Microbial pesticidal compositions and production thereof

Abstract

The present disclosure relates to methods for producing a composition comprising one or more lipopeptides such as e.g. fengycin, iturin or surfactin, comprising the steps a) initiating a fermentation process by introducing bacteria belonging to Bacillus spp. into a suitable fermentation medium; b) maintaining a low level of dissolved oxygen (DO) in the fermentation medium, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides, wherein the low level of dissolved oxygen in step b) is preferably a dissolved oxygen level in the range of 5-35%. A composition obtained by said methods and its use for controlling, preventing or ameliorating an infection in a plant caused by a fungal or bacterial pathogen is also claimed.

Description

[0001] MICROBIAL PESTICIDAL COMPOSITIONS AND PRODUCTION THEREOF

[0002] The present invention relates to novel and improved methods for producing lipopeptide compositions by fermenting bacteria. In a specific embodiment the lipopeptide composition further contains biomass produced in the fermentation. The lipopeptide composition and the lipopeptide- biomass composition both exhibit antifungal and antibacterial activity against various phytopathogenic agents.

[0003] BACKGROUND

[0004] Among the microorganisms 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 [12, 13, 14, 15],

[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 fortheir antifungal activity

[0015] ,

[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. EP2311936 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] US2011 / 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] The fungicidal activity of the isolated biomass (cells and spores) after harvesting from the culture medium is not satisfactory from a commercial view, and therefore the objection of the present disclosure is to provide an improved process for production of lipopeptides and biomass with antifungal activity.

[0011] US5470827 discloses that iturin A can be harvested from the culture medium by extraction with a solvent, or alternatively the culture medium may be filtered through a filter membrane or the like. If desired, the culture medium may be brought into contact with an active charcoal, powdery cellulose, adsorbing resin or the like carriers so that the produced iturin A is adsorbed to the carrier, and thereafter the product may be desorbed therefrom by elution.

[0012] SUMMARY

[0013] In its broadest aspect, the present disclosure relates to a method of for producing one or more lipopeptides, the method comprising fermentation of a bacteria belonging to the Bacillus spp under low dissolved oxygen conditions.

[0014] In another aspect, the present disclosure relates to a method for producing fengycin, the method comprising fermentation of a bacteria belonging to the Bacillus spp under low dissolved oxygen conditions.

[0015] In a further aspect, the present disclosure relates to a method of for producing one or more lipopeptides and a biomass, the method comprising fermentation of a bacteria belonging to the Bacillus spp under low dissolved oxygen conditions. In an additional aspect, the present disclosure relates to a method for producing fengycin and a biomass, the method comprising fermentation of a bacteria belonging to the Bacillus spp under low dissolved oxygen conditions.

[0016] In another aspect, the present disclosure relates to compositions comprising fengycin, one or more lipopetides, fengycin and a biomass, or, one or more lipopeptides and a biomass.

[0017] In another different aspect, the present disclosure relates to the use of the fengycin, the one or more lipopeptides, the fengycin and a biomass, the one or more lipopeptides and a biomass, and / or the compositions comprising these as antifungal agents.

[0018] In a further different aspect, the present disclosure relates to the use of the fengycin, the one or more lipopeptides, the fengycin and a biomass, the one or more lipopeptides and a biomass, and / or the compositions comprising these for controlling, preventing or ameliorating infections caused by fungal pathogens in plants.

[0019] As used herein, a bacterial strain identified by the corresponding DSM number (as e.g. DSM 27033) is intended to mean the strain as deposited with the DSMZ under the specific DSM number.

[0020] FIGURES

[0021] Figure 1 shows DO profiles during fermentation trials. DO.SP:10, Fermentation made with a DO setpoint at 10%; DO.SP:20, , Fermentation made with a DO setpoint at 20%; DO.SP:30, Fermentation made with a DO setpoint at 30%.

[0022] Figure 2 shows a Zoomed-in view of the DO profiles in the first 20 hours of the fermentation trials. DO.SP:10 is a fermentation made with a DO setpoint at 10%; DO.SP:20 is a fermentation made with a DO setpoint at 20%; and DO.SP:30 is a fermentation made with a DO setpoint at 30%.

[0023] Figure 3 shows CTR and OTR profiles during fermentation trials. DO.SP:10 is a fermentation made with a DO setpoint at 10%; DO.SP:20 is a fermentation made with a DO setpoint at 20%; DO.SP:30 is a fermentation made with a DO setpoint at 30%.

[0024] Figure 4 shows concentration of fengycins during fermentation. DO.SP:10 is a Fermentation made with a DO setpoint at 10%; DO.SP:20 is a fermentation made with a DO setpoint at 20%;

[0025] DO.SP:30 is a fermentation made with a DO setpoint at 30%. Figure 5: shows bacterial growth and lipopeptide production during a batch cultivation. A: optical density (OD); B: vegetative cells measured by flow cytometry; C: spore counts measured by flow cytometry; D: Surfactins; E: Iturins; F: Fengycins.

[0026] Figure 6: shows bacterial growth and lipopeptide production during two batch cultivations, one reference (solid line) and one with a brief oxygen depletion with dissolved oxygen going to 0% DO (dotted line). Recorded parameters are dissolved oxygen (DO) (figure 6 A), biomass (optical density, OD620) (figure 6 B), counts of vegetative cells (figure 6 C), counts of spores (figure 6 D), carbon dioxide transmission rate (CTR) (figure 6 E), and lipopeptides, i.e., surfactins (figure 6 F), Iturins (figure 6 G) and Fengycins (figure 6 H).

