Improving fermentation output by media supplementation

Abstract

The present disclosure relates to methods for improving the fermentation process to produce the antifungal lipopeptides fengycin and iturin by supplementation of the fermentation medium with glutamate using Bacillus amyloliquefaciens.

Description

[0001] TITLE: Improving fermentation output by media supplementation

[0002] The present disclosure relates to methods withing the field of improving yields in fermentation processes by supplementation of fermentation media.

[0003] BACKGROUND

[0004] Among the microorganisms used for biological control, bacteria of Bacillus sp. genus have received much attention due to the wide variety of antibiotic compounds they produce, their long shelf life, their fast growth in culture, and their ability to colonize leaf surfaces [1 , 2, 3, 4],

[0005] In particular, certain species of Bacillus such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thuringiensis show antimicrobial activity.

[0006] The antimicrobial activity of these bacteria is due to their ability to produce lipopeptides e.g. of the surfactin, iturin, and fengycin families, which differ in the amino acid sequence and the branching of the fatty acid chain. Surfactins exhibit high antibacterial activity, whereas iturins and fengycins are recognized for their antifungal activity [4],

[0007] The prior art describes the use of B. subtilis and B. amyloliquefaciens to control various diseasecausing microorganisms in a wide variety of crops, including fruit and vegetable crops such as blackberry, grape, raspberry, strawberry, tomato, cucumber, black pepper, orange, melon, apple, peach, custard apple, banana, papaya, mango, and kiwi. EP231 1936 discloses a B. subtilis strain KS1 (NITE BP-569) as a biological control agent to counteract several phytopathogenic microorganisms in vine crops. WO 98 / 21968 discloses an antibiotic produced by B. subtilis A0153 (ATCC 55614) effective against bacterial and fungal infections and also as method for protecting plants that comprises the application of these antibiotic compounds.

[0008] WO9850422, WO9909819, and W00029426 disclose antibiotic compounds produced by the B. subtilis strain AQ713 (equivalent to strain QST713, deposited as NRRL B-21661) and its mutants which exhibit insecticidal, nematidical, antifungal, and antibacterial activity.

[0009] US201 1 / 0318386 describes methods for inducing systemic resistance against various pathogens through the use of biological controllers of the Bacillus genus, specifically of the isolated B. mojavensis 203-7 and isolated B. mycoides species. In turn, ES 2345969 describes a phytostrengthener for application on banana and plantain pseudostems, which includes B. subtilis, Trichoderma viride, and B. megaterium var phosphaticum. WO14178032 discloses a process for increasing the production of biomass of microorganisms of the Bacillus genus, including Bacillus subtilis EA-CB0015 and Bacillus amyloliquefaciens EA- CB0959. The biomass obtained by the process can be separated from the culture medium using conventional methods of centrifugation or microfiltration, whereas the active metabolites can be obtained by extraction with solvents, precipitation, adsorption, or chromatography. In a preferred embodiment of the invention, the amount of biomass of microorganisms of Bacillus sp. obtained can range between 3.0 and 20.0 g / L.

[0010] However, fermentation processes are expensive and time-consuming to setup and therefore maximizing the output from the fermentation processes is key in ensuring the profitability of the materials produced in the fermentation processes and the development of optimized fermentation strategies are highly desired.

[0011] SUMMARY

[0012] In one aspect, the present disclosure relates to methods for increasing production of one or more lipopeptides in a fermentation process.

[0013] In a different aspect, the present disclosure relates to a composition comprising a fermentation broth or one or more lipopeptides produced according to the methods disclosed herein.

[0014] In an additional aspect, the present disclosure relates to the use of a fermentation broth, or a composition as disclosed herein for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.

[0015] In a further aspect, the present disclosure relates to a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering a fermentation broth, or a composition produced by the methods disclosed herein, to the plant.

[0016] DETAILED DESCRIPTION

[0017] It has been surprisingly shown herein that supplementing a fermentation medium with glutamate (glutamic acid) and fermenting bacteria belonging to the species Bacillus amyloliquefaciens, resulted in an increased production of lipopeptides and specifically an increased production of the anti-fungal lipopeptides fengycin and iturin (see example 1 and figure 1). As demonstrated in example 1 and figure 1 , the supplementation of LC medium with glutamate in concentrations of 7 and 10 g / L respectively resulted in an increase in iturin production in the range of 5-11 % and an increase of fencygin production in the range of 10-17%.

[0018] It is further anticipated in the present disclosure that such an increase of in production of lipopeptides will also be achieved by fermentation of bacteria belonging to Bacillus spp. in general. Thus, the present disclosure relates to methods for increasing lipopeptide production in fermentations comprising a Bacillus spp.

