Microorganism culture, fermentation methods, method for reducing the necessary fermentation time, use of a culture, set of culture parameters, and method for producing one or more substances.

Description

1 / 54 “MICROORGANISM CULTURE, FERMENTATION METHODS, METHOD FOR REDUCING THE NECESSARY FERMENTATION TIME, USE OF A CULTURE, SET OF CULTURE PARAMETERS AND METHOD FOR PRODUCING ONE OR MORE SUBSTANCES”

[001] The invention relates to microorganisms, genes, materials and methods for improving microbial activity. In particular, the invention provides methods for preparing microorganisms to improve alignment and / or metabolic activity, metabolically activated and / or aligned microorganisms, and methods for using them, particularly in industrial fermentation processes. Background of the Invention

[002] The objective of industrial fermentation processes is typically to produce, in the shortest possible time, a desired product using the lowest cost resources. In these processes, a culture of microorganisms or cell-free extract converts one, or typically more, metabolically accessible nutrients into a desired product. Typical industrial fermentation processes are submerged fermentations. In these processes, the culture of microorganisms is suspended in a fermentation medium that normally provides all the nutrients necessary for the production of the desired product; gaseous nutrients are usually supplied by means of bubble columns or other forms of aeration.

[003] Fermentation processes can be conducted as discontinuous fermentations (also called batch fermentation) or continuous fermentations. In a discontinuous (batch) fermentation, a fermentation medium is inoculated with a seed culture of production microorganisms. For the actual fermentation, the inoculated medium is then treated in a fermenter under conditions conducive to the production of the desired product. At the end of fermentation, the fermentation medium Petition 870250087381, dated 09 / 26 / 2025, page 49 / 113 2 / 54 containing the desired product is collected. Typically, the fermenter is then cleaned and prepared for another fermentation batch. A fed-batch fermentation (also referred to as fed-batch fermentation) differs from a continuous fermentation by the addition of other substances (feed) during fermentation. The feed may or may not have the same composition as the fermentation medium used at the beginning of fermentation. In a continuous fermentation, all or a sufficient portion of the microorganism culture is retained in the fermenter, where the fermenter is continuously or intermittently fed with new feed, and portions of the fermenter contents containing the product are collected (partial collection or repeated batch process).

[004] The growth of microorganisms during fermentation typically goes through several stages. During the lag phase (see Monod, Ann. Rev. Microbiol 1949, 371-394), after inoculation, the doubling time of a microorganism culture is high. It is believed that microorganisms need time to germinate and can adapt their metabolism to changes in the composition of the medium. The lag phase is followed by an exponential growth phase (“logarithmic phase”), in which cell division occurs at a roughly constant and high rate. For example, doubling times of only 20 minutes have been observed for Salmonella enterica during the logarithmic phase. During exponential growth, the rate of microbial nutrient consumption is higher. It is believed that not all nutrients are consumed all the time at the same rate.Instead, it is believed that some complex nutrients require the synthesis of specialized enzymes for consumption (“diauxic growth,” see Salvy et al., Proc. Natl. Acad. Sci. USA 2021, doi:10.1073 / pnas.2013836118). This can result in one or more periods of temporarily reduced growth rate, during which the necessary changes occur. Petition 870250087381, dated 09 / 26 / 2025, p. 50 / 113 3 / 54 enzyme concentrations. With even less easily consumable nutrients remaining, growth enters a stationary phase, in which the doubling time increases significantly again. It is believed that microorganisms in the stationary phase enter a dormant stage with severely reduced metabolic activity and / or that microorganisms in the stationary phase subsist on the remains of dead cells, so that the cell doubling rate more or less equals the mortality rate. Once the stationary phase can no longer be maintained, the microorganism culture enters a decline stage, in which the mortality rate is greater than the doubling rate.

[005] In industrial fermentations, it is desirable for microorganisms to enter the exponential phase as early as possible and for as long as possible to maximize product formation. Bertrand et al. (J. Bacteriol. 2019, https: / / doi.org / 10.1128 / JB.00697-18) reviewed the factors influencing the duration of the lag phase. They found that the duration of the lag phase is influenced not only by the history of the culture used for inoculation, but also by the number of bacteria in a culture. As the number of cells increases, the duration of the lag phase decreases. Caipo et al. (J. App. Microbiology 2002, 879-884) tested the effect of varying inoculum sizes (10E3 to 10E8 spores / mL) from a Bacillus megaterium spore bank by microscopic assessments. Their results indicate that higher concentrations of Bacillus megaterium spores stimulate faster germination and result in more spores germinating.According to the authors, this is consistent with the theory of Woese et al. (Proc. Natl. Acad Sci USA 1968, 869875), according to which Bacillus spores can contain a variable number of "germination molecules," so that at higher densities of Bacillus spores, a greater percentage of rapidly germinating spores will be present. Thus, a larger inoculum size is positively implied. Petition 870250087381, dated 09 / 26 / 2025, page 51 / 113 4 / 54 correlated with faster germination. As the completion of germination is a precondition for the entry into exponential growth for spore-forming microorganisms, a larger inoculum size is considered beneficial for industrial fermentations. Similarly, Webb et al. (Food Microbiol. 2012, 104-109) observe that the concentration of spores in the inoculation significantly affected the germination time of Clostridium botulinum; reducing the size of the C. botulinum spore inoculum increased the time for toxin formation in food products. The authors propose that molecules from germinated spores could trigger the germination of dormant neighbors.

[006] Inoculation of the fermenter is typically performed by preparing a starter or seed culture of the production microorganism and adding a defined quantity of this to the fermentation medium in the fermenter or pre-culture vessel. The seed culture is prepared by inoculating a volume of a cell bank flask or colonies from an agar plate culture into a propagation medium (seed medium) that may or may not have the same composition as the fermentation medium, and then incubating the inoculated propagation medium under conditions suitable for the growth of the microorganism culture. Typically, the fermentation medium is inoculated with a seed culture volume of 2 to 10% v / v of the fermenter volume (see documents WO2006096164 and WO2017068012).

[007] Attempts have been made to eliminate the seed culture preparation step. For example, document WO2006096164 describes a Clostridium botulinum fermentation for the production of botulinum toxin, in which the seed culture preparation step is omitted, so that the fermenter is inoculated directly with the contents of the cell bank flask. No positive effect was attributed to this inoculation scheme, and the Petition 870250087381, dated 09 / 26 / 2025, page 52 / 113 5 / 54 authors recommend that approximately 2-4% of a culture medium containing C. botulinum from a seed culture growth phase be used to inoculate the fermentation medium. Similarly, document WO2017068012 describes a fermentation of Trichoderma reesei for the production of beta-xylosidase, in which the fermenter is inoculated directly from the contents of a cell bank flask or from the surface of a PDA agar plate. The authors speculate that such an inoculation scheme could be used not only for Ascomycota but also for bacteria. However, there is no data shown to support this speculation and, in preferred embodiments, the microorganism is a filamentous fungus.

[008] The quality of an industrial fermentation process depends not only on the duration of the lag phase, but also on the yield of the desired product and the degree of formation of undesirable by-products, such as mucus or viscous biofilm (slime) or foam. Furthermore, the reproducibility of fermentation results is essential to avoid the occurrence of surprisingly long lag phases, surprisingly short logarithmic phases, or unforeseen nutrient demands.

[009] Therefore, the objective of the present invention was to provide improved materials and methods for industrial fermentations, in particular with regard to microorganisms of the Paenibacillaceae family. Brief Description of the Invention

[010] The invention provides a culture of microorganisms, wherein the microorganism is of the family Paenibacillaceae, preferably of the genus Paenibacillus, in which the cells in the culture are: - metabolically aligned and / or - in an activated metabolic state.

[011] The invention also provides a fermentation method, comprising: Petition 870250087381, dated 09 / 26 / 2025, page 53 / 113 6 / 54 i) to provide a fermentation broth comprising a culture of microorganisms according to the invention in a fermentation medium, and ii) to cultivate the fermentation broth under suitable conditions to produce a target substance, and iii) preferably, to recover the target substance.

[012] In addition, the invention provides a method for reducing the fermentation time required to produce a desired quantity of a target substance and / or for reducing viscosity during fermentation and / or for increasing the accuracy of repeatability (reproducibility) of the fermentation, comprising the steps of: i) to provide a culture according to the invention in a fermentation medium, and ii) to cultivate the culture in the fermentation medium.

[013] The invention provides a use of a culture, according to the invention, to reduce the fermentation time required to produce a desired quantity of a target substance, and / or to reduce viscosity during fermentation, and / or to increase the accuracy of repeatability (reproducibility) of the fermentation.

[014] The invention also provides a set of cultivation parameter data for controlling a fermentation, comprising: - a designation for a set of applicable fermentation methods, - a designation for a set of applicable samples of microorganisms, - a designation of fermentation conditions conducive to obtaining a culture according to the invention by cultivating an applicable sample in an applicable fermentation medium, wherein the conditions Petition 870250087381, dated 09 / 26 / 2025, p. 54 / 113 7 / 54 of fermentation preferably includes at least one of the following: Type of culture medium, quantity of culture medium, desired culture temperature range, desired culture aeration parameter ranges, desired quantity of cells produced, desired culture duration, desired pH during culture, yield or desired quantity of a target substance.

[015] And the invention provides a computer-implemented fermentation method comprising: - to receive, via a computing device, a set of cultivation parameter data according to the invention, - to receive a fermentation medium that conforms to the set of cultivation parameters, - receive a sample volume of microorganisms in accordance with the set of culture parameters, and - To control, via the computing device, a fermenter to cultivate microorganisms and / or their spores in the fermentation medium under the fermentation conditions of the culture parameter dataset.

[016] In addition, the invention provides a method for producing one or more substances that increase viscosity, comprising: i) inoculate a fermentation medium with a concentration of a microorganism culture according to the invention at a predetermined concentration sufficient to obtain, during cultivation, a desired viscosity, and ii) cultivate the culture obtained in step i), and iii) preferably recover one or more viscosity-increasing substances or viscosifiers. Brief Description of the Figures

[017] Fig. 1: shows the impact of inoculation volume on the growth of Paenibacillus polymyxa LU21132 in shake flasks. A Petition 870250087381, dated 09 / 26 / 2025, page 55 / 113 8 / 54 Biomass formation is shown as ODgqq [UA] after 24 hours of culture in shake flasks containing PX-125 complex medium. A variety of different volumes of Working Cell Bank 1041 (WCB1Q41) were used for inoculation (Q,1 - 4% v / v of the total culture volume, as listed in Table 2) of the culture medium. With increasing amounts of cells used for inoculation (i.e., increasing volume of WCB spore composition), the optical density at 24 hours post-inoculation decreases, indicating slower growth in densely inoculated culture broths. The fastest growth is obtained at inoculation concentrations of Q,2 - Q,4% (v / v); at lower concentrations, the growth rate is again reduced. [Q18] Fig. 2: shows the impact of inoculation volume on the growth of P. polymyxa LU2Q754 in 2 mL deep-well plates. The culture was performed in a BioLector device (m2p labs, Germany), using a 48-position deep-well plate and shaking at 9QQ rpm. Biomass formation was periodically assessed by light scattering at 62Q nm ex / em. Different volumes of a working cell bank of spores of the LU2Q754 strain were used for inoculation (Q,6 - 1.8% v / v of the total culture volume). The higher the inoculum concentration, the greater the delay until biomass reached 2Q UA. [Q19] Fig. 3A: shows microscopic images (left) and particle outlines (right) deduced from P. polymyxa LU21552 cultures in shake flasks. Images were taken after 28 hours of culture. The shake flasks were inoculated with Q,2% v / v volume of inoculum using the WCB1Q59 spore (prepared as described in Table 8). [Q2Q] Fig. 3B: shows microscopic images (left) and particle outlines (right) deduced from cultures of P. polymyxa LU21552 in shake flasks. The images were taken after 28 hours of Petition 870250087381, dated 09 / 26 / 2025, page 56 / 113 9 / 54 culture. The shaken flasks were inoculated with 1% v / v volume of inoculum using the WCB1059 spore (prepared as described in Table 8).

[021] Fig. 4: shows the corpuscular lengths of the cells determined from the particle outlines in figures 3A and 3B.