[0027] DETAILED DESCRIPTION

[0028] In the present disclosure, it has been surprisingly found that when Bacillus amyloliquefaciens is subjected to a fermentation process under conditions with low dissolved oxygen, the amount of fengycin produced is increased dramatically.

[0029] It is shown in example 1 and figure 4 that reducing the dissolved oxygen levels from 30% to 10% in a fermentation process comprising Bacillus amyloliquefaciens surprisingly doubles the amount of fengycin produced. Additionally, reducing the dissolved oxygen levels from 20% to 10% leads to about a 50% increase in the amount of fengycin produced.

[0030] As demonstrated in example 1 and shown figures 1-2, and 4 maintaining anaerobic conditions of about 0% DO levels in the fermentation medium for 0,5 hours triggers a moderate increase in fengycin production, whereas maintaining anaerobic conditions of about 0% DO levels in the fermentation medium for 1 ,5 hours triggers a much greater increase of fengycin production.

[0031] As is also shown in example 3 and figure 6, the production of fengycin is increased 3-fold following a short period of oxygen depletion in a fermentation culture with a DO set point of 20%, compared to a culture that did not experience oxygen depletion.

[0032] Thus, it is clear from the results presented herein that the mechanisms that govern lipopeptide (e.g. fengycin) production in the Bacillus species are complicated and that production efficiency can be increased by subtle changes in fermentation conditions. It is thought that an important trigger for increased production of fengycin is that the dissolved oxygen (DO) levels in the fermentation medium drops into anaerobic conditions for a period of time during the early growth phase, before the DO levels are returned to the desired concentration.

[0033] Generally, for a fermentation process where it is desired to maintain a DO level in the fermentation medium of about 5-25%, an increased production of fengycin is triggered by allowing the DO level in the fermentation medium to drop below 10% and as low as 0% DO for a period of time during the exponential phase of the fermentation process and then returning the DO levels to the desired setting within the range of 5-25% DO.

[0034] In other words, for a fermentation process where a DO level in the fermentation medium of about 20% is maintained in the fermentation medium, an increased production of fengycin is triggered by allowing the DO level in the fermentation medium to drop below 10% and as low as 0% DO for a period of time during the exponential phase of the fermentation process and then returning the DO levels in the fermentation medium to a DO level of 20% for the remaining time of the fermentation process.

[0035] Alternatively, for a fermentation process where a DO level in the fermentation medium is maintained at about 10% DO in the fermentation medium, an increased production of fengycin is triggered by allowing the DO level in the fermentation medium to drop below 10% and as low as 0% DO for a period of time during the exponential phase of the fermentation process, before returning the DO levels to about 10 % for the remaining time of the fermentation process.

[0036] The increased production of fengycin in low dissolved oxygen fermentation processes as discussed herein is a promising and important step towards improving biologically produced antifungal compositions, as an increased fengycin potentially leads to a more potent antifungal lipopeptide compositions.

[0037] Moreover, as fermentation processes are time-consuming to set up, an increased production of fengycin also increases yield from downstream processes such recovery and purification and thereby leads to a more cost-efficient production. However, as fengycin is not the only antifungal lipopeptide produced by Bacillus, the present disclosure is not only concerned with production of fengycin alone. Many Bacillus species (including Bacillus amyloliquefaciens) produce a range of lipopeptides including fengycin, iturin and surfactin. Thus, the methods presented herein can also be used to produce and recover compositions of these lipopeptides that have an increased content of fengycin compared to the other lipopeptides produced by Bacillus spp.. Furthermore, Bacillus spp. cells and spores have also been shown to have a beneficial antimicrobial properties. Therefore, the methods disclosed herein can also be used to produce and recover one or more lipopeptides together with a biomass produced during the fermentation process, and / or to produce and recover fengycin together with a biomass produced during the fermentation process.

[0038] Thus, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process by introducing bacteria belonging to Bacillus spp. into a suitable fermentation medium, b) maintaining a low level of dissolved oxygen (DO) in the fermentation medium, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0039] The low level of dissolved oxygen (DO) in step b) is maintained at a dissolved oxygen level of 5- 35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5-15%, 6-14%, 7-13%, 8-12%, 9- 11 % or 10% dissolved oxygen.

[0040] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 10-30%.

[0041] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 5-25%.

[0042] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 10-20%.

[0043] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 5-20%. In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 20%.

[0044] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 5-15%.

[0045] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 6-14%.

[0046] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 7-13%.

[0047] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 8-12%.

[0048] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 9-11%.

[0049] In one or more exemplary, embodiments the low level of dissolved oxygen (DO) maintained in step b) is a dissolved oxygen level of 10%.

[0050] Methods for the recovery of lipopeptides (and other compounds of interest in general) 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 lipopeptides are either recovered from the fermentation medium or together with the fermentation medium during or at the end of fermentation. A biomass may also be recovered from the fermentation medium or together with the fermentation in addition to the lipopeptides.

[0051] Accordingly, the recovery step, step c) comprises performing one or more of the following steps i)- iii) i) recovering one or more lipopeptides from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising one or more lipopeptides at the end of the fermentation process, iii) recovering the fermentation medium comprising one or more lipopeptides throughout the fermentation process. In one or more exemplary embodiments the recovery steps, step i)-iii) comprises recovering a biomass in addition to the one or more lipopeptides.

[0052] As previously mentioned, it is shown in examples 1 and 3 that the lipopeptide composition and fengycin content of the lipopeptide composition is altered when the dissolved oxygen levels of the fermentation process dips very low for a short period of time.

[0053] Additionally, in one or more exemplary embodiments, the method further comprises a step a1) that is performed after step a) and prior to step b), wherein the step a1) consists of a1) allowing the dissolved oxygen (DO) level in the fermentation medium to drop below the low level of dissolved oxygen (DO) introduced in step b) for a period of time during the early growth phase of the fermentation process.