[0019] A method for improving lipopeptide yield in Bacillus fermentations

[0020] In the present disclosure it was surprisingly found that supplementing a fermentation medium with glutamate (glutamic acid) and fermenting bacteria belonging to the species Bacillus amyloliquefaciens in such a supplemented medium, resulted in an increased production of lipopeptides and specifically an increased production of the anti-fungal lipopeptides fengycin and iturin.

[0021] This increased production is achieved by providing a Bacilllus spp., providing a suitable fermentation medium, supplementing the fermentation medium with glutamate, and initiating and running a fermentation process by introducing the provided Bacillus spp. into the supplemented fermentation medium.

[0022] Thus, the present disclosure relates to a method for increasing production of one or more lipopeptides in a fermentation process comprising, the method comprising the steps a) providing a bacillus spp. b) providing a suitable fermentation medium c) providing a supplemented fermentation medium by supplementing the suitable fermentation medium with glutamate d) initiating a fermentation process by introduction of the bacillus spp. provided in step a) into the supplemented fermentation medium provided in step c), and e) running the fermentation process, thereby providing a fermentation broth with an increased content of one or more lipopeptides.

[0023] As shown in figure 1 , glutamate supplementation of the fermentation medium clearly demonstrated an increased production of fengycin and iturin. Therefore, in one or more exemplary embodiments, the one or more lipopeptides is selected as one or more of fengycin and iturin.

[0024] The key to achieving an increased production of fengycin and iturin lies in the supplementation medium with glutamate. Methods for producing and supplementing fermentation media with desirable additional nutrients are commonly described in the prior art. Accordingly, the use of such methods for obtaining a supplemented fermentation medium is considered merely a routine task for the person skilled in the art.

[0025] In the present context the supplementation of the fermentation medium with aspartate relates to supplementing a fermentation medium with 3-30 g / L glutamate, 5-25 g / L glutamate, 7-20 g / L glutamate, 10-15 g / L glutamate, 7-15 g / L glutamate, 10-20 g / L glutamate, 5-15 g / L glutamate, 5-13 g / L glutamate, 7-13 g / L glutamate, 9-11 g / L glutamate, 5 g / L glutamate, 6 g / L glutamate, 7 g / L glutamate, 8 g / L glutamate, 9 g / L glutamate, 10 g / L glutamate, 11 g / L glutamate, 12 g / L glutamate or 13 g / L glutamate, 14 g / L glutamate, or 15 g / L glutamate.

[0026] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 3 g / L glutamate, at least 4 g / L glutamate, at least 5 g / L glutamate, 6 g / L glutamate, at least 7 g / L glutamate, at least 8 g / L glutamate, at least 9 g / L glutamate, at leastl 0 g / L glutamate, up to 15 g / L glutamate, up to 18 g / L glutamate, up to 20 g / L glutamate, up to 22 g / L glutamate, up to 24 g / L glutamate, up to 26 g / L glutamate, up to 28 g / L glutamate, or up to 30 g / L glutamate. The method according to claim 41-42, wherein the suitable fermentation medium is supplemented with at least 3 g / L glutamate.

[0027] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 3 g / L glutamate.

[0028] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 5 g / L glutamate.

[0029] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 7 g / L glutamate. In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 8 g / L glutamate.

[0030] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 9 g / L glutamate.

[0031] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with at least 10 g / L glutamate.

[0032] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 3-30 g / L glutamate, 5-25 g / L glutamate, 7-20 g / L glutamate, 10-15 g / L glutamate, 7-15 g / L glutamate, 10-20 g / L glutamate, 5-15 g / L glutamate, 5-13 g / L glutamate, 7-13 g / L glutamate, 9-11 g / L glutamate, 5 g / L glutamate, 6 g / L glutamate, 7 g / L glutamate, 8 g / L glutamate, 9 g / L glutamate, 10 g / L glutamate, 1 1 g / L glutamate, 12 g / L glutamate or 13 g / L glutamate, 14 g / L glutamate, or 15 g / L glutamate.

[0033] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-15 g / L glutamate, I Q- 20 g / L glutamate, 5-15 g / L glutamate, 7-13 g / L glutamate, or 9-11 g / L glutamate.

[0034] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 10-20 g / L glutamate.

[0035] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-15 g / L glutamate.

[0036] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 5-15 g / L glutamate.

[0037] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 5-13 g / L glutamate.

[0038] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-13 g / L glutamate.

[0039] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 7-10 g / L glutamate.

[0040] In one or more exemplary embodiments, step c) of the method disclosed herein relates to supplementing the suitable fermentation medium provided in step b) with 10-15 g / L glutamate.

[0041] Methods for the recovery of lipopeptides from a fermentation process or from a fermentation medium are well known in the prior art, and the choice of a suitable method of recovery is considered a routine choice that the skilled person will make in accordance with circumstances. In the present context it is generally contemplated that the one or more lipopeptides are recovered alone from the fermentation medium during or at the end of fermentation.