[022] Fig. 5: shows the impact of inoculation volume in shake flasks on fusaricidin production from P. polymyxa LU21132. The fusaricidin titer is shown after 24 hours of culture in shake flasks containing PX-125 complex medium and using different inoculation volumes from the working cell bank (WCB) (0.1 to 4% v / v of the total culture volume, Table 2) of the WCB1041 working cell bank. WCB1041 consists of 1,6E+08 (endospores) / mL [cv% 9.5]. Culture conditions: 250 mL shake flasks with baffles, 30 mL of medium, 150 rpm, orbital shaker with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h. At inoculation volumes greater than 0.4% (v / v), the fusaricidin yield drops drastically by a factor of approximately 50 times compared to the fusaricidin yield obtained at an inoculation volume of 0.2% (v / v).

[023] Fig. 6: shows the impact of inoculation volume in shake flasks on the growth of P. polymyxa LU21132 using working cell banks (WCBs) of six different preparation procedures, as described in Table 8. Biomass formation is shown as ODgqq [UA] after 24 hours of culture in shake flasks containing PX125 complex medium. Two different WCB spore inoculation volumes (0.3 and 0.6% v / v of the total culture medium volume) were used to test the growth properties of each of the six working cell banks. Culture conditions: 250 mL shake flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h. Regardless of the working cell bank preparation, the growth rate indicated by the optical density at 600 nm 24 hours after the Petition 870250087381, dated 09 / 26 / 2025, page 57 / 113 10 / 54 inoculation at 0.3% (v / v) is approximately twice that of corresponding crops inoculated at 0.6% (v / v).

[024] Fig. 7: shows the impact of the inoculation volume in shake flasks on fusaridin production from P. polymyxa LU21132 using six different working cell banks (WCBs) prepared under varying conditions (described in Table 8). The fusaridin titer is shown after 24 hours of culture in shake flasks containing PX-125 complex medium and using two WCB inoculation volumes (0.3 and 0.6% v / v of the total culture volume). Culture conditions: 250 mL shake flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h. Regardless of the preparation of the working cell bank, fusaricidin levels for cultures inoculated with 0.3% (v / v) are approximately 3 to 25 times higher than for cultures inoculated with 0.6% (v / v) of the WCB spore composition.

[025] Fig. 8: shows that the initial inoculation volume in a pre-culture shake flask affects fusaricidin production in the main culture in a two-stage process: fusaricidin A, B, and D levels in main cultures in shake flasks with PX-143 medium as a function of pre-culture inoculation volume. Pre-culture flasks were inoculated with 0.3 and 0.6% (v / v) of different types of WCB (see Table 8). Cultures from Example 3 were transferred after 24 hours of culture to main culture flasks using a 2% (v / v) inoculation volume. The fusaricidin level of the main cultures that were inoculated with the pre-culture flasks with a 0.6% (v / v) pre-culture inoculation volume is shown relative to the fusaricidin level detected in the parallel main cultures of the 0.3% pre-culture inoculation variant.Culture conditions: 250 mL shaker flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h pre-culture, 48 h main culture. The content of... Petition 870250087381, dated 09 / 26 / 2025, page 58 / 113 11 / 54 fusaricidin accumulated in 48 h of fermentation is approximately 10% lower in fermentations inoculated with 2% (v / v) of a preculture inoculated with 0.6% (v / v) WCB spores compared with corresponding fermentations inoculated with 2% (v / v) of a preculture inoculated with 0.3% (v / v) WCB spores (whose absolute fusaricidin content was approximately the same, regardless of the WCB preparation and, in each case, greater than 2,000 mg / L).

[026] Fig. 9: shows that a smaller inoculum volume in the preculture synchronizes growth in the main culture (two-stage process). The effect is demonstrated for six different working cell banks (WCBs) of P. polymyxa LU21132 (prepared according to Table 8). Preculture flasks (PX-125 medium) were inoculated with 0.3 and 0.6% (v / v) of different types of WCBs of P. polymyxa LU21132 from Table 8. The precultures were transferred after 24 hours of cultivation to main culture flasks (PX-143 medium) using 2% (v / v) of the inoculation volume. The OD600 (UA) of each culture of the six WCBs from Table 8 was measured after 48 hours of cultivation in the main culture medium. Culture conditions: 250 mL shaker flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h pre-culture, 48 h main culture.

[027] Fig. 10: shows that the inoculation volume of the main crop determines the viscosity of the culture broth in soybean-based medium (EPS-03) in shake flask cultures (250 mL shake flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5). Viscosity was evaluated after 16 hours of culture. As indicated by OD600, EPS formation is partially dissociated from growth, with particular emphasis on inoculation volumes of 2, 4, and 6%. Detailed Description of the Invention

[028] The technical teaching of the invention is expressed in this Petition 870250087381, dated 09 / 26 / 2025, page 59 / 113 12 / 54 document by linguistic means, in particular by means of scientific and technical terms. However, a person skilled in the art understands that, however detailed and precise the linguistic means may be, they can only approximate the complete content of the technical teaching, because there are multiple ways of expressing a teaching, and each of them inevitably fails to fully capture all the nuances and conceptual interconnections, since each expression must necessarily reach its limit. With this in mind, a person skilled in the art understands that the subject matter of the invention consists of the sum of the individual technical concepts described or expressed in this document, and, inevitably, these concepts are presented in a pars-pro-toto manner, due to the inherent limitations of a written description.In particular, those skilled in the art will understand that the meaning of individual technical concepts is given in this document as an abbreviation for each possible combination of concepts, as far as is technically sensible, so that, for example, the disclosure of three concepts or embodiments A, B, and C is an abbreviated notation of the concepts A+B, A+C, B+C, A+B+C. Specifically, alternative positions for features are described in this document in terms of lists of convergent alternatives or specific examples. Unless otherwise indicated, the invention described herein comprises any combination of such alternatives. The choice of more or less preferred elements from these lists is part of the invention and is due to the preference of those skilled in the art for a minimum degree of realization of the advantage or advantages afforded by the respective features. Such multiple combinations represent suitably preferred embodiments of the invention.

[029] Insofar as references are made to database entries, for example, Uniprot entries, the entries are those published on 10 / 01 / 22 at 10:00 CET. This also applies to sequences published under the corresponding database entry identifiers. Petition 870250087381, dated 09 / 26 / 2025, pp. 60 / 113 13 / 54

[030] As used in this invention, singular terms and singular forms such as “a” and “the” include plural referents unless the content clearly indicates otherwise. Thus, for example, the use of the term “a nucleic acid” optionally includes, in practice, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules. Furthermore, as used in this application, the word “comprising,” or variations such as “comprises” or “containing,” will be understood as implying the inclusion of a stated element, whole or step, or group of elements, whole or step, but not the exclusion of any other element, whole or step, or group of elements, whole or step.

[031] As used in the present invention, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the aforementioned associated items, as well as the lack of combinations when interpreted as alternative (“or”). The term “comprising” also encompasses the term “consisting of”.

[032] The term “about”, when used in reference to a measurable value, for example, a quantity of mass, dose, time, temperature, sequence identity and the like, refers to a variation of ± 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% of the specified value, as well as the specified value. Thus, if a given composition is described as comprising “about 50% of X,” it should be understood that, in some embodiments, the composition comprises 50% of X, while in other embodiments it may comprise from 40% to 60% of X (i.e., 50% ± 10%).

[033] The term “microorganism”, according to the invention, designates both individual cells of microorganisms and pluralities thereof. Petition 870250087381, dated 09 / 26 / 2025, pp. 61 / 113 14 / 54 cells, the latter also being called a “population” or, when combined with a medium, a “culture.” Preferably, the microorganism is of a single taxonomic species and, even more preferably, a single strain of a species. Thus, microbiologically pure cultures are preferred according to the present invention. Microorganism populations may also be consortia of microorganism cells from different strains of a common species and / or may be consortia comprising different taxonomic species or genera. Such mixed populations and cultures are, however, less preferred. The microorganisms may, in principle, be any cell type of the taxonomic family Paenibacillaceae.The microorganism may be a wild-type organism, i.e., a microorganism essentially unchanged from the organism originally isolated from nature, or it may be a modified microorganism, which has been modified by one or more technical procedures, such as mutagenesis. Samples of wild-type microorganisms are preferably registered as type strains in culture collections. Genetic modification may be effected by random mutagenesis, for example, NTG chemical mutagenesis, UV irradiation or transposon mutagenesis, or by targeted mutagenesis, for example, incorporation of heterologous plasmids or homologous recombination with heterologous nucleic acids and / or by site-directed mutagenesis, for example, using meganucleases, TALEN-type mutagenesis or CRISPR. For example, preferred methods of mutagenesis of Bacillus and Paenibacillus are described in document WO2017117395, which is incorporated in its entirety into this application.

[034] The microorganism used in the invention belongs to the taxonomic family Paenibacillaceae. Microorganisms of the taxonomic family Paenibacillaceae are defined by their similarity to 16S rDNA (De Vos, P., W. Ludwig, KH Scheifer and WB Whitman 2009. Family IV. “Paenibacillaceae”. Bergey's Manual of Systematic Bacteriology. P. De Vos, G. Petition 870250087381, dated 09 / 26 / 2025, p. 62 / 113 15 / 54 M. Garrity, D. Jones et al. New York, Springer. 3: 269). The genome size is typically 3 to 9 Mb. In general, these microorganisms are straight or curved rods, small to medium in size, about 0.5 to 1.0 x 2 to 6 μm. They typically exhibit a Gram-positive cell wall structure and are also capable of producing spores. Members of this family have been isolated from various habitats, from Alaska to Antarctica, including deep marine sediments, ice cores, desert brines, sandstone biofilms, grassland soils, forest soils, and roots of agronomic plants. Due to the diversity of this family, taxonomic rearrangements are expected to occur in the future. The preferred genera and species according to the invention are described below in more detail.

[035] Preferably, the microorganism is from any of the genera Ammoniibacillus, Ammoniphilus, Aneurinibacillus, Oxalophagus, Brevibacillus, Candidatus, Reconcilbacillus, Chengkuizengella, Cohnella, Fontibacillus, Gorillibacterium, Longirhabdus, Marinicrinis, Paenibacillus, Paludirhabdus, Saccharibacillus, Thermobacillus or Xylanibacillus, most preferably of the genus Paenibacillus.

