PROPIONIC ACID PRODUCTION IN BACTERIAL

CULTURE AND USES THEREOF

INCORPORATION BY CROSS-REFERENCE

[001] This Application claims priority from Australian Provisional Patent Application Number 2022901476 entitled “Propionic acid production in bacterial culture supernatants and uses thereof’ filed on 31 May 2022, the entire content of which is incorporated herein by cross reference.

FIELD OF THE DISCLOSURE

[002] The present disclosure relates to the production of propionic acid by microorganisms. Other forms of the disclosure relate to uses of propionic acid derived from microbial culture, microbial culture supernatants or propionic acid purified from microbial culture supernatants. [003] Although the present disclosure will be described hereinafter with reference to its preferred embodiment, it will be appreciated by those of skill in the art that the spirit and scope of the disclosure may be embodied in many other forms.

BACKGROUND OF THE DISCLOSURE

[004] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[005] Propionic acid (PA; CH3CH2CO2H) is a naturally occurring three-carbon fatty acid used in the agricultural, pharmaceutical and food industries. It is used in the manufacture of herbicides, fine chemical intermediates, rubber chemicals, emulsions, environmentally friendly solvents for coating formulations, artificial fruit flavours, pharmaceuticals, and modified synthetic cellulose fibres. It inhibits the growth of mould, fungi, and some bacteria and is also industrially used as a preservative. Further, commercially available synthetically produced PA can be used to facilitate emulsion polymerisation with, for example, lecithin and a surfactant. Such emulsions can reduce the surface tension of pesticides and increase their spreadability. [006] Demand for PA is high. PA can be produced synthetically, or biosynthetically by particular microorganisms. However, PA is costly to produce synthetically, with variable or limited worldwide supplies. Alternative ways to produce PA or PA-containing solutions that are more efficient, less costly or more environmentally acceptable are desired. [007] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[008] The present inventors have realised that biosynthetic production of PA by microorganisms may be enhanced by improving fermentation parameters and methods. Moreover, they have surprisingly found that clarified bacterial culture supernatants containing PA and PA purified from such supernatants may provide a suitable alternative source of PA for industrial use.

[009] Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

DEFINITIONS

[010] In describing and defining the present invention, the following terminology will be used in accordance with the definitions set out herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only and is not intended to be limiting.

[OH] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the disclosure pertains.

[012] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[013] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter.

[014] With respect to the terms “comprising”, “consisting of’ and “consisting essentially of’, where one of these three terms are used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of’.

[015] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about”, having regard to normal tolerances in the art. The examples are not intended to limit the scope of the disclosure. In what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.

[016] The term “substantially” as used herein shall mean comprising more than 50%, where relevant, unless otherwise indicated.

[017] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[018] The terms “preferred” and “preferably” refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the disclosure.

[019] It must also be noted that, as used in the specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[020] The person skilled in the art would appreciate that the embodiments described herein are exemplary only and that the electrical characteristics of the present application may be configured in a variety of alternative arrangements without departing from the spirit or the scope of the disclosure.

[021] Although exemplary embodiments of the disclosed technology are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosed technology be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosed technology is capable of other embodiments and of being practised or carried out in various ways.

[022] The term “bacterial culture” or “culture” as used herein, is intended to refer to a liquid medium which is suitable to support the growth of the bacteria of interest, after it has been inoculated with a suitable bacterial inoculum and incubated under conditions suitable to support growth of the bacteria for a suitable period of time such that the bacteria has undergone at least some growth, as would be readily understood by the person skilled in the art.

[023] The term “bacterial culture supernatant” as used herein, would readily be understood by a person skilled in the art to refer to the liquid or soluble component of a bacterial culture obtained when bacteria are grown in liquid culture media, after the culture has undergone a separation step, such as centrifugation, filtration, and/or precipitation step, that reduces or substantially removes insoluble material from the culture.

[024] The term “effectiveness” as used herein with respect to an industrial agent, for example, a pesticide, a herbicide, etc, refers to the activity of the industrial agent. For example, a herbicide with enhanced effectiveness would have an increased ability to kill plants, for example, it may kill plants more quickly, a particular amount of the herbicide may be used to treat more plants, or a particular number of plants may be killed using a lower concentration of the herbicide, when compared to a control herbicide.

[025] As used herein, the term “clarified”, such as in “a clarified bacterial supernatant” is intended to describe a liquid substance (e.g., a bacterial culture supernatant) that has undergone a method step, such as centrifugation, filtration, and/or precipitation, to reduce or substantially remove insoluble material from the liquid.

[026] The term “emulsion” as used herein refers to a mixture of two or more liquids that are normally immiscible owing to liquid-liquid phase separation; for example, one liquid may be present as a dispersion of minute droplets within the other liquid. This would readily be understood by a person skilled in the art

[027] The term “insoluble matter” as referred to herein with respect to the bacterial culture is intended to refer to solid particulate material as would readily be understood by a person skilled in the art. For example, the insoluble matter of a bacterial culture would include the non-liquid components such as bacterial cells, cellular matter, cellular fragments, membrane fragments, and under some circumstances may include smaller components such as insoluble proteins, etc.

[028] The term “purified” as referred to herein would readily be understood by a person skilled in the art to describe a substance that has undergone a treatment that removes at least some contaminating components from the substance.

[029] The term “spreadability” as used herein is intended to refer to the characteristic of a liquid to spread when applied to a surface, which can be determined by measuring the surface area that is covered by a particular volume of a liquid. Spreadability is a function of the surface tension of a liquid. A liquid with decreased surface tension has increased spreadability and would accordingly cover a higher amount of surface area when compared to a liquid with lower spreadability. Spreadability is a relative term that can be construed qualitatively or quantitatively by comparison against a control. Qualitative comparison may invite a visual comparison between two spray streams, with that displaying enhanced spreadability apparent merely upon inspection and comparison. A quantitative comparison may involve calculation of the respective areas (e.g., m ² ) covered by two sprays, with the stream displaying enhanced spreadability determined mathematically.

SUMMARY OF THE DISCLOSURE

[030] In a first aspect, the present disclosure provides a bacterial culture comprising propionic acid (PA) for use as a component of an emulsion. In an embodiment, the bacterial culture is obtained from culturing a bacterial strain selected from the group consisting of Propionibacterium spp., Acidipropionibacterium spp., and Cutibacterium spp. In an embodiment, the bacterial culture comprises between 0.2% (v/v) and 2% (v/v) propionic acid. In an embodiment, the bacterial culture is irradiated. In an embodiment, the bacterial culture is gamma-irradiated.

[031] In a second aspect, the present disclosure provides clarified bacterial culture supernatant obtained from the bacterial culture of the first aspect. In an embodiment, the clarified bacterial culture supernatant comprises between 0.2% (v/v) and 2% (v/v) propionic acid.

[032] In a third aspect, the present disclosure provides purified propionic acid for use as a component of an emulsion, wherein the purified propionic acid has been purified by extraction and/or distillation of the clarified bacterial culture supernatant of the second aspect.

[033] In a fourth aspect, the present disclosure provides the bacterial culture of the first aspect, the clarified bacterial culture supernatant of the second aspect, or the purified propionic acid of the third aspect, wherein the emulsion is adapted to increase the spreadability of an industrial agent. In an embodiment, the industrial agent is a herbicide or a pesticide.

[034] In a fifth aspect, the present disclosure provides a method of preparing a clarified bacterial culture supernatant comprising propionic acid, the method comprising the following steps:

(i) inoculating a suitable culture medium with a bacterial strain that releases propionic acid into the culture medium;

(ii) culturing the bacterial strain in the culture medium under suitable conditions for a suitable length of time to obtain a bacterial culture comprising propionic acid; and

(iii) separating the culture supernatant from insoluble particulate matter of the bacterial culture by centrifugation and/or filtration to obtain a clarified culture supernatant comprising at least 0.1% v/v propionic acid.

[035] In an embodiment, the bacterial strain is selected from the group consisting of Propionibacterium spp., Acidipropionibacterium spp., and Cutibacterium spp. In an embodiment, the clarified culture supernatant comprises between 0.2 (v/v) and 2% (v/v) propionic acid. In an embodiment, the culture medium has a neutral pH at step (i). In an embodiment, the suitable conditions in step (ii) comprise a temperature between 30 C and 34 C. In an embodiment, the suitable conditions in step (ii) comprise a room temperature or an environmental temperature. In an embodiment, the suitable conditions in step (ii) comprise a temperature between 18 °C and 28 C. In an embodiment, the suitable conditions in step (ii) comprise microaerophilic conditions.

[036] In a sixth aspect, the present disclosure provides the method of the fifth aspect, further comprising the following step:

(iv) purifying the propionic acid from the clarified culture supernatant by extraction and/or distillation to obtain purified propionic acid.

[037] In an embodiment, the pH of the bacterial culture is adjusted to less than 5.0 immediately prior to step (iv).

[038] In a seventh aspect, the present disclosure provides a clarified bacterial culture supernatant comprising propionic acid obtained using the method of the fifth aspect.

[039] In an eighth first aspect, the present disclosure provides a purified propionic acid obtained using the method of the sixth aspect.

[040] In a ninth aspect, the present disclosure provides the purified propionic acid of the third or eight aspects, wherein the concentration of propionic acid is at least 2% (v/v).

[041] In a tenth aspect, the present disclosure provides an emulsion comprising the bacterial culture of the first aspect or the clarified bacterial culture supernatant of the second or seventh aspects, a lecithin and a surfactant. In an embodiment, the emulsion when mixed with an industrial agent increases the spreadability of the industrial agent.

[042] In an eleventh aspect, the present disclosure provides an industrial agent composition comprising an industrial agent and the emulsion of the tenth aspect having enhanced spreadability compared to the industrial agent in the absence of the emulsion.

[043] In a twelfth aspect, the present disclosure provides an industrial agent composition comprising an industrial agent and the clarified bacterial culture supernatant of the second or seventh aspects having enhanced spreadability compared to the industrial agent in the absence of the clarified bacterial culture supernatant.

[044] In a thirteenth aspect, the present disclosure provides an industrial agent composition of the eleventh or twelfth aspects, wherein the industrial agent composition has enhanced effectiveness compared to the industrial agent in the absence of the emulsion.

[045] In some embodiments of the disclosure, the clarified bacterial culture supernatant comprises about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9% (v/v) propionic acid. In some embodiments of the disclosure, the bacterial culture comprises about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9% (v/v) propionic acid. In some embodiments, the concentration of the purified propionic acid is at least 3, 4, 5, 6, 7, 8, 9 or 10% (v/v).

BRIEF DESCRIPTION OF THE DRAWINGS

[046] A preferred embodiment of the present disclosure will now be described having regard to the accompanying drawings/figures in which:

[047] Figure 1 provides schematic diagrams of the propionic acid fermentation pathways; (A) 1,2-propanediol pathway (entry points directly through sugars such as fucose, glycolytic intermediate glycerone phosphate or lactate), (B) acrylate pathway, (C) Wood-Werkman pyruvate transcarboxylase pathway (D) methylmalonyl-CoA decarboxylase pathway.

[048] Figure 2 provides an example of quantifying the spread of the liquid on leaves from controls and tests using Adobe Photoshop 2022. An area of 158 px x 158 px adjusted using “Brightness/Contrast” and liquid droplets were selected using the “lasso” tool.

[049] Figure 3 provides photographs representative of growth of colonies of (A) HI broth P. freudenreichii culture and (B) yeast extract lactose (YEL) P. freudenreichii culture, respectively, at passage two during cell density testing.

[050] Figure 4 provides a plot of optical density (OD) measurements of passage four P. freudenreichii at 600 nm (open circles) and 650 nm (closed circles) from 72 hours to 144 hours at 24-hour intervals as a measurement of culture growth. Culturing occurred in glass vials containing yeast extract lactose (YEL) media at 37 °C in microaerophilic conditions following 1 : 1000 inoculation.

[051] Figure 5 provides a plot of optical density (OD) measurements for P. freudenreichii at passage six from 24 hours to 216 hours at 24-hour intervals (except at 72 hours) cultured in glycerol-pep broth (GPB) at 37 °C in microaerophilic conditions in (open diamond) glass vial following a 1 :20 inoculation; (closed square) glass vial following 1 : 1000 inoculation; (closed triangle) plastic vial following a 1 :20 inoculation; (cross) plastic vial following 1 :1000 inoculation. The plastic 1 :20 sample was not tested after 192 hours due to insufficient sample. [052] Figure 6 (A, B) provides an image showing patch plates of unique morphological microorganisms originating from mammalian milk or intestinal samples on Brain Heart infusion (HI) agar and glycerol-PEP agar with bromocresol purple isolated from spread plates.

[053] Figure 7 provides an image of thin layer chromatography (TLC) of (A; left to right) 10% propionic acid (PA), 10% lactic acid (LA), 10% acetic acid (AA) and a bacterial culture supernatant derived from a camel isolate termed “Sophie Anaerobic 4”; and (B; left to right) 10% propionic acid (PA), bacterial culture supernatant derived from P. freudenreichii, 10% lactic acid (LA), and bacterial culture supernatant derived from Lactococcus lactis. A bromophenol blue indicator solution was used to detect a change in colour with the presence of an acidic property.

[054] Figure 8 provides images of spectral results retrieved from HPLC-UV line at wavelength of 210 nm. (A) PA Standard at 20 ppm, (B) Distilled PA product showing PA and impurities, (C) Glycerol-peptone broth (negative) control (1 : 10 dilution), (D) clarified supernatant of P. freudenreichii at 6 days of incubation (1 : 10 dilution), and (E) clarified supernatant of P. freudenreichii at 14 days of incubation (1 : 10 dilution). A characteristic PA peak was detected at 3.3 min elution time for A, B, D, E (indicated by an arrow on the respective figures).

[055] Figure9 provides an image of a standard curve created using the area (UV*sec) and standard concentrations (ppm) to achieve the quantified HPLC-UV results shown in Table 10. [056] Figure 10 provides images of spectral results retrieved from HPLC-UV line at wavelength of 210 nm. (A) PA Standard at 20 ppm, (B) Glycerol-peptone broth (negative) control (1 : 10 dilution), (C) clarified supernatant of P. freudenreichii (1 : 10 dilution) having PA at 10 ppm, and (D) clarified supernatant of P. freudenreichii (1 : 10 dilution) having PA at 7.5 ppm. A characteristic PA peak was detected at 1.8 min elution time for A, C, D (indicated by an arrow on the respective figures).

[057] Figure 11 provides images of (A) Gram stained P. freudenreichii isolated using the methods described herein, and (B) reference photograph of Gram stained P. freudenreichii. [058] Figure 12 provides photographic images of (A) a P. freudenreichii culture grown in a 5 L drum under stagnant, environmental conditions of approximately 20 to 25°C, and (B) close up of the Nalgene plasticware which prevents gas-deformation when growing the culture shown in (A). DETAILED DESCRIPTION

[059] The present disclosure relates to the biosynthetic production of propionic acid, and the industrial uses of bacterial cultures containing propionic acid (PA), clarified bacterial culture supernatants containing propionic acid and PA purified from such supernatants. Further, the present disclosure relates to bacterially produced PA for industrial use.

[060] The bacterially produced PA of the present disclosure may comprise a whole bacterial culture comprising propionic acid, a clarified bacterial culture supernatant comprising propionic acid derived from the bacterial culture, or purified propionic acid derived from the bacterial culture or from the clarified bacterial culture supernatant as described herein. In an embodiment, the purified propionic acid has been purified by extraction and/or distillation the clarified bacterial culture supernatant.

[061] The bacterially produced PA of the present disclosure may be used for various industrial applications. For example, the present inventors have realised that, surprisingly, a clarified bacterial culture supernatant comprising PA, or PA purified from such supernatants, may be used as a component of an emulsion, replacing synthetically produced PA to facilitate emulsion polymerisation. Moreover, the present inventors have surprisingly realised that the clarified culture supernatant comprising propionic acid alone may be used to enhance the spreadability of an industrial agent, such as a herbicide.

[062] Accordingly, the present disclosure provides a bacterial culture comprising PA for use as a component of an emulsion. The present disclosure also provides a clarified bacterial culture supernatant comprising PA for use as a component of an emulsion. In an embodiment, a clarified bacterial culture supernatant comprising PA as disclosed herein is a component of an emulsion.

[063] An emulsion of the present disclosure may have any suitable components, providing that one of the components is a bacterially produced PA and that the components undergo emulsification when mixed under suitable conditions, such as agitation and/or heating. In an embodiment, the emulsion may comprise a bacterially produced PA, a lecithin, and a surfactant. [064] A lecithin of the present disclosure may be any lecithin that is suitable to promote emulsification. The lecithin may be a mixture of glycerophospholipids. For example, the lecithin may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid or a mixture comprising any two or more such components. In an embodiment, the lecithin is soy lecithin (e.g. soyal phospholipids). Soy lecithins are commercially available from a range of suppliers, including from Ortho Chemicals, Victoria, Australia. Those skilled in the art will appreciate that other lecithins may be suitable for use in the emulsion of the present disclosure.

[065] As used herein, the term “surfactant” refers to an agent, usually an organic chemical compound that is at least partially amphiphilic (i.e., typically containing a hydrophobic tail group and hydrophilic polar head group). Given their structure, surfactants are generally capable of lowering the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Further to this, these properties typically allow solubility of the surfactant in organic solvents as well as in water, and allow the surfactant to promote solubilization or at least dispersal of fatty/waxy materials in water and water-containing solutions. In this regard, a surfactant may act as a detergent, a wetting agent, an emulsifying agent, a foaming agent, a solubilising agent and/or a dispersing agent.

[066] A surfactant of the present disclosure may be any suitable surfactant that promotes emulsification. Suitable surfactants are commercially available from a range of suppliers, including Ortho Chemicals, Victoria, Australia. In an embodiment, the surfactant may be Surfactant 12A3 (polyoxyethylene lauryl ether (C12-C15 alcohols, ethoxylated with 3 moles ethylene oxide).

[067] Those skilled in the art will appreciate that other surfactants may be suitable for use in the emulsion of the present disclosure. The surfactant may be any surfactant that is suitable for use in the industrial agent composition of the present disclosure. The surfactant provided herein is preferably an agriculturally acceptable surfactant. The term “agriculturally acceptable surfactant” as used herein refers to a surfactant that is not unacceptably damaging to a plant and/or its environment, and/or not unsafe to the user or others that may be exposed to the surfactant when used as described herein.

[068] For example, the surfactant may be selected from the group consisting of non-ionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, cation-anion composite surfactants and any combination thereof. Suitable non-ionic surfactants may include polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polyoxypropylene ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkyl esters, polyoxyethylene polyoxypropylene block copolymers, sorbitan alkyl esters, higher fatty acid alkanolamides and any combination thereof. Suitable cationic surfactants may include alkylamine salts, quaternary ammonium salts and combinations thereof. Further, suitable anionic surfactants may include naphthalenesulfonic acid polycondensate, alkenylsulfonate, naphthalenesulfonate, formalin condensate of a naphthalenesulfonate, formadehyde condensate of an alkylnaphthalenesulfonate, lignin sulfonate, alkylarylsulfonate, alkylaryl sulfonate sulfate, polystyrene sulfonate, polycarboxylate, polyoxyethylenealkyl ether sulfate, polyoxyethylenealkylaryl ether sulfate, alkyl sulfosuccinate, alkyl sulfate, alkyl ether sulfonate, a higher fatty acid alkali salt and any combination thereof.

[069] In some embodiments, the surfactant is present in an amount of at least about 30 g/L. In an embodiment, the surfactant is present in an amount ranging between about 30 g/L to about 100 g/L.

[070] In an embodiment, the surfactant is selected from the group consisting of an alkylphenol, an alkylbenzene sulphonate, an alkoxylated styryl phenol, a vegetable oil ethoxylate, and any combination thereof. In an embodiment, the surfactant is selected from the group consisting of an alkoxylated alkylphenol, calcium Cio-16 alkylbenzyl sulphonate, an ethoxylated nonylphenol and any combination thereof.

[071] Accordingly, in particular embodiments, the surfactant is or comprises an emulsifying agent. As generally used herein, the term “emulsifying agent” or “emulsifier” refers to a chemical agent, compound, or substance capable of producing an emulsion by reducing the interfacial tension between the two insoluble liquids. In some embodiments, the surfactant may be a solubilising agent and act as a solvent in the formulations described herein. In such embodiments, the surfactant may replace at least a portion of the solvent required to form a stable formulation.

[072] Exemplary surfactants include an alkylphenol alkoxylate (e.g., Termul 200), a polyoxyalkylene ether (e.g., Termul 203), an alkoxylated oil (e.g., ~54 x ethoxylated (e.g., Termul 1285)), an alkylphenol alkoxylate (e.g., ~10 x ethoxylated (e.g., TericNIO)), an alkoxylated alcohol (Alcohol C12 - C15, ~23 x ethoxylated (e.g. Teric 12A23); Alcohol C12, ~3 x ethoxylated (e.g. Surfactant 12A3)), an alkoxylated polyaryl phenol (e.g., Tristyryl Phenol, ~16 x ethoxylated (e.g. TSP15)), an alkoxylated block co-polymer (Ethoxylate-Propoxylate copolymer (e.g. Teric PE64)), an alkylbenzenesulphonate (e.g., Calcium CIO-16 alkylbenzyl sulphonate, branched or linear chain in solvent (e.g. Ninate 60E, NANSA EVM70/2E, Kemmat HF60)), an alcohol ether sulphate (e.g. Toximul TANS-5), an alkylamine ethoxylate (e.g. Toximul TAABS-5), a silicone polymer, inclusive of polysiloxanes (e.g., Evonik Break-Thru OE 446, Evonik Break-Thru AF 9903, Jiangxi Tiansheng QS-302) and silicone glycol copolymers (e.g., Xiameter OFX-5211), a block co-polymer (e.g., Teric PE64), an alkoxylated nonylphenol (e.g., Teric N10 or Teric N30), an alkoxylated alcohol (e.g., Teric BL8), an alkyl polyglucoside (e.g., Croda AL-2575), an ethoxylate-propoxylate copolymer, a CIO-16 alkylbenzenesulphonate (branched or linear in solvent), a branched alcohol ether sulphate, a tallowamine alkoxylated salt, and a mineral oil (e.g., Rhodoline DF5888).

[073] The emulsion may be formed by emulsifying together the bacterially produced PA, lecithin and the surfactant in a defined ratio. For example, the ratio of commercially available PAdecithin: surfactant may be combined in a ratio of 1 : 1 : 1. Additionally, in an embodiment, the 1 : 1 : 1 ratio is a suitable ratio for the emulsion when the PA is the purified PA of the present disclosure. However, persons skilled in the art will appreciate that other ratios will be suitable to form an emulsion. For example, any one of the components of the emulsion may be present in a ratio of about 0.5 to 1.5 with respect to any other component. For instance, the PA may be present in a v/v ratio of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or about 1.5 with respect to either the lecithin or the surfactant. In an embodiment, the PA may be present in a v/v ratio of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or about 20 with respect to either the lecithin or the surfactant. In an embodiment, the volume of the clarified bacterial culture supernatant may be increased to compensate for reduced concentration of PA in the clarified culture supernatant compared to the purified PA or the commercially available PA. For example, if the concentration of PA in the clarified culture supernatant is 0.5% (v/v), then 60 mL of clarified culture supernatant may be mixed with 0.3 mL lecithin and 0.3 mL of the surfactant. The surfactant may also be a surfactant blend or mixture comprising one or more suitable surfactants.

[074] In an embodiment, the emulsion may be formed (that is, emulsified) using any suitable method known to those skilled in the art, for example, by mixing the components, agitating the components, etc. In an embodiment, the emulsifying occurs at an environmental temperature (e.g., room temperature or an ambient outdoors temperature of between 10 °C and 38 °C). In an embodiment, the emulsifying occurs at a temperature between 10 °C and 40 °C. In an embodiment, the emulsifying may occur at a temperature between 20 °C and 38 °C. In an embodiment, the emulsifying may occur at a temperature between 15 °C and 20 °C. In an embodiment, the emulsifying may occur at a temperature between 20 °C and 25 °C. In an embodiment, the emulsifying may occur at a temperature between 25 °C and 30 °C. In an embodiment, the emulsifying may occur at a temperature between 30 °C and 35 °C. In an embodiment, the emulsifying may occur at a temperature between 35 °C and 40 °C. In an embodiment, the emulsifying occurs at a temperature between 18 °C and 30°C.

[075] The emulsion of the present disclosure may be used to reduce the surface tension of an industrial agent and/or increase the spreadability of the industrial agent. The reduced surface tension may facilitate enhanced effectiveness or usefulness of the industrial agent. For example, for a herbicide that is sprayed upon the leaves of a weed, the herbicide may form droplets due to the high surface tension of the herbicide. Increasing the spreadability of the herbicide may facilitate an increase in surface area of the leaf that the herbicide comes into contact with, which many in turn enhance the effectiveness of the herbicide. In an embodiment, the emulsion is adapted to increase the spreadability of an industrial agent. In an embodiment, the emulsion when mixed with an industrial agent increases the spreadability of the industrial agent. In an embodiment, the industrial agent is any suitable pesticide. In an embodiment, the industrial agent is a herbicide. In an embodiment, the herbicide is glyphosphate. However, the herbicide may be any suitable herbicide, such as 2,4-D, MCPA, Metsulfuron-methyl, etc. The herbicide may be used in a range of concentrations, for example, as specified by the manufacturer.

[076] In an embodiment, the emulsion is diluted 1 : 100 in the industrial agent. However, when the emulsion comprises the clarified culture supernatant comprising PA, the emulsion volume may be changed to compensate for the reduced PA concentration. For example, where the PA concentration in the clarified bacterial culture supernatant is 0.5% (v/v), then the emulsion comprising 60 mL of the clarified culture supernatant, 0.3 mL lecithin and 0.3 mL surfactant may be added to approximately 40 mL of the industrial agent. Persons skilled in the art will appreciate that different dilutions of the emulsion in the industrial agent may be suitable, for example, 1 : 10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, 1 : 100, 1 : 110, 1 : 120, 1 : 130, 1 : 140, 1 : 150, 1 : 160, 1 : 170, 1 : 180, 1 : 190, 1 :200, etc.