[0042] In some cases, it is desired to separate the one or more lipopeptides from the fermentation broth. In these cases, the method disclosed herein comprises an additional step, step f) that relates to the recovery of the one or more lipopeptides from the fermentation broth.

[0043] In one or more exemplary embodiments, the method as disclosed herein, further comprises a recovery step f) for recovering the one or more lipopeptides from the fermentation broth.

[0044] In one or more exemplary embodiments, the recovery step f) for recovering one or more lipopeptides from the fermentation broth comprises a centrifugation or filtration step.

[0045] In some cases, it is desired to purify the one or more lipopeptides produced according to the method disclosed herein. In these instances, the method disclosed herein comprises an additional step, step g) that relates to the purification of the one or more lipopeptides from the fermentation broth.

[0046] In one or more exemplary embodiments, the method as disclosed herein, further comprises a purification step g) for providing one or more purified lipopeptides.

[0047] In some cases, it is desired to both include a recovery step and a purification step in the method as disclosed herein. Thus, in one or more exemplary embodiments the method as disclosed herein comprises both a recovery step f) and a purification step g).

[0048] In one or more exemplary embodiments, an increased production of one or more lipopeptides of the method a production of one or more lipopeptides that is increased by 5-30%, 5-25%, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.

[0049] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 8- 18%.

[0050] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 10- 16%.

[0051] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by 12- 14%.

[0052] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0053] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 8%.

[0054] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 9%.

[0055] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by at least 10%.

[0056] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0057] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 12%.

[0058] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 14%.

[0059] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 16%.

[0060] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 18%.

[0061] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 20%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 22%.

[0062] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 24%.

[0063] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of one or more lipopeptides that is increased by up to 26%.

[0064] In one or more exemplary embodiments, the one or more lipopeptides is selected from the group consisting of fengycin and iturin.

[0065] In one or more exemplary embodiments, the one or more lipopeptides is selected as fengycin and iturin.

[0066] In one or more exemplary embodiments, the one or more lipopeptides is selected as fengycin.

[0067] In one or more exemplary embodiments, an increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 5-30%, 5-25%, 8- 22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.

[0068] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 8-18%.

[0069] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 10-16%.

[0070] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by 12-14%.

[0071] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0072] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 8%.

[0073] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 9%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by at least 10%.

[0074] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0075] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 12%.

[0076] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 14%.

[0077] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 16%.

[0078] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 18%.

[0079] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 20%.

[0080] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 22%.

[0081] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 24%.

[0082] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of fengycin that is increased by up to 26%.

[0083] In one or more exemplary embodiments, the one or more lipopeptides is selected as iturin.

[0084] In one or more exemplary embodiments, an increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 4-10%, 5-12%, 5-14%, 5-30%, 5-25%, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.

[0085] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 4-10%.

[0086] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 5-12%.

[0087] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by 5-14%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0088] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 5%.

[0089] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 6%.

[0090] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 7%.

[0091] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 8%.

[0092] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 9%.

[0093] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by at least 10%.

[0094] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0095] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 12%.

[0096] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 14%.

[0097] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 16%.

[0098] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 18%.

[0099] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 20%. In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 22%.

[0100] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 24%.

[0101] In one or more exemplary embodiments, the increased production of one or more lipopeptides of the method as disclosed herein is a production of iturin that is increased by up to 26%.

[0102] The present disclosure also relates to compositions with an increased content of one or more lipopeptides as produced according to the method as disclosed herein.

[0103] Thus, in one or more exemplary embodiments, the present disclosure relates to a composition comprising a fermentation broth produced according to the method as disclosed herein, a composition comprising one or more lipopeptides recovered in step f) of the method as disclosed herein, or a composition comprising the purified one or more lipopeptides obtained in step g) of the method as disclosed herein.

[0104] Thus, in one or more exemplary embodiments, the present disclosure relates to a composition comprising a fermentation broth produced according to the method as disclosed herein, a composition comprising fengycin and / or iturin recovered in step f) of the method as disclosed herein, or a composition comprising the purified fengycin and / or iturin obtained in step g) of the method as disclosed herein.

[0105] In one or more exemplary embodiments, the present disclosure relates to the use of a fermentation broth as produced by the method as disclosed herein, the use of the one or more lipopeptides as produced by the method as disclosed herein, the use of a composition comprising a fermentation broth produced according to the method as disclosed herein, the use of a composition comprising one or more lipopeptides recovered in step f) of the method as disclosed herein, and / or the use of a composition comprising the purified one or more lipopeptides obtained in step g) of the method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.