[036] Microorganisms of one of the following species are particularly preferred: Paenibacillus species: P. abekawaensis, P. abyssi, P. aceris, P. aceti, P. aestuarii, P. agarexedens, P. agaridevorans, P. alba, P. albidus, P. albus, P. alginolyticus, P. algorifonticola, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. antibioticophila, P. antri, P. apiaries, P. apiarius, P. apis, P. aquistagni, P. arachidis, P. arcticus, P. assamensis, P. aurantiacus, P. azoreducens, P. azotifigens, P. baekrokdamisoli, P. barcinonensis, P. barengoltzii, P. beijingensis, P. borealis, P. bouchesdurhonensis, P. bovis, P. brasilensis, P. brassicae, P. bryophyllum, P. caespitis, P. camelliae, P. camerounensis, P. campinasensis, P. castaneae, P. Petition 870250087381, dated 26 / 09 / 2025, p. 63 / 113 16 / 54 catalpae, P. cathormii, P. cavernae, P. cellulosilyticus, P. cellulositrophicus, P. chartarius, P. chibensis, P. chinensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. chungangensis, P. cineris, P. cisolokensis, P. contaminans, P. cookii, P. crassostreae, P. cucumis, P. curdlanolyticus, P. daejeonensis, P. dakarensis, P. darangshiensis, P. darwinianus, P. dauci, P. dendritiformis, P. dongdonensis, P. donghaensis, P. doosanensis, P. durus, P. edaphicus, P. ehimensis, P. elgii, P. elymi, P. endophyticus, P. enshidis, P. esterisolvens, P. etheri, P. eucommiae, P. faecis, P. favisporus, P. ferrarius, P. filicis, P. flagellatus, P. fonticola, P. forsythiae, P. frigoriresistens, P. fujiensis, P. fukuinensis, P. gansuensis, P. gelatinolyticus, P. ginsengagri, P. ginsengarvi, P. ginsengihumi, P. ginsengiterrae, P. glacialis, P. glebae, P. glucanolyticus, P. glycanilyticus, P. gorillae, P. graminis, P. granivorans, P. guangzhouensis, P. harenae, P. helianthi, P.hemerocallicola, P. herberti, P. hispanicus, P. hodogayensis, P. hordei, P. horti, P. humicus, P. hunanensis, P. ihbetae, P. ihuae, P. ihumii, P. illinoisensis, P. insulae, P. intestini, P. jamilae, P. jilunlii, P. kobensis, P. koleovorans, P. konkukensis, P. konsidensis, P. koreensis, P. kribbensis, P. kyungheensis, P. lactis, P. lacus, P. larvae, P. lautus, P. lemnae, P. lentimorbus, P. lentus, P. liaoningensis, P. limicola, P. lupini, P. luteus, P. lutimineralis, P. macerans, P. macquariensis, P. marchantiophytorum, P. marinisediminis, P. marinum, P. massiliensis, P. maysiensis, P. medicaginis, P. mendelii, P. mesophilus, P. methanolicus, P. mobilis, P. montanisoli, P. montaniterrae, P. motobuensis, P. mucilaginosus, P. nanensis, P. naphthalenovorans, P. nasutitermitis, P. nebraskensis, P. nematophilus, P. nicotianae, P. nuruki, P. oceanisediminis, P. odorifer, P. oenotherae, P. oralis, P. oryzae, P. oryzisoli, P. ottowii, P. ourofinensis, P. pabuli, P. paeoniae, P. panacihumi, P.panacisol, P. panaciterrae, P. paridis, P. pasadenensis, P. pectinolyticus, P. peoriae, P. periandrae, P. phocaensis, P. phoenicis, P. phyllosphaerae, P. physcomitrellae, P. pini, P. pinihumi, P. pinisoli, P. pinistramenti, P. pocheonensis, P. polymyxa, Petition 870250087381, dated 26 / 09 / 2025, p. 64 / 113 17 / 54 P. polysaccharolyticus, P. popilliae, P. populi, P. profundus, P. prosopidis, P. protaetiae, P. provencensis, P. psychroresistens, P. pueri, P. puernese, P. puldeungensis, P. purispatii, P. qingshengii, P. qinlingensis, P. quercus, P. radicis, P. relictisesami, P. residui, P. rhizoplanae, P. rhizoryzae, P. rhizosphaerae, P. rigui, P. ripae, P. rubinfantis, P. ruminocola, P. sabinae, P. sacheonensis, P. salinicaeni, P. sanguinis, P. sediminis, P. segetis, P. selenii, P. selenitireducens, P. senegalensis, P. senegalimassiliensis, P. seodonensis, P. septentrionalis, P. sepulcri, P. shenyangensis, P. shirakamiensis, P. shunpengii, P. siamensis, P. silagei, P. silvae, P. sinopodophylli, P. solanacearum, P. solani, P. soli, P. sonchi group, P. sophorae, P. spiritus, P. sputi, P. stellifer, P. susongensis, P. swuensis, P. taichungensis, P. taihuensis, P. taiwanensis, P. taohuashanense, P. tarimensis, P. telluris, P. tepidiphilus, P. terrae, P. terreus, P. terrigena, P. tezpurensis, P.thailandensis, P. thermoaerophilus, P. thermophilus, P. thiaminolyticus, P. tianmuensis, P. tibetensis, P. timonensis, P. translucens, P. tritici, P. triticisoli, P. tuaregi, P. tumbae, P. tundrae, P. turicensis, P. tylopili, P. typhae, P. tyrfis, P. uliginis, P. urinalis, P. validus, P. velaei, P. vini, P. vortex, P. vorticalis, P. vulneris, P. wenxiniae, P. whitsoniae, P. wooponensis, P. woosongensis, P. wulumuqiensis, P. wynnii, P. xanthanilyticus, P. xanthinilyticus, P. xerothermodurans, P. xinjiangensis, P. xylanexedens, P. xylaniclasticus, P. xylanilyticus, P. xylanisolvens, P. yanchengensis, P. yonginensis, P. yunnanensis, P. zanthoxyli, P. zeae, preferentially P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. barcinonensis, P. borealis, P. brasiliensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P.curdlanolyticus, P. daejeonensis, P. dendritiformis, P. ehimensis, P. elgii, P. favisporus, P. glucanolyticus, P. glycanilyticus, P. graminis, P. granivorans, P. hodogayensis, P. illinoisensis, P. jamilae, P. Petition 870250087381, dated 26 / 09 / 2025, p. 65 / 113 18 / 54 kobensis, P. koleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. macerans, P. macquariensis, P. massiliensis, P. mendelii, P. motobuensis, P. naphthalenovorans, P. nematophilus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. popilliae, P. rhizosphaerae, P. sanguinis, P. stellifer, P. taichungensis, P. terrae, P. thiaminolyticus, P. timonensis, P. tylopili, P. turicensis, P. validus, P. vortex, P. vulneris, P. wynnii, P. xylanilyticus, being particularly preferred the species Paenibacillus peoriae, Paenibacillus jamilae, Paenibacillus brasiliensis, Paenibacillus kribbensis, Paenibacillus koreensis, Paenibacillus rhizosphaerae, Paenibacillus polymyxa, Paenibacillus amylolyticus, Paenibacillus terrae, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov.spec epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei, more preferably Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nova spesie (nov. spec.) epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei, even more preferably Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae.

[037] Some suitable strains of Paenibacillus are described and information on the filing of these strains is provided in the following international patent application; spores of such microorganisms or pesticidally active variants of any of them may be incorporated as spores into the composition according to the invention: document WO 2019155253: DSM32460 (Paenibacillus polymyxa strain VMC10 / 96), NCBI accession number: NR_117743 (Paenibacillus peoriae DSM320), NCBI accession number: HG324076 (Paenibacillus polymyxa DSM 36, clone 13), NCBI accession number: Petition 870250087381, dated 09 / 26 / 2025, page 66 / 113 19 / 54 AJ271157 (Paenibacillus jamilae CECT 5266), NCBI accession no.: NR_025169 (Paenibacillus kribbensis AM49), NCBI accession no.: NR_025106 (Paenibacillus brasiliensis PB172), NCBI accession no.: AF391124 (Paenibacillus terrae AM141); WO2020181053: Paenibacillus sp. NRRL B-67721, Paenibacillus sp. NRRL B-67723, Paenibacillus sp. NRRL B-67724, Paenibacillus sp. NRRL B50374.

