USE OF CATIONIC HYDROXYALKYL GUARS FOR MICROORGANISMS GROWTH

Abstract

The present invention relates to the in vitro use of a cationic hydroxyalkyl guar for maintaining or increasing the growth rate of microorganisms, wherein said cationic hydroxyalkyl guar has an average molecular weight of between 2,000 Daltons and 90,000 Daltons.

Claims

1. A method, comprising maintaining or increasing the growth rate of microorganisms by using a cationic hydroxyalkyl guar in vitro or on a plant, on a seed or in soil, wherein said cationic hydroxyalkyl guar has an average molecular weight of between 2,000 Daltons and 90,000 Daltons.

2. (canceled)

3. The method of claim 1, wherein the microorganisms are fungi or bacteria.

4. The method of claim 1, the growth rate of microorganisms is increased.

5. The method of claim 1, wherein the cationic hydroxyalkyl guar is obtained by chemically modifying a guar with a cationic etherifying agent.

6. The method of claim 1, wherein the cationic hydroxyalkyl guar is selected from the group consisting of: cationic hydroxyethyl guar, cationic hydroxypropyl guar, cationic hydroxybutyl guar.

7. The method of claim 1, wherein the cationic hydroxyalkyl guar has a Degree of Substitution of between 0.005 and 1.

8. The method of claim 1, wherein the cationic hydroxyalkyl guar has an average molecular weight of between 5,000 Daltons and 90,000 Daltons.

9. The method of claim 1, wherein the cationic hydroxyalkyl guar has a degree of hydroxyalkylation comprised between 0.1 and 1.7.

10. The method of claim 1, comprising a step of contacting at least one seed with a cationic hydroxyalkyl guar having an average molecular weight of between 2,000 Daltons and 90,000 Daltons.

11. (canceled)

12. A biostimulant composition comprising at least one microorganism and at least one cationic hydroxyalkyl guar having an average molecular weight of between 2,000 Daltons and 90,000 Daltons.

13. A kit comprising at least one microorganism and at least one cationic hydroxyalkyl guar having an average molecular weight of between 2,000 Daltons and 90,000 Daltons.

14. (canceled)

15. A seed coated with the biostimulant composition of claim 12.

16. The method of claim 5, wherein the cationic etherifying agent is a quaternary ammonium salt selected from the group consisting of: 3-chloro-2-hydroxypropyl trimethyl ammonium chloride, 2,3-epoxypropyl trimethyl ammonium chloride, diallyldimethyl ammonium chloride, and trimethylammoniumpropyl methacrylamide.

17. The method of claim 6, wherein the cationic hydroxyalkyl guar is guar hydroxypropyltrimonium chloride.

18. The method of claim 7, wherein the cationic hydroxyalkyl guar has a Degree of Substitution of between 0.12 and 0.5.

19. The method of claim 8, wherein the cationic hydroxyalkyl guar has an average molecular weight of between between 5,000 Daltons and 60,000 Daltons.

20. The method of claim 9, wherein the cationic hydroxyalkyl guar has a degree of hydroxyalkylation comprised between 0.2 and 1.0.

21. The method of claim 10, wherein the microorganisms are bacteria.

22. The biostimulant composition of claim 12, wherein the at least one microorganism is a bacterium.

Description

EXAMPLES

Example 1

[0102] The following materials are used in the experiments:

[0103] Guar: a guar hydroxypropyltrimonium chloride having an average molecular weight between 5,000 and 25,000 Daltons, a DS of 0.2, and a MS between 0.2 and 1.0, available from Solvay (provided as a powder)

[0104] Bacteria strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil. [0105] Bacillus subtilis CCT 0089 [0106] Bacillus megaterium CCT 0536 [0107] Bradyrhizobium japonicum CCT 4065

[0108] All strains were stored at −80° C. in the appropriated culture media, containing 15% of glycerol.

[0109] Two different culture media were used in the experiments: [0110] NA media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only) [0111] YMA media containing per liter: 0.5 g of monobasic potassium phosphate, 0.2 g of magnesium sulphate; 0.1 g of sodium chloride; 0.5 g of yeast extract; 10 g of mannitol (for inoculum and solid media only); 5 mL of a 5% bromothymol blue solution and 15 g of agar (solid media only).
For strains Bacillus subtilis and Bacillus megaterium, NA media was used. For strain Bradyrhyzobium japonicum, YMA media was used. These media were selected according to strains supplier.

[0112] A 250 mL shake flask containing 100 mL of NA or YMA culture media, was inoculated with 1 mL of the stock culture and incubated at 30° C., 150 rpm for 72 hours.

