Iron chelates for inducing and enhancing biological processes in microorganisms and plants

Abstract

The present invention relates to iron chelates, in particular iron(III)-chelates ferric quinate, ferric citrate, ferric lactate and ferric tyrosine, for enhancing and improving various biological processes such as microbial growth and diverse metabolites production, and seed germination and growth.

Description

[0001] Iron chelates for inducing and enhancing biological processes in microorganisms and plants

[0002] Technical field

[0003] The present invention relates to iron chelates, in particular iron(l I l)-chelates , for enhancing and improving various biological processes such as microbial growth and diverse metabolites production, and seed germination and growth.

[0004] Background

[0005] Induction

[0006] Induction is a process that involves the activation of dormant or latent genetic material in cells (Ptashne & Gann, 1997). The process is triggered by environmental cues or external stimuli that activate a specific genetic pathway, leading to the expression of previously silent genes. One of the most well-known examples of induction is the process of lysogeny in bacteriophages, which are viruses that infect bacteria (Little & Shepley, 1989).

[0007] Induction is not limited to bacteriophages; many (micro)organisms have complex regulatory pathways that allow them to respond to changes in their environment (Lee & Palsson, 2010). Some bacteria can for example sense the presence of nutrients, such as sugars or amino acids, and activate the expression of genes involved in their uptake and metabolism. Others can sense the presence of toxins or other stressors and activate genes that allow them to resist or detoxify these compounds.

[0008] The molecular mechanisms underlying induction are complex and vary depending on the specific genetic pathway involved. It can involve the binding of a regulatory protein to a specific DNA sequence (Stock, Robinson, & Goudreau, 2000) or the production of small signalling molecules, such as quorum-sensing molecules or cyclic AMP (Waters & Bassler, 2005).

[0009] Bacterial induction in particular has important implications for both basic research and practical applications. In the laboratory, it can be used to study the regulation of gene expression and the function of specific genes (Joska, Mashruwala, Boyd, & Botsford, 2014). It is also utilized in biotechnology and medicine to produce large quantities of recombinant proteins (Kim & Hegde, 2019). Recombinant protein expression involves cloning a gene of interest into a bacterial expression vector, which contains regulatory sequences that can be induced to activate gene expression. This enables the production of the desired protein (de Carvalho & da Fonseca, 2005).

[0010] Moreover, bacterial induction is being explored as a potential therapeutic approach for the treatment of bacterial infections. Small molecules can be used to activate the expression of genes involved in bacterial killing or virulence attenuation (Mulcahy, Charron-Mazenod, Lewenza, & Baird, 2008). For example, inducing the expression of bacteriocins, small peptides that can kill bacteria, or virulence factors that trigger an immune response against the bacteria, can help clear the infection (Lopez, Sierra, Castano, & Rios, 2004).

[0011] Lactobacillus and yeast strains are important microorganisms widely utilized in various industries due to their ability to produce valuable compounds through fermentation processes. Inducing their growth and metabolic activities are crucial factors in the efficiency and quality of industrial applications such as food and beverage production, biofuel generation, and pharmaceutical manufacturing (De Vuyst and Weckx, 2016; Fleet, 2003; Liu et al., 2020).

[0012] Biofilm formation

[0013] The strong biofilm formation capability of Bacillus subtilis has significant impacts on various industries and thus offers a wide range of potential applications across industries, providing benefits such as enhanced productivity, improved sustainability, and increased efficiency in various processes. Below are some of the notable effects (Branda et al. 2006):Agriculture and Plant Growth Promotion: B. subtilis biofilms can enhance plant growth by promoting nutrient uptake, stimulating root development, and protecting plants against pathogens. This has applications in agriculture, horticulture, and sustainable farming practices.

[0014] Bioremediation and Environmental Applications: B. subtilis biofilms can aid in the degradation of organic pollutants, heavy metals, and hydrocarbons, making them valuable in bioremediation efforts for contaminated soil, water, and industrial waste treatment.

[0015] Food and Beverage Production: B. subtilis biofilms have been utilized in various aspects of food and beverage production. They can improve fermentation processes, enhance flavours, and contribute to the development of desirable textures in fermented foods. Additionally, they can provide protection against foodborne pathogens.

[0016] Biotechnology and Enzyme Production: Biofilms of B. subtilis serve as a platform to produce enzymes, bioactive compounds, and other valuable biomolecules. Their robustness and stability make them suitable for large-scale biotechnological applications.

[0017] Biofouling Prevention: While biofilms can be problematic in certain industrial settings, B. subtilis biofilms can be harnessed to prevent biofouling. By forming a protective layer on surfaces, they can inhibit the attachment and growth of unwanted microorganisms, reducing maintenance requirements and improving the lifespan of equipment in industries such as water treatment, oil and gas, and marine industries.

[0018] Biomedical and Pharmaceutical Applications: B. subtilis biofilms have shown potential in various biomedical applications, including wound healing, drug delivery, and probiotic formulations. Their ability to adhere to surfaces and withstand harsh conditions make them promising candidates for targeted drug delivery and bioactive compound release.

[0019] Biofuel and Biochemical Production: B. subtilis biofilms can be used in the production of biofuels and biochemicals through fermentation processes. Their ability to efficiently convert renewable feedstocks into valuable products makes them attractive for sustainable energy and chemical production.

[0020] Seed germination and growth

[0021] Light is a crucial environmental factor that plays a significant role in plant growth and development. The quality and quantity of light can influence various physiological processes in plants, including seed germination, photosynthesis, and hormone regulation. Different wavelengths of light, such as red, blue, green, yellow, and white, have distinct effects on plant growth due to their differential absorption and utilization by photoreceptors.

[0022] Additionally, light conditions can impact the microbial communities present in the soil, including the composition and activity of beneficial bacteria. The soil microbiome plays a vital role in nutrient cycling, disease suppression, and plant health. Thus, understanding the relationship between light conditions and soil bacterial flora is important for optimizing soil fertility and promoting sustainable agriculture.

[0023] Summary

[0024] The present invention provides methods and compositions for inducing, increasing and / or enhancing various biological processes. The invention does for example provide methods and compositions for inducing and / or increasing growth of microorganisms and plants; biofilm formation; microbial production of various metabolites.

[0025] The inventor has surprisingly discovered that certain iron(l I l)-chelates can be used to induce, increase and / or enhance various biological processes, such as growth of microorganisms and plants; biofilm formation; microbial production of various metabolites.

[0026] Hence, in one aspect, the present invention provides a method for inducing and / or increasing growth of a microorganism selected from bacteria and / or yeast, said method comprising incubating the microorganism in a medium comprising an iron chelate according to the invention, thereby inducing and / or increasing growth of the microorganism; wherein theincrease in growth is as compared to the growth of a microorganism incubated in the absence of an iron chelate, but otherwise cultivated under the same conditions; and wherein the iron chelate is selected from the group consisting of ferric citrate (iron(lll) citrate), ferric quinate (iron(lll) quinate), ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof.

[0027] In another aspect, the invention provides a method for inducing and / or increasing metabolism of a microorganism selected from bacteria and / or yeast, said method comprising incubating the microorganism in a medium comprising an iron chelate according to the invention, thereby inducing and / or increasing metabolism of the microorganism;

[0028] wherein the metabolism is increased as compared to the metabolism of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions; and wherein the iron chelate is selected from the group consisting of ferric quinate, ferric lactate, and ferric citrate.

[0029] The invention also provides a composition comprising:

[0030] a. ferric quinate and ferric lactate;

[0031] b. ferric quinate and ferric citrate;

[0032] c. ferric lactate and ferric citrate; or

[0033] d. ferric quinate, ferric lactate and ferric citrate;

[0034] optionally wherein the composition comprises:

[0035] a. between 10 and 90% of ferric quinate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric quinate; and / or b. between 10 and 90% of ferric lactate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric lactate; and / or c. between 10 and 90% of ferric citrate, such as between such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric citrate;

[0036] optionally wherein the composition comprises:

[0037] a. between 25 and 35% ferric quinate, between 25 and 35% ferric lactate, and between 25 and 35% ferric citrate; or

[0038] b. between 45 and 55% ferric citrate and between 45 and 55% ferric lactate; or c. between 45 and 55% ferric citrate and between 45 and 55% ferric quinate; or d. between 45 and 55% ferric quinate and between 45 and 55% ferric lactate; with the proviso that the sum of the percentage of iron chelates is not >100%.

[0039] The invention further provides a composition comprising:

[0040] a. an iron chelate according to the invention; and

[0041] b. isopropyl p-d-1-thiogalactopyranosid (IPTG)

[0042] optionally wherein the composition comprises:

[0043] c. between 10 and 90% of iron chelate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of iron chelate; and d. between 10 and 90% of IPTG, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of IPTG;

[0044] optionally wherein the composition comprises:

[0045] e. between 5 and 15% of ferric lactate; and

[0046] f. between 30% and 50% of ferric quinate; and

[0047] g. between 30% and 50% of IPTG;

[0048] with the proviso that the sum of the percentage of iron chelate and IPTG is not >100%. The invention also provides a method for inducing and / or increasing biofilm formation by a microorganism selected from bacteria and / or yeast, such as a bacteria, in particular a non-pathogenic bacteria, said method comprising incubating the bacteria in a media comprising aniron chelate according to the invention, thereby inducing and / or increasing biofilm by the bacteria;

[0049] wherein the increase in biofilm formation is as compared to the biofilm formation of a bacteria incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions; wherein the iron chelate is selected from the group consisting of ferric quinate, ferric lactate, ferric tyrosine and ferric citrate;

[0050] optionally wherein the bacteria is gram positive, such as wherein the bacteria is of the genus Bacillus, such as for example Bacillus subtilis

[0051] optionally wherein the concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 50 pM and 750 pM, such as between 25 pM and 50 pM, such as between 50 pM and 250 pM, such as between 100 pM and 400 pM, such as between 150 pM and 350 pM, such as between 200 pM and 300 pM, such as between 300 pM and 500 pM, such as between 350 pM and 450 pM, such as between 150 pM and 200 pM;

[0052] optionally wherein the bacteria are incubated:

[0053] a. in light conditions, optionally wherein the light is selected from the group consisting of white light, blue light and red light; or

[0054] b. in dark conditions, i.e. in the absence of light; and / or

[0055] c. at a temperature of between 15°C and 35°C, such as between 20°C and 30°C, such as between 20°C and 25°C; and / or

[0056] d. for at least 1 day, such as at least 2 days, such as at least 3 days; and / or e. under static conditions.

[0057] The invention relates in particular embodiments to methods wherein the microorganism, such as the bacteria, are non-pathogenic bacteria, as described under Microorganisms.

[0058] Description of drawings

[0059] Figure 1. The induction of soluble plasmid protein PTA by complexes FeQ, Fe-cit, Fe-lac, or IPTG alone.

[0060] Figure 2. The induction of soluble plasmid protein YQFL by complexes Fe-cit, FeQ, Fe-lac, or IPTG alone.

[0061] Figure 3. Overexpression of soluble plasmid protein induced using complexes FeQ and Fe-lac in combination with IPTG in E. coH carrying the plasmid culture.

[0062] Figure 4. Overexpression of soluble plasmid proteins by complexes (FeQ and Fe-lac) in combination with IPTG.

[0063] Figure 5. The expression of TolC protein naturally produced by enteropathogenic E. coH (EPEC) wild type and the effect of complexes FeQ and Fe-cit on TolC protein expression.

[0064] Figure 6. Increased concentration of induction compounds applied to bacterial (B. subtilis) samples generally lead to an increase in biofilm formation and bacterial growth. The data presented is based on two repeated experiments.

[0065] Figure 7. Examination of compounds (FeQ, Fe-lac, Fe-cit and Fe-tyr) effects on B subtilis biofilm formation in a concentration dependent manner. Data was generated from Crystal Violet Assays.

[0066] Figure 8. Biofilm formation of Bacillus subtilis cultured on minimal salts glutamate glycerol (MSgg) + 0.5% glutamate agar at 30°C.

[0067] Figure 9. Biofilm formation of B. subtilis supplemented with ferric quinate (FeQ), ferric lactate (Fe-lac), ferric citrate (Fe-cit) and ferric tyrosine (Fe-tyr).

[0068] Figure 10. Bacillus subtilis cells cultivated for a duration of 14 days on LBGM plates containing nutrients that promote biofilm formation. These plates were subjected to different light conditions, including white, blue, red, and no light (dark), in the presence and absence of FeQ at a concentration of 400 pM.Figure 11. B. subtilis floating biofilm (pellicle) formation in different concentrations of iron chelate.

[0069] Figure 12. Biofilm formation on MSgg, 0.25 % glutamate. B. subtilis cells were cultured on MSgg agar supplemented with 0.25% glutamate.

[0070] Figure 13. B. subtilis biofilm formation. The effects of different iron chelates at concentrations ranging from 200 to 400 pM were assessed on biofilm formation.

[0071] Figure 14. Influence of light and incubation time on the occurrence of a reddish compound from B. subtilis biofilm.

[0072] Figure 15. Influence of culture volume on the occurrence of a reddish compound from B. subtilis biofilm.

[0073] Figure 16. Effect of FeQ induction on biofilm formation investigated by using a bead assay. Figure 17. Score plots of principal component analysis (PCA) generated to analyse the metabolites in LBMG media from B. subtilis and the reddish material isolated from the biofilm after 3 days of incubation in the dark.

[0074] Figure 18. Score plots of principal component analysis (PCA) generated to analyse the metabolites in LBMG media from B. subtilis and the reddish material isolated from the biofilm after 3 days of incubation in the dark

[0075] Figure 19. Effect of Fe-cit in combination with Fe-lac (a total of 85pM) on the growth rate of B. subtilis under anaerobic conditions (square points) as compared to control (FeCI3; round points). Figure 20. Effect of Fe-cit in combination with Fe-lac and FeQ (a total of 85 pM) on the growth rate of B. subtilis under anaerobic conditions.

[0076] Figure 21. Effect of Fe-cit in combination with Fe-lac and FeQ (a total of 170 pM) on the growth rate of B. subtilis under anaerobic conditions.

[0077] Figure 22. Effect of Fe-cit in combination with FeQ (a total of 170 pM) on the growth rate of B. subtilis under anaerobic conditions

[0078] Figure 23. Effect of Fe-lac in combination with FeQ (a total of 170 pM) on the growth rate of B. subtilis under anaerobic conditions

[0079] Figure 24. Score plots of principal component analysis (PCA) performed on metabolites obtained from B. subtilis induced with FeQ, with FeCI3 used as the control.

[0080] Figure 25. Bacterial growth analysis of Lactobacillus strain AD4550 / 01 (14492 ) to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium.

[0081] Figure 26. Bacterial growth analysis of Lactobacillus delbrueckii LA-67 (14499 ) to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium.

[0082] Figure 27. Growth analysis of yeast strain 14502 to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium.

[0083] Figure 28. Bacterial growth analysis of Lactobacillus delbrueckii LA-67 (14499) phenotype Aci to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium.

[0084] Figure 29. Bacterial growth analysis of Lactobacillus strain 14492 phenotype Hel to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium.

[0085] Figure 30. Total gas production during fermentation of BSG residue using Lactobacillus strain (14492).

[0086] Figure 31. Total gas production during fermentation of BSG residue using Lactobacillus strain Lactobacillus delbrueckii LA-67 (14499).

[0087] Figure 32. Total gas production during fermentation of BSG residue using yeast strain 14502. Figure 33. Score plot principal (class-3 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Lactobacillus 14492.Figure 34. Score plot principal (class-3 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Lactobacillus delbrueckii LA-67 (14499). Figure 35. Score plot principal (class-3 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Kluyveromyces marxianus 14502.

[0088] Figure 36. Score plot principal (class-2 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Lactobacillus 14492.

[0089] Figure 37. Score plot principal (class-2 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Lactobacillus Lactobacillus delbrueckii LA-67 (14499).

[0090] Figure 38. Score plot principal (class-2 OPLS-DA modelling) showing a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Spent media was collected from Kluyveromyces marxianus (14502).

[0091] Figure 39. Mean shoot length of 48 Vigna radiata plants that were supplemented with iron chelate and exposed to white light through a transparent cellophane covering.

[0092] Figure 40. Mean shoot length of 48 Vigna radiata plants that were supplemented with iron chelate and exposed to red light through a transparent cellophane covering.

[0093] Figure 41. Mean shoot length of 48 Vigna radiata plants that were supplemented with iron chelate and exposed to blue light through a transparent cellophane covering.

[0094] Figure 42. Mean shoot length of 48 Vigna radiata plants that were supplemented with iron chelate and exposed to yellow light through a transparent cellophane covering.

[0095] Figure 43. Mean shoot length of 48 Vigna radiata plants that were supplemented with iron chelate and exposed to green light through a transparent cellophane covering.

[0096] Figure 44. Mean shoot length of 90 Vigna radiata plants that were supplemented with different concentrations of FeQ and exposed to different colours of light through cellophane covering. Figure 45. Mean shoot length of 90 Vigna radiata plants that were supplemented with different concentrations of Fe-L and exposed to different colours of light through cellophane covering. Figure 46. Ferric Quinate

[0097] Detailed description

[0098] The present invention provides methods and compositions for inducing and / or enhancing various biological processes. The invention does for example provide methods and compositions for inducing and / or increasing growth of microorganisms and plants; biofilm formation; recombinant protein production; and microbial production of various metabolites. Definitions

[0099] The term “blue light” as used herein refers to light with a wavelength of approximately 450-495 nm.

[0100] The terms “exponential growth”, “exponential growth phase” and “exponential phase” are used interchangeably herein and refers to the part of the microbial growth phase characterized by cell doubling. It may also be referred to as “log phase” or “logarithmic phase”. The number of new microorganisms appearing per unit of time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells, i.e. the concentration of cells, i.e. the cellular density, and the rate of population increase doubles with each consecutive time-period. Preferably, for this type of exponential growth, plotting the natural logarithm of cellular density against time produces a straight line, where the slope of this line is the specific growth rate of the microorganism, which is a measure of the number of divisions percell per unit of time. The generation time (GT) and the maximum growth rate (max) is calculated during the exponential phase using methods known in the art, such as the equation below. In Nt- In No = p(t - to)

[0101] p = In2 / GT;

[0102] max = In2 / GT at time when cell growth is maximal

[0103] wherein

[0104] No= Number of cells (directly or indirectly, i.e. cellular density) at time to of an early stage of the exponential phase

[0105] Nt= Number of cells or (directly or indirectly, i.e. cellular density) at time t of a later stage of the exponential phase

[0106] Instead of using the cellular density, another parameter indicative of the number of cells may be used for both No and Nt

[0107] GT=generation time

[0108] = specific growth rate

[0109] Pmax = maximal (i.e. maximum) specific growth rate

[0110] The pmax is commonly calculated by fitting a linear regression to a subset of data with the steepest log-linear increase. The pmax is then defined as the 1stderivative of log-transformed y-values, sometimes after smoothing.

[0111] The term “green light” as used herein refers to light with a wavelength of approximately 500-565 nm.

[0112] The term “growth” as used herein refers to the growth of microorganisms or plants, such as yeast, bacteria or plants. When referring to growth of a microorganism, i.e. microbial growth, the term growth as used herein encompass various stages of growth of said microorganism, hereunder growth at different growth phases including lag phase and exponential phase.

[0113] Microbial growth may be determined using methods known in the art, such as for example by measuring the optical density, i.e. absorbance, at 600 nm (OD600) at regular time intervals over time.

[0114] The term “growth rate” as used herein refers to the number of microbial generations per unit of time, such as generations per hour or per minute. Preferably, the growth rate is the growth rate during the exponential growth phase of the microorganism, such as the generation time or the maximum growth rate (pmax). In some embodiments herein, the growth rate is the generation time of the microorganism, such as the amount of hours it takes for a population of microorganisms to double, wherein said doubling may be a doubling in the number of cells, such as a doubling of the cell density, i.e. a doubling of the concentration of cells.

[0115] The term “increasing growth” as used herein refers to increasing the growth of microorganisms or plants, such as yeast, bacteria or plants. When referring to growth of a microorganism, said term may refer to increasing growth during the growth rate, decreasing the duration of the lag phase, and / or increasing the growth rate during exponential phase, such as the maximum growth rate (pmax). Preferably the growth is increased as compared to the growth rate of a similar microorganism or plant incubated in the absence of iron chelate according to the invention but otherwise grown under the same conditions. Using the methods and compositions of the present invention, growth of a microorganism can for example be increased by decreasing the lag phase and / or increasing the growth rate in the exponential phase (such as the maximum (i.e. the maximal) growth rate (pmax)).

[0116] The term “lag phase” as used herein refers to the period of time it takes from contacting a microorganism with a media, until the microorganism begins to grow, such as until exponential growth is started. In other words, it is the time it takes for the microorganism to adapt to one or more components of the growth medium, such as the time it takes for the microorganism to redirect their metabolism to be able to utilize, i.e. grow on, one or more nutrient sources of thegrowth medium. Generally, the microorganism does not grow during the lag phase, and thus, the number of cells or parameters indicative of the number of cells, preferably the cell density, does not change from the time of contacting the microorganism with a media, until the microorganism begins to grow, i.e. divide.

[0117] The term “pmax” as used herein refers to the maximum growth rate, i.e. the growth rate in the exponential phase, such as the maximum growth rate of a microorganism on a specific media. The term “red light” as used herein refers to light with a wavelength of approximately 625-750 nm.

[0118] The term “yellow light” as used herein refers to light with a wavelength of approximately 565-590 nm.

[0119] The term “white light” as used herein refers to light with a wavelength of approximately 400-700 nm.

[0120] Note that any lower range limit described herein may be combined with any upper range limit described herein to define additional ranges.

[0121] The term “Industrial microorganism” refers to microorganisms used in industrial fermentation or biotechnological production processes.

[0122] Inducing and increasing microbial growth, production and biofilm formation Microbial growth

[0123] Provided herein is a method for inducing and / or increasing growth of a microorganism selected from bacteria and / or yeast and combinations thereof, said method comprising the step of:

[0124] a. incubating the microorganism in a media comprising an iron chelate according to the invention; thereby inducing and / or increasing growth of the microorganism. The invention also relates to a method for producing a microorganism culture displaying increased growth as compared to a control, said method comprising the step of

[0125] a. incubating a microorganism in a medium comprising iron chelate according to the invention;

[0126] thereby producing a microorganism culture displaying increased growth.

[0127] References herein to inducing and / or increasing growth of a microorganism may also be understood as referring to modulation of growth phase kinetics of said microorganisms, including for example changes in lag phase, the maximum specific growth rate, exponential growth rate, biomass accumulation, biomass accumulation prior to stationary phase and / or stationary phase.

[0128] The invention thus provides a method for modulating growth phase kinetics of a microorganism (in particular a non-pathogenic microorganism), characterized in that the microorganism is cultured in a medium comprising a ferric iron(lll) chelate for example an iron chelate selected from ferric quinate, ferric citrate or ferric lactate, where the growth kinetics are modulated as compared to relative to culturing the same microorganism under identical conditions in the presence of an equimolar amount of Fe3+supplied as ferric chloride.

[0129] Particular embodiments relate to where the modulation of growth phase kinetics are one or more of the following: (a) the duration of the lag phase is reduced; (b) the maximum specific growth rate (pmax) is increased; and / or (c) increased biomass accumulation prior to stationary phase.

[0130] In some embodiments, the microorganism is a non-pathogenic microorganism (qv). In some embodiments the ferric iron chelate is provided in amount in the range from 5 to 400 pM (i.e., microM), for example at least 5, In certain embodiments, the amount is from 5 to 300 pM, 5 to 200 pM, 5 to 100 pM, 10 to 400 pM, 25 to 400 pM, 50 to 400 pM, or 100 to 400 pM. In further embodiments, the amount may be from 10 to 300 pM, 10 to 200 pM, 25 to 300 pM, 25 to 200 pM, 50 to 300 pM, or 50 to 200 pM. The medium in which the microorganisms is cultured may be any suitable medium, particular embodiments relate to where the medium is a liquid culturemedium. The method is particularly useful in industrial applications, for example, industrial fermentations. “Industrial fermentation” refers to the cultivation of microorganisms under controlled conditions for the large-scale or commercial production of biomass or one or more fermentation products, typically carried out in fermentation vessels or bioreactors, rather than small-scale laboratory culturing performed solely for analytical or research purposes.

[0131] Thus, particular embodiments of the invention relate to a method for modulating growth phase kinetics of a microorganism used in industrial fermentation,

[0132] characterised in that the microorganism is cultured in a medium, preferably liquid medium, comprising a ferric iron(lll) chelate selected from ferric quinate, ferric citrate or ferric lactate at a concentration of 25-400 pM Fe3+, wherein: (a) the duration of the lag phase is reduced; and / or (b) the maximum specific growth rate (pmax) during exponential phase is increased relative to culturing the same microorganism under identical conditions in the presence of an equimolar amount of Fe3+supplied as ferric chloride. Further particular embodiments relate to where the (a) the duration of the lag phase is reduced; and (b) the maximum specific growth rate (pmax) during exponential phase is increased, relative to culturing the same microorganism under identical conditions in the presence of an equimolar amount of Fe3+supplied as ferric chloride. The microorganism according to the methods of the invention is selected from any bacteria, yeast, or combinations thereof.

[0133] The methods of the invention relate to non-therapeutic methods.

[0134] Also provided herein is the use of an iron chelate according to the invention as described herein, for inducing and / or increasing growth of a microorganism.

[0135] Also provided herein is the use of an iron chelate as described herein for producing a microorganism culture displaying increased growth.

[0136] In one embodiment, the increase in growth is as compared to the growth of the corresponding microorganism when incubated in the absence of the iron chelate according to the invention but otherwise cultivated under the same conditions.

[0137] In one embodiment, the increase in growth is as compared to the growth of the corresponding microorganism when incubated in the absence of the iron chelate according to the invention, but in the presence of ferric chloride (FeCI3), and otherwise cultivated under the same conditions. The term “the same conditions” comprises that the compared medium comprises an equimolar amount of iron to the method of the invention, but that this iron is provided by compounds other than the iron chelate of the invention.

[0138] The method may comprise inducing and / or increasing growth in anyway possible and / or suitable. In one embodiment, the method comprises at least one of:

[0139] a. decreasing the duration of the lag phase of the microorganism;

[0140] b. increasing the growth rate of the microorganism;

[0141] c. increasing the cell density of the microorganism at the end of the exponential phase and / or at the end of incubation.

[0142] The duration of the lag, phase, the growth rate and the cell density may be determined using methods known in the art, for example by measuring the absorbance at 600 nm (OD600) using a spectrophotometer, such as at regular intervals during incubation.

[0143] The control used for comparison in any embodiment of the invention may be a microorganism (or microorganism culture respectively) incubated in the absence of iron chelate according to the invention, but otherwise cultivated under the same conditions,

[0144] Preferably, the control is incubated in the presence of inorganic ferric complex, such as FeCI3, but otherwise cultivated under the same conditions.