[038] More preferably, the microorganism is a microorganism of the taxonomic genus Paenibacillus and is selected from any of the species Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae. According to the invention, the following type strains are preferred. Table 1 Species Deposit Paenibacillus abyssi DSM 26238, GMCC 1.12987 Paenibacillus aestuarii DSM 23861, CM 15521, ACC 13125 Paenibacillus agarexedens DSM 1327, IP 107437 Paenibacillus agaridevorans DSM 1355, IP 107436 Paenibacillus alginolyticus DSM 5050, RRL-NRS 1347 Paenibacillus algorifonticola DSM 26746, CM 16598, J259, GMCC 1.10223 Paenibacillus alkaliterrae DSM 17040, CTC 3956, IP 109150 Paenibacillus alvei DSM 29, TCC 6344, CM 2051, FO 3343, MAB B-3-4, MG 13253, CIB 9371, CTC 6352, BRC 3343 Paenibacillus amylolyticus DSM 15211, RRL NRS-290 Paenibacillus anaericanus DSM 15890, TCC BAA-844, IP 109447, CTC 13749 Paenibacillus apiarius DSM 5581, TCC 29575, RRL-NRS 1438, CIMB 13506 Paenibacillus arcticus DSM 108811, CTC 33985, CM 30981, ME2_R6, AMC 28731 Paenibacillus azoreducens DSM 13822, CIMB 13761 Paenibacillus azotifigens DSM 104138, ACC 18967, MG 29963 Paenibacillus azotofixans DSM 5976, TCC 35681, MG 14658 Paenibacillus barcinonensis DSM 15478,ECT 7022 Paenibacillus barengoltzii DSM 22255, BRC 101215, TCC BAA-1209, IP 109354 Paenibacillus beijingensis DSM 24997, CCC 03082 Paenibacillus borealis DSM 13188, CUG 43137 Paenibacillus bovis DSM 28815, GMCC 8333 Paenibacillus brasilensis DSM 14914, TCC BAA-413, CTC 3812, IP 110686 Paenibacillus brassicae DSM 24983, CCC 01125 Paenibacillus campinasensis DSM 21989, CM 11200, CTC 0364BP, CRC 17341, Petition 870250087381, dated 26 / 09 / 2025, p. 67 / 113 20 / 54 Species Deposit Paenibacillus castaneae DSM 19417, ECT 7279 Paenibacillus catalpae DSM 24714, GMCC 1.10784 Paenibacillus cavernae DSM 100100, CTC 33652 Paenibacillus cellulosilyticus DSM 21372, ECT 5696, MG 22232 Paenibacillus chartarius DSM 28439, CUG 55240, CM 7759 Paenibacillus chibensis DSM 11731, RRL B-142, CM 9905, FO 15958, BRC 15958 Paenibacillus chinjuensis DSM 15045, CTC 8951, CM 10939 Paenibacillus chitinolyticus DSM 11030, FO 15660, BRC 15660 Paenibacillus chondroitinus DSM 5051, RRL-NRS 1351 Paenibacillus chungangensis DSM 28440, CUG 59129, CTC 13717 Paenibacillus cineris DSM 16945, IP 108109, MG 18439 Paenibacillus cisolokensis DSM 101873, ICC B-42, RRL B-65368 Paenibacillus cookii DSM 16944, IP 108110, MG 18419 Paenibacillus cucumis DSM 101601, CM 8655, MG 29222 Paenibacillus curdlanolyticus DSM 10247, FO 15724, BRC 15724 Paenibacillus daejeonensis DSM 15491, CTC 3745, CM 11236 Paenibacillus darwinianus DSM 27245, CMP 19912 Paenibacillus dendritiformis DSM 18844,IP 105967 Paenibacillus dongdonensis DSM 27607, CTC 33221 Paenibacillus donghaensis DSM 22278, MG 23780, CTC 13049 Paenibacillus durus DSM 1735, TCC 27763, MG 15707 Paenibacillus ehimensis DSM 11029, FO 15659, BRC 15659 Paenibacillus elgii DSM 22254, BRC 100335, CTC 10016BP, IP 108552 Paenibacillus elymi DSM 106581, CTC 33853 Paenibacillus etheri DSM 29760, ECT 8558 Paenibacillus eucommiae DSM 26048, CTC 33054 Paenibacillus faecis DSM 23593, IP 101062 Paenibacillus favisporus DSM 17253, MG 20987, ECT 5760 Paenibacillus filicis DSM 23916, CTC 13693, CM 16417, ACC 14197 Paenibacillus fonticola DSM 21315, MG 23577, CRC 17579 Paenibacillus forsythiae DSM 17842, CBAU 10203, IP 110608 Paenibacillus frigoriresistens DSM 25554, CM 18141, CTCC AB 2011150 Paenibacillus gansuensis DSM 16968, CTC 3950, IP 109446 Paenibacillus ginsengarvi DSM 18677, CTC 13059, IP 109800 Paenibacillus ginsengihumi DSM 21568, CM 14928, CTC 13141 Paenibacillus glucanolyticus DSM 5162, TCC 49278, FO 15330, CIB 12809,93, BRC 15330 Paenibacillus glycanilyticus DSM 17608, CM 11221, IP 107742, CTC 3808, RRL B23455, S-1 Paenibacillus gorillae DSM 26181, SUR P205 Paenibacillus graminis DSM 15220, TCC BAA-95, MG 19080 Paenibacillus harenae DSM 16969, CTC 3951 Paenibacillus hodogayensis DSM 22253, CM 12520, CTC 3919 Paenibacillus hongkongensis DSM 17642, CUG 49571, IP 107898, Petition 870250087381, dated 26 / 09 / 2025, p. 68 / 113 21 / 54 Species Deposit Paenibacillus humicus DSM 18784, BRC 102415, MG 23886, IP 110609 Paenibacillus hunanensis DSM 22170, CCC 10718, GMCC 1.8907, IP 110610 Paenibacillus ihumii DSM 100664, SUR P1981, SUR 1981 Paenibacillus illinoisensis DSM 11733, RRL NRS-1356, CM 9907, FO 15959, CIMB 13573, BRC 15959 Paenibacillus jilunlii DSM 23019, GMCC 1.10239, IP110611 Paenibacillus kobensis DSM 10249, FO 15729, BRC 15729 Paenibacillus konkukensis DSM 104139, ACC 18876, MG 29568 Paenibacillus konsidensis DSM 21992, CM 14798, CTC 13165 Paenibacillus kribbensis DSM 27933, CM 11465, CTC 0766BP Paenibacillus lactis DSM 15596, MG 21940, IP 108827 Paenibacillus larvae DSM 7030, TCC 9545, MG 9820, ed-540, RRL B-2605 Paenibacillus lautus DSM 3035, TCC 43898, MG 11157, CIMB 12780 Paenibacillus lentus DSM 25539, TCC BAA-2594 Paenibacillus macerans DSM 24, TCC 8244, CM 2012, AM 12467, MG 13281, CIB 9368, CTC 6355, IP 66.19 Paenibacillus macquariensis subsp.defensor DSM 23149, CM 14954, CIMB 14397 Paenibacillus macquariensis subsp. macquariensis DSM 2, TCC 23464, MG 6935, IP 103269, CTC 10419 Paenibacillus marchantiophytorum DSM 29850, GMCC 1.15043 Paenibacillus massiliensis subsp. massiliensis DSM 16942, IP 107939, CUG 48215 Paenibacillus mendelii DSM 19248, CM 4839, MG 23002 Paenibacillus motobuensis DSM 18200, CM 12774, CUG 50090, TC 1835 Paenibacillus mucilaginosus DSM 24461, IP 105815, SCC 1605, CTC 3870, KM B1480D, KPM B-7519 Paenibacillus nanensis DSM 22867, CTC 13044, ISTR 1828, CU 276 Paenibacillus naphthalenovorans DSM 14203, TCC BAA-206 Paenibacillus nebraskensis DSM 103623, IP 111179, MG 29764 Paenibacillus nematophilus DSM 13559, IP 108049 Paenibacillus nicotianae DSM 28018, RRL B-59112, GMCC 1.12819 Paenibacillus odorifer DSM 15391, TCC BAA-93, MG 19079 Paenibacillus ottowii DSM 107750, TCC TSD-165 Paenibacillus pabuli DSM 3036, TCC 43899, MG 11158, CIMB 12781 Paenibacillus panacisoli DSM 21345, CTC 13020, MG 23405 Paenibacillus pasadenensis DSM 19293, BRC 101214, TCC BAA-1211 Paenibacillus pectinolyticus DSM 28340, ECT 7358, CTC 13222 Paenibacillus peoriae DSM 8320, MG 14832, RRL B-14750 Paenibacillus phoenicis DSM 27463, BRC 106274, RRL B-59348 Paenibacillus phyllosphaerae DSM 17399, MG 22192, ECT 5862 Paenibacillus physcomitrellae DSM 29851, GMCC 1.15044 Paenibacillus pinihumi DSM 23905, CTC 13695, CM 16419, ACC 14199 Paenibacillus piri DSM 105496, ACC 19385 Paenibacillus pocheonensis DSM 23906, CTC 13941, MG 23404. Petition 870250087381, dated 26 / 09 / 2025, p. 69 / 113 22 / 54 Species Deposit Paenibacillus polymyxa DSM 36, TCC 842, UCSAV 162, CM 1459, CM 2507, MG 13294, CIB 8158, CTC 10343 Paenibacillus popilliae DSM 22700, IP 106066, RRL B-2309, TCC 14706, CUG 28881, CCB 75017 Paenibacillus prosopidis DSM 22405, MG 25259, ECT 7506 Paenibacillus provencensis DSM 22280, CUG 53519, IP 109358 Paenibacillus pueri DSM 22870, CTC 13223, ECT 7360 Paenibacillus puldeungensis DSM 27603, CTC 13718, CUG 59189 Paenibacillus purispatii DSM 22991, IP 110057 Paenibacillus qingshengii DSM 100926, CM 30613, CTCC AB 2014290 Paenibacillus radicis DSM 100762, GMCC 1.15286 Paenibacillus relictisesami DSM 25385, CM 18068 Paenibacillus residui DSM 22072, CUG 57263 Paenibacillus rhizophilus DSM 103168, GMCC 1.15699 Paenibacillus rhizoplanae DSM 103963, MG 29875, CM 8725 Paenibacillus rhizosphaerae DSM 17254, MG 21955, ECT 5831 Paenibacillus ripae DSM 104672, MG 29834, CTCC AB 2014276, MG 28639 Paenibacillus sabinae DSM 17841, CBAU 10202, IP 109632, CTC 13697 Paenibacillus sacheonensis DSM 23054, ACC 14895, IP 110612 Paenibacillus sanguinis DSM 16941, IP 107938, CUG 48214 Paenibacillus sediminis DSM 23491, T-H3, MG 25635, IP 110613 Paenibacillus segetis DSM 28014, GMCC 1.12769 Paenibacillus senegalensis DSM 25958, SUR P157 Paenibacillus sepulcri DSM 27042, MG 19508, CM 7311, eyrman R-514, cha6024 Paenibacillus shirakamiensis DSM 26806, BRC 109471, CTC 33126, IP 110571 Paenibacillus silagei DSM 101953, CM 30974 Paenibacillus silvae DSM 28013, GMCC 1.12770 Paenibacillus solani DSM 100999, CTCC AB 2015207 Paenibacillus soli DSM 21316, MG 23604, CTC 13010 Paenibacillus sonchi DSM 28159, ECT 7330, CGB 1313 Paenibacillus sophorae DSM 23020, GMCC 1.10238, IP 110614 Paenibacillus sputi DSM 22699, CTC 13252 Paenibacillus stellifer DSM 14472 Paenibacillus taichungensis DSM 19942, CRC 17757, IP 110615 Paenibacillus taiwanensis DSM 18679, AM 15414, MG 23799 Paenibacillus taohuashanense DSM 25809, GMCC 1.12175 Paenibacillus tarimensis DSM 19409, CTCC AB 206108, IP 110616 Paenibacillus terrae JCM:11466, KCCM:41557 Paenibacillus terreus DSM 100035, ACC 18491, CTCC AB 2015273 Paenibacillus terrigena DSM 21567, CTCC AB206026, AM 15291, CM 21741 Paenibacillus thailandensis DSM 22866, CTC 13043, ISTR 1827, CU 275 Paenibacillus thermoaerophilus DSM 26310, CM 18657 Paenibacillus thiaminolyticus DSM 7262, CM 8360, RRL B-4156 Paenibacillus tianmuensis DSM 22342, GMCC 1.8946, IP 110617. Petition 870250087381, dated 26 / 09 / 2025, p. 70 / 113 23 / 54 Species Deposit Paenibacillus tibetensis DSM 29321, CCC 19728 Paenibacillus timonensis DSM 16943, IP 108005, CUG 48216 Paenibacillus triticisoli DSM 25425, GMCC 1.12045 Paenibacillus tundrae DSM 21291, RRL B-51094, IP 110036 Paenibacillus turicensis DSM 14349, CCB 100011 Paenibacillus tylopili DSM 18927, MG 23975 Paenibacillus typhae DSM 25190, GMCC 1.11012, IP 110618 Paenibacillus uliginis DSM 21861, MG 24790 Paenibacillus urinalis DSM 22281, CUG 53521, IP 109357 Paenibacillus validus DSM 3037, TCC 43897, MG 11161, CIMB 12782, RRL- NRS 1000 Paenibacillus vulneris DSM 27954, CM 18268, CUG 53270 Paenibacillus wenxiniae DSM 100576, GMCC 1.15007 Paenibacillus woosongensis DSM 16971, CTC 3953, IP 110595 Paenibacillus wulumuqiensis DSM 29194, PCC 100602, CM 30284 Paenibacillus wynnii DSM 18334, IP 108306, MG 22176 Paenibacillus xerothermodurans DSM 520, TCC 27380 Paenibacillus xinjiangensis DSM 16970, CTC 3952, IP 109466 Paenibacillus xylanexedens DSM 21292, RRL B-51090, IP 110619 Paenibacillus xylaniclasticus DSM 26531, BRC 106381, CTC 13719, ISTR 1914 Paenibacillus xylanilyticus DSM 17255, MG 21957, ECT 5839 Paenibacillus xylanisolvens DSM 25299, CTC 13042, CU 311, ISTR 1829 Paenibacillus xylanivorans DSM 107920, CIMB 15123 Paenibacillus zanthoxyli DSM 18202, CBAU 10243, IP 109595, CTC 13696.

[039] As described above, there is a widespread belief in the art, supported by evidence from various species of microorganisms of the genus Firmicutes, that a high inoculation titer is beneficial for industrial fermentations, in particular to reduce the duration of the lag phase and ensure rapid spore germination. Contrary to this belief, the inventors have found that a low inoculation titer is beneficial for industrial fermentations. At the same time, the inventors have found that very low inoculation titers, as suggested, for example, in documents WO2006096164 and WO2017068012, are not beneficial in industrial fermentations of Paenibacillaceae. Although useful inoculation titers are described in the present invention, particularly in the examples section, it is understood that the ideal inoculation titers are also influenced, to some degree, by Petition 870250087381, dated 09 / 26 / 2025, pp. 71 / 113 24 / 54 composition of the medium. The invention, correspondingly, provides a method for selecting an inoculation quantity of a microorganism from the family Paenibacillaceae, more preferably from the genus Paenibacillus.

[040] According to the invention, the terms “spore”, “spores”, “endospore” and “endospores” are used interchangeably.

[041] The invention provides a culture of microorganisms, wherein the microorganism is of the family Paenibacillaceae, preferably of the genus Paenibacillus. The term “culture”, according to the invention, means a collection or set of microorganisms in which at least 50% of all live cells and spores belong to the family Paenibacillaceae, preferably to the genus Paenibacillus. More preferably, the culture is a pure culture of microorganisms belonging to the family Paenibacillaceae, preferably to the genus Paenibacillus.Furthermore, a “culture”, according to the present invention, can be either a liquid culture, in which microorganisms are suspended in a liquid medium suitable for their growth, or a preparation of microorganisms in a form suitable for inoculation into a liquid culture, preferably in the form of a cell bank flask for cryopreservation, preferably containing an antifreeze and / or stabilizing agent, for example, glycerol and / or sugar. The means and methods for creating and maintaining cell bank flasks are known to those skilled in the art.

[042] The cells in the culture are metabolically aligned and / or in an activated metabolic state. A particular advantage of the present invention is that such cultures are first supplied in industrially relevant quantities, i.e., in liquid culture volumes exceeding 10 liters (L), more preferably from 10 to 1,000 L, even more preferably from 20 to 1,000 L, even more preferably from 50 to 1,000 L, even more preferably from 100 to 1,000 L. However, an advantage Petition 870250087381, dated 09 / 26 / 2025, pp. 72-113 A particular advantage of the method is that the volume of medium required for carrying out the method can be small, preferably from 10 to 500 milliliters (mL), more preferably from 15 to 250 mL, and even more preferably from 20 to 100 mL. As described in this invention, the invention is advantageously applicable to industrial fermentations. Thus, the fermentation tank preferably has an average volume greater than 200 L, preferably 300 L, more preferably 400 to 1,000 L, more preferably 500 L, more preferably 1,000 to 15,000 L, more preferably 2,000 to 15,000 L, and more preferably 50,000 to 150,000 L. A particular advantage is that the fermenter can have an average volume of 50 to 300 m3, fermentation volumes frequently found in existing industrial fermentation facilities. Thus, the invention advantageously avoids the need for re-adaptation, in particular increasing or decreasing the size of existing fermentation equipment.

[043] Cell cultures of metabolically aligned and / or metabolically activated microorganisms offer numerous advantages in fermentation processes, as described in more detail below. In particular, such cultures allow for a reduction in the fermentation time required to produce a desired quantity of a target substance and / or allow for a reduction in viscosity during fermentation and / or allow for an increase in the reproducibility of the fermentation. It is particularly surprising that such advantages can be obtained by maintaining suitable cultivation conditions for Paenibacillaceae microorganisms, particularly species belonging to the genus Paenibacillus. Such microorganisms have been used in various applications in the past.However, to date, genetic modifications of these microorganisms have been considered the only viable way to significantly reduce the fermentation times required to produce a desired amount of target substance and / or reduce viscosity during the process. Petition 870250087381, dated 09 / 26 / 2025, pp. 73 / 113 26 / 54 fermentation and / or increase the accuracy of repeatability (increase the reproducibility) of fermentation compared to a wild-type cell. Another advantage of the present invention is that the necessary properties of a microorganism culture for seeding an industrial fermentation are easily observable using standard laboratory practices and equipment. Furthermore, the conditions for producing a microorganism culture according to the present invention are robust, so that variations in operation typical of a laboratory or production environment can be tolerated. In particular, the time between inoculation of the growth medium and collection of the culture can vary by several hours. Thus, the properties that identify the microorganism culture are, in practice, easy to determine and can be reliably obtained using standard media and equipment.

