WO1994006732A1 - Method for obtaining a wettable powder inoculant for use with leguminous crops - Google Patents

Method for obtaining a wettable powder inoculant for use with leguminous crops Download PDF

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Publication number
WO1994006732A1
WO1994006732A1 PCT/BR1993/000032 BR9300032W WO9406732A1 WO 1994006732 A1 WO1994006732 A1 WO 1994006732A1 BR 9300032 W BR9300032 W BR 9300032W WO 9406732 A1 WO9406732 A1 WO 9406732A1
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inoculant
formulation
peat
obtaining
wettable powder
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PCT/BR1993/000032
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French (fr)
Inventor
José Joaquim Rodrigues ABRAÃO
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Geratec S/A - Biotecnologia Aplicada
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Priority to AU49372/93A priority Critical patent/AU4937293A/en
Publication of WO1994006732A1 publication Critical patent/WO1994006732A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like

Definitions

  • the invention refers to a method for obtaininga wettable powder inoculant elaborated from Rhizobium spp and formulated as a wettable powder for use with leguminous crops, whose useful life is increased up to 25 months, contrary to other formulations made with peat supports, which have a useful life of only 6 months.
  • the inoculant according to the present invention is also characterized by having a greater degree of stickness to the seeds and by not requiring storage at low temperatures.
  • the inoculant according to the present invention is a biological fertilizer having a peat support and comprising more than 50% of organic material, enriched with a Rhizobium type bacteria.
  • the inoculation method according to the present invention comprises mixting the inoculant with the seeds slightly prior to sowing them in order to promote ⁇ higher degree of symbiosis between the Rhizobium spp and the seeds, which results, in the formation of nodules in the roots of the plants through which the nitrogen present in the atmosphere is absorbed and transferred to the plant, therefore partially or completely substituting with atmospheric nitrogen the nitrogen-based fertilizers presently used with leguminous crops.
  • the present inventors started iron a Rhizobium spp culture , leavened for 72 hours, centrifugated and lyophilized Thereafter, the lyophile was formulated as at wettable powder wherein the peat inoculant bath was replaced by a lyophile and the peat replaced by inert supports, rich in polysaecharides, associated to a celular protector, which imparts to the new formulation the following characteristics: (1) it easily adhres, to the seeds, because it has adhesives in the formulation; (1) it is economic arid easy to use because it provides, after it has been jellied in water, a direct mixing with the seeds in the planting box: (3) it can be formulated with inert supports which do not cause; any harm to the sowing machines because they do not have art/ abrading substances in their composition; (4) it has a storage useful life of up to 25 months, therefore well superior to the 6 months useful life of the inoculants supported by peat or peat; (5) it maintained
  • the concentration of the Rhizobium spp during the fermentation was evaluated by counting the cells in a Neybauer chamber with a bioculau microscope, at 400X, and also by inoculating a Petry's plaque with an agaric culture-media and a Congo red marker. After the bath, comprising a mixture of SEMIA 587 and 5019 from Rhizobium japonicum, had fermented and reached a logarithmic growth stage with concentration of 1.7 ⁇ 10 9 viable cells/ml o f the bath, it was submitted to centrifugation.
  • a criogenic protective solution comprising 3.0% of glycerol, 2.0% of gelatin, 2.5% of casein, and 100 ml ofde-ionized water qsq, with an adjusted pH of 6.8 and sterilized at 120 oC for 15 minutes was used to treat thebiological mass.
  • the biological mass was twice immersed in the criogenic protective solution with a 50% proportion in order to protect the cells during the method of lyophilization-drying.
  • the drying procedure was, conducted an a FIC lyophilizer model 50-L for 26 hours,, and it finally had a concentration of 1.5 ⁇ 10 11 viable cells/gram of the lyophile with a water content of 1% .
  • each new dose of the inoculant comprises;
  • the substances were dried at 105 oC, untill a constant weight was obtained, and thereafter the formulation was prepared obeying the following steps: (A) the lyophile and the celular protector (saccharose) were mixed together with the dispersant (sodium sulphate lauril); (B) the inert (kaolin) and the adhesive (carboxymethil cellulose) were mixed. After the formulation had been balanced, mixtures A and B were mixed, in a V shaped stainless mixer having a capacity of 100 liters mechanically agitated for 20 minutes. The wettable powdor-formulation was then tightly packaged in 50 ml bottles or polypropilene bags.
  • each dose corresponds to 2.5 g of the wettable powde- formulation, which is to be diluted in 120 ml of water up to six hours prior to be used.
  • the gelification of the product in the water forms a gel which easily adhere to the seeds required to cover half hectare.
  • soy-bean Glycine max
  • each dose (2.5 g of the formulation + 120 ml of water) shall be applyed to 50 Kg of seeds, which amount corresponds to about half hectare.
  • Example 1 Three months after the lyophilization process, the analysis of the lyophile showed it contained 1.5 ⁇ 10 11 viable cells of lyophile/gram (Example 1), which survival rate was maintained for up to 25 months.
  • Example 2 shows the results of experiences conducted at the Green House of the Institute de pesquisas Agronpmicas da Secretaria da Agriculture (IPAGRO-RS). It can be noticed that the results of the formulation of the wettable powder inoculant was superior to that with the same results obtained with a peat-supported inoculant from IPAGRO (official results), in both types of soil, both with respect to the formation of dry nodules as with respect to t he in crease of weig ht of t he abo v e ground part of t he p l an t .
  • IPAGRO official results
  • Example 3 shows the average results of the grain yield, dry weight of the nodules and the final population of the plants, as well as a comparation of the averages according to Ducan test at 1% and the coefficients of the variations.
  • the inoculant according to the present invention formulated as a wettable powder, in addition to not losing its agronomical efficiency, provides a series of advantages when compared with the traditionally used peat-supported inoculants of the prior art, such as:
  • the peats normally used have a high content of sand and silica which increase, the wear of the sowing dis. s in the machines, which does not happen with the wettable powder inoculating formulation according to the present invention which does not have any abrading substances in its formulation.
  • Example 4 Effect of storage temperature of inoculant with respect to the survival of the Rhizobium spp
  • NMP offocial method for analysing dilution and infection in plants