[0145] In one embodiment, the growth rate is increased during exponential phase. In one embodiment, the maximum specific growth rate (pmax) is increased as compared to a control.In one embodiment, the growth rate, such as the maximum specific growth rate, is increased at least 1.1-fold, such as at least 1.2-fold, such as at least 1.3-fold, such as at least 1.4-fold, such as at least 1.5-fold, such as at least 1.75-fold, such as at least 2-fold, such as at least 2.5-fold, such as at least 3-fold, such as at least 4-fold, such as at least 5-fold, such as at least 10-fold as compared to a control.

[0146] In one embodiment, the growth rate, such as the maximum growth rate, is increased between 1.1 -fold and 10-fold, such as between 1.2-fold and 5-fold, such as between 1.2-fold and 3-fold, such as between 1.5-fold and 3-fold as compared to a control.

[0147] In one embodiment, the duration of the lag phase is decreased by at least 15 min, such as at least 30 min, such as at least 60 min, such as at least 90 min, such as by at least 3 h, such as by at least 5 h, such as by at least 10 h as compared to as compared to a control, the duration of the lag phase of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0148] In some embodiments of the method according to the invention, the maximum specific growth rate is increased by at least 5%, or for example at least 10% In some embodiments, the maximum specific growth rate increased by from 10% to 80%, such as from 10% to 70%, from 10% to 60%, from 10% to 50%, from 20% to 80%, from 30% to 80%, from 40% to 80%, or from 50% to 80%. In certain embodiments, the increase is from 20% to 70%, 20% to 60%, 30% to 70%, 30% to 60%, or 40% to 70%.

[0149] In one embodiment, the duration of the lag phase is decreased by between 15 min and 10 h, such as by between 1 h and 10, such as by between 5 h and 10 h, such as by between 1 h and 5 h, such as by between 15 min and 90 min, such as by between 30 min and 90 min, such as by between 15 min and 60 min as compared to a control.

[0150] In some embodiments of the method according to the invention, the duration of the lag phase is decreased by at least 5%, or for example at least 10% In some embodiments, the dureation of lag phase is reduced by from 10% to 80%, such as from 10% to 70%, from 10% to 60%, from 10% to 50%, from 20% to 80%, from 30% to 80%, from 40% to 80%, or from 50% to 80%. In certain embodiments, the reduction is from 20% to 70%, 20% to 60%, 30% to 70%, 30% to 60%, or 40% to 70%.

[0151] In particular embodiments, the duration of the lag phase is decreased and the growth rate is increased in the method of the invention.

[0152] In the context of the present disclosure, increased cell density at the end of the exponential phase may also be described as increased biomass accumulation prior to the stationary phase, as both refer to the amount of biomass generated during exponential growth. Thus, in some embodiments of the method of the invention, the biomass accumulation prior to stationary phase is increased relative to ferric chloride control, for example at least 10%. In some embodiments of the method according to the invention, the biomass accumulation prior to stationary phase is increased by at least 5%, or for example at least 10% In some embodiments, increase is from 10% to 80%, such as from 10% to 70%, from 10% to 60%, from 10% to 50%, from 20% to 80%, from 30% to 80%, from 40% to 80%, or from 50% to 80%. In certain embodiments, the reduction is from 20% to 70%, 20% to 60%, 30% to 70%, 30% to 60%, or 40% to 70%.

[0153] In one particular embodiment, the duration of the lag phase is decreased; the growth rate is increased; and the cell density is increased as compared to the duration of the lag phase; the growth rate; and the cell density, of a microorganism incubated in the absence of an iron chelate of the invention, but otherwise cultivated under the same conditions.

[0154] In a further particular embodiment, the duration of the lag phase is decreased; the growth rate is increased; and the cell density is increased as compared to the duration of the lag phase; the growth rate; and the cell density of a microorganism, incubated in the absence of an ironchelate of the invention, but in the presence of ferric chloride (FeCI3), and otherwise cultivated under the same conditions.

[0155] Decreasing the lag phase and / or increasing the growth rate may increase the productivity and / or decrease the overall time for any bioprocess in which the microorganism is involved. Thus, in one embodiment, the present invention also provides a method for increasing productivity and / or reducing processing time of a bioprocess involving a microorganism selected from bacteria and / or yeast, said method comprising the step of:

[0156] a. incubating said microorganism in a media comprising an iron chelate of the invention;

[0157] thereby increasing the productivity and / or reducing the processing time of the bioprocess as compared to the productivity and / or processing time of a bioprocess involving the same microorganism incubated in the absence of an iron chelate of the invention; or, compared to the same microorganism incubated in presence of FeCI3, but otherwise cultivated under the same conditions.

[0158] The productivity may for example be measured as the amount of microbial cells generated per litre and hour (e.g. g cells H h1); the amount of metabolites produced per litre and hour (e.g. g metabolite H h1); and / or the amount of recombinant protein produced per hour (e.g. g protein H h-1).

[0159] The metabolite may be any type of metabolite, such as for example any metabolite described in the section “Microbial metabolite production”.

[0160] The iron chelate according to the invention is as described in the sections “Iron chelate” and “Compositions comprising iron chelates”. In one embodiment, the iron chelate is selected from the group consisting of ferric quinate, ferric citrate and ferric lactate. In one embodiment, concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 100 pM and 1000 pM, such as between 250 pM and 750 pM, such as between 500 pM and 1000 pM, such as between 50 pM and 500 pM, such as between 25 pM and 100 pM, such as between 200 pM and 500 pM, such as between 50 pM and 100 pM, such as between 150 pM and 200 pM such as between 350 pM and 450 pM.

[0161] The microorganism may be any type of bacteria and / or yeast. In one embodiment, the microorganism is as described in the section “Microorganism”. Hence, in one embodiment, the microorganism is a non-pathogenic microorganism, such as a non-pathogenic bacteria and / or a non-pathogenic yeast.

[0162] Culture of microorganisms in broth / liquid media encourages planktonic (free-floating) growth, and recovery of biomass, metabolites and / or any recombinant proteins produced is facilitated. Thus, some embodiments of the present invention relate to where incubation of microorganism is in a liquid medium.

[0163] In some embodiments, the method of the invention relates to wherein the wherein the microorganism is cultured in a batch, fed-batch, or continuous fermentation system. In other embodiments, the method of the invention relates to wherein the microorganism is cultured under aerobic or anaerobic conditions. Other embodiments relate to immobilized cell reactors. One embodiment relates to a method for inducing and / or increasing growth of one or more microorganisms, said method comprising incubating the one or more microorganisms in medium comprising a ferric iron (III) chelate, thereby inducing and / or increasing growth of the one or more microorganisms; wherein the increase in growth is as compared to the growth of the one or more microorganisms when incubated in medium lacking the ferric iron chelate but comprising equimolar amount of iron, and otherwise cultivated under the same conditions.; wherein the one or more microorganisms are selected from the group consisting of Kluweromyces, Lactobacillus and Bacillus subtillis- wherein the ferric iron (III) chelate is selected from the group consisting of ferric quinate, ferriclactate and ferric citrate; and

[0164] wherein the equimolar amount of iron in control is provided in form of ferric chloride, optionally wherein the concentration of selected ferric iron chelate in the media is between 10 pM and 1000 pM, such as between 50 pM and 750 pM, such as between 25 pM and 50 pM, such as between 50 pM and 250 pM, such as between 100 pM and 400 pM, such as between 150 pM and 350 pM, such as between 200 pM and 300 pM, such as between 300 pM and 500 pM, such as between 350 pM and 450 pM, such as between 150 pM and 200 pM.

[0165] Microbial metabolite production

[0166] The present invention further provides a method for inducing and / or increasing metabolism of a microorganism selected from bacteria and / or yeast, said method comprising the step of:

[0167] a. incubating the microorganism in a media comprising an iron chelate according to the invention; thereby inducing and / or increasing metabolism of the microorganism.

[0168] Also provided herein is the use of an iron chelate according to the invention as described herein, for inducing and / or increasing bacterial and / or yeast metabolism.

[0169] In one embodiment, the metabolism is increased as compared to the metabolism of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0170] In one embodiment, the metabolism is increased as compared to the metabolism of a microorganism incubated in the absence of an iron chelate according to the invention, but in the presence of ferric chloride (FeCI3), and otherwise cultivated under the same conditions.

[0171] The method may comprise inducing and / or increasing any part of the metabolism. In one embodiment, the method comprises at least one of:

[0172] a. inducing and / or increasing the production of one or more metabolites; b. inducing and / or increasing the utilization of one or more carbon sources; and / or c. inducing and / or increasing the expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division.

[0173] One embodiment relates to a method according to the invention wherein intracellular metabolic profiles differ relative to ferric chloride control as determined by liquid chromatography-mass spectrometry (LC-MS) analysis. One embodiment relates to a method according to the invention, wherein metabolites associated with central carbon metabolism differ relative to ferric chloride control. One embodiment relates to a method according to the invention, wherein at least one metabolite selected from pyruvate, citrate, a-ketoglutarate, succinate, lactate, or NADH differs relative to ferric chloride control.

[0174] The production of one or more metabolites and / or utilization of one or more carbon sources may be measured using methods known in the art. For example, the concentration of metabolites and / or carbon sources may be measured using metabolomics, HPLC, LC-MS, GC-MS, or other analytic techniques suitable for the specific metabolite and / or carbon source. The person skilled in the art will know which technique to choose based on the metabolite to be measured.

[0175] The expression of one or more genes may be measured using methods known in the art. For example, the amount of RNA transcribed from the one or more genes may be measured using real-time quantitative PCR and / or the amount of protein encoded by the one or more genes may be measured using proteomics and / or Western blot.

[0176] Preferably, the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division is increased as compared to the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding involved in nutrient uptake, metabolism and / or cell division of a microorganismincubated in the absence of an iron chelate of the invention, but otherwise cultivated under the same conditions.

[0177] In one embodiment, the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division is increased as compared to the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division of a microorganism incubated in the absence of an iron chelate according to the invention, but in the presence of ferric chloride (FeCI3), and otherwise cultivated under the same conditions.

[0178] In one embodiment, the one or more metabolites are selected from the group consisting of metabolites involved in nutrient transport; metabolites involved in biosynthesis pathways; metabolites involved in cell cycle regulation; secondary metabolites; amino acids; organic acids; and energy molecules. In a preferred embodiment, the one or more metabolites are secondary metabolites and / or organic acids.

[0179] One embodiment of the invention relates to a method according to the invention, for inducing and / or increasing growth of a microorganism selected from bacteria and / or yeast, said method comprising the step of:

[0180] a. incubating the microorganism in a media comprising an iron chelate according to the invention;

[0181] thereby inducing and / or increasing metabolism of the microorganism.

[0182] The microorganism is selected from any bacteria and / or yeast. In one embodiment, the microorganism is as described in the section “Microorganism”. Hence, in one embodiment, the microorganism is a non-pathogenic microorganism, such as a non-pathogenic bacteria and / or a non-pathogenic yeast.

[0183] In one embodiment, the microorganism is of the genus Bacillus, such as of the species Bacillus subtilis. In these embodiments, the one or more metabolites may be selected from the group consisting of mugineic acid, malonate, coformycin, sorbate, sinipate, cyclohexanecarboxylic acid, orotate, lespeol, 2-oxoglutarate, 1-(3,4)-dimathylphenylethanone, fluocinolone acetonide, biotin sulfoxide, L-ornithine, hexazinone, mefenacet, 3-(2-carboxyethenyl)-cis,cis-muconate, dihydrobiopterin, 2-amino-2-deoxy-D-gluconate, valdecoxib, 3-dehydroshikimic acid, 3-hydroxy-cis-5-tetradecenoylcarnitine, streptamine, N(omega)-hydroxyarginine, 4-hydroxy-2-nonenal, homocysteine thioacetone, 9-hydroxy-hexadecan-1, 16-dioic acid, dehydro juvabione, phosphoglycerol (15:0 / 0:0), 3-beta-D-galactosyl-sn-glycerol, urea-1 -carboxylate, juvenile hormone III diol, 3”-deamino-3”-oxonicotianamine, 12-methyl-tetradecanoic acid, 3-oxo-2-pentyl-cyclopentanehexanoic acid, 9-hexadecenoic acid (9Z-palmitoleic acid), 4-amino-4-deoxychorismate, eriodictyol and sorbic acid. In one embodiment, the metabolite is sorbic acid. In one embodiment, the microorganism is of the genus Lactobacillus, such as one or more of the species Lactobacillus delbrueckii, Lactobacillus delbrueckii LA-67 or Lactobacillus AD4550 / 01. In this embodiment, the one or more metabolites may be one or both of ornithine and adenosine.

[0184] In one embodiment, the microorganism is of the genus Kluyveromyces, such as of the species Kluyveromyces marxianus. In this embodimentthe one or more metabolites may be selected from the group consisting of uridine, pseudouridine, uracil and phenylethylamine.

[0185] In one embodiment, the one or more carbon source is maltose. Thus, further provided herein is a method for inducing and / or increasing maltose utilization by microorganism such as bacteria, said method comprising the step of:

[0186] a. incubating microorganism in a media comprising an iron chelate of the invention; thereby inducing and / or increasing maltose utilization by the microorganism.The iron chelate may be any iron chelate according to the invention capable of inducing and / or increasing metabolism. Preferably, the iron chelate is as described in the sections “Iron chelate” and “Compositions comprising iron chelates”. In one embodiment, the iron chelate is selected from the group consisting of ferric quinate, ferric citrate and ferric lactate. In one embodiment, concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 100 pM and 1000 pM, such as between 250 pM and 750 pM, such as between 500 pM and 1000 pM, such as between 50 pM and 500 pM, such as between 25 pM and 100 pM, such as between 200 pM and 500 pM, such as between 50 pM and 100 pM, such as between 150 pM and 200 pM such as between 350 pM and 450 pM.

[0187] Recombinant protein production

[0188] In particular, the inventor has demonstrated that the method of the invention can on its own or in synergy with other promotors, re-direct microorganism metabolism to produce increased amounts of recombinant protein.

[0189] Thus, further provided herein is a method for inducing and / or increasing recombinant protein production by a microorganism capable of producing a recombinant protein, said microorganism being selected from bacteria and / or yeast, said method comprising the step of:

[0190] a. incubating the microorganism in a media comprising an iron chelate according to the invention; thereby inducing and / or increasing recombinant protein production by the microorganism.

[0191] Also provided herein is the use of an iron chelate according to the invention as described herein, for inducing and / or increasing recombinant protein production in bacteria and / or yeast. Preferably, recombinant protein production is increased as compared to the recombinant protein production by a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0192] Particular embodiments relate to a method for increasing recombinant protein production in a bacterium, for example a non-pathogenic bacteria, characterised in that the bacterium is cultured in a medium comprising a ferric iron(lll) chelate selected from ferric quinate, ferric citrate or ferric lactate, wherein recombinant protein yield is increased relative to culturing the bacterium under identical conditions in the presence of an equimolar amount of Fe3+supplied as ferric chloride.

[0193] In some embodiments the ferric iron chelate is provided in amount in the range from 1 to 400 pM (i.e., microM), for example at least 5, In certain embodiments, the amount is from 5 to 300 pM, 5 to 200 pM, 5 to 100 pM, 10 to 400 pM, 25 to 400 pM, 50 to 400 pM, or 100 to 400 pM. In further embodiments, the amount may be from 10 to 300 pM, 10 to 200 pM, 25 to 300 pM, 25 to 200 pM, 50 to 300 pM, or 50 to 200 pM.

[0194] In one embodiment, the method comprises increasing recombinant protein production by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100% as compared to a control. In some embodiments the increase is from 10% to 80%, such as from 10% to 70%, from 10% to 60%, from 10% to 50%, from 20% to 80%, from 30% to 80%, from 40% to 80%, or from 50% to 80%. In certain embodiments, the reduction is from 20% to 70%, 20% to 60%, 30% to 70%, 30% to 60%, or 40% to 70%.

[0195] In one embodiment, the method comprises increasing recombinant protein production by between 5% and 150%, such as between 15% and 100%, such as between 15% and 60%, such as between 20% and 40% as compared to a control.

[0196] Production of recombinant protein may be measured using methods known in the art. For example, the amount of recombinant protein may be measured using Western blot and / or proteomics.The method may comprise increasing and / or inducing production of any type of recombinant protein. In one embodiment, the recombinant protein is an enzyme. In one embodiment, the recombinant protein is at least 15 kilodaltons (kDa), such as at least 25 kDa, such as at least 35 kDa, such as at least 55 kDa, such as at least 75 kDa, such as at least 95 kDa. In one embodiment, the recombinant protein is between 15 and 100 kDa, such as between 25 and 85 kDa, such as between 25 and 35 kDa, such as between 75 and 85 kDa.

[0197] In one embodiment, the gene encoding the recombinant protein is comprised on a vector or a plasmid. In one embodiment, the gene encoding the recombinant protein is integrated into the genome of the bacteria and / or yeast.

[0198] The gene encoding the recombinant protein may be under the control of any type of promoter suitable for recombinant protein production. In one embodiment, the gene encoding the recombinant protein is under the control of an inducible promoter. In one embodiment, the gene encoding the recombinant protein is under control of an isopropyl p-d-1 -thiogalactopyranosid (IPTG)-inducible promoter. For example, the gene may be under the control of a lactose-inducible promoter or a hybrid promoter thereof which is induced by addition of IPTG. Promoters inducible by IPTG are well known in the art and include, for example, the T7-lac promoter (see also Examples).

[0199] In one embodiment, the method further comprises the step of adding IPTG to the media, such as between 0.1 mM and 1 mM of IPTG, for example between 0.5 mM and 1 mM IPTG.

[0200] The microorganism may be any type of bacteria and / or yeast. In one embodiment, the microorganism is as described in the section “Microorganism”. In one embodiment, the microorganism is Bacillus subtilis. In one embodiment, the microorganism is Escherichia coli. Preferably, the microorganism is a non-pathogenic microorganism, such as a non-pathogenic E. coli capable of recombinant protein production, for example an E. coli as described in the section “Microorganism”.

[0201] The iron chelate may be any suitable iron chelate according to the invention capable of inducing and / or increasing recombinant protein production. Preferably, the iron chelate is as described in the sections “Iron chelate”, “Compositions comprising iron chelates” and / or “Compositions comprising iron chelates and IPTG”. In one embodiment, the iron chelate is selected from the group consisting of ferric quinate, ferric citrate and ferric lactate. In one embodiment, concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 100 pM and 1000 pM, such as between 250 pM and 750 pM, such as between 500 pM and 1000 pM, such as between 50 pM and 500 pM, such as between 25 pM and 100 pM, such as between 200 pM and 500 pM, such as between 50 pM and 100 pM, such as between 150 pM and 200 pM such as between 350 pM and 450 pM.

[0202] Biofilm formation

[0203] Further provided herein is a method for inducing and / or increasing biofilm formation by a microorganism, such as a bacteria and / or yeast, said method comprising the step of:

[0204] a. incubating bacteria and / or yeast in a media comprising an iron chelate according to the invention; thereby inducing and / or increasing biofilm by the bacteria.

[0205] Thus, also provided herein is the use of an iron chelate according to the invention as described herein for inducing and / or increasing biofilm formation of a bacteria.

[0206] The invention in one particular embodiment relates to a method for modulating biofilm formation of a non-pathogenic bacterium, characterised in that the bacterium is cultured in the presence of a ferric iron (III) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof, at 1-400 pM Fe3+,

[0207] wherein biofilm biomass differs relative to that obtained using an equimolar amount of Fe3+supplied as ferric chloride, for example where the biofilm biomass is increased by at least 10%In some embodiments the ferric iron chelate is provided in amount in the range from 1 to 400 pm (i.e., microM), such as from 5 to 400 pM), for example at least 5. In some embodiments, the amount of iron chelate is from 5 to 400 pM, such as from 5 to 300 pM, 5 to 200 pM, 5 to 150 pM, 5 to 100 pM, 5 to 75 pM, 5 to 50 pM, 10 to 400 pM, 25 to 400 pM, 50 to 400 pM, 75 to 400 pM, 100 to 400 pM, 150 to 400 pM, 200 to 400 pM, 10 to 300 pM, 10 to 200 pM, 25 to 300 pM, 25 to 200 pM, 50 to 300 pM, 50 to 200 pM, 75 to 300 pM, or 100 to 300 pM.

[0208] In one embodiment, the increase in biofilm formation is as compared to the biofilm formation of a bacteria incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0209] In one embodiment, the increase in biofilm formation is as compared to the biofilm formation of a bacteria incubated in the absence of an iron chelate, but in the presence of ferric chloride (FeCI3), and otherwise cultivated under the same conditions.

[0210] In one embodiment, the media is supplemented with glutamate, such as between 0.1% and 1.0% w / v of glutamate.

[0211] The bacteria may be incubated in any type of condition suitable for biofilm formation.

[0212] In one embodiment, the bacteria are incubated in light conditions, optionally wherein the light is selected from the group consisting of white light, blue light and red light.

[0213] In one embodiment, the bacteria are incubated in dark conditions, i.e. in the absence of light. In one embodiment, the bacteria are incubated at a temperature of between 15°C and 35°C, such as between 20°C and 30°C, such as between 20°C and 25°C.

[0214] In one embodiment, the bacteria are incubated for at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 day, such as at least 5 days. In one embodiment, the bacteria are incubated for between 1 and 6 days, such as between 2 and 5 days, such as between 3 and 5 days.

[0215] In one embodiment, the bacteria are incubated under static conditions. In one embodiment, the bacteria are incubated on and / or over a surface.

[0216] In one embodiment, the bacteria is Gram-positive. In one embodiment, the bacteria is of the genus Bacillus. In one embodiment, the bacteria is Bacillus subtilis.

[0217] In one embodiment, the microorganism is incubated under limited oxygen conditions. In one embodiment, the microorganism is incubated under microaerophilic conditions.

[0218] In some embodiments, the microorganism is a non-pathogenic microorganism as described herein below (see heading Microorganism), for example at least one non-pathogenic bacteria. The iron chelate may be any suitable iron chelate according to the invention capable of inducing and / or increasing biofilm formation. Preferably, the iron chelate is as described in the sections “Iron chelate” and “Compositions comprising iron chelates”. In one embodiment, the iron chelate is selected from the group consisting of ferric quinate, ferric citrate ferric tyrosine and ferric lactate. In one embodiment, the concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 50 pM and 750 pM, such as between 25 pM and 50 pM, such as between 50 pM and 250 pM, such as between 100 pM and 400 pM, such as between 150 pM and 350 pM, such as between 200 pM and 300 pM, such as between 300 pM and 500 pM, such as between 350 pM and 450 pM, such as between 150 pM and 200 pM, or for example in the range from 0.25 to 10 pm, such as from 0.25 to 8 pm, from 0.25 to 5 pm, from 0.25 to 5 pm. Microorganism

[0219] The microorganism described herein is selected from bacteria, yeast, and combinations thereof. For example, the microorganism may be a microbial consortium comprising bacteria and / or yeast. Preferably, the microorganism is a non-pathogenic microorganism, such as a non-pathogenic bacteria or a non-pathogenic yeast.

[0220] The terms “non-pathogenic microorganism”, “non-pathogenic bacteria”, “non-pathogenic yeast” as used herein refers to microorganisms, such as bacteria and / or yeast, that do not causedisease, harm or death to another organism. In particular, the term refers to a microorganism unable to cause disease in a healthy human, animal and / or plant.

[0221] Non-pathogenic microorganisms are any microbial strain / species that have not been observed to cause disease or produce harmful toxins in healthy, immunocompetent humans under normal conditions of exposure.

[0222] Non-pathogenic microorganisms typically display one or more of the following: lack virulence factors, do not produce endotoxins, lack Type III, IV, VI secretion systems.

[0223] In contrast, pathogenic bacteria typically display at least one of the following: specialized virulence factors, thick capsule, highly immumogenic endoxoin, adhesins for host attachment and invasionion, secretions systems, invasins, toxins, immune evasion proteins.

[0224] Examples of non-pathogenic microorganisms are those listed on the EFSA’s QPS list and / or FDA GRAS notices. Thus, in some embodiments, the microorganisms according to the invention are one or more microorganisms that the European food safety authority’s Qualified presumption of safety (QPS) recognizes as safe and / or the FDA has granted GRAS status (with no unresolved safety concerns), as notified by the respective body.

[0225] Examples of non-pathogenic microorganisms suitable for the invention are one or more selected from the group consisting of Cupriavidus necator, Gluconobacter oxydans, Komagataeibacter sucrofermentans, Agrobacterium radiobacter, Xanthomonas campestris, Acidipropionibacterium acidipropionici, Bifidobacterium adolescentis , Bifidobacterium animalis, Bifidobacterium bifidum , Bifidobacterium breve, Bifidobacterium longum, Carnobacterium divergens, Companilactobacillus alimentarius , Companilactobacillus farciminis , Corynebacterium ammoniagenes, Corynebacterium glutamicum , Fructilactobacillus sanfranciscensis, Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis , "Lacticaseibacillus , paracasei", Lacticaseibacillus rhamnosus , Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum , Lactiplantibacillus pentosus , Lactiplantibacillus plantarum , Lactobacillus acidophilus , Lactobacillus amylolyticus , Lactobacillus amylovorus, Lactobacillus crispatus , Lactobacillus delbrueckii, Lactobacillus gallinarum , Lactobacillus gasseri , Lactobacillus helveticus , Lactobacillus johnsonii , Lactobacillus kefiranofaciens, Lactococcus lactis, Lapidilactobacillus dextrinicus, Latilactobacillus curvatus , Latilactobacillus sake! , Lentilactobacillus buchneri , Lentilactobacillus diolivorans, Lentilactobacillus hilgardii , Lentilactobacillus kefiri , Lentilactobacillus parafarraginis, Leuconostoc citreum, Leuconostoc lactis , Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Levilactobacillus brevis , Ligilactobacillus animalis, Ligilactobacillus aviarius, Ligilactobacillus salivarius , Limosilactobacillus fermentum , Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis , Limosilactobacillus reuteri , Loigolactobacillus coryniformis, Microbacterium imperials, Oenococcus oeni, Pediococcus acidilactici, Pediococcus parvulus, Pediococcus pentosaceus, Propionibacterium freudenreichii, Secundilactobacillus collinoides, Streptococcus thermophilus, Bacillus amyloliquefaciens , Bacillus atrophaeus , Bacillus licheniformis , Bacillus mojavensis, Bacillus paralicheniformis , Bacillus pumilus , Bacillus smithii, Bacillus subtilis , Bacillus vallismortis, Bacillus velezensis , Clostridium tyrobutyricum , Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Heyndrickxia coagulans, Lederbergia lenta, Lysinibacillus fusiformis , Niallia circulans, Paenibacillus illinoisensis, Parageobacillus thermoglucosidasius, Pasteuria nishizawae, Priestia flexa, Priestia megaterium, Shouchella clausii , Chlamydomonas reinhardtii , Euglena gracilis, Haematococcus lacustris , Tetraselmis chuii, Aurantiochytrium limacinum, Cyberlindnera jadinii, Debaryomyces hansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromyces marxianus, Komagataella pastoris , Komagataella phaffii, Limtongozyma cylindracea, Ogataea angusta, Ogataea polymorpha, Phaffia rhodozyma, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus ,Schizosaccharomyces pombe, Wickerhamomyces anomalus, Yarrowia lipolytica, Zygosaccharomyces rouxii,

[0226] In one embodiment, the microorganism is a gram positive bacteria. In alternative embodiments, the microorganism is a gram-negative bacteria.

