accu- mulation of potassium, phosphorus and nitrogen in the stems and leaves of Chinese cabbage.

Example 3

The effect of the poly-y-glutamic acid (with the average mo- lecular weight of 10,000 kDa) on the yield of Chinese cabbage cul- tured in the nutrient pots via root irrigation

Experiment treatment: six groups are set up. One group is not added with the poly-y-glutamic acid, and the remaining five groups are added with the poly-y-glutamic acid in the concentration of 25 mg/kg of soil, 50 mg/kg of soil, 100 mg/kg of soil, 200 mg/kg of soil and 400 mg/kg of soil, respectively. Four groups are set up according to the application rate of the nutrient elements of the base fertilizer, and one of the four groups is applied with 0.20 g nitrogen, 0.065 g phosphorus and 0.16 g potassium per kilogram of soil that the fertilizers are commercially available urea, calcium superphosphate and potassium chloride. While the application rate of the nutrient elements in the remaining groups is reduced to 75%, 50% and 33% sequentially.

Experimental steps: The fertilizer is dissolved in water to mix with the poly-y-glutamic acid solution (with the average mo- lecular weight of 10,000 kDa), then the obtained mixture is evenly mixed with 1 kg of soil per pot, and put into the pots. There are total 24 experiments, repeating 3 times. Each pot is seeded with 15 uniform and plump seeds of Chinese cabbage (with the variety of

Brassica campestris L., Shanghaiging), and 10 seedlings at the two-leaves-and-one-bud stage (the early growth stage) are planted and watered irregularly. The seedlings at the stage of 7-9 leaves are harvested, and the above-ground parts are harvested to dry at 70 °C and then weighed.

The experimental results are shown in Table 4.

Table 4 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 10,000 kDa) in different concentrations on the yield of Chinese cabbage (pot culture experiment), with the unit of g

Concentration of fertilizer 33% 50% 75% 100%

Concentration of poly-y-glutamic acid 0 0.36 0.42 O5 0.e2 25 mg/kg of soil 0.38 0.45 0.55 0.64 50 mg/kg of soil 0.42 0.49 0.59 0.e8 100 mg/kg of soil 0.48 0.55 0.6L 0.7 200 mg/kg of soil 0.55 0.6 0.62 0.71 400 mg/kg of soil 0.53 0.61 0.63 0.7 lizer is 100%, the addition of 25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg of the poly-y-glutamic acid increases the yield of Chinese cabbage by 3%, 10%, 13%, 15% and 13% respectively compared with the control. When the concentration of the fertiliz- er is reduced to 33%, 50%, 75% and 100%, the addition of 100 mg/kg of the poly-y-glutamic acid increases the yield of Chinese cabbage by 33%, 31%, 22% and 13% respectively compared with the control, and the addition of 200 mg/kg of the poly-y-glutamic acid increas- es the yield of Chinese cabbage by 53%, 43%, 24% and 15% respec- tively compared with the control. This experiment demonstrates that the poly-y-glutamic acid (with the average molecular weight of 10,000 kDa) may promote the growth of Chinese cabbage. When the fertilizer is in shortage, the effect of the poly-y-glutamic acid on the yield of Chinese cabbage is particularly obvious. When 200 mg/kg of the poly-y-glutamic acid is added, the growth of Chinese cabbage applied with the fertilizer in concentration of 50% is al- most equivalent to that in concentration of 100%, indicating that the application of the poly-y-glutamic acid may save nearly 50% of the fertilizer under suitable conditions.

Example 4

The effect of the poly-y-glutamic acid (with the average mo- lecular weight of 10 kDa) on the yield of Chinese cabbage cultured in the field plot via root irrigation

Experimental area: the experiment is carried out in a field with an area of 2.0 m x 5.0 m.

Experiment steps: The uniform and plump seeds of Chinese cab- bage (with the variety of Brassica campestris L., Shanghaiding) are selected to sow evenly, and the seedlings at the two-leaves- and-one-bud stage are planted with 10 cm spacing and watered ir- regularly. The root irrigation is carried out once after planting 10 d with the application rate of 40 mL/plant, and the spraying is carried out once after planting 18 d with the application rate of 10 mL/plant. The application rate of the poly-y-glutamic acid (with the average molecular weight of 50 kDa) in the spraying is 1/4 of that in the root irrigation. There are three repetitions with random arrangement in blocks. The seedlings at the stage of 7-9 leaves are harvested, and the above-ground parts are collected to obtain the fresh weight.

There are three experiment treatments: the concentration of the poly-y-glutamic acid applied is 0, 100 mg/L and 300 mg/L.

The experiment results are shown in Table 5.