[077] Biosynthetic production of PA can utilise a number of different PA producing microorganisms. Accordingly, the bacterial culture or clarified bacterial culture supernatant may be obtained from a culture of a bacterial strain that produces a suitable amount of PA within the culture supernatant.

[078] “Propionic acid bacteria” (PAB) is an umbrella term for a specific group of bacteria that can produce propionic acid, namely, Propionibacterium spp., Acidipropionibacterium spp., and Cutibacterium spp., which are all Gram-positive, non-spore-forming and aerotolerant bacteria. These genera of bacteria are phenotypically similar and were initially considered as a single genus, Propionibacterium. Prior to the currently valid classification of PAB into three genera in 2016, all PAB were assigned to the genus Propionibacterium. Accordingly, some bacteria previously referred to as Propionibacterium spp., have since been re-classified as Acidipropionibacterium spp. Or Cutibacterium spp. Examples of species falling under the PAB umbrella are shown in Table 1.

Table 1: Propionic acid bacteria - Propionibacteriaceae

[079] Gram-negative bacteria that produce PA include Veillonella spp., Fusobacterium spp., Selenomonas riiminalium, Anaerovibrio lipolytica and Bacteroides fragilis.

[080] Accordingly, in an embodiment, the bacterial culture or clarified bacterial culture supernatant of the present disclosure is obtained from culture of a bacterial strain is selected from Veillonella spp., Fusobacterium spp., Selenomonas riiminalium. Anaerovibrio lipolytica, Bacteroides fragilis, Propionibacterium spp., Acidipropionibacterium spp., and Cutibacterium spp. In an embodiment, the bacterial strain is a Propionic acid bacteria (PAB). In an embodiment, the bacterial strain is selected from the group consisting of Propionibacterium spp., Acidipropionibacterium spp., and Cutibacterium spp. In an embodiment, the bacterial strain is Propionibacterium spp. In an embodiment, the bacterial strain P. freudenreichii. [081] The major anaerobic metabolic pathways for the production of PA by bacteria are the propanediol, acrylate, Wood-Werkman and succinate pathways (see, Figure 1 A, Figure IB, Figure 1C, Figure ID, respectively). PAB are considered to possess three classes of PA production pathways: primary, catabolic, and anabolic. Primary fermentation pathways produce propionate from original carbon sources which are metabolised via the acrylate and Wood- Werkmen cycle; the latter being the most energy-efficient pathway in producing propionate that is currently known. Catabolic pathways use amino acids to produce propionate via degradation. Anabolic pathways are useful in producing propionate using pyruvate and carbon dioxide.

[082] Substrate selection can impact production of propionate. Glucose may be less efficient in achieving PA production as many external components are required to balance the reduction and oxidation of the reactant; whereas glycerol may be used to promote PA production as it is a more reduced substrate compared to glucose. Nonetheless, glycerol may be economically unfavourable.

[083] Primary production pathway for PA in Propionibacterium spp. Are linked to the presence of 1,2-propanediol as an intermediate to facilitate the acryloyl-CoA, methylmalonyl- CoA and succinate pathways.

[084] The acrylate pathway converts D-lactic acid, D-lactoyl-CoA, acryloyl-CoA, and propionyl-CoA to propionate using NADH and ATP. These pathways are not limited to Propionibacterium spp. As they are also found in Clostridium propionicum, Megasphaera elsdenii, and Prevotella ruminicola. For this pathway, glucose does not serve as a substrate to produce PA.

[085] The Wood-Werkman and succinate pathways offer a convenient catabolic production of propionate using the tricarboxylic acid (TCA) cycle. The TCA cycle allows the production of propionate via two mechanisms. Under anaerobic conditions, there is less available ATP to produce CO2 and pyruvate into oxaloacetate, which is compensated by NADH dehydrogenase and fumarate reductase to act as an anaerobic electron transport chain. With the absence of the PFL enzyme in most PAB, the production of succinate and acetate occurs due to the energy deficit. Some organisms have adapted to the energy deficit by removing the carboxyl group on succinate (decarboxylase) to form propionate. The two aforementioned mechanisms are (1) sodium facilitated methylmalonyl-CoA decarboxylase and (2, Wood-Werkman cycle) methylmalonyl-CoA:pyruvate transcarboxylase.

[086] Using succinate, the methylmalonyl-CoA decarboxylase pathway produces propionyl- CoA using two sodium ions that are transported across the cell membrane which allows a gain in energy of approximately 0.25 ATP. This shows the first pathway being energetically favourable and produces a 2: 1 propionate: acetate equivalent. This pathway is found in Propionigenium modestum. For microorganisms containing the PFL enzyme, the ATP yield increases to 3.25 per glucose molecule which results in a 1 : 1 production of propionate:acetate. [087] The concentration of the propionic acid in the culture supernatant and in the purified propionic sample may be measured by any suitable means known to persons skilled in the art. For example, the presence of acid can be qualitatively measured using agar containing bromocresol purple (BP). The presence of propionic acid can be qualitatively determined using thin-layer chromatography and bromophenol blue indicator; and quantitated using high performance liquid chromatography (HPLC). [088] In an embodiment, the whole bacterial culture undergoes a treatment to kill the bacteria prior to using the bacterial culture as a source of propionic acid. The bacterial of the present disclosure may be killed using any suitable methods known in the art, providing that the killed bacteria are suitable for use as a composition of the present disclosure. For example, the PAB may be killed by suitable protocols for chemical treatment, thermal treatment, irradiation treatment, high hydrostatic pressure, pulsed electric field, ultrashort pulsed laser, ultrasound under pressure, UV-irradiation, or microbial inactivation.

[089] In an embodiment, the cultured PAB of the present disclosure may be treated by irradiation. The term “irradiation” will be understood to encompass UV-radiation (i.e. ultraviolet rays), gamma-radiation (i.e., gamma-rays) and X-radiation (i.e., X-rays). Methods of irradiating cultures are known to those skilled in the art. In an embodiment, the irradiation is using UV radiation. In an embodiment, the irradiation is gamma irradiation. In an embodiment, the irradiation is X-ray irradiation.

[090] In an embodiment, the irradiating is performed by cobalt 60 gamma irradiation using standard techniques. However, any suitable source of gamma-radiation may be used. Suitable gamma emitters include, but are not limited to Ba ¹³⁷ , Co ⁶⁰ , Cs ¹³⁷ , Ir ¹⁹² , U ²³⁵ , Se ⁷⁵ and Yb ¹⁶⁹ . [091] Gamma-irradiation of the streptococcal bacteria of the disclosure may be performed using commercially available devices, for example, a Gammacell irradiator manufactured by Atomic Energy of Canada Ltd., Canada (e.g., Gammacell 40 Irradiator, Gammacell 220 Irradiator, Gammacell 1000 irradiator, Gammacell 3000 irradiator), a gamma-irradiator manufactured by J. L. Shepherd and Associates (San Fernando, California, USA), or a Nordi on Gamma Cell- 1000 irradiator manufactured by Nordi on Inc. (Kanata, Ontario, Canada). Other devices may be suitable.

[092] Additionally or alternatively, streptococcal bacteria of the disclosure may be X- irradiated. As known to those of ordinary skill in the art, X-rays are identical to gamma-rays except they are emitted by the passage of electrons through an electric field of a nucleus rather than the nucleus itself upon radioactive decay. Any suitable source of X-radiation may be used. Suitable sources of X-radiation include, but are not limited to, the eXelis® sterilization X-ray machine manufactured by IBA Industrial (Louvain-la-Neuve, Belgium). Other suitable devices include for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).

[093] By way of non-limiting example only, a to become gamma-irradiated, the bacterial culture may be subjected to photon-radiation at energies of at least O.OlMeV, at least 0. IMeV, at least 0.5MeV, between O.OlMeV and 0.5MeV, between O.OlMeV and IMeV, between O.O1 MeV and lOMeV, between 0.5MeV and 20MeV, between 0.5MeV and 15MeV, between 0.5MeV and lOMeV, between 0.5MeV and 5MeV, between 0.5MeV and 2MeV, or between IMeV and 2MeV (e.g., 1.25MeV).

[094] As known to those of ordinary skill in the art, a measure for an absorbed dose of radiation is the gray (Gy), which is defined as 1 joule of energy deposited in 1 kilogram of mass. An old unit of measure for this is the rad, which stands for “radiation absorbed dose”, where 1 Gy = 100 rad. In an embodiment, the bacterial culture of the present disclosure is exposed to a total dose of radiation (e.g., X-radiation and/or gamma-radiation) of between about 6.5 x 10 ⁴ rad and about 2 x 10 ⁷ rad (about 0.65 kGy to about 200 kGy). In other embodiments of the disclosure, the bacterial culture is exposed to a total photon-radiation dose of about 10 kGy to about 12 kGy, about 12 kGy to about 14 kGy, about 14 kGy to about 16 kGy, about 10 kGy to about 20 kGy, about 14 kGy to about 20 kGy, about 20 kGy to about 30 kGy, about 20 kGy to about 25 kGy, about 25 kGy to about 30 kGy, about 30 to 35 kGy, about 10 kGy, about 11 kGy, about 12 kGy, about 13 kGy, about 14 kGy, about 15 kGy, about 16 kGy, about 17 kGy, about 18 kGy, about 19 kGy, about 20 kGy, about 21 kGy, about 22 kGy, about 23 kGy, about 24 kGy, about 25 kGy, about 26 kGy, about 27 kGy, about 28 kGy, about 29 kGy, about 30 kGy, about 31 kGy, about 32 kGy, about 33 kGy, about 34 kGy, about 35 kGy, about 20 kGy, about 20 kGy, more than 10 about 12 kGy to about 14 kGy, more than 12kGy, more than 14 kGy, more than 16 kGy, more than 18 kGy, more than 20 kGy, more than 22 kGy, more than 24 kGy, more than 26 kGy, more than 28 kGy, more than 30 kGy, more than 35 kGy, more than 40 kGy, 1.26 x 10 ⁶ rad (12.6 kGy), a total photon-radiation dose of about 1 x 10 ⁶ rad (about 10 kGy) photon-rays, or a total photon-radiation dose of about 1 x 10 ⁵ rad (1 KGy).

[095] The optimal dose of photon-radiation (e.g., gamma-radiation and/or X-radiation) may be influenced by factors such as the medium in which the bacterial culture of the present disclosure are present, the number of bacteria present to be treated, the temperature of the bacteria present to be treated, water availability, oxygen availability and/or the subtype or strain under treatment. Accordingly, the total dose of photon-radiation, the exposure time and/or the level of photonradiation applied over the period of exposure may be optimised to enhance the effectiveness of the treatment.

[096] The total dose of photon-radiation (e.g., X-radiation and/or gamma-radiation) may be administered to the bacterial culture of the present disclosure cumulatively over a period of time. For example, radiation may be administered to the bacterial culture of the present disclosure at a level lower than that of the total dose, over a time period sufficient to achieve the total dose of photon-radiation required.

[097] In an embodiment, the bacterial culture or clarified bacterial culture supernatant comprises a sufficient amount of propionic acid to be industrially useful. For example, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.01% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified culture supernatant may comprise at least 0.05% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.1% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.2% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.25% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.3% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.4% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.6% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.7% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.8% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 0.9% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 1.0% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 1.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 2.0% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 2.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 5% (v/v) propionic acid.

[098] In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.1% (v/v) and 2.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.2% (v/v) and 2% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.3% (v/v) and 1.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.4% (v/v) and 1% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.4% (v/v) and 0.8% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.5% (v/v) and 1% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 1% (v/v) and 1.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.5% (v/v) and 1.5% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.1% (v/v) and 0.6% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.2% (v/v) and 0.3% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.2% (v/v) and 0.4% (v/v) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 0.2% (v/v) and 0.7% (v/v) propionic acid.

[099] In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 1 parts per million (ppm) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 2 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 3 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 4 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 5 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 6 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 7 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 8 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise at least 10 ppm propionic acid.

[0100] In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 1 and 30 parts per million (ppm) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 2 and 15 ppm propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 3 and 8 parts per million (ppm) propionic acid. In an embodiment, the bacterial culture or clarified bacterial culture supernatant may comprise between 2 and 4 ppm propionic acid.

[0101] In an embodiment, the purified propionic acid may comprise at least 0.5% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 1% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 1.5% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 2% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 2.5% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 3% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 4% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise at least 5% (v/v) propionic acid.

[0102] In an embodiment, the purified propionic acid may comprise between 0.5% (v/v) and 5% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise between 1% (v/v) and 4% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise between 2% (v/v) and 3% (v/v) propionic acid. In an embodiment, the purified propionic acid may comprise between 2% (v/v) and 2.5% (v/v) propionic acid.

[0103] In an embodiment, the purified propionic acid may comprise at least 5 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 10 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 15 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 20 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 25 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 30 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 40 ppm propionic acid. In an embodiment, the purified propionic acid may comprise at least 50 ppm propionic acid.