[0227] Due to recent taxonomic revisions, several species previously classified within the genus Lactobacillus have been reassigned to new genera. Accordingly, references herein to Lactobacillus are intended to encompass bacteria historically classified within the genus Lactobacillus as well as species currently classified in related genera including, for example, Lacticaseibacillus and Lactiplantibacillus.

[0228] In particular embodiments, the microorganism comprises one or more bacterial strains selected from Bacillus spp., Escherichia coli, Lactobacillus spp., Lacticaseibacillus spp., and Lactiplantibacillus spp, in particular where said strain is suitable for industrial fermentation processes. These bacteria are examples of non-pathogenic microorganisms

[0229] In another embodiment, the microorganism is at least one bacteria of a genus selected from the group consisting of Bacillus, Lactobacillus and Escherichia, such as for example Bacillus subtilis, Lactobacillus delbrueckii, Lactobacillus delbrueckii LA-67 , Lactobacillus AD4550 / 01 or Escherichia coli.

[0230] In one embodiment, the microorganism is a non-pathogenic Escherichia coli strain, such as an E. coli strain that does not contain genes encoding or transcribing products that are toxic and / or cause disease or harm in humans and / or animals. Hence, in one embodiment, the microorganism is an E. coli strain that does not produce shiga-toxin. In one embodiment, the E. coli strain is a derivative of E. coli K or E. coli B. In one embodiment, the bacteria is an E. coli strain selected from the group consisting of E. coli K12 and derivatives thereof, E. coli MG1655, E. coli DH5alpha, E. coli DH10B E. coli 25922, E. coli Nissle, E. coli Castellani and Chalmers ATCC 700728

[0231] In one embodiment, the microorganism is a yeast. In one embodiment, the microorganism is a yeast of the genus Kluyveromyces, such as for example Kluyveromyces marxianus.

[0232] In one embodiment, the microorganism comprises at least two different types of microorganisms, such as for example two different genera and / or two different species. In one embodiment, the microorganism is a yeast and a bacteria, such as for example a yeast of the genus Kluyveromyces and a bacteria of the genus Lactobacillus.

[0233] Non-pathogenic microorganisms are used in industrial processes, such as industrial fermentation and biotechnology processes.

[0234] Iron chelates

[0235] The term “iron chelate” refers to a complex formed by a ligand that can form ring(s) by bonding to an iron atom at two or more points.

[0236] The iron chelates of the invention relate to ferric (Fe3+) complexes, in particular iron(lll)-chelates of organic compounds. In one embodiment, the iron chelates of the invention are ferric complexes with carboxylic acids (ferric carboxylates). In some embodiments the iron chelates may be selected from group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof. Ferric quinate, ferric citrate, ferric lactate, and ferric tyrosine in turn refers to any complex formed upon binding any conjugate base derived from citric acid, quinic acid, lactic acid and tyrosine, respectively, with ferric ion(s).

[0237] The terms “ferric citrate”, “Fe-citrate” and “Fe-cit” are used interchangeably herein; the terms “ferric lactate”, “Fe-lactate” and “Fe-lac” are used interchangeably herein; the terms “ferric quinate”, “Fe-quinate” and “FeQ” are used interchangeably herein; and the terms “ferric tyrosine”, “Fe-tyrosine” and “Fe-tyr” are used interchangeably herein.In particular embodiments, the iron chelates of the invention are ferric carboxylates, such as selected from the group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), and ferric lactate (iron(lll) lactate) and combinations thereof.

[0238] In further particular embodiments, the iron chelates of the invention comprise or consist of ferric quinate.

[0239] In one embodiment, the iron chelates according to the invention comprises at least two different iron chelates of the invention, such as at least three different iron chelates of the invention. Hence, in one embodiment, the media comprises at least two iron chelates, such as at least three iron chelates, such as for example:

[0240] a. ferric quinate and ferric lactate;

[0241] b. ferric quinate and ferric citrate;

[0242] c. ferric lactate and ferric citrate;

[0243] d. ferric quinate, ferric lactate and ferric citrate.

[0244] In one embodiment, the media comprises:

[0245] a. between 40 and 45 pM ferric citrate and between 40 and 45 pM ferric lactate; or b. between 80 and 90 pM ferric citrate and between 80 and 90 pM ferric quinate; or c. between 80 and 90 pM ferric lactate and between 80 and 90 pM ferric quinate; or d. between 25 and 35 pM ferric citrate, between 25 and 35 pM ferric lactate and between 25 and 35 pM ferric quinate; or

[0246] e. between 50 and 60 pM ferric citrate, between 50 and 60 pM ferric lactate and between 50 and 60 pM ferric quinate.

[0247] In one embodiment, the media comprises between 10 pM and 1000 pM iron chelate, such as between 100 pM and 1000 pM, such as between 250 pM and 750 pM, such as between 500 pM and 1000 pM, such as between 50 pM and 500 pM, such as between 25 pM and 100 pM, such as between 200 pM and 500 pM, such as between 50 pM and 100 pM, such as between 150 pM and 200 pM such as between 350 pM and 450 pM ion chelate or for example in the range from 0.25 to 10 pm, such as from 0.25 to 8 pm, from 0.25 to 5 pm, from 0.25 to 5 pm.

[0248] The iron chelate according to the invention may be supplemented to the media at any point in time, such as before, after and / or simultaneously as incubating the microorganism in the media. Hence, in one embodiment, the iron chelate is supplemented to the media:

[0249] a. after;

[0250] b. simultaneously as; and / or

[0251] c. before;

[0252] incubating the microorganism in said media.

[0253] In one embodiment, the iron chelate of the invention is supplemented to the media at least once, such as at least twice, such as at least three times.

[0254] Compositions comprising iron chelates

[0255] The present invention provides iron chelate compositions suitable for enhancing, increasing and / or inducing biological processes as described herein.

[0256] Thus, provided herein is a composition comprising:

[0257] a. ferric quinate and ferric lactate;

[0258] b. ferric quinate and ferric citrate;

[0259] c. ferric lactate and ferric citrate; or

[0260] d. ferric quinate, ferric lactate and ferric citrate.

[0261] In some embodiments, the iron chelate compositions according to the invention comprise ferric quinate. In further embodiments, said compositions further comprise one or both of ferric citrate and ferric lactate.

[0262] In one embodiment, the composition comprises at least two iron chelates selected from the group consisting of ferric quinate, ferric lactate and ferric citrate, said composition comprising:a. between 10 and 90% of ferric quinate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric quinate; and / or b. between 10 and 90% of ferric lactate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric lactate; and / or c. between 10 and 90% of ferric citrate, such as between such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric citrate;

[0263] with the proviso that the sum of the percentage of iron chelates is not >100%.

[0264] In one embodiment, the composition comprises:

[0265] a. between 25 and 35% ferric quinate, between 25 and 35% ferric lactate, and between 25 and 35% ferric citrate; or

[0266] b. between 45 and 55% ferric citrate and between 45 and 55% ferric lactate; or c. between 45 and 55% ferric citrate and between 45 and 55% ferric quinate; or d. between 45 and 55% ferric quinate and between 45 and 55% ferric lactate; with the proviso that the sum of the percentage of iron chelates is not >100%.

[0267] In one embodiment, the composition comprises:

[0268] a. approximately 33% ferric quinate, approximately 33% ferric lactate, and approximately 33% ferric citrate; or

[0269] b. approximately 50% ferric citrate and approximately 50% ferric lactate; or c. approximately 50% ferric citrate and approximately 50% ferric quinate; or d. approximately 50% ferric quinate and approximately 50% ferric lactate; with the proviso that the sum of the percentage of iron chelates is not >100%.

[0270] Compositions comprising iron chelates and IPTG

[0271] The present invention provides compositions suitable for enhancing, increasing and / or inducing recombinant protein production as described herein.

[0272] Further provided herein is a composition comprising:

[0273] a. an iron chelate according to the invention; and

[0274] b. isopropyl p-d-1-thiogalactopyranosid (IPTG).

[0275] In one embodiment, the iron chelate is as defined herein in the section “Iron chelates”. Hence, preferably the iron chelate is selected from the group consisting of ferric quinate, ferric lactate, ferric citrate and ferric tyrosine.

[0276] In one embodiment, the composition comprises:

[0277] a. between 10 and 90% of iron chelate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of iron chelate; and b. between 10 and 90% of IPTG, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of IPTG;

[0278] with the proviso that the sum of the percentage of iron chelate and IPTG is not >100%.

[0279] In one embodiment, the composition comprises:

[0280] a. between 5 and 15% of ferric lactate; and

[0281] b. between 30% and 50% of ferric quinate; and

[0282] c. between 30% and 50% of IPTG;

[0283] with the proviso that the sum of the percentage of ferric lactate, ferric quinate and IPTG is not >100%.

[0284] In one embodiment, the composition comprises:

[0285] a. approximately 10% of ferric lactate; and

[0286] b. approximately 45% of ferric quinate; and

[0287] c. approximately 45% of IPTG;

[0288] with the proviso that the sum of the percentage of ferric lactate, ferric quinate and IPTG is not >100%.Inducing and increasing seed germination and growth

[0289] Provided herein is a method for inducing and / or increasing germination of plant seeds and / or growth of a plant, said method comprising the steps of:

[0290] a. planting plant seeds in a growth media comprising an iron chelate according to the invention;

[0291] b. subjecting the planted plant seeds to light;

[0292] thereby inducing and / or enhancing germination of the plant seeds and / or growth of the plant. Thus, also provided herein is the use of an iron chelate according to the invention for inducing and / or increasing germination and / or growth of plant seeds.

[0293] In one embodiment, the increase in germination and / or growth is as compared to the germination of plant seeds and / or growth of a plant planted in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0294] Particular embodiments relate to where ethe iron is provided in equimolar amounts as ferric chloride.

[0295] In one embodiment, the iron chelate is as described herein in the section “Iron chelates”. In one embodiment, the iron chelate is a ferric carboxylate, such as one or more selected from the group consisting of ferric quinate, ferric lactate and ferric citrate. In one embodiment, the iron chelate is at least two different iron chelates, such as at least three different iron chelates. Hence, in one embodiment, the media comprises at least two iron chelates, such as at least three iron chelates, such as for example:

[0296] a. ferric quinate and ferric lactate;

[0297] b. ferric quinate and ferric citrate;

[0298] c. ferric lactate and ferric citrate;

[0299] d. ferric quinate, ferric lactate and ferric citrate.

[0300] In one embodiment, the media comprises at least two iron chelates, such as at least three iron chelates, such as for example:

[0301] a. ferric quinate and ferric lactate;

[0302] b. ferric quinate and ferric citrate;

[0303] c. ferric lactate and ferric citrate; or

[0304] d. ferric quinate, ferric lactate and ferric citrate.

[0305] In one embodiment, the iron chelate is supplemented to the growth media:

[0306] a. after;

[0307] b. simultaneously as; and / or

[0308] c. before;

[0309] planting the plant seeds in said growth media.

[0310] In one embodiment, the iron chelate is supplemented to the growth media 5 days, 8 days and 15 days after planting the seeds.

[0311] In one embodiment, between 0.5 pM and 50 pM of iron chelate in is supplemented to the growth media, such as between 0.5 pM and 15 pM, such as between 1 pM and 10 pM, such as between 1 pM and 5 pM or for example in the range from 0.25 to 10 pm, such as from 0.25 to 8 pm, from 0.25 to 5 pm, from 0.25 to 5 pm.

[0312] In one embodiment, between 0.015 pmol and 5 pmol of iron chelate per plant seed is supplemented to the growth media, such as between 0.015 pmol and 1 pmol, such as between 0.015 pmol and 0.5 pmol, such as between 0.015 pmol and 0.05 pmol.

[0313] The iron chelate of the invention may be supplemented to the growth media at any point in time, such as before, after and / or simultaneously as planting the plant seeds in the growth media. Hence, in one embodiment, the iron chelate is supplemented to the growth media:

[0314] a. after;

[0315] b. simultaneously as; and / orc. before;

[0316] planting the plant seeds in said media.

[0317] In one embodiment, the iron chelate is supplemented to the media at least once, such as at least twice, such as at least three times.

[0318] The light may be any type of light suitable for growing the plant seeds. In one embodiment, the light is selected from the group of light consisting of blue light, red light, yellow light, green light and white light.

[0319] Further, the growth media may be any type of growth media suitable for growing the plant seeds. In one embodiment, the growth media is selected from the group consisting of soil, rockwool, peat moss, perlite, vermiculite, sponge, rice hulls, oasis cubes, clay pebbles, floral foam and coco coir.

[0320] In one embodiment, the present invention provides a method for inducing and / or increasing germination of plant seeds and / or growth of a plant, said method comprising the steps of:

[0321] a. planting plant seeds in a growth media comprising an iron chelate according to the invention;

[0322] b. subjecting the planted plant seeds to light;

[0323] thereby inducing and / or enhancing germination of the plant seeds and / or growth of the plant, optionally wherein the growth media is soil; and

[0324] optionally wherein the light is selected from white light and green light: and

[0325] optionally wherein the growth media is supplemented with between 0.015 pmol and 0.05 pmol of iron chelate per plant seed after planting;

[0326] optionally wherein the iron chelate is selected from ferric lactate and ferric quinate.

[0327] The plant may be any type of plant. In one embodiment, the plant is of the family Fabaceae, for example of the genus Vignia, such as of the species Vignia radiata.

[0328] The increase in germination of the plant seeds and / or growth of the plant may be measured using methods known in the art. For example, the increase in growth of the plant may comprise an increase in the shoot length of the plant. Thus, in one embodiment, the method comprises increasing the shoot length of the plant, wherein the increase in shoot length is as compared to the shoot length of a plant planted in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0329] In one embodiment, the growth media is soil and the method comprises increasing the growth of soil bacteria, preferably beneficial soil bacteria, preferably wherein the soil bacteria is of the genus Rhizobacter, Gluconacetobacter or Bacillus, such as Gluconacetobacter diazotrophicus or Bacillus subtilis.

[0330] Thus, also provided herein is a method for inducing and / or increasing growth of Rhizobacter, Gluconacetobacter and / or Bacillus in soil said method comprising the steps of:

[0331] a. planting plant seeds in soil comprising:

[0332] a. an iron chelate according to the invention; and

[0333] b. Rhizobacter, Gluconacetobacter and / or Bacillus’

[0334] b. subjecting the planted plant seeds to light;

[0335] thereby inducing and / or increasing growth of Rhizobacter, Gluconacetobacter and / or Bacillus in the soil.

[0336] The invention further provides a method for modulating at least one plant growth or developmental parameter in a plant, characterised in that a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof is applied to a plant, plant seed or plant growth substrate at a concentration of 1-400 pM Fe3+, wherein the least one plant growth or developmental parameter selected from shoot length, root length, biomass accumulation or germination rate differs relative to that obtained using an equimolar amount of Fe3+provided as ferric chloride, under otherwise identical conditions.Without wishing to be bound by theory, the inventors believe that the observed plant growth enhancement is mediated, at least in part, through modulation of the rhizosphere microbiome. Thus, the invention further provides a method for modifying rhizosphere microbial composition associated with a plant, characterised in that a ferric iron(lll) chelate selected from ferric quinate, ferric citrate or a combination thereof is applied to soil containing the plant at 1-400 pM Fe3+, wherein the rhizosphere microbial composition differs relative to soil treated with an equimolar amount of Fe3+supplied as ferric chloride. This difference may be for example increase of the relative abundance of plant growth promoting organisms, and / or the reduction of plant pathogenic microorganisms. Examples of plant beneficial microorganisms or plant growth promoting organisms include Psuedomonas, Azospirillum, Rhizobium and Bradyrhizobium bacteria. Plant pathogenic microorganism may be for example one or more selected from Pseudomonas (e.g., P. syringae), Xanthomonas, Erwinia (e.g., Erwinia amylovora), Ralstonia (e.g., R. solanacearum) and Agrobacterium.

[0337] The invention also provides a method for enhancing plant growth via modulation of rhizosphere microbial composition, characterised in that a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof is applied to soil or plant growth substrate, wherein the application results in a shift in microbial composition associated with the rhizosphere, and wherein said shift is associated with an increase in plant growth relative to treatment with an equimolar amount of Fe3+supplied as ferric chloride.

[0338] In addition to bacterial microorganisms, the rhizosphere microbiome may also include fungal and oomycete organisms. In certain embodiments, the application of ferric iron(lll) chelates may influence fungal or oomycete populations present in the rhizosphere, including microorganisms belonging to genera such as Fusarium, Alternaria, Botrytis, Rhizoctonia, Verticillium, Phytophthora, and Pythium.

[0339] Without wishing to be bound by theory, such effects may arise indirectly through modulation of bacterial communities, changes in nutrient availability, or alterations in the physicochemical environment of the rhizosphere. However, the primary demonstrated effects relate to bacterial community modulation.

[0340] The invention further provides a composition comprising: a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof, for enhancing plant growth and / or modifying rhizosphere microbiota in agriculture.

[0341] The invention further provides the use of a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof for: (a) enhancing plant growth; and / or (b) modifying rhizosphere microbial composition.

[0342] Items

[0343] 1. A method for inducing and / or increasing growth of a microorganism selected from bacteria and / or yeast, said method comprising the step of:

[0344] a. incubating the microorganism in a medium comprising an iron (III) chelate selected from the group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof;

[0345] thereby inducing and / or increasing growth of the microorganism.

[0346] 2. The method according to any one of the preceding items, wherein the iron chelate is selected from the group consisting of ferric quinate, ferric lactate, and ferric citrate. 3. The method according any of the preceding items wherein the microorganism is incubated in a liquid culture medium.

[0347] 4. The method of any of the preceding items 1 to 2 wherein the microorganism is incubated in a medium which is soil.5. The method according to any of the preceding items wherein the increase in growth is as compared to the growth of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0348] 6. The method according to any of the preceding items, wherein the increase in growth is as compared to the growth of a microorganism incubated in the absence of said iron (III) chelate, with inorganic ferric complex, such as ferric chloride, as control.

[0349] 7. The method according to any of the previous items wherein the incubation of microorganism is in a batch culture, fed-batch culture, or continuous fermentation system.

[0350] 8. The method according to any of the preceding items wherein the method comprises at least one of:

[0351] a. decreasing the duration of the lag phase of the microorganism;

[0352] b. increasing the growth rate of the microorganism;

[0353] c. increasing the cell density of the microorganism at the end of the exponential phase and / or at the end of incubation;

[0354] wherein the decrease of a, increase of b and / or of c are as compared to a microorganism incubated in the absence of the iron chelates of items 1 or 2, but otherwise cultivated under the same conditions.

[0355] 9. The method according to any one of preceding items, wherein the method comprises at least one of :

[0356] a. decreasing the duration of the lag phase of the microorganism;

[0357] b. increasing the growth rate of the microorganism;

[0358] c. increasing the cell density of the microorganism at the end of the exponential phase and / or at the end of incubation;

[0359] wherein the decrease of a, increase of b and / or increase of c are as compared to a microorganism incubated in the in the absence of the iron chelates of items 1 or 2 but with inorganic ferric complex, such as FeCI3, as control, and otherwise cultivated under the same conditions.

[0360] 10. The method any of the preceding items wherein the duration of the lag phase is decreased by at least 15 min, such as at least 30 min, such as at least 60 min, such as at least 90 min, such as by at least 3 h, such as by at least 5 h, such as by at least 10 h as compared to the duration of the lag phase of a microorganism incubated in the absence of the iron chelates of items 1 or 2 and otherwise cultivated under the same conditions

[0361] 11. A method for inducing and / or increasing metabolism of a microorganism selected from bacteria and / or yeast, said method comprising the step of:

[0362] a. incubating the microorganism in a media comprising an iron chelate being a ferric complex with a carboxylic acid, for example selected from group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), , ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof ; thereby inducing and / or increasing metabolism of the microorganism

[0363] 12. The method according to item 11 , wherein the metabolism is increased as compared to the metabolism of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0364] 13. The method according to any one of the preceding items, wherein the method comprises at least one of:

[0365] a. inducing and / or increasing the production of one or more metabolites;

[0366] b. inducing and / or increasing the utilization of one or more carbon sources;c. inducing and / or increasing the expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division.

[0367] 14. The method according to item 13 wherein the method comprises a, b and c .

[0368] 15. The method according to item 14, wherein the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding proteins involved in nutrient uptake, metabolism and / or cell division is increased as compared to the production of one or more metabolites; utilization of one or more carbon sources; and / or expression of one or more genes encoding involved in nutrient uptake, metabolism and / or cell division of a microorganism incubated in the absence of an iron chelate according to the invention, but otherwise cultivated under the same conditions.

[0369] 16. The method according to any one of items 13 to 15, wherein the one or more metabolites are selected from the group consisting of metabolites involved in nutrient transport; metabolites involved in biosynthesis pathways; metabolites involved in cell cycle regulation; secondary metabolites; amino acids; organic acids; and energy molecules.

[0370] 17. The method according to any one of items 11 to 16, wherein the microorganism is of the genus Bacillus, such as of the species Bacillus subtilis, and the one or more metabolites are selected from the group consisting of mugineic acid, malonate, coformycin, sorbate, sinipate, cyclohexanecarboxylic acid, orotate, lespeol, 2-oxoglutarate, 1-(3,4)- dimathylphenylethanone, fluocinolone acetonide, biotin sulfoxide, L-ornithine, hexazinone, mefenacet, 3-(2-carboxyethenyl)-cis,cis-muconate, dihydrobiopterin, 2- amino-2-deoxy-D-gluconate, valdecoxib, 3-dehydroshikimic acid, 3-hydroxy-cis-5- tetradecenoylcarnitine, streptamine, N(omega)-hydroxyarginine, 4-hydroxy-2-nonenal, homocysteine thioacetone, 9-hydroxy-hexadecan-1, 16-dioic acid, dehydro juvabione, phosphoglycerol (15:0 / 0:0), 3-beta-D-galactosyl-sn-glycerol, urea-1 -carboxylate, juvenile hormone III diol, 3”-deamino-3”-oxonicotianamine, 12-methyl-tetradecanoic acid, 3-oxo- 2-pentyl-cyclopentanehexanoic acid, 9-hexadecenoic acid (9Z-palmitoleic acid), 4- amino-4-deoxychorismate, eriodictyol and sorbic acid.

[0371] 18. The method according to any one of items 11 to 17, wherein the microorganism is of the genus Lactobacillus, and the one or more metabolites are selected from the group consisting of ornithine and adenosine.

[0372] 19. The method according to any one of items 11 to 18 wherein the microorganism is of the genus Kluyveromyces, such as of the species Kluyveromyces marxianus, and the one or more metabolites are selected from the group consisting of uridine, pseudouridine, uracil and phenylethylamine.

[0373] 20. The method according to item 13, wherein the one or more carbon sources is maltose.

[0374] 21. The method according to any one of the preceding items, wherein growth and / or metabolism is induced and / or increased during fermentation.

[0375] 22. A method for inducing and / or increasing recombinant protein production by a microorganism capable of producing a recombinant protein, said microorganism being selected from bacteria and / or yeast, said method comprising the step of:

[0376] a. incubating the microorganism in a media comprising an iron chelate which is a ferric complex with a carboxylic acid;

[0377] thereby inducing and / or increasing recombinant protein production by the microorganism.

[0378] 23. The method according to item 22 wherein the iron chelate iron chelates is selected from group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), , ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof.24. The method according to item 22 or 2322, wherein the recombinant protein production is increased as compared to the recombinant protein production by a microorganism incubated in the absence of an iron chelate of the invention, but otherwise cultivated under the same conditions.

[0379] 25. The method according to any one of items 22 to 24, wherein the gene encoding the recombinant protein is under control of an isopropyl p-d-1-thiogalactopyranosid (IPTG)- inducible promoter.

[0380] 26. The method according to any one of items 22 to 24, wherein said method further comprises the step of adding IPTG to the media, such as between 0.1 mM and 1 mM of IPTG.

[0381] 27. The method according to any one of items 22 to 26, wherein the gene encoding the recombinant protein is:

[0382] a. comprised on a vector or a plasmid; or

[0383] b. integrated into the genome of the bacteria and / or yeast.

[0384] 28. The method according to any one of items 22 to 27, wherein the microorganism is Escherichia coli.

[0385] 29. The method according to any one of the preceding items, wherein the concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 100 pM and 1000 pM, such as between 250 pM and 750 pM, such as between 500 pM and 1000 pM, such as between 50 pM and 500 pM, such as between 25 pM and 100 pM, such as between 200 pM and 500 pM, such as between 50 pM and 100 pM, such as between 150 pM and 200 pM such as between 350 pM and 450 pM.

[0386] 30. The method according to any one of the preceding items, wherein the microorganism is a non-pathogenic microorganism, such as a non-pathogenic bacteria and / or a non- pathogenic yeast

[0387] 31. The method according to any one of the preceding items, wherein the microorganism is a gram positive bacteria.

[0388] 32. The method according to any one of the preceding items, wherein the microorganism is a bacteria, such as a bacteria of a genus selected from the group consisting of Bacillus, Lactobacillus and Escherichia, such as for example Bacillus subtilis, Lactobacillus delbrueckii, Lactobacillus delbrueckii LA-67 , Lactobacillus AD4550 / 01 or Escherichia coli.

[0389] 33. The method according to any one of the preceding items, wherein the microorganism is a yeast, such as a yeast of the genus Kluyveromyces, such as for example Kluyveromyces marxianus.

[0390] 34. A method for inducing and / or increasing biofilm formation by a bacteria, said method comprising the step of:

[0391] a. incubating bacteria in a media comprising an iron chelate which is a ferric complex with a carboxylic acid;

[0392] thereby inducing and / or increasing biofilm by the bacteria.

[0393] 35. The method of item 34 wherein the bacteria is non-pathogenic.

[0394] 36. The method according to item 34 or 35, wherein the increase in biofilm formation is as compared to the biofilm formation of a bacteria incubated in the absence of an iron chelate, but otherwise cultivated under the same conditions.