Abstract

The invention refers to a method for obtaining a wettable powder inoculant formulation for use with leguminous crops, comprising the industrialization of an inoculant for leguminous from a centrifugated biological mass of Rhizobium spp twice immersed in a cryogenic protective solution rich in sugar and protein which is subjected to a lyophilization method. After a lyophile having a concentration of 1.5 x 1011 of viable cells/gram has been obtained, it is formulated with substances rich in polysaccharides and associated with a cellular protector which substitutes the previously used peat support. The new formulation is advantageous when compared with the peat-supported inoculants because it has higher adherence to the seeds, it can be directly applied to the seeds in the sowing box, it does not cause an increased wear of the sowing machines, and it has a storage useful life of up to 25 months even when stored at room temperature.

Description

Specification
"METHOD FDR DETAINING A WETTABLE POWDER INOCULANT FOR USE WITH LEGUMINOUS CROPS" Field of the Invention
The invention refers to a method for obtaininga wettable powder inoculant elaborated from Rhizobium spp and formulated as a wettable powder for use with leguminous crops, whose useful life is increased up to 25 months, contrary to other formulations made with peat supports, which have a useful life of only 6 months. The inoculant according to the present invention is also characterized by having a greater degree of stickness to the seeds and by not requiring storage at low temperatures.
Background of the Invention
The inoculant according to the present invention is a biological fertilizer having a peat support and comprising more than 50% of organic material, enriched with a Rhizobium type bacteria.
The inoculation method according to the present invention comprises mixting the inoculant with the seeds slightly prior to sowing them in order to promote α higher degree of symbiosis between the Rhizobium spp and the seeds, which results, in the formation of nodules in the roots of the plants through which the nitrogen present in the atmosphere is absorbed and transferred to the plant, therefore partially or completely substituting with atmospheric nitrogen the nitrogen-based fertilizers presently used with leguminous crops.
Notwithstanding the positive effect which can easily be confirmed by the higher productivity of the leguminous crops, the use d an inoculant manufactured with a peat support still find a strong resistance from Iirazilian farmers due to the "following reasons: its application requires the use of additional peoples it increases the wear of the sowing machines; it does not easily adhere to the seeds; it requires special storage and conservation conditions, with temperatures below 15 ºC; the increases in productivity can not be easily confirmed; and its storage life of 6 months. On the other hand., industries also find it difficult to use peat due to; the unevoness and low quality of the peat (an acid fibrous peat with a high content of sand and a small capacity of retaining water); the need of extracting great volumes of peat which have to be dried and treated, the treatment usually employing methods that generate extremely unhealthy environmental conditions; the continuous extraction of the peat might cause irreversible damages to the environment; not to mention the above-cited problems related to its formulation and application.