[0104] In an embodiment, the purified propionic acid may comprise between 5 ppm and 50 ppm propionic acid. In an embodiment, the purified propionic acid may comprise between 10 ppm and 40 ppm propionic acid. In an embodiment, the purified propionic acid may comprise between 20 ppm and 30 ppm propionic acid. In an embodiment, the purified propionic acid may comprise between 20 ppm and 25 ppm propionic acid.

[0105] The clarified bacterial culture or clarified bacterial culture supernatant comprising propionic acid and/or the purified propionic acid may be obtained following culturing of a propionic acid producing bacteria. In another form of the disclosure, there is provided a method of preparing a clarified bacterial culture supernatant comprising propionic acid, the method comprising the following steps:

(i) inoculating suitable culture medium with a bacterial strain that releases propionic acid into the culture medium;

(ii) culturing the bacterial strain in the culture medium under suitable conditions for a suitable length of time to obtain a bacterial culture comprising propionic acid; and

(iii) separating the culture supernatant of the bacterial culture from insoluble particulate matter of the bacterial culture by centrifugation and/or filtration to obtain a clarified culture supernatant comprising at least 0.1% v/v propionic acid.

[0106] The centrifugation and/or filtration steps can utilise any suitable centrifugation and/or filtration methods known to those skilled in the art.

[0107] The bacteria of the present disclosure may be cultured in any suitable liquid culture medium, providing that it contains suitable nutrients to facilitate the production of a suitable amount of PA as disclosed herein. For example, the bacteria may be cultured in commercially available media such as SMS (0.125 g/litre casein, 0.1 g/litre starch from potato, 1 g/litre Casamino Acids (Sigma and Difco)), and Difco™ Lactobacilli MRS media (Thermo Fisher Scientific, Massachusetts), yeast extract lactose (YEL), and glycerol-peptone broth (GPB). In an embodiment, the culture medium of the present disclosure is YEL or GPB. In an embodiment, the culture medium of the present disclosure is GPB.

[0108] PAB bacteria such as P.freudenreichii may be culturable within a pH range of 4.5 to 8, although increased growth conditions occur at approximately 37 °C, with optimal PA production observed between 30-32 °C. However, PA may be produced at an environmental temperature (e.g., room temperature or at an ambient outdoors temperature of between 10 C and 38 C). Glycerol and glucose may both be used as a substrate for PA production, although glycerol has been observed to improve PA production. P. freudenreichii is a facultative anaerobe therefore can be grown in aerobic conditions; however, microaerophilic (i.e., 2-10% oxygen) or anaerobic conditions may facilitate fermentation of PA. Accordingly, the culturing of the present disclosure may occur under aerated, microaerophilic (that is, between approximately 2% and 10% oxygen) or anerobic conditions. In an embodiment, the culturing occurs in microaerophilic conditions.

[0109] The PAB bacteria may be inoculated into fresh culture media to initiate growth of a new culture (i.e., a sub-culture). The inoculum can contain PAB from an actively growing culture, an exhausted culture or from frozen stock. The number of bacteria in the inoculum can vary as would be appreciated by those skilled in the art. The inoculum may be diluted by any suitable factor into the fresh culture media, for example 1 : 10; 1 :20; 1 :50: 1 :100; 1 :200; 1 :250; 1 :300; 1 :400: 1 :500; 1 : 1000: 1 :2000, etc.

[0110] PAB bacteria such as P. freudenreichii tend to suffer from the effects of end-product inhibition when producing PA. That is, the production of the acid decreases the pH of the culture until it reaches a pH where bacterial growth is inhibited. End-product inhibition tends to occur at approximately pH 4.5 for PAB such as P. freudenreichii . Accordingly, the pH of the culture medium may be any suitable pH that results in a suitable amount of PA as disclosed herein. In an embodiment, the pH of the culture medium maybe between approximately 5.0 and 7.5. In an embodiment, the pH of the culture medium is between 5.5 and 6.0. In an embodiment, the pH of the culture medium is between 6.0 and 6.5. In an embodiment, the pH of the culture medium is between 6.5 and 7.0. In an embodiment, the pH of the culture medium is between 6.0 and 7.0. In an embodiment, the pH of the culture medium is between 7.0 and 7.4. In an embodiment, the pH of the culture medium is between 7.1 and 7.3. In an embodiment, the pH of the culture medium is approximately 7.2. In an embodiment, the pH of the culture medium has a neutral pH. For example, the pH may be approximately pH 7, or near pH 7, for example, in a range between about pH 6.5 to 7.5, or between about pH 6.8 and 7.2.

[OHl] The culturing of the present disclosure may occur at any suitable temperature providing that a suitable amount of PA is produced as disclosed herein. In an embodiment, the culturing may occur between 30 °C and 37 °C. In an embodiment, the culturing may occur at a temperature between 30 °C and 34 °C. In an embodiment, the culturing may occur at a temperature between 27 °C and 33 °C. In an embodiment, the culturing may occur at 28 °C. In an embodiment, the culturing may occur at 30 °C. In an embodiment, the culturing may occur at 31 °C. In an embodiment, the culturing may occur at 32 °C. In an embodiment, the culturing may occur at 33 °C. In an embodiment, the culturing may occur at 34 °C. In an embodiment, the culturing may occur at 35 °C. In an embodiment, the culturing may occur at 36 °C. In an embodiment, the culturing may occur at 37 °C.

[0112] In an embodiment, the culturing may occur at an environmental temperature (e.g., room temperature or at an ambient outdoors temperature of between 10 C and 38 C). In an embodiment, the culturing may occur between 10 °C and 30 °C. In an embodiment, the culturing may occur at a temperature between 15 °C and 28 °C. In an embodiment, the culturing may occur at a temperature between 18 °C and 26 °C. In an embodiment, the culturing may occur at a temperature between 20 °C and 25 °C. In an embodiment, the culturing may occur at a temperature between 25 °C and 30 °C. In an embodiment, the culturing may occur at a temperature between 18 °C and 33 °C. In an embodiment, the culturing may occur at a temperature between 20 °C and 30 °C. In an embodiment, the culturing may occur at 20 °C. In an embodiment, the culturing may occur at 21 °C. In an embodiment, the culturing may occur at 22 °C. In an embodiment, the culturing may occur at 23 °C. In an embodiment, the culturing may occur at 24 °C. In an embodiment, the culturing may occur at 25 °C. In an embodiment, the culturing may occur at 26 °C. In an embodiment, the culturing may occur at 27 °C. In an embodiment, the culturing may occur at 28 °C. In an embodiment, the culturing may occur at 29 °C.

[0113] The culturing of the bacterial strain may occur under suitable conditions for a suitable length of time to obtain a bacterial culture comprising propionic acid. The suitable length of time may vary depending on the conditions the bacterial strain is cultured in, for example, depending upon which media is selected, which temperature the culturing occurs at, the oxygen concentration the culturing occurs at, whether the culturing is under stagnant or agitated conditions; the number of bacteria present in the inoculum; and whether the bacteria were actively growing when they were inoculated into the culture . However, the culturing should occur for sufficiently long to facilitate production of a suitable concentration of PA. In an embodiment, the culturing may occur for between 2 and 35 days. For example, the culturing may occur for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days. In an embodiment, the culturing occurs for two days. In an embodiment, the culturing occurs for at least three days. In an embodiment, the culturing occurs for at least four days. In an embodiment, the culturing occurs for at least five days. In an embodiment, the culturing occurs for at least six days. In an embodiment, the culturing occurs for at least seven days. In an embodiment, the culturing occurs for at least eight days. In an embodiment, the culturing occurs for at least nine days. In an embodiment, the culturing occurs for at least ten days. In an embodiment, the culturing occurs for at least 14 days. In an embodiment, the culturing occurs for at least 21 days. In an embodiment, the culturing occurs for at least 28 days. In an embodiment, the culturing occurs for between 5 and 15 days. In an embodiment, the culturing occurs for between 12 and 20 days. In an embodiment, the culturing occurs for between 6 and 18 days. In some embodiment, the cultures are sub-cultured into fresh media periodically, for example, every 5 to 10 days, for example, every 7 days. [0114] Reducing the pH of the culture immediately prior the centrifuging and/or filtration described in step (iii) of the method may remove carboxylic acid from the bacterial culture supernatant or the purified propionic acid. Accordingly, in an aspect, the pH of the bacterial culture is adjusted to less than 5.0 immediately prior to step (iii). In an aspect, the pH of the bacterial culture is adjusted to less than 4.0 immediately prior to step (iii). In an aspect, the pH of the bacterial culture is adjusted to less than 3.0 immediately prior to step (iii). In an aspect, the pH of the bacterial culture is adjusted to less than 2.5 immediately prior to step (iii).

[0115] The bacterial culture supernatant comprising propionic acid may be separated from the insoluble matter of the culture (that is, bacterial cells, cellular matter, cellular fragments, membrane fragments, and under some circumstances may include smaller components such as insoluble proteins) by centrifugation and/or filtration. Routine centrifugation and filtrations steps that separate the culture supernatant from the insoluble matter of a bacterial culture supernatant are known to persons skilled in the art. For example, the culture may be centrifuged at 5 °C, 12,000 rpm for 15 minutes to separate the cells and larger proteins from the supernatant, and then filtered using a 0.22 pm filter. Persons skilled in the art will appreciate that alternative centrifugation and filtration methods can be substituted, providing that the culture supernatant is substantially separated from the insoluble components of the culture.

[0116] In a preferred embodiment, the method further comprises the following step: (iv) purifying propionic acid from the clarified culture supernatant by extraction and/or distillation to obtain purified PA.

[0117] The purified PA may be purified from the clarified culture supernatant using a primary extraction procedure, such as reactive extraction, followed by distillation, as described herein. However, persons skilled in the art will appreciate that alternative methods of purifying propionic acid may be utilised.

[0118] In another form, the present disclosure provides a bacterial culture comprising propionic acid obtained using the method described herein.

[0119] In another form, the present disclosure provides a clarified bacterial culture supernatant comprising propionic acid obtained using the method described herein.

[0120] In another form, the present disclosure provides a purified propionic acid obtained using the method described herein.

[0121] In another form, the present disclosure provides an emulsion comprising the bacterial culture described herein, a lecithin and a surfactant. [0122] In another form, the present disclosure provides an emulsion comprising the clarified bacterial culture supernatant described herein, a lecithin and a surfactant.

[0123] In another form, the present disclosure provides an emulsion comprising the purified propionic acid described herein, a lecithin and a surfactant.

[0124] In another form, the present disclosure provides an emulsion as described herein, wherein the emulsion when mixed with an industrial agent increases the spreadability of the industrial agent. In an embodiment, the industrial agent is a pesticide. In an embodiment, the industrial agent is a herbicide.

[0125] In another form, the present disclosure provides a industrial agent composition comprising an industrial agent and the emulsion as described herein having enhanced spreadability compared to the industrial agent in the absence of the emulsion.

[0126] In another form, the present disclosure provides an industrial agent composition comprising an industrial agent as described herein, wherein the industrial agent composition has enhanced effectiveness compared to the industrial agent in the absence of the emulsion. In an embodiment, the industrial agent is a herbicide. In an embodiment, the industrial agent is a pesticide. In embodiment, the pesticide component is a pesticide disclosed herein.

Example 1 - Screening mammalian samples for novel microbial species on their ability to produce propionic acid and agricultural applications of Propionibacterium freudenreichii fermentation supernatants

Introduction

[0127] This study investigated PA production from an historical P. freudenreichii strain, and then examined the agricultural potential of the PA-containing culture supernatant as an emulsion component and compared this to a solvent-purified PA standard. Purified and extracted microbial PA or the culture supernatant was used to form an emulsion with lecithin and surfactant, and the spreading ability of the resulting emulsion was determined.

[0128] Fermentation was performed in 2 L glycerol-pep broths from which PA was extracted. An PA concentration of 0.59% (v/v) was discovered within the supernatant and 2.3% (v/v) in the distilled product by HPLC-UV. The culture supernatant emulsion had a spreadability which was comparable to the commercial PA emulsion positive control.

[0129] The study also investigated PA production from microbes isolated from milk and manure samples from cows, goats and camels using thin-layer chromatography to detect the presence of PA. A species was discovered from the natural isolates that potentially produced PA alongside other carboxylic acids. Materials and Methods

Sources of samples

[0130] A lyophilised culture of P. freudenreichii was retrieved from Public Health England (PHE) National Collection of Culture Types (NCTC).

[0131] Milk and manure samples were retrieved from cows and goats from a chemical-free farm in Nimbin, NSW, Australia. The gastrointestinal tract (GIT) (clean and dirty) of two camels (2.5 years and 6 months of age) was obtained from the company Qcamel, an organic and biodynamic farm, in the Glass House Mountains, Queensland, Australia. Retrieved samples were stored at 4°C.

