use

Abstract

This invention relates to the use of one or more nicotine-degrading microorganisms or an extract thereof for the degradation of one or more alkaloids on an alkaloid-contaminated object, wherein at least one of the nicotine-degrading microorganisms is Pseudomonas putida S16.

Description

[0001] USE

[0002] FIELD OF INVENTION

[0003] The present disclosure relates to the use of one or more nicotine-degrading microorganisms; and a method of degrading one or more alkaloids on an alkaloid-contaminated object.

[0004] BACKGROUND OF THE INVENTION

[0005] Many alkaloids are considered to be toxic and hazardous to human health and the environment when released in sufficient quantities. For example, nicotine is classed as a toxic and hazardous waste and must be disposed of through permitted means in many countries.

[0006] Various waste products are produced throughout the lifecycle of tobacco products from production waste, through to consumer waste following use of the products. Alkaloids, such as nicotine, often remain on these waste products, even after the tobacco is no longer present. Therefore, novel ways of reducing unwanted alkaloids to safe levels for disposal, recycling, or re-use are required.

[0007] SUMMARY OF THE INVENTION

[0008] In one aspect there is provided a use of one or more alkaloid degrading (e.g. nicotinedegrading microorganisms) for the degradation of one or more alkaloids on an alkaloid- contaminated object, wherein at least one of the nicotine-degrading microorganisms is Pseudomonas putida S16.

[0009] Suitably, the alkaloid-degrading microorganism described herein may be a nicotinedegrading microorganism. Suitably, the nicotine-degrading microorganism described herein may degrade nicotine. Suitably, the nicotine-degrading microorganism described herein may degrade nicotine and / or other alkaloids.

[0010] In one aspect, there is provided a method of degrading one or more alkaloids on an alkaloid- contaminated object, wherein the method comprises: a) inoculating the alkaloid-contaminated object with alkaloid degrading ( e.g. nicotinedegrading microorganisms), to decontaminate the object wherein at least one of the nicotine-degrading microorganisms is Pseudomonas putida S16.. Suitably, the microorganisms (e.g. bacteria) may be pre-cultured. For example the microorganisms (e.g. bacteria) may be pre-cultured in media to grow the bacteria. Suitably, the microorganisms (e.g. bacteria) may be pre-cultured with or without one or more alkaloids.

[0011] Suitably, the microorganisms (e.g. bacteria) may be pre-cultured with at least one alkaloid e.g. nicotine. Suitably, the microorganisms (e.g. bacteria) may be pre-cultured with at least one alkaloid e.g. nicotine before inoculating the alkaloid-contaminated object.

[0012] Suitably, prior to step (a) the alkaloid-contaminated object may be suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloid-contaminated object provides the main carbon and nitrogen source when added to the liquid media.

[0013] Suitably, the method may further comprise: b) incubating the inoculated alkaloid-contaminated object until it is decontaminated.

[0014] Suitably, the inoculated alkaloid-contaminated object may be incubated for a minimum of 1 hour.

[0015] Suitably, the inoculated alkaloid-contaminated object may be incubated for between about 1 hour and about 120 hours.

[0016] Suitably, the method may further comprise: c) drying the decontaminated object.

[0017] Suitably, the method may further comprise: d) either

[0018] (i) further processing the decontaminated object for subsequent use; or

[0019] (ii) disposing of the decontaminated object.

[0020] The initial nicotine-degrading microorganism cell density in step (a) may be between about 2.0 x 106CFU / ml and about 1.0 x 109CFU / ml.

[0021] The concentration of the one or more alkaloids may be reduced to a final concentration of equal to or less than 2.7 mg / L, wherein the one or more alkaloid is nicotine. The nicotine-degrading bacteria may degrade nicotine through the pyrrolidine pathway.

[0022] Suitably, the one or more alkaloids may be selected from the group consisting of: nicotine, nornicotine, anabasine, anatabine, myosmine, cotinine and pseudooxynicotine.

[0023] Suitably at least one of the alkaloids may be nicotine.

[0024] The alkaloid-contaminated object may be selected from the group consisting of: combustible aerosol provision systems, non-combustible aerosol provision systems, aerosol-free delivery systems, and consumable or non-consumable components thereof.

[0025] The alkaloid-contaminated object may be a consumable or non-consumable component of a non-combustible aerosol provision system, wherein the component is a vaping cartridge.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Embodiments of the invention will now be described, by way of example only, with reference to accompanying drawings, in which:

[0028] Figure 1 : shows the nicotine content present in the liquid solution containing re-suspended bacteria and nicotine contaminated objects. Three different treatments are shown; mock (no microbes inoculated), single (single application at Oh) and multiple microbial applications (microbial applications at Oh, 24h,48h,72h and 96h). Samples taken at Oh, 48h, 72h, 96h and 168 hours (7days).