[044] In a culture of the present invention, microorganisms of the family Paenibacillaceae, preferably of the genus Paenibacillus, are preferably characterized by any of the following parameters: a) the median corpuscular length of the cells is 0.8 to 2.9 micrometers (µm), more preferably 1 to 2.5 µm, even more preferably 1.3 to 2 µm, even more preferably 1.3 to 1.8 µm, and / or b) the 75th percentile of the corpuscular length of the cells is 1.3 to 3 pm, more preferably 1.3 to 2.9 pm, more preferably 1.5 to 2.5 pm, and / or c) the 25th percentile of the corpuscular length of the cells is 0.8 to 2.5 pm, more preferably 0.8 to 1.9 pm, and most preferably 0.8 to 1.5 pm.

[045] Thus, 75% of cell bodies preferentially have a length of at most 3 pm, more preferably up to 2.9 pm, most preferably at most 2.5 pm; and / or 25% of Petition 870250087381, dated 09 / 26 / 2025, pp. 74 / 113 27 / 54 cell bodies have a length of at most 2.5 pm, more preferably 1.9 pm, even more preferably 0.8-1.5 pm.

[046] Microorganisms of the Paenibacillaceae family form rod-shaped corpuscles in liquid culture. According to the invention, the “corpuscular length of the cell” is the extent, along the longest axis, of this corpuscle. The accompanying examples and figures illustrate these corpuscles in greater detail and describe methods for determining the corpuscular length of the cells. A particular advantage of the present invention is that microorganisms of the Paenibacillaceae family produce two clearly different types of growth in liquid culture. In the common type observed in state-of-the-art fermentations, the cells are quite large, with a median corpuscular length of 3.4 µm. However, the inventors have discovered that there is a second type of growth that offers the advantages of the present invention.Cells growing in this type of growth are here referred to as “metabolically aligned” or “metabolically activated state” cells, since their fermentation properties in liquid fermentations are significantly and consistently different from the properties of microorganisms growing in the first type of growth, as described in the present invention. A particular advantage is that microorganisms of the second type of growth, the “metabolically aligned” or “metabolically activated state” microorganisms, can be differentiated from those of the first type of growth using simple microscopy. For a trained user, the transition from the first to the second type of growth in liquid culture is easily observable due to the profound change in the median corpuscular length of the cells.

[047] A culture of Paenibacillaceae microorganisms of the present invention is preferably obtained, or may be obtained, by inoculating a culture medium with a concentration of microorganisms Petition 870250087381, dated 09 / 26 / 2025, pp. 75 / 113 28 / 54 sufficient to produce the aforementioned crop, where the concentration is preferably determined: i) by cultivating a series of cultures inoculated with different inoculation concentrations for a sufficient time to differentiate the cultures by a parameter indicative of metabolic activity, wherein the parameter is preferably selected from any of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH course, gas formation, oxygen uptake rate, CO2 transfer rate and variance of any of the parameters mentioned above, and ii) by determining one or more parameters, and iii) by selecting an inoculation concentration based on one or more determined parameters.

[048] A particular advantage of the present invention is that the transition from the first type of growth to the second, metabolically aligned or activated type, can be induced by selecting a sufficiently low inoculation concentration. Inoculation can be carried out by seeding a fresh growth medium with cells and / or spores of microorganisms. By varying the inoculation concentration, it is possible to find the ideal concentration to achieve the transition from the first to the second type of growth within a desired cultivation time.

[049] Another advantage of the present invention is that, instead of the corpuscular length of the cells, other parameters can also be observed to indicate the transition to a culture of metabolically aligned microorganisms and / or microorganisms in an activated metabolic state. As described in more detail below, the microorganism cultures of the present invention advantageously provide increased metabolic activity compared to wild-type cells in the first type of Petition 870250087381, dated 09 / 26 / 2025, pp. 76 / 113 29 / 54 growth. Preferably, this increased metabolic activity is determined from any of the following factors: growth rate, cell size, viscosity or production of an indicator substance, optical density, cell count, pH course, gas formation, oxygen uptake rate, and CO2 transfer rate. It is also a particular advantage that the microorganism culture of the present invention comprises metabolically aligned cells and therefore allows for highly consistent results in repeated fermentations. Thus, in addition to or instead of measuring any of the aforementioned parameters, the variance of any of the aforementioned parameters can also be determined. Low variance indicates a successful transition to the second type of growth.

[050] For example, the production of an indicator substance is preferably determined by the concentration at a predetermined time or by the time required to reach a predetermined concentration. The indicator substance may be chosen at will. For example, the indicator substance may be a colored substance, a metabolic byproduct (e.g., acetoin, 2,3 BDO, glutamate) or a target substance, preferably a fusaricidin, paeniserin, paeniprolixin or polymyxin. Thus, batches are preferably compared by any of the following criteria: - time required to achieve a predefined yield of a target substance, preferably a fusaricidin, paeniserin, paeniprolixin or polymyxin, - production of a target substance, preferably a fusaricidin, paeniserin, paeniprolixin or polymyxin at a predetermined time after the start of step ii), preferably within 8 to 48 hours, more preferably 12 to 24 hours. Petition 870250087381, dated 09 / 26 / 2025, page 77 / 113 30 / 54

[051] In view of the advantages mentioned above, the invention also provides a fermentation method comprising: i) provide a fermentation broth comprising a culture of microorganisms of the present invention in a fermentation medium, and ii) cultivate the fermentation broth under suitable conditions to produce a target substance.

[052] The fermentation method of the present invention offers surprising benefits compared to fermentation methods that do not use microorganisms of the Paenibacillaceae family in an activated and / or aligned metabolic state: one advantage is the increased fermentation rate. In particular, the fermentation method allows for a significantly reduced lag phase after the transfer of the inoculum to the fermentation medium. Furthermore, the fermentation method allows microorganisms to reach half of their maximum cell and / or optical density in less time. In addition, the fermentation method allows for higher productivity, i.e., yield per unit time, and total yield of target substances. For Paenibacillus microorganisms, this allows for an advantageously increased yield of fusaricidin, paeniserin, paeniprolixin, and / or polymyxin.Furthermore, the development of high viscosity can be advantageously delayed or controlled. This avoids high energy consumption for agitation of the medium and facilitates the purification of the target substances produced.

[053] It is particularly advantageous and surprising that the fermentation proceeds with high reproducibility. This is preferably verified by a very low variability of the optical density at any given time for repeated fermentations. Thus, the fermentation method according to the invention is particularly suitable for automation, since, once a fermentation protocol is established, the Petition 870250087381, dated 09 / 26 / 2025, pp. 78 / 113 31 / 54 The need for interfering guidance by a user to combat unforeseen divergent developments in fermentation can be reduced or completely avoided. Consequently, the fermentation method of the present invention allows the production of a target substance of reliable quality over several batches.

[054] Another effect of the significantly increased reproducibility in fermentations is that variations in the growth and metabolic behavior of microbes during fermentation, caused by small variations in the properties of the cell bank samples, are avoided. In contrast, by initiating an industrial fermentation with a culture of microorganisms in a defined state, i.e., a culture of microorganisms of the present invention, the fermentation is protected from such random disturbances.

[055] In step i), the fermentation broth is preferably prepared by inoculating a cell-free fermentation medium with a microorganism culture of the present invention. Thus, the fermentation medium is inoculated with Paenibacillaceae microorganisms, preferably with a pure culture of Paenibacillus microorganisms, which are already metabolically aligned and / or in an activated metabolic state. When using the microorganism culture of the present invention, only a short adaptation time is required before the microorganisms become productive for the production of a target substance. Preferably, when producing the microorganism culture of the present invention by the production method above, the microorganism culture is already cultivated in the fermentation medium of step i) of the fermentation method.This is even more suitable for reducing adaptation time and achieving particularly high productivity, i.e., the amount of recoverable target substance produced in step ii) in a given fermentation time and volume is particularly high. The fermentation broth can also be prepared by inoculating a fermentation medium with cells. Petition 870250087381, dated 09 / 26 / 2025, pp. 79 / 113 32 / 54 purified from a culture of microorganisms of the present invention.

[056] Preferably, the fermentation medium is inoculated in step i) with a sufficient quantity of microorganisms to maintain the metabolically aligned and / or activated state in step ii). A particular advantage of the present invention is that the optimization of the inoculation quantity can be done by observing any of the parameters mentioned above, such as growth, cell size, viscosity, and production of an indicator substance and / or its variation. Typically, for microorganisms of the genus Paenibacillus in a metabolically aligned or activated state, the addition of at least 2E4 cfu / mL to the fermentation medium is necessary in industrial fermentations. Preferably, a fermentation or culture medium is inoculated with 2E05 to 2E06 cfu / mL (200,000 - 2,000,000 cfu / mL).

[057] A particular advantage of the present invention is that the microorganisms maintain their metabolically aligned and / or activated state throughout multiple passages. For example, a microorganism culture of the present invention can be poured as such into a new fermentation medium for inoculation, or the microorganism culture cells can be purified, preferably by centrifugation, and introduced into the new fermentation medium. In either case, the cells will continue to grow according to the second type of growth, as described in the present invention. Thus, the invention avoids, for the inoculation of a large volume of industrial fermentation, the need to prepare the required microorganism population in step i) in a single step, which would likely require the storage of a large quantity of inoculation material in cell banks.Instead, microorganisms from a cell bank flask can first be cultured in a small, agitated liquid culture vessel of approximately 50 mL to 2,500 mL until they have transitioned to the second type of growth and thus reached a state. Petition 870250087381, dated 09 / 26 / 2025, pp. 80 / 113 33 / 54 metabolically aligned and / or activated. Then, the cells are transferred (in the fermentation broth or after purification and, preferably, resuspension in a new fermentation medium) to successively larger fermentation volumes.

[058] In step ii), the fermentation broth is fermented under suitable conditions for the production of a target substance. A particular advantage is that fermentation using the microorganism culture of the present invention is not limited to specific conditions. Instead, suitable conditions can be chosen by a person skilled in the art, particularly in view of the oxygen and nutrient demand, viscosity development and / or the production of a target substance. In addition, additional components can be added to or removed from the fermentation medium. Preferably, the pH of the fermentation broth is adjusted by adding appropriate amounts of acids or bases. Preferably, the oxygen concentration is adjusted by increasing or decreasing aeration, for example, by bubbling oxygen into the fermentation broth and / or adjusting the speed of a mixer.The temperature is preferably adjusted by heating or cooling the fermenter mantle and / or increasing or decreasing the concentration and / or type of available nutrients. Furthermore, preferably, the medium comprises or is supplied during fermentation with an antifoaming agent in sufficient quantity to prevent or reduce foam formation during stage ii) of fermentation.

[059] Preferably, the fermentation method of the present invention further comprises: iii) recover the target substance from the cultured fermentation broth of step ii).

[060] The target substance according to the present invention is preferably selected from: Petition 870250087381, dated 09 / 26 / 2025, pp. 81 / 113 34 / 54 - microorganisms, preferably viable cells and / or spores, and / or - one or more metabolic products, preferably at least one fusaricidin, paeniserin, paeniprolixin or polymyxin, preferably at least two or more fusaricidins, paeniserins, paeniproxilins or polymyxins, more preferably from 3 to 120 fusaricidins, wherein one or more fusaricidins comprise any one of fusaricidin A, B or D and / or surfactin and / or iturin.

[061] In particular, the target substance is preferably any of the following: - a composition comprising spores and / or cells of the microorganism, preferably a composition comprising or consisting of viable cells and spores in a ratio of no more than 4:1, more preferably 3:1 to 0.2:1, even more preferably less than 0.2:1, even more preferably containing only spores without viable cells, - a composition comprising or consisting of at least one fusaricidin, paeniserin, paeniprolixin or polymyxin, preferably at least two or more fusaricidins, paeniserins, paeniproxilins or polymyxins, more preferably from 3 to 120 fusaricidins, wherein one or more fusaricidins comprise any one of fusaricidin A, B or D, and / or surfactin and / or iturin, and / or - a composition comprising or consisting of at least one viscosity-increasing or viscosifying substance, preferably selected from exopolysaccharides, proteoglycans, and polygamma-glutamic acid.

[062] A particular advantage of the present invention is that the target substance is not limited to one specific type of substance. As Petition 870250087381, dated 09 / 26 / 2025, pp. 82-113 35 / 54 As demonstrated in the examples, the use of a fermentation method of the present invention allows for reproducibly obtaining higher productivity and / or higher yields for various target substances, compared to a fermentation method in which the fermentation medium is not inoculated with a population of metabolically aligned and / or activated microorganisms.