[0106] In one or more exemplary embodiments, the present disclosure relates to the use of a fermentation broth as produced by the method as disclosed herein, the use of the fengycin and / or iturin as produced by the method as disclosed herein, the use of a composition comprising a fermentation broth produced according to the method as disclosed herein, the use of a composition comprising the fengycin and / or iturin recovered in step f) of the method as disclosed herein, and / or the use of a composition comprising the purified fengycin and / or iturin obtained in step g) of the method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen. The fermentation broth, the compositions and / or the one or more lipopeptides produced according to the method can also be used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.

[0107] Thus, in one or more exemplary embodiments, the fermentation broth, the compositions, or the one or more lipopeptides produced according to the method are used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering one or more of the fermentation broth, the compositions, or the one or more lipopeptides produced according to the method to the plant.

[0108] Thus, in one or more exemplary embodiments, the fermentation broth, the compositions, or the fengycin and / or iturin produced according to the method are used in a method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering one or more of the fermentation broth, the compositions, or the fengycin and / or iturin produced according to the method to the plant.

[0109] Recovery and purification steps

[0110] The recovery step f) and the purification step g) of the method as disclosed herein comprises one or more of the following steps

[0111] 1) a filtration step,

[0112] 2) a centrifucation step,

[0113] 3) a pH adjustment step

[0114] 4) an adsorption step comprising adsorption of one or more lipopeptides onto an adsorption column,

[0115] 5) an adsorption step comprising adsorption of one or more lipopeptides onto a geopolymer,

[0116] 6) a chromatography step

[0117] 7) a CaCh flocculation step

[0118] 8) a solvent extraction step

[0119] Geopolymers that are useful in the methods of the present disclosure comprises kieselguhr, diatomite, diatomaceous earth, Kaolin (Chinese clay), bentonite, talcum, volcanic ash, volcanic rock, clay, perlite, lignin, drilling mud, diatomic earth and synthetic silica. In one or more embodiments of the present disclosure, the purification step g) is performed on the material recovered in step f) of the methods as disclosed herein.

[0120] In one or more exemplary embodiments, the pH is adjusted to between pH 4.5 and 8.0 prior to adsorption onto a geopolymer or an adsorption column.

[0121] Adjustment of pH in step f) or step g) comprises adjusting the pH to 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or adjusting the pH to within the ranges 4.5-8.0 pH, 5.0-7.0 pH or 5.5-7.0.

[0122] Bacillus spp.

[0123] A bacteria belonging to the Bacillus spp. in the context of the present disclosure is any bacteria that belongs within the Bacillus genus. The Bacillus genus is well defined in the prior art and the selection and identification of bacterial species belonging to the Bacillus genus is a routine task for the person skilled in the art.

[0124] Thus, in one or more exemplary embodiments of the present disclosure, a bacteria belonging to the Bacillus spp. is any bacteria that belongs to the Bacillus genus.

[0125] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method as disclosed herein is a bacteria belonging to a species selected from the list consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, Bacillus thuringiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, and Bacillus tequilensis.

[0126] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method as disclosed herein is a bacteria belonging to the Bacillus amyloliquefaciens species.

[0127] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is a bacteria selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with the DSMZ accession number DSM 25840, B. amyloliquefaciens strain with the DSMZ accession number DSM 27032, B. amyloliquefaciens strain with the DSMZ accession number DSM 27033 and B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.

[0128] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is a Bacillus amyloliquefaciens strain selected from the list consisting of DSM 25840, DSM 27032, DSM 27033 and DSM 34003.

[0129] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is a Bacillus amyloliquefaciens strain selected from the list consisting of DSM 25840, DSM 27032 and DSM 27033. In one or more exemplary embodiments of the present disclosure, the Bacillus spp. as used in the method M1 is Bacillus amyloliquefaciens strain DSM 34003.

[0130] Deposits and expert solution

[0131] The applicant requests that a sample of the deposited micro-organisms stated below may only be made available to an expert, until the date on which the patent is granted.

[0132] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03 April

[0133] 2012, under the accession No. DSM 25840.

[0134] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 21 March

[0135] 2013, under the accession No. DSM 27032.

[0136] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 21 March 2013, under the accession No. DSM 27033.

[0137] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 24 August 2021 , under the accession No. DSM 34003.

[0138] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0139] BRIEF DESCRIPTION OF THE FIGURES

[0140] Figure 1

[0141] Fig. 1 shows lipopeptide concentrations and bioactivity measured in terms of ID50 (The half maximal inhibitory dilution of the fermentates) in end-of-fermentation fermentates from cultures of Bacillus amyloliquefaciens DSM 34003 incubated in BioLector Pro, at 1500 rpm, 33°C, pH 7.8-8.0, for 40 hours, in 0.8 mL LC medium supplemented with glutamate and / or aspartate as depicted in table 1 .