[0113] For each strain, 10 mL of the reactivation media were then transferred into a 250 mL shake flask containing 100 mL of the same media, with the addition of guar powder (at 1 wt % in the incubation media); and incubated at 30° C., 150 rpm, for 96 hours. An experiment without addition of guar powder is also performed for each strain as a control.

[0114] 100 μL samples of each experiments were taken after 0 h, 24 h, 48 h, 72 h and 96 h of incubation. These samples were diluted (the dilutions were variable according to strain growth, being from 1×10.sup.−5 up to 1×10.sup.−15) and the dilutions plated in solid NA or YMA media. The plates were incubated at 30° C. until appearance of colonies. After incubation, the number of colonies present in each dilution was counted and used to evaluate bacterial growth.

[0115] For bacterial growth rate determination, a graph of the log.sub.10 (number of colonies) versus time of incubation was constructed. The straight line in this graph represents the exponential phase of bacterial growth and the angular coefficient represents the bacterial growth rate (O.

[0116] The μ value was used to compare all the experiments and to evaluate the influence of guar addition on bacterial growth.

[0117] For this set of experiments the ratio of microorganisms and guar is equal to 3.50×10.sup.4 CFU/g. The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 1:

TABLE-US-00001 TABLE 1 Bacteria growth Composition rate (h.sup.−1) Bacillus subtilis CCT 0089 0.0647 Bacillus subtilis CCT 0089 + guar 0.0739 Bacillus megaterium CCT 0536 0.0605 Bacillus megaterium CCT 0536 + guar 0.0690 Bradyrhyzobium japonicum CCT 4065 0.0891 Bradyrhyzobium japonicum CCT 0.0880 4065 + guar

[0118] For the three strains, a comparable or higher value of bacteria growth rate is obtained in presence of guar. The addition of guar permits to maintain or increase the growth rate of these different strains of bacteria. In Table 2 are reported the relative increase or decrease of bacteria growth rate with the addition of guar compared to the control for each strain. An increase of bacteria growth rate of +14% is observed for the two gram positive bacteria (Bacillus subtilis and Bacillus megaterium), whereas a nil value is observed for Bradyrhyzobium japonicum, which correspond to a comparable growth rate of bacteria with and without guar.

TABLE-US-00002 TABLE 2 Relative increase of bacteria growth rate Strain with guar addition Bacillus subtilis CCT 0089 14% Bacillus megaterium CCT 0536 14% Bradyrhyzobium japonicum CCT 4065  0%

[0119] A similar experiment was conducted at a ratio bacteria/guar equals to 7.00×10.sup.5 on the same bacteria species, a comparable or higher value of bacteria growth is obtained in presence of guar.

Example 2

[0120] The following materials are used in the experiments:

[0121] Guar: a guar hydroxypropyltrimonium chloride having an average molecular weight between 5,000 and 25,000 Daltons, a DS of 0.2, and a MS between 0.2 and 1.0, available from Solvay (provided as a powder)

[0122] Liquid guar formulation: an aqueous formulation of the powder guar at 25% from Solvay

[0123] Bacteria strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil. [0124] Bacillus subtilis CCT 0089 [0125] Bacillus megaterium CCT 0536 [0126] Bradyrhizobium japonicum CCT 4065

[0127] All strains were stored at −80° C. in the appropriated culture media, containing 15% of glycerol.

[0128] Two different culture media were used in the experiments: [0129] NA media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only) [0130] YMA media containing per liter: 0.5 g of monobasic potassium phosphate, 0.2 g of magnesium sulphate; 0.1 g of sodium chloride; 0.5 g of yeast extract; 10 g of mannitol (for inoculum and solid media only); 5 mL of a 5% bromothymol blue solution and 15 g of agar (solid media only).

[0131] For strains Bacillus subtilis and Bacillus megaterium, NA media was used. For strain Bradyrhyzobium japonicum, YMA media was used. These media were selected according to strains supplier.

[0132] A 250 mL shake flask containing 100 mL of NA or YMA culture media, was inoculated with 1 mL of the stock culture and incubated at 30° C., 150 rpm for 72 hours.

[0133] For each strain, 10 mL of the reactivation media were then transferred into a 250 mL shake flask containing 100 mL of the same media, with the addition of guar powder or guar liquid formulation; and incubated at 30° C., 150 rpm, for 96 hours. An experiment without addition of guar powder is also performed for each strain as a control.