[0029] Figure 2: shows the nicotine content drop observed in the liquid solution containing re-suspended inoculated bacteria and nicotine contaminated objects for a period of two days. Samples taken at 0, 2h, 4h, 6h and 48h.

[0030] Figure 3: shows the comparison of different samples on HPTLC run. Isolated band shown in the picture corresponds to Nicotine band. Different intensities correspond to different concentrations. Absence of nicotine band indicates that the concentration of nicotine is below detection levels.

[0031] Figure 4: shows the nicotine content drop observed in the liquid of the incubated solution containing vaping cartridges and Pseudomonas putida S16. Figure 5: shows nicotine content and hours of incubation XY Exponential Decay (Non linear regression) curve built as a simulation from collected data at different sampling points (0,2,4 6 and 24h).

[0032] Figure 6: shows the change in pH in tobacco extracts across time for the different cultures inoculated. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SEM . Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0033] Figure 7: shows cell growth evolution in extracts across time for the different cultures inoculated. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SEM. Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0034] Figure 8: shows nicotine concentration in tobacco extracts inoculated with different cultures. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SD. Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0035] Figure 9: shows anabasine concentration in tobacco extracts inoculated with different cultures. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SD. Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0036] Figure 10: shows pseudooxynicotine concentration in tobacco extracts inoculated with different cultures. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SD. Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0037] Figure 11 : shows anatabine concentration in tobacco extracts inoculated with different cultures. Data analysis was performed by Two-way Anova (repeated measures, (GraphPad prism). Data are shown as the means + SD. Significance (p < 0.05, n = 9) among groups is denoted by different letters (a to d).

[0038] Figure 12: shows the inhibition zone (mm) for the different dilutions various e-liquids, Golden Tobacco, Mint, Tobacco Marvel, Menthol and Blueberry ice, in deionized water for the three bacterial strains, with H2O2 5% as positive control. 6 mm is represented as the minimal measurement covered by all test disks (6 mm). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data are shown as the means + SEM. Significance (p < 0.0001 , n = 3) against the positive control is indicated by asterisks

[0039] Figure 13: shows nicotine biodegradation in various e-liquids from initial inoculation with three different strains at Oh and an incubation period of 24h. Different dilutions of Golden Tobacco e-liquid (16.87 mg / ml nicotine), Mint e-liquid (16.52 mg / ml nicotine) and Tobacco Marvel e-liquid (33.9 mg / ml nicotine) in IS - media. H2O2 5% and Luria Broth are shown as positive control. Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data are shown as the means + SEM. Significance against the mock is indicated by asterisks (p<0.01,*; P<0,05, **; p<0,005,*** and p < 0.0001 , ****).

[0040] Figure 14: Cell growth of P. putida S16 in Luria Broth containing no nicotine (LB -) and nicotine 4 mg / ml (LB+) compared to a positive control (H2O2 5%). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significant difference at 24h.

[0041] Figure 15: Nicotine degradation by P. putida S16 exposed to nicotine (LB+) and nonexposed (LB-) in Luria Broth containing nicotine 4 mg / ml compared to mock treatment (not inoculated). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significant difference at 24h

[0042] Figure 16: Cell growth of P. nicotinovorans in Luria Broth containing no nicotine (LB -) and nicotine 4 mg / ml (LB+) compared to a positive control (H2O2 5%). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significant difference at 24h. Figure 17: Nicotine degradation by P.nicotinovorans exposed to nicotine (LB+) and nonexposed (LB-) in Luria Broth containing nicotine 4 mg / ml compared to mock treatment (not inoculated). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significant difference at 24h.

[0043] Figure 18: Cell growth of P. ureafaciens in Luria Broth containing no nicotine (LB -) and nicotine 4 mg / ml (LB+) compared to a positive control (H2O2 5%). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significant difference at 24h.

[0044] Figure 19: Nicotine degradation by P. ureafaciens exposed to nicotine (LB+) and nonexposed (LB-) in Luria Broth containing nicotine 4 mg / ml compared to mock treatment (not inoculated). Data analysis was performed by Two-way Anova (repeated measures, GraphPad prism). Data is shown as the means + SEM. Significance (p < 0.001 , n = 9) of the differences between the three treatments is represented by letters (a,b,c). Data compiled under the same letter does not show significance.

[0045] DEFINITIONS

[0046] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[0047] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0048] “Alkaloid-degrading microorganisms” are microorganisms capable of degrading one or more alkaloids. For example, alkaloid-degrading microorganisms may be capable of degrading nicotine and / or other alkaloids. Nicotine-degrading microorganisms may be capable of degrading nicotine and / or other alkaloids. The term “alkaloid-contaminated object” refers to any object that has unwanted alkaloids present on or within it. The alkaloids may from any source, for example they could be synthetically produced and provided in a cartridge such as nicotine in a vaping cartridge which requires decontamination before disposal. In another example, the alkaloids may be naturally produced, such as by tobacco, and deposited on an object such as a cigarette filter through use, which requires decontamination to prevent the deposited alkaloids from leaching into the environment when disposed of.