In order to overcome the above-listed disadvantages related with the industrialization and application of inoculants manufactured from peat, researchers have developed other promissing techniques for manufacturing the inoculant, among which liophilization. using as support a vegetal or mineral oil, peat, organic waste from crops, vegetal or mineral coal and clays (talcum, bentonite, vermiculits). All the above re f erre d supports:, however, suffer from problems related to the application en the product or the survival of the Rhizobium. The following works corroborate the aboveaffirmative: Kremer & Petersen (1983), in the united States, noticed that the Rhizobium spp under a lyophilized form and using an oil or a vegetable coal as support still would have, after 125 days stored at a temperature o f 35 ºC a survival rate well above the one presented by the Rhizobium spp supported on grounded peat. In another work, Kremer at al ( 1982 ) also noticed similar results in an inoculant supported by an oil stored both at 35 ºC and 65 ºC.
Additionally, a lyophilized inoculant supported by an oil is now being manufactured in Brazil, but with some problems with respect to the stability of the suspension and of the survival of the Rhizobιum spp. This is confirmed by Scholles et al (1990), which have analyzed several commercially available Brazilian products comprising lyophilized Rhizobium spp supported in oil, only to notice that from 6 samples none presented the minimum concentration required by brazilian, law.
Notwithstanding the benefits which result from inoculation, it is believed that over 50% of the farmers, do not use inoculants because of the poor quality of the inoculants and to the above cited problems. This fact has caused an effort from researchers to provide improved inoculant formulations for use with leguminous crops which reduce to a minimum the problems related to manufacturing, application and agronomical efficiency.
With this Purpose in mind, the present inventors" started iron a Rhizobium spp culture , leavened for 72 hours, centrifugated and lyophilized Thereafter, the lyophile was formulated as at wettable powder wherein the peat inoculant bath was replaced by a lyophile and the peat replaced by inert supports, rich in polysaecharides, associated to a celular protector, which imparts to the new formulation the following characteristics: (1) it easily adhres, to the seeds, because it has adhesives in the formulation; (1) it is economic arid easy to use because it provides, after it has been jellied in water, a direct mixing with the seeds in the planting box: (3) it can be formulated with inert supports which do not cause; any harm to the sowing machines because they do not have art/ abrading substances in their composition; (4) it has a storage useful life of up to 25 months, therefore well superior to the 6 months useful life of the inoculants supported by peat or peat; (5) it maintained its agronomical efficiency after it had been manufactured and also maintened its concentration of RHizobium spp in a level higher than the provided by the Brazilian law, for a period of up to 25 months, which is the greatest ad vantage provided by the inoculant according to the present invention. mainly because up to now it was not known that any commercially available Rhizobium spp product could survive "for so long stored at normal conditions.
Manufacturing Process
SΕMIA 557 and 5019 stocks, usually recommended for soy-bean crops were multiplied for 72 hours in self- sterilining stainless reactors with a capacity of 1500 liters and aerated with i V/V up to reaching thes tage of iogarithmic growth. The media of the culture was thensterilized at 120 ºC or about 1 hour and kept at 28 ºC with constant agitation at 350 rpm and comprised the following substances:
Man ino l . . . . . . . . . . . . .. .. . . . . . . . . . . .. .. 10 . 0 g / l
Yeast water . . . . . . . . . .. .. . . . . . . . . .. .. 100.0 g / l
Potassium phosphate, dibasic . . . . . . . . . . 0.5 g / l
Magnesium sulphate . . . . . . . . . .. .. .. . . . . . . 0.2 g / l
Ammonium phosphate, monobasic . . . . . . . . . 0.5 g / l
Potassium nitrate . . . . . . . . . .. . .. . . . . . . . 1.0 g / l
Sodium chloride . . . . . . . . . .. .. . . . . . . . . .. 0.2 g / l
Iron sulphate . . . . . . . . . .. . . . . . . . . . . .. . 0.01 g / l
Manganese sulphate . . . . . . . . . .. . . .. . . .. 0.01 g / l