[063] The target substance may be cells and / or spores of the microorganism. In agriculture, bacterial spores have been used in plant pest control compositions, reducing or preventing phytopathogenic fungal or bacterial diseases. Spore-based bioproducts are also applied to improve plant resistance against biotic and abiotic stresses, to accelerate plant growth and increase yield during the harvest of plants, fruits or legumes. Spore products (bioproducts) have been applied to leaves, shoots, fruits, roots or plant propagation material, as well as to the substrate where the plants are to grow (Toyota K. Bacillus-related Spore Formers: Attractive Agents for Plant Growth Promotion. Microbes Environ. 2015;30(3):205-207. doi:10.1264 / jsme2.me3003rh). Bochow, H., et al. “Use of Bacillus subtilis as a Biocontrol Agent. IV. Salt-Stress Tolerance Induction by Bacillus subtilis FZB24 Seed Treatment in Tropical Vegetable Field Crops, and Its Mode of Action / Die Nutzung von Bacillus subtilis zur biologischen Bekämpfeung. IV.duktion einer Salzstress-Toleranz durch Application von Bacillus subtilis FZB24 bei australim Feldgemüse und sein Wirkungsmechanik”. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz / Journal of Plant Diseases and Protection, vol. 108, n°. 1, 2001, págs. 21-30. JSTOR, www.jstor.org / stable / 43215378. Accessed on December 14, 2020.)(Hashem, Abeer & Tabassum, B. & Abd_Allah, Elsayed. (2019). Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences. 26. 10.1016 / j.sjbs.2019.05.004). Petição 870250087381, de 26 / 09 / 2025, pág. 83 / 113 36 / 54

[064] Furthermore, bacterial spores have been applied in the area of ​​nanobiotechnology and chemical construction, such as self-healing concrete (crack healing), mortar stability and reduced water permeability [JY Wang, H. Soens, W. Verstraete, N. De Belie, Self-healing concrete by use of microencapsulated bacterial spores, Cement and Concrete Research, Volume 56, 2014, 139-152, ISSN 0008-8846, https: / / doi.org / 10.1016 / j.cemconres.2013.11.009] [Ricca E, Cutting SM. Emerging Applications of Bacterial Spores in Nanobiotechnology. J Nanobiotechnology. 2003;1(1):6. Published December 15, 2003. doi:10.1186 / 1477-3155-1-6].

[065] Furthermore, bacterial spores have been applied in the area of ​​cleaning products, such as for cleaning clothes, hard surfaces, sanitation and odor control (Caselli E. Hygiene: microbial strategies to reduce pathogens and drug resistance in clinical settings. Microb Biotechnol. September 2017;10(5):1079-1083. doi: 10.1111 / 1751-7915.12755. Epub July 5, 2017) in clinical and domestic settings. For example, spores have been used in cosmetic compositions, such as skin cleansers (US20070048244), dishwashing agents (WO2014 / 107111), pipe degreasers (DE19850012), odor control for clothing (WO2017 / 157778 and EP3430113), or allergen removal (US20020182184). Spores can also be incorporated into non-biogenic matrices to catalyze their subsequent decomposition.

[066] In addition, bacterial spores have been applied in the area of ​​human and animal nutrition and health. As an example, different bacterial strains have been applied to broiler chickens as part of an antibiotic replacement strategy (Neveling, DP, Dicks, LM Probiotics: an Antibiotic Replacement Strategy for Healthy Broilers and Productive Rearing. Probiotics & Antimicro. Prot. 13, 1-11 (2021). https: / / doi.org / 10.1007 / s12602-020-09640-z). Others Petition 870250087381, dated 09 / 26 / 2025, pp. 84 / 113 37 / 54 examples include aquaculture, swine, among others (Nayak, SK (2021), Multifaceted applications of probiotic Bacillus species in aquaculture with special reference to Bacillus subtilis. Rev. Aquacult., 13: 862-906. (https: / / doi.org / 10.1111 / raq.12503). The applications of bacterial spores for human health are also widely described (e.g., US document 20180289752; and Lee, NK., Kim, WS. & Paik, HD. Bacillus strains as human probiotics: characterization, safety, microbiome and probiotic carrier. Food Sci Biotechnol 28, 1297-1305 (2019). https: / / doi.org / 10.1007 / s10068-019-00691-9).

[067] The target substance may also be a fusaricidin, paeniserin, paeniprolixin or polymyxin. In each case, it is particularly preferred that one or more of the fusaricidins comprise any of the fusaricidins A, B or D. As described below, fusaricidins, paeniserins, paeniproxilins or polymyxins have numerous applications, particularly in promoting plant health (phytosanitary) and / or controlling plant pests.

[068] The target substance may also be a viscosity-increasing or viscosifying substance, such as Paenan and / or Levan. Microbial exopolysaccharides have numerous applications, such as oil drilling, thickening in cosmetics, in paints, food, as rheology modifiers, in pharmaceuticals and in household care. Several applications are listed, for example, in Liyaskina EV, Rakova NA, Kitykina AA, Rusyaeva VV, Toukach PV, Fomenkov A, Vainauskas S, Roberts RJ, Revin VV. Production and characterization of exopolysaccharide from Paenibacillus polymyxa 2020 strain. PLoS One. Jul 6, 2021; 6;16(7):e0253482. doi: 10.1371 / journal.pone.0253482. PMID: 34228741; PMCID: PMC8259973.

[069] In addition, the microorganism or its spores may be the target substance. As described, for example, by Grady EN, MacDonald J, Liu L, Richman A, Yuan ZC. Current knowledge and perspectives on Paenibacillus: a review. Microb Cell Fact. December 1, 2016;15(1):203. doi: Petition 870250087381, dated 09 / 26 / 2025, page 85 / 113 38 / 54 10.1186 / s12934-016-0603-7. PMID: 27905924; PMCID: PMC5134293, many Paenibacillus species can promote crop growth directly through biological nitrogen fixation, phosphate solubilization, production of the phytohormone indole-3-acetic acid (IAA), and release of siderophores that allow iron uptake. They can also offer protection against herbivorous insects and phytopathogens, including bacteria, fungi, nematodes, and viruses.

[070] The microorganism culture in step i) of the fermentation method of the present invention is preferably obtained, or may be obtained by: ii) inoculation of a pre-culture fermentation medium with a quantity of microorganisms and i.ii) cultivating the crop from stage ii) in order to provide a crop with: a) the median corpuscular length of the cells from 0.8 to 2.9 micrometers (µm), more preferably from 1 to 2.5 µm, even more preferably from 1.3 to 2 µm, even more preferably from 1.3 to 1.8 µm, and / or b) the 75th percentile of the corpuscular cell length of 1.3 to 3 pm, more preferably 1.3 to 2.9 pm, more preferably 1.5 to 2.5 pm, and / or c) the 25th percentile of corpuscular cell length from 0.8 to 2.5 pm, more preferably from 0.8 to 1.9 pm, and most preferably from 0.8 to 1.5 pm.

[071] As described above, cell bodies with such corpuscular length are those derived from a culture of microorganisms of the present invention, in which microorganisms of the family Paenibacillaceae, preferably of the genus Paenibacillus, are Petition 870250087381, dated 09 / 26 / 2025, page 86 / 113 39 / 54 metabolically aligned and / or in an activated metabolic state. As described above, such microorganisms can also be described by other parameters, such as growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH variation, gas formation, oxygen uptake rate, CO2 transfer rate, and variance of any of these parameters.

[072] The invention also provides a method for reducing the fermentation time required to produce a desired quantity of a target substance and / or for reducing viscosity during fermentation and / or for increasing the reproducibility of fermentation, comprising the steps of: i) to provide a culture according to the invention in a fermentation medium, and ii) to cultivate the culture in the fermentation medium.

[073] As described in the present invention, in particular in the attached figures and examples, an advantage of the microorganism culture of the present invention is that the microorganisms are in a metabolic state that allows the production of a target substance much more rapidly than those that grow according to the first type of growth. Thus, the culture of the present invention, when used in a fermentation method, allows a certain quantity of the target substance to be produced more rapidly, or allows a greater quantity of the target substance to be produced in a given time. A particular advantage is that this faster fermentation can be achieved while simultaneously obtaining a reduction in the viscosity of the fermentation medium compared to a microorganism culture that grows according to the first type of growth. Petition 870250087381, dated 09 / 26 / 2025, p. 87 / 113 40 / 54

[074] Correspondingly, a microorganism culture of the present invention is preferably used to reduce the fermentation time required to produce a desired quantity of a target substance and / or to reduce viscosity during fermentation and / or to increase the accuracy of repeatability (reproducibility) of the fermentation.

[075] As described herein, the invention particularly improves the reproducibility of fermentations using microorganisms of the Paenibacillaceae family, preferably of the Paenibacillus genus, thereby facilitating automated fermentations. Correspondingly, the invention provides a set of cultivation parameter data for controlling a fermentation, comprising: - a designation (specification) of a set of applicable fermentation media, - a designation for a set of applicable samples of microorganisms, - a designation of fermentation conditions conducive to obtaining a culture according to the invention, by cultivating an applicable sample in an applicable fermentation medium, wherein the fermentation conditions preferably comprise at least one of the following: type of culture medium, amount of culture medium, desired culture temperature range, desired culture aeration parameter ranges, desired quantity of cells produced, desired duration of cultivation, desired pH during cultivation, yield or desired quantity of a target substance.

[076] The set of culture parameters data can be advantageously used to guide a fermentation of microorganisms from the Paenibacillaceae family, preferably from the Paenibacillus genus. Due to the reproducibility obtained with the use of a culture of Petition 870250087381, dated 09 / 26 / 2025, pp. 88 / 113 41 / 54 microorganisms of the present invention, the set of culture parameters data is sufficient to guide such fermentation in order to produce a target substance.

[077] The invention also provides a computer-implemented fermentation method, comprising, comprising: - to receive, via a computing device, a set of crop parameter data according to the present invention, - to receive a fermentation medium that conforms to the set of cultivation parameters, - receive a sample volume of microorganisms in accordance with the set of culture parameters, and - To control, via the computing device, a fermenter to cultivate microorganisms and / or their spores in the fermentation medium under the fermentation conditions of the culture parameter dataset.

[078] As described in the present invention, fermentation according to the set of cultivation parameters results in the production of a culture of microorganisms of the present invention and / or in the maintenance of such Paenibacillaceae microorganisms, preferably microorganisms of the genus Paenibacillus, in a metabolically aligned and / or activated state, thus ensuring the advantages of the present invention. In particular, the computer-implemented fermentation method allows for the production of a target substance reliably, more quickly and / or with a higher yield compared to microorganisms cultivated according to the first type of growth.

[079] Control preferably includes: - to determine one or more parameters indicative of metabolic activity, where the parameter is preferentially selected. Petition 870250087381, dated 09 / 26 / 2025, pp. 89 / 113 42 / 54 among any of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH course, gas formation, oxygen uptake rate, CO2 transfer rate, and the variance of any of the aforementioned parameters. - compare one or more determined parameters with the target fermentation conditions, preferably one or more parameters from the culture parameter dataset, and - Adjust the fermentation conditions to promote compliance of one or more determined parameters with the aforementioned target fermentation conditions.

[080] In a related note, the invention also provides a method for producing one or more substances that increase viscosity. The method comprises: i) inoculate a fermentation medium with a concentration of a microorganism culture according to the invention at a predetermined concentration sufficient to obtain, during cultivation, a desired viscosity, and ii) cultivate the culture obtained in step i), and iii) preferably recover one or more viscosity-increasing substances or viscosifiers.

[081] Thus, the method begins with a culture of microorganisms of the present invention. However, maintaining the cells in the second type of growth leads to a reduction in viscosity during fermentation. Thus, the method typically involves cultivating, in step ii), a culture at a higher cell density. A particular advantage of the present invention is that the culture of microorganisms of Petition 870250087381, dated 09 / 26 / 2025, pp. 90 / 113 43 / 54 The present invention allows for improved reproducibility and yield of a target substance, including substances that increase viscosity, even if the culture is used only to inoculate an industrial fermentation without maintaining the conditions for growth according to the second type of growth.

[082] The invention is described below by means of accompanying examples and figures, none of which are intended to limit the scope of the invention. Examples Example 1 Impact of Inoculation Volume in the Shaker Bottle on the Growth of P. polymyxa LU21132 (Fig. 1)

[083] The impact of the initial inoculation volume of the pre-culture on the growth of P. polymyxa was investigated in shaken flask cultures using the LU21132 strain of Paenibacillus.