[0142] EXAMPLES

[0143] Materials and methods

[0144] The experiments described herein were performed with Bacillus amyloliquefaciens DSM 34003 in BioLector Pro, flower-type plates (MF32C-BOH2), at 1500 rpm, 33°C, pH 7.8-8.0, for 40 h, in 0.8 mL LC medium. The LC-medium was supplemented with a concentration corresponding to 0-10 g / L glutamate and / or aspartate in the LC-medium as described in table 1 .

[0145] Table 1

[0146] Table 1 : Glutamate and / or aspartate supplementation in the variants of LC medium in the experiments conducted with Bacillus amyloliquefaciens DSM 34003 in BioLector Pro, at 33°C, pH 7.8-8.0 for 40 h.

[0147] Example 1 - Lipopeptide yield of Bacillus amyloliquefaciens cultured in supplemented LC medium

[0148] The lipopeptide contents of the fermentate from each of the cultures described in example 1 culture was also analyzed and quantified and the iturin and fengycin contents are shown in figure 1 . The supplementation of LC medium with glutamate clearly increases the production of lipopeptides while aspartate supplementation results in a lower production of lipopeptides.

[0149] Supplementation with 7.0 and 10 g / L glutamate (LC-var1 and LC-var2) yielded 410 and 400 mg / L iturin compared to 380 and 370 mg / L in cultures grown in supplemented with 0 and 3.4 g / L glutamate (LC-varO and LC medium) representing an increase in iturin yield of at least 5% and up to 11%, while the iturin yield in cultures supplemented with only aspartate were much lower at around 260-310 mg / L iturin (LC-var3, LC-var4 and LC-var5).

[0150] Supplementation with 7.0 and 10 g / L glutamate (LC-var1 and LC-var2) yielded 280 and 290 mg / L fengycin compared to 240 / 250 mg / L in cultures grown in supplemented with 0 and 3.4 g / L glutamate (LC-varO and LC-medium) which is an increase of at least 10% and up to an increase of 17%, while the fengycin yield in cultures supplemented with only aspartate were much lower at around 160-200 mg / L fengycin (LC-var3, LC-var4 and LC-var5).

[0151] Example 2 - Bioactivity against Fusarium graminearum

[0152] Fermentate samples from Bacillus amuliquefaciens cultures that were cultivated in LC media supplemented with glutamate and aspartate respectively as described in table 1 and under the conditions described in the materials and methods, were used for preparation of serial dilutions (10 samples in total plus a blank isopropanol sample as negative control) in triplicate using isopropanol as solvent. These serial dilutions were then tested fortheir inhibitory potency (bioactivity) against Fusarium graminearum and the inhibitory potency (bioactivity) was determined in terms of the ID50 (The half maximal inhibitory dilution of biological samples) of each fermentate.

[0153] The inhibitory potency was assessed by filling microtiter plates with 200 pl Fusarium Graminearum conidia media mixture in a total concentration of 5.0x105F. graminearum conidia and supplementing this mixture with 50 pl sample. Following mixing of media and samples, the microtiter plates are transferred to an incubator or robotic incubator and OD measurements at OD620 is performed for each sample every 20 hours for 120 hours, measuring the increase in OD overtime. Antibiotics were added to prevent the outgrowth of Bacillus (spores and / or vegetative cells) in the samples over the course of the experiment.

[0154] Calculating the ID50 of the Bacillus amyloliquefaciens fermentate samples

[0155] The data from the dilution series is exported to MS Excel, where the datapoints are vertically sorted per plate and horizontally sorted per time interval. Sorted data is presented in a new file, where the OD increase is calculated from time point t=20 hours to the different time intervals. Uninhibited samples are inspected for their growth and typically the OD620 of the uninhibited sample (negative control / isopropanol only) is between 1 .3 and 1 .8 at time point T=110 hours. The background OD increase is used as a normalization step for the specific time interval (e.g. T=20 hours or T=40 hours). Samples with OD620 values of 1 .8 and above are disqualified as the measurement becomes imprecise and very often is an indicator of a contaminated sample.

[0156] A dose-response curve with OD / increase in OD is plotted using data from the dilution series for each fermentate at a specific time interval. First, the slope of the dose-response curve for the fermentate dilution series is normalized against the averaged slope of the uninhibited sample (negative control / isopropanol only) and multiplied by 100. Then a sigmoidal model is fitted onto the logistic relationship between the inhibition and no inhibition state. The half maximal inhibitory dilution (ID50) of the sample fermentate is defined in Equation 1.

[0157] 100 Equation 1: Y =1 + e(_^(B-c))

[0158] Equation 1 : Sigmoidal formula describing the dose-response of F. graminearum growth inhibition at different dilutions. A is the origin of the function, also the ID50; B describes the dilution factor; C stands for the slope of the function. The fit of the sigmoidal curve to the data is controlled by visual inspection and poor fits are considered as outlier and excluded.