[0134] 100 μL samples of each experiments were taken after 0 h, 24 h, 48 h, 72 h and 96 h of incubation. These samples were diluted (the dilutions were variable according to strain growth, being from 1×10.sup.−5 up to 1×10.sup.−15) and the dilutions plated in solid NA or YMA media. The plates were incubated at 30° C. until appearance of colonies. After incubation, the number of colonies present in each dilution was counted and used to evaluate bacterial growth.

[0135] For bacterial growth rate determination, a graph of the log.sub.10 (number of colonies) versus time of incubation was constructed. The straight line in this graph represents the exponential phase of bacterial growth and the angular coefficient represents the bacterial growth rate (μ).

[0136] The μ value was used to compare all the experiments and to evaluate the influence of the two guars addition on bacterial growth. For this set of experiments the ratio of microorganisms and guar was set at 1.0×10.sup.10 CFU/g. The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 3:

TABLE-US-00003 TABLE 3 Bacteria growth Composition rate (h.sup.−1) Bacillus subtilis CCT 0089 0.0862 Bacillus subtilis CCT 0089 + guar powder 0.0931 Bacillus subtilis CCT 0089 + guar 0.0975 liquid formulation Bacillus megaterium CCT 0536 0.0834 Bacillus megaterium CCT 0536 + guar powder 0.1018 Bacillus megaterium CCT 0536 + guar 0.0958 liquid formulation Bradyrhyzobium japonicum CCT 4065 0.0915 Bradyrhyzobium japonicum CCT 4065 + guar powder 0.0936 Bradyrhyzobium japonicum CCT 4065 + guar 0.0913 liquid formulation

[0137] For the three strains, a comparable or higher value of bacteria growth rate is obtained in presence of the guar under powder form and the guar in aqueous formulation. The addition of guar permits to maintain or increase the growth rate of these different strains of bacteria. In Table 4 are reported the relative increase or decrease of bacteria growth rate with the addition of the two guar grades compared to the control for each strain. An increase of bacteria growth rate ranging from 8% to 22% is observed for the two gram positive bacteria (Bacillus subtilis and Bacillus megaterium), whereas a nil value or 2% is observed for Bradyrhyzobium japonicum, which correspond to a comparable growth rate of bacteria with and without guar.

TABLE-US-00004 TABLE 4 Relative increase of bacteria growth rate Strain with guar addition Bacillus subtilis CCT 0089 + guar  8% powder Bacillus subtilis CCT 0089 + guar 13% liquid formulation Bacillus megaterium CCT 0536 + guar 22% powder Bacillus megaterium CCT 0536 + guar 15% liquid formulation Bradyrhyzobium japonicum CCT 4065 + guar  2% powder Bradyrhyzobium japonicum CCT 4065 + guar  0% liquid formulation

Example 3

[0138] The following materials are used in the experiments:

[0139] Guar: a guar hydroxypropyltrimonium chloride having an average molecular weight between 5,000 and 25,000 Daltons, a DS of 0.2, and a MS between 0.2 and 1.0, available from Solvay (provided as a powder)

[0140] Bacteria strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil: [0141] Bacillus thuringiensis CCT 2335 [0142] Pseudomonas putida CCT 2357

[0143] All strains were stored at −80° C. in the appropriate culture media, containing 15% of glycerol.

[0144] Only one culture media was used for both strains [0145] NA media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only)

[0146] A 250 mL shake flask containing 100 mL of NA or YMA culture media, was inoculated with 1 mL of the stock culture and incubated at 30° C., 150 rpm for 72 hours.

[0147] For each strain, 10 mL of the reactivation media were then transferred into a 250 mL shake flask containing 100 mL of the same media, with the addition of guar powder or guar liquid formulation; and incubated at 30° C., 150 rpm, for 96 hours. An experiment without addition of guar powder is also performed for each strain as a control.

[0148] 100 μL samples of each experiments were taken after 0 h, 24 h, 48 h, 72 h and 96 h of incubation. These samples were diluted (the dilutions were variable according to strain growth, being from 1×10.sup.−5 up to 1×10.sup.−15) and the dilutions plated in solid NA media. The plates were incubated at 30° C. until appearance of colonies. After incubation, the number of colonies present in each dilution was counted and used to evaluate bacterial growth.

[0149] For bacterial growth rate determination, a graph of the log.sub.10 (number of colonies) versus time of incubation was constructed. The straight line in this graph represents the exponential phase of bacterial growth and the angular coefficient represents the bacterial growth rate (μ).