[0395] 37. The method according to any one of items 34 to 36, wherein the bacteria is gram positive, such as wherein the bacteria is of the genus Bacillus, such as for example Bacillus subtilis.

[0396] 38. The method according to any one of items 34 to 37, wherein the concentration of iron chelate in the media is between 10 pM and 1000 pM, such as between 50 pM and 750pM, such as between 25 pM and 50 pM, such as between 50 pM and 250 pM, such as between 100 pM and 400 pM, such as between 150 pM and 350 pM, such as between 200 pM and 300 pM, such as between 300 pM and 500 pM, such as between 350 pM and 450 pM, such as between 150 pM and 200 pM or for example in the range from 0.25 to 10 pm, such as from 0.25 to 8 pm, from 0.25 to 5 pm, from 0.25 to 5 pm.

[0397] 39. The method according to any one items 34 to 38, wherein the bacteria are incubated in light conditions, optionally wherein the light is selected from the group consisting of white light, blue light and red light.

[0398] 40. The method according to any one of items 34 to 39, wherein the bacteria are incubated in dark conditions, i.e. in the absence of light.

[0399] 41. The method according to any one of items 34 to 40, wherein the bacteria are incubated at a temperature of between 15°C and 35°C, such as between 20°C and 30°C, such as between 20°C and 25°C.

[0400] 42. The method according to any one of items 34 to 41, wherein the bacteria are incubated for at least 1 day, such as at least 2 days, such as at least 3 days.

[0401] 43. The method according to any one of items 34 to 42, wherein the media is supplemented with sugars, such as glucose, and / or glutamate, such as between 0.1% and 1.0% w / v of glutamate.

[0402] The method according to any one of items 34 to 43, wherein the bacteria are incubated under static conditions.

[0403] 45. The method according to any one of 34 to 44, wherein the iron chelate is selected from group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), , ferric lactate (iron(lll) lactate) , ferric tyrosine (iron(lll) tyrosine) and combinations thereof. 46. The method according to any one of items 34 to 45, wherein the iron chelate is selected from the group consisting of ferric quinate, ferric lactate, ferric tyrosine and ferric citrate.

[0404] 47. The method according to any one of the preceding items, wherein the media comprises at least two iron chelates, such as at least three iron chelates, such as for example: a. ferric quinate and ferric lactate;

[0405] b. ferric quinate and ferric citrate;

[0406] c. ferric lactate and ferric citrate;

[0407] d. erric quinate, ferric lactate and ferric citrate.

[0408] 48. The method according to any one of the preceding items, wherein the media comprises:

[0409] a. between 40 and 45 pM ferric citrate and between 40 and 45 pM ferric lactate; or b. between 80 and 90 pM ferric citrate and between 80 and 90 pM ferric quinate; or c. between 80 and 90 pM ferric lactate and between 80 and 90 pM ferric quinate; or d. between 25 and 35 pM ferric citrate, between 25 and 35 pM ferric lactate and between 25 and 35 pM ferric quinate; or

[0410] e. between 50 and 60 pM ferric citrate, between 50 and 60 pM ferric lactate and between 50 and 60 pM ferric quinate.

[0411] 49. The method according to any one of the preceding items, wherein the iron chelate is supplemented to the media:

[0412] a. after;

[0413] b. simultaneously as; and / or

[0414] c. before;

[0415] incubating the microorganism in said media.

[0416] 50. A composition comprising:

[0417] a. an iron chelate; and

[0418] b. isopropyl p-d-1-thiogalactopyranosid (IPTG).51. The composition according to item 50, wherein the iron chelate is as defined in any one of the preceding items.

[0419] 52. The composition according to any one of items 50 to 51 , wherein the composition comprises:

[0420] a. between 10 and 90% of iron chelate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of iron chelate; and b. between 10 and 90% of IPTG, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of IPTG;

[0421] with the proviso that the sum of the percentage of iron chelate and IPTG is not >100%.

[0422] 53. The composition according to any one of items 50 to 52, wherein the composition comprises:

[0423] a. between 5 and 15% of ferric lactate; and

[0424] b. between 30% and 50% of ferric quinate; and

[0425] c. between 30% and 50% of IPTG;

[0426] with the proviso that the sum of the percentage of ferric lactate, ferric quinate and IPTG is not >100%.

[0427] 54. A composition comprising:

[0428] a. ferric quinate and ferric lactate;

[0429] b. ferric quinate and ferric citrate;

[0430] c. ferric lactate and ferric citrate; or

[0431] d. ferric quinate, ferric lactate and ferric citrate.

[0432] 55. The composition according to item 54, wherein the composition comprises at least two iron chelates selected from the group consisting of ferric quinate, ferric lactate and ferric citrate, said composition comprising:

[0433] a. between 10 and 90% of ferric quinate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric quinate; and / or b. between 10 and 90% of ferric lactate, such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric lactate; and / or c. between 10 and 90% of ferric citrate, such as between such as between 20 and 80%, such as between 30 and 70%, such as between 40 and 60% of ferric citrate;

[0434] with the proviso that the sum of the percentage of iron chelates is not >100%.

[0435] 56. The composition according to any one of items 54 to 55, wherein the composition comprises:

[0436] a. between 25 and 35% ferric quinate, between 25 and 35% ferric lactate, and between 25 and 35% ferric citrate; or

[0437] b. between 45 and 55% ferric citrate and between 45 and 55% ferric lactate; or c. between 45 and 55% ferric citrate and between 45 and 55% ferric quinate; or d. between 45 and 55% ferric quinate and between 45 and 55% ferric lactate; with the proviso that the sum of the percentage of iron chelates is not >100%.

[0438] 57. A method for inducing and / or increasing germination of plant seeds and / or growth of a plant, said method comprising the steps of:

[0439] a) planting plant seeds in a growth media comprising an iron chelate which is ferric complex with a carboxylic acid;

[0440] b) subjecting the planted plant seeds to light;

[0441] thereby inducing and / or enhancing germination of the plant seeds and / or growth of the plant.58. The method according to item 57 wherein the iron chelate is selected from group consisting of ferric quinate (iron(lll) quinate), ferric citrate (iron(lll) citrate), , ferric lactate (iron(lll) lactate) , and ferric tyrosine (iron(lll) tyrosine) and combinations thereof.

[0442] 59. The method according to item 57 or 5857, wherein the increase in germination and / or growth is as compared to the germination of plant seeds and / or growth of a plant planted in the absence of an iron chelate, but otherwise cultivated under the same conditions.

[0443] 60. The method according to any one of items 57 to 59, wherein the iron chelate is selected from the group consisting of ferric quinate, ferric lactate and ferric citrate.

[0444] 61. The method according to any one of items 57 to 60, wherein the media comprises at least two iron chelates, such as at least three iron chelates, such as for example:

[0445] a. ferric quinate and ferric lactate;

[0446] b. ferric quinate and ferric citrate;

[0447] c. ferric lactate and ferric citrate; or

[0448] d. ferric quinate, ferric lactate and ferric citrate.

[0449] 62. The method according to any one of items 57 to 61, wherein the iron chelate is supplemented to the growth media:

[0450] a. after;

[0451] b. simultaneously as; and / or

[0452] c. before;

[0453] planting the plant seeds in said growth media.

[0454] 63. The method according to any one of items 57 to 62, wherein the iron chelate is supplemented to the growth media 5 days, 8 days and 15 days after planting the seeds.

[0455] 64. The method according to any one of items 57 to 63, wherein between 0.5 pM and 50 pM of iron chelate in is supplemented to the growth media, such as between 0.5 pM and 15 pM, such as between 1 pM and 10 pM, such as between 1 pM and 5 pM or for example in the range from 0.25 to 10 pm, such as from 0.25 to 8 pm, from 0.25 to 5 pm, from 0.25 to 5 pm..

[0456] 65. The method according to any one of items 57 to 64, wherein between 0.015 pmol and 5 pmol of iron chelate per plant seed is supplemented to the growth media, such as between 0.015 pmol and 1 pmol, such as between 0.015 pmol and 0.5 pmol, such as between 0.015 pmol and 0.05 pmol.

[0457] 66. The method according to any one of items 57 to 65, wherein the light is selected from the group of light consisting of blue light, red light, yellow light, green light and white light.

[0458] 67. The method according to any one of items 57 to 66, wherein the growth media is selected from the group consisting of soil, rockwool, peat moss, perlite, vermiculite, sponge, rice hulls, oasis cubes, clay pebbles, floral foam and coco coir.

[0459] 68. The method according to any one of items 57 to 67, wherein the plant is of the family Fabaceae, for example of the genus Vignia, such as of the species Vignia radiata. 69. The method according to any one of items 57 to 68, wherein the method comprises increasing the shoot length of the plant, wherein the increase in shoot length is as compared to the shoot length of a plant planted in the absence of an iron chelate, but otherwise cultivated under the same conditions.

[0460] 70. The method according to any one of items 57 to 69, wherein the growth media is soil and wherein the method comprises increasing the growth of soil bacteria, preferably wherein the soil bacteria is of the genus Rhizobacter, Gluconacetobacter or Bacillus, such as Gluconacetobacter diazotrophicus or Bacillus subtilis.Further Items - Microorganisms

[0461] 1. A method for modulating growth phase kinetics of a microorganism used in industrial fermentation, characterized in that

[0462] the microorganism is cultured in a medium comprising a ferric (III) iron chelate selected from ferric quinate, ferric citrate, ferric lactate, or combinations thereof in a concentration of from 1 to 400 pM Fe3+,

[0463] wherein the duration of the lag phase is reduced relative to a control culturing the same microorganism in the presence of an equimolar amount of Fe3+provided as ferric chloride, under otherwise identical culture conditions.

[0464] 2. The method according to item 1 , wherein the maximum specific growth rate (pmax) during exponential phase is increased relative to a control culturing the same microorganism in the presence of an equimolar amount of Fe3+supplied as ferric chloride, under otherwise identical culture conditions.

[0465] 3. The method according to item 1 or 2, wherein the duration of the lag phase is decreased by at least 10% relative to ferric chloride control.

[0466] 4. The method according to any of the preceding items wherein the maximum specific growth rate (pmax) is increased by at least 10% relative to ferric chloride control.

[0467] 5. The method according to any of the preceding items, wherein biomass accumulation prior to stationary phase differs relative to a control culturing the same microorganism in the presence of an equimolar amount of Fe3+provided as ferric chloride, under otherwise identical culture conditions.

[0468] 6. The method according to any of the preceding items wherein the microorganism is a non- pathogenic microorganism, a plant growth promoting microorganism, or an industrial microorganism.

[0469] 7. The method according to any of the preceding items, wherein the microorganism is selected from bacteria or yeast.

[0470] 8. The method according to any of the preceding items, wherein the microorganism is a bacterium selected from Bacillus spp., Escherichia coll, Lactobacillus spp., Lacticaseibacillus spp., Lactiplantibacillus spp, or combinations thereof, for example wherein the bacterium is Bacillus subtilis.

[0471] 9. The method according to any of the preceding items, wherein the microorganism is cultured in a liquid medium.

[0472] 10. A method for modulating metabolism of a microorganism, characterised in that the microorganism is cultured in a medium comprising a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, or combinations thereof in a concentration of from 1 to 400 pM Fe3+,

[0473] wherein intracellular metabolic profiles differ relative to a control culturing the same microorganism in the presence of an equimolar amount of Fe3+supplied as ferric chloride, under otherwise identical culture conditions.

[0474] 11. The method according to item 10, wherein the metabolic profile is determined by LC-MS.

[0475] 12. The method according to item 10 or 11 , wherein metabolites associated with central carbon metabolism differ relative to ferric chloride control.

[0476] 13. The method according to any of items 10 to 12, wherein production of one or more metabolites is increased relative to ferric chloride control.14. The method according to any of items 10 to 13, wherein utilisation of one or more carbon sources is increased relative to ferric chloride control.

[0477] 15. The method according to any of items 10 to 14, wherein expression of one or more genes associated with metabolism or cell division is increased relative to ferric chloride control 16. A method for increasing recombinant protein production in a microorganism, characterised in that

[0478] a. the microorganism comprises a heterologous nucleic acid encoding a recombinant protein operably linked to a promoter, and

[0479] b. the microorganism is cultured in a medium comprising a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, or combinations thereof at a concentration of from 1 to 400 pM Fe3+,

[0480] wherein recombinant protein production is increased relative to a control culturing the microorganism in the presence of an equimolar amount of Fe3+supplied as ferric chloride, under otherwise identical culture conditions.

[0481] 17. The method according to item 16, wherein the promoter is a T7 promoter.

[0482] 18. The method according to item 17, wherein expression of the recombinant protein is mediated by T7 RNA polymerase.

[0483] 19. The method according to item 18, wherein expression of T7 RNA polymerase is under control of a lac-derived regulatory system.

[0484] 20. The method according to any of items 16 to 19, wherein the increase in recombinant protein production occurs independently of lac operon-mediated de-repression.

[0485] 21. The method according to any of items 16 to 20, wherein the method further comprises addition of isopropyl p-D-1 -thiogalactopyranoside (IPTG).

[0486] 22. The method according to item 21, wherein ferric iron(lll) chelate and IPTG are present at equal molar concentrations.

[0487] 23. The method according to any of items 16 to 22, wherein recombinant protein yield is increased by at least 10% relative to ferric chloride control.

[0488] 24. The method according to items 21 to 23, wherein recombinant protein production is maintained or increased relative to IPTG alone.

[0489] 25. The method according to any of items 16 to 24, wherein the bacterium is Escherichia coll.

[0490] 26. The method according to any of items 16 to 25, wherein the recombinant protein is expressed under control of an inducible promoter.

[0491] 27. A method for modulating biofilm formation of a microorganism, characterised in that the microorganism is cultured in the presence of a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof, at a concentration of from 1 to 400 pM Fe3+,

[0492] wherein biofilm biomass differs relative to that obtained in a control using an equimolar amount of Fe3+supplied as ferric chloride, under otherwise identical culture conditions. 28. The method according to item 27, wherein biofilm biomass is increased by at least 10% relative to ferric chloride control.

[0493] 29. The method according to item 27 or 28, wherein the microorganism is a bacterium of the genus Bacillus.30. The method according to item 29 wherein the bacterium is Bacillus subtilis.

[0494] 31. A composition comprising a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof, for use in a method of:

[0495] (a) modulating growth phase kinetics of a microorganism; and / or

[0496] (b) increasing recombinant protein production in a microorganism; and / or (c) modulating biofilm formation of a microorganism.

[0497] 32. The composition according to item 31, further comprising isopropyl [3-D-1- thiogalactopyranoside (IPTG).

[0498] 33. The composition according to item 31 or 32, comprising at least two ferric iron(lll) chelates.

[0499] 34. The composition according to any of items 31 to 33 for use in fermentation.

[0500] 35. The composition according to any of items 31 to 34, for use in recombinant protein production recombinant protein production.

[0501] 36. The composition according to any of items 31 to 35, wherein the ferric iron(lll) chelate is present at a concentration corresponding to 1-400 pM Fe3+in use.

[0502] Further items - Plants

[0503] 1. A method for modulating at least one plant growth or developmental parameter in a plant, characterised in that

[0504] a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof is applied to a plant, plant seed or plant growth substrate at a concentration of 1-400 pM Fe3+,

[0505] wherein the least one plant growth or developmental parameter selected from shoot length, root length, biomass accumulation or germination rate differs relative to that obtained in a control using an equimolar amount of Fe3+provided as ferric chloride, under otherwise identical conditions.

[0506] 2. The method according to item 1 , wherein the plant growth or developmental parameter is increased by at least 10% relative to ferric chloride control.

[0507] 3. The method according to item 1 or 2, wherein the ferric iron chelate is ferric quinate. 4. The method according to any of the preceding items, wherein the plant is cultivated under light selected from blue light (450-495 nm), red light (625-750 nm), or white light (400-700 nm).

[0508] 5. The method according to any of the preceding items, wherein the plant is a legume. 6. The method according to item 5 wherein the plant is Vigna radiata.

[0509] 7. The method according to any of the preceding items, wherein the ferric iron chelate is applied to the growth substrate, such as to the soil.

[0510] 8. A method for modifying rhizosphere microbial composition associated with a plant, characterised in that

[0511] a ferric iron(lll) chelate selected from ferric quinate, ferric citrate or a combination thereof is applied to soil containing the plant at 1-400 pM Fe3+,

[0512] wherein the rhizosphere microbial composition differs relative to a control where the soil is treated with an equimolar amount of Fe3+supplied as ferric chloride.9. The method according to item 8, wherein relative abundance of plant growth-promoting microorganisms (PGPM) is increased compared to ferric chloride control.

[0513] 10. The method according to item 8 or 9, wherein relative abundance of plant pathogenic microorganisms is reduced compared to ferric chloride control.

[0514] 11. The method according to any of items 8 to 10, wherein microbial composition differences are determined by next-generation sequencing (NGS) of 16S rRNA gene amplicons. 12. The method according to any of items 9 to 11 , wherein at least one bacterial taxon associated with plant growth promotion is increased relative to ferric chloride control. 13. The method according to item 12, wherein the increased taxon belongs to the genus Bacillus, Pseudomonas, or Lactobacillus.

[0515] 14. The method according to any of items 8 to13 wherein alpha-diversity or beta-diversity of the rhizosphere microbiome differs relative to ferric chloride control.

[0516] 15. A method for enhancing plant growth via modulation of rhizosphere microbial composition,

[0517] characterised in that a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof is applied to soil or plant growth substrate,

[0518] wherein the application results in a shift in microbial composition associated with the rhizosphere,

[0519] and wherein said shift is associated with an increase in plant growth relative to a control treatment with an equimolar amount of Fe3+supplied as ferric chloride.

[0520] 16. The method according to item 15, wherein the rhizosphere microbial composition is determined by next-generation sequencing (NGS).

[0521] 17. The method according to item 15 or 16, wherein the relative abundance of beneficial microorganisms is increased relative to ferric chloride control.

[0522] 18. The method according to items 15 to 17, wherein the abundance of plant pathogenic microorganisms is reduced relative to ferric chloride control.

[0523] 19. A composition comprising: a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine, or combinations thereof, for enhancing plant growth and / or modifying rhizosphere microbiota in agriculture.

[0524] 20. The composition according to item 19, formulated for a carrier selected from water, soil additives, fertilizers, or seed coating materials.

[0525] 21. Use of a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof for:

[0526] (a) enhancing plant growth; and / or

[0527] (b) modifying rhizosphere microbial composition.

[0528] Examples

[0529] Induction methods have been shown to have a significant impact on various biological processes. In this study, we investigated the effects of a novel and natural bacterial and yeast inducer on the promotion of bacterial biofilm, growth, expression of recombinant protein, plant seed growth, and fermentation efficiency of plant-based materials. Our findings demonstrate that induction leads to an increase in bacterial biofilm formation, reduction of lag phase, and promotion of anti-pathogenic effects. Moreover, induction was found to be effective in promotingthe expression of recombinant proteins in bacteria. Additionally, the colour of light was found to have a significant effect on the induction of plant seed growth. Furthermore, induction was found to significantly increase the efficiency of fermentation of various plant-based materials. Overall, our study suggests that induction methods can be applied in various biological processes to enhance efficiency and promote desired outcomes.

[0530] It was found that the Iron chelates compounds can be used to modulate regulatory proteins, ensuring the activation of target genes and the induction of previously silent genes. This unique approach offers a high level of selectivity and precision in promoting bacterial growth, enabling targeted manipulation of metabolic pathways and gene expression. By utilizing these compounds, on may exert fine control over the desired cellular processes, leading to enhanced understanding and manipulation of bacterial behaviour and function.

[0531] The method was tested in a variety of bacterial strains, including Escherichia coll and Bacillus subtilis, Lactobacillus spp and was been shown to accelerate the growth of bacterial populations. The iron chelates were optimized for stability and potency, and their effectiveness demonstrated at low concentrations.

[0532] The novel method has potential applications in a range of fields, including biotechnology, agriculture, and medicine. In biotechnology, the method can be used to optimize bacterial growth and metabolic processes to produce biomolecules, such as enzymes and biofuels. In agriculture, the method can be used to enhance plant growth by manipulating the bacterial communities in the soil.

[0533] The method offers several advantages over traditional methods for inducing bacterial growth. First, the iron chelates are highly specific, allowing for targeted manipulation of metabolic pathways and gene expression. Second, the method is fast acting and can accelerate bacterial growth within hours, rather than days or weeks. Third, the method is versatile and can be applied to a wide range of bacterial strains and environmental conditions.

[0534] The iron chelates can be delivered to bacterial cells through the outer membrane / membrane proteins by addition to the culture media.

[0535] In summary, the present invention provides a novel method for inducing bacterial growth using specific iron chelates that specifically activates genes involved in bacterial metabolism and growth. The method offers several advantages over traditional methods for inducing bacterial growth, including specificity, speed, and versatility. The invention has potential applications in biotechnology, agriculture, and medicine, and provides a valuable tool for researchers and industry professionals seeking to optimize bacterial growth and metabolic processes.

[0536] Examples - Induction of microbial growth and production

[0537] 1. Enhancing recombinant protein expression

[0538] Introduction

[0539] A novel and natural bacterial inducer, e.g., Ferric quinate, Ferric citrate, Ferric lactate is shown herein to hold great promise for enhancing recombinant protein expression. By utilizing naturally occurring compounds with specific regulatory properties, the process becomes more sustainable and environmentally friendly. This innovative approach aims to harness the inherent capabilities of bacteria by activating their endogenous gene expression machinery. The natural inducer is shown to act as a signal to trigger the production of recombinant proteins, leading to higher yields and improved protein quality. This breakthrough not only reduces reliance on synthetic inducers but also ensures the preservation of biological systems and enables the development of more efficient and cost-effective protein expression systems.

[0540] Thus, herein is demonstrated a novel method for bacterial induction using a different iron chelates such as Ferric quinate, Ferric citrate, Ferric lactate that binds to a regulatory protein and activates genes involved in recombinant bacterial protein expression system. The method accelerates the expression of recombinant proteins and soluble secreted proteinsRecombinant protein expression in bacteria often requires the use of inducers to activate the expression of target genes. Isopropyl p-D-1 -thiogalactopyranoside (IPTG) is a widely used inducer. However, exploring novel inducers can offer advantages such as improved expression levels and reduced toxicity. In this study, we combined IPTG with a novel inducer to enhance recombinant protein expression in bacteria.

[0541] Materials and Methods

[0542] 1. Pre-culture and Inoculation:

[0543] • Pick a single colony from the transformed cells and inoculate it into a small volume of LB medium supplemented with the appropriate antibiotic.

[0544] • Incubate the pre-culture overnight at the optimal temperature with shaking to obtain a dense cell culture (e.g., OD = 0.4 to 0.7).

[0545] 2. Main Culture and Induction:

[0546] • Inoculate the main culture by transferring a small aliquot of the pre-culture into a larger volume of fresh LB medium supplemented with the appropriate antibiotic.

[0547] • Add the novel inducer at a suitable concentration (0.1 to 1mM) determined in preliminary experiments (Figure 1-2).

[0548] • While IPTG is commonly associated with inducing the lac promoter, it can also be used to induce other promoters in certain bacterial strains or expression systems. Like IPTG the effectiveness of novel inducers as an inducer for a specific promoter depends on factors such as the genetic background of the host strain and the regulatory elements associated with the promoter sequence. Therefore, it is possible to utilize Iron chelates as an inducer for promoters other than the lac promoter, provided that the specific promoter and bacterial strain are compatible with IPTG induction.

[0549] • Add IPTG to a final concentration within the range commonly used for induction (e.g., 0.1-1 mM) to activate promoter.

[0550] • Control cultures without the novel inducer or without IPTG was included for comparison (Figure 1-5)

[0551] • Incubate the main culture at the optimal temperature with shaking.

[0552] 3. Sampling and Analysis:

[0553] • Take periodic samples at specific time intervals to monitor bacterial growth and recombinant protein expression.

[0554] • Measure the optical density at a suitable wavelength to assess bacterial growth.

[0555] • Harvest a portion of the culture for protein extraction and purification.

[0556] • Analyse the expression level and quality of the recombinant protein using techniques such as SDS-PAGE, western blotting.

[0557] Results and discussion

[0558] The plasmid used in our experiments is based on a T7 promoter system, not a classical lac promoter. In this setup: The recombinant gene is under control of a T7 promoter. Expression is driven by T7 RNA polymerase, which is typically encoded in the host strain (E. coli BL21(DE3)). The T7 RNA polymerase itself is under control of a lac-derived regulatory system (e.g. lacUV5) and is induced by IPTG. Therefore, IPTG acts indirectly, by inducing T7 RNA polymerase, which then drives transcription from the T7 promoter.

[0559] Our experimental observations show that ferric iron chelates (e.g. ferric quinate, ferric citrate) increase recombinant protein production in this system. lmportantly:The effect is maintained when combined with IPTG (including at equal molar concentrations), and No inhibition is observed, which indicates that the chelates do not compete at the level of lac repressor or promoter binding. This strongly suggests that the chelates act downstream of transcriptional induction, likely via modulation of cellular metabolism, redox balance, or translational capacity.The effect of the novel inducer alone or in combination with IPTG on bacterial recombinant protein expression was evaluated. The results were compared with control cultures to assess the effectiveness of the novel inducer in enhancing protein expression. Production of recombinant proteins (PTA and YFQL; Figure 1-4) was promoted by minimum of 10% compare with IPTG, using only the novel inducer, i.e., Ferric quinate at 0.5 mM concentration. In addition, the expression of PTA increased between 22-45% depending on used IPTG concentration in combination with different Iron chelates, i.e., Ferric quinate and ferric lactate. Moreover, the similar pattern was observed concerning YQFL (Figure 3-4).

[0560] Figure 1 shows the induction of soluble plasmid protein PTA by complexes FeQ, Fe-cit, Fe-lac, or IPTG alone. Samples were collected from a LB culture inoculated with a 1 / 100 dilution of a saturated starter culture that reached stationary phase within a few hours. It should be noted that the expression of a recombinant protein may impose a metabolic burden on the microorganism, leading to a considerable decrease in generation time. The samples were induced with the respective inducers from the time of inoculation. The E. coli culture was either left uninduced, induced with 0.5 mM IPTG, or induced with a 0.5 mM concentration of the individual complexes. The PTA protein band was individually quantified three times for each band, considering the size and colour density. Overall, an increased expression level of the PTA protein (+10%) was observed when induced by the FeQ complex compared with IPTG.

[0561] Interestingly, Fe-cit and Fe-lac resulted in 11% and 21% lower expression levels, respectively, compared to IPTG alone. However, the self-sustaining induction by the complexes increased the expression of PTA compared to uninduced E. coli, indicating that the complexes act as weak inducers compared with IPTG.