[0054] In step a1) the dissolved oxygen (DO) level is allowed to drop to below 10%, below 9%, below 8%, below 7%, below 6%, below 5%, below 4%, below 3%, below 2%, below 1% or allowed to drop to 0% DO.

[0055] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10%.

[0056] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5%.

[0057] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0%.

[0058] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 10-25%.

[0059] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 20%.

[0060] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 15%.

[0061] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 8-12%.

[0062] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 9-11%. In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 10% followed by maintaining a dissolved oxygen (DO) level in step b) of 10%.

[0063] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 5-30%, 10- 30%, 5-25%, 10-25%, 10-20%, 5-20%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11 %, 20%, 15% or 10% dissolved oxygen (DO).

[0064] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 5-25%.

[0065] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 20%.

[0066] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 15%.

[0067] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 8-12%.

[0068] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 9-11 %.

[0069] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to below 5% followed by maintaining a dissolved oxygen (DO) level in step b) of 10%.

[0070] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 5-30%, 10-30%, 5- 25%, 10-25%, 10-20%, 5-20%, 5-15%, 6-14%, 7-13%, 8-12%, 9-11 %, 20%, 15% or 10% dissolved oxygen (DO).

[0071] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 5-25%.

[0072] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 20%.

[0073] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 10-20%.

[0074] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 15%.

[0075] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 6-14%. In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 7-13%.

[0076] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 8-12%.

[0077] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 9-11%.

[0078] In one or more exemplary embodiments, the dissolved oxygen (DO) level in step a1) is allowed to drop to 0% followed by maintaining a dissolved oxygen (DO) level in step b) of 10%.

[0079] As discussed in example 1 , the period of time that the Bacillus fermentation is subjected to the dissolved oxygen (DO) level of step a1) also has an impact on fengycin production (example 1 and figure 4) and prolonging the period of time spent at this dissolved oxygen level from 0.5 hours to 1.5 hours resulted in a considerable increase in fengycin production.

[0080] Therefore, in step a1) the period of time is selected as one of the group consisting of at least 0.5 hours, at least 1 .0 hour, at least 1 .5 hours, at least 2.0 hours, at least 2.5 hours, at least 3.0 hours, between 0.5-3.0 hours, between 0.5-2.5 hours, between 0.5-2.0 hours, between 0.5-1 .5 hours, between 0.5-1 .0 hours, at the most 3.0 hours, at the most 2.5 hours, at the most 2.0 hours, at the most 1 .5 hours, and at the most 1 .0 hours.

[0081] In one or more exemplary embodiments, the period of time in step 1a) is at least 0.5 hours.

[0082] In one or more exemplary embodiments, the period of time in step 1a) is at least 1.0 hours.

[0083] In one or more exemplary embodiments, the period of time in step 1a) is at least 1 .5 hours.

[0084] In one or more exemplary embodiments, the period of time in step 1a) is 0.5-1 .5 hours.

[0085] In one or more exemplary embodiments, the period of time in step 1 a) is 0.5-2.0 hours.

[0086] In one or more exemplary embodiments, the period of time in step 1a) is 1 .0-2.0 hours.

[0087] In one or more exemplary embodiments, the period of time in step 1a) is 0.5-2.5 hours.

[0088] In one or more exemplary embodiments, the period of time in step 1a) is 0.5-3.0 hours.

[0089] In one or more exemplary embodiments, the composition comprising one or more lipopeptides further comprises a biomass. In one or more exemplary embodiments, the one or more lipopeptides is fengycin and the composition is a composition comprising fengycin.

[0090] In one or more exemplary embodiments, the composition comprising fengycin further comprises a biomass.

[0091] In one or more exemplary embodiments, the methods disclosed herein further comprises a purification step d) following the recovery step c).

[0092] The recovery step c) and the purification step d) comprises one or more of the following steps

[0093] 1) a filtration step,

[0094] 2) a centrifucation step,

[0095] 3) a pH adjustment step

[0096] 4) an adsorption step comprising adsorption of one or more lipopeptides onto an adsorption column,

[0097] 5) an adsorption step comprising adsorption of one or more lipopeptides onto a geopolymer,

[0098] 6) a chromatography step

[0099] 7) a CaCh flocculation step

[0100] 8) a solvent extraction step

[0101] 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.

[0102] In one or more embodiments of the present disclosure, the purification step d) is performed on the material recovered in step c) of the methods as disclosed herein.

[0103] 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. Adjustment of pH in step c) or step d) 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.

[0104] For recovery step c) and / or purification step d) of one or more lipopeptides or fengycin together with a biomass, adjustment of the pH to 4-6, 4.5-5.5, or 4.5-5.0 is preferred.

[0105] Producing a composition comprising one or more lipopeptides

[0106] While the methods disclosed herein result in increased production of the lipopeptide fengycin, the fermentation processes disclosed herein still produce a range of lipopeptides. As such these other lipopeptides are also of interest when recovered together with the increased proportion of fengycin produced by the methods disclosed herein as these other lipopeptides are known to also have antimicrobial activity. Therefore, the methods disclosed herein also relates to methods for producing a composition comprising one or more lipopeptides.