Water qsq . . . . . . . . . .. . . . . . . . . . . .. . . . . . 1.000 ml pH corrected to . . . . . . . . . .. . . . . . . . . . . .. .. . . 7.0
The concentration of the Rhizobium spp during the fermentation was evaluated by counting the cells in a Neybauer chamber with a bioculau microscope, at 400X, and also by inoculating a Petry's plaque with an agaric culture-media and a Congo red marker. After the bath, comprising a mixture of SEMIA 587 and 5019 from Rhizobium japonicum, had fermented and reached a logarithmic growth stage with concentration of 1.7 × 109 viable cells/ml o f the bath, it was submitted to centrifugation. A tubular centrifugue IMA, model T-7 with a continuous flow and a capacity of 300 1/hour was used in the centrifugation process, where the supernatant was eliminated and the precipitate presented an yield of 8.0 g/l of biclogical mass with a water content of 75% .
A criogenic protective solution comprising 3.0% of glycerol, 2.0% of gelatin, 2.5% of casein, and 100 ml ofde-ionized water qsq, with an adjusted pH of 6.8 and sterilized at 120 ºC for 15 minutes was used to treat thebiological mass. The biological mass was twice immersed in the criogenic protective solution with a 50% proportion in order to protect the cells during the method of lyophilization-drying. The drying procedure was, conducted an a FIC lyophilizer model 50-L for 26 hours,, and it finally had a concentration of 1.5 × 1011 viable cells/gram of the lyophile with a water content of 1% .
After obtaining the lyophile, a study of several wettable powder formulations using different special supports with higher adherence to the seeds and higher useful life of the Rhizobium spp was conducted. This was determined by using supports rich in sugars, proteins and a celular protector to enhance the rehydration of the bacteria. The following substances can be used in order to achieve this objective. As celular protectors: saccharose, glucose, casein and peptone. As adhesives: carboxymethilcellulose, powdered arable gum. As dispersants: tween 35, sodium sulphate lauril. As inerts: caulim, bentonite and silieium bioxide.
The amount of lyophile. to be used in the formulation depends on the concentration of viable cells, In the formulation of the wettable powder according to the present invention 0.2 g/dose of the lyophilo were used, each dose of 120 ml of the inoculant. Therefore, each new dose of the inoculant comprises;
Lyophile (1.5 × 1011 viable cells/g) ...... 0.2 g Saccharose ( celular protector) ............ 0.2 g Sodium sulphate lauril (dispersant) ....... 0.5 g
Kaolin (inert) ............................ 0.6, g
Carboxymethil cellulose (adhesive) ........ 1.0 g
Total ..................................... 2.5 g
Initially, the substances were dried at 105 ºC, untill a constant weight was obtained, and thereafter the formulation was prepared obeying the following steps: (A) the lyophile and the celular protector (saccharose) were mixed together with the dispersant (sodium sulphate lauril); (B) the inert (kaolin) and the adhesive (carboxymethil cellulose) were mixed. After the formulation had been balanced, mixtures A and B were mixed, in a V shaped stainless mixer having a capacity of 100 liters mechanically agitated for 20 minutes. The wettable powdor-formulation was then tightly packaged in 50 ml bottles or polypropilene bags.
From a point of view of in-site final use, each dose corresponds to 2.5 g of the wettable powde- formulation, which is to be diluted in 120 ml of water up to six hours prior to be used. The gelification of the product in the water forms a gel which easily adhere to the seeds required to cover half hectare. In the case of soy-bean (Glycine max), each dose (2.5 g of the formulation + 120 ml of water) shall be applyed to 50 Kg of seeds, which amount corresponds to about half hectare. Agronomical Results Obtained with the Wet cable Powder Inoculant Formulation