[0562] Figure 2 shows the induction of soluble plasmid protein YQFL by complexes Fe-cit, FeQ, Ferric lactate, or IPTG alone. Samples were collected from a LB culture inoculated with a 1 / 100 dilution of a saturated starter culture that reached stationary phase within a few hours. It should be noted that the expression of a recombinant protein may impose a metabolic burden on the microorganism, leading to a considerable decrease in generation time. The samples were induced with the respective inducers from the time of inoculation. The E. coli carrying the plasmid culture was either left uninduced, induced with 0.5 mM IPTG, or induced with a 0.5 mM concentration of the individual complexes. The YGFL protein band was individually quantified three times for each sample, considering the size and colour density. Overall, increased expression levels of proteins were observed when induced by the complexes Fe-cit and FeQ. The results indicated a 4% and 10% higher expression by Fe-cit and FeQ, respectively, compared to IPTG alone. Interestingly, Fe-lac resulted in a 20% decrease in expression levels compared to IPTG alone. However, the self-sustaining induction by the complexes increased the expression compared to uninduced E. coli, suggesting that the complexes act as weak inducers. Additionally, the profile of unrelated E. coli proteins was comparable across all samples.

[0563] Figure 3 shows overexpression of soluble plasmid protein was induced using complexes FeQ and Fe-lac in combination with IPTG in E. coll carrying the plasmid culture. FeCI3(ferric chloride) is used as a control. The culture was inoculated with a 1 / 100 dilution of a saturated starter culture and reached stationary phase within a few hours. However, it should be noted that the expression of a recombinant protein may impose a metabolic burden on the microorganism, leading to a significant decrease in generation time.

[0564] SDS-PAGE was conducted to analyse the protein expression levels after 3 hours of induction for PTA expression under different treatments. The plasmids were left uninduced, induced with 0.5 mM IPTG alone, or induced with 0.5 mM IPTG along with an equal molar concentration of the individual complexes or in tandem. One exception was the treatment with 0.25 mM IPTG plus 0.25 mM FeQ. Overall, we observed increased expression levels of proteins induced by thecomplexes FeQ, Fe-lac, in combination with IPTG. The results indicated a 20-45% higher expression by FeQ + IPTG and Fe-lac + IPTG compared to IPTG alone. Interestingly, FeQ + Fe-lac + IPTG or IPTG + FeCI3combinations resulted in a 19% and 34% decrease in expression levels, respectively, suggesting competition between FeQ and Fe-lac for the same binding site. A similar observation was made with the FeCI3+ IPTG combination. The PTA protein band was individually quantified for each treatment three times, considering the size and colour density.

[0565] Figure 4 shows overexpression of soluble plasmid proteins by complexes (FeQ, and Fe-lac) in combination with IPTG. SDS-PAGE was performed to analyse the protein expression levels after 3 hours of induction for YQFL expression. The plasmids were left uninduced, induced with 0.5 mM IPTG alone, or induced with 0.5 mM IPTG along with an equal molar concentration of the individual complexes or in tandem. Exceptions were the treatments with 0.25 mM IPTG plus 0.25 mM FeQ and IPTG (0.5 mM) plus FeQ (0.5 mM) plus Fe-lac (0.125 mM) in the fourth lane from the left. Overall, an increase in expression levels of the protein was observed (8-25%) with the complexes FeQ, and Fe-lac in combination with IPTG. Interestingly, when the Fe-lac concentration was lowered to 0.125 mM in the combination of IPTG (0.5mM) plus FeQ (0.5mM) plus Fe-lac (0.125 mM), the expression increased by 18%, confirming the competition between FeQ and Fe-lac. In contrast, the combination of IPTG (0.5mM) plus FeQ (0.5mM) plus Fe-lac (0.5 mM) resulted in a 17% decrease in expression levels, indicating a competition between FeQ and Fe-lac for the same binding site. Regarding PTA, a slight increase (8-25%) in induced expression levels was observed with the various complex combinations compared to IPTG alone. The highest increase in overexpression (25%) was observed with the FeQ (0.5 mM) plus IPTG (0.5 mM) combination. The YQFL protein band was individually quantified for each treatment three times, considering the size and colour density.

[0566] Figure 5 shows the expression of TolC protein naturally produced by enteropathogenic E. coli (EPEC) wild type. The effect of complexes FeQ and Fe-cit (both at 0.5 mM concentration) on TolC protein expression was investigated. A LB culture inoculated with a 1 / 100 dilution of a saturated starter culture (overnight) was used, and the culture reached stationary phase within a few hours. The samples were collected and induced with the respective inducers from the time of inoculation. It should be noted that the EPEC used in this study is a wild-type bacteria without any plasmid.

[0567] The EPEC culture was either left uninduced or induced with 0.5 mM FeQ or 0.5 mM Fe-cit. It was observed that the complexes FeQ and Fe-cit induced an increase in protein expression levels ranging from 6% to 21%. Regarding TolC protein, a slight increase in expression levels (6%) was observed. However, the highest increase in overexpression was recorded with the FeQ inducer (21%). These results suggest that the induction of EPEC by the complexes is a specific mechanism and not a result of the complexes themselves. The TolC protein band was individually quantified for each treatment three times, taking into account the size and colour density.

[0568] In conclusion, the induction method provides an effective way to promote recombinant protein expression in bacteria. The use of these inducers alone or in combination with IPTG allow for the controlled and efficient expression of the protein of interest, leading to high yields and purity. The method is versatile and can be adapted to a range of bacterial strains and expression systems. Additionally, the method has potential applications in various fields, such as biotechnology, pharmaceuticals, and research. The described method represents a significant improvement over current methods for recombinant protein expression and has the potential to revolutionize the field.2. Enhancing biofilm formation

[0569] Introduction

[0570] Biofilm formation plays a crucial role in the survival and adaptability of Bacillus subtilis, offering numerous benefits across various industries (Branda et al., 2006). In this study, we investigate the utilization of a novel induction system by iron chelates, i.e., Ferric quinate for promoting biofilm formation in B. subtilis, considering the influence of light as an additional factor. By employing this system, we aim to enhance the industrial applications and harness the advantages associated with robust biofilm development. Through controlled experiments, we evaluate the effectiveness of the novel inducer in stimulating biofilm formation and characterizing its impact on biofilm architecture, structural integrity, functionality, and the influence of light as a modulator. Our findings demonstrate that the novel induction system, in combination with light, significantly promotes biofilm formation in B. subtilis, surpassing the capabilities of conventional inducers. Furthermore, we explore the potential applications of enhanced biofilm formation in industries such as agriculture, bioremediation, food production, and biomedical engineering, considering the added effects of light. This research highlights the promising potential of the novel induction system and the modulatory role of light in maximizing the advantages of B. subtilis biofilms, opening new avenues for industrial applications and providing a foundation for future studies in biofilm engineering and optimization.

[0571] Material and methods

[0572] Non-pathogenic B. subtilis was grown overnight in MSgg containing 0.5% glutamate in a shaking incubator at 200 rpm and 30°C. To study the effects of different compounds (Ferric quinate (FeQ), ferric lactate (Fe-lac), Ferric citrate (Fe-cit) and Ferric tyrosine, (Fe-tyr)) on biofilm formation at different concentrations, the crystal violet assay was performed using 12-well plates (with three replicates). MSgg containing 0.5% glutamate was supplemented with the different compounds or FeCI3 (as a control) at concentrations of 50, 100, and 250 pM. Half of the wells in the plates were filled with 1970 pl of media and 30 pl of the overnight bacterial culture. The crystal violet assay was employed to study the establishment of bacterial biofilms in the presence and absence of different Iron chelates.

[0573] Colonies of B. subtilis were cultured on MSgg + 0.5% glutamate agar at 30C to form biofilms in the presence and absence of different Iron chelates at various concentrations (50-250 pM). FeCI3 was used as a control.

[0574] Results and discussion

[0575] The study aimed to evaluate the effectiveness of an inducer in promoting biofilm formation by Bacillus subtilis under different light conditions (white, Blue, Red and dark), as well as variations in temperature (ranging from 21-30°C) and duration of incubation (2.5-14 days). Controlled experiments were conducted to compare the biofilm growth dynamics of B. subtilis in the presence and absence of the inducer.

[0576] Initially, the biofilm formation capability of B. subtilis was assessed under different light conditions without the inducer. The bacteria displayed limited biofilm formation, characterized by a thin and discontinuous layer on the substrate surface, regardless of the light conditions. The biofilm development was relatively slow, and the resulting biomass was sparse.

[0577] Next, the effect of the inducer on B. subtilis biofilm formation was investigated under both light and dark conditions. The addition of the inducer, i.e., Ferric quinate (400 pM), to the growth medium had a profound impact on biofilm development, particularly in the dark. In the absence of light, the bacteria exhibited enhanced adherence to the substrate and robust biofilm growth compared to the control group (FeCI3was used) without FeQ induction. The biofilms formed were visibly thicker, more structured, and displayed a higher biomass.

[0578] To further assess the impact of the inducer on biofilm formation, a pellicle assay was conducted. Pellicles are biofilms that form at the air-liquid interface and provide an additionalmeasure of biofilm formation. The results showed that the inducer significantly promoted pellicle formation by B. subtilis, under white light condition. The pellicles formed in the presence of the FeQ inducer were visibly thicker and more robust compared to those formed without induction. Microscopic analysis confirmed the substantial increase in pellicle formation in the presence of the FeQ inducer. Macroscopy images revealed dense and well-structured pellicle architecture with a higher biomass compared to the control group. The pellicles formed under dark conditions at 21°C for 2.5 days appeared to be more extensive, suggesting increased extracellular polymeric substance (EPS) production and better intercellular connectivity.

[0579] Moreover, observations demonstrated that the pellicles formed in the presence of the FeQ inducer exhibited accelerated maturation, increased resistance to physical disruption, maintaining their integrity even under mild agitation.

[0580] The inducer was found to upregulate genes associated with biofilm formation, including those involved in EPS synthesis, adhesion, and biofilm regulatory networks, regardless of the light conditions. This suggests that the inducer promotes the activation of biofilm-related genes, leading to enhanced pellicle formation.

[0581] Figure 6 shows increased concentration of induction compounds applied to bacterial samples generally lead to an increase in biofilm formation and bacterial growth. The data presented is based on two repeated experiments. Non-pathogenic B. subtilis was grown overnight in MSgg containing 0.5% glutamate in a shaking incubator at 200 rpm and 30°C. To study the effects of different compounds (Ferric quinate (FeQ), ferric lactate (Fe-lac), Ferric citrate (Fe-cit) and Ferric tyrosine, (Fe-tyr)) on biofilm formation at different concentrations, the crystal violet assay was performed using 12-well plates (with three replicates). MSgg containing 0.5% glutamate was supplemented with the different compounds or FeCI3 (as a control) at concentrations of 50, 100, and 250 pM. Half of the wells in the plates were filled with 1970 pl of media and 30 pl of the overnight bacterial culture.

[0582] Figure 7 shows examination of compounds (FeQ, Fe-lac, Fe-cit and Fe-tyr) and their effects on B subtilis biofilm formation in a concentration dependent manner. The figure shows the comparison between those treated with different Iron-chelates at different concentrations. Data was generated from Crystal Violet Assays. B subtilis biofilm formation is significantly promoted by all iron chelates compared with control FeCI3. However, the Fe-lac at 100pM wasn’t significant. Overall the data showed a specific effect by different Iron-chelates in these format of Iron complexation.

[0583] Biofilm formation is a social behaviour that enables sustained survival in the natural environment. In the Gram-positive bacterium Bacillus subtilis, this process involves differentiation of cell fate within an isogenic population and production of communal goods that form the biofilm matrix. Figure 8 shows biofilm formation of colonies of B. subtilis were cultured on MSgg + 0.5% glutamate agar at 30°C. to form biofilms in the presence and absence of different Iron chelates at various concentrations (50-250 pM). FeCI3 was used as a control. Among the different compounds tested, FeQ and Ferric lactate at 250 pM treatment resulted in the strongest biofilm.

[0584] Figure 9 shows biofilm formation of B. subtilis grown overnight in MSgg containing 0.5% glutamate in a shaking incubator at 200 rpm and 30°C. To study the effects of different compounds (Ferric quinate (FeQ), ferric lactate (Fe-lac), Ferric citrate (Fe-cit) and Ferric tyrosine, (Fe-tyr)) on biofilm formation at different concentrations, the crystal violet assay was performed using 12-well plates (with three replicates). MSgg containing 0.5% glutamate was supplemented with the different compounds or FeCI3 (as a control) at concentrations of 200 and 400 pM. Half of the wells in the plates were filled with 1970 pl of media and 30 pl of the overnight bacterial culture.Figure 10 shows Bacillus subtilis cells cultivated for a duration of 14 days on LBGM plates containing nutrients that promote biofilm formation. These plates were subjected to different light conditions, including white, blue, red, and no light (dark), in the presence and absence of FeQ at a concentration of 400 pM. Notably, the inclusion of FeQ resulted in the production of higher quantities of extracellular polymeric substances (EPSs) by the bacteria, which serve as a binding material to facilitate cell-cell and cell-surface adhesion.

[0585] An interesting observation was made regarding the effect of red light on the biofilm formation. Bacteria grown under red light exhibited the development of a distinct red ring around the colony biofilm. In contrast, the impact of white light on the formation of the red ring around the biofilm was less pronounced. Furthermore, it was observed that the formation of biofilm in plates supplemented with FeQ was more prominent in the absence of light (dark conditions), suggesting enhanced EPS expression.

[0586] Transmitted light images were captured to provide detailed insights into the microstructure of the biofilms. The control group depicted a smooth surface of the bacterial colony, while a closer examination of the biofilm formed in x-supplemented plates revealed rougher surfaces indicative of the presence of EPS-rich structures.

[0587] These findings highlight the role of light conditions and the presence of FeQ in influencing the formation and characteristics of B. subtilis biofilms. The red light-induced red ring and the enhanced biofilm formation under dark conditions suggest potential applications in the development of strategies for controlling biofilm-related issues and promoting biofilm-mediated protective effects against other bacteria.

[0588] Figure 11 shows a floating biofilm, known as a pellicle, generated with a unique macroscopic architecture. Cells were grown in LBGM over the weekends at 30°C. Interestingly, in the presence of 250 pM of FeQ, a noticeable red coloration was observed in the biofilm. It is worth noting that the red coloration cannot be attributed to the iron supplement, as the other samples, including the control supplemented with iron-chelates, did not exhibit a similar colour change. Once again, the FeQ-treated samples demonstrated the strongest pellicle formation among all the tested conditions.

[0589] Figure 12 shows biofilm formation on MSgg (minimal salts glycerol glutamate), 0.25 % glutamate. B. subtilis cells were cultured on MSgg agar supplemented with 0.25% glutamate, a crucial factor for biofilm formation. The cultures were incubated at 30°C to allow colony biofilm formation in the presence and absence of different compounds at various concentrations (50-250 pM). FeCI3 was used as the control. Remarkably, even in the presence of the essential 0.25% glutamate, the FeQ and Fe-cit treated samples exhibited the strongest biofilm formation. These findings highlight the potent biofilm-promoting effects of the FeQ and Fe-cit, demonstrating their ability to enhance biofilm formation in B. subtilis even under conditions where biofilm formation relies on the presence of glutamate.

[0590] Figure 13 shows B. subtilis cells cultured on LBGM agar at 30°C for a duration of approximately 2.5 days to allow the formation of colony biofilms. The effects of different Iron chelates at concentrations ranging from 200 to 400 pM were assessed on biofilm formation, with FeCI3 serving as the control. Notably, the FeQ and Fe-cit exhibited the most robust induction of colony biofilm formation compared to the control. Furthermore, the biofilms displayed distinct characteristics, including a thick ring pattern and a wrinkled appearance, indicative of elevated production of extracellular polymeric substances (EPS). These observations imply that the presence of the FeQ and Fe-cit stimulates EPS overexpression, thereby enhancing biofilm formation in B. subtilis.

[0591] Figure 14 shows the influence of light and incubation time on the occurrence of a reddish compound. B. subtilis was incubated for 3 and 6 days under dark conditions. The bacterial cultures were induced with FeQ at 400 pM or FeCI3(as a control). The results demonstratedthat B. subtilis produced a coloured compound at both 3 and 6 days of incubation. Moreover, the intensity of the coloration increased overtime. The architectural complexity of the pellicles also increased, indicating a higher production of secreted extracellular polymeric substances (EPS). The separated reddish compound was subsequently analysed for the identification and characterization of metabolites.

[0592] Figure 15 shows the influence of culture volume on the occurrence of a reddish compound. To investigate the influence of culture volume on the occurrence of a reddish compound, B. subtilis was induced with FeQ at concentrations of 250 and 400 pM and incubated for three consecutive overnights. The findings revealed that B. subtilis produced a coloured compound at both concentrations when the culture volume was lower. However, in the 4 ml culture, the red coloration was observed only at 250 pM. The separated reddish compound was subsequently analysed to identify and characterize the metabolites released by the bacteria.

[0593] The supernatant obtained from the 250 pM FeQ-treated condition exhibited a distinct reddish coloration on a filter membrane. To recover the sample, it was flushed with 50% methanol, and subsequently subjected to further analysis for the identification and characterization of metabolites.

[0594] Further experiments showed that the reddish coloration observed around Bacillus subtilis biofilms is due to the production of a pigment called pulcherrimin. This pigment is synthesized through a specific biochemical pathway where two molecules of tRNA-charged leucine are cyclized to form cyclo(L-leucine-L-leucine). This dipeptide is then oxidized by the enzyme CypX to produce pulcherriminic acid, a water-soluble compound. Once secreted, pulcherriminic acid chelates extracellular ferric iron (Fe3+), resulting in the formation of the insoluble, red-coloured pigment pulcherrimin.

[0595] Pulcherrimin serves multiple functions in B. subtilis. It acts as an iron chelator, limiting the availability of free iron in the environment, which can inhibit the growth of competing microorganisms. Additionally, pulcherrimin has been shown to protect B. subtilis against oxidative stress during biofilm development, enhancing the resilience and stability of the biofilm community. The biosynthesis of pulcherrimin is tightly regulated by multiple transcription factors, including ScoC, AbrB, and PchR, which coordinate to control the timing, rate, and amount of pulcherrimin produced, especially during the transition from exponential growth to stationary phase.

[0596] In summary, the reddish hue around B. subtilis biofilms is attributed to pulcherrimin, a pigment that plays a crucial role in iron sequestration and oxidative stress protection, thereby contributing to the ecological success of B. subtilis in various environments.

[0597] Figure 16 shows the effect of FeQ induction on biofilm formation investigated by using a bead assay. B. subtilis cells were induced with FeQ at a concentration of 40 pM, and the impact on biofilm formation was compared to a control group treated with FeCI3. The results revealed a significant increase in biofilm formation in the FeQ-induced samples, with an approximately 5000% enhancement compared to the untreated control. In contrast, the FeCI3-treated control exhibited a 370% increase in biofilm formation. Importantly, the biofilm formation induced by FeQ was approximately 14 times higher than that induced by FeCI3 at the same concentration. These findings indicate the specificity of the x induction complex and highlight the role of extracellular polymeric substances (EPS) overexpression in promoting biofilm formation. The data presented in this study are based on a collection of 12 replicates performed at two different occasions, ensuring the reliability and reproducibility of the results.

[0598] Figure 17 shows score plots of principal component analysis (PCA) generated to analyse the metabolites in LBMG media from B. subtilis and the reddish material isolated from the biofilm after 3 days of incubation in the dark. The study included 6 biological replicates. Metabolomics,which involves the comprehensive analysis of the metabolome, is a powerful tool for global metabolite profiling (Fiehn, 2002).

[0599] The score plots clearly illustrate the separation of the three groups. Each data point represents 6 replicate samples of the control and the isolated material (represented by the blue substrate). The distance between data points indicates the similarity between samples. The control group represents samples without any additives, while the second control group was supplemented with FeCI3. The FeQ-treated samples clustered in the upper left quadrant, while their corresponding FeCI3 samples clustered in the lower left quadrant. This indicates a significant change in metabolic levels between these two groups, with the magnitude of the changes dependent on the specificity of Fe-chelates (FeQ vs. FeCI3) in affecting B. subtilis for the expression of extracellular polymeric substances (EPS).

[0600] Figure 18 shows score plots of principal component analysis (PCA) were generated to analyse the metabolites in LBMG media from B. subtilis and the reddish material isolated from the biofilm after 3 days of incubation in the dark. The study included 6 biological replicates.

[0601] The score plots clearly show distinct separation of the three groups. Each data point represents six replicate samples of the control and the isolated material. The distance between points reflects the similarity between samples. The control group represents samples without any additives. The second control group was supplemented with FeCI3 as an iron compound control.

[0602] The FeQ-treated samples clustered in the upper left quadrant of the plot, while their corresponding FeCI3 samples clustered in the lower left quadrant. This indicates a significant change in the metabolic levels between these two groups. The magnitude of the changes depended on the specificity of FeQ in comparison to FeCI3, which affected B. subtilis metabolism including anabolism and catabolism.

[0603] These findings highlight the impact of the induction method on the metabolic profile of B. subtilis, suggesting alterations in key metabolic pathways and potential modulation of cellular processes.

[0604] In summary, the inducer proved highly effective in promoting biofilm formation by Bacillus subtilis, with a stronger effect observed under dark conditions. The inducer stimulated robust and structured biofilm growth, enhancing biomass production and stability. The inducer's impact was observed across different temperature ranges and incubation durations, highlighting its versatility. These findings provide valuable insights into optimizing inducer application for biofilm-related applications under varying environmental conditions.

[0605] In conclusion, our study demonstrates the efficacy of a novel induction method in promoting biofilm formation by non-pathogenic bacteria. Specifically, the use of FeQ and Fe lactate compounds significantly enhances biofilm formation by Bacillus subtilis. Among the tested compounds, FeQ proves to be the most effective in facilitating the formation of strong biofilms by B. subtilis. We observed a concentration-dependent effect of these compounds, with specific concentrations showing significant impact on biofilm formation (100-400 pM). Notably, even at the lowest concentration of 0.25% glutamate, FeQ continues to promote biofilm formation. Interestingly, the pellicle formation, indicative of robust biofilm growth, is most pronounced when using the iron chelates at concentration 250 pM. Additionally, we found that biofilm formation in plates supplemented with FeQ is more prominent under dark conditions, suggesting an enhanced expression of extracellular polymeric substances (EPS) in the absence of light. These findings highlight the potential of the novel induction method in facilitating strong biofilm formation and provide insights into the influence of compound concentration and light conditions on biofilm development.Overall, our results show that the induction method for the promotion of B. subtilis biofilm formation have potential applications in various industries, such as wastewater treatment and bioremediation.

[0606] 3. Enhancing growth, reducing lag phase and enhancing metabolism

[0607] Introduction

[0608] Herein is described a novel method for inducing B. subtilis growth, which involves the use of Iron chelates that activates specific genes involved in bacterial metabolism and growth. The iron chelates have been shown to significantly reduce the lag phase of bacterial growth, leading to faster and more efficient growth of bacterial populations. Furthermore, the induction method promotes the production of anti-pathogenic compounds by the bacteria, providing potential applications in the field of biotechnology, agriculture, and medicine. The patent application provides detailed information on examples of its use in bacterial induction experiments. Overall, the induction method offers a promising approach to enhancing bacterial growth and increasing the production of bioactive compounds.

[0609] The application of the inducers, i.e., Ferric quinate, Ferric lactate, Ferric citrate or a combination of inducers, in promoting the log phase and shortening the lag phase of Bacillus subtilis was investigated in this study. Through controlled experiments, the growth dynamics of B. subtilis in the presence of the inducer were compared to those without induction and only FeCI3was used.

[0610] Results and discussion

[0611] Initially, the growth kinetics of B. subtilis without induction were determined. The bacterial culture exhibited a characteristic lag phase, where the cells acclimated to the new environment and prepared for exponential growth. Following the lag phase, the cells entered the log phase, characterized by rapid growth and division.

[0612] Next, the effect of the inducer on B. subtilis growth was evaluated. The addition of the inducer to the growth medium resulted in notable changes in the growth behaviour of the bacteria. Firstly, the lag phase duration was significantly reduced (by 60 minutes) compared to the control group (FeCI3) without induction. The cells exhibited a quicker transition from the lag phase to the log phase, indicating a more rapid adaptation to the growth conditions.

[0613] Furthermore, the log phase of B. subtilis was greatly enhanced in the presence of the inducer. The cells displayed a higher growth rate and reached higher cell densities compared to the control group. This improvement in growth dynamics can be attributed to the inducer's ability to activate specific genes and metabolic pathways that promote cell division and biomass production.

[0614] To further elucidate the impact of the inducer on B. subtilis metabolism, various metabolic parameters were analysed. The inducer was found to enhance the utilization of carbon sources and the production of key metabolites essential for growth (listed below). Metabolic profiling by using Metabolomic analysis (QC / LCMS) revealed increased production of amino acids, organic acids, and energy molecules, indicating enhanced metabolic activity and cellular energy generation.

[0615] Additionally, the inducer influenced the expression of genes associated with nutrient uptake, metabolism, and cell division. Metabolomic analysis revealed upregulation of key metabolites involved in nutrient transport, biosynthesis pathways, and cell cycle regulation. These findings indicate that the inducer not only accelerates the growth rate but also facilitates the overall metabolic activity of B. subtilis.

[0616] We used growth assays for B. subtilis in an anaerobic condition to investigate how different inducer effect the bacterial growth over 1000 minutes (about 17 hours). To extract the growth rate in each condition, we fitted the growth curves with a modified Gompertz growth Equation. Where Kz is the absolute growth rate (i.e. tangent to the curve) and T Lag represents the time between recovery of the microbial population from being transferred to a new habitat and theoccurrence of substantial cell division. In the present case, the dependent variable W(t) represents the change in OD 600 as a function of time. The advantage of this reparameterisation is that the growth rate coefficient (Kz) constitutes the absolute growth rate at inflection, and that A (the upper asymptote) does not affect this parameter. Three individual growth curves were analysed in each condition.

[0617] Figure 19 shows the effect of Fe-cit in combination with Fe-lac (a total of 85pM) on the growth rate of B. subtilis under anaerobic conditions. The experiments were conducted with and without the presence of Fe-cit and Fe-lac, while FeCI3was used as a control. The growth curves of B. subtilis (n=3) averaged overtime showed a significantly reduced lag phase of approximately 120 minutes when Fe-cit and Fe-lac were present, compared to the medium supplemented with FeCI3, where the lag phase was about 180 minutes (GR; 0.0151 vs 0.0115, resp).

[0618] Interestingly, Fe-lac alone as a supplement did not show any effect on the lag phase, but when Fe-cit was added, the effect was enhanced. Notably, at a concentration of 42.5pM, Fe-cit did not exhibit the same strong effect on the lag phase as the combination of Fe-cit and Fe-lac. These results indicate that the combination of Fe-cit and Fe-lac has a synergistic effect on reducing the lag phase of B. subtilis growth under anaerobic conditions.