[0132] The surfactant used in these experiments was Surfactant 12 A3 (Dodecyl Alcohol, ethoxylated; Polyoxyethylene Lauryl Ether; Polyethylene Monododecyl Ether; Polyethylene glycol dodecyl etherolyoxyethylene lauryl ether (C12-C15 alcohols, ethoxylated with 3 moles ethylene oxide) obtained from Ortho Chemicals Australia Pty. Ltd., Victoria, Australia. The lecithin used in these experiments was commercially available Ortho Lecithin AG (U) which is a high-end soyal lecithins at 350 g/L obtained from Ortho Chemicals Australia Pty. Ltd., Victoria, Australia. Where commercially available propionic acid was used, it was obtained form Ortho Chemicals Australia Pty. Ltd. at an initial concentration of 350 g/L.

Media used for culturing

[0133] The media listed in Table 2 were the main sources of nutrients for all the bacteria in this investigation. For the fresh goat, cow and camel isolates, Brain Heart Infusion (HI) (Sigma Aldrich, Missouri), SMS, and Difco™ Lactobacilli MRS media (Thermo Fisher Scientific, Massachusetts) were commonly used to help isolate and identify bacteria. F or P. freudenreichii, brain heart infusion (HI), yeast extract lactose (YEL), and glycerol-peptone media were commonly used for growth and PA production. HI was more commonly used for growth of the microorganism as it did not have the necessary nutrients to allow production of PA. YEL media was used for the production of PA, however, this soon changed to glycerol-peptone media (also known as glycerol-peptone broth, GPB; Sigma) after a greater yield of PA was visualised qualitatively using bromocresol purple dyes. This indicator’s purpose is to detect acidic metabolic products caused by the change pH from respective cultures. This indicator did not provide any quantitative results. Table 2: Culture media used in this investigation

Each component is listed in g/L. SMS and MRS were used in agar form

Revival and growth of P. freudenreichii

[0134] A lyophilised P. freudenreichii strain was bought from PHE NCTC and revived using the procedures described herein. Passages were made until abundant growth was detected, and to avoid any major mutations within the species, passage seven was the final growth stage used. This enhanced purity of the cultures and consistency of results.

[0135] Lactococcus lactis was set as a positive control in parallel to the recovery of P. freudenreichii. Cultures were incubated under microaerophilic conditions (aiming for between 2-10% oxygen presence) at 37 °C. Microaerophilic conditions were facilitated by 1 injecting CO2 (supplied by CO2 cannisters) in zip-lock bags containing the samples. Under these microaerophilic conditions, most freeze-dried cultures grow out in a few days.

[0136] 0.5 mL of liquid medium was aseptically added to the freeze-dried lyophilised

P. freudenreichii sample with a sterile Pasteur pipette, and the total mixture was then transferred to a test tube containing 2 mL of HI broth.

[0137] For the first passage, lyophilised cultures were revived using the standard operating procedure (SOP) provided by American Type Culture Collection (ATCC). The stagnant culture in half strength HI from the first passage was used to inoculate the second passage.

[0138] For the second passage, a 1 : 1000 dilution of P. freudenreichii from the first passage was inoculated in a 15 mL glass vial and incubated at 37 °C in microaerophilic conditions both shaking and stagnant in half strength HI broth; or in YEL media, which was incubated at 37 °C in stagnant microaerophilic conditions. Improved growth in half strength HI and in YEL media in the second passage was observed compared to the first passage. Production of PA was qualitatively examined using Lac-pep broth and bromocresol purple (BP) indicator.

[0139] To create the third passage, the second passage YEL media culture was used to inoculate both YEL and half strength HI broths in 15 mL glass vials which was incubated at 37 °C in stagnant, microaerophilic conditions.

[0140] The fourth passage was inoculated with a 1 :1000 inoculum from the third passage. This culture underwent a growth kinetics test to determine the most suitable conditions to enhance the growth of P. freudenreichii, as explained herein, but briefly, this passage was cultured in 15 mL of YEL broth at 37 °C for at least six days.

[0141] The fifth and sixth passages were grown in HI broth and used for precursors of the seventh passage. They were incubated at 37 °C under microaerophilic conditions for approximately one week.

[0142] The seventh passage was the last passage for all testing procedures. All major testing for PA production was done with this seventh passage, usually in glycerol-peptone broth. The culturing conditions for PA production are described elsewhere herein.

[0143] The colony forming units (cfu) were calculated on revived cells to determine the concentration of cells within a medium. Three serial dilutions of the homogenous culture medium from 10' ⁶ to 10' ⁸ were made, and 25 pL of each dilution were plated in duplicate. The plate was briefly dried and then incubated under microaerophilic conditions at 37 °C. After approximately 96 hours, the total number of colonies was recorded for each dilution and the concentration cfu/mL of the culture was determined using the following equation cfu. Ml' ¹ = 1 / SDF x PDF x TSDF where : SDF = sample dilution factor = 1, PDF = plate dilution factor = 25 pL/1000 pL, and TSDF = total serial dilution factor = 1/10 ⁶ = 10' ⁶ (due to all colony counts being standardised to IO' ⁶ ).

Growth kinetics of revived P. freudenreichii

[0144] The purpose of this experiment was to determine how efficiently the revived P. freudenreichii obtained from lyophilised stock was growing.

[0145] 15 pL of P. freudenreichii (passage four) was inoculated in 15 mL YEL broth (1 : 1000). Controls of HI and YEL broth were also prepared. A 0-hour time point reading of optical density (OD) was taken, and this was compared to readings taken at 72, 96, 120, and 144 hours to determine growth. Readings were measured at wavelengths of 600 nm and 650 nm. Collected OD data was graphed in order to analyse the time taken to reach the stationary phase of growth. [0146] A secondary growth kinetics test was performed on passage six within glass and plastic vials with glycerol-pep broth to observe which growth containment vial provided better results over time, which in turn might allow further production of PA. Media in both plastic and glass vials were inoculated with the fourth passage at either 1 : 1000 or 1 :20. Tubes were placed in 37 °C under microaerophilic conditions (aiming for 2-10% oxygen). Every 24 hours, the OD was taken at a 600 nm wavelength. All OD readings were taken with a cuvette spectrophotometer.

Creating a library of bacteria derived from goat and cow milk and manure and camel intestinal samples

[0147] Within a Laminar Flow hood, the milk samples obtained were separated into 3 different sections (top, middle, and bottom; visualised from the bottle it was retrieved in) which allows for a range of bacteria to be cultured from the different layers. Each layer was diluted from 10' ³ to 10' ⁸ in lx phosphate-buffered saline (PBS). For intestinal samples, 2 cm was cut and placed into a falcon tube containing lx PBS. From this, dilutions from 10' ³ to 10' ⁸ were made in lx PBS. Manure samples were weighed to 1 g with 10 mL of lx PBS to provide a 10' ¹ dilution factor, which was further diluted to 10' ⁶ .

[0148] Samples were plated using either a spread technique onto different media using sterile glass beads to disperse the bacteria within the plate or with a pour technique which will initiate growth within the agar. Incubation occurred at either 30 °C or 37 °C under either aerobic (21% oxygen) or microaerophilic (2-10% oxygen) conditions until distinguished colonies were visible on each plate. Post-incubation, plates were stored at 4 °C until patch plates could be made.

[0149] From the spread and pour plates, unique appearing and individual colonies were patched onto HI agar in a grid formation to allow more growth for those selections. Plates were incubated again for up to ~72 hrs (or until colonies had expanded without interfering with neighbouring colonies). Plates were stored at 4 °C until glycerol stocks could be made.

[0150] Glycerol stocks were made by inoculating unique appearing colonies into Falcon tubes containing 5 mL HI broth which was left to incubate at its respective temperature and oxygen conditions for ~16 hrs. Post-incubation, Falcon tubes were centrifuged at 4000 rpm for 8 mins at 4 °C to form a pellet. The supernatant was discarded, and the pellet was resuspended in 3 mL of 20% glycerol in half strength HI broth. In triplicate, 1 mL of each sample was transferred to 1.5 mL Eppendorf tubes and stored overnight at -20 °C. Samples were then stored cryogenically at - 80 °C.

PA production, extraction, purification, and concentration

[0151] The inventors herein sought to determine whether biosynthetically produced PA will function as a natural ingredient in a biopesticide. A method was formulated to produce, purify, concentrate, and test bacterially derived PA.

Culturing bacteria

[0152] Throughout the protocol development, P. freudenreichii was found to have a high yield of PA in glycerol-peptone broth (GPB), and this media was then used as the main culture medium for PA production. After comparing various growth parameters as described herein, the optimal protocol was found to involve inoculating passage six P. freudenreichii in autoclaved GPB (pH adjusted to approximately 7.2) in 2 or 4 1 L or 2 L bottles with aerobic and anerobic quality controls (QCs) (Table 3).

Table 3: Cultures for PA production

[0153] The quality control cultures were set up and incubated for a few days (e.g., four days) to ensure media was contamination-free. The 2x1 L bottles were then inoculated. Cultures were incubated in stagnant microaerophilic conditions at 32 °C. 16S sequencing was performed by Australian Genome Research Facility (AGRF) for passage seven P. freudenreichii to ensure the absence of contamination.

DNA extraction and 16S sequencing

[0154] This procedure helps discern the purity of the culture that is grown. A specialised procedure for the extraction of DNA from Gram positive bacteria was utilised. The type of DNA that is being extracted is genomic DNA (gDNA). The procedure used was based on the PureLink DNA Extraction Kit which was amended to improve the extraction.

[0155] To lyse the cells, the water bath was initially set to 37 °C in preparation for the incubation step. On day 8 of the culture’s growth, 1 mL of broth culture from each bottle was transferred into a 1.5 mL Eppendorf tube, in duplicate, before being centrifuged for 10,000 g for 5 minutes. The supernatant was discarded ensuring the pellet was not disturbed. The pellet from the first duplicate tube was resuspended in 200 pL of 1% Triton X-100 Digestion buffer and 200 pL of lysozyme. This mixture was then used to resuspend the pellet from the second duplicate tube in order to obtain a higher concentration of the gDNA. The Eppendorf tubes were then incubated at 37 °C for 30 minutes. After removing the samples from the water bath, the temperature was set to 55 °C and allowed to heat up. Proteinase K, 20 pL, was added to the Eppendorf tubes, mixed, followed by 200 pL of genomic lysis/binding buffer before being mixed again. The tubes were returned to the water bath and incubated for 30 minutes at 55 °C. After removal, 20 pL of Rnase was added and mixed before being incubated at room temperature for 2 minutes. An addition of 200 pL of 100% ethanol was made and mixed for 5 minutes until the solution became homogenous.

[0156] After lysing the cells, the gDNA is bound as follows. Approximately 640 pL of the lysate solution was added to a PureLink Spin Column (Thermo Fisher) and centrifuged for 1 minute. The collection tube was discarded before the column was placed in a fresh collection tube. The DNA was then washed by adding 500 pL of wash buffer 1, followed by centrifugation for 1 minute at 10,000 g. The collection tube was again discarded and replaced with a fresh one. 500 pL of wash buffer 2 was added to the spin column, followed by centrifugation at 10,000 g for 3 minutes. The final elution step was performed by discarding the collection tube and replaced by a fresh 1.5 mL Eppendorf Tube. 50 pL of filtered DEPC-water was added to the spin column which was incubated at room temperature for 1 minute. The tube was then centrifuged at 14,000 rpm for 1 minute. The column was discarded, and the Eppendorf tube was sealed, labelled, and placed at -20 °C for storage prior to sequencing. The samples were sent to the Australian Genome Research Facility Ltd (Melbourne, Australia) for 16S sequencing.

Clarification ofP.freudenreichii culture supernatant

[0157] Passage 7 P. freudenreichii was cultured as described herein. The culture supernatant was clarified as follows. The pH of the culture medium was adjusted to <2.5 using H2SO4 to facilitate carboxylic acid to become a carboxylate salt. The culture was then centrifuged at 5 °C, 12,000 rpm for 15 minutes to separate the cells and larger proteins from the supernatant; and filtered to remove further impurities including the carboxylic salt as follows. The supernatant was transferred to a borosilicate glass bottle filled up half-way. A vacuum filtration mechanism was setup to allow the supernatant to be further clarified in an efficient manner using a 0.22 pm filter, which further removes impurities including cell debris and some insoluble proteins that may have been transferred with the supernatant. The clarified PA containing culture supernatant was used in further experiments.

PA extraction and purification

[0158] For some experiments, PA was purified from the clarified culture supernatant using a primary extraction procedure. Reactive extraction was deduced as the most economical, and feasible method. The organic portion of the reactive extraction mechanism was prepared using tri-n-octylamine (TOA) and oleyl alcohol (OA). A homogenous solution was prepared with the two components: 40% TOA and 60% OA. The organic component was placed within a borosilicate glass bottle with the filtered supernatant (the “aqueous phase”) in a 1 : 1 ratio. The bottle was shaken vigorously before being incubated at 32 °C at 150 rpm for 12 hours. Postincubation, the organic and aqueous phase were transferred to a separating funnel and incubated at room temperature for up to 4 hours to allow the layers to separate. On the occasion where three layers were present, the middle layer (emulsion) was broken down with a glass stirring rod. After incubation, the bottom aqueous layer was discarded.