[084] The LU21132 strain is a polymyxin-free mutant of the wild-type isolate P. polymyxa LU17007 and is derived from a random mutagenesis approach. The strain was chosen as an example to demonstrate the impact of pre-cultured inoculation volume in one- and two-stage cultures, but the same phenomenon has also been demonstrated in the wild-type strain P. polymyxa LU17007 and its mutant successors, such as strains LU20754 and LU21552, as well as in public Paenibacillus strains, such as strains P. polymyxa DM365 or P. terrae DSM15891.

[085] Different proportions (% v / v) of a working cell bank of the LU21132 strain (0.1 to 4.0% v / v) were used to inoculate pre-culture flasks in order to analyze their impact on LU21132 biomass formation after 24 hours of growth in PX-125 medium. The inoculation volumes tested are listed in Table 2. Petition 870250087381, dated 09 / 26 / 2025, pp. 91 / 113 44 / 54 Table 2 Inoculation Volumes in Shaker Bottles Used for Testing Impact of Inoculation Density on the Growth of P. polymyxa in 250 mL Shaker Bottles Containing 30 mL of PX-125 Medium % vol. of inoculation [v / v] in culture medium mL of WCB used for inoculation of 30 mL of medium 0.1% 0.03 0.2% 0.06 0.4% 0.12 0.6% 0.18 0.8% 0.24 1.0% 0.3 2.0% 0.6 4.0% 1.2

[086] The WCB1041, used in this culture, was generated after 72 h of culture in a shaken flask using a complex soy-based medium. Before filling, microscopic evaluations were performed to identify the progress of sporulation in the culture. Under the microscope, spores and cells containing endospores were detected. Subsequently, the WCB of the P. polymyxa LU21132 strain was filled into 2 mL cryoculture flasks with 25% glycerol and 5% sucrose. The preparation procedure for all WCBs used in this study is listed in Table 8. Growing conditions

[087] The composition of PX-125 is listed in Table 3. The components of the stock solution were dissolved in distilled water and sterilized by filtration or autoclaving at 121 °C, 1 bar overpressure for 60 min. The antifoaming agent was added to the main solution shortly before starting the autoclaving process. After mixing the stock solutions, the pH of the medium was adjusted to 6.5 with 25% (w / w) ammonia solution or 40% (w / w) phosphoric acid. Petition 870250087381, dated 09 / 26 / 2025, pp. 92 / 113 45 / 54 Table 3 Composition of the PX-125 Complex Medium with the Specification For Storage (Room Temperature (RT) or 4 °C) and Method of Sterilization (Sterile Filtration(s) or Autoclaving(a)) of the Solution Stock Stock Solution Component Concentration in medium [g / L] Main solution (TA, a) Citric acid monohydrate 3.21 Dipotassium hydrogen phosphate 1.00 Ammonium sulfate 1.07 Magnesium sulfate heptahydrate 1.62 Yeast extract 5.00 Soy flour 10.00 Antifoaming agent 0.2 Calcium nitrate tetrahydrate (4 °C, s) Calcium Nitrate Tetrahydrate 0.342 Maltose (TA, a) Vitamin solution (4 °C, s) Maltose monohydrate 63.00 Thiamine hydrochloride 0.005 Nicotinic acid 0.005 Riboflavin 0.0002 Biotin 0.00005 Calcium pantothenate 0.001 Pyridoxine hydrochloride 0.005 Vitamin B12 0.00005 Lipoic acid 0.00005 Trace element solution (4 °C, s) Manganese sulfate monohydrate 0.013 Copper sulfate pentahydrate 0.0046 Sodium molybdate dihydrate 0.0028 Iron sulfate monohydrate 0.015 Citric acid monohydrate 0.4 Water Add 1L pH 6.5.

[088] The culture was carried out in 250 mL shaker flasks with baffles containing 30 mL of PX-125 culture medium sealed with breathable silicone stoppers. The culture was carried out at 33 °C, 150 rpm and 25 mm shaking frequency for 24h.

[089] To determine bacterial growth, measuring Petition 870250087381, dated 09 / 26 / 2025, pp. 93 / 113 Optical density (OD600) at 600 nm was determined using the corresponding uninoculated medium as a blank. The OD of the culture experiment with different inoculation volumes is shown in Figure 1. Example 2 Growth Curve of Microtiter Plate Scale (MTP) Cultures at Different Inoculation Volumes (Fig. 2)

[090] Paenibacillus LU20754 cultures were performed in 48-well microtiter plates with a flower geometry, sealed with a sterile m2p labs membrane (F-GPR48-10, gas-permeable sealing sheet with evaporation-reducing layer). The culture parameters were a shaking frequency of 900 rpm, a shaking diameter of 3 mm, a total well volume of 2.4 mL, and a liquid volume of 1.2 mL per well, 85% humidity, and 35 °C. Biomass formation was evaluated by light scattering analysis of each well at excitation and emission wavelengths of 620 nm, respectively. Different inoculation rates (0.6, 1.2, and 1.8% v / v) of a WCB spore (7.95E + 07 spores / mL) of the P. polymyxa LU20754 strain were used. Example 3 Cell Size and Arrangement (Figs. 3 + 4) Depending on Inoculation Volume

[091] The LU21552 strain of P. polymyxa was cultivated in a 1 L shaker flask without baffles, using 100 mL of soy-based medium. Two different inoculation volumes, 0.2% and 1% (v / v), respectively, were tested using the WCB1059 spore generated after 72 h of fermentation (see Table 8). Cultivation was carried out at 33 °C, 250 rpm, and orbital shaking with a diameter of 5 cm. Optical microscopy evaluation was performed at 1000x magnification after 28 h of cultivation.

[092] The corpuscular length of the cells was determined Petition 870250087381, dated 09 / 26 / 2025, pp. 94 / 113 47 / 54 using the particle analysis function implemented in Fiji (Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012, 9: 676-682. DOI: 10.1038 / nmeth.2019) and calculating the Feret diameter of each particle recognized by the particle analyzer (see images with particle outlines for selected cells). First, a threshold was applied to the RGB microscopy image using the standard thresholding method to generate a binary image. Subsequently, the segmented image was used as input for the particle analysis function, and the corpuscular lengths of all analyzed cells are displayed in the images, showing the cell outlines as well as their number, in the analysis report.The following list shows the corpuscular lengths of each cell corpuscle identified in Figures 3A and 3B. Table 4 Fig. 3B Corpuscular cell lengths [pm] Fig. 3A Corpuscular cell lengths [pm] 8.183 3 2.87 2 2.26 2 1.96 1 1.80 2 1.64 6 1.51 1 1.41 2 1.31 1.17 4 1.02 6 7.259 2.746 2.71 2.21 5 1.94 1 1.80 1 1.63 2 1.49 4 1.41 2 1.28 9 1.17 4 0.98 5 6.469 2.728 2.70 5 2.16 6 1.93 7 1.80 1 1.62 3 1.49 4 1.38 7 1.28 9 1.17 2 0.97 3 6.319 2.687 2.59 5 2.12 8 1.92 8 1.80 1 1.60 1 1.48 6 1.37 6 1.28 9 1.16 4 0.97 3 5.848 2.676 2.56 7 2.12 8 1.90 3 1.79 4 1.60 1 1.48 1.37 6 1.28 1.16 4 0.97 3 5.385 2.617 2.56 1 2.1 1.9 1.78 4 1.58 6 1.48 1.37 1.26 9 1.15 4 0.92 6 5.294 2.617 2.51 5 2.07 8 1.9 1.77 1 1.58 6 1.47 8 1.37 1.26 2 1.14 4 0.89 7 4.954 2.53 2.51 2 2.07 1.88 6 1.75 6 1.57 8 1.47 8 1.37 1.25 2 1.13 6 0.86 4.123 2.463 2.50 6 2.07 1.87 8 1.75 6 1.56 9 1.47 1.36 3 1.25 2 1.13 6 0.79 2 4,077 2,463 2,49 6 2,06 4 1,86 1 1,75 4 1,56 9 1,46 1,36 3 1,25 2 1,13 6 Petition 870250087381, dated 09 / 26 / 2025, pp. 95 / 113 48 / 54 Fig. 3B Corpuscular cell lengths [pm] Fig. 3A Corpuscular cell lengths [pm] 4.033 2.415 2.48 1 2.06 4 1.86 1 1.75 4 1.56 9 1.45 1 1.36 1 1.21 6 1.12 3.586 2.139 2.46 3 2.03 7 1.85 3 1.75 4 1.55 8 1.45 1 1.36 1 1.21 6 1.12 3.523 1.978 2.41 5 2.03 2 1.85 3 1.75 4 1.55 6 1.44 5 1.36 1 1.21 6 1.12 3.438 2.40 9 2.03 1 1.84 9 1.72 9 1.54 8 1.43 7 1.34 3 1.21 6 1.10 1 3.427 2.38 6 2.02 4 1.82 8 1.69 9 1.52 7 1.43 1 1.34 3 1.21 6 1.09 3 3.388 2.34 3 2.02 4 1.82 4 1.68 9 1.51 7 1.41 8 1.32 8 1.21 6 1.06 9 3.379 2.34 3 2.01 3 1.81 1 1.68 7 1.51 5 1.41 8 1.32 3 1.17 7 1.06 9 3.101 2.30 9 2.00 6 1.81 1 1.68 1.51 5 1.41 8 1.32 3 1.17 7 1.04 6 3.048 2.29 5 1.98 5 1.80 2 1.68 1.51 5 1.41 4 1.31 4 1.17 4 1.04 6 3.043 2.26 4 1.97 1.80 2 1.64 6 1.51 1 1.41 4 1.31 1.17 4 1.04 6

[093] Thus, the cutoff values ​​for corpuscular cell length in μm for the respective percentiles are: Table 5 Percentile Fig. 3B Fig. 3A 0% (=minimum) 2.0 0.8 25% (=25th percentile of corpuscular cell length) 2.7 1.3 50% (=median) 3.4 1.5 75% (=75th percentile of corpuscular cell length) 4.1 1.9 100% (=maximum) 8.2 2.9 Example 4 Impact of Inoculation Volume in the Shaker Bottle on Fusaricin Production by P. polymyxa LU21132 (Fig. 5)

[094] Fusaricin production was evaluated in the culture of example 1 after 24 hours of cultivation. For this, 50 pL of culture broth were mixed with 950 pL of acetonitrile-water mixture (1:1) for extraction. The sample was treated for 30 minutes at 20 °C in an ultrasonic bath. The sample was then centrifuged for 5 min at 14,000 rpm and the supernatant filtered into an HPLC vial for measurement. The fusaricidin concentration was determined by HPLC. Petition 870250087381, dated 09 / 26 / 2025, page 96 / 113 49 / 54 UV-VIS, as listed in Table 6 and Table 7. Table 6 HPLC Configuration for Quantification of Fusaricidin A, B, and C in Culture Broth Samples Column: Aqua C18, 250x4.6mm (Phenomenex) Pre-column: Aqua C18 Temperature: 40 °C Flow rate: 1.00 mL / min Injection volume: 2.0 pL Detection: UV 200nm Maximum pressure: 400 bar Holding time: 20 min Eluent A: H2O with 0.1% H3PO4 Eluent B: acetonitrile Table 7 Solvent Gradient for HPLC-Based Quantification of Fusaricidin A, B, and C in Culture Broth Samples Time [min] A [%] B [%] Flow [mL / min] 0.0 70.0 30.0 1.00 6.0 60.0 40.0 1.00 12.0 0.00 100.0 1.00 16.0 0.00 100.0 1.00 16.10 70.0 30 1.00

[095] The production of Fusaricidin A, B and D in the Example culture is shown in Figure 2. Example 5 The effect of WCB inoculum density is independent of the WCB preparation procedure (Figs. 6 + 7).

[096] Several working cell bank (WCB) preparation procedures were tested to identify strain sensitivity to inoculation volumes in pre-cultures in shake flasks. The WCB preparation procedures used are listed in Table 8. Petition 870250087381, dated 09 / 26 / 2025, pp. 97 / 113 50 / 54 Table 8 WCB Preparation Protocols WCB No. Strain Preparation medium Scale Culture time Cell type before collection Cryopreservation 1003 LU207 54 PX-48 1L bioreactor 48h Spores 10% glycerol 5% sucrose -80°C 1041 Cryo LU211 32 PX-99 1L shaker flask 72h Cells with endospores, free spores 25% glycerol 5% sucrose; -80°C 1041 Lyo LU211 32 Lyophilization of culture broth, TA 1048 LU211 32 PX-105 1L bioreactor 48h Viable cells with and without endospores 25% glycerol 5% sucrose; -80 °C 1049 LU211 32 PX-125 1050 LU211 32 PX-105 72h Cells with endospores, free spores 1051 LU211 32 PX-125 1059 LU215 52 PX-162 1L bioreactor 72h Spores 25% glycerol 5% sucrose; -80 °C

[097] 0.3% and 0.6% (v / v, see Table 2) of six different WCBs of the LU21132 strain (see Table 8) were collected to initiate shake flask cultivation. The WCB flasks were heated to 60 °C for 30 minutes before inoculation with PX-125 preculture medium. Cultivation was performed in a 250 mL shake flask, according to the procedure of Example 1.