[0619] Figure 20 shows the effect of Fe-cit in combination with Fe-lac and FeQ (a total of 85 pM) on the growth rate of B. subtilis under anaerobic conditions. The experiments were conducted with and without the presence of these iron chelates combination, while FeCI3was used as a control. The growth curves of B. subtilis (n=3) averaged over time showed a significantly reduced lag phase in the presence of these iron chelates supplementation. After 60 minutes in the culture supplemented with Fe-cit, Fe-lac, and FeQ, the bacteria completed the lag phase and entered the exponential growth phase, while in the FeCI3supplemented medium containing 85 pM FeCI3, the lag phase was prolonged to approximately 3 hours. Additionally, the growth rate was higher in these iron chelates supplemented bacterial culture compared with FeCI3control (0.0175 vs 0.0115, resp.).

[0620] Interestingly, Fe-lac as a stand-alone supplement did not show any effect on the lag phase, but when Fe-cit and FeQ were added, the effect was enhanced. Notably, neither Fe-cit nor FeQ at 28 pM alone exhibited the same strong effect on the lag phase.

[0621] These results indicate that the combination of Fe-cit, Fe-lac, and FeQ has a synergistic effect on reducing the lag phase and enhancing the growth rate of B. subtilis under anaerobic conditions.

[0622] Figure 21 shows the effect of Fe-cit in combination with Fe-lac and FeQ (a total of 170 pM) on the growth rate of B. subtilis under anaerobic conditions. The experiments were conducted with and without the presence of these iron chelates combined, while FeCI3was used as a control. The growth rate curves of B. subtilis (n=3) averaged overtime showed a significantly reduced lag phase in the presence of these Iron chelates supplementation. After 120 minutes in the culture supplemented with Fe-cit, Fe-lac, and FeQ, the bacteria completed the lag phase and entered the exponential growth phase, while in the FeCI3supplemented medium containing 85 pM FeCI3, the lag phase was prolonged to approximately 3 hours (0.0150 vs 0.0115, respectively).

[0623] Interestingly, Fe-lac as a stand-alone supplement did not show any effect on the lag phase, but when Fe-cit and FeQ were added, the effect was enhanced. Notably, neither Fe-cit nor FeQ at 56 pM exhibited the same strong effect on the lag phase. In contrast, the effect was identical when Fe-cit and Fe-lac were combined.

[0624] These results suggest that the combination of Fe-cit, Fe-lac, and FeQ at a total concentration of 170 pM has a synergistic effect on reducing the lag phase and enhancing the growth rate of B. subtilis under anaerobic conditions.Figure 22 shows the bacterial growth curve of B. subtilis analysed by monitoring the absorbance at 600 nm every 20 minutes for a period of 20 hours. The average growth curves (n=3) revealed that the maximum population density of B. subtilis was significantly influenced by the presence of Fe-cit combined with FeQ (a total of 170 pM) in the culture medium.

[0625] Interestingly, in the presence of Fe-cit and FeQ, the logarithmic phase of growth was prolonged compared to the control (FeCI3). This suggests that the combination of Fe-cit and FeQ has a stimulatory effect on the growth of B. subtilis. However, the FeCI3control did not exhibit the same effect on the growth curve.

[0626] These findings highlight the potential role of Fe-cit and FeQ in enhancing the growth dynamics of B. subtilis.

[0627] Figure 23 shows the bacterial growth analysis of B. subtilis by monitoring the absorbance at 600 nm every 20 minutes for a period of 20 hours. The averaged growth curves (n=3) indicated that the maximum population density of B. subtilis was influenced by the presence of FeQ combined with Fe-lac (a total of 170 pM) in the culture medium.

[0628] Interestingly, in the presence of FeQ and Fe-lac, the logarithmic phase of growth in B. subtilis was prolonged compared to the control group treated with FeCI3. This suggests that the combination of FeQ and Fe-lac has a stimulatory effect on the growth dynamics of B. subtilis. Figure 24 shows principal component analysis (PCA) performed on metabolites obtained from B. subtilis induced with FeQ, with FeCI3 used as the control. Metabolic footprinting by mass spectrometry was employed to analyse the extracellular metabolites. After pre-processing steps, including removal of unvalued peaks and combining peaks from the same metabolite, a total of 1449 metabolites were identified in the samples.

[0629] To assess the reproducibility of the measurements across different biological replicates, the clean data were normalized and subjected to PCA. The results demonstrated that the biological replicates within each group clustered closely together, indicating high reproducibility. Notably, the quality control (QC) samples exhibited exceptionally high reproducibility, suggesting the overall stability and reliability of the experimental protocol and analysis methods.

[0630] In terms of the group comparisons, the PCA plots showed distinct separation between the FeQ-treated samples and their corresponding FeCI3-treated samples. The FeQ-treated samples clustered towards the upper left quadrant, while the FeCI3-treated samples clustered towards the lower left quadrant. This indicates significant changes in metabolic levels between the two groups, and the extent of these changes depended on the specificity of FeQ-chelates in influencing the expression of extracellular polymeric substances (EPS) in B. subtilis.

[0631] Each sample in the study included three biological replicates, further reinforcing the robustness of the findings.

[0632] Overall, the use of the inducer demonstrated significant effects on promoting the log phase and shortening the lag phase of B. subtilis. The inducer facilitated a faster adaptation of the cells to the growth conditions, resulting in reduced lag phase duration. Moreover, it stimulated enhanced growth during the log phase, leading to higher cell densities and improved overall growth rates. The inducer also modulated the metabolism of B. subtilis, promoting increased nutrient utilization and metabolic activity. These results highlight the potential of the inducer in optimizing bacterial growth and metabolism, with implications for various biotechnological and industrial applications.

[0633] Metabolomic analysis of Bacillus subtilis after induction with ferric quinate (FeQ) revealed a significant release of beneficial metabolites as shown below. These findings highlight the potential of the FeQ compound in modulating the metabolic profile of B. subtilis for various applications.

[0634] Mugineic acid: The overexpression of Mugineic acid by B. subtilis resulted in increased production of this specialized metabolite. Mugineic acid is known for its role in iron acquisitionand its potential application in improving iron bioavailability in plants or as a chelating agent in various industries.

[0635] Malonate: The overexpression of Malonate by B. subtilis led to a significant increase in the production of this metabolite. Malonate has been recognized for its potential applications in various industries, such as biotechnology and pharmaceuticals (Bryce-Smith, 1986; Albert, 1973).

[0636] Coformycin: Microbes (fungi, bacteria and actinomycetes) to provide an excellent source of natural herbicide products. Coformycin is a potent phytotoxins from microbes with potential herbicidal properties.

[0637] Sorbate: Induction of B. subtilis by FeQ led to a significant increase in the production of this compound. Sorbate has been recognized for its antimicrobial properties and potential applications in food preservation and pharmaceutical industries (Sahni, 1984). Karabulut and coworkers (Karabulut et al., 2005) reported that a single application of potassium sorbate applied to harvested grape berries partially controlled subsequent gray mold during cold storage.

[0638] Sinapate: Sinapate is known for its antioxidant and antimicrobial properties, making it valuable in various industries such as food, cosmetics, and pharmaceuticals.

[0639] Cyclohexanecarboxylic acid: it is formed from shikimic acid through a series of dehydrations and double-bond reductions in which no intermediate is ever aromatic. Cyclohexanecarboxylic acid has potential applications in various industries, including pharmaceuticals and bioplastics.

[0640] Orotate (OA): The expression of Orotate by B. subtilis resulted in the production of this important metabolite. Orotate plays a crucial role in nucleotide biosynthesis and has applications in fields such as biotechnology and pharmaceuticals. Also, the cytokinin-like behaviour of OA detected in plants suggest that OA has a role in regulating gene transcription in a range of different organisms.

[0641] Lespeol: strong antioxidant activity against DPPH radical (DPPH is a common abbreviation for the organic chemical compound 2,2-diphenyl-1-picrylhydrazyl). Lespeol has been recognized for its potential applications in various industries, including pharmaceuticals and agriculture.

[0642] 2-oxoglutarate: Inorganic nitrogen assimilation (a reductant), in the form of ammonium, onto carbon skeletons to produce amino acids is one of the most important biochemical processes in plants. Although nitrogen is present in the atmosphere, it is often a limiting nutrient for plant growth as only some plants can use this N source. Ammonium is the reduced nitrogen form available to plants for assimilation into amino acids. This is achieved by the GSuGOGAT pathway that requires carbon skeletons in the form of 2-oxoglutarate. Ammonium can be toxic to plant function and so it must be rapidly assimilated into non-toxic organic compounds.

[0643] Daphnoretin: Daphnoretin has been studied for its effects on biofilm formation and growth of Bacillus subtilis. It has been found that Daphnoretin exhibits inhibitory properties against biofilm formation of B. subtilis. Biofilms are structured communities of bacteria that can be difficult to eradicate and contribute to various infections. Daphnoretin has shown the ability to disrupt the formation of biofilms, thereby potentially reducing their pathogenicity. Interestingly the induction of B. subtilis by FeQ significantly reduced the expression of Daphnoretin compared with control (FeCI3). In addition to its impact on biofilms, Daphnoretin has also been investigated for its effects on the growth of B. subtilis. Studies have demonstrated that Daphnoretin exhibits antimicrobial activity against B. subtilis, inhibiting its growth.

[0644] 1-(3,4)-dimethylphenyl)ethanone: 1-(3,4-dimethylphenyl)ethanone is a chemical compound with a molecular structure consisting of a phenyl group substituted with two methyl groups. It is an aromatic ketone that may have various applications in organic synthesis and pharmaceutical research.Fluocinolone acetonide: it is in a blend of refined peanut and mineral oils and has been used as treatment for scalp psoriasis and for atopic dermatitis.

[0645] Biotin sulfoxide: it obtained as intermediate oxidation products between biotin and its sulphone. Pimelic acid plays an important role in biotin biogenesis in higher plants and has been shown that it is a degradation product.

[0646] L-Ornithine: it is a biproduct of arginase enzyme on L-arginine which is a central part of the urea cycle, and allows for the disposal of excess nitrogen.

[0647] Hexazinone: it is an organic compound that is used as a broad-spectrum herbicide.

[0648] Mefenacet: It is a member of benzothiazoles and a tertiary carboxamide. It has a role as an antimitotic and a herbicide. Since 1984 mefenacet with molinate at 30-60 kg / ha has given good control of weeds of rice in Spain, especially Echinochloa crus-galli, when applied pre-sowing or from the 3-leaf stage.

[0649] 3-(2-Carboxyethenyl)-cis,cis-muconate: Overexpression of 3-(2-carboxyethenyl)-cis,cis-muconate by Bacillus subtilis refers to the increased production of the compound 3-(2-carboxyethenyl)-cis,cis-muconate through induction by FeQ in B. subtilis. 3-(2-carboxyethenyl)-cis.cis-muconate is an intermediate compound in the degradation pathway of aromatic compounds, such as catechol and protocatechuate. B. subtilis possesses the ability to degrade aromatic compounds, and overexpression of the enzyme(s) involved in the production of 3-(2-carboxyethenyl)-cis,cis-muconate can enhance its production. The specific applications or motivations for overexpressing 3-(2-carboxyethenyl)-cis,cis-muconate in B. subtilis can vary. It may be done to enhance the efficiency of aromatic compound degradation, to study the metabolic pathways involved, or to produce 3-(2-carboxyethenyl)-cis,cis-muconate for industrial or research purposes.

[0650] Dihydrobiopterin (BH2): it is a pteridine compound produced in the synthesis of L-DOPA, dopamine, norepinephrine and epinephrine. It is restored to the required cofactor tetrahydrobiopterin by dihydrobiopterin reductase.

[0651] 2-Amino-2-deoxy-D-gluconate: Controlling the selectivity for metal ions during complexation is of great importance for practical purposes, for example, application to medicines, antibiotics, ion exchangers, etc. For over a quarter century, a great number of macrocyclic ligands, such as crown ethers, cryptands and sepulchrates have been developed and some of them are valid for the discrimination of metal ion size.

[0652] Valdecoxib: it is a new anti-inflammatory drug that is highly selective for inhibition of the inducible form of cyclooxygenase (COX-2). In recent years, many studies, focused on biotransformation of valdecoxib on medicinal plants. The production of pharmaceutical compounds like Valdecoxib typically involves complex chemical processes, while B. subtilis induced with FeQ synthesise it.

[0653] 3-Dehydroshikimic acid: It is a chemical compound related to shikimic acid. 3-DHS is available in large quantity through engineering of the shikimic acid pathway. The enzyme 3-dehydroquinate dehydratase uses 3-dehydroquinate to produce 3-dehydroshikimate. 3-Dehydroshikimate is then reduced to shikimic acid by the enzyme shikimate dehydrogenase, which uses nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor.

[0654] 3-Hydroxy-cis-5-tetradecenoylcarnitine: it belongs to the class of organic compounds known as beta hydroxy acids and derivatives. It is a derivative of Carnitine, essential cofactor of fatty acid metabolism; required for the transport of fatty acids through the inner mitochondrial membrane.

[0655] Streptamine: There are several related structural classes of aminoglycoside

[0656] antibiotics. Uniquely differentiated from the majority of aminoglycosides is streptomycin, which contains a guanylated streptamine core, as opposed to the deoxy-streptamine core of the other aminoglycosides.N(omega)-Hydroxyarginine: appears to interfere with cell proliferation / cell growth by inhibiting arginase, a binuclear Mn(2+) metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea.

[0657] 4-Hydroxy-2-nonenal (HNE): a cytotoxic product of lipid peroxidation, inhibits O2consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. A range of biotic and abiotic stresses raise oxygen species levels in plants due to perturbations of metabolism and the generation of AOS in defence responses (Bouchereau et al., 1991). Changes in the abundance and / or activities of a variety of plant mitochondrial proteins have been linked with response to AOS. Synthesis of the alternative oxidase was induced by H2O2treatment or addition of respiratory inhibitors that raised mitochondria AOS production (Padmanabhan, 2009).

[0658] Homocysteine thioacetone: Homocysteine (Hey) 1 -thiolactone, a cyclic thioester of Hey, was discovered by serendipity almost 70 years ago as a byproduct of the digestion of methionine with hydriodic acid, a procedure used then for the determination of protein methionine Hcythiolactone (Neuburger, 1996). It has been suggested to be a positive effector of the stationary phase response in Escherichia coli (Goodrich-Blair and Kolter, 1994) and is also likely to be involved in the regulation of methionine synthase gene expression in E. coli (Huisman and Kolter, 1994).

[0659] 9-hydroxy-hexadecan-1, 16-dioic acid: During evolution, plants have developed a variety of strategies to protect themselves against desiccation. One of the strategies is the formation of suberin, a cell specific, wall-associated biopolymer, during plant growth and development as well as in response to stress conditions such as wounding, where it serves as a physical barrier to protect against water loss and pathogen attack.

[0660] Dehydro juvabione: Certain plants contain substances with insect hormone like activity. These hormone analogues may influence insect-plant interactions like some other factors such as nutritional requirements, chemoreception, toxicity, etc. Experimental evidence indicates that an external supply of the hormonal substances, which comes from the plant without respect to the physiological control mechanisms, disturbs the precise synchronization of insect development and leads to the appearance of malformed creatures unable to survive and reproduce. The best-known example of a plant-derived JH mimic is juvabione or the “paper factor” which was identified after it was observed that fifth in stars of the linden bug, Pyrrhocoris apterus, from Europe, failed to mature into normal adults and instead metamorphosed into sixth instar supernumerary intermediates when reared in the United States.

[0661] Extracts from these paper towels were only active against the Hemipteran insect, P. apterus, but inactive against pupae of the silkworms, Hylophora cecropia, H. gloveri, Antheraea mylitt and Sarnia cynthia, and two other Hemipteran insects, Rhodnius prolixus and Oncopeltus fasciatus. Therefore, it appeared that only insects in the family Pyrrhocoridae are sensitive to this JH analogue.

[0662] Phosphoglycerol (15:0 / 0:0), PG: PG, and PI are important lipids that function as integral parts of thylakoid membranes. Maintenance of lipid composition may be used as a strategy in species with high resistance to long-term moderate heat, whereas species with less resistance show lipid degradation.

[0663] 3-beta-D-Galactosyl-sn-glycerol: There are two types of it, type A and type B. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGDI gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGDI is therefore likely devoted tothe massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network.

[0664] Urea-1 -carboxylate: Urea, also known as carbamide, is an organic compound. More than 90% of world industrial production of urea is destined for use as a nitrogenrelease fertilizer. Urea has the highest nitrogen content of all solid nitrogenous fertilizers in common use. Therefore, it has a low transportation costs per unit of nitrogen nutrient.

[0665] Juvenile hormone III diol: Juvenile hormones (JHs) are insect hormones which promote the retentionn of larval structures when present at larval-larval moults. One defensive strategy of plants against insect herbivory is the production of secondary metabolites which interfere with the physiology of the insect. It is thought that targeting the insect endocrine system makes it difficult for the insect to evolve counter adaptive strategies. Insect juvenile hormones (JHs) are sesquiterpenoids which are involved in the regulation of insect metamorphosis and reproduction. In some insect species, these hormones are also involved in the regulation of other physiological processes including diapause and behaviour. Plants have been shown to contain compounds which can either mimic JH activity or act as antagonists by inhibiting JH biosynthesis.

[0666] 3"-Deamino-3"-oxonicotianamine: a 30-deamino-30-oxonicotianamine reductase reduces the 30-oxo group to an alpha-hydroxy group to make deoxymugineic acid. Finally, this product can be hydroxylated to produce mugineic acid by a 29-deoxymugineic-acid 29-dioxygenase.

[0667] Mugineic acid and deoxymugineic acid are secreted by roots and then reassimilated via transporters that are family members of the Yellow Stripe 1 (YS1) group of proteins. The yellow stripe-like (YSL) transporter family has paved the way to decipher the mechanisms of longdistance transport of Fe.

[0668] 12-methyl-tetradecanoic acid: Many types of bacteria and marine organisms produce anteiso-fatty acids. Some bacteria have iso- but not anteiso-fatty acids while others have anteiso- but not iso-fatty acids. Some bacteria handle stress (such as heat and toxicity) by changing the ratio of anteiso / iso-fatty acids in the cell membrane. Although anteiso-fatty acids are not usually found in plant oils the waxy surface of leaves can contain significant amounts of these fatty acids. Branched chain fatty acids are critical for the regulation of fluidity in membranes and in membrane transport for many types of bacteria due to their having a significantly lower transition temperature than straight chain fatty acids.

[0669] 3-oxo-2-pentyl-cyclopentanehexanoic acid: Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. It is safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation.

[0670] 9-Hexadecenoic acid, (9Z)- (Palmitoleic acid): Palmitic acid, or hexadecanoic acid in IUPAC nomenclature, is the most common saturated fatty acid found in animals, plants and microorganisms. It is a major component of the oil from the fruit of oil palms (palm oil). Palmitic acid is used to produce soaps, cosmetics, and industrial mould release agents.

[0671] 4-Amino-4-deoxychorismate: The pyridoxal 5'-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. Folates are present throughout plant cells in the mitochondria, plastids, cytosol, and vacuoles but are synthesized only in mitochondria. The pterin and pABA precursors are made in the cytosol and plastids, respectively. Although neither 4-methyleneglutamine nor4-methyleneglutamic acid is found in free or bound form in ungerminated peanut seeds (Arachis hypogaea L), both non-protein amino acids are formed soon after germination. 4-Meglu appears within 2 days (slightly before 4-Megln is seen) and its concentration remains low. The level of 4-Megln, in contrast, risesrapidly between 2 and 10 days of germination and declines very slowly thereafter. In mature peanut plants, 4-Megln occurs in all parts except developing pods. It is virtually the only free amino acid in the xylem sap (constituting about 70% / o of the total soluble nitrogen) and appears to be the primary nitrogen carrier from roots to leaves in this species.

[0672] Eriodictyol: a flavonoid isolated from the Chinese herb Dracocephalum rupestre. It was found to significantly enhance fibre development through accumulating and maintaining the temporal auxin gradient in developing unicellular cotton fibres. It has long been known that fungal pathogens like Fusarium and Alternaria spp. produce toxins (mycotoxin) to kill plant cells. These mycotoxins have been shown to perturb the plant sphingolipid biosynthesis pathway, resulting in the necrotic cell death of plant cells. This cell death process is blocked by the SPT inhibitor, myriocin, suggesting that the increase in the cellular sphingolipid intermediates is crucial for the observed cell death.

[0673] Sorbic acid, or 2,4-hexadienoic acid, is a natural organic compound used as a

[0674] food preservative. The traditional route to sorbic acid involves condensation of malonic acid and trans-butenal. Sorbic acid and its salts, such as sodium sorbate, potassium sorbate, and calcium sorbate, are antimicrobial agents often used as preservatives in food and drinks to prevent the growth of mould, yeast, and fungi.

[0675] In conclusion, the use of FeQ-inducer has demonstrated significant effects on the expression of a series of very useful metabolites without the need for genetic modification. This finding holds great promise to produce valuable compounds in a more sustainable and efficient manner. By leveraging the properties of FeQ-inducer, it becomes possible to stimulate the production of desired metabolites in a non-invasive manner, avoiding the complexities and potential drawbacks associated with genetic manipulation. This approach opens new possibilities to produce valuable compounds, offering a potential alternative to traditional methods that rely on genetic modification. Further research and optimization of FeQ-inducer-based strategies are warranted to fully harness its potential for industrial applications.

[0676] In conclusion, the induction method described in this study has proven to be effective in promoting bacterial growth and reducing the lag phase. The use of this method may have practical applications in various fields such as biotechnology and microbiology, where efficient and rapid bacterial growth is crucial. Overall, this induction method provides a valuable tool for researchers to explore the metabolic capabilities of bacteria and may lead to the discovery of novel applications in different fields.

[0677] 4. Enhancing growth and secondary metabolism

[0678] Introduction

[0679] Next, we investigated the promotion of Lactobacillus’ and yeast strains’ growth by FeQ inducer, highlighting its role in metabolism and potential applications in various industries. Lactobacillus strains and yeast strain (Kluyveromyces marxianus, Yeast culture Kefir- 14, internal reference nr 14502), Lactobacillus delbrueckii LA-67 internal reference 14499) and Lactobacillus AD4550 / 01 (internal reference nr 14492). Cultures were treated with varying concentrations of FeQ inducer, and their growth kinetics and metabolic profiles were analysed. Results demonstrate that FeQ inducer significantly enhances the growth of Lactobacillus and yeast strains, leading to increased biomass production and improved metabolic activity. Moreover, FeQ inducer influences key metabolic pathways, such as carbohydrate utilization and organic acid production, contributing to enhanced fermentation processes. The findings underscore the potential of FeQ inducer as a valuable tool for optimizing microbial growth and metabolic performance in industrial applications, including food and beverage production, biofuel generation, and pharmaceutical manufacturing.

[0680] The promotion of Lactobacillus and yeast strains’ growth has been an area of interest for researchers aiming to optimize their industrial applications. The present approach involves theuse of specific inducers, such as FeQ inducer, which can enhance microbial growth and metabolism. These inducers act as modulators of key metabolic pathways, influencing the production of desirable metabolites and improving overall fermentation efficiency.

[0681] Understanding the role of FeQ inducer on the growth and metabolism of Lactobacillus and yeast strains are important for harnessing their full potential in various industrial sectors. By elucidating the effects of FeQ inducer on microbial physiology, including biomass production, metabolic pathways, and product synthesis, we can identify strategies to enhance productivity and optimize industrial processes.

[0682] Furthermore, the application of FeQ inducer in the industry has the potential to revolutionize production systems. By promoting the growth and metabolic activity of Lactobacillus and yeast, it can contribute to higher yields, improved product quality, and reduced processing times. Additionally, the modulation of metabolic pathways by FeQ inducer offers opportunities to tailor specific product profiles and optimize production for various applications.

[0683] In this study, we aim to investigate the promotion of Lactobacillus’ and yeast strains’ growth by FeQ inducer, focusing on its impact on microbial metabolism and its potential applications in diverse industries. By elucidating the underlying mechanisms and characterizing the metabolic changes induced by FeQ or mimic structures, we can provide insights into its role as a valuable tool for enhancing microbial performance in industrial settings. This knowledge will contribute to the development of more efficient and sustainable production processes, benefiting industries reliant on Lactobacillus and yeast-based fermentations.

[0684] Materials and Methods

[0685] Microorganisms: Lactobacillus strains and yeast strain (Kluyveromyces marxianus Yeast culture Kefir-14 (internal reference 14502), Lactobacillus delbrueckii LA-67 (internal reference 14499) and Lactobacillus AD4550 / 01 (internal reference 14492) were used in this study.

[0686] Growth Media: The growth medium used was MRS (De Man, Rogosa, and Sharpe) broth for Lactobacillus and yeast.

[0687] Preparation of Inducer Solution: FeQ-inducer was prepared as a stock solution with a concentration of 100 mM in sterile water.

[0688] Induction Experiment: A range of concentrations of FeQ-inducer (10-100 pM) was added to the MRS, to create the induction media. The final concentrations of FeQ-inducer in the induction media were adjusted accordingly.

[0689] Inoculum Preparation: Lactobacillus and yeast strains were cultured separately in their respective basal media (MRS) overnight at 37°C.

[0690] Inoculation and Incubation: The overnight cultures were inoculated into the induction media containing the desired concentration of FeQ-inducer, maintaining an appropriate dilution to achieve an initial optical density (OD) of 0.07-0.120. The cultures were incubated at 37°C with constant agitation.

[0691] Growth Measurement: The growth of Lactobacillus and yeast strains were monitored by measuring the optical density at a specific wavelength (e.g., 600 nm) at regular intervals using a spectrophotometer. Growth curves were plotted by recording the OD values overtime.

[0692] Metabolic Analysis: At specific time points during the growth, samples were taken from the cultures for metabolic analysis. This included measurements of pH, glucose consumption, organic acid production, and other relevant parameters using appropriate analytical techniques (e.g., high-performance liquid chromatography).

[0693] Statistical Analysis: The experimental data were analysed using appropriate statistical methods, such as analysis of variance (ANOVA) ort-test, to determine significant differences between control and induced cultures.

[0694] Replicates: All experiments were performed in triplicate or more to ensure the reliability and reproducibility of the results.Controls: Control cultures without the addition of FeQ-inducer was included to assess the baseline growth and metabolic activity of Lactobacillus and yeast.

[0695] Data Presentation: The results were presented as growth curves, metabolic profiles, and statistical analysis to illustrate the impact of FeQ-inducer on Lactobacillus and yeast strains’ growth and metabolism

[0696] Results and discussion

[0697] Effect of FeQ Inducer on Growth of Lactobacillus and Yeast: The addition of FeQ inducer to the growth media significantly promoted the growth of both Lactobacillus and yeast strains. Growth curves of the induced cultures showed higher optical density values compared to the control cultures without the FeQ inducer. The optimal effect was at 80 pM concentration of FeQ-inducer leading to greater growth enhancement.