[0159] The half maximal inhibitory concentration (IC50) of the sample fermentate is defined in Equation 2.

[0160] 100

[0161] Equation 2: Y =1 + e(- -B-c))

[0162] Equation 2: Sigmoidal formula describing the dose-response of F. graminearum growth inhibition at different dilutions. A is the origin of the function, also the ID50; B describes the dilution factor; C stands for the slope of the function. The fit of the sigmoidal curve to the data is controlled by visual inspection and poor fits are considered as outlier and excluded.

[0163] The deviations between calculated responses from the samples and the model are minimized by a script to optimize the model fit. Model fitting is constrained to minimize false interpretations caused by outliers. Therefore, the variables A, B and C are >0; while A is < 500; B is <1 and C is <2.

[0164] The ID50 values are calculated for each fermentate dilution series and a comparison of these ID50 values are represented by the curve shown in figure 1 . As can be seen on figure the ID50 of fermentates from Bacillus amyloliquefaciens cultured in LC medium supplemented with glutamate in concentrations of 7 and 10 g / L (LC-var1 and LC-var2) is greater than when the LC medium is supplemented with 0 or 3.4 (LC-varO and LC medium) and much greater than when LC medium is supplemented with aspartate (LC-var3-6). These results show that the fermentates from cultures supplemented with glutamate produce a fermentate that has increased bioactivity / higher potency against fusarium graminearum.

[0165] In conclusion, from the examples disclosed herein it is clear that aspartate supplementation reduces lipopeptide production and bioactivity against fungi, whereas glutamate supplementation increases lipopeptide production and bioactivity against fungi. Therefore, supplementation of the growth medium with glutamate is useful in any fermentation process where the lipopeptide yield and in particular fengycin and iturin yields are important.

[0166] It also is clear from the data presented in example 2 that the I D50 / bioacti vity of fermentates from bacillus amyloliquefaciens cultured in glutamate supplemented medium possess greater bioactivity and thereby potency against fusarium graminearum than medium lacking glutamate or supplemented with aspartate. These fermentates or compositions comprising these fermentates are useful in antifungal applications and could because of their improved potency potentially be applied directly to a plant in need.

[0167] Finally, it is interesting to note that supplementation with each of the two amino acid results in improvements that are different for each amino acid and sometimes directly opposite (e.g. aspartate reducing lipopeptide content and glutatmate increasing lipopeptide content), indicating that the effects seen herein is a direct result of the impact these amino acids have on Bacillus metabolism and not just a general nutritional boost caused by the addition of an amino acid to a fermentation medium.

[0168] ITEMS

[0169] 1 . A method for increasing production of one or more lipopeptides in a fermentation process comprising, the method comprising the steps a) providing a bacillus spp. b) providing a suitable fermentation medium c) providing a supplemented fermentation medium by supplementing the suitable fermentation medium with glutamate d) initiating a fermentation process by introduction of the bacillus spp. provided in step a) into the supplemented fermentation medium provided in step c), and e) running the fermentation process, thereby providing a fermentation broth with an increased content of one or more lipopeptides.

[0170] 2. The method according to item 1 , wherein the one or more lipopeptides is one or more of fengycin and / or iturin.

[0171] 3. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L glutamate, at least 4 g / L glutamate, at least 5 g / L glutamate, 6 g / L glutamate, at least 7 g / L glutamate, at least 8 g / L glutamate, at least 9 g / L glutamate, 10 g / L glutamate.

[0172] 4. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 3 g / L glutamate. 5. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 5 g / L glutamate.

[0173] 6. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 7 g / L glutamate.

[0174] 7. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with at least 10 g / L glutamate.

[0175] 8. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with up to 15 g / L glutamate, up to 18 g / L glutamate, up to 20 g / L glutamate, up to 22 g / L glutamate, up to 24 g / L glutamate, up to 26 g / L glutamate, up to 28 g / L glutamate, or up to 30 g / L.

[0176] 9. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 3-30 g / L glutamate, 5-25 g / L glutamate, 7-20 g / L glutamate,

[0177] 10-15 g / L glutamate, 7-15 g / L glutamate, 10-20 g / L glutamate, 5-15 g / L glutamate, 5-13 g / L glutamate, 7-13 g / L glutamate, 9-11 g / L glutamate, 5 g / L glutamate, 6 g / L glutamate, 7 g / L glutamate, 8 g / L glutamate, 9 g / L glutamate, 10 g / L glutamate, 11 g / L glutamate, 12 g / L glutamate or 13 g / L glutamate, 14 g / L glutamate, or 15 g / L glutamate.