[0159] A distillation setup was used to purify the PA from the organic solution. In a roundbottom flask, the organic solution was added so it occupied approximately half the total volume. Sufficient boiling chips were placed inside the flask to prevent bumping when the solution is heated to high temperatures. The flask was placed on a specialised heating element with the rest of the apparatus being fitted to suit specifications. All air holes in the apparatus were sealed using glue-tack to ensure entrapment of vapours. Water was used as a coolant mechanism for the condenser which was supplied by a laboratory tap. The organic solution was slowly heated until a temperature of approximately 120 °C. As the solution began to bubble the water tab for the condenser was turned on to allow cooling of the vapours. This process was conducted until bubbles were no longer present in the organic solution. The purified product was then stored at 4 °C before quantification by HPLC-UV.

Qualitative indication of PA by indicator dye in media - bromocresol purple

[0160] Agar and broth media was prepared with 0.3 g/L of bromocresol purple (BP). The purpose of the BP indicator is to identify the presence of acidic properties where a change in colour from purple to yellow occurs at approximately pH 5.2.

[0161] For agar media, colonies are transferred from patch plates created for the bacteria library and grown in aerobic and microaerophilic conditions at 37 °C. Colonies were picked for their unique growth and patched as described using the same format as for colonies when making the bacteria library.

[0162] For liquid culture media (broth media), colonies from patch plates and revived P. freudenreichii cultures were inoculated into 5-10 mL of the broth containing BP. After approximately 1 week under the desired culture conditions, cultures were fully grown. If acid was produced, the media would change colour to yellow. These samples are then stored at 4 °C to be further tested by thin-layer chromatography (TLC). Qualitative detection of PA by thin-layer chromatography (TLC)

[0163] Detection of PA produced by a pre-cultured organism was conducted using thin-layer chromatography (TLC). To prepare cell cultures, 1 mL of cultured broths was transferred to an Eppendorf tube and centrifuged at 10,000 rpm for 10 min to separate the cells from the supernatant.

[0164] The chromatography chamber was constructed using a 200 mL glass beaker. Within the chamber, a 50 mL solvent was developed consisting of acetone (Sigma Aldrich, Missouri), water, chloroform (RCI Labscan, Bangkok), ethanol, and ammonium hydroxide (RCI Labscan, Bangkok) in a 60:2:6: 10:22 respective format. The chambers were covered with aluminium foil to contain the vapours. Standard solutions were made as controls for PA (Ortho Chemicals, Victoria), LA (Sigma Aldrich, Missouri), AA (Sigma Aldrich, Missouri), and butyric acid (BA) (Sigma Aldrich, Missouri) at 10% w/v or v/v.

[0165] TLC plates made of Merck silica gel 60 F254 (Merck, Darmstadt) were cut to 8x10 cm to standardise all tests. Approximately 5-7 mm from the bottom short-end of the plate, a horizontal line was gently drawn with a pencil to indicate where the test supernatant and standard solutions should be placed. For each test, 15 pL of PA, LA, and AA/BA standards marking and 20 pL of the test supernatant were spotted along the horizontal line. The spots were allowed to air-dry before being placed in the chromatography chamber for 20 min to achieve approximately 7 cm of migration. The plates were removed from the chamber and placed overnight at 37 °C.

[0166] A bromophenol blue indicator solution was used to detect a change in colour with the presence of an acidic property. 0.25 g of bromophenol blue powder was added to 100 mL of 70% methanol diluted with reverse osmosis (RO) water. The indicator was added to the molten agar prior to pouring onto plates, which was conducted following manufacturer specifications.

Acid-base titrations

[0167] Acid-base titrations can be a preliminary method in determining the percentage of acid concentration within a solution. Three drops of phenolphthalein were used as an indicator in 5 mL of the clarified culture supernatant which was placed in an Erlenmeyer flask. 0.1 N NaOH was then slowly titrated into the Erlenmeyer flask until a light pink colour was consistent within the solution. This marked the endpoint of the titration. The following calculation was then used to determine the percentage of acid concentration. N . y . mw % ^acW = v. iooo - ¹ ™ where N = normality = 0.1, mw = molecular weight of PA = 74.08 g/mol, V = starting volume = 5 mL, y = volume of NaOH used.

HPLC-UV quantification

[0168] Propionic acid was detected and quantified using an HPLC-UV system. The analysis was carried out using Arc-HPLC system (Waters: Milford, Massachusetts) connected to UV detector and Empower 3 software (Waters, Massachusetts). For the column, separations were carried out using C18 3.5 pm Xbridge column (4.6 x 50 mm; Waters, Massachusetts) with the temperature set at 45 °C. H3PO4 was added to improve the resolution of the organic acid and to lower the pH, with 0.1% H3PO4 set as the mobile phase condition. The mobile phase was composed of A (0.1% H3PO4 in water) and B (Acetonitrile) with the following gradient elution: 0-5 min 2%B, followed by 5-10 min with 30%B. Ten microliters of sample were injected, flow rate was set to 0.5 mL/min, and propionic peak was detected using a wavelength of 210 nm. Water and Acetonitrile used were LC-MS grade and purchased from Sigma, H3PO4 was purchased from Fluka (85%: HPLC grade) and propionic acid analytical standard from Sigma. To quantify the results, software tools were used to retrieve the area of the PA peak which was then substituted into the equation of the standard curve to retrieve a concentration value.

Microbial-derived PA for agricultural use

[0169] Synthetically derived propionic acid is commercially available. It can be emulsified with lecithin and a surfactant to form an emulsion, which can be mixed with an established pesticide. When these substances are mixed together, the surface tension of a preferred pesticide is reduced, which facilitates spreading of the liquid due to a reduced contact angle between the spray and foliage. Lecithin is a viscous substance which is usually not miscible in water. The addition of PA promotes emulsification such that a homogenous mixture is formed. The surfactant unifies the composition of the mixture. The emulsion can be used with a range of pesticides. In this experiment, water was used in the place of a pesticide to demonstrate the ability of microbial-derived PA to reduce surface tension of a mixture and enhance its spreadability. Preparing emulsion containing microbial PA or clarified PA-containing culture supernatants [0170] Agriculturally, commercially available PA, lecithin and surfactant are mixed in a 1 : 1 : 1 volume ratio to form an emulsion, and this formulation is used to enhance spreadability of a pesticide. For example, the emulsion containing commercially available PA would be used in a diluted format to allow efficient spreading of glyphosate.

[0171] Emulsions were prepared with lecithin, the surfactant and either commercially available PA, the purified microbial-derived PA or the clarified, PA-containing microbial culture supernatant detailed herein. For the commercially available PA and purified microbial-derived PA, the emulsions were prepared using the 1 : 1 : 1 formula as herein, without first diluting the components. However, due to the lecithin, the mixture is highly viscous. To combat this, the mixture was incubated at 32 °C under agitation for up to 24 h until emulsification occurred.

[0172] The commercially available PA or the purified microbial-derived PA emulsions were then diluted 1 : 100 in water (as an industrial agent in place of a pesticide) to test the whether the emulsion in a diluted form, as used agriculturally, enhanced spreadability of the water. Accordingly, the PA (that is, the commercially available PA or purified microbial-derived PA), lecithin, and surfactant were present at 0.33% volume in the diluted emulsion.

[0173] The clarified PA-containing culture supernatant emulsion was made using a different method. It was found using acid-base titrations that there is an approximate acid concentration of 0.5% within the clarified supernatant. Using this value, the supernatant volume was adjusted accordingly to achieve proportional acid concentration compared to the commercial PA. Briefly, the acid levels in the emulsion made with commercially available PA was used as a standard to compare to the emulsion made with the purified microbial-derived PA or clarified PA- containing culture supernatant.

[0174] For example, to maintain a 1 : 1 : 1 ratio of PAlecithin: surfactant when the PA is provided by the clarified bacterial culture supernatant, a 100 mL of the mixture would contain 60 mL of supernatant, 0.3 mL of lecithin, 0.3 mL of surfactant, with the remaining volume topped up with water (acting as an industrial agent in the place of a pesticide such as glyphosate). 60 mL of clarified supernatant was a suitable amount of PA to emulsify the amount of lecithin which was added. This mixture was placed in agitated conditions at 32 °C until emulsification occurred.

Plant trials

[0175] The PA containing emulsions can be used agriculturally to increase the spreadability of a pesticide such that it spreads more evenly across the leaves of weeds. Some pesticides are considered to have low absorptivity on weed leaves due to the waxy surface of the leaf. The waxy surface is hydrophobic which prevents polar liquids (such as water and various pesticides) from contacting the upper surface of the leaf effectively. Further, the surface tension of the pesticide results in the formation of pesticide droplets upon the leaves, rather than spreading of the pesticide over the leaves.

[0176] Plants were selected that had leaves with a width of more than 1 cm (to accurately observe droplets) upon which water formed droplets. Plants with leaves with width of at least 1 cm were tested for suitability using a spray bottle was filled with water to test whether droplets formed. Three suitable plants were found that prevented liquids with a large surface tension from dispersing. The chosen plants were Viburnum odoratissimum, Tibouchina heteromalla, Duranta sheena.

[0177] Plants were sprayed with control and test variables using three squirts of the diluted emulsion samples from a spray bottle; adjusted to standardise angle of dispersion, from a distance of approximately 20 cm and then the leaf was photographed from the same angle for all variables to reduce bias during quantitative analysis. The controls were water only, medium only, clarified culture supernatant only, and commercial PA- lecithin-surfactant mix (1%) in water. The test samples were P. freudenreichii supernatant - lecithin-surfactant 15% mix in water and P. freudenreichii supernatant - lecithin-surfactant 1% mix in water.

[0178] Pictures were then uploaded to Adobe Photoshop 2022 to quantify the dispersion of droplets from each sample (Figure 2). A leaf was chosen that had an approximate distance standardisation to leaves in other photos. A 158px x 158px area was then selected and isolated to remove interference from the background. The contrast and brightness of the photo was adjusted so the droplets could be discerned, and these were selected using the “lasso tool”. This “histogram” function was used to gather information on the number of pixels selected, which indicates the amount of spread by the droplets. Pixel results were received from Adobe Photoshop 2022 and converted into a coverage percentage.

Results and Discussion

Revival of P. freudenreichii from lyophilised stocks

[0179] The revival of P. freudenreichii cultures was efficient with prominent growth visualised by passage two post-revival after 96 hours under microaerophilic conditions at 37 °C in both YEL and HI media. This growth was strengthened over the course of further subculturing. Lyophilised cells usually take time to regain their original growth characteristics therefore subculturing is helpful in the recovery process. By the seventh passage, a high cell density P. freudenreichii culture was obtained.

[0180] A cell density test of P. freudenreichii cultured in HI broth or yeast extract lactose (YEL) was performed using the cfu/mL method at passage two (Figure 3)with growth analysed on agar plates. The presence of approximately 20 colonies was representative of good cell growth at passage two. At this stage in recovery, growth of the colonies was the primary aim. YEL and HI medium resulted in a similar cell density which was sufficient growth for subculturing to make passage three to continue revival (Table 4). The colonies were more prominent when grown in YEL media compared to HI (Figure 3).

Table 4: Cell density of passage two P. freudenreichii samples using the colony forming units (cfu) method

Growth of P. freudenreichii in YEL and glycerol-pep media at 32 or 37 °C [0181] The growth of passage four P. freudenreichii from 72 hours to 144 hours in yeast extract lactose (YEL) media at 37 °C in microaerophilic conditions following a 1 : 1000 inoculation was measured at 24-hour intervals using optical density (Figure 4). As this growth curve was done on a low passage, growth strength was low. Between 72 and 96 hours, it was seen that P. freudenreichii was in an exponential growth phase, reaching an OD of 0.180. By 120 hours, the culture had entered the decline phase, with an OD of 0.062 and 0.140 for 600 nm and 650 nm respectively. The culture may have entered a stationary phase before 120 hours that was not detected. The decline phase seemed to further continue over the next 24 hours for the 650 nm OD reading (OD = 0.048) at 144 hours, but increased for the 600 nm reading (OD = 0.078) (Figure 4). Based on the overall shape of the graph, the 650 nm OD curve more closely followed the expected general shape of a bacterial growth curve which may indicate a higher reliability over the 600 nm OD readings (Figure 4). As the peak of OD reading was approximately 0.180 for both wavelengths, it is possible that the culture was not fully revived at passage four to undergo abundant growth.

[0182] The growth of later passages showed much improved growth (Figure 5). Growth of P. freudenreichii at passage six from 24 hours to 216 hours post-inoculation was measured at ODeoonm at 24-hour intervals (except at 72 hours) cultured in glycerol-pep broth (GPB) at 37 °C in microaerophilic conditions in either glass or plastic vials following a 1 :20 inoculation or 1 : 1000 inoculation. The lag phase lasted approximately 24 hours for the 1 :20 samples which is evident of a greater capacity to enter the exponential phase earlier compared to the 1 : 1000 samples (Figure 5). The 1 : 1000 samples had a lag phase which lasted approximately 96 hours post-inoculation before entering the exponential phase. A longer lag phase in the 1 : 1000 inoculation compared to the 1 :20 inoculation can be correlated to the inoculation ratio itself. There are more cells being transferred into the medium in the 1 :20 sample which allows the cultures to “jumpstart” the exponential phase compared to the 1 : 1000 sample.