[0698] Figure 25 shows bacterial growth analysis conducted on Lactobacillus strain 14492, with averaged growth curves (n=3, 4 replicates at each occasion), to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium. The growth was monitored by measuring the absorbance at 600 nm every 30 minutes for 6 hours.

[0699] The analysis of the growth curves for Lactobacillus strain 14492 showed that the presence of FeQ prolonged the logarithmic phase of growth, while FeCI3did not have the same effect. The slopes of the growth curves and the ratio were calculated. In the logarithmic growth curve, the slope was steeper in the FeQ-treated bacteria (0.16940) compared to the non-treated bacteria (0.08676), and the ratio of slopes between the treated and non-treated bacteria was 1.9523. This indicates that the bacterial population is growing more rapidly by a factor of 1.9523, and the time taken for cell division is reduced.

[0700] Figure 26 shows bacterial growth analysis of Lactobacillus delbrueckii LA-67 (14499 ), with averaged growth curves (n=3, 4 replicates at each occasion), to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium. The growth was monitored by measuring the absorbance at 600 nm every 30 minutes for 6 hours.

[0701] The analysis of the growth curves for Lactobacillus delbrueckii LA-67 14499 showed that the presence of FeQ prolonged the logarithmic phase of growth, while FeCI3did not have the same effect. The slopes of the growth curves and the ratio were calculated. In the logarithmic growth curve, the slope was steeper in the FeQ-treated bacteria (0.64376) compared to the non-treated bacteria (0.39910), and the ratio of slopes between the treated and non-treated bacteria was 1.6130 This indicates that the bacterial population is growing more rapidly by a factor of 1.6130, and the time taken for cell division is reduced.

[0702] Figure 27 shows growth analysis conducted on yeast strain 14502, with averaged growth curves (n=3, 4 replicates at each occasion), to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium. The growth was monitored by measuring the absorbance at 600 nm every 30 minutes for 6 hours.

[0703] The analysis of the growth curves for yeast strain (14502) showed that the presence of FeQ prolonged the logarithmic phase of growth, while FeCI3did not have the same effect. The slopes of the growth curves and the ratio were calculated. In the logarithmic growth curve, the slope was steeper in the FeQ-treated bacteria (0.71434) compared to the non-treated bacteria (0.48784), and the ratio of slopes between the treated and non-treated bacteria was 1.46429 This indicates that the bacterial population is growing more rapidly by a factor of 1.46429, and the time taken for cell division is reduced.

[0704] Figure 28 shows bacterial growth analysis conducted on Lactobacillus delbrueckii LA-67 (14499) phenotype Aci, with averaged growth curves (n=3, 4 replicates at each occasion), to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium. The growth was monitored by measuring the absorbance at 600 nm every 30 minutes for 6 hours.The analysis of the growth curves for Lactobacillus delbrueckii LA-67 (14499) phenotype Aci showed that the presence of FeQ prolonged the logarithmic phase of growth, while FeCI3did not have the same effect. The slopes of the growth curves and the ratio were calculated. In the logarithmic growth curve, the slope was steeper in the FeQ-treated bacteria (0.68060) compared to the non-treated bacteria (0.33396), and the ratio of slopes between the treated and non-treated bacteria was 2.0379 This indicates that the bacterial population is growing more rapidly by a factor of 2.0379, and the time taken for cell division is reduced.

[0705] Figure 29 shows bacterial growth analysis conducted on Lactobacillus strain (14492) phenotype Hel with averaged growth curves (n=3, 4 replicates at each occasion), to investigate the effect of Ferric Quinate (FeQ, 80 pM) in the MRS culture medium. The growth was monitored by measuring the absorbance at 600 nm every 30 minutes for 6 hours.

[0706] The analysis of the growth curves for Lactobacillus strain (14492) phenotype Hel showed that the presence of FeQ prolonged the logarithmic phase of growth, while FeCI3did not have the same effect. The slopes of the growth curves and the ratio were calculated. In the logarithmic growth curve, the slope was steeper in the FeQ-treated bacteria (0.24374) compared to the non-treated bacteria (0.13553), and the ratio of slopes between the treated and non-treated bacteria was 1.79834. This indicates that the bacterial population is growing more rapidly by a factor of 1.79834, and the time taken for cell division is reduced.

[0707] In order to verify the effect of induction by FeQ and Fe-lac on the efficiency of fermentation using plant-based side-stream, the total gas production was measured. The BPC bioprocess, Gas Endeavour (automatic Gas Flow measuring system), was utilized to measure the total gas released during fermentation.

[0708] Lactobacillus strain (14492) was subjected to fermentation of BSG (brewers' spent grain), with or without FeQ and Fe-lac induction, under air-limited conditions (figure 30). The results shown in figure 30 demonstrate a significant effect on the total gas release, indicating a more efficient fermentation and adaptation of the strains to the new environment, i.e., different types of plantbased side-streams.

[0709] As depicted in figure 30, which provides an example using BSG, the fermentation process promoted well beyond that of the control group. This observation suggests that the fermentation was more prolonged and efficient in the induced groups. The two-way analysis of variance (ANOVA) test is employed for comparing the means of multiple groups when there are two independent variables, also known as factors.

[0710] Further, Lactobacillus delbrueckii LA-67 (14499) was subjected to fermentation of BSG (brewers' spent grain), with or without FeQ and Fe-lac induction, under air-limited conditions (figure 31). The results demonstrated a significant effect on the total gas release, indicating a more efficient fermentation and adaptation of the strains to the new environment, i.e., different types of plant-based side-streams.

[0711] As depicted in figure 31, which provides an example using BSG, the fermentation process promoted well beyond that of the control group. This observation suggests that the fermentation was more prolonged and efficient in the induced groups. The two-way analysis of variance (ANOVA) test is employed for comparing the means of multiple groups when there are two independent variables, also known as factors.

[0712] Further, yeast strain 14502 subjected to fermentation of BSG (brewers' spent grain), with or without FeQ and Fe-lac induction, under air-limited conditions (figure 32). The results demonstrated a significant effect on the total gas release, indicating a more efficient fermentation and adaptation of the strains to the new environment, i.e., different types of plantbased side-streams (figure 32).

[0713] As depicted in figure 32, which provides an example using BSG, the fermentation process promoted well beyond that of the control group. This observation suggests that the fermentationwas more prolonged and efficient in the induced groups. The two-way analysis of variance (ANO A) test is employed for comparing the means of multiple groups when there are two independent variables, also known as factors.

[0714] Metabolomic Analysis: Metabolomic analysis revealed significant alterations in the metabolic pathways of both Lactobacillus and yeast strains upon induction with FeQ inducer. The induction resulted in changes in the production and utilization of various metabolites, including sugars, organic acids, amino acids and secondary metabolites.

[0715] Figure 33 shows data representing a class-3 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Lactobacillus 14492 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0716] The Score plot principal (figure 33) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.

[0717] A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites, indicating a shift in metabolic profile.

[0718] Figure 34 shows data representing a class-3 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Lactobacillus delbrueckii LA-67 14499 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0719] The Score plot principal (figure 34) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.

[0720] A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites.

[0721] Figure 35 shows data representing a class-3 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Kluyveromyces marxianus 14502 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0722] The Score plot principal (figure 35) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.

[0723] A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites.

[0724] Figure 36 shows data representing a class-2 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Lactobacillus 14492 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0725] The Score plot principal (figure 36) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites.

[0726] Figure 37 shows data representing a class-2 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Lactobacillus delbrueckii LA-67 14499 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0727] The Score plot principal (figure 37) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.

[0728] A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites.

[0729] Figure 38 shows data representing a class-2 OPLS-DA modelling for the metabolomic analysis was performed using QC / LCMS (Quality Control / Liquid Chromatography-Mass Spectrometry). The spent media collected from Kluyveromyces marxianus 14502 strain cultured in MRS media, as well as MRS media alone (used as a control), were subjected to analysis.

[0730] The Score plot principal (figure 38) revealed a significant difference between the material induced with FeQ and the non-induced material when compared to the MRS media control. Three replicate samples cultured at different occasions were analysed using LCMS and QC analysis.

[0731] A comparison between the non-induced and induced strains, relative to the MRS media control, demonstrated significant differences in a series of useful metabolites, including organic acids, carbohydrate utilization, and secondary metabolites.

[0732] Carbohydrate Utilization: The induction of Lactobacillus and yeast with FeQ inducer led to increased utilization of carbohydrates such as Maltose, as evidenced by reduced levels of residual sugars in the growth media. This indicates enhanced metabolic activity and more efficient conversion of sugars into energy and other metabolic products.

[0733] Organic Acid Production: Metabolomic analysis also revealed changes in the production of organic acids by Lactobacillus and yeast. Induction with FeQ inducer resulted in increased production of specific organic acids, such as citric acid and isocitric acid, indicating enhanced metabolic pathways involved in acid production.

[0734] Secondary Metabolites: Furthermore, the induction with FeQ inducer influenced the production of secondary metabolites in both Lactobacillus and yeast strains. Metabolomic profiling identified the presence of specific secondary metabolites, such as antimicrobial compounds or flavour-enhancing molecules, which could have potential applications in the food and beverage industry.

[0735] Statistical Analysis: Statistical analysis, such as t-test, confirmed the significant impact of FeQ inducer on the metabolic pathways of Lactobacillus and yeast. The differences observed in the metabolomic profiles between induced and control cultures were statistically significant, highlighting the specific effects of FeQ inducer on the metabolic activities of these microorganisms.

[0736] Overall, the results demonstrate that the addition of FeQ inducer promotes the growth of Lactobacillus and yeast strains and significantly affects their metabolic pathways. The enhanced growth and altered metabolomic profiles suggest that FeQ inducer acts as a modulator of key metabolic pathways, influencing carbohydrate utilization, organic acid production, and secondary metabolite synthesis. These findings have implications for various industrial applications, particularly in the food and beverage industry, where the promotion of microbialgrowth and modulation of metabolic pathways are desired for product development and optimization.

[0737] Examples of released metabolites that were significantly reduced or increased:

[0738] Pantothenic acid was significantly reduced in Lactobacillus delbrueckii strain 14499Pantothenic acid, also known as vitamin B5, plays an essential role in the growth and metabolism of Lactobacillus and other microorganisms. Here are some key aspects of the role of pantothenic acid for Lactobacillus growth:

[0739] Coenzyme A (CoA) synthesis: Pantothenic acid is a precursor for the synthesis of coenzyme A (CoA), an important cofactor involved in various metabolic pathways. CoA is essential for the metabolism of carbohydrates, lipids, and amino acids. Lactobacillus requires CoA for the synthesis and activation of fatty acids, energy production, and other key metabolic processes. Fatty acid synthesis: Pantothenic acid, as a component of CoA, is involved in the synthesis of fatty acids. CoA carries acetyl groups, which are essential building blocks for fatty acid synthesis. Fatty acids are important for cell membrane formation, energy storage, and the production of lipids required for the growth and viability of Lactobacillus.

[0740] Acetate metabolism: Lactobacillus species can metabolize acetate, a common byproduct of carbohydrate fermentation. Pantothenic acid is necessary for the activation of acetate into acetyl-CoA, which can then enter various metabolic pathways for energy production and biosynthesis. The availability of pantothenic acid influences the efficiency of acetate utilization by Lactobacillus.

[0741] Stress tolerance: Pantothenic acid is involved in stress response mechanisms in Lactobacillus. It plays a role in the synthesis of stress-related compounds, such as polyamines, which are important for cell protection, DNA stability, and adaptation to environmental stressors. Adequate pantothenic acid availability supports the stress tolerance and survival of Lactobacillus under challenging conditions.

[0742] Growth promotion: Pantothenic acid acts as a growth-promoting factor for Lactobacillus. Its presence in the growth medium or substrate is necessary to support the optimal growth rate, biomass production, and overall viability of Lactobacillus cultures. Insufficient pantothenic acid availability can lead to growth impairment and metabolic disturbances in Lactobacillus.

[0743] In summary, pantothenic acid is vital for the growth, metabolism, stress tolerance, and overall physiological functions of Lactobacillus. It is required for the synthesis of CoA, fatty acids, and stress-related compounds, supporting energy production, membrane formation, and adaptation to changing environments. Providing an adequate supply of pantothenic acid is crucial for maintaining the robust growth and viability of Lactobacillus strains in various applications, including probiotics, food fermentation, and biotechnology.

[0744] Uridine / pseudouridine was significantly increased in FeQ-induced yeast strain 14502 (Kluyveromyces marxianus).

[0745] Uridine and pseudouridine are nucleosides that can have various impacts on yeast metabolism. Here are some general effects of these compounds on yeast metabolism:

[0746] RNA synthesis and stability: Uridine and pseudouridine are essential components of RNA molecules. Uridine is incorporated into RNA during transcription and is important for RNA synthesis and stability. Pseudouridine, on the other hand, is a modified nucleoside that can be enzymatically introduced into specific RNA molecules. Both uridine and pseudouridine play roles in maintaining proper RNA structure and function, which are crucial for gene expression and overall cellular metabolism.

[0747] Nucleotide metabolism’. Uridine is also involved in nucleotide metabolism, serving as a precursor for the synthesis of other nucleotides. It can be converted to uridine monophosphate (UMP), which is further phosphorylated to form uridine triphosphate (UTP). UTP is essential forRNA synthesis and acts as a precursor for the synthesis of other nucleotides, including cytidine triphosphate (CTP).

[0748] Mitochondrial function’. Uridine can have specific effects on mitochondrial function in yeast. Uridine supplementation has been shown to rescue mitochondrial DNA (mtDNA) defects in certain yeast mutants. It can help restore proper mitochondrial protein synthesis and function, which are important for cellular energy production and metabolism.

[0749] Stress response’. Uridine and pseudouridine can impact yeast stress response pathways. They may influence the expression of stress-responsive genes, such as those involved in oxidative stress or heat shock response. Additionally, uridine has been implicated in modulating cellular responses to nutrient availability and growth conditions.

[0750] Ornithine was significantly increased by FeQ-induced Lactobacillus delbrueckii 14499.

[0751] Ornithine is an amino acid that can have important implications for the growth and metabolism of Lactobacillus. Here are some key aspects of the relationship between ornithine and Lactobacillus growth:

[0752] Polyamine biosynthesis: Ornithine is a precursor in the biosynthesis of polyamines, such as putrescine, spermidine, and spermine. Polyamines are essential compounds involved in various cellular processes, including cell growth, proliferation, and stress response. Lactobacillus requires polyamines for optimal growth and viability, and ornithine availability is crucial for the synthesis of these polyamines.

[0753] Acid resistance: Lactobacillus species are often exposed to acidic conditions in their natural environments, such as the gastrointestinal tract or fermented food products. Ornithine decarboxylase, an enzyme that converts ornithine to putrescine, is involved in acid resistance mechanisms in Lactobacillus. Putrescine can help protect Lactobacillus cells against acid stress by maintaining cellular pH homeostasis and stabilizing cell membranes.

[0754] Nitrogen metabolism: Ornithine is also involved in nitrogen metabolism in Lactobacillus. It serves as a precursor for the biosynthesis of other amino acids, such as arginine and proline, which are important for protein synthesis and various metabolic processes. Adequate ornithine availability supports nitrogen assimilation and utilization by Lactobacillus, promoting efficient growth and metabolic activities.

[0755] Stress response and adaptation: Ornithine and polyamines play roles in the stress response and adaptation mechanisms of Lactobacillus. They are involved in protecting cells against various environmental stressors, including oxidative stress, heat stress, and osmotic stress. Polyamines, derived from ornithine, can act as antioxidants, stabilize DNA, and enhance cell survival under adverse conditions.

[0756] Overall, ornithine is an important metabolite for Lactobacillus growth and survival. It contributes to polyamine biosynthesis, acid resistance, nitrogen metabolism, and stress response mechanisms. Providing an adequate supply of ornithine supports the robust growth, metabolism, and stress tolerance of Lactobacillus strains in various applications, including probiotics, food fermentation, and biotechnology.

[0757] Uracil was significantly increased by FeQ-induction in yeast 14502 (Kluyveromyces marxianus).

[0758] Uracil is a pyrimidine nucleobase that can have important implications for yeast growth and metabolism. Here are some key aspects of the relationship between uracil and yeast growth: Nucleotide synthesis: Uracil is a key component in the biosynthesis of RNA and DNA. It serves as a building block for the synthesis of uridine triphosphate (UTP), which is crucial for RNA synthesis. Without an adequate supply of uracil, yeast may experience impaired nucleotide synthesis, leading to growth inhibition.

[0759] Pyrimidine salvage pathway: Yeast can also salvage uracil through a pyrimidine salvage pathway. In this pathway, uracil is taken up from the environment and converted intonucleotides, bypassing the de novo synthesis pathway. The availability of uracil in the growth medium can affect the efficiency of the salvage pathway and the overall growth rate of yeast. Protein synthesis and metabolism: Uracil is involved in the synthesis of proteins and various metabolic processes in yeast. It contributes to the formation of transfer RNA (tRNA), which is essential for protein translation. Additionally, uracil is a precursor for the synthesis of other metabolically important compounds, such as thymine and certain coenzymes.

[0760] Stress response: Uracil metabolism and availability can influence the stress response and adaptive mechanisms in yeast. Adequate uracil levels are important for yeast to respond to various stressors, including nutrient limitation, temperature changes, and oxidative stress. Uracil depletion can compromise yeast's ability to mount an efficient stress response, leading to growth impairment.

[0761] In summary, uracil is vital for yeast growth, nucleotide synthesis, protein translation, and stress response mechanisms. Ensuring an adequate supply of uracil or appropriate conditions for uracil salvage supports the robust growth and metabolic activities of yeast.

[0762] Phenylethylamine (PEA) was significantly increased in FeQ-induced yeast strain 14502 (Kluyveromyces marxianus).

[0763] Phenylethylamine (PEA) is a biogenic amine that can have various effects on yeast growth and metabolism. Here are some key aspects of the relationship between phenylethylamine and yeast growth:

[0764] Growth promotion: Phenylethylamine has been reported to stimulate yeast growth in certain conditions. It can act as a growth-promoting factor by providing a carbon and nitrogen source for yeast metabolism. This effect may be more pronounced when phenylethylamine is present at low concentrations and yeast cells are in a nutrient-limited environment.

[0765] Metabolic regulation: Phenylethylamine can influence yeast metabolism by modulating the activity of specific enzymes and pathways. It has been shown to affect the expression of genes involved in carbohydrate and lipid metabolism, as well as the production of secondary metabolites. This modulation of metabolic pathways can impact yeast growth and overall cellular physiology.

[0766] Stress response: Phenylethylamine may have a role in the yeast stress response. It has been reported to enhance yeast tolerance to certain stress conditions, such as high temperatures and oxidative stress. Phenylethylamine can act as an antioxidant and protect yeast cells from oxidative damage, thereby promoting their survival and growth under stress.

[0767] Biofilm formation: Phenylethylamine has been implicated in the regulation of yeast biofilm formation. Biofilms are structured communities of microorganisms attached to surfaces, and they play a role in various processes, including fermentation and infection. Phenylethylamine can affect biofilm development in yeast by influencing cell adhesion and biofilm-related gene expression.

[0768] Alpha-aminobutyric acid (AABA) was significantly reduced in FeQ-induced yeast strain (Kluyveromyces marxianus).

[0769] Alpha-aminobutyric acid (AABA) can have varying effects on the growth of Kluyveromyces marxianus, a yeast species commonly used in industrial applications. Here are some key aspects of the relationship between AABA and the growth of Kluyveromyces marxianus’.

[0770] Growth promotion: In certain conditions, AABA has been reported to promote the growth of Kluyveromyces marxianus. AABA can serve as an alternative nitrogen source for yeast cells, supporting their metabolic activities and biomass production. When Kluyveromyces marxianus is grown in a medium with limited nitrogen sources, AABA may be utilized as an additional nitrogen resource to enhance growth.

[0771] Nitrogen metabolism: AABA can influence nitrogen metabolism in Kluyveromyces marxianus. It can be taken up by yeast cells and metabolized through various enzymatic reactions involved inamino acid metabolism. AABA can contribute to the synthesis of other amino acids, such as glutamate and glutamine, which are essential for protein synthesis and cellular functions.

[0772] Stress response: AABA exposure can trigger stress responses in Kluyveromyces marxianus. Similar to other yeast species, AABA-induced stress responses may involve the activation of stress-related genes and the production of stress proteins, such as chaperones and antioxidant enzymes. These stress responses help Kluyveromyces marxianus adapt to the presence of AABA and maintain growth and viability under challenging conditions.

[0773] Alanine was significantly reduced in FeQ-induced yeast strain 14502 (Kluyveromyces marxianus).

[0774] Alanine is an amino acid that can play a significant role in yeast growth and metabolism. Here are some key aspects of the relationship between alanine and yeast growth:

[0775] Protein synthesis: Alanine is one of the 20 standard amino acids used in protein synthesis. It serves as a building block for the production of proteins in yeast cells. Adequate availability of alanine is crucial for protein synthesis and the maintenance of optimal growth and cellular functions.

[0776] Nitrogen source: Alanine can serve as a nitrogen source for yeast growth. It can be utilized by yeast cells as a precursor for the synthesis of other amino acids and cellular components. When other nitrogen sources are limited, yeast cells can metabolize alanine to meet their nitrogen requirements and support growth.

[0777] Energy metabolism: Alanine participates in various metabolic pathways related to energy production in yeast. It can be converted to pyruvate through transamination reactions, and pyruvate serves as a central metabolite in glycolysis and other energy-generating pathways. Alanine metabolism contributes to the production of ATP and other energy-rich molecules that are essential for yeast growth.

[0778] Stress response: Alanine metabolism and availability can impact the yeast stress response. During stress conditions, such as nutrient limitation or exposure to toxic substances, the intracellular levels of alanine can change, triggering adaptive responses. Alanine can be involved in the synthesis of stress-related proteins and contribute to the overall stress tolerance and survival of yeast cells.

[0779] Leucine was significantly reduced in both yeast (Kluyveromyces marxianus) and Lactobacillus 14492 strains.

[0780] Leucine, an essential amino acid, can have important effects on the growth of both yeast and Lactobacillus species. Here's an overview of its relationship with the growth of these microorganisms:

[0781] Yeast Growth:

[0782] Protein synthesis: Leucine is a key component in protein synthesis and is required to produce functional proteins in yeast cells. It is one of the amino acids incorporated into nascent polypeptide chains during translation, supporting the formation of properly folded proteins and facilitating yeast growth.

[0783] Nutrient signalling: Leucine plays a vital role in nutrient signalling pathways, particularly through the target of rapamycin complex 1 (TORC1) signalling pathway. Leucine availability can activate TORC1, which regulates various cellular processes, including protein synthesis, nutrient uptake, and metabolism, ultimately promoting yeast growth.

[0784] Stress response: Leucine metabolism and availability can influence yeast stress responses. Leucine has been shown to be involved in the regulation of stress-related genes and pathways, contributing to the cellular adaptation and survival of yeast under stress conditions.

[0785] Lactobacillus Growth:Amino acid Leucine: Leucine is metabolized by Lactobacillus species as a source of energy and carbon. These bacteria possess enzymes capable of catabolizing leucine to generate energy and intermediate metabolites necessary for growth.

[0786] Acid tolerance: Leucine has been reported to enhance the acid tolerance of certain Lactobacillus strains. It can help these bacteria withstand acidic conditions, such as those encountered in the gastrointestinal tract, enabling their survival and growth in such environments.

[0787] Glycine and Creatinine were significantly reduced in Lactobacillus (14492) induced with FeQ.

[0788] When Lactobacillus 14492 was induced with a FeQ compound, it resulted in a significant reduction in glycine and Creatinine concentration in the growth media, suggesting that the compound may affect glycine or Creatinine metabolism or utilization by the bacteria. Here are some possible scenarios that could explain this observation:

[0789] Glycine or Creatinine consumption: The FeQ induced Lactobacillus strain 14492 may have an increased capacity to consume glycine from the growth media. The compound could trigger metabolic pathways or regulatory mechanisms that enhance the utilization of glycine or Creatinine by the bacteria. This increased uptake and utilization of glycine and Creatinine would lead to a reduction in its concentration in the media.

[0790] Metabolic alteration: The FeQ compound may cause metabolic changes in the Lactobacillus strain 14492, affecting its ability to synthesize or recycle glycine or Creatinine. It could inhibit enzymes or pathways involved in glycine or Creatinine biosynthesis or interfere with the conversion of other metabolites into glycine or Creatinine. As a result, the bacteria may rely more on external glycine or Creatinine sources, depleting its concentration in the media.

[0791] Secretion or conversion: The FeQ induced Lactobacillus strain 14492 may secrete enzymes or metabolites that interact with glycine or Creatinine, leading to its conversion or degradation. The compound could induce the production of enzymes that modify glycine / Creatinine or promote its breakdown, resulting in a reduced glycine / Creatinine concentration in the media.

[0792] Transporter modulation: The compound may affect the expression or activity of glycine / Creatinine transporters in Lactobacillus strain 14492. It could alter the uptake or efflux of glycine / Creatinine, influencing its availability within the bacteria and causing a decrease in glycine / Creatinine concentration in the media.

[0793] The specific mechanism underlying the observed reduction in glycine / Creatinine concentration would depend on the inducer used and its interaction with the Lactobacillus strain 14492.

[0794] Aspartic acid and Cysteine were significantly reduced in Lactobacillus (14492) induced with FeQ.

[0795] Aspartic acid and Cysteine, amino acids, can have various effects on the growth of Lactobacillus species. Here's an overview of its relationship with Lactobacillus growth:

[0796] Nutrient source: Lactobacillus species can utilize aspartic acid as a source of carbon and nitrogen. Aspartic acid / Cysteine serves as a nutrient for Lactobacillus growth, providing the necessary building blocks for protein synthesis and other cellular processes.

[0797] Acid tolerance: Aspartic acid has been reported to enhance the acid tolerance of certain Lactobacillus strains. It can act as a pH buffer, helping to maintain intracellular pH and protect Lactobacillus cells against acidic conditions, such as those encountered in the gastrointestinal tract. This increased acid tolerance can promote the survival and growth of Lactobacillus in challenging environments.

[0798] Metabolism regulation: Aspartic acid is involved in various metabolic pathways in Lactobacillus, including amino acid metabolism and energy production. It participates in the synthesis of other amino acids and contributes to the formation of key metabolic intermediates. Proper regulation of aspartic acid metabolism is crucial for optimal growth and metabolism of Lactobacillus.Citrulline was significantly reduced in FeQ induced Lactobacillus strain 14492.