[0178] 10. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L glutamate, 10-20 g / L glutamate, 5-15 g / L glutamate, 7-13 g / L glutamate, or 9-11 g / L glutamate.

[0179] 11 . The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 10-20 g / L glutamate.

[0180] 12. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L glutamate.

[0181] 13. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-15 g / L glutamate.

[0182] 14. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 5-13 g / L glutamate. 15. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-13 g / L glutamate.

[0183] 16. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-10 g / L glutamate.

[0184] 17. The method according to any one of the preceding items, wherein the suitable fermentation medium is supplemented with 7-15 g / L glutamate.

[0185] 18. The method according to any one of the preceding items, the method further comprising a recovery step f), for recovering the one or more lipopeptides from the fermentation broth.

[0186] 19. The method according to any one of the preceding items, wherein the recovery step f) comprises a centrifugation step or filtration step.

[0187] 20. The method according to any one of the preceding items, the method further comprising a purification step g) for providing one or more purified lipopeptides.

[0188] 21. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by 5-30, 5-25, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14-16%.

[0189] 22. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by 8-18%.

[0190] 23. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by 10-16%.

[0191] 24. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by 12-14%.

[0192] 25. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. 26. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by at least 8%.

[0193] 27. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by at least 9%.

[0194] 28. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by at least 10%.

[0195] 29. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0196] 30. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 12%.

[0197] 31. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 14%.

[0198] 32. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 16%.

[0199] 33. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 18%.

[0200] 34. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 20%.

[0201] 35. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 22%. 36. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 24%.

[0202] 37. The method according to any one of the preceding items, wherein production of one or more lipopeptides is increased by up to 26%.

[0203] 38. The method according to any one of the preceding items, wherein the one or more lipopeptide is selected from the group consisting of fengycin and iturin.

[0204] 39. The method according to any one of the preceding items, wherein the one or more lipopeptide is fengycin and iturin.

[0205] 40. The method according to according to any one of the preceding items, wherein the one or more lipopeptide is fengycin.

[0206] 41. The method according to according to any one of the preceding items, wherein production of fengycin is increased by 5-30, 5-25, 8-22%, 10-20%, 12-18%, 12-16%, 12-14% or 14- 16%.

[0207] 42. The method according to any one of the preceding items, wherein production of fengycin is increased by 8-22%.

[0208] 43. The method according to any one of the preceding items, wherein production of fengycin is increased by 10-20%.

[0209] 44. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0210] 45. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 8%.

[0211] 46. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 9%. 47. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 10%.

[0212] 48. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 11 %.

[0213] 49. The method according to any one of the preceding items, wherein production of fengycin is increased by at least 12%.

[0214] 50. The method according to any one of the preceding items, wherein production of fengycin is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0215] 51. The method according to any one of the preceding items, wherein production of fengycin is increased by up to 20%.

[0216] 52. The method according to any one of the preceding items, wherein production of fengycin is increased by up to 22%.

[0217] 53. The method according to any one of the preceding items, wherein production of fengycin is increased by up to 24%.

[0218] 54. The method according to any one of the preceding items, wherein production of fengycin is increased by up to 26%.

[0219] 55. The method according to any one of the preceding items, wherein the one or more lipopeptide is iturin.

[0220] 56. The method according to any one of the preceding items, wherein production of iturin is increased by 4-10%, 5-12%, 5-14%, 5-30%, 5-25%, 8-22%, 10-20%, 12-18%, 12-16%, 12- 14% or 14-16%. 57. The method according to any one of the preceding items, wherein production of iturin is increased by 4-10%.

[0221] 58. The method according to any one of the preceding items, wherein production of iturin is increased by 5-12%.

[0222] 59. The method according to any one of the preceding items, wherein production of iturin is increased by 5-14%.

[0223] 60. The method according to any one of the preceding items, wherein production of iturin is increased by at least 5%, 6, % 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, or 25%.

[0224] 61 . The method according to any one of the preceding items, wherein production of iturin is increased by at least 5%.

[0225] 62. The method according to any one of the preceding items, wherein production of iturin is increased by at least 6%.

[0226] 63. The method according to any one of the preceding items, wherein production of iturin is increased by at least 7%.

[0227] 64. The method according to any one of the preceding items, wherein production of iturin is increased by at least 8%.

[0228] 65. The method according to any one of the preceding items, wherein production of iturin is increased by at least 9%.

[0229] 66. The method according to any one of the preceding items, wherein production of iturin is increased by at least 10%.

[0230] 67. The method according to any one of the preceding items, wherein production of iturin is increased by up to 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%. 68. The method according to any one of the preceding items, wherein production of iturin is increased by up to 12%.

[0231] 69. The method according to any one of the preceding items, wherein production of iturin is increased by up to 14%.

[0232] 70. The method according to any one of the preceding items, wherein production of iturin is increased by up to 16%.