[098] Bacterial growth, assessed by OD600 analyses, was evaluated after 24 h of culture and the results are shown in Figure 6. In addition, the production of the secondary metabolite Fusaricidin was quantified after 24 h of growth using the method described in Example 2. The level of Fusaricidin as a function of the WCB inoculation volume is shown in Figure 7. Petition 870250087381, dated 09 / 26 / 2025, pp. 98 / 113 51 / 54 Example 6 The Volume of Pre-Culture Inoculum Affects the Fusaricin Titer in the Main Culture (Two-Stage Process) (Fig. 8)

[099] After 24 h of cultivation of the precultures in example 3, 600 pL of each approach were transferred to the main culture medium PX-143 to investigate the effect of the preculture inoculation volume on performance in a two-stage process.

[0100] Main culture medium: PX-143. Table 9 Composition of PX-143 Complex Medium with Specification for Storage (Room Temperature (RT) or 4 °C) and Sterilization Method (Sterile Filtration (s) or Autoclaving (a)) of the Solution Stock Component Concentration in medium [g / L] Main solution (TA, a) Citric acid monohydrate 3.21 Dipotassium hydrogen phosphate 1.00 Ammonium sulfate 1.07 Magnesium sulfate heptahydrate 1.62 Yeast extract 10.00 Soy flour 13.00 Antifoaming agent 0.2 Calcium nitrate tetrahydrate (4 °C, s) Maltose (TA, a) Calcium nitrate tetrahydrate 0.342 Maltose syrup (50%) 156.00 Vitamin solution (4 °C, s) Thiamine hydrochloride 0.005 Nicotinic acid 0.005 Riboflavin 0.0002 Biotin 0.00005 Calcium pantothenate 0.001 Pyridoxine hydrochloride 0.005 Vitamin B12 0.00005 Lipoic acid 0.00005 Trace element solution (4 °C, s) Manganese sulfate monohydrate 0.013 Copper sulfate pentahydrate 0.0046 Sodium molybdate dihydrate 0.0028 Petition 870250087381, dated 09 / 26 / 2025, pp. 99 / 113 52 / 54 Component Concentration in medium [g / L] Ferrous sulfate monohydrate 0.015 Citric acid monohydrate 0.4 Buffer (4 °C, s) 2-(N-morpholino)ethanesulfonic acid 100mM Water Add 1L pH: 6.5.

[0101] Once again, fusaricidin production in the main cultures was evaluated after 48 hours of cultivation using the method described in Example 2. The fusaricidin levels detected in the culture of Example 4 are shown in Figure 5. Here, the fusaricidin level of the main cultures that were inoculated with the pre-culture flasks with a pre-culture inoculation volume of 0.6% is shown in relation to the fusaricidin level detected in the parallel main cultures of the 0.3% pre-culture inoculation variant. Example 6 The volume of pre-culture inoculum synchronizes growth in the main culture (two-stage process) of several WCBs (Fig. 9).

[0102] Biomass formation in the main crops of Example 5 was analyzed by OD600 measurements in samples of the final culture broth after 48 h of cultivation. Figure 6 shows the variation of OD600 [AU] values ​​of the culture using the six WCBs from Table 8 at 0.3% and 0.6% of the inoculation volume. Culture conditions: 250 mL shake flasks with baffles, 30 mL of medium, 150 rpm, orbital shaking with a diameter of 2.5 cm, 33 °C, pH 6.5, 24 h pre-culture, 48 h main culture. Example 7 The Inoculation Volume of the Main Culture Influences the Viscosity of the Culture Broth (Fig. 10)

[0103] The impact of inoculation volume on the viscosity of the culture broth was analyzed in soy-based exopolysaccharide production medium (Table 10). Petition 870250087381, dated 09 / 26 / 2025, pages 100 / 113 53 / 54 Table 10 Composition of the Exopolysaccharide Production Medium With the Specification for Storage (Room Temperature (RT) or 4 °C) and Sterilization Method (Sterile Filtration (s) or Autoclaving (a)) of Stock Solutions Component Concentration in medium [g / L] Main solution (TA, a) Dipotassium hydrogen phosphate 1.67 Calcium chloride dihydrate 0.05 Magnesium sulfate heptahydrate 1.33 Soy peptone 5 Glucose (TA, a) Glucose monohydrate 50 Vitamin solution (4 °C, s) Thiamine hydrochloride 0.005 Nicotinic acid 0.005 Riboflavin 0.0002 Biotin 0.00005 Calcium pantothenate 0.001 Pyridoxine hydrochloride 0.005 Vitamin B12 0.00005 Lipoic acid 0.00005 Trace element solution (4 °C, s) Manganese sulfate monohydrate 0.013 Copper sulfate pentahydrate 0.0046 Molybdate Sodium dihydrate 0.0028g Ferrous sulfate monohydrate 0.015g Citric acid monohydrate 0.4g Buffer (4°C, s) 2-(N-morpholino)ethanesulfonic acid 100mM Water Add 1L pH: 7

[0104] The cultivation of P. polymyxa LU21132 was carried out in 250 mL shake flasks with baffles, filled with 30 mL of medium (Table 10) at 150 rpm, orbital agitation with a diameter of 2.5 cm, 33 °C and pH 6.5. Viscosity was evaluated after 16 h of cultivation using an Anton Paar rheometer, type MCR302, by rotary rheology, using a cylindrical geometry. Measurements were performed at T = 33 °C. Initially, the samples were preconditioned in a pre-shear experiment at a constant shear rate of 10 s⁻¹ for 100 s. 10 data points are recorded every 10 s. After preconditioning, viscosity is measured as a function of shear rate. Petition 870250087381, dated 09 / 26 / 2025, pages 101 / 113 54 / 54 Therefore, the shear rate varied from 1 s-1 to 100 s-1. Twenty-five (25) data points are recorded logarithmically. As indicated by OD600, EPS formation is, in part, dissociated from growth, with special emphasis on inoculation volumes of 2%, 4% and 6%. Petition 870250087381, dated 09 / 26 / 2025, pages 102 / 113

Claims

1 / 5 Claims 1. MICROORGANISM CULTURE, characterized by the microorganism being from the Paenibacillaceae family, preferably from the Paenibacillus genus, in which the culture cells are: - metabolically aligned and / or - in an activated metabolic state.

2. CULTURE, according to claim 1, characterized: a) by the median corpuscular length of the cells being from 0.8 to 2.9 micrometers (µm), more preferably from 1 to 2.5 µm, even more preferably from 1.3 to 2 µm, even more preferably from 1.3 to 1.8 µm, and / or b) by the 75th percentile of the corpuscular length of the cells being from 1.3 to 3 µm, more preferably from 1.3 to 2.9 µm, more preferably from 1.5 to 2.5 µm, and / or c) by the 25th percentile of the corpuscular length of the cells being from 0.8 to 2.5 µm, more preferably from 0.8 to 1.9 µm, and more preferably from 0.8 to 1.5 µm.

3. CULTURE, according to any one of claims 1 or 2, characterized in that it is obtained, or can be obtained, by inoculation, in a culture medium, of microorganisms in a concentration sufficient to produce said culture, wherein the concentration is preferably determined: i) by cultivating a series of cultures inoculated with different inoculation concentrations for a time sufficient to differentiate the cultures by a parameter indicative of metabolic activity, wherein the parameter is preferably selected from any of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH course, Petition 870250087381, dated 09 / 26 / 2025, p.103 / 113 2 / 5 gas formation, oxygen uptake rate, CO2 transfer rate and variance of any of the parameters mentioned above, and ii) by determining one or more parameters, and iii) by selecting an inoculation concentration based on one or more determined parameters.

4. FERMENTATION METHOD, characterized by comprising: i) providing a fermentation broth comprising a culture of microorganisms, as defined in any one of claims 1 to 3, in a fermentation medium, and ii) cultivating the fermentation broth under suitable conditions to produce a target substance, and iii) preferably, recovering the target substance.

5. FERMENTATION METHOD, according to claim 4, characterized in that the microorganism culture in step i) is obtained, or can be obtained, by: ii) inoculating a pre-culture fermentation medium with a quantity of microorganisms, and i.ii) cultivating the culture of step ii) so as to provide: a) the median cell length of 0.8 to 2.9 micrometers (µm), more preferably from 1 to 2.5 µm, even more preferably from 1.3 to 2 µm, even more preferably from 1.3 to 1.8 µm, and / or b) the 75th percentile of the cell length of 1.3 to 3 µm, more preferably from 1.3 to 2.9 µm, more preferably from 1.5 to 2.5 µm, and / or c) the 25th percentile of the cell length of 0.8 to 2.5 pm, more preferably from 0.8 to 1.9 pm, and more preferably from Petition 870250087381, dated 09 / 26 / 2025, page 104 / 113 3 / 5 0.8 to 1.5 pm.

6. FERMENTATION METHOD, according to any one of claims 4 to 5, characterized in that the target substance is selected from: - microorganisms, preferably viable cells and / or spores, - one or more metabolic products, preferably at least one fusaricidin, paeniserin, paeniprolixin or polymyxin, preferably at least two or more fusaricidins, paeniserins, paeniproxilins or polymyxins, more preferably from 3 to 120 fusaricidins, wherein one or more fusaricidins comprise any one of fusaricidin A, B or D and / or surfactin and / or iturin.

7. METHOD FOR REDUCING THE FERMENTATION TIME REQUIRED to produce a desired quantity of target substance and / or to reduce viscosity during fermentation and / or to increase the reproducibility of fermentation, characterized by comprising the steps of: i) providing, in a fermentation medium, a culture, as defined in any of claims 1 to 3, and ii) cultivating the culture in the fermentation medium.

8. USE OF A CULTURE, as defined in any one of claims 1 to 3, characterized in that it reduces the fermentation time required to produce a desired quantity of a target substance and / or reduces viscosity during fermentation and / or increases the reproducibility of fermentation.

9. CULTURE PARAMETER DATA SET for controlling a fermentation, characterized by comprising: - a designation of a set of applicable fermentation media, Petition 870250087381, dated 26 / 09 / 2025, page 105 / 113 4 / 5 - a designation of a set of applicable microorganism samples, - a designation of fermentation conditions conducive to obtaining a culture, as defined in any of claims 1 to 3, by cultivating an applicable sample in an applicable fermentation medium, wherein the fermentation conditions preferably comprise at least one of the following: type of culture medium, quantity of culture medium, desired culture temperature range, desired culture aeration parameter ranges, desired quantity of cells produced, desired duration of culture, desired pH during culture, desired yield or quantity of a target substance.

10. COMPUTER-IMPLEMENTED FERMENTATION METHOD, characterized by comprising: - receiving, by a computing device, a set of cultivation parameter data, as defined in claim 9, - receiving a fermentation medium conforming to the set of cultivation parameter data, - receiving a volume of a sample of microorganisms conforming to the set of cultivation parameter data, and - controlling, by the computing device, a fermenter to cultivate the microorganisms and / or their spores in the fermentation medium under the fermentation conditions of the set of cultivation parameter data.

11. A computer-implemented fermentation method according to claim 10, characterized in that the control comprises: - determining one or more parameters indicative of metabolic activity, wherein the parameter is preferably selected from any of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH course, gas formation, oxygen uptake rate, CO2 transfer rate and the variance of any of the aforementioned parameters, - comparing one or more determined parameters with the target fermentation conditions, and - adjusting the fermentation conditions to promote the conformity of one or more determined parameters with said target fermentation conditions.

12. METHOD FOR PRODUCING ONE OR MORE SUBSTANCES that increase viscosity, characterized by comprising: i) inoculating a fermentation medium with a concentration of a microorganism culture, as defined in any one of claims 1 to 3, at a predetermined concentration sufficient to obtain, during cultivation, a desired viscosity, and ii) cultivating the culture obtained in step i), and iii) preferably, recovering one or more substances that increase viscosity. Petition 870250087381, dated 09 / 26 / 2025, pp. 107 / 113

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