[0150] The μ value was used to compare all the experiments and to evaluate the influence of the two guars addition on bacterial growth. For this set of experiments the ratio of microorganisms and guar was set at 1.0×10.sup.5 CFU/g. The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 5:

TABLE-US-00005 TABLE 5 Bacteria growth Composition rate (h.sup.−1) Bacillus thuringiensis CCT 2335 0.0898 Bacillus thuringiensis CCT 2335 + guar 0.1028 Pseudomonas putida CCT 5357 0.1133 Pseudomonas putida CCT 5357 + guar 0.1282

[0151] For the two strains, a higher value of bacteria growth rate is obtained in the presence of guar than without guar addition. The addition of guar permits to increase the growth rate of these different strains of bacteria. In Table 6 are reported the relative increase of bacteria growth rate with the addition of guar compared to the control for each strain. An increase of bacteria growth rate ranging from 13% to 14% is observed for the two strains of bacteria.

TABLE-US-00006 TABLE 6 Relative increase of bacteria growth rate Strain with guar addition Bacillus thuringiensis CCT 2335 14% Pseudomonas putida CCT 5357 13%

Example 4

[0152] The following materials are used in the experiments:

[0153] Guar: a guar hydroxypropyltrimonium chloride having an average molecular weight between 5,000 and 25,000 Daltons, a DS of 0.2, and a MS between 0.2 and 1.0, available from Solvay (provided as a powder)

[0154] Liquid guar formulation: an aqueous formulation of the powder guar at 25% from Solvay

[0155] All microorganisms strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil, some of them have reference in American Type Culture Colection (ATCC). [0156] Trichoderma harzianum CCT 4790 [0157] Aspergillus niger ATCC 16404

[0158] All strains were stored at −80° C. in the appropriate culture media, containing 20% of glycerol.

[0159] Culture media used in the experiments: [0160] Nutrient broth (NA) media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only) [0161] Oatmeal Agar (OA) containing per liter: 25 g of oat flakes or flour and 15 g of agar [0162] Sabouraud dextrose agar (SDA) containing per liter: 40 g of glucose, 10 g of peptone and 20 g agar

[0163] The media SDA and OA were used for reactivation of the strains A. niger and T. harzianum respectively, according to supplier's recommendation.

[0164] For the experiments with guar, only NA media was used.

Reactivation of Microorganisms:

[0165] A Petri dish containing 20 mL of SDA or OA media was used for the reactivation of the strains A. niger and T. harzianum, respectively.

[0166] The stock culture was used to inoculate the solid media for each strain and the petri dishes were incubated at 25° C. until complete growth.

Incubation with Guar:

[0167] From the reactivation media on petri dish, the spores of fungi were recovered and a spore solution was prepared.

[0168] 500 μL of the spore solution (approximately 1×10.sup.10 CFU/mL) were transferred to Erlenmeyer flasks containing 50 mL of media (controls and NA media with guar) and incubated at 25° C. Samples were taken at 48 h, 120 h and 168 h, filtered on filter paper and incubated at 60° C. before weighing [0169] Control media=NA without guar addition

Growth Evaluation:

[0170] The dry biomass recovery after each sample was plotted in a graphic dry biomass vs time and the growth curve could be obtained.

[0171] The growth rate (g) was calculated considering only the exponential phase of the growth and compared with the control.

[0172] The μ value was used to compare all the experiments and to evaluate the influence of guar addition on fungi growth. The microorganisms growth rate (μ) obtained for the different experiments are summarized in Table 7:

TABLE-US-00007 TABLE 7 Fungi growth Composition rate (h.sup.−1) Trichoderma harzianum CCT 4790 0.0009 Trichoderma harzianum CCT 4790 + guar 0.0013 powder Trichoderma harzianum CCT 4790 + guar 0.0015 liquid formulation Aspergillus niger ATCC 16404 0.0008 Aspergillus niger ATCC 16404 + guar 0.0014 powder Aspergillus niger ATCC 16404 + guar 0.0015 liquid formulation

[0173] For the two strains, a higher value of growth rate is obtained in presence of the guar under powder form and the guar in aqueous formulation compared to control. Hence, the addition of guar permits to increase the growth rate of these different strains of fungi. In Table 8 are reported the relative increase of growth rate with the addition of the two guar grades compared to the control for each strain. An increase of bacteria growth rate ranging from 44% to 88% is observed for the two strains of fungi.

TABLE-US-00008 TABLE 8 Relative increase of fungi growth rate Strain with guar addition Trichoderma harzianum CCT 4790 + guar 44% powder Trichoderma harzianum CCT 4790 + guar 67% liquid formulation Aspergillus niger ATCC 16404 + guar 75% powder Aspergillus niger ATCC 16404 + guar 88% liquid formulation