[0107] Embodiment A

[0108] Thus, in one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0109] Embodiment B

[0110] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopetides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0111] Embodiment C

[0112] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen (DO) for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0113] Embodiment D

[0114] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 2-18%, 4-17%, 5-15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0115] Embodiment E

[0116] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen (DO) for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

[0117] Embodiment F

[0118] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen (DO) for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 2-18%, 4-17%, 5-15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides. In one or more exemplary embodiments of the present disclosure, the recovery step, step c) of embodiments A-F, further comprises performing one or more of the following steps i)-iii) i) recovering one or more lipopeptides from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising one or more lipopeptides at the end of the fermentation process, and iii) recovering the fermentation medium comprising one or more lipopeptides throughout the fermentation process.

[0119] Producing a composition comprising fengycin

[0120] As the present disclosure is also concerned with increased production of the lipopeptide fengycin, which is a compound that possesses anti-fungal activity, the methods disclosed herein also relates to methods for producing a composition comprising fengycin.

[0121] Embodiment G

[0122] Thus, in one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin.

[0123] Embodiment H

[0124] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin.

[0125] Embodiment I

[0126] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin.

[0127] Embodiment J

[0128] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin.

[0129] Embodiment K

[0130] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin.

[0131] Embodiment L

[0132] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin thereby providing a composition comprising fengycin. In one or more exemplary embodiments of the present disclosure, the recovery step, step c) of embodiments G-L, further comprises performing one or more of the following steps i)-iii) i) recovering fengycin from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising fengycin at the end of the fermentation process, and iii) recovering the fermentation medium comprising fengycin throughout the fermentation process.

[0133] Producing a composition comprising one or more lipopeptides and a biomass

[0134] As previously discussed, compositions comprising one or more lipopeptides are also of interest in the methods disclosed herein. However, in addition to the lipopeptides, the biomass produced in the fermentation process is also of interest as the spores of the bacillus spp. contribute to the total antimicrobial effect. Therefore, the present disclosure also relates to methods for producing a composition comprising one or more lipopeptides and a biomass.

[0135] Embodiment M

[0136] Thus, in one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising one or more lipopeptides and a biomass.

[0137] Embodiment N

[0138] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising the one or more lipopeptides and a biomass.

[0139] Embodiment O

[0140] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising one or more lipopeptides and a biomass.

[0141] Embodiment P

[0142] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising the one or more lipopeptides and a biomass.

[0143] Embodiment Q

[0144] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising one or more lipopeptides and a biomass.

[0145] Embodiment R

[0146] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising one or more lipopeptides and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11 % or 10%, and c) performing a recovery step to recover the one or more lipopeptides and a biomass thereby providing a composition comprising the one or more lipopeptides and a biomass.

[0147] In one or more exemplary embodiments of the present disclosure, the recovery step, step c) of embodiments M-R, further comprises performing one or more of the following steps i)-iii) i) recovering one or more lipopeptides and a biomass from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising one or more lipopeptides and a biomass at the end of the fermentation process, and iii) recovering the fermentation medium comprising one or more lipopeptides and a biomass throughout the fermentation process.

[0148] Producing a composition comprising fengycin and a biomass

[0149] As previously discussed the present disclosure relates to methods for producing a composition comprising fengycin. However, in addition to fengycin, the biomass produced in the fermentation process is also of interest as the spores of the bacillus spp. contribute to the total antimicrobial effect. Therefore, the present disclosure also relates to methods for producing a composition comprising fengycin and a biomass.

[0150] Embodiment S

[0151] Thus, in one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11 % or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0152] Embodiment T

[0153] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0154] Embodiment U

[0155] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0156] Embodiment V

[0157] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop below 10%, 9%, 8%, 7%, 6% or 5% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0158] Embodiment l / l /

[0159] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-35%, 5-30%, 10-30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5- 15%, 8-12%, 9-11% or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0160] Embodiment X

[0161] In one or more exemplary embodiments, the present disclosure relates to a method for producing a composition comprising fengycin and a biomass, comprising the steps a) initiating a fermentation process with bacteria belonging to the Bacillus spp. in a bioreactor containing a suitable fermentation medium a1) allowing the dissolved oxygen (DO) level in the fermentation medium of step a) to drop to 0% dissolved oxygen for a period of time during the early growth phase of the fermentation process, before performing step b) b) maintaining in the fermentation medium a low level of dissolved oxygen (DO) in the range of 5-15%, 6-14%, 7-13%, 8-12%, 9-11 % or 10%, and c) performing a recovery step to recover the fengycin and a biomass thereby providing a composition comprising fengycin and a biomass.

[0162] In one or more exemplary embodiments of the present disclosure, the recovery step, step c) of embodiments S-X, further comprises performing one or more of the following steps i)-iii) i) recovering fengycin and a biomass from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising fengycin and a biomass at the end of the fermentation process, and iii) recovering the fermentation medium comprising fengycin and a biomass throughout the fermentation process.

[0163] Exponential phase

[0164] In the present context the exponential phase is the period of time wherein the bacteria in a fermentation process undergo exponential growth. Exponential growth in a fermentation process can for example be detected through OD measurements over time, cell counts, or through indicators of cell growth such as the carbon dioxide transfer rate (CTR) and oxygen transfer rate (OTR). Time periods with high levels of CTR and OTR are indicative of the exponential phase in fermentation processes comprising bacteria.

[0165] Early growth phase

[0166] In the present context, the early growth phase is the period of time when bacteria in a fermentation process leave the lag phase and start growing, thus entering the exponential phase. The exact length of the lag-phase and the timing of the early growth phase depends on multiple factors, including the fitness of the inoculum used for initiating the fermentation process and whether a preinoculum was used. However, the growth phases of bacillus are well known in the prior art and making adaptions for Bacillus growth in accordance with the circumstances would only amount to routine task for the person skilled in the art.