[0233] 71. The method according to any one of the preceding items, wherein production of iturin is increased by up to 18%.

[0234] 72. The method according to any one of the preceding items, wherein the Bacillus spp. is selected as one from the list consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thurigiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis.

[0235] 73. The method according to any one of the preceding items, wherein the Bacillus spp. is Bacillus amyloliquefaciens.

[0236] 74. The method according to any one of the preceding items, wherein the Bacillus spp. is a Bacillus amyloliquefaciens strain selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with the DSMZ accession number DSM 25840, B. amyloliquefaciens strain with the DSMZ accession number DSM 27032, B. amyloliquefaciens strain with the DSMZ accession number DSM 27033, and B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.

[0237] 75. The method according to any one of the preceding items, wherein the Bacillus spp. is B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.

[0238] 76. A composition comprising a fermentation broth produced according to the method of any one of the preceding items, a composition comprising the one or more lipopeptides recovered in step f) according to the method of any one of the preceding items, or a composition comprising the purified one or more lipopeptides obtained in step g) according to the method of any one of the preceding items. Use of a fermentation broth produced by a method according to any one of the items 1-75 or a composition according to item 76 for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen. The use according to item 77, wherein the infection in a plant is caused by a fungal pathogen. A method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering a fermentation broth produced by the method according to any one of items 1-75 or a composition according to item 76 to the plant. The method according to item 79 wherein the pathogen is a fungal pathogen.

[0239] References

[0240] 1 . M. Shoda: Bacterial Control of Plant Disease, Journal of Bioscience and Bioengineering, pp. 515-521 , 200.

[0241] 2. H. P. Bais, R. Fall and .J M. Vivanco: Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas s y r i n g e is facilitated by biofilm formation and surfactin production, Plant Physiology, vol. 134, pp. 307-319, 2004.

[0242] 3. T Stein: Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, vol. 56, pp. 854-857, 2005.

[0243] 4. M. Ongena and P. Jacques: Bacillus lipopeptides: versatile weapons for plant disease biocontrol, Applied Microbiology and Biotechnology, vol. 16, No. 3, pp. 115-125, 2008.

Claims

CLAIMS1 . A method for increasing production of one or more lipopeptides in a fermentation process comprising, the method comprising the steps a) providing a bacillus spp. b) providing a suitable fermentation medium c) providing a supplemented fermentation medium by supplementing the suitable fermentation medium with glutamate d) initiating a fermentation process by introduction of the bacillus spp. provided in step a) into the supplemented fermentation medium provided in step c), and e) running the fermentation process, thereby providing a fermentation broth with an increased content of one or more lipopeptides.

2. The method according to claim 1 , wherein the one or more lipopeptides is one or more of fengycin and / or iturin.

3. The method according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with at least 7 g / L glutamate.

4. The method according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with at least 10 g / L glutamate.

5. The method according to any one of the preceding claims, wherein the suitable fermentation medium is supplemented with 7-15 g / L glutamate.

6. The method according to any one of the preceding claims, the method further comprising a recovery step f), for recovering the one or more lipopeptides from the fermentation broth.

7. The method according to any one of the preceding claims, the method further comprising a purification step g) for providing one or more purified lipopeptides.

8. The method according to any one of the preceding claims, wherein production of one or more lipopeptides is increased by at least 8%.

9. The method according to any one of the preceding claims, wherein production of fengycin is increased by at least 10%.

10. The method according to any one of the preceding claims, wherein production of iturin is increased by at least 5%.11 . The method according to any one of the preceding claims, wherein the Bacillus spp. is selected as one from the list consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mycoides, Bacillus circulans, Bacillus megaterium, Bacillus pumilus, Bacillus mojavensis, and Bacillus thurigiensis, Bacillus simplex, Bacillus safensis, Bacillus atrophaeous, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis.

12. The method according to any one of the preceding claims, wherein the Bacillus spp. is a Bacillus amyloliquefaciens strain selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with the DSMZ accession number DSM 25840, B. amyloliquefaciens strain with the DSMZ accession number DSM 27032, B. amyloliquefaciens strain with the DSMZ accession number DSM 27033, and B. amyloliquefaciens strain with the DSMZ accession number DSM 34003.

13. A composition comprising a fermentation broth produced according to the method of any one of the preceding claims, a composition comprising the one or more lipopeptides recovered in step f) according to the method of any one of the preceding claims, or a composition comprising the purified one or more lipopeptides obtained in step g) according to the method of any one of the preceding claims.

14. Use of a fermentation broth produced by a method according to any one of the claims 1-12 or a composition according to claim 13 for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.

15. A method for controlling, preventing, or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering a fermentation broth produced by the method according to any one of claims 1-12 or a composition according to claim 13 to the plant.

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