Three months after the lyophilization process, the analysis of the lyophile showed it contained 1.5 × 1011 viable cells of lyophile/gram (Example 1), which survival rate was maintained for up to 25 months.
From Examples 1 and 4 it can be noticed that the results of infection in plants (NMP), 25 months after the lyophile had been obtained, show a minimum of 1.7 × 10° viable cells/gram and that the wettable powder "formulation resulting therefrom, initially with 4.5 × 109 viable cells/gram, only shows a tendency to reduce the concentration of Rhizobium spp after 25 months of storage tame at a temperature of 35 ºC. It is advisable to remind that the conventional peat-based inoculants have a 6 months storage time and should present at least 10 × 106 viable cells/gram, six months after it has been manufactured. Therefore, even with 25 months, the wettable-powder formulation according to the present invention is well above the legal provisions about Fertilizers, Correctives and Inoculants.
Example 2 shows the results of experiences conducted at the Green House of the Institute de pesquisas Agronpmicas da Secretaria da Agriculture (IPAGRO-RS). It can be noticed that the results of the formulation of the wettable powder inoculant was superior to that with the same results obtained with a peat-supported inoculant from IPAGRO (official results), in both types of soil, both with respect to the formation of dry nodules as with respect to t he in crease of weig ht of t he abo v e ground part of t he p l an t .
In an experience conducted by the Centro de Biotecnologia e Faculdade de Agronomia da UFRCS (Grafico 1), Silva et al (1992) also noticed increases in over 1.0 t of soy-bean grains, when using the wettable powder inoculant formulation according to the present invention applyed through the seeds,,, when compared to the treatment without the use of the inoculant. Example 3 shows the average results of the grain yield, dry weight of the nodules and the final population of the plants, as well as a comparation of the averages according to Ducan test at 1% and the coefficients of the variations., It can be noticed from comparing the averages that the average grain yield after the treatment with the wettable powder according to the present invention, according to Ducan test at 1% shows that it was statistically similar to that of the peat-based inoculants, but superior ro the same? result without inoculant.
The analysis of the results of the previously shown experiences conducted allow us to conclude that the concentration of viable cells of the Rhizobium spp an the wettable powder inoculant formulation according to the present invention, 25 months after it was first manufactured, has a concentration above the minimum provided by law, which is 10 × 106 viable cells/gram at the last useful day of validity of the product. and this reflected positively in the grain yield of the soy-bean, when compared to the traditionally used peat-supported inoculants.
Advantages
The inoculant according to the present invention formulated as a wettable powder, in addition to not losing its agronomical efficiency, provides a series of advantages when compared with the traditionally used peat-supported inoculants of the prior art, such as:
a) The peat-supported inoculants require the use of additional people, while the wettable powder inoculating formulation according to the present invention does not require the use of additional people because it can be directly applied to the seeds in the planting box.
b) The commercially available inoculants have to be prepared and used when the seeds are being sowed, while the wettable powder inoculating formulation according to the present invention can be prepared up to 6. hours prior to its use.
c) The peats normally used have a high content of sand and silica which increase, the wear of the sowing dis. s in the machines, which does not happen with the wettable powder inoculating formulation according to the present invention which does not have any abrading substances in its formulation.