[0183] The 1 :20 samples were in an exponential phase until 144 hours post-inoculation where the glass vial sample seemed to enter a stationary phase at an OD of 1.827 (Figure 5). The plastic vial with a 1 :20 inoculation did not have sufficient sample to show a confirmed entrance into the stationary phase, however, if extrapolated, the stationary phase is expected to have started at around 168 hours post-inoculation. For the culture in a plastic vial 1 : 1000 inoculation, the exponential phase lasting approximately 72 hours and then a slow growth phase was entered until the end of the trial. For the 1 : 1000 inoculations, the plastic vial sample ended on a higher OD after the exponential growth phase compared to the glass vial sample (1.732 compared to 1.377) with the OD at the end of the trial being higher for the plastic vial (1.905 compared to 1.580), indicating stronger growth in the plastic vial. Clearly, the growth kinetics of passage six is much higher than that of passage four which indicates an improvement in the quality of cultures.

[0184] There is an anomaly in the results with the glass vial, 1 : 1000 inoculation between 120 and 144 hours (Figure 5), which may indicate an error in procedure during the recording of the results. However, the results as a whole were not largely affected as most curves on the graph seem to follow similar principles to a standard growth curve.

Optimised culturing method for producing PA-containing culture supernatant

[0185] The conditions for performing the final culturing step for PA production were to inoculate 2 L GPB media at pH 7.2 with a 1 :20 dilution (e.g., 100 mL) of P. freudenreichii culture showing good growth and incubate the culture under stagnant, microaerophilic conditions at 32 °C for 14 days prior to harvesting the culture supernatant. Al : 100 dilution of a P. freudenreichii culture into fresh media also consistently resulted in a successful subculture. However, as detailed elsewhere herein, other culture conditions also produced PA within the bacterial culture supernatant. Moreover, while not wanting to be bound by theory, it is likely that other similar PAB bacteria species would also produce suitable amounts of PA under these conditions.

Screening for acid production by mammalian derived bacteria

[0186] Propionic acid is found in milk and the gastrointestinal system, and certain propionic acid producing bacteria are found within the gastrointestinal GIT and milk of various mammalian species. We investigated whether we could identify novel propionic acid producing bacteria from samples obtained from cow milk, goat milk and camel gut samples. Isolated bacterial colonies originating from mammalian milk or intestinal samples recovered from spread plates prepared from the samples were transferred to Brain Heart infusion (HI) agar and glycerol-pep agar with bromocresol purple patch plates (Figure 6A, B). One patch plate could contain colonies from up to 3 spread plate origins. Acid production was detected for several colonies, as indicated by the change of colour of glycerol-pep agar with bromocresol purple from purple to yellow.

[0187] TLC and 16S sequencing were performed on these samples to detect propionic acid and attempt to identify the species.

Thin layer chromatography (TLC) results for P. freudenreichii and mammalian derived bacteria [0188] TLC was performed on the mammalian derived bacterial samples to identify the production of PA. A bromophenol blue indicator solution was used to detect a change in colour due to the presence of an acidic property. The bacteria from the respective sources were cultured in both aerobic and microaerophilic conditions in an attempt to find a PA-producing bacteria. From the tests conducted, one isolate from a camel gut was able to show traces of PA (purple, Rf = 0.63) production as well as lactic acid (LA) (yellow, Rf = 0.75) and potentially other organic acids (Figure 7 and Table 5). This bacterium was sent for 16S sequencing to AGRF; however, the results came back inconclusive. Further tests would need to be conducted on the isolate to evaluate its classification. Table 5: Rf values for TLC results and colour of the spot

[0189] TLC tests were also conducted on P. freudenreichii to confirm the production of PA. When comparing to controls, it can be seen that PA is being produced by P. freudenreichii with an Rf value of 0.55 (Table 5). To further confirm that the supernatant was not showing a spontaneous purple spot, a Lactococcus lactis culture supernatant was used as a control for LA which was evident at Rf value 0.51.

[0190] This result shows that the spots are not due to influences from the growth media. The P. freudenreichii culture was shown to produce PA. P. freudenreichii culture could now be scaled up for higher PA yield and extraction.

16S sequencing results for P. freudenreichii

[0191] The 16S sequencing results shows that all samples of P. freudenreichii sequenced were P. freudenreichii (Table 6).

Table 6: 16S sequencing results

(a) passage five P. freudenreichii in yeast extract lactose (YEL) media

(b) passage five P. freudenreichii in brain heart infusion (HI) media

(c) passage six P. freudenreichii in glycerol-pep broth

(d) passage seven P. freudenreichii in glycerol-pep broth

[0192] The samples that were sent to AGRF for sequencing were passage five, six, and seven. As passage seven was the working passage, it allowed an insight on whether the sample contaminated, and if so, whether the contamination was in the previous passages. 16S sequencing also helped to determine mutations which occur across passages. Between passage five and six, the appearance of subspecies globosum, as part of the P. freudenreichii species, was present (Table 6). This shows that as the passages increase, there were more subspecies present. Accordingly, the inventors did not allow culturing to proceed past passage seven. [0193] The camel sample, which was positive for PA production by the TLC method, was unable to be identified by 16S sequencing. Further study is required to determine whether this is a truly novel organism. Scale up of P. freudenreichii cultures for PA production

[0194] Larger volumes of P. freudenreichii were cultured in an attempt to produce larger amounts of PA. To reduce the chance of contamination, all bottles were inoculated in a LaminarFlow which contains negative pressure and high-efficiency particulate absorbing (HEP A) filters to purify the air within the working environment.

[0195] For PA production, quality controls (QCs) for the fermentation process were prepared 4 days before inoculation of the large-scale cultures to check for contamination of the media. Then, passage 6 of P. freudenreichii was inoculated in 2x or 4x 1 L bottles of autoclaved GPB. Cultures were incubated in these bottles with a 1 : 1000 inoculation and left at 37 °C for 14 days. The procedure obtained 4 mL of distilled product from 500 mL of supernatant.

[0196] Means for improving yield of PA were considered. The pH of the initial starting culture medium was approximately 5.92 for the four bottles. Accordingly, the culture can only decrease in pH by approximately 1.4 before acid production stops, because of end-product inhibition, which tends to occur at approximately pH 4.5 in order to maintain a viable environment for the bacteria. It was realised that this problem might be averted by adjusting the pH of the culture media to approximately 7. This pH is still viable for culture growth, and it might allow more PA to be produced before end-product inhibition would occur.

[0197] Further, it was considered that the reactive extraction could be improved. As the pK _a value of PA is 4.88, at a pH of 4.88 the concentration of carboxylate salt and carboxylic acid is equal. To counteract this, an addition of acid prior to extraction might further reduce the pH during the extraction procedure to enhance PA yield. Ideally, this acid should be aqueous, such as sulphuric acid, which can be removed from the aqueous phase during reactive extraction.

Acid-base titration of P. freudenreichii culture supernatants

[0198] Acid concentration in /< freudenreichii culture supernatants was determined using acidbase titration as a marker of PA concentration. The volume of NaOH required to neutralise the acid in the culture supernatants was measured to calculate the concentration of PA present in the clarified culture supernatant (Table 7). Table 7: Acid-base titrations for 3 different . freudenreichii supernatant replicates

Simple distillation of PA

[0199] After 14 days of culture incubation, the process of distillation of PA from the clarified culture supernatant (i.e., aqueous phase) was performed using TOA and OA composites to extract PA out of the aqueous phase. In early stages of investigating simple distillation techniques, small-scale trials were conducted to purify PA from the organic phase. This trial was conducted using vacuum distillation, which lowers the boiling point (bp) of the compound to prevent the onset of superheating and a “bumping” phenomenon where product can be lost.

[0200] However, it was found that although the bp of PA was reduced, so was the bp of both TOA and OA, such that there is a smaller margin in which the product can be efficiently extracted. It was accordingly considered more efficient to revert to a simple distillation method, where there is a temperature range of 190 °C before the bp of oleyl alcohol is reached and then a further 30 °C for TOA.

[0201] To prevent or reduce bumping caused by superheating, boiling chips or glass beads were utilised. Further, the inventors found that by insulating (for example, with aluminium foil) the neck of the round-bottom flask leading towards the entry point of the condenser to maintain temperature further reduced the chance of bumping. This prevented rapid changes in temperature due to atmospheric conditions, which facilitates the environment within the distillation unit to be more uniform. The insulation also prevented or reduced the vapours from condensing prematurely (before reaching the entry point of the condenser) due to the more uniform temperature within that passageway.

Effect of pH on PA production

[0202] P. freudenreichii production of PA can be enhanced under certain conditions, with temperature and pH found to of particular relevance. pH was observed to affect acid production and the final acid composition within the solution. When starting from a near neutral pH, the PA production was enhanced. P. freudenreichii stops producing acids at a pH of 4.5 due to endproduct inhibition. When GPB which is not pH adjusted (pH = 5.9) was used to produce PA, the pH can only decrease by approximately 1.4 before acid production is halted due to end-product inhibition (Table 8). When using a pH adjusted GPB (pH = 7.2), the pH can be reduced by approximately 2.7 before acid production is inhibited. Using culture media with a neutral or near neutral pH resulted in a higher volume of PA obtained.

Table 8: Volume of final distilled product under different starting pH

[0203] Additionally, pH may prevent disassociation of the acid. Acid dissociation is marked by pK _a where the presence of salt and acid is equal. In the case of PA, the pK _a value is 4.87, suggesting that a pH lower than 4.87 will yield the most PA. Accordingly, acidifying the supernatant (e.g., with H2SO4) before extraction may assist to convert propionate ions into PA.

HPLC quantification of PA in distilled PA and clarified . /rezzz/ez rezc/zzz culture supernatants [0204] Samples cultured under different conditions were analysed by HPLC-UV line to quantify PA. The PA standards used to create a standard curve were also used to determine the concentration of PA in the test samples and detect impurities (Figure 8 and Table 9).

Table 9: Concentration of PA in distilled PA and clarified P. freudenreichii culture supernatants

[0205] When testing standards, elution time for PA usually was around 3.3 minutes at a 210 nm UV wavelength, which indicated likely timing to detecting PA in the distilled product and clarified culture supernatants as seen in Figure 8. It is evident from Figure 8C, which shows the HPLC profile of glycerol-peptone broth alone, that there is constant noise present. This gives an indication of the number of other components, such as nutrients, found within the broth. To improve costs, further experiments could aim to see the effects of experimenting with half strength glycerol-pep media.

[0206] A comparison between Figure 8D and Figure 8E shows the PA peak doubling in concentration in the clarified culture supernatants between days 6 and 14 of incubation, which is further quantified in Table 9. Figure 8B shows the HPLC-UV spectrum for distilled PA. It can be seen that there are a total of six peaks, five of which are contaminants. The source of this contamination may be metabolic products/by-products from the bacterium or remnants of the medium within the final product. By comparing the spectra in Figure 8B, Figure 8C, and Figure 8E, it can be seen that some of the peaks present in the distilled phase spectra are present in the other figures.

[0207] The concentration of PA within the clarified P. freudenreichii culture supernatant following 14 days of incubation was calculated at approximately 0.6% and 5.9 ppm. The concentration of 0.6% correlates well with the concentration of approximately 0.6% found using the acid-base titration method (compare Table 7 and Table 9). The distilled PA has a PA concentration of 2.39% or 23.8 ppm (Table 9). This concentration is lower than synthetically produced PA than is commercially available.

Plant trials to determine the suitability of microbial PA to be used agriculturally

[0208] The plant Duranta sheena qualitatively resembled the characteristics of weeds currently targeted by a herbicide composition mixed with an emulsion consisting of lecithin, a surfactant and commercially available PA. The plant has a waxy upper leaf area, upon which water sits in droplets that maintain their surface tension, rather than spreading over the leaves. The herbicide also sits in droplets on these types of waxy leaves, which reduces efficacy. The herbicide is more effective when the droplets spread out to cover more of the leaf surface area.

[0209] This experiment investigated whether the clarified P. freudenreichii supernatant can be used as a component for emulsion polymerisation with lecithin and a surfactant to facilitate the reduction of surface tension in the occupying liquid, such as a pesticide. In this experiment, water was used in place of a pesticide as an indicator of an industrially useful agent, as water is a high-surface tension liquid. Negative controls were used to establish parameters of the test samples that included all the necessary components of the final product. Positive controls were used to establish parameters that would resemble the results of the test subject. Controls and tests were replicated to confirm results for spreading ability. The results for this trial are shown in Table 10. Figure 9 provides an image of a standard curve created using the area (UV*sec) and standard concentrations (ppm) to achieve the quantified HPLC-UV results shown in Table 10

Table 10: Quantitative results for trials with clarified P. freudenreichii supernatant in an emulsion with lecithin, surfactant on Duranta sheena

[0210] The first trial consisted of Viburnum odoratissimum, Tibouchina heteromalla, Duranta sheena which investigated the spreading ability of the commercial PA-lecithin-surfactant emulsion product when mixed with water, which was used as a model of a compound that has a high surface tension. Only the results from Duranta sheena are shown in Table 10. The commercial PA-lecithin-surfactant emulsion product mixed with water was visualised to have a lack of water droplets and accordingly spread more than the water only control.

[0211] A second trial was conducted with more controls and testing parameters to see the effects of the emulsion consisting of clarified PA containing P. freudenreichii supernatant, lecithin and surfactant. The spreading ability of the clarified PA containing P. freudenreichii supernatant itself was seen to have similar effects to that of the media control and water control. However, when the clarified PA containing P. freudenreichii supernatant was mixed with lecithin and surfactant, there was a spreading ability that was comparative to the commercial PA-lecithin-surfactant complex. It was noted, however, that the supernatant-lecithin-surfactant mixture contained 85% water, whereas the commercial PA-lecithin-surfactant mixture contained 99% water. The clarified PA containing P. freudenreichii supernatant-lecithin-surfactant mixture nonetheless showed promise, with the clarified PA containing P. freudenreichii supernatant providing effective PA function, which is potentially comparative to the commercial PA-lecithin-surfactant product. It was noticed that only two of the three plants trialled showed a high dispersion of the commercial product, with the exception being Viburnum odoratissimum. Following this, this plant was not used in further trials.

[0212] For the third trial, it was observed that there was a high spreading ability for the clarified PA containing P. freudenreichii supernatant-lecithin-surfactant mix at 15% concentration in water showed comparative results to the commercial PA-lecithin-surfactant mix at 30% concentration in water.

[0213] The fourth trial incorporated clarified P. freudenreichii supernatant-lecithin-surfactant mix at 1% concentration in water, and this produced a result that was comparative to the commercial PA mix (at 1%). After comparing it to controls, there were spreading similarities between the standardised commercial PA mix and clarified P. freudenreichii supernatant mix. Comparing the 1% P. freudenreichii supernatant mix to 15% clarified P. freudenreichii supernatant mix, it was realised that the higher concentrated mix is more effective in spreading, but the lower concentrated mix still achieves spreading that is comparative to commercial standards. This shows that from an experimental standpoint, there is a possibility of replacing the commercial PA with clarified P. freudenreichii supernatant within an emulsion for increasing the spreading of pesticides.

[0214] The fifth trial was a repeat of the fourth trial to help further establish the findings through repetition.

[0215] When the results of all of the trials were considered, the water-only control provided in 24.05% of leaf coverage; whereas the commercial PA-lecithin-surfactant mix at 1% in water provided 70.68% coverage. Meanwhile, the P. freudenreichii supernatant- lecithin-surfactant mix at 15% in water provided 96.13% coverage, while P. freudenreichii supematant-lecithin- surfactant mix at 1% in water provided 61.52% leaf coverage (Table 10). Accordingly, the P. freudenreichii supematant-lecithin-surfactant mix at 1% provided 37.47% more coverage than water alone. This shows that the lowest concentrated microbial mix sample that was tested had the ability to spread over the leaves of the plant better than water.

[0216] The commercial PA- lecithin-surfactant mix at 1% in water provided 9.16% more coverage than P. freudenreichii supematant-lecithin-surfactant mix at 1% in water (Table 10); however, increasing the concentration of the P. freudenreichii supernatant- lecithin-surfactant mix to 15% in water was superior to the current commercial standard (Commercial PA-lecithin- surfactant mix at 1% in water). This may indicate that the culture supernatant is not inhibiting or interfering with an increase in spreadability. This result accordingly shows that /< freudenreichii supernatant-lecithin-surfactant mix at both 1% and 15% facilitate emulsion polymerisation to a similar extent to commercial PA- lecithin-surfactant mix at 1%, which is considered the gold standard for such experiments.

[0217] Accordingly, these experiments portray a proof of concept where an emulsion polymer comprising a clarified P. freudenreichii supernatant is able to spread the dissolving liquid at a similar manner as the gold standard for spreading a herbicide upon waxy leaves. As the emulsion using P. freudenreichii supernatant showed similar results to the emulsion using commercial PA when mixed with water, and given that it is already known that the emulsion using commercial PA provides enhanced spreadability to a herbicide, it follows that an emulsion using P. freudenreichii supernatant to provide the PA will result in enhanced spreadability of that herbicide.

Example 2 - Optimising P. freudenreichii culturing conditions for optimal PA production Introduction

[0218] To further evaluate the conditions of P. freudenreichii growth for enhanced PA production, factors including nutrient concentration, length of culture, pH adjustment of the cell culture, and culturing in strictly anaerobic vs. non-strictly anaerobic conditions were investigated.

Materials and Methods

[0219] P. freudenreichii culturing conditions were tested in a checkerboard format with a number of variables:

• PEP Glycerol media at lx, 0.5x or 0.25 x concentration,

• With or without pH adjustment to approximately pH 6.5; and

• Aerobic, non-strictly anaerobic or strictly anerobic conditions (in an anerobic incubator).

[0220] PEP glycerol media was autoclaved in glass bottles. A P. freudenreichii was inoculated into each bottle using a syringe in a ratio 1 : 1000 or 1 : 100 of total media volume and incubated at 32°C as described herein. Culturing occurred for 7, 14, 21 or 28 days. All cultures became turbid. After 7 days, 4.5 ml of the culture was used to determine CFU count and HPLC analysis was used to determine the amount of PA in the culture supernatants. The HPLC analysis was performed using standard conditions with Cl 8 columns with an ACQUITY Arc Liquid Chromatography System", as a LC model from Waters Corporation. The conditions were , and as described elsewhere herein, except that to better separate peaks, voltage and current was reduced and injection time was increased. The HPLC samples were stored in deep freezer to preserve the PA concentration prior to analysis.

[0221] Anaerobic culturing was performed in an anerobic incubator. Culturing under non- strictly anerobic conditions was performed by minimising oxygen contact with the culture as much as possible, but outside of an anerobic incubator as described elsewhere herein.

[0222] At day 7, the pH of the aerobic culture with 0.25x PEP Glycerol media and no pH adjustment was approximately 4.6. The pH of cultures undergoing pH adjustment was adjusted to approximately pH 6.5 using 10 drops of NaOH at days 7,14, and 21 (where culturing was continuing).

Results and Discussion

Effect of culturing P. freudenreichii in strictly vs non-strictly anaerobic conditions

[0223] The aim of this experiment was to test whether strictly anaerobic conditions or non- strictly anaerobic conditions at different PEP glycerol media concentrations (lx, 0.5x or 0.25x) and culture length optimised PA production. An example of HPLC results showing different amounts of PA production is shown in Figure 10. PA likely eluted at a different time to the earlier experiments due to the different HPLC conditions used.

[0224] The tabular results are shown herein in Table 11. Samples were cultured for 7, 14, 21 or 28 days; in 0.25x, 0.5x, or lx PEP glycerol media concentration; under strictly or non-strictly aerobic conditions.

Table 11: PA production following P. freudenreichii culturing under variable conditions

[0225] The results shown here indicate PA production was higher in in strictly anaerobic conditions except for the value marked with an asterisk. In addition, lowering the media components (nutrient composition) has lowered the amount of PA produced.

Effect of culturing P. freudenreichii by adjusting the pH of the culture

[0226] Production of PA during the culturing of the P. freudenreichii significantly reduces pH which potentially has an inhibitory effect on the P. freudenreichii growth and PA production. This experiment tested the samples adjusted to approximately pH 6.5, compared with samples for which pH was not adjusted. The results are presented in the Table 12 herein.

Table 12: PA production (ppm) following P. freudenreichii culturing under variable conditions

Samples were cultured for 7, 14, 21 or 28 days; in 0.25x, 0.5x, or lx PEP glycerol media concentration; under strictly or non-strictly aerobic conditions, with or with pH adjustment to approximately pH 6.5.

[0227] Table 12 shows that using IxPEP glycerol media typically produced higher PA levels that any other media concentration. Additionally, culturing the samples for 21 or 28 days, compared to 14 days, did not appear to increase PA production by an amount that can justify the extra time and resources required. Moreover, culturing P. freudenreichii under strictly or non- strictly anaerobic conditions did not consistently change the PA amount produced Similarly, the pH adjustment did not consistently change the amount of PA produced. As this may have been due to the crude method used to adjust pH, this experiment is to be repeated with a more precise pH adjustment step.

Example 3: Laboratory and Field upscaling of PA production

Phase 1: Inoculation and sterility testing

[0228] P. freudenreichii was inoculated into 50 ml batches using the techniques described herein, under conditions that replicate the agricultural environment. Cultures were grown under stagnant growth conditions at room or environmental temperatures of approximately 20 to 25°C. Minimal air space in the culture vessels is used to create an anerobic culture.

[0229] The procedures to be used in up-scaling will undergo sterility testing. Different equipment and sterile conditions are to be tested to determine optimal P. freudenreichii growth conditions, and optimal conditions for PA production. Quality control of the microbial cultures will be undertaken to analyse PA production (e.g. using HLPC as described herein) and population purity (e.g., Gram staining as shown in Figure 11).

Phase 2: Upscaling P. freudenreichii culture and optimizing PA production

[0230] P. freudenreichii was inoculated into and cultured in 5 L drums in under agricultural conditions using methods herein. Cultures were grown under stagnant growth conditions at room or environmental temperatures of approximately 20 to 25°C. Minimal air space in the culture vessels is used to create an anerobic culture. Nalgene plasticware prevents gas-induced deformation. Cultures grown in the dark for 4 weeks at 20-25°C showed heavy turbidity (Figure 12A). Nalgene plasticware prevents gas-induced deformation (Figure 12B).

[0231] Methods for the sterile transfer and inoculation of cultures in an agricultural setting and quality control of the samples will be tested. Next, P. freudenreichii w be inoculated into and cultured in 50 L drums outdoors to replicate realistic industry conditions. Methods for obtaining the supernatant and removing debris will be validated.

Phase 3: Sterilization of the supernatant and field trials

[0232] Filtration and other means of sterilization will be tested. Quality control testing of the produced supernatant will be undertaken, for example, the amount of PA produced will be detected using HLPC. Example 4: Testing the effectiveness of P. freudenreichii derived PA in field trials

[0233] The effectiveness of clarified P. freudenreichii derived PA obtained under field conditions will be tested in emulsions containing the P. freudenreichii derived PA, a lecithin and a surfactant using a checkboard approach that varies the concentrations of the components; the ratios of the components; the lecithin and surfactant used; and the methods used to form an emulsion (for example, by investigating different temperatures). Commercially available surfactant, propionic acid and surfactant will be utilised as detailed herein.

[0234] In one trial, various emulsions containing irradiated P. freudenreichii cultures grown as described herein will be used to form emulsions and mixed with a pesticide such as glyphosphate in a range of concentrations as shown in Table 11. The trials will utilise commercially available PA, lecithin and surfactant from Ortho Chemicals, Victoria, Australia. [0235] The P. freudenreichii cultures will be irradiated by cobalt blue gamma irradiation using a standard protocol. No centrifugation or filtration will be conducted. Accordingly, the effect of the PA will be tested within a whole bacterial culture, rather than in a clarified bacterial supernatant.

[0236] The spreadability of the emulsion-pesticide mixture will be assessed using methods described herein. The killing ability of the emulsion-pesticide mixtures on plants in the field will be tested by applying to plants using standard techniques. Trials will be conducted on 3x16 m ² plots in two distinct areas across New South Wales, Australia, for compliance with requirements for regulatory approval, which may or may not be substantiated by an additional trial in a different season.

Table 11: Ratios of lecithin, surfactant, Propionic acid testing in triplicate field trial, testing the ability of the propionic acid (PA)-producing probiotic Propionibacterium freudenreichii, in replacing the use of a commercial-grade chemical PA (solvents-purified) as a non-living irradiated whole culture

[0237] The trials will investigate whether irradiated P. freudenreichii cultures can effectively replace chemically synthesized PA, and whether the amount of glyphosphate can be reduced in the presence of the irradiated culture. It is anticipated that an irradiated P. freudenreichii culture will have approximately the same amount of propionic acid as a clarified P. freudenreichii culture supernatant, as would be understood by those skilled in in the art.

Example 5: Laboratory Observations

[0238] Within one-week post-inoculation, P. freudenreichii cultures typically reach maximum cell density. Within one further week of incubation, P. freudenreichii cultures reached acceptable, maximum or near-maximum PA concentration in culture. While 28 days sometimes provides a higher PA concentration, 14 days was considered to provide a suitable culturing time as the additional PA increase seen at 28 days of culture is not substantial enough to justify an additional 2-week incubation at 32°C.

[0239] A 1 :20 dilution of a P. freudenreichii culture as an inoculum typically induced strongest growth of a sub-culture. However, a 1 : 100 dilution of a P. freudenreichii culture provided a reliable and useful inoculum for a further culture. When a 1 : 1000 dilution of a P. freudenreichii was used as an inoculum, culture growth was typically delayed in comparison. INDUSTRIAL APPLICABILITY

[0240] The industrial applicability of the present disclosure can be readily envisaged by way of commercial products and services employing the claimed method/s and/or use/s.