[0799] While citrulline may not be an essential metabolite for Lactobacillus growth, its utilization or presence in the growth medium can still have potential physiological effects on Lactobacillus strains. For example, citrulline can be involved in arginine metabolism, and its availability can impact the synthesis of other metabolites and cellular processes.

[0800] Glutamic acid was significantly reduced in FeQ induced Lactobacillus strain 14492.

[0801] Glutamic acid, an amino acid, plays an essential role in the growth and metabolism of Lactobacillus species. Overall, glutamic acid is an important metabolite for Lactobacillus growth, serving as a nutrient source, contributing to energy metabolism, aiding in acid resistance, and influencing flavour development during fermentation. The availability and utilization of glutamic acid can impact the growth and physiology of Lactobacillus species, highlighting its significance in their overall metabolism.

[0802] Phenylalanine was significantly reduced in FeQ induced Lactobacillus strain 14492. Overall, phenylalanine serves as an important amino acid for Lactobacillus growth, contributing to protein synthesis, aromatic amino acid metabolism, flavour development, and stress response. The availability and utilization of phenylalanine can influence the growth, metabolism, and functional properties of Lactobacillus species.

[0803] Homocysteine was significantly reduced in FeQ induced Kluyveromyces marxianus. If Kluyveromyces marxianus is induced with a specific compound and exhibits a significant reduction in homocysteine concentration in the culture medium, it suggests that the compound affects homocysteine metabolism or utilization by the yeast. The compound may induce changes in the yeast's ability to consume, synthesize, recycle, or utilize homocysteine. Possible explanations for the observed reduction in homocysteine concentration include increased homocysteine consumption by the induced strain, metabolic alterations affecting homocysteine synthesis or conversion, utilization of homocysteine as a carbon or nitrogen source, or secretion and degradation of homocysteine by the yeast.

[0804] Glycerol 3-phosphate (G3P) was significantly reduced in FeQ induced Lactobacillus strain 14492.

[0805] Overall, G3P is a versatile metabolite that serves as an energy source, participates in redox reactions, contributes to membrane lipid synthesis, provides osmoprotection, and engages in metabolic crosstalk in Lactobacillus. Understanding the role of G3P in Lactobacillus metabolism is essential for elucidating the bacterial physiology, optimizing fermentation processes, and exploring potential applications in various biotechnological and industrial contexts.

[0806] Citric acid and isocitric acid were significantly reduced in FeQ induced Lactobacillus strain 14492.

[0807] In summary, citric acid and isocitric acid play essential roles in energy production, biosynthesis of metabolites, pH regulation, and stress response in Lactobacillus. Their involvement in the TCA cycle highlights their significance in central metabolism and cellular homeostasis.

[0808] Understanding the roles of citric acid and isocitric acid in Lactobacillus metabolism can provide insights into bacterial physiology, fermentation processes, and potential applications in various industries, including food and biotechnology.

[0809] Lysine was significantly reduced in FeQ induced Kluyveromyces marxianus

[0810] In summary, when Kluyveromyces marxianus is induced with a specific compound and exhibits a significant reduction in lysine concentration in the culture medium, it suggests that the compound affects lysine metabolism or utilization by the yeast. Possible explanations include enhanced lysine consumption by the induced strain, metabolic alterations affecting lysine synthesis or conversion, or secretion and degradation of lysine by the yeast.

[0811] Glucose 6-phosphate was significantly reduced in FeQ induced Lactobacillus strain 14492.When the induction of Lactobacillus results in a significant reduction in glucose 6-phosphate concentration in the culture medium, it indicates that the induction has an impact on the metabolism or utilization of glucose 6-phosphate by the bacteria. Understanding the impact of induction on glucose 6-phosphate metabolism in Lactobacillus can provide valuable insights into the bacteria's metabolic response, energy utilization, and biosynthetic processes. It can also have implications for various applications, including strain engineering and optimization of Lactobacillus-based bioprocesses.

[0812] Maltose / lsomaltose was significantly reduced in FeQ induced Lactobacillus strain 14492.

[0813] Lactobacillus induced with FeQ show a significant reduction in maltose concentration compared to non-induced conditions, suggesting that the induction affects the metabolism or utilization of maltose by the bacteria.

[0814] Enhanced maltose utilization: The induction of Lactobacillus may trigger the upregulation of maltose transporters and enzymes involved in maltose metabolism. This enhanced expression and activity enable the bacteria to consume maltose more efficiently, leading to a reduction in its concentration in the culture medium.

[0815] Induction of alternative carbon sources: The specific factor used for induction may provide an alternative carbon source that is preferentially utilized by Lactobacillus over maltose. The bacteria may shift their metabolic focus towards utilizing the induced compound, leading to a decreased reliance on maltose and subsequent reduction in its concentration.

[0816] Metabolic redirection: The induction could cause metabolic changes in Lactobacillus, resulting in a redirection of carbon flux away from maltose metabolism. The induced factor may activate enzymes or pathways that compete with maltose utilization, causing a decreased utilization of maltose and a subsequent reduction in its concentration in the medium.

[0817] Regulatory effects: The induction may have regulatory effects on gene expression and metabolic pathways in Lactobacillus. This could result in the downregulation of enzymes or transporters involved in maltose metabolism, leading to a decreased utilization of maltose and a significant reduction in its concentration.

[0818] It is important to conduct further investigations to determine the specific mechanism responsible for the observed reduction in maltose concentration and to understand the implications of the induction on Lactobacillus metabolism. Studying the induced factor's impact on gene expression, enzyme activity, and metabolic pathways can provide valuable insights into its mode of action and its potential applications in controlling or modulating carbohydrate metabolism in Lactobacillus.

[0819] Adenosine was significantly increased in FeQ induced Lactobacillus delbrueckii LA-67 14499.

[0820] Lactobacillus exhibits a significant increase in adenosine concentration after induction, it suggests that the induction has a positive effect on both the growth of the bacteria and adenosine production.

[0821] Enhanced growth: The induction of Lactobacillus promotes its growth and metabolic activity. This can result in increased biomass production, leading to higher adenosine production as a by-product of the bacteria's metabolic processes.

[0822] Metabolic rewiring: The induction may trigger metabolic changes in Lactobacillus, redirecting its metabolic pathways towards the synthesis or accumulation of adenosine. It could upregulate enzymes involved in adenosine biosynthesis or activate specific pathways that contribute to adenosine production.

[0823] Induced adenosine synthesis: The induction may specifically activate or upregulate enzymes responsible for adenosine synthesis in Lactobacillus. This can lead to increased conversion of precursor molecules into adenosine, resulting in its elevated concentration in the culture medium.Regulatory effects: The induction could have regulatory effects on gene expression and metabolic pathways in Lactobacillus. It may induce the expression of genes involved in adenosine metabolism, resulting in enhanced adenosine production by the bacteria.

[0824] Adenosine was significantly reduced in FeQ induced Kluyveromyces marxianus.

[0825] Kluyveromyces marxianus shows a significant reduction in adenosine concentration after induction, suggesting that the induction influences both the growth of the yeast and adenosine metabolism.

[0826] Altered adenosine utilization: The induction of Kluyveromyces marxianus may lead to changes in its metabolic pathways, causing a shift in adenosine utilization. The yeast may exhibit enhanced adenosine consumption for energy production or biosynthetic processes, resulting in a significant reduction in adenosine concentration in the culture medium.

[0827] Induced adenosine degradation: The induction could activate enzymes or pathways involved in adenosine degradation in Kluyveromyces marxianus. This enhanced degradation can lead to a significant reduction in adenosine levels as the yeast actively breaks down adenosine into its constituent components.

[0828] Metabolic rerouting: The induction may cause metabolic rerouting in Kluyveromyces marxianus, directing the flow of carbon and energy away from adenosine synthesis or utilization. This rerouting can result in reduced adenosine production or increased conversion of adenosine into other metabolites.

[0829] Regulatory effects: The induction may have regulatory effects on gene expression and metabolic pathways in Kluyveromyces marxianus. It may downregulate genes involved in adenosine synthesis or upregulate genes involved in adenosine degradation, leading to a significant reduction in adenosine concentration.

[0830] Examples - Improving seed germination and growth

[0831] Introduction

[0832] The present study investigates the impact of different filters for light, i.e., white, red, green, yellow, blue filters on seed germination and growth, along with the effects of FeQ and Fe-lac inducers at different concentrations (1, 5, 10 pM). Seeds of Vigna radiata were exposed to light filtered through all filters and treated with varying concentrations of FeQ or Fe-lac inducers. Germination rates, seedling development, and shoot growth were evaluated. Results indicate that the white filter required the lowest concentration of either FeQ or Fe-lac inducers, specifically 1 pM, to promote shoot growth in the seedlings. While the red filter require for the best performance the highest concentration of either FeQ or Fe-lac, specifically 10 pM.

[0833] However, for the green, yellow and blue filters the concentration of FeQ and Fe-lac inducers did not have a direct impact on shooting, although their presence significantly enhanced shoot growth compared to water alone. These findings highlight the role of light wavelengths and inducer concentrations in influencing seedling development and provide insights for optimizing growth conditions in agricultural and horticultural practices. Further research is warranted to elucidate the underlying mechanisms and explore the potential applications of light filtering and inducer treatments in plant cultivation.

[0834] We further hypothesized that inducers, such as FeQ and Fe-lac compounds, can influence plant growth and development by modulating physiological processes. These inducers may have direct effects on seed germination, root development, and shoot growth, as well as indirect effects on the surrounding microbial communities. Studying the combined effects of different light sources, inducers, and bacterial flora in soil can provide insights into the intricate interactions within the plant-soil ecosystem.

[0835] In this study, we aimed to investigate the effects of different light sources (including red, blue, white, green and yellow filters) on seed germination, growth, and the promotion of normal bacterial flora in the soil. Additionally, we will explored the influence of FeQ and Fe-lac inducersat various concentrations on seedling development and their potential impact on soil microbial communities. By elucidating these relationships, we can gain a deeper understanding of the complex interplay between light, inducers, and soil bacteria, paving the way for innovative approaches to optimize plant growth and enhance soil health.

[0836] Results and discussion

[0837] Effect of Different Light Sources on Seed Germination and Growth

[0838] When exposed to different light sources, the seeds exhibited variations in germination rates and seedling growth. The white filtered light treatment resulted in the highest germination percentage, followed by lowest concentration of either FeQ or Fe-lac at 1 pM. While the red filtered light required the highest concentration of either FeQ or Fe-lac. The yellow, blue filters had moderate effects, however significant while the unfiltered control group showed the lowest germination rates. Regarding seedling growth, the green-filtered light treatment promoted the most pronounced shoot development using Fe-lac inducer, as evidenced by longer and more robust shoots compared to other light treatments. Conversely, the yellow, blue and red-filtered light treatment showed moderate effects, while the control group exhibited the weakest shoot growth, however significant compare with water alone with normal light.

[0839] Impact of FeQ and Fe-lac Inducers on Seedling Development

[0840] The presence of FeQ and Fe-lac inducers significantly influenced seedling development.

[0841] Interestingly, the concentration of FeQ and Fe-lac inducers at the lowest 1 pM did have a direct impact on shoot development while the shooting was reduced at 10 pM using the white filtered light, while a diverse effect was observed using the red filtered light, best shooting at the highest concentration of FeQ. However, compared to the control group (water treatment alone), the presence of FeQ and Fe-lac inducers at any concentration resulted in significantly enhanced shoot growth, suggesting their positive effect on overall seedling vigor.

[0842] Figure 39 represents processed data of the mean shoot length (cm ± 0.05 cm) of 48 Vigna radiata plants that were exposed to white light through a transparent cellophane covering. The shoot lengths were measured six days after being planted. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Ferric Quinate or Ferric Lactate) at three different concentrations (1 pM, 5 pM, 10 pM) as well as tap water for the control on day 5. The results show a significant increase in shoots length for treated groups. Standard Error of Mean (cm ± 0.05 cm).

[0843] Figure 40 represents processed data of the mean shoot length (cm ± 0.05 cm) of 48 Vigna radiata plants that were exposed to red light through a transparent cellophane covering. The shoot lengths were measured six days after being planted. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Ferric Quinate or Ferric Lactate) at three different concentrations (1 pM, 5 pM, 10 pM) as well as tap water for the control. The results show a significant increase in shoots length for treated groups compared with water alone. SME (cm ± 0.05 cm).

[0844] Figure 41 represents processed data of the mean shoot length (cm ± 0.05 cm) of 48 Vigna radiata plants that were exposed to blue light through a transparent cellophane covering. The shoot lengths were measured six days after being planted. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Ferric Quinate or Ferric Lactate) at three different concentrations (1, 5, 10 pM) as well as tap water for the control. The results show a significant increase in shoots length for treated groups compared with water alone.

[0845] Standard Error of Mean (cm ± 0.05 cm).

[0846] Figure 42 represents processed data of the mean shoot length (cm ± 0.05 cm) of 48 Vigna radiata plants that were exposed to yellow light through a transparent cellophane covering. The shoot lengths were measured six days after being planted. The Vigna radiata seeds were given35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Ferric Quinate or Ferric Lactate) at three different concentrations (1 pM, 5 pM, 10 pM) as well as tap water for the control. The results show a significant increase in shoots length for treated groups compared with water alone. Standard Error of Mean (cm ± 0.05 cm).

[0847] Figure 43 represents processed data of the mean shoot length (cm ± 0.05 cm) of 48 Vigna radiata plants that were exposed to green light through a transparent cellophane covering. The shoot lengths were measured six days after being planted. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Ferric Quinate or Ferric Lactate) at three different concentrations (1 pM, 5 pM, 10 pM) as well as tap water for the control. The results show a significant increase in shoots length for treated groups compared with water alone. Standard Error of Mean (cm ± 0.05 cm).

[0848] Figure 44 depicts the mean shoot length (cm ± 0.05 cm) of 90 Vigna radiata plants that were exposed to different light colours (White, Red, Green, Blue Yellow) through cellophane coverings of the same colours. The shoot lengths measured on days five, six and twenty-one after being planted have been combined to create an overall mean shoot length at different concentrations of Ferric Quinate. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (FeQ) at three different concentrations (1, 5, 10 pM) on days 5, 8 and 15. The error bars represent the SEM, Standard Error of Mean. The concentration dependent nature of the shoot length in white light that has been previously noted is compared to other colours in order to establish whether or not different light colours in combination with the water supplement (FeQ) also have an impact on the shoot length, through the removal of certain wavelengths of light. It is interesting to see that overall, whilst shoot length increases as concentration decreases in white light with FeQ, in red light one can see the opposite pattern. One can also see that on average the shoot lengths are significantly lower overall in the colours Green, Blue and Yellow with no concentration or colour dependent relationship.

[0849] Figure 45 depicts the mean shoot length (cm ± 0.05 cm) of 90 Vigna radiata plants that were exposed to different light colours (White, Red, Green, Blue Yellow) through cellophane coverings of the same colours. The shoot lengths measured on days five, six and twenty-one after being planted have been combined to create an overall mean shoot length at different concentrations of Ferric Lactate. The Vigna radiata seeds were given 35 ml (± 0.05 ml) of tap water on the day of planting and 10 ml (± 0.05 ml) of water supplement (Fe-L) at three different concentrations (1, 5, 10 pM) on days 5, 8 and 15. The error bars represent the SEM, Standard Error of Mean. The concentration dependent nature of the shoot length in white light that has been previously noted is compared to other colours in order to establish the nature of Ferric Lactate as a control. Similarly, to FeQ the Blue and Yellow light colours in combination with the water supplement (Fe-L) did not have a significant impact on the shoot length or demonstrate a concentration or colour dependent relationship. However, the results show that shoot length remained relatively constant at all concentrations when exposed to the Red Filter, further highlighting how the antithetical relationship between White and Red light that was evident in FeQ is not mimicked in Fe-L. Furthermore, it is also clear that the Green filter demonstrates similar but not exactly the same results to the White light in Fe-L. In both White and Green light, the lowest concentration of 1 pM is the optimum concentration for maximum shoot growth, with the other two higher concentrations of 5 pM and10 pM proving to be less effective.

[0850] Effect of Light Sources and Inducers on Soil Bacterial Flora:

[0851] The combination of different light sources and inducers had discernible effects on the bacterial flora in the soil. Specifically, the blue-filtered light treatment, along with FeQ and Fe-lac inducers, promoted the growth of beneficial soil bacteria, including promoting rhizobacteria (PGPR). This finding highlights the potential of the blue-filtered light and inducer combination toenhance the population and activity of PGPR in the soil, which are known to contribute to plant growth promotion and disease suppression. Conversely, the red-filtered light treatment required the highest concentration of FeQ (10 pM) to elicit a comparable effect on the growth of PGPR and other beneficial soil bacteria. The white and yellow filters showed moderate impacts on the soil bacterial flora, albeit to a lesser extent compared to the blue-filtered light treatment.

[0852] Overall, these results demonstrate the interplay between different light sources, inducers, seedling development, and the promotion of beneficial soil bacteria, including PGPR (plant growth promoting rhizobacteria) or PGPM (plant growth promoting microorganisms. The blue-filtered light treatment, combined with 1-10 pM concentrations of FeQ and Fe-lac inducers, exhibited the most favourable outcomes in terms of seedling shoot growth and the promotion of PGPR populations in the soil. These findings provide valuable insights for optimizing plant growth conditions, enhancing soil health, and harnessing the benefits of PGPR in agricultural and horticultural practices. Further analysis and investigations are warranted to uncover the underlying mechanisms governing these interactions.

[0853] In conclusion, our studies provide a new understanding of tri-trophic (Bacteria-plant-specific compound(s)) interactions and their role in the induction of defence mechanisms, promoting the germination of seeds, growth, and reducing the need of fertiliser as a source for nitrogen. The results suggest that the incorporation of one, or a combination of ferric compounds in fermentation and formulation procedures will have a significant impact on the survival and colonization of both Gluconacetobacter diazotrophicus and B. subtilis on seeds. This new formulation is an invaluable source of Iron nutrition that can help the plant to protect itself even under stress condition due to the seasonal changes from potential threats and promote the plant growth by using the atmospheric nitrogen.

[0854] Material and Methods

[0855] 1. 300 Vigna radiata seeds were left to soak in a plastic bowl filled with tap water for 60 minutes. The water was measured out using a 500 ml plastic bottle.

[0856] 2. 64 transparent plastic cups were filled with 12g of potting soil. The soil-filled cups were weighed using a balance in order to ensure that the same volume of soil was in each cup.

[0857] 3. The soil-filled cups were placed in the carboard cup-holders. The cup-holders were labelled with the relevant colours, concentrations and compounds using a marker pen. There were 2 boxes, 18 cups and 36 seeds in total for each colour. For the white filter, there were 2 boxes, 16 cups and 48 seeds in total (one box for Fe-L with 6 cups compound, 2 cups water and 24 seeds and one box for FeQ with 6 cups compound, 2 cups water and 24 seeds).

[0858] 4. The Vigna radiata seeds were drained of water using a colander.

[0859] 5. A ceramic plate was covered with FeQ powder until the surface of the plate was evenly covered in compound.

[0860] 6. Using a pair of sterile plastic tweezers, 90 seeds were individually coated in FeQ powder. Care was taken to evenly coat each seed in powder. Excess powder was tapped off on the side of the plate. 90 of the best (unsplit, green) seeds were chosen, as the soaked seeds were prepared in excess to ensure a good selection to choose from.

[0861] 7. A 1 cm diameter plastic tube was marked with a line to indicate a 2cm depth using a marker pen and a ruler (± 0.05cm).

[0862] 8. The marked plastic tube was used to create three wells with a 2 cm depth in each cup that was marked as FeQ. Care was taken to ensure that the distribution of the wells was as even as possible and that they were not too close to the edges of the cup.

[0863] 9. Three coated seeds were planted into the three wells in the soil (one seed per well). 10. Steps 4-10 were repeated using Fe-L powder.11. 12 non-coated seeds (4 cups) were planted using steps 7-10. The non-coated seeds were planted in the cups that were marked as controls.

[0864] 12. Every cup was watered with 35 ml (± 0.05ml) of tap water using a measuring tube. 13. The freshly planted and watered cups were left for 24 hours with no covering to allow some of the water to evaporate. This was to ensure that the seeds would have the highest possible chance of successful germination, due to the fact that the cups had no drainage system.

[0865] 14. After the initial 24 -hour evaporation period, the rolls of cellophane were cur to size to produce sheets using a pair of scissors. The cellophane sheets of different colours (Red, Blue, Green, Yellow and transparent) were placed over the relevant cup holders. The cellophane was not fastened to the cup holders in order to ensure that a flow of air / ventilation system could be established.

[0866] 15. The cup holders covered in cellophane were placed in a well-lit, indoor are with a relatively stable temperature of around 26°C.

[0867] 16. The plants were left to germinate.

[0868] 17. On the fifth day after planting the seeds, the plants were watered with complex. Thus, 10 / zM stock solutions of Ferric Quinate and Ferric Lactate were prepared.

[0869] 18. Two stock solutions of 10 / zM were prepared by mixing 0.0063 g of FeQ powder and 0.0024g of Fe-L powder (weighed using a digital balance [± 0.000001]) in 1.00 litre of water using a 500 ml plastic bottle. The stock solutions were serially diluted to achieve 5 / zM (500 ml stock + 500 ml water) and 1 / zM (100 ml stock + 900 ml water).

[0870] 19. 10 ml of FeQ and Fe-L solution were used to water the plants at three different concentrations (10 / zM, 5 / zM and 1 / zM) using a measuring tube (± 0.05 ml). The control plants were given 10 ml of tap water.

[0871] 20. Five days after planting the seeds, the shoots were deemed suitable for measuring after achieving a stable growth rate. The shoots were measured in cm using a ruler (± 0.05 cm) and the data was recorded along with other qualitative observations, such as: stem quality and leaf number. The number of seeds that did not germinate was also taken note of. Care was taken when measuring the shoot height to be gentle when handling to prevent damage.

[0872] 21. At Day 6, it was noted that the shoots had rapidly doubled in height overnight, thus, step 20 was repeated.

[0873] 22. Step 26 was repeated until the next watering days (8 days after planting and 15 days after planting).

[0874] 23. The plants continued to be observed until the 21 -day mark.

[0875] 24. The plants were harvested, their roots were cut. Qualitative observations were made regarding the quality of the dead stems and the bagged roots.

[0876] References

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[0881] Stock, A. M., Robinson, V. L., & Goudreau, P. N. (2000). Two-component signal transduction. Annual Review of Biochemistry, 69(1), 183-215.Kim, J., & Hegde, M. (2019). Biotechnological production of recombinant proteins: current trends and future perspectives. Journal of Applied Microbiology, 126(1), 54-69.

[0882] Joska, T. M., Mashruwala, A. A., Boyd, J. M., & Botsford, J. L. (2014). Evidence for a second class of AraC DNA-binding proteins. PLoS One, 9(5), e97177.

[0883] Mulcahy, H., Charron-Mazenod, L, Lewenza, S., & Baird, R. C. (2008). High-throughput screening of Staphylococcus aureus genes required for bacterial growth during infection of host tissues. FEMS Immunology & Medical Microbiology, 54(1), 18-24.

[0884] Lopez, J. E., Sierra, S. A., Castano, L., & Rios, L. (2004). Induction of bacteriocin production by coculture is widespread among plantaricin-producing Lactobacillus plantarum strains with different regulatory operons. Journal of Bacteriology, 186(2), 615-624.

[0885] de Carvalho, C. C., & da Fonseca, M. M. (2005). Bacteriocins from lactic acid bacteria: purification, properties and use as biopreservatives. Brazilian Journal of Microbiology, 36(3), 251-256.

[0886] Branda, S. S., Chu, F., Kearns, D. B., Losick, R., & Kolter, R. (2006). A major protein component of the Bacillus subtilis biofilm matrix. Molecular microbiology, 59(4), 1229-1238. De Vuyst, L., Weckx, S. (2016). The cocoa bean fermentation process: From ecosystem analysis to starter culture development. Journal of Applied Microbiology, 121(1), 5-17.

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Claims

69CLAIMS1. A method for modulating at least one plant growth or developmental parameters in a plant, characterised in thata ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof is applied to a plant, plant seed or plant growth substrate at a concentration of 1-400 pM Fe3+,wherein the at least one plant growth or developmental parameter selected from shoot length, root length, biomass accumulation or germination rate differs relative to that obtained in a control using an equimolar amount of Fe3+supplied as ferric chloride under otherwise identical conditions.

2. The method according to claim 1 , wherein the plant growth or developmental parameter is increased by at least 10% relative to ferric chloride control.

3. The method according to claim 1 or 2, wherein the ferric iron chelate is ferric quinate.

4. The method according to any of the preceding claims, wherein the plant is cultivated under light selected from blue light (450-495 nm), red light (625-750 nm), or white light (400-700 nm).

5. The method according to any of the preceding claims, wherein the plant is a legume, such as wherein the plant is Vigna radiata.

6. The method according to any of the preceding claims, wherein the ferric iron chelate is applied to the growth subtrate, such as to the soil.

8. A method for modifying rhizosphere microbial composition associated with a plant, characterised in thata ferric iron(lll) chelate selected from ferric quinate or ferric citrate is applied to soil containing the plant at 1-400 pM Fe3+,wherein the rhizosphere microbial composition differs relative to a control where the soil is treated with an equimolar amount of Fe3+supplied as ferric chloride.

9. The method according to claim 8, wherein relative abundance of plant growth-promoting microorganisms (PGPM) is increased compared to ferric chloride control.

10. The method according to claim 8 or 9, wherein relative abundance of plant pathogenic microorganisms is reduced compared to ferric chloride control.

11. The method according to any of claims 8 to 10, wherein microbial composition differences are determined by next-generation sequencing (NGS) of 16S rRNA gene amplicons.

12. The method according to any of claims 9 to 11 , wherein at least one bacterial taxon associated with plant growth promotion is increased relative to ferric chloride control, for example wherein the increased taxon belongs to the genus Bacillus, Pseudomonas, or Lactobacillus.

13. The method according to any of claims 8 to12, wherein alpha-diversity or beta-diversity of the rhizosphere microbiome differs relative to ferric chloride control.

14. A method for enhancing plant growth via modulation of rhizosphere microbiota, characterised in that a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof is applied to soil or plant growth substrate,wherein the application results in a shift in microbial composition associated with the rhizosphere,and wherein said shift is associated with an increase in plant growth relative to a control treatment with an equimolar amount of Fe3+supplied as ferric chloride.7015. A composition comprising: a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate, ferric tyrosine or combinations thereof, for enhancing plant growth and / or modifying rhizosphere microbiota in agriculture.

16. Use of a ferric iron(lll) chelate selected from ferric quinate, ferric citrate, ferric lactate or ferric tyrosine and combinations thereof for:(a) enhancing plant growth; and / or(b) modifying rhizosphere microbial composition.

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