d) The good adherence of the peat-supported inoculants of the prior art to the seeds depends on the use of adhesives and powder mixing machines, while- the wettable powder inoculating formulation according to the present invention easily adheres to the seeds because it has adherents in its composition. e) The commercially available peat-supported inoculants have a useful life of 6 months while the wettable powder inoculating formulation according to the present invention has a useful life of up to 25 months. Example 1:
Tests conducted in a Green House by the official method of diluting and infection in plants (NMP) in a lyophilized bath (lyophile) and an the wettable powder inoculating formulation (PM).
Storage Time Tests in Plants (NMP)*
(Months) Lyophile Wettable Powder Peat
Formulation Inoculant
(cells/g) (cells/g/p.c.) 3 1.5 × 1011 4 . 5 × 109 5.8 × 108 6 - 4 . 5 × 109 6.9 × 107 6 - + 1 . 4 × 109 5.9 × 105 12 1.5 × 1011 1.7 × 1010 0** 15 - 1 . 4 × 109 0
18 - 4 . 5 × 109 0
21 1.5 × 1011 1 . 7 × 108 0
25 1.5 × 1011 1 . 7 × 108 0 * Average of four repetitions
cells/g/p.c. - amount of viable cells per gram of wettable powder formulation product.
** - Low concentration in which the official method did not detect the presence of Rhizoblum, spp Example 2 - Experiment conducted by IPAGRO-RS; in a green house from UFRGS, with two different types of arenous soil, involving inoculatn formulation for soy-bean. The harvesting of the nodules (mg/vase) and of the upper part of the soy-bean (g/vase) was, conducted in January/1991 (Jo o Kolling et al, 1991, unpublished).
Treatments Dry Weight of Nodules Plant Weight Soil 1 Soil 2 Aver. Soil 1 Soil 2 Aver.
IPAGRO
Inoculant* 131 183 157 2.48 2.94 2.66
No inoculant 9 9 9 2.10 2.36 2.23
Moist, powder**
Formulation 147 231 189 2.72 3.05 2.89
Oil-supported**
Formulation 166 110 138 2.66 2.69 2.68
Soil 1 - Vacacai Soil
Soil 2 - Såo Jeronimo Soil
* Peat-supported inoculant prepared by IPAGRO
** Wettable powder formulation and oil-supported f ormu l ati on con tain ing l yophi l i zed Rhi zobium j apon i cum Example 3 - Average yield of 5 repetitions of soy-bean grains (Kg/ha), dry weight of the nodules (g), final population of plants/linear meter of variety, Cobb, according to the inoculant used,
Treatments Grain Yield Dry Weight Final
Aver. % No Inoc. % W/Inoc. Nodules Population
No Inoc. 1915 C 100 61.40 0.98 B 20.4 A Inoc.¹ 3117 B 102.8 100 2.03 A 21.6 A
Sterilized Peat-supported
Inoc. 3315 BA 173.1 106.40 1.92 A 19.8 A
Wettable powder
Form.** 3223 BA 168.3 183.40 2.11 A 22.8 A Variation 5.835 - - 10.870 8.815
1- Commercially available peat-supported inoculant
2- Wettable powder formulation
Averages followed by the same letter do net differ from each other acording to Ducan test at 1% probability
Example 4 - Effect of storage temperature of inoculant with respect to the survival of the Rhizobium spp
Storage Time Peat Inoc.* Moist. Powder Inoc.
(Months) 15 ºC 35 ºC Room 15 ºC 35ºC Room
3 5.8×108 5.8×108 5.8×108 4.5×109 4.5×109 4.5×109
6 6.9×107 5.8×105 6.9×107 1.7×109 4.5×109 4.5×109
9 5.9×106 - 1.8×105 4.5×109 1.4×109 1.4×109
12 3.1×105 - - 9.3×109 1.7×10101.7×101
1 5 - - - 7.0×109 1.4×109 1.4×109
18 - - - 4.5×109 1.8×109 4.5×109
21 - - - 1.8×109 1.7×108 1.7×109
25 - - - 4.5×109 1.7×108 1.7×108
* - offocial method for analysing dilution and infection in plants (NMP): number of viable cells/gram of formulate:., product
Figure imgf000017_0001
Graphic 1 - Grain yield in the evaluation experience of the application of the inoculant in covering soy-bean
(Silva et al. 1992, M.B., 1992)
(1) - Official Result
(2) - Peat-supported inoculant through the seeds
(3) - Wettable powder inoculant formulation through the seeds
(4) - Wettable powder inoculant formulation coverage
(5) - Treatment 1 + treatment 2
(6) - Treatment 3 + treatment 4

Claims

CLA IMS
1 A method for obtaining a wettable-pownder inoculant for use with leguminous crops, chsracterized by the industrialization, from a biological mass of Rhizobium spp twice immersed in a solution rich in sugars and proteins, comprising casein, glycerol and gelatin, which concentration varies from 1.5. × 1011 to 1. 7 × 1011 of viable cells/gram of lyophile, after it has been lyophilized, which is used to substitute the bath for manufacturing the new formulation
2. A method for obtaining a wettable-powder inoculant for use with leguminous crops, according to claim 1, wherein the peat support is substituted by one of the following supports rich in polysaccharides associated with a celular protector: saccharide. sodium sulphate lauril, kaolin and carboxymethil cellulose, which impart to the new formulation a higher adhenence to the seeds after it has been jellied in water, which Formulation can be preparated up to 6 hours prior to its use
3. A method for obtaining a wettable powder inoculant comprising 3 lyophile, a celular protector, a dispersant, and adhesive and an inert support for use with leguminous crops, according to claim 1, characterized in that the inoculant has a storage useful life of up 25 months while still having a minimum concentration 1.7. × 108 viable cells/gram of the formulation
4. A method for obtaining a wettable powder inoculant comprising a lyophile a celular protector, a dispersant, an adhesive and an inert support for use with leguminous crops, according to claim 1, characterized in that the Rizobium SPP present in the formulation has a storage useful life of up to 25 months even when stored at room temperature.
PCT/BR1993/000032 1991-09-11 1993-09-10 Method for obtaining a wettable powder inoculant for use with leguminous crops WO1994006732A1 (en)

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BRPI9103905 1992-09-11

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CN103288536A (en) * 2013-05-29 2013-09-11 王龙潮 Probiotic wettable powder fertilizer
WO2014160827A1 (en) 2013-03-28 2014-10-02 Novozymes Bioag A/S Compositions and methods for enhancing microbial stability
EP3702441A4 (en) * 2017-10-27 2020-12-02 Kumiai Chemical Industry Co., Ltd. Microorganism lyophilized composition

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EP2126043A2 (en) * 2007-06-21 2009-12-02 Saturnina Halos Stable organic-carrier-based microbial inoculants and method for producing the same
EP2126043A4 (en) * 2007-06-21 2010-07-28 Saturnina Halos Stable organic-carrier-based microbial inoculants and method for producing the same
WO2014160827A1 (en) 2013-03-28 2014-10-02 Novozymes Bioag A/S Compositions and methods for enhancing microbial stability
US9986735B2 (en) 2013-03-28 2018-06-05 Novozymes Bioag A/S Compositions and methods for enhancing microbial stability
US20180242576A1 (en) * 2013-03-28 2018-08-30 Novozymes Bioag A/S Compositions and methods for enhancing microbial stability
US11369110B2 (en) 2013-03-28 2022-06-28 Novozymes Bioag A/S Compositions and methods for enhancing microbial stability
CN103288536A (en) * 2013-05-29 2013-09-11 王龙潮 Probiotic wettable powder fertilizer
CN103288536B (en) * 2013-05-29 2014-07-02 王龙潮 Probiotic wettable powder fertilizer
EP3702441A4 (en) * 2017-10-27 2020-12-02 Kumiai Chemical Industry Co., Ltd. Microorganism lyophilized composition

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