[0167] In one or more exemplary embodiments of the present disclosure, the early growth phase is a time interval from the start of fermentation that is selected as one of 8-20 hours, 8-18 hours, 10-18 hours, 10-16 hours, 10-14 hours, 12-14 hours, 13-15 hours and / or 12-16 hours following start of fermentation.

[0168] In one or more exemplary embodiments of the present disclosure, the early growth phase is the time interval 12-16 hours from start of fermentation.

[0169] In one or more exemplary embodiments of the present disclosure, the early growth phase is the time interval 13-15 hours from start of fermentation.

[0170] In one or more exemplary embodiments of the present disclosure, the early growth phase is the time interval 10-14 hours from start of fermentation.

[0171] In one or more exemplary embodiments of the present disclosure, the early growth phase is the time interval 8-16 hours from start of fermentation.

[0172] Anaerobic conditions and dissolved oxygen

[0173] The terms anaerobic conditions, pseudo-anaerobic conditions, anaerobic phase, pseudo-anaerobic phase, pseudo-anaerobic period and oxygen depletion are used interchangeably in the present disclosure and refer to conditions in a fermentantion process where the dissolved oxygen (DO) level in the fermentation medium drops to 0% for a period of time.

[0174] A dissolved oxygen level of 0% in the fermentation medium as used in the present disclosure refers to conditions in the fermentation medium wherein the dissolved oxygen levels are so low that they drop below the detection limit of the detectors used to detect dissolved oxygen levels. For example, if the dissolved oxygen level is detected in the exhaust gas from a bioreactor by a sensor, then a dissolved oxygen level of 0% in the exhaust gas of the bioreactor represents a dissolved oxygen level that is below the detection level of the sensor. In the present context, DO is used as an abbreviation of dissolved oxygen and the two terms are used interchangeably.

[0175] Biomass

[0176] Biomass as used herein refers to either bacterial cells or spores of bacterial cells that are present in the fermentation medium during a fermentation process and / or at the end of a fermentation process. For example, when a fermentation process is performed using bacteria belonging to the species Bacillus amyloliquefaciens, the biomass of that fermentation process is bacterial cells or spores of bacterial cells belonging to the Bacillus amyloliquefaciens species. Thus, in the present context, recovering a biomass refers the recovery of cells and spores from a fermentation medium, either during or at the end of a fermentation process.

[0177] Bacillus spp.

[0178] The methods of the embodiments disclosed herein may be performed using any species belonging to the Bacillus genus. Examples of suitable bacteria belonging to the Bacillus spp. includes 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.

[0179] Bacteria belonging to the species Bacillus amyloliquifaciens are of particular interest for use with the methods disclosed in embodiments A-R and these bacteria includes the strains of Bacillus amyloliquefaciens selected from the list consisiting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens strain with DSMZ accession number DSM 25840, B. amyloliquefaciens strain with DSMZ accession number DSM 27032, B. amyloliquefaciens strain with DSMZ accession number DSM 27033, and B. amyloliquefaciens strain with DSMZ accession number DSM 34003.

[0180] In or more exemplary embodiments, the fermentation process of step a) is initiated using bacteria belonging to the bacillus spp.. In or more exemplary embodiments, the fermentation process of step a) is initiated using a bacillus spp. selected as one or more from the list consisting of Bacillus spp. includes 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.

[0181] In or more exemplary embodiments, the fermentation process of step a) is initiated using a bacillus spp. selected as one or more from the list consisting of Bacillus spp. includes 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.

[0182] In or more exemplary embodiments, the fermentation process of step a) is initiated using bacteria belonging to a B. amyloliqufaciens species.

[0183] In or more exemplary embodiments, the fermentation process of step a) is initiated using bacteria belonging to a B. amyloliqufaciens species selected from the list consisting of Bacillus amyloliquefaciens subsp. amyloliquefaciens, Bacillus amyloliquefaciens subsp. plantarum, B. amyloliquefaciens DSM 25840, B. amyloliquefaciens strain DSM 27032, B. amyloliquefaciens DSM 27033 and B. amyloliquefaciens strain with DSMZ accession number DSM 34003.

[0184] In or more exemplary embodiments, the fermentation process of step a) is initiated using bacteria belonging to the B. amyloliqufaciens strain with DSMZ accession number DSM 34003.

[0185] Deposits and expert solution

[0186] 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.

[0187] The strain Bacillus amyloliquefaciens was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03 April 2012, under the accession No. DSM 25840. 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 27032.

[0188] 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.

[0189] 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.

[0190] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0191] Compositions and their use

[0192] The methods disclosed in the present disclosure are used to produce compositions that comprise either one or more lipopeptides, one or more lipopeptides and a biomass, fengycin or fengycin and a biomass. These compositions are particularly useful as antibacterial and antifungal agents against pathogens that infect plants. Therefore, these compositions are applied to plants to control, prevent or ameliorate infections caused by a fungal or bacterial pathogen.

[0193] In particular, these compositions are used for controlling, preventing or ameliorating infections caused by fungal pathogens because of their high content of fengycin and / or their high ratio of fengycin to surfactin and iturin.

[0194] In one or more exemplary embodiments, the present disclosure relates to the use of a composition comprising one or more lipopeptides produced by the methods disclosed herein as an antifungal agent.

[0195] The present disclosure also relates to the use of a composition comprising one or more lipopeptides produced by the methods disclosed herein for controlling, preventing or ameliorating infections in plants caused by fungal pathogens. In one or more exemplary embodiments, the present disclosure relates to the use of a composition comprising one or more lipopeptides and a biomass produced by the methods disclosed herein as an antifungal agent.

[0196] The present disclosure also relates to the use of a composition comprising one or more lipopeptides and a biomass produced by the methods disclosed herein for controlling, preventing or ameliorating infections in plants caused by fungal pathogens.

[0197] In one or more exemplary embodiments, the present disclosure relates to the use of a composition comprising fengycin produced by the methods disclosed herein as an antifungal agent.

[0198] The present disclosure also relates to the use of a composition comprising fengycin produced by the methods disclosed herein for controlling, preventing or ameliorating infections in plants caused by fungal pathogens.

[0199] In one or more exemplary embodiments, the present disclosure relates to the use of a composition comprising fengycin and a biomass produced by the methods disclosed herein as an antifungal agent.

[0200] The present disclosure also relates to the use of a composition comprising fengycin and a biomass produced by the methods disclosed herein for controlling, preventing or ameliorating infections in plants caused by fungal pathogens.

[0201] In one or more exemplary embodiments, the present disclosure relates to the use of a composition comprising an increased ratio of fengycin to iturin and / or surfactin produced by the methods disclosed herein as an antifungal agent.

[0202] The present disclosure also relates to the use of a composition comprising an increased ratio of fengycin to iturin and / or surfactin produced by the methods disclosed herein for controlling, preventing or ameliorating infections in plants caused by fungal pathogens. In one or more exemplary embodiments, the present disclosure relates a method for controlling, preventing or ameliorating an infection in a plant caused by a fungal pathogen, the method comprising administering a composition comprising an increased ratio of fengycin to iturin and / or surfactin produced by the methods disclosed herein, to the plant.

[0203] In one or more exemplary embodiments, the present disclosure relates a method for controlling, preventing or ameliorating an infection in a plant caused by bacterial pathogen, the method comprising administering a composition comprising an increased ratio of fengycin to iturin and / or surfactin produced by the methods disclosed herein, to the plant.

[0204] EXAMPLES

[0205] Materials and Methods

[0206] Strain

[0207] A Bacillus amyloliquefaciens strain (DSM 34003) was used for the cultures in this work. Single colonies were used to inoculate shake flask cultures for making aliquots as the primary inoculation material (PIM).

[0208] Fermentation conditions

[0209] The fermentations were made essentially as disclosed before (1 , 2).

[0210] The fermentation trials were made in 2L parallel bioreactors (DASGIP, Eppendorf, GE) with control units for temperature, pH, and dissolved oxygen (DO). Acid and base used for pH control were H2SO4 and NaOH, respectively. The bioreactors are equipped with level sensors for controlling foam by adding antifoam reagent. DO was controlled around desired levels via cascading to agitation and airflow during fermentation. Carbon dioxide Exhausting Rate (CER) and Oxygen Transfer Rate (OTR) were calculated with online measurements of DO, CO2 and 02 concentrations in the exhaust gas from fermenters

[0211] Analytical methods

[0212] Samples were taken during fermentation and kept at -20°C until lipopeptide analysis. The lipopeptides, such as surfactins, fengycins, and iturins, are analyzed using a method reported before (3).

[0213] Example 1 1ncreasing production of fengycins by including an anaerobic period in exponential phase

[0214] Three batches of cultures were made with different DO set points, i.e., 10% (DO.SP:10), 20% (DO.SP:20), and 30% (DO.SP:30), respectively. As shown in Figure 1 , the three batches reached DO set points after ca. 20 hours with some oscillation.

[0215] The effect of different DO set-points on the actual culture conditions can be better seen in Figure 2, which is a zoomed-in view of the DO profiles in the first 20 hours. The cultures of batches DO.SP:10 and DO.SP:20 experienced a period where the DO levels were below the detection limit of the DO sensors at around 15 hours. The cells in the cultures thus experienced a “pseudo”- anaerobic condition. The "low DO" period was longer in the DO.SP:10 batch (ca. 1.5 hours) than that in the DO.SP:20 batch ( less than 0.5 hours), whereas the DO.ST:30 batch didn't have an anaerobic phase. This happened during the “peak” period in the Carbon-dioxide Transfer Rate (CTR) and Oxygen Transfer Rate (OTR) profiles (Figure 3), which indicates that the cells were in the exponential phase of the cultures. Therefore, the DO set points at low, middle, and high levels resulted in long, short, and no “pseudo-anaerobic” phase during the exponential growth phase.

[0216] These culture conditions had significant effect on the production of fengycins (Figure 4). Specifically, the concentration of fengycins in the cultures showed a clear correlation with the DO setpoints, i.e., the fengycin concentrations were the highest in the culture made at DO.SP:10 (long pseudo-anaerobic period during the exponential phase), and the lowest fengycin concentration among these tests were found in the culture made at DO.SP:30 (no pseudo-anaerobic period during the exponential phase). This difference was already observed in the first sample taken for lipopeptide analysis (at ca. 20 hours), and it became even more clear in the following samples (taken at 24 and 46 hours).

[0217] The increased production of fengycins under the described culture conditions can be a result of combined effects of upregulated expression levels of responsible genes, and the metabolic profile in the cell that enables improved supply of energy and precursors for the fengycin synthesis. Fengycins are synthesized by a non-ribosomal peptide synthetases (NRPSs), composed of multiple modules, such as fenA, fenB, fenC, fenD, and fenE, arranged in a specific spatial order that identifies and integrates specific substrates into the nascent skeleton [4,5], Bacillus cells possess a high-level regulatory architecture and respond to environmental changes by adjusting their transcriptomes in a complex manner [6], The expression of the fen operon is regulated by complex mechanisms such as DegU and SpoOA [7, 8], The oxygen depletion is sensed by a [4Fe- 4S]2 +cluster containing Fnr (fumarate and nitrate reductase) and the two-component regulatory system ResDE [9], The expression of Fnr is upregulated under anaerobic conditions in Bacillus

[0010] , The oxygen conditions as well as the expression levels of the Fnr or ResDE regulators affects the overall metabolic network and energy metabolism in the cells

[0011] , which consequentially affected the production levels of fengycins in the described trials.

[0218] Example 2 Subsequent onset of lipopeptide production during the growth phase

[0219] A batch cultivation at set-point 20 % was conducted.

[0220] It was observed that the lipopeptides, surfactins, iturins, and fengycins were not produced at the same time. Instead, the start of the production of surfactins was detected first during the early growth phase (Figure 5 D). Subsequently, iturins were started to be produced (Fig. 5 E). A few hours later, also fengycins were produced (Fig. 5 F). All lipopeptides were produced during the growth phase while the OD and vegetative cells were increasing (Fig. 5 A-B).

[0221] It was concluded that the production of these lipopeptides was not initiated at the same time. The production of specific lipopeptides can hence be modulated by amending the process conditions accordingly in the right time of the cultivations. For example, the cultivation conditions could be changed at the on-set of the fengycin production to increase the titer of those further. The correct timing to balance the bacterial growth and lipopeptide production will be crucial to achieve an optimal product.

[0222] Example 3 Fengycin increased after oxygen depletion

[0223] A batch cultivation at set-point 20 % dissolved oxygen (DO) was conducted. Various process parameters dissolved oxygen (DO), biomass (optical density, OD620), counts of vegetative cells, counts of spores, carbon dioxide transmission rate (CTR), and lipopeptides, i.e., surfactins, Iturins, and Fengycins were measured and are shown in figure 6 A-H.

[0224] The cultivation was run in duplicates with one batch being a reference batch (solid line) and the other batch being subjected to a short period of oxygen depletion (dotted line) with dissolved oxygen going to 0% DO.

[0225] It was observed that a short period of oxygen depletion (dotted line) increased production of fengycins (figure 6 H) by three-fold in comparison to a non-interrupted cultivation (solid line), meanwhile other lipopeptides, surfactins (figure 6 F) and iturins (figure 6 G), diverged by less than 1.2 fold.

[0226] It was concluded that the production of fengycins was stimulated by low oxygen availability, so in accordance with example 3, regulating the dissolved oxygen (DO) levels can be used to control the speciation of lipopeptides.

[0227] References

[0228] 1. W017 / 081201

[0229] 2. W014178032A

[0230] 3. Smyth, T., et. al. 2010. Isolation and Analysis of Lipopeptides and High Molecular Weight Biosurfactants. In KN. Timmis (Ed.), Handbook of Hydrocarbon and Lipid Microbiology. Vol. 5: 3688-3704. Springer. ttps: / / doi.org / 10.1007 / 978-3-540-77587-4_290

[0231] 4. Hofemeister, J. et al. 2004. Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1 / 3. Molecular Genetics and Genomics. 272:363-378.

[0232] 5. Chen et al., 2008. More than Anticipated - Production of Antibiotics and Other Secondary metabolites by Bacillus amyloliquefaciens FZB42. Journal of Molecular Microbiology and Biotechnology. 16 (1-2): 14-24.

[0233] 6. Nicolas, P. et al. 2012. Science, 335( 6072): 1103-1106

[0234] 7. Verhamme D.T. et al., 2009. Degll and SpoOA jointly control transcription of two loci required for complex colony development by Bacillus subtilis.

[0235] 8. Sun J. et al. 2021 . CodY, ComA, Degll and SpoOA controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ. Journal of Applied Microbiology, 131 (3): 1289— 1304.

[0236] 9. Elisabeth Hartig and Dieter Jahn. 2012. Regulation of the Anaerobic Metabolism in Bacillus subtilis. Advances in Microbial Physiology. 61 : 195-216

[0237] 10. Ramos H.C. et al. 1995. EMBO J. 14(23): 5984-5994.

[0238] 11. Zhu J et al., 2006. Effect of the global redox sensing / regulation networks on Escherichia coli and metabolic flux distribution based on C-13 labeling experiments. Metabolic Engineering. 8(6):619-627.

[0239] 12. M. Shoda: Bacterial Control of Plant Disease, Journal of Bioscience and Bioengineering, pp. 515-521 , 200.

[0240] 13. H. P. Bais, R. Fall and .J M. Vivanco: Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringe is facilitated by biofilm formation and surfactin production, Plant Physiology, vol. 134, pp. 307-319, 2004.

[0241] 14. T Stein: Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, vol. 56, pp. 854-857, 2005.

[0242] 15. 1 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. PCT

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[0245] PCT

[0246] (Original in Electronic Form)

[0247] (This sheet is not part of and does not count as a sheet of the international application) PCT

[0248] (Original in Electronic Form)

[0249] (This sheet is not part of and does not count as a sheet of the international application)

[0250] FOR RECEIVING OFFICE USE ONLY

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Claims

CLAIMS1 . A method for producing a composition comprising one or more lipopeptides, comprising the steps a) initiating a fermentation process by introducing bacteria belonging to Bacillus spp. into a suitable fermentation medium b) maintaining a low level of dissolved oxygen (DO) in the fermentation medium, and c) performing a recovery step to recover the one or more lipopeptides thereby providing a composition comprising the one or more lipopeptides.

2. The method according to any one of the preceding claims, wherein the recovery step, step c) comprises performing one or more of the following steps i)-iii) i) recovering one or more lipopeptides from the fermentation medium during the fermentation process, ii) recovering the fermentation medium comprising one or more lipopeptides at the end of the fermentation process, and iii) recovering the fermentation medium comprising one or more lipopeptides throughout the fermentation process.

3. The method according to claim 2, wherein the recovery steps i)-iii) further comprises recovering a biomass in addition to the one or more lipopeptides.

4. The method according to any one of claims 1-3, wherein the composition comprising one or more lipopeptides further comprises a biomass.

5. The method according to any one of claims 1-4, wherein the composition comprising one or more lipopeptides contains an increased ratio of fengycin compared to iturin and surfactin.

6. The method according to any one of claims 1-5, wherein the one or more lipopeptides is selected as fengycin.

7. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 5-35%, 5-30%, 10- 30%, 15-25%, 10-25%, 10-20%, 5-25%, 5-20%, 5-15%, 6-14%, 7-13% 8-12%, 9-11 % or 10% dissolved oxygen (DO).

8. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 10-30%, 15-25%, 10- 25%, 10-20%, 5-25%, 5-20%, 5-15%, 8-12%, 9-11 % or 10% dissolved oxygen (DO).

9. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 5-25%, 5-20%, 5-15%, 8-12%, 9-11 % or 10% dissolved oxygen (DO).

10. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 5-15%, 6-14%, 7-13% 8-12%, 9-11 % or 10% dissolved oxygen (DO).

11. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 7-13% 8-12%, 9-11 % or 10% dissolved oxygen (DO).

12. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 5-25%.

13. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 5-20%.

14. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 10-20%.

15. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 6-14%.

16. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 7-13%.

17. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 8-12%.

18. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level in the range of 9-11%.

19. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is a dissolved oxygen (DO) level at a about 10%.

20. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is introduced at the exponential phase of the fermentation.

21. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is introduced at the early growth phase of the fermentation.

22. The method according to any one of the preceding claims, wherein the low level of dissolved oxygen (DO) in step b) is maintained from the early growth phase until the end of the fermentation process.

23. The method according to any one of the preceding claims, the method further comprises an additional step a1) that is performed after step a) and prior to step b), wherein the step a1) consists of a1) allowing the dissolved oxygen (DO) level in the fermentation medium to drop below the low level of dissolved oxygen (DO) introduced in step b) for a period of time during the early growth phase of the fermentation process.

24. The method according to claim 23, wherein the level of dissolved oxygen (DO) in step a1) is allowed to drop below 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % dissolved oxygen (DO) for a period of time at the early growth phase of the fermentation.

25. The method according to any one of claims 23-24, wherein the level of dissolved oxygen (DO) in step a1) is allowed to drop below 10% dissolved oxygen (DO) for a period of time at the early growth phase of the fermentation.

26. The method according to any one of claims 23-25, wherein the level of dissolved oxygen (DO) in step a1) is allowed to drop below 5%, 4%, 3%, 2% or 1 % dissolved oxygen (DO) for a period of time at the early growth phase of the fermentation.

27. The method according to any one of claims 23-26, wherein the level of dissolved oxygen (DO) in step a1) is allowed to drop below 5% dissolved oxygen (DO) for a period of time at the early growth phase of the fermentation.

28. The method according to any one of claims 23-27, wherein the level of dissolved oxygen (DO) in step a1) is allowed to drop to 0% dissolved oxygen (DO) for a period of time at the early growth phase of the fermentation.

29. The method according to any one of claims 23-28, wherein the period of time in step a1 is at least 0.5 hour.

30. The method according to any one of claims 23-29, wherein the period of time in step a1 is at least 1 .0 hour.

31. The method according to any one of claims 23-30, wherein the period of time in step a1 is at least 1 .5 hour.

32. The method according to any one of claims 23-31 , wherein the period of time in step a1 is between 0.5-3.0 hours.

33. The method according to any one of claims 23-32, wherein the period of time in step a1 is between 0.5-2.0 hours.

34. The method according to any one of claims 23-33, wherein the period of time in step a1 is between 0.5-1 .5 hours.

35. The method according to any one of claims 23-34, wherein the period of time in step a1 is between 1.0-2.0 hours.

36. The method according to any one of the preceding claims, further comprising a purification step d).

37. The method according to anyone of the preceding claims wherein step c) and / or step d) comprises one or more of the following steps1) a filtration step,2) a centrifugation step,3) a pH adjustment step,4) an adsorption step comprising adsorption of one or more lipopeptides onto an adsorption column,5) an adsorption step comprising adsorption of one or more lipopeptides onto a geopolymer,6) a chromatography step,7) a CaCh flocculation step, and / or8) a solvent extraction step.

38. The method according to any one of the preceding claims, wherein the Bacillus spp. is selected as one or more 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.

39. The method according to any one of the preceding claims, wherein the Bacillus spp. is selected as one or more from the list consisting of 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.

40. The method according to any one of the preceding claims, wherein the Bacillus spp. is Bacillus amyloliquefaciens.41 . 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 DSM 25840, B. amyloliquefaciens strain DSM 27032, B. amyloliquefaciens strain DSM 27033, and B. amyloliquefaciens strain DSM 34003.

42. The method according to any one of the preceding claims, wherein the Bacillus spp. is the Bacillus amyloliquefaciens strain DSM 34003.

43. A composition obtained by a method according to any one of the preceding claims.

44. Use of a composition according to claim 43 for controlling preventing or ameliorating an infection in a plant caused by a fungal or bacterial pathogen.

45. The use according to claim 44, wherein the pathogen is a fungal pathogen.

46. A method for controlling, preventing or ameliorating an infection in a plant caused by a fungal or bacterial pathogen comprising administering the composition according to claim 43 to the plant.

47. The method according to claim 46, wherein the pathogen is a fungal pathogen.

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