Table 5 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 10 kDa) on the yield of Chinese cabbage via root irrigation

Concentration Average plant Average plant Growth rate of poly-yY- weight of each weight of each in yield glutamic acid repetition (9) treatment (9) (3) 1 2 3

© 0 (clear wa- 26.46 27.08 22.86 25.47 ter) 100 mg/L 34.50 37.20 30.30 34.00 33.50 300 mg/L 36.56 33.30 31.72 33.86 32.94 © The results show that: The addition of 100 mg/L and 300 mg/L of the poly-y-glutamic acid (with the average molecular weight of 10 kDa) increases the yield of Chinese cabbage by 33.5% and 32.94% respectively compared with the control. It is more appropriate to apply the poly-y-glutamic acid solution in the concentration of 100-300 mg/L for the root irrigation in the field experiments.

Example 5

The effect of the poly-y-glutamic acid with the average mo- lecular weight of 200 kDa on the yield of Chinese cabbage cultured in the field plot via foliar application

To be close to the actual field experiment, the field plot (2.0m x 5.0 m) experiment is adopted in this experiment. The con- ventional fertilization is applying 0.20 g nitrogen, 0.065 g phos- phorus and 0.16 g potassium per kilogram of soil that the ferti- lizers are commercially available urea, calcium superphosphate and potassium chloride. The foliar application of the aqueous solution of the poly-y-glutamic acid is adopted, and the data from differ- ent treatments is obtained to statistically analyze the effect of the poly-y-glutamic acid on the growth of Chinese cabbage via fo- liar application.

Experiment treatments: 1. Conventional fertilization, and irrigation with clear wa- ter (as the blank control); 2. Conventional fertilization, and performing the foliar ap- plication of the poly-y-glutamic acid solution in the concentra- tion of 40 mg/L once, with the application rate of 200 L/666 m? and the application rate of the poly-y-glutamic acid being 5 g/666 m’; 3. Conventional fertilization, and performing the foliar ap- plication of the poly-y-glutamic acid solution in the concentra- tion of 60 mg/L once, with the application rate of 200 L/666 m? and the application rate of the poly-y-glutamic acid being 25 g/666 m’; 4. Conventional fertilization, and performing the foliar ap- plication of the poly-y-glutamic acid solution in the concentra-

tion of 80 mg/L once, with the application rate of 200 L/666 m? and the application rate of the poly-y-glutamic acid being 50 g/666 m.

The experiment results are shown in Table 6.

Table 6 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) on the yield of Chinese cabbage via the foliar application

Treatments Average plant weight (g) Yield increased by (3) 1 25 -- 2 28 12 3 30 20 4 32 28 © Through the above ezperiment, the yield of Chinese cabbage is increased according to the increase in the application rate of the poly-y-glutamic acid under the conditions that the foliar applica- tion of the poly-y-glutamic acid in different concentrations is performed to Chinese cabbage. What’s more, the leaf area is in- creased, and the color is greener, indicating that the foliar ap- plication of the poly-y-glutamic acid may greatly promote the growth of Chinese cabbage.

Example 6

The effect of fermented pig manure containing the poly-y- glutamic acid (with the average molecular weight of 2000 kDa) as a base fertilizer on the yield of Chinese cabbage cultured in the field plot

Experimental treatment: The plot area is 4.0 m x 5.0 m. In the field of 300 g dry weight/m?, the fertilizer containing fer- mented pig manure and the fertilizer containing unfermented pig manure are air-dried and applied. The application design of the base fertilizer is shown in the following table.

Table 7 The application design of the base fertilizer (CK)

Composition unfermented fermented pig fermented pig pig manure manure : unfer- manure : unfer- mented pig ma- mented pig ma-

EE nure =1 : 450 nure = 1 : 150 (w/w) (w/w)

Content of OO 160 300 poly-y- glutamic ac- id (mg/kg of fertilizer) © Experimental steps: The uniform and plump seeds of Chinese cabbage (with the variety of Erassica campestris L., Shanghaiqing) are selected to sow evenly, and the seedlings at the two-leaves- and-one-bud stage are planted with 10 cm spacing and watered ir- regularly. The seedlings at the stage of 7-9 leaves are harvested, and the above-ground parts are collected to obtain the fresh weight. The experiment results are shown in Table 8.

Table 8 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 2000 kDa) in different concentrations in the base fertilizer on the yield of Chinese cabbage

Treatments Average plant Average plant Yield in- weight of each weight of each creased by repetition (g) treatment (g) (%) 1 2 3

Fertilizer I 10.68 11.24 11.75 11.22

Fertilizer II 14.80 13.69 15.72 14.74 31.56

Fertilizer III 15.68 16.32 14.98 15.66 39.57 © Fertilizer II (containing 100 mg/kg of the poly-y-glutamic acid) and fertilizer III (containing 300 mg/kg of the poly-y- glutamic acid) may increase the yield of Chinese cabbage by 31.56% and 39.57% respectively compared with fertilizer I (control), in- dicating that the poly-y-glutamic acid may significantly increase the yield of Chinese cabbage.