[0049] The term “inoculating” refers to the introduction of bacteria to the matter to be treated. The matter in the context of this disclosure is an alkaloid-contaminated object.

[0050] The term “microorganism extract” refers to any product that has been isolated from a microorganism. For example, the product may be an enzyme extracted from a microorganism. Alternatively, the microorganism extract may be a gene isolated from the genome of a microorganism which is then inserted and expressed by a different cell or organism, such as a plant, virus, or alternative bacteria.

[0051] The term “bacterial extract” refers to any product that has been isolated from bacteria. For example, the product may be an enzyme extracted from bacteria. Alternatively, the bacterial extract may be a gene isolated from the genome of a bacterium which is then inserted and expressed by a different cell or organism, such as a plant, virus, or alternative bacteria.

[0052] Other definitions of terms may appear throughout the specification. Before the exemplary aspects and embodiments are described in more detail, it is important to understand that this disclosure is not limited to particular aspects or embodiments described, which as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects or embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0053] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an alkaloid-contaminated object” includes a plurality of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

[0054] It should also be noted that as used herein and in the appended claims, that the term “about” refers to the stated value and values that can be rounded to that value. For example, 1.0 would encompass values between 0.95 and 1.04; 1 would encompass values between 0.5 and 1.4; 10 would encompass values between 9.5 and 10.4; and so on and so forth. Where a value is expressed in terms of ‘a’ x 10y; the term about applies to ‘a’. For example, 1.0 x 103would encompass values between 0.95 x 102and 1.04 x 103.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention employs the use of alkaloid degrading e.g. nicotine-degrading microorganisms to decontaminate objects that have unwanted alkaloids present on them. Various objects associated with the consumption of tobacco and tobacco alternatives become contaminated with nicotine and other unwanted alkaloids. These objects, such as vaping cartridges, filters, and single-use vaping devices, present a problem when it comes to disposal as the nicotine and / or other alkaloids present on the objects can leach into the surrounding environment and have the potential to cause harm. By decontaminating the objects, the present invention allows for them to be safely disposed or recycled.

[0057] Use

[0058] In one aspect there is provided a use of one or more alkaloid-degrading microorganisms e.g. nicotine-degrading microorganisms, or an extract thereof, for the degradation of one or more alkaloids on an alkaloid-contaminated object.

[0059] In an embodiment, the one or more alkaloid-degrading microorganisms are one or more nicotine-degrading microorganisms.

[0060] In an embodiment, the one or more nicotine-degrading microorganisms are one or more nicotine-degrading bacteria.

[0061] In a further embodiment, the nicotine-degrading bacteria degrade nicotine through the pyridine pathway or the pyrrolidine pathway.

[0062] In yet a further embodiment, the nicotine-degrading bacteria is one or more of Pseudomonas putida S16, Paenarthrobacter nicotinovorans, and Paenarthrobacter ureafaciens.

[0063] In a further embodiment, the nicotine-degrading bacteria degrade nicotine through the pyrrolidine pathway. In yet a further embodiment, the nicotine-degrading bacteria is Pseudomonas putida S16.

[0064] In one embodiment, there is provided use of a bacterial extract for the degradation of one or more alkaloids on an alkaloid-contaminated object.

[0065] In a further embodiment the bacterial extract is an enzyme derived from an alkaloiddegrading bacteria (e.g. a nicotine-degrading bacteria) that degrades alkaloids through the pyrrolidine or pyridine pathways. Suitably, the alkaloid-degrading bacteria may be a nicotine degrading bacteria.

[0066] In yet a further embodiment, the enzyme is one or more of the group comprising or consisting of nicotine oxidoreductase (NicA), pseudooxynicotine amine oxidase (PNAO), 3- succinoylsemialdehyde pyridine dehydrogenase (SAPD), 6-hydroxy-3-succinoyl pyridine hydroxylase (HspA and / or HspB), nicotine dehydrogenase (NDH), 6-hydroxy-L-nicotine oxidase (6-HLNO), 6-hydroxy-D-nicotine oxidase (6HDNO), ketone oxidase (KO), ketone dehydrogenase (KDH), 2-6-dihyroxypseudooxynicotine hydrolase (2,6-DHPONH), 2,6- dihydroxypyridine-3-hydroxylase (2,6-DHPH), y-N-methylaminobutyrate oxidase (MABO), monoamine oxidase (MAO), amine oxidase (AO), methylenetetrahydrofolate dehydrogenase / cyclohydrolase (FolD); formyltetrahydrofolate deformylase (Purll), succinic semialdehyde dehydrogenase (SsaDH), or variants thereof.

[0067] In one embodiment, there is provided use of a bacterial extract from Pseudomonas putida S16, Paenarthