Methods for improving one or more process parameters of a fermentation process

Abstract

The present disclosure relates to methods for improving one or more process parameters of a fermentation process comprising Bacillus spp.

Description

[0001] TITLE: Methods for improving one or more process parameters of a fermentation process

[0002] The present invention relates to novel and improved methods for fermentation of bacteria that produces lipopeptides and / or bacteria belonging to the bacillus spp.

[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 [1 , 2, 3, 4],

[0005] 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],

[0006] 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.

[0007] However, because lipopeptides are amphiphilic molecules that consist of a cyclic hydrophilic peptide part and a hydrophobic tail part, lipopeptide secretion during fermentation of lipopeptide producing bacteria causes foaming.

[0008] When foaming occurs in bioreactors, the effective volume of the fermentation medium in the bioreactor increases. This can lead to loss of culture liquid and microorganisms through the air exhaust or lead to seepage into bearings and other installations, which further leads to sterility and containment problems. Foaming can also affect the physical conditions of the fermentation medium as foaming also reduces gas transfer rates from a bioreactor head space to a culture liquid.

[0009] Therefore, efforts are often made to limit foaming in fermentation processes. A common way to manage foaming is addition of antifoaming agents or defoamers. However, this approach can demand addition of a lot of antifoam / defoamer, which dilutes the fermentation process and can have a negative impact on the efficiency of processes such as recovery and purification processes downstream of the fermentation process. Moreover, the oxygen transfer rate is often decreased due to the addition of defoamers, leading to a higher oxygen supply demand, which might be limited.

[0010] SUMMARY

[0011] In its broadest aspect, the present disclosure relates to methods for improving one or more process parameters of a fermentation process comprising bacillus spp., comprising introduction of a suitable fermentation medium with cyclodextrin prior to fermentation start, or introduction of cyclodextrin to a bioreactor after fermentation start.

[0012] In another aspect, the present disclosure relates to the use of cyclodextrin as an anti-foaming agent in a fermentation process.

[0013] In a further aspect, the present disclosure relates to the use of cyclodextrin as a defoaming agent in a fermentation process.

[0014] In an additional aspect, the present disclosure relates to a defoaming composition comprising cyclodextrin.

[0015] In an additional aspect, the present disclosure relates to an anti-foaming composition comprising cyclodextrin.

[0016] DETAILED DESCRIPTION

[0017] Bacillus amyloliquefaciens is utilized in spore producing cultivations, where lipopeptides and other non-characterized components are produced by the organisms. The lipopeptides are nonribosomal cyclic lipopeptides including iturins, fengycins, and surfactins. These have antifungal properties which is highly useful in fighting for example the wheat plant disease fusarium head blight.

[0018] However, the lipopeptide secretion by Bacillus spp. during the fermentation causes foaming, because the lipopeptides are amphiphilic molecules consisting of a cyclic hydrophilic peptide part, and a hydrophobic tail part. In fermentation processes, foaming is an issue because it demands a lot of antifoam / defoamer additions, which is not desirable as it reduces the oxygen transfer from the gas phase to the broth and dilutes the fermentation medium, negatively influences efficiency of downstream processes and adds further costs.

[0019] Surprisingly, it was found that (see figure 1) introducing alpha-cyclodextrin in the fermentation medium of a fermentation process containing Bacillus amyloliquefaciens improved several process parameters when compared to an identical reference fermentation process. As shown in example 1 (figure 1 , Level sensor [pS]), a considerable reduction of foaming was observed, and a commercially available defoamer was introduced 55 times in the reference bioreactor, while the bioreactor containing 2.0 w / w% alpha-cyclodextrin only required introduction of commercially available defoamer 44 times. In total 60 % less defoamer was needed in the bioreactor containing 2.0 w / w % alpha-cyclodextrin (figure 1 , defoamer [mL]).

[0020] Thus, while 2.3 % (w / w) commercially available defoamer were added to the reference fermentation process, only 0.9 % (w / w) were added to the bioreactor during the cultivation with alpha-cyclodextrin. Both bioreactors had 0.1 % (w / w) antifoam added initially to the medium.

[0021] In addition, the maximum measured conductance was considerably lower with alpha-cyclodextrin, when the foam reached the sensor, indicating that the foam disappeared immediately once a tiny amount of defoamer had been added (figure 1 , Level sensor [pS]).

[0022] Also, the lower stirrer speed during the maximum growth phase (16 h) in the fermentation process where alpha-cyclodextrin was used as defoamer compared to the reference fermentation process, could be attributed to the addition of less defoamer, as defoamer is known to interfere with the oxygen transfer rate of fermentation processes.

[0023] Indeed, as can be seen from figure 1 (DO [%]) the fermentation process that received alpha- cyclodextrin had a dissolved oxygen (DO) level, that had less severe spikes in dissolved oxygen levels over time compared to the reference fermentation process.

[0024] All in all, the addition of 2.0 % alpha-cyclodextrin reduced the demand for commercially available defoamer during fermentation and led to a significant decrease in the measurable lipopeptides. It has not been investigated whether the decrease in lipopeptide content was due to the lipopeptides being “hidden / covered / masked” by the 2% alpha-cyclodextrin or whether less lipopeptides were produced when 2% alpha-cyclodextrin was present in the fermentation medium.

[0025] It is clear from the data presented in the present disclosure that addition of alpha-cyclodextrin reduced variations in the oxygen transfer rate over time (i), reduced the amount of foaming (ii), reduced the amount of foaming caused by lipopeptides (iii), reduced volume of defoamer required (iv), and improved fermentation productivity and cost-efficiency (v) due a larger fermentation volume and reduced defoamer usage.

[0026] Thus, the present disclosure relates to a method for improving one or more process parameters in a fermentation process performed in a bioreactor comprising bacteria belonging to bacillus spp., the method comprising the steps a) providing a suitable bacillus spp., b) providing a bioreactor containing a suitable fermentation medium, c) initiating a fermentation process by introducing the bacillus spp. provided in step a into the bioreactor of step b, d) allowing the fermentation process to proceed, and, either in a step a1) that is performed prior to step b) introducing cyclodextrin into a suitable fermentation medium to be contained in the bioreactor in step b), and / or in a step c1) that is performed after step b) but prior to step d) introducing cyclodextrin into the bioreactor, and / or in a step e) introducing cyclodextrin into the bioreactor when a threshold value of a relevant process indicator related to the fermentation process taking place in the bioreactor is measured, thereby improving one or more process parameters in a bioreactor fermentation process comprising bacillus spp.

[0027] The one or more process parameters of a fermentation reaction that is improved by the addition of cyclodextrin in step a1) and / or c1) and / or step e) is selected as one or more of i)-v) as defined in the following i. reducing variation of dissolved oxygen (DO) in the fermentation medium of the fermentation process over time, ii. reducing the amount of foaming caused by fermentation of bacilllus spp. over time, iii. reducing the amount of foaming caused by lipopeptides produced in a fermentation process comprising bacillus spp., iv. reducing the volume of defoamer required to be used over the course of a fermentation process from start to end, and, v. improving fermentation productivity and cost-efficiency. Thus, in one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as i).

[0028] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as ii).

[0029] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as iii).

[0030] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as iv).

[0031] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as v).

[0032] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as i), ii), iii), iv) and v).

[0033] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as one of i), ii), iii) and iv), or i), ii), iii) and v), or i), ii), iv), and v), or i), iii), iv) and v).

[0034] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as one of i), ii) and iii) or i), ii), and iv), or i), ii), and v), or i), iii), and iv), or i), iii), and v), or i), iv) and v).

[0035] In one or more exemplary embodiments of the present disclosure, the one or more process parameters is selected as one of i) and ii), or i) and iii), or i) and iv), or i) and v), or ii) and iii), or ii) and iv), or ii) and v), or iii) and iv), or iii) and v), or iv) and v).

[0036] It is contemplated herein that the beneficial effects of cyclodextrin on fermentation process parameters are present regardless of whether cyclodextrin is introduced into the fermentation medium prior to fermentation start (step a1)), but not step e)), or whether it is introduced into the bioreactor after fermentation start (step e)) but not step a1)). It is also contemplated that this effect is present if cyclodextrin is both introduced into the fermentation medium prior to fermentation start and introduced into the bioreactor after fermentation start (step a1)) and step e)). It will be a routine matter for the skilled person to choose in accordance with circumstances how cyclodextrin is added and in what concentrations.

[0037] In one or more exemplary embodiments of the present disclosure, the method for improving one or more process parameters includes step a1) but not step e).

[0038] In one or more exemplary embodiments of the present disclosure, the method for improving one or more process parameters includes step e) but not step a1).

[0039] In one or more exemplary embodiments of the present disclosure, the method for improving one or more process parameters includes both step e) and step a1).

[0040] Introducing cyclodextrin in step a1)

[0041] In step a1) cyclodextrin is introduced into the suitable fermentation medium to pre-emptively address and prevent the foaming that occurs over time during the fermentation process initiated in step c. This pre-emptive and preventive use of cyclodextrin is within the present context considered as the use of cyclodextrin as an anti-foaming agent.

[0042] The cyclodextrin that is introduced in step a1) is introduced in a concentration of 1 .0 w / w %, 1 .1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w %, 2.0 w / w %, 2.1 w / w % or 2.2 w / w %, at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w %, at least 2.0 w / w %, at most 1 .0 w / w %, at most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, in a range of 0.5-2.5 w / w %, in a range of 0.5-2.0 w / w %, in a range of 0.5-1 .5 w / w %, in a range of 0.5-1 .0 w / w %, in a range of 1 .0-2.5 w / w %, in a range of 1 .0-2.0 w / w %, in a range of 1 .1-2.0 w / w %, in a range of 1 .2-2.0 w / w %, in a range of 1 .3-2.0 w / w %, in a range of 1 .4-2.0 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .6-2.0 w / w %, in a range of 1 .7-2.0 w / w %, in a range of 1 .8- 2.0 w / w %, in a range of 1 .9-2.0 w / w %, in a range of 1 .0-1 ,5 w / w %, in a range of 1 .5-2.5 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .3-2.2 w / w %, in a range of 1 .4-2.1 w / w %, in a range of 1 .6-1 .9 w / w %, or in a range of 1 .7-1 .8 w / w %, in a range of 0.5-3.5 w / w%, in a range of 1 .0-3.0 w / w%, in a range of 1 .5-3.5 w / w%, or in a range of 2.0-3.5 w / w%. In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w % or at least 2.0 w / w %.

[0043] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of at most 1 .0 w / w %, at most 1 .5 w / w %, at most 2.0 w / w %, at most

[0044] 2.5 w / w %, at most 3.0 w / w %, or at most 3.5 w / w %.

[0045] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of1 .0 w / w %, 1.1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %,

[0046] 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

[0047] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a range of 0.5-2.5 w / w %, in a range of 0.5-2.0 w / w %, in a range of 0.5-1 .5 w / w %, in a range of 0.5-1 .0 w / w %, in a range of 1 .0-2.5 w / w %, in a range of 1 .0-2.0 w / w %, in a range of 1 .1 - 2.0 w / w %, in a range of 1 .2-2.0 w / w %, in a range of 1 .3-2.0 w / w %, in a range of 1 .4-2.0 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .6-2.0 w / w %, in a range of 1 .7-2.0 w / w %, in a range of 1 .8-2.0 w / w %, in a range of 1 .9-2.0 w / w %, in a range of 1 .0-1 ,5 w / w %, in a range of 1 .5-2.5 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .3-2.2 w / w %, in a range of 1 .4-2.1 w / w %, in a range of 1 .6-1 .9 w / w %, or in a range of 1 .7-1 .8 w / w %, in a range of 0.5-3.5 w / w%, in a range of 1 .0-3.0 w / w%, in a range of 1 .5-3.5 w / w%, or in a range of 2.0-3.5 w / w%.

[0048] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration in a range of 1 .0-2.0 w / w%, in a range of 1 .0-1 .5 w / w%, in a range of 1 .5-2.0 w / w%, in a range of 1 .0-2.5 w / w%, in a range of 1 .0-3.0 w / w%, or in a range of 1 .0-3.5 w / w%.

[0049] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration in a range of 1 .0-2.0 w / w%, in a range of 1 .0-1 .5 w / w%, in a range of 1 .5-2.0 w / w%, or in a range of 1 .0-2.5 w / w%.

[0050] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration in a range of 1 .0-2.0 w / w%.

[0051] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration in a range of 1 .0-1 .5 w / w%.

[0052] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration in a range of 1 .5-2.0 w / w%. In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 0.5 w / w %, 1 .0 w / w%, 1 .5 w / w% or 2.0 w / w %.

[0053] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 0.5 w / w %.

[0054] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 1 .0 w / w%.

[0055] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 1 .5 w / w%.

[0056] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 2.0 w / w %.

[0057] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 2.5 w / w %.

[0058] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 3.0 w / w %.

[0059] In one or more exemplary embodiments of the present disclosure, the cyclodextrin is introduced in step a1) in a concentration of 3.5 w / w %.

[0060] In the present context weight percent cyclocedextrin is calculated using the weight of the cyclodextrin per the total weight of the suitable fermentation medium. For example in a suitable fermentation medium containing 2 w / w% cyclodextrin, then 2% of the total weight of the suitable fermentation medium is cyclodextrin. Thus, if the suitable fermentation medium containing 2% cyclodextrin weighs 1 kg in total, then the fermentation medium contains 20 g cyclodextrin.

[0061] Introducing cyclodextrin in step c1)

[0062] In step c1) cyclodextrin is introduced into the suitable fermentation medium to pre-emptively address and prevent the foaming that occurs overtime during the fermentation process initiated in step c. This pre-emptive and preventive use of cyclodextrin is within the present context considered as the use of cyclodextrin as an anti-foaming agent.

[0063] The cyclodextrin that is introduced in step c1) is introduced in a concentration of 1 .0 w / w %, 1 .1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w %, 2.0 w / w %, 2.1 w / w % or 2.2 w / w %, at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w %, at least 2.0 w / w %, at most 1 .0 w / w %, at most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, in a range of 0.5-2.5 w / w %, in a range of 0.5-2.0 w / w %, in a range of 0.5-1 .5 w / w %, in a range of 0.5-1 .0 w / w %, in a range of 1 .0-2.5 w / w %, in a range of 1 .0-2.0 w / w %, in a range of 1 .1-2.0 w / w %, in a range of 1 .2-2.0 w / w %, in a range of 1 .3-2.0 w / w %, in a range of 1 .4-2.0 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .6-2.0 w / w %, in a range of 1 .7-2.0 w / w %, in a range of 1 .8- 2.0 w / w %, in a range of 1 .9-2.0 w / w %, in a range of 1 .0-1 ,5 w / w %, in a range of 1 .5-2.5 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .3-2.2 w / w %, in a range of 1 .4-2.1 w / w %, in a range of 1 .6-1 .9 w / w %, or in a range of 1 .7-1 .8 w / w %, in a range of 0.5-3.5 w / w%, in a range of 1 .0-3.0 w / w%, in a range of 1 .5-3.5 w / w%, or in a range of 2.0-3.5 w / w%.

[0064] Introducing cyclodextrin in step e)

[0065] In step e) cyclodextrin is introduced into the suitable fermentation medium to address, prevent and eliminate the foaming that occurs over time during the fermentation process initiated in step c. This use of cyclodextrin is within the present context considered as the use of cyclodextrin as an antifoaming agent.

[0066] The cyclodextrin that is introduced in step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %, 1.1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w %, 2.0 w / w %, 2.1 w / w % or 2.2 w / w %, at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w %, at least 2.0 w / w %, at most 1 .0 w / w %, at most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, in a range of 0.5-2.5 w / w %, in a range of 0.5-2.0 w / w %, in a range of 0.5-1 .5 w / w %, in a range of 0.5-1 .0 w / w %, in a range of 1 .0-2.5 w / w %, in a range of 1 .0-2.0 w / w %, in a range of 1 .1-2.0 w / w %, in a range of 1 .2-2.0 w / w %, in a range of 1 .3-2.0 w / w %, in a range of 1 .4- 2.0 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .6-2.0 w / w %, in a range of 1 .7-2.0 w / w %, in a range of 1 .8-2.0 w / w %, in a range of 1 .9-2.0 w / w %, in a range of 1 .0-1 ,5 w / w %, in a range of 1 .5-2.5 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .3-2.2 w / w %, in a range of 1 .4-2.1 w / w %, in a range of 1 .6-1 .9 w / w %, or in a range of 1 .7-1 .8 w / w %, in a range of 0.5-3.5 w / w%, in a range of 1 .0-3.0 w / w%, in a range of 1 .5-3.5 w / w%, or in a range of 2.0-3.5 w / w%.

[0067] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w %, at least 2.0 w / w %, at least 2,5 w / w% or at least 3.0 w / w%.

[0068] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of at most 1 .0 w / w %, at most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, at most 3.0 w / w %, or at most 3.5 w / w %.

[0069] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %, 1.1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

[0070] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration in a range of 0.5-2.5 w / w %, in a range of 0.5- 2.0 w / w %, in a range of 0.5-1 .5 w / w %, in a range of 0.5-1 .0 w / w %, in a range of 1 .0-2.5 w / w %, in a range of 1 .0-2.0 w / w %, in a range of 1 .1-2.0 w / w %, in a range of 1 .2-2.0 w / w %, in a range of 1 .3-2.0 w / w %, in a range of 1 .4-2.0 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .6-2.0 w / w %, in a range of 1 .7-2.0 w / w %, in a range of 1 .8-2.0 w / w %, in a range of 1 .9-2.0 w / w %, in a range of 1 .0-1 ,5 w / w %, in a range of 1 .5-2.5 w / w %, in a range of 1 .5-2.0 w / w %, in a range of 1 .3- 2.2 w / w %, in a range of 1 .4-2.1 w / w %, in a range of 1 .6-1 .9 w / w %, in a range of 1 .7-1 .8 w / w %, in a range of 0.5-3.5 w / w%, in a range of 1 .0-3.0 w / w%, in a range of 1 .5-3.5 w / w%, or in a range of 2.0-3.5 w / w%.

[0071] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 0.5 w / w %, 1 .0 w / w%, 1 .5 w / w%, 2.0 w / w %, 2.5 w / w%, 3.0w / w% or 3.5 %w / w.

[0072] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 0.5 w / w %, 1 .0 w / w%, 1 .5 w / w% or 2.0 w / w %.

[0073] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 0.5 w / w %.

[0074] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w%.

[0075] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .5 w / w%.

[0076] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.0 w / w %.

[0077] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.5 w / w %.

[0078] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.0 w / w %.

[0079] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.5 w / w %.

[0080] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually.

[0081] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually to reach a total cyclodextrin concentration. In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually in response to the relevant process indicator.

[0082] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually to the bioreactor whenever the threshold value of the relevant process indicator is measured.

[0083] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually in response to the relevant process indicator to reach a total cyclodextrin concentration.

[0084] In one or more exemplary embodiments of the present disclosure, the cyclodextrin of step e) is introduced gradually to the bioreactor whenever the threshold value of the relevant process indicator is measured to reach a total cyclodextrin concentration.

[0085] In the present context, the phrase "introduced gradually" refers to an incremental introduction of cyclodextrin to the bioreactor overtime. When "introduced gradually" is used together with "up to a total concentration", then this refers to an incremental introduction of cyclodextrin to the bioreactor up to a total cyclodextrin concentration selected as specified above. For example, if the total concentration of cyclodextrin is selected as 2 w / w%, then cyclodextrin is introduced incrementally to the bioreactor over time until a total concentration of 2 w / w% cyclodextrin has been introduced to bioreactor.

[0086] In some cases, the methods herein comprise both step a1) and step e) and in these cases any cyclodextrin added in the step a1). Therefore, if 2w / w% cyclodextrin is added in step a1) and it is desired to reach a total concentration of 2w / w% cyclodextrin in step e), then no further cyclodextrin is introduced to the bioreactor in step e). However, if 1 w / w% cyclodextrin is added in step a1) and it is desired to reach a 2 w / w% concentration in step e), then an additional 1 w / w% cyclodextrin is introduced in step e). The additional 1 w / w% cyclodextrin of step e) is introduced either at once, in response to a relevant process indicator, gradually or combinations thereof as discussed above.

[0087] Managing cyclodextrin introduction of step e) in a fermentation process

[0088] A key step in managing fermentation processes in bioreactors is timing the addition of defoamer. Because defoamer is costly by itself, takes up precious volume in the bioreactor and impacts physical parameters such as the oxygen transfer rate of oxygen into the fermentation medium, it is important to properly manage addition of defoamerto a fermentation process. To properly dose a defoamer in a fermentation process it is therefore necessary to monitor the fermentation process and identify when the defoamer needs to be introduced into the fermentation medium. In the present context, it is equally important to manage introduction of cyclodextrin, as the cyclodextrin must be introduced timely and in suitable concentrations to allow a reduced use of commercial defoamer.

[0089] A desired concentration of cyclodextrin is reached by either introducing an amount of cyclodextrin at once, that corresponds to the desired concentration, or gradually introducing cyclodextrin to reach a total concentration of cyclodextrin. Preferably, cyclodextrin may be introduced before or at the beginning of the fermentation process.

[0090] In one embodiment cyclodextrin is added at once before the fermentation process start. In another embodiment cyclodextrin is added at once at the beginning of the fermentation process.

[0091] In cases where cyclodextrin is introduced gradually it is required to monitoring relevant process indicator(s) related to the fermentation medium to identify when further cyclodextrin is required in the bioreactor. When the relevant process indicator(s) reaches a threshold value, it is an indication that cyclodextrin is needed and should be introduced into the bioreactor.

[0092] Thus, the cyclodextrin of step e) is introduced into the bioreactor when the relevant process indicator reaches a threshold value.

[0093] The relevant process indicator can for example be one of a function of time from fermentation start, a function of fermentation stage, optical density (OD) of the fermentation, optical density (OD) increase of the fermentation, oxygen transfer rate, carbon dioxide transfer rate (CTR), bioreactor foam level and bioreactor pressure. Selection of a relevant process indicator and monitoring of the relevant process indicator is routine work for the skilled person, who will make this selection in accordance with the circumstances.

[0094] In one or more exemplary embodiments of the present disclosure, the relevant process indicator is selected from the list consisting of a function of time from fermentation start, a function of fermentation stage, optical density (OD) of the fermentation, optical density (OD) increase of the fermentation, oxygen transfer rate, carbon dioxide transfer rate (CTR), bioreactor foam level and bioreactor pressure.

[0095] In one or more exemplary embodiments of the present disclosure, the relevant process indicator is selected as one or more from the list consisting of a function of time from fermentation start, OD of a fermentation process, OD increase in a fermentation process, and bioreactor foam level.

[0096] In one or more exemplary embodiments of the present disclosure, the relevant process indicator is a function of time.

[0097] When the relevant process indicator is a function of time from fermentation start, the threshold value is a single time point or a series of time points. In this context, a time point could be fermentation start, an hour from fermentation start or 4.5 hours from fermentation start. An example of a series of time points is 0.5 hours from fermentation start, 1 hour from fermentation start, 2, hours from fermentation start, 4 hours from fermentation start, 8 hours form fermentation start.

[0098] Time is measured from the start of fermentation and the selection of a single time point or a series of time points as threshold value is a routine task well known to the person skilled in the art.

[0099] When the threshold value is a single time point, then cyclodextrin is introduced up to a desired total cyclodextrin concentration once the single time point is reached.

[0100] When the threshold value is a series of time points, then cyclodextrin is introduced each time a time points is measured in a concentration resulting in a desired total cyclodextrin concentration in the bioreactor once the series of time points has been completed. The amount of cyclodextrin introduced at each time point in a series of time points can be an identical amount for every time point or it can vary according to a pre-determined schedule leading to a desired final concentration of cyclodextrin in the bioreactor.

[0101] In one or more embodiments of the present disclosure, the relevant process parameter is a function of time and the threshold value a single time point.

[0102] In one or more embodiments of the present disclosure, the relevant process parameter is a function of time and the threshold value a series of time points.

[0103] In one or more exemplary embodiments of the present disclosure, the relevant process indicator is bioreactor foam level.

[0104] Measuring foaming in a fermentation process

[0105] One way of managing foaming in bioreactors is monitoring the foam level in a bioreactor and when foaming reaches a certain level in the bioreactor adding a defoamer. Therefore, in the context of the method described herein, the foam level in a bioreactor can be used as the relevant process indicator and a specific level of foam in the bioreactor used as threshold value.

[0106] In one or more exemplary embodiments of the present disclosure, the relevant process indicator is the foam level in the bioreactor.

[0107] Foaming in a bioreactor can be measured in several ways including, but not limited to, use of conductivity sensors, conductivity switches, impedance detection probes or even visual inspection of the bioreactor and it is considered routine work for the person skilled in the art to choose a suitable method for measuring the foam level in a bioreactor in accordance with the circumstances.

[0108] In one or more exemplary embodiments of the present disclosure, the foam level in a bioreactor is measured using a conductivity sensor. In one or more exemplary embodiments of the present disclosure, the foam level in a bioreactor is measured by a conductivity sensor disposed in the head space of a bioreactor. Conductivity is then measured continuously by the sensor and the conductivity measured will increase as the foam levels in the bioreactor increase and reach the conductivity sensor disposed in the head space of the bioreactor.

[0109] When the foam level in a bioreactor is measured in terms of conductivity in the head space of a bioreactor, then the threshold value is expressed in terms a conductance value.

[0110] Thus, in one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value between 30-70, 35-65, 40-60, 45-55 or 48-52 pS.

[0111] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value of at least 30, at least 35, at least 40, at least 45 or at least 48, at the most 52, at the most 55, at the most 60, at the most 65 and at the most 70 pS.

[0112] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value selected as one from the list consisting of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63 , 64, 65, 66, 67, 68, 69 or 70 pS.

[0113] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value selected as one from the list consisting of 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60 pS.

[0114] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value selected as one from the list consisting of 40, 45, 48, 50, 52, 55, and 60 pS.

[0115] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value selected as one from the list consisting of 45, 48, 50, 52, and 55 pS. In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value selected as one from the list consisting of 48, 50, and 52 pS.

[0116] In one or more exemplary embodiments of the present disclosure, the threshold value is a conductance value of 50 pS.

[0117] Cyclodextrins

[0118] The cyclodextrins are a group of cyclic oligosaccharides formed by glucopyranose (glucose) monomers linked via a-1 ,4-glycosidic bonds. Alpha-cyclodextrin contains six glucose monomers, beta-cyclodextrin contains seven glucose monomers and gamma-cyclodextrin contains eight glucose monomers. Cyclodextrins form a truncated cone shape with a hydrophilic outer wall and a less hydrophilic inner wall, the latter wall forming a more apolar internal cavity. Because of this shape, cyclodextrins are soluble in water and can host lipophilic guest molecules while in aqueous solution, thus increasing solubility of the lipophilic guest molecule. The ability of cyclodextrins to increase solubility of lipophilic molecules makes them useful as defoamers and / or anti-foaming agents in fermentation processes that comprise bacteria, lipopeptides, lipopeptide producing bacteria, and / or bacteria belonging to the bacillus species, such as Bacillus amyloliquefaciens.

[0119] Alpha-, beta- and gamma-cyclodextrins are all soluble in water and consequently they will all be soluble in most culture media. The exact ease of solubilization will depend on the specific components of the culture medium. Gamma-cyclodextrin has the highest water solubility of the three and Beta-Cyclodextrin has the lowest water solubility. However, with stirring, warming, or the addition of solubilizing agents, beta-cyclodextrin can still be solubilized in most culture media, though it may require more time or adjustments compared to alpha and gamma.

[0120] As described above, alpha-, beta- and gamma-cyclodextrins have a cavity effective for encapsulating smaller hydrophobic compounds, the cavity, vary in size, with alpha being the smallest. With this cavity these cyclodextrins can reduce foam by interacting with surface-active compounds in the medium, encapsulating them, and thereby reducing their ability to stabilize bubbles. Since all three cyclodextrins (alpha, beta, and gamma) share this property of encapsulating hydrophobic molecules, they are all able to disrupt surface activity and reduce foam formation during fermentation.

[0121] As demonstrated herein foaming in fermentation processes comprising Bacillus amyloliquefaciens is partially prevented and mitigated by introduction of alpha-cyclodextrin to a suitable fermentation medium prior to fermentation start. Given the high structural similarities between the cyclodextrins, it is likely that other cyclodextrins are also capable of mitigating foaming in fermentation processes.

[0122] As such, in the present context, any one of the cyclodextrins is considered suitable for use as a defoaming agent or anti-foaming agent in a fermentation process.

[0123] Thus, in one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is any cyclodextrin.

[0124] In one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is one or more cyclodextrins.

[0125] In one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin. In one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is alpha-cyclodextrin.

[0126] In one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is beta-cyclodextrin.

[0127] In one or more exemplary embodiments, the cyclodextrin referred to in the present disclosure is gamma-cyclodextrin.

[0128] Use of cyclodextrins as anti-foaming agent and / or defoaming agent

[0129] As demonstrated herein foaming in fermentation processes comprising Bacillus amyloliquefaciens is mitigated by introduction of alpha-cyclodextrin. Given the high structural similarities between the cyclodextrins, it is likely that other cyclodextrins are also capable of mitigating foaming in fermentation processes.

[0130] Therefore, in one or more exemplary embodiments, the present disclosure relates to the use of one or more cyclodextrin(s) as anti-foaming agent(s) or defoaming agent(s) in a fermentation process.

[0131] In one or more exemplary embodiments, the present disclosure relates to the use of one or more cyclodextrin (s) as anti-foaming agent(s) or defoaming agent(s) in a fermentation process comprising bacteria.

[0132] In one or more exemplary embodiments, the present disclosure relates to the use of one or more cyclodextrin(s) as anti-foaming agent(s) or defoaming agent(s) in a fermentation process comprising lipopeptides.

[0133] In one or more exemplary embodiments, the present disclosure relates to the use of one or more cyclodextrin(s) as anti-foaming agent(s) or defoaming agent(s) in a fermentation process comprising lipopeptide producing bacteria.

[0134] In one or more exemplary embodiments, the present disclosure relates to the use of one or more cyclodextrin(s) as anti-foaming agent(s) or defoaming agent(s) in a fermentation process comprising bacteria belonging to the Bacillus spp.

[0135] In one or more exemplary embodiments of the present disclosure, the cyclodextrin(s) used as antifoaming agent(s) or defoaming agent(s) is selected as one or more from the list consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin. In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent(s) or defoaming agent(s) is alpha-cyclodextrin.

[0136] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent(s) or defoaming agent(s) is beta-cyclodextrin.

[0137] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent(s) or defoaming agent(s) is gamma-cyclodextrin.

[0138] In one or more exemplary embodiments of the present disclosure, the cyclodextrin(s) used as antifoaming agent(s) is selected as one or more from the list consisting alpha-cyclodextrin, beta- cyclodextrin, and gamma-cyclodextrin.

[0139] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent is alpha-cyclodextrin.

[0140] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent is beta-cyclodextrin.

[0141] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as antifoaming agent is gamma-cyclodextrin.

[0142] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as defoaming agent(s) is selected as one or more from the list consisting of alpha-cyclodextrin, beta- cyclodextrin, and gamma-cyclodextrin.

[0143] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as defoaming agent is alpha-cyclodextrin.

[0144] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as defoaming agent is beta-cyclodextrin.

[0145] In one or more exemplary embodiments of the present disclosure, the cyclodextrin used as defoaming agent is gamma-cyclodextrin.

[0146] Anti-foaming and defoaming compositions comprising cyclodextrins

[0147] It is envisioned that cyclodextrins, such as for example alpha-, beta- and gamma-cyclodextrin, are suitable for use in defoaming and / or anti-foaming compositions. Formulations of defoaming and anti-foaming compositions is generally known in the prior art and such compositions commonly contain but are not limited to ingredients such as insoluble oils, stearates, glycols, alcohols, fatty alcohols and polyethers. As it has been shown herein that alpha-cyclodextrin is also useful as an anti-foaming agent, it is contemplated that cyclodextrin can be formulated together with other suitable ingredients to form defoaming and anti-foaming compositions. The specific formulation of anti-foaming and defoaming compositions is considered a routine task for the person skilled in the art.

[0148] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition or a defoamer composition comprising one or more cyclodextrins.

[0149] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition or a defoamer composition comprising one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

[0150] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition comprising one or more cyclodextrins selected from the list consisting of alpha- cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

[0151] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition comprising alpha-cyclodextrin.

[0152] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition comprising beta-cyclodextrin.

[0153] In one or more exemplary embodiments, the present disclosure relates to an anti-foaming composition comprising gamma-cyclodextrin.

[0154] In one or more exemplary embodiments, the present disclosure relates to a defoamer composition comprising one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta- cyclodextrin and gamma-cyclodextrin.

[0155] In one or more exemplary embodiments, the present disclosure relates to a defoamer composition comprising alpha-cyclodextrin.

[0156] In one or more exemplary embodiments, the present disclosure relates to a defoamer composition comprising beta-cyclodextrin.

[0157] In one or more exemplary embodiments, the present disclosure relates to a defoamer composition comprising gamma-cyclodextrin.

[0158] Defoaming agents and anti-foaming agents

[0159] A defoamer or anti-foaming agent as used in the present context is a chemical additive that reduces and hinders the formation of foam in fermentation processes. The chemical additive is used to prevent formation of foam or is added to break a foam already formed. The terms defoamer or anti-foaming agents can refer to the same compound and the main difference between a defoamer and an anti-foaming agent lies in the time of application. In the present context a defoamer or defoaming agent is considered a compound that is introduced to a bioreactor during a fermentation process to reduce the amount of foam already present in the fermentation process, whereas an anti-foaming agent is a compound that is introduced into a fermentation medium prior to autoclavation / sterilization of the fermentation medium in order to prevent and / or reduce the rate by which foam occurs when the fermentation medium is used in a fermentation process.

[0160] For example, an anti-foaming agent used in a fermentation process in the present context relates to the addition or introduction of an agent to a suitable fermentation medium prior to the start of a fermentation process, where the agent suppresses or prevents foaming in the fermentation process once the fermentation process is started.

[0161] Bacillus spp.

[0162] 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 of bacterial species belonging to the Bacillus genus is a routine task for the person skilled in the art.

[0163] 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.

[0164] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. 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.

[0165] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. is a bacteria belonging to the Bacillus amyloliquefaciens species.

[0166] In one or more exemplary embodiments of the present disclosure, the Bacillus spp. 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 and B. amyloliquefaciens strain with the DSMZ accession number DSM 27033. In one or more exemplary embodiments of the present disclosure, the Bacillus spp. is a Bacillus amyloliquefaciens strain selected from the list consisting of DSM 25840, DSM 27032 and DSM 27033.

[0167] Bioreactors

[0168] A bioreactor in the context of the present disclosure is a manufactured device, container or system that supports a biologically active environment. Examples of suitable manufactured devices, containers and systems include but is not limited to glass flasks, glass containers, stainless steel containers and vessels. Such devices also comprise systems that contain multiple containers or vessels connected in series that are designed to support a biologically active environment. An example of such a biologically active environment is a fermentation medium wherein a fermentation process is ongoing.

[0169] Bioreactor head space and head space volume

[0170] A head space within the context of bioreactors as defined herein, is a space inside the volume of a bioreactor that contains air and is located between the liquid surface of the fermentation medium volume inside the bioreactor and the roof of the bioreactor.

[0171] In cases where the bioreactor does not have a roof, the head space is instead a space inside the volume of a bioreactor that is delimited by the side wall(s) of the bioreactor, the liquid surface of the fermentation medium and the opening in the bioreactor defined by the upper edge of the bioreactor side wall(s). The head space volume of a bioreactor is then the volume of the head space.

[0172] Deposits and expert solution

[0173] 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.

[0174] The strain Bacillus amyloliquefaciens that 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.

[0175] The strain Bacillus amyloliquefaciens that 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. The strain Bacillus amyloliquefaciens that 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.

[0176] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0177] BRIEF DESCRIPTION OF THE FIGURES

[0178] Figure 1

[0179] Fig. 1 shows a comparison of dissolved oxygen (DO) %, stirrer rounds per minute (RPM), Carbon dioxide transfer rate (CTR), conductance measured in terms of pS by a sensor, and defoamer addition in mL measured overtime in two fermentation process comprising Bacillus amyloliquefaciens. One fermentation process is a reference process (B1 shown on the left hand side) and one wherein the fermentation medium was supplemented with 2 w / w% alphacyclodextrin prior to fermentation start (B5 shown on the right hand side).

[0180] EXAMPLES

[0181] Example 1 - addition of 2 w / w% alpha-cyclodextrin to a fermentation medium

[0182] Two fermentation processes, named B1 and B5, with Bacillus amyloliquefaciens were set up in 0,8 L bioreactors and cultivated under 1 vmm gas flow rate and a dissolved oxygen (DO) set-point of 20 % with the only difference being the addition of 2 w / w% alpha-cyclodextrin to the fermentation medium used in reactor B5 prior to autoclavation. During fermentation the DO was maintained by adjusting the stirrer speed. Both fermentations were similar and especially the carbon transfer rate (CTR), which indicates the aerobic metabolic activity was comparable between the two bioreactors.

[0183] The most striking observation was that the defoamer (Antifoam food grade SB509) addition was considerably different. While 2.3 % (w / w) defoamer were added to the reference process, only 0.9 % were added to the bioreactor with alpha-cyclodextrin (B5). The defoamer addition is triggered in lab and production scale by an electrical circuit, which is closed when the foam touches a metal sensor in the headspace of the bioreactor. Raising foam in the bioreactor will eventually touch the sensor and trigger the defoamer addition once a threshold of 50 pS conductance is exceeded. Analyzing the trigger events of the sensor in both experiments, underlines the observation of less defoamer demand with alpha-cyclodextrin. In the reference process (B1), the defoamer addition was triggered 55 times, while with alpha-cyclodextrin (B5) 44 times. In addition, the maximum measured conductance was considerably lower with alpha- cyclodextrin, when the foam reached the sensor, indicating that the foam disappeared immediately once a tiny amount of defoamer had been added (figure 1 , Level sensor [pS]). Further, the lower stirrer speed (figure 1 , stirrer [rpm]) required in fermentation process B5 could be attributed to the lower content of defoamer that had been added to the cultivation, because defoamer reduces the oxygen transfer rate. Nevertheless, to maintain the DO, more mixing power needed to be introduced.

[0184] All-in-all, In the performed Bacillus amyloliquefaciens cultivation in 0.8-L laboratory scale, surprisingly a considerable reduction of foaming was observed, and 60 % less defoamer was needed in the bioreactor (B5) where 2 w / w% alpha-cyclodextrin had been added to the fermentation medium.

[0185] Example 2 - Effect of alpha-cyclodextrin addition on Bacillus amyloliquefaciens cells and lipopeptide detection

[0186] After 43.5 h, the cultivation broth was analyzed. Vegetative cells and spores as well as lipopeptides were quantified in the cultivation broth. The number of vegetative cells and spores were the same considering the variation of the analytical method (ca. 15 %). Around 2.3x109veg. cells / mL and 7.2x109spores / mL were quantified (table 1). This furthermore stressed that both cultivations were not affected on the metabolic activity due to the addition of alpha-cyclodextrin.

[0187] Considerably less lipopeptides were measured when alpha-cyclodextrin was added: 811 mg / L Iturins compared to 1266 mg / L in the reference; 342 mg / L fengycins compared to 1721 mg / L; 1929 mg / L compared to 4051 mg / L (table 1). Due to the amphiphilic nature of the lipopeptides they act as surfactants and form foam. The reduction of lipopeptides hence matched the observation that less defoamer was required.

[0188] Table 1 : Comparison of vegetative cells, spores, and measured lipopetides in the fermentation broth after 43.5 hours of fermentation in a reference culture (B1) and a culture wherein the fermentation medium had been supplemented with 2 w / w% alpha-cyclodextrin prior to fermentation start (B5).

[0189] Alpha-cyclodextrin was added with the intention to increase the bioactivity. However, an in vitro bioactivity assay, showed a very low antifungal bioactivity against Fusarium graminearum with alpha-cyclodextrin added. The ability of alpha-cyclodextrin to engage with and reduce foaming of lipopeptides probably contributed to this reduced bioactivity.

[0190] Concluding remarks

[0191] The addition of 2.0 % alpha-cyclodextrin reduced the demand for defoamer and lead to a significant decrease in the measurable lipopeptides. It has not been investigated whether the “hidden” lipopeptides are hidden by alpha-cyclodextrin or whether less lipopeptides were produced under these conditions. In either case it is clear from the data that the amount of foaming is reduced in the fermentation process wherein alpha-cyclodextrin was added. The addition of alpha-cyclodextrin can potentially reduce the demand for defoamer in many cultivations drastically. This would on the one hand reduce the amount of used defoamer, leading to cost reductions, and might improve the downstream efficiency. Additionally, reducing the amount of defoamer used and reducing the amount of foaming will free up space in a suitable bioreactor and allow for cultivations with a larger filling volume, which leads to an increase in capacity and productivity.

[0192] Items

[0193] 1 . A method for improving one or more process parameters in a fermentation process performed in a bioreactor comprising bacteria belonging to bacillus spp., the method comprising the steps a) providing a suitable bacillus spp., b) providing a bioreactor containing a suitable fermentation medium, c) initiating a fermentation process by introducing the bacillus spp. provided in step a into the bioreactor of step b, d) allowing the fermentation process to proceed, and either in a step a1) that is performed prior to step b) introducing cyclodextrin into a suitable fermentation medium to be contained in the bioreactor in step b), and / or in a step e) introducing cyclodextrin into the bioreactor when a threshold value of a relevant process indicator related to the fermentation process taking place in the bioreactor is measured, thereby improving one or more process parameters in a bioreactor fermentation process comprising bacillus spp.

[0194] 2. The method of any one of the preceding items, wherein improving one or more process parameters is selected as one or more of i. reducing variation of dissolved oxygen (DO) in the fermentation medium of the fermentation process overtime, ii. reducing the amount of foaming caused by fermentation of bacilllus spp. over time, iii. reducing the amount of foaming caused by lipopeptides produced in a fermentation process comprising bacillus spp., iv. reducing the volume of defoamer required to be used over the course of a fermentation process from start to end, and, v. Improving fermentation productivity and cost-efficiency.

[0195] 3. The method according to any one of the preceding items, wherein the cyclodextrin is selected as one or more from the list consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

[0196] 4. The method according to any one of the preceding items, wherein the cyclodextrin is alpha-cyclodextrin.

[0197] 5. The method according to any one of the preceding items, wherein the cyclodextrin is beta- cyclodextrin.

[0198] 6. The method according to any one of the preceding items, wherein the cyclodextrin is gamma-cyclodextrin.

[0199] 7. The method according to any one of the preceding items, wherein the method comprises step a1) but not step e).

[0200] 8. The method according to any one of the preceding items, wherein the method comprises step e) but not step a1).

[0201] 9. The method according to any one of the preceding items, wherein the method comprises step a1) and step e). 0. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w% or at least 2.0 w / w %. 11 . The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of at the most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, at most 3.0 w / w%, or at most 3.5 w / w%.

[0202] 12. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .0 w / w %, 1.1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

[0203] 13. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .0 w / w %.

[0204] 14. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .5 w / w %.

[0205] 15. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 2.0 w / w %.

[0206] 16. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 2.5 w / w %.

[0207] 17. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 3.0 w / w %.

[0208] 18. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 3.5 w / w %.

[0209] 19. The method according to any one of the preceding items, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration in a range of 0.5-2.5 w / w %, 0.5-2.0 w / w %, 0.5-1 .5 w / w %, 0.5-1 .0 w / w %, 1 .0-2.5 w / w %, 1 .0-2.0 w / w %, 1 .1-2.0 w / w %, 1 .2-2.0 w / w %, 1 .3-2.0 w / w %, 1 .4-2.0 w / w %, 1 .5-2.0 w / w %, 1 .6-2.0 w / w %, 1 .7-2.0 w / w %, 1 .8- 2.0 w / w %, 1 .9-2.0 w / w %, 1 .0-1 ,5 w / w %, 1 .5-2.5 w / w %, or 1 .5-2.0 w / w %, 1 .3-2.2 w / w %, 1 .4-2.1 w / w %, 1 .6-1 .9 w / w %, 1 .7-1 .8 w / w %, 0.5-3.5 w / w%, 1 .0-3.0 w / w%, 1 .5-3.5 w / w% or 2.0-3.5 w / w%. 20. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w%, at least 2.0 w / w %.

[0210] 21 . The method according to any one of the preceding items, wherein the cyclodextrin of step e) introduced up to a total cyclodextrin concentration of at the most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, at most 3.0 w / w %, or at most 3.5 w / w %.

[0211] 22. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %, 1 .1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

[0212] 23. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration in the range of 0.5-2.5 w / w %, 0.5- 2.0 w / w %, 0.5-1 .5 w / w %, 0.5-1 .0 w / w %, 1 .0-2.5 w / w %, 1 .0-2.0 w / w %, 1 .1-2.0 w / w %,

[0213] 1 .2-2.0 w / w %, 1 .3-2.0 w / w %, 1 .4-2.0 w / w %, 1 .5-2.0 w / w %, 1 .6-2.0 w / w %, 1 .7-2.0 w / w %, 1 .8-2.0 w / w %, 1 .9-2.0 w / w %, 1 .0-1 ,5 w / w %, 1 .5-2.5 w / w %, or 1 .5-2.0 w / w %, 1 .3-2.2 w / w %, 1 .4-2.1 w / w %, 1 .6-1 .9 w / w %, 1 .7-1 .8 w / w %, 0.5-3.5 w / w%, 1 .0-3.0 w / w%, 1 .5-3.5 w / w% or 2.0-3.5 w / w%.

[0214] 24. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %.

[0215] 25. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .5 w / w %.

[0216] 26. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.0 w / w %.

[0217] 27. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.5 w / w %.

[0218] 28. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.0 w / w %. 29. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.5 w / w %.

[0219] 30. The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced gradually in response to the relevant process indicator to reach a total cyclodextrin concentration overtime.

[0220] 31 . The method according to any one of the preceding items, wherein the cyclodextrin of step e) is introduced gradually to the bioreactor whenever the threshold value of the relevant process indicator is measured.

[0221] 32. The method according to any one of the preceding items, wherein the relevant process indicator of step e) is measured inside the bioreactor.

[0222] 33. The method according to any one of the preceding items, wherein the relevant process parameter is selected as one from the list consisting of a function time, fermentation stage, optical density (OD), optical density (OD) increase, foam level, bioreactor pressure, oxygen transfer rate, carbon dioxide transfer rate (CTR), and conductance.

[0223] 34. The method according to any one of the preceding items, wherein the relevant process indicator is a function of time.

[0224] 35. The method according to any one of the preceding items, wherein the relevant process indicator is measured in the bioreactor.

[0225] 36. The method according to any one of the preceding items, wherein the relevant process parameter is measured by a sensor.

[0226] 37. The method according to item 36, wherein sensor is located in a headspace of the bioreactor.

[0227] 38. The method according to any one of items 33-37, wherein the relevant process indicator is conductance.

[0228] 39. The method according to item 38, wherein conductance is measured by a sensor in a headspace of the bioreactor. 40. The method according to any of the preceding items, wherein the relevant process indicator is conductance and the threshold value is a conductance value between 30-70, 35-65, 40-60, 45-55, or 48-52 pS.

[0229] 41 . The method according to any of the preceding items, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected from the list consisting of a conductance value of at least 30, at least 35, at least 40, at least 45 or at least 48, at the most 52, at the most 55, at the most 60, at the most 65 and at the most 70 pS.

[0230] 42. The method according to any one of the preceding items, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected as one from the list consisting of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63 , 64, 65, 66, 67, 68, 69 or 70 pS.

[0231] 43. The method according to any one of the preceding items, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected as one from the list consisting of 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60 pS.

[0232] 44. The method according to any one of the preceding items, wherein the relevant process indicator is conductance and the threshold value is a conductance value of 50 pS.

[0233] 45. The method according to any one of the preceding items, wherein improving one or more process parameters is selected as i), ii), iii), iv) and v).

[0234] 46. The method according to any one of the preceding items, wherein improving one or more process parameters is selected as i).

[0235] 47. The method according to any one of the preceding items, wherein improving one or more process parameters is selected as ii).

[0236] 48. The method according to any one of the preceding items, wherein improving one or more process parameters is selected as iii). The method according to any one of the preceding items, wherein improving one or more process parameters is selected as iv). The method according to any one of the preceding items, wherein improving one or more process parameters is selected as v). The method according to any one of the preceding items, 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. The method according to any one of the preceding items, wherein the Bacillus spp. is Bacillus amyloliquefaciens. 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, and B. amyloliquefaciens strain with the DSMZ accession number DSM 27033. 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 DSM 25840, DSM 27032 and DSM 27033. Use of a cyclodextrin as an anti-foaming agent or a defoaming agent in a fermentation process. Use according to item 55, wherein the antifoaming agent is part of an anti-foaming composition and the defoaming agent is part of a defoaming composition. Use according to any one of items 55-56 in a fermentation process comprising lipo peptides. 58. Use according to any one of item 55-57 in a fermentation process comprising bacteria belonging to the genus Bacillus spp.

[0237] 59. The use according to any one of items 58 in a fermentation process 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.the Bacillus amyloliquefaciens.

[0238] 60. The use according to item 59 wherein the Bacillus spp. is Bacillus amyloliquefaciens.

[0239] 61 . The use according to any one of items 58-60 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, and B. amyloliquefaciens strain with the DSMZ accession number DSM 27033.

[0240] 62. The use according to any one of items 55-61 , wherein the cyclodextrin is used as antifoaming agent.

[0241] 63. The use according to any one of items 55-61 , wherein the cyclodextrin is used as defoaming agent.

[0242] 64. The use according to any one of items 55-63, wherein the cyclodextrin is one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta-cycloedextrin, and gamma-cyclodextrin.

[0243] 65. The use according to any one of items 55-64, wherein the cyclodextrin is alpha- cyclodextrin.

[0244] 66. The use according to any one of items 55-64, wherein the cyclodextrin is beta-cyclodextrin.

[0245] 67. The use according to any one of items 55-64, wherein the cyclodextrin is gamma- cyclodextrin.

[0246] 68. An anti-foaming composition comprising cyclodextrin. 69. An anti-foaming composition according to item 68, wherein the cyclodextrin is selected as one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta- cycloedextrin, and gamma-cyclodextrin.

[0247] 70. An anti-foaming composition according to any one of items 68-69, wherein the cyclodextrin is alpha-cyclodextrin.

[0248] 71 . An anti-foaming composition according to any one of items 68-69, wherein the cyclodextrin is beta-cyclodextrin.

[0249] 72. An anti-foaming composition according to any one of items 68-69, wherein the cyclodextrin is gamma-cyclodextrin.

[0250] 73. A defoaming composition comprising cyclodextrin.

[0251] 74. A defoaming composition according to item 73, wherein the cyclodextrin is selected as one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta- cyclodextrin, and gamma-cyclodextrin.

[0252] 75. A defoaming composition according to any one of items 73-74, wherein the cyclodextrin is alpha-cyclodextrin.

[0253] 76. A defoaming composition according to any one of items 73-74, wherein the cyclodextrin is beta-cyclodextrin.

[0254] 77. A defoaming composition according to any one of items 73-74, wherein the cyclodextrin is gamma-cyclodextrin.

[0255] References

[0256] 1 . M. Shoda: Bacterial Control of Plant Disease, Journal of Bioscience and Bioengineering, pp. 515-521 , 200.

[0257] 2. H. P. Bais, R. Fall and .J M. Vivanco: Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production, Plant Physiology, vol. 134, pp. 307-319, 2004.

[0258] 3. T Stein: Bacillus subtilis antibiotics: structures, syntheses and specific functions, Molecular Microbiology, vol. 56, pp. 854-857, 2005.

[0259] 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.

[0260] (Original in Electronic Form)

[0261] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0262] (This sheet is not part of and does not count as a sheet of the international application)

[0263] FOR RECEIVING OFFICE USE ONLY

[0264] FOR INTERNATIONAL BUREAU USE ONLY -5 This form was rec international Bure -5- Authorized officer

Claims

CLAIMS1 . A method for improving one or more process parameters in a fermentation process performed in a bioreactor comprising bacteria belonging to bacillus spp., the method comprising the steps a) providing a suitable bacillus spp., b) providing a bioreactor containing a suitable fermentation medium, c) initiating a fermentation process by introducing the bacillus spp. provided in step a into the bioreactor of step b, d) allowing the fermentation process to proceed, and either in a step a1) that is performed prior to step b) introducing cyclodextrin into a suitable fermentation medium to be contained in the bioreactor in step b), and / or in a step c1) that is performed after step b) but prior to step d) introducing cyclodextrin into the bioreactor, and / or in a step e) introducing cyclodextrin into the bioreactor when a threshold value of a relevant process indicator related to the fermentation process taking place in the bioreactor is measured, thereby improving one or more process parameters in a bioreactor fermentation process comprising bacillus spp.

2. The method of any one of the preceding claims, wherein improving one or more process parameters is selected as one or more ofi. reducing variation of dissolved oxygen (DO) in the fermentation medium of the fermentation process overtime, ii. reducing the amount of foaming caused by fermentation of bacilllus spp. over time, iii. reducing the amount of foaming caused by lipopeptides produced in a fermentation process comprising bacillus spp., iv. reducing the volume of defoamer required to be used over the course of a fermentation process from start to end, and, v. Improving fermentation productivity and cost-efficiency.

3. The method according to any one of the preceding claims, wherein the cyclodextrin is selected as one or more from the list consisting of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

4. The method according to any one of the preceding claims, wherein the cyclodextrin is alpha-cyclodextrin.

5. The method according to any one of the preceding claims, wherein the cyclodextrin is beta- cyclodextrin.

6. The method according to any one of the preceding claims, wherein the cyclodextrin is gamma-cyclodextrin.

7. The method according to any one of the preceding claims, wherein the method comprises step a1) but not step e).

8. The method according to any one of the preceding claims, wherein the method comprises step e) but not step a1).

9. The method according to any one of the preceding claims, wherein the method comprises step a1) and step e).

10. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w% or at least 2.0 w / w %.11 . The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of at the most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, at most 3.0 w / w%, or at most 3.5 w / w%.

12. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .0 w / w %, 1.1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

13. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .0 w / w %.

14. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 1 .5 w / w %.

15. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 2.0 w / w %.

16. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 2.5 w / w %.

17. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 3.0 w / w %.

18. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration of 3.5 w / w %.

19. The method according to any one of the preceding claims, wherein the cyclodextrin of step a1) is introduced in a cyclodextrin concentration in a range of 0.5-2.5 w / w %, 0.5-2.0 w / w %, 0.5-1 .5 w / w %, 0.5-1 .0 w / w %, 1 .0-2.5 w / w %, 1 .0-2.0 w / w %, 1 .1-2.0 w / w %, 1 .2-2.0 w / w %, 1 .3-2.0 w / w %, 1 .4-2.0 w / w %, 1 .5-2.0 w / w %, 1 .6-2.0 w / w %, 1 .7-2.0 w / w %, 1 .8- 2.0 w / w %, 1 .9-2.0 w / w %, 1 .0-1 ,5 w / w %, 1 .5-2.5 w / w %, or 1 .5-2.0 w / w %, 1 .3-2.2 w / w %,1 .4-2.1 w / w %, 1 .6-1 .9 w / w %, 1 .7-1 .8 w / w %, 0.5-3.5 w / w%, 1 .0-3.0 w / w%, 1 .5-3.5 w / w% or 2.0-3.5 w / w%.

20. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of at least 0.5 w / w %, at least 1 .0 w / w %, at least 1 .5 w / w%, at least 2.0 w / w %.

21. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) introduced up to a total cyclodextrin concentration of at the most 1 .5 w / w %, at most 2.0 w / w %, at most 2.5 w / w %, at most 3.0 w / w %, or at most 3.5 w / w %.

22. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %, 1 .1 w / w %, 1 .2 w / w %, 1 .3 w / w %, 1 .4 w / w %, 1 .5 w / w %, 1 .6 w / w %, 1 .7 w / w %, 1 .8 w / w %, 1 .9 w / w % or 2.0 w / w %, 2.1 w / w % or 2.2 w / w %.

23. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration in the range of 0.5-2.5 w / w %, 0.5- 2.0 w / w %, 0.5-1 .5 w / w %, 0.5-1 .0 w / w %, 1 .0-2.5 w / w %, 1 .0-2.0 w / w %, 1 .1-2.0 w / w %,1 .2-2.0 w / w %, 1 .3-2.0 w / w %, 1 .4-2.0 w / w %, 1 .5-2.0 w / w %, 1 .6-2.0 w / w %, 1 .7-2.0 w / w %, 1 .8-2.0 w / w %, 1 .9-2.0 w / w %, 1 .0-1 ,5 w / w %, 1 .5-2.5 w / w %, or 1 .5-2.0 w / w %, 1 .3-2.2 w / w %, 1 .4-2.1 w / w %, 1 .6-1 .9 w / w %, 1 .7-1 .8 w / w %, 0.5-3.5 w / w%, 1 .0-3.0 w / w%, 1 .5-3.5 w / w% or 2.0-3.5 w / w%.

24. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .0 w / w %.

25. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 1 .5 w / w %.

26. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.0 w / w %.

27. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 2.5 w / w %.

28. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.0 w / w %.

29. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced up to a total cyclodextrin concentration of 3.5 w / w %.

30. The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced gradually in response to the relevant process indicator to reach a total cyclodextrin concentration overtime.31 . The method according to any one of the preceding claims, wherein the cyclodextrin of step e) is introduced gradually to the bioreactor whenever the threshold value of the relevant process indicator is measured.

32. The method according to any one of the preceding claims, wherein the relevant process indicator of step e) is measured inside the bioreactor.

33. The method according to any one of the preceding claims, wherein the relevant process parameter is selected as one from the list consisting of a function time, fermentation stage, optical density (OD), optical density (OD) increase, foam level, bioreactor pressure, oxygen transfer rate, carbon dioxide transfer rate (CTR), and conductance.

34. The method according to any one of the preceding claims, wherein the relevant process indicator is a function of time.

35. The method according to any one of the preceding claims, wherein the relevant process indicator is measured in the bioreactor.

36. The method according to any one of the preceding claims, wherein the relevant process parameter is measured by a sensor.

37. The method according to claim 36, wherein sensor is located in a headspace of the bioreactor.

38. The method according to any one of claims 33-37, wherein the relevant process indicator is conductance.

39. The method according to claim 38, wherein conductance is measured by a sensor in a headspace of the bioreactor.

40. The method according to any of the preceding claims, wherein the relevant process indicator is conductance and the threshold value is a conductance value between 30-70, 35-65, 40-60, 45-55, or 48-52 pS.41 . The method according to any of the preceding claims, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected from the list consisting of a conductance value of at least 30, at least 35, at least 40, at least 45 or at least 48, at the most 52, at the most 55, at the most 60, at the most 65 and at the most 70 pS.

42. The method according to any one of the preceding claims, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected as one from the list consisting of 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63 , 64, 65, 66, 67, 68, 69 or 70 pS.

43. The method according to any one of the preceding claims, wherein the relevant process indicator is conductance and the threshold value is a conductance value selected as one from the list consisting of 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 and 60 pS.

44. The method according to any one of the preceding claims, wherein the relevant process indicator is conductance and the threshold value is a conductance value of 50 pS.

45. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as i), ii), iii), iv) and v).

46. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as i).

47. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as ii).

48. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as iii).

49. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as iv).

50. The method according to any one of the preceding claims, wherein improving one or more process parameters is selected as v).51 . 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.

52. The method according to any one of the preceding claims, wherein the Bacillus spp. is Bacillus amyloliquefaciens.

53. 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, and B. amyloliquefaciens strain with the DSMZ accession number DSM 27033.

54. 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 DSM 25840, DSM 27032 and DSM 27033.

55. Use of a cyclodextrin as an anti-foaming agent or a defoaming agent in a fermentation process.

56. Use according to claim 55, wherein the antifoaming agent is part of an anti-foaming composition and the defoaming agent is part of a defoaming composition.

57. Use according to any one of claims 55-56 in a fermentation process comprising lipo peptides.

58. Use according to any one of claim 55-57 in a fermentation process comprising bacteria belonging to the genus Bacillus spp.

59. The use according to any one of claims 58 in a fermentation process 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.the Bacillus amyloliquefaciens.

60. The use according to claim 59 wherein the Bacillus spp. is Bacillus amyloliquefaciens.61 . The use according to any one of claims 58-60 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 and B. amyloliquefaciens strain with the DSMZ accession number DSM 27033.

62. The use according to any one of claims 55-61 , wherein the cyclodextrin is used as antifoaming agent.

63. The use according to any one of claims 55-61 , wherein the cyclodextrin is used as defoaming agent.

64. The use according to any one of claims 55-63, wherein the cyclodextrin is one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin.

65. The use according to any one of claims 55-64, wherein the cyclodextrin is alpha- cyclodextrin.

66. The use according to any one of claims 55-64, wherein the cyclodextrin is beta- cyclodextrin.

67. The use according to any one of claims 55-64, wherein the cyclodextrin is gammacyclodextrin.

68. An anti-foaming composition comprising cyclodextrin.

69. An anti-foaming composition according to claim 68, wherein the cyclodextrin is selected as one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta- cycloedextrin, and gamma-cyclodextrin.

70. An anti-foaming composition according to any one of claims 68-69, wherein the cyclodextrin is alpha-cyclodextrin.71 . An anti-foaming composition according to any one of claims 68-69, wherein the cyclodextrin is beta-cyclodextrin.

72. An anti-foaming composition according to any one of claims 68-69, wherein the cyclodextrin is gamma-cyclodextrin.

73. A defoaming composition comprising cyclodextrin.

74. A defoaming composition according to claim 73, wherein the cyclodextrin is selected as one or more cyclodextrins selected from the list consisting of alpha-cyclodextrin, beta- cycloedextrin, and gamma-cyclodextrin.

75. A defoaming composition according to any one of claims 73-74, wherein the cyclodextrin is alpha-cyclodextrin.

76. A defoaming composition according to any one of claims 73-74, wherein the cyclodextrin is beta-cyclodextrin.

77. A defoaming composition according to any one of claims 73-74, wherein the cyclodextrin is gamma-cyclodextrin.

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