Example 7

The effect of applying urea containing the poly-y-glutamic acid (with the average molecular weight of 200 kDa) at the trumpet stage of corn on the yield of corn cultured in the field

The experimental design is as follows: 1. Four treatments are set up:

A. Applying 7.5 kg of urea per 666 m’; B. Applying 7.5 kg of urea and 10 g of the poly-y-glutamic acid per 666 m?;

C. Applying 15 kg of urea per 666 m’; D. Applying 15 kg of urea and 10 g of the poly-y-glutamic acid per 666 m°. 2. Area: each plot has an area of 64 m? and 4 rows, and each row is 16 m in length and 1 m in width. 3. The fertilization is carried out on 23%, June (at the trumpet stage of corn). 4. The application method is spraying urea and the poly-y- glutamic acid after being dissolved and mixed in water.

The experimental results are shown in Table 9.

Table 9 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) on the yield of corn via the root irrigation

Treatments A ~~ B Cc DOD

Plot yield 40.5 45.5 52 53.5 (kg)

Plant 184 192.6 195.2 194.6 height (cm)

Increased 12.3 28.4 32.1 by than A (3)

Increased 2.9 by than C (5)

The experiment results are as follows: 1. The poly-y-glutamic acid may significantly increase the yield of corn under low fertilization levels, and the yield of corn is increased by 12.3%; 2. The poly-y-glutamic acid does not increase the yield of corn significantly under high fertilization levels, and the yield of corn is only increased by 2.9%.

Example 8

The effect of the poly-y-glutamic acid (with the average mo- lecular weight of 200 kDa) on increasing the fertilizer use effi- ciency in tobacco culture

Experimental steps: There are three growth stages of tobacco:

resettling growth stage, fast-growing stage and maturing stage.

Based on the actual conditions of base fertilizer and topdressing, the poly-y-glutamic acid with different application rates is ap- plied via the root irrigation at three different growth stages.

And 12.5 kg of pure nitrogen is applied per 666 m* and the ratio of pure nitrogen to potassium and phosphorus is 1 : 1 : 2. The appli- cation rate of the base fertilizer is 50% nitrogen fertilizer, 50% potassium fertilizer and 100% phosphorus fertilizer, and the base fertilizer is the ternary compound fertilizer of nitrogen, potas- sium and phosphorus + calcium superphosphate and potassium sul- fate. The topdressing is ammonium nitrate and potassium sulfate.

The experimental results are shown in Table 10.

Table 10 The effect of the poly-y-glutamic acid (with the av- erage molecular weight of 200 kDa) on the yield of tobacco in to- bacco culture

Treatments Yield per Yield in- 666 m° (kg) creased by (%)

Topdressing + 10 g/mu of poly-y- 170.4 6.4 glutamic acid via root irrigation at the resettling growth stage

Topdressing + 20 g/mu of poly-y- 177.8 11.1 glutamic acid via root irrigation at the resettling growth stage

Topdressing + 40 g/mu of poly-y- 185.7 16.0 glutamic acid via root irrigation at the resettling growth stage

Topdressing + 10 g/mu of poly-y- 156.5 2.2 glutamic acid via root irrigation at the fast-growing stage

Topdressing + 20 g/mu of poly-y- 159.3 -0.5 glutamic acid via root irrigation at the fast-growing stage

Topdressing + 40 g/mu of poly-y- 161.0 0.6 glutamic acid via root irrigation at the fast-growing stage

Topdressing + 10 g/m of poly-y- 158.5 -1.0 glutamic acid via root irrigation at the maturing stage

Topdressing + 20 g/mu of poly-y- 160.6 0.3 glutamic acid via root irrigation at the maturing stage

Topdressing + 40 g/mu of poly-y- 161.0 0.6 glutamic acid via root irrigation at the maturing stage

Topdressing + clear water via root 160.1 0.0 irrigation (control) © Overall, the application of the poly-y-glutamic acid via the root irrigation at the resettling growth stage increases the yield of tobacco mostly, and the application rate of 10 g/666 m’, with the yield-increasing rate of 15.9%, has the best yield-increasing effect, which is 25.5 kg higher than that of the control. Perform- ing the root irrigation at the fast-growing stage and maturing stage has no effect on increasing the yield. According to the con- centration of the poly-y-glutamic acid for the root irrigation at the resettling growth stage, the yield and output value of tobacco are gradually increased with the increase of the concentration of the poly-y-glutamic acid.

Claims

CONCLUSIONS

1. Application of poly-γ-glutamic acid as a fertilizer uptake promoter in agricultural planting, wherein the poly-γ-glutamic acid is a high polymer formed by D-glutamic acid (D-Glu) and L-glutamic acid (L-Glu) via o-amino groups and y-carboxyl groups linked in the form of peptide bonds, with a free carboxyl bond attached to an o-carbon atom of each repeating unit; wherein an average molecular weight of the poly-γ-glutamic acid is 10,000-10,000,000 Da; the poly-γ-glutamic acid is used as the promoter for the uptake of nitrogen, phosphorus, potassium or a combination thereof of fertilizers; and the fertilizer uptake promoter of the poly-γ-glutamic acid is a poly-γ-glutamic acid solution by fermentation in the liquid state, or is a bio-organic fertilizer containing the poly-γ-glutamic acid by fermentation in the solid state .

The use of the poly-γ-glutamic acid as the fertilizer absorption promoter on planting in agriculture according to claim 1, wherein the use of the poly-γ-glutamic acid as the fertilizer absorption promoter on planting in agricultural hydroponics includes: diluting a standard nutrient solution in a ratio of 1:3 to irrigate after a seed germination 3 d; diluting the standard nutrient solution in a ratio of 1:1 to irrigate after seed germination 7 d; transplanting uniform seedlings into @ 7 cm x 15 cm hydroponic pots when the uniform seedlings are at a two-leaf-and-one-bud stage (an early growth stage); adding 400 ml of the standard nutrient solution and 4-40 mg of the fertilizer absorption promoter of the poly-γ-glutamic acid dissolved in each of the hydroponic pots.

3. Application of the poly-γ-glutamic acid as the fertilizer absorption promoter in agricultural planting according to con-

claim 1, wherein the use of the poly-γ-glutamic acid as the fertilizer absorption promoter in agricultural planting includes root irrigation: mixing a ternary compound fertilizer in a normal dosage for different crops with the poly -γ-glutamic acid to dissolve in water, carrying out the root irrigation in a good growth stage of crops after planting the crops, wherein a dosage of the poly-γ-glutamic acid is in the range of 10 - 50 g/666 m'.

The use of the poly-γ-glutamic acid as the promoter of fertilizer absorption at planting in agriculture according to claim 1, wherein the use of the poly-γ-glutamic acid as the promoter of fertilizer absorption at planting in the agricultural foliar application includes: mixing the ternary compound fertilizer in the normal dosage for different crops with the poly-y-glutamic acid to dissolve in water, carrying out the foliar application at the right growth stage of the crop sen after planting the crops, wherein a dosage of the poly-γ-glutamic acid is in the range of 10 - 50 g/666 m°.

The use of the poly-γ-glutamic acid as the absorption promoter of fertilizer in the agricultural plantation according to claim 1, wherein the planted crops include vegetables, grains, soybeans and economic crops.

The use of the poly-γ-glutamic acid as the fertilizer absorption enhancer in the agricultural plantation according to claim 1, wherein the solid-state fermentation of the bio-organic fertilizer containing the poly-γ-glutamic acid comprises the following steps: { 1} preparation of seminal fluid: activating Bacillus subtilis (CCTCC202048) as a strain, and then inoculating the activated strain in a culture medium of the seminal fluid; wherein the culture medium is composed of a bovine extract 10.0 g, peptone 5.0 g, NaCl 10.0 g and distilled water 1000 ml, and a pH value of the culture medium is 7.2 to 7.4; the seed is during-

the cultured at 30°C for 4.5 hours and the strain growing to a mid-log phase (OD600nm = 1.4) is used to obtain the seminal fluid;

(2) preparation of a culture medium for amplified culture in the solid state fermentation, wherein the culture medium is composed of the following components by weight:

formulation 1: fresh pig manure 62.5%, soybean meal 25.0%, wheat bran 5.0%, glutamic acid 5.0% and citric acid 2.5%; adding water to the culture medium so that the moisture content of the culture medium is 60%, and adjusting the pH value to 9.0;

formulation 2: fresh cow manure 56.32%, soybean meal 23.35%, wheat bran 16.08% and glutamic acid 4.25%; adding water to the culture medium so that the moisture content of the culture medium is 60%, and adjusting the pH value to 9.0;

(3) inoculating the seminal fluid obtained in step (1) into the culture medium prepared in step (2) for the solid-state fermentation;

(4) conducting the solid-state fermentation under the following conditions: a loading amount of a fermenter

mentor is 10 to 30%, an inoculation amount is 83, an initial pH value is 9.0; cultivating at 40°C and conducting the solid-state fermentation for 48 hours in an aerated state to obtain a fermented product containing 5 to 6% of the poly-γ-glutamic acid; and

(5) drying the fermented product obtained in step (4) at 80°C to make the moisture content less than 5%, to obtain the bio-organic fertilizer containing 10 to 12% of the poly-γ-glutamic acid by the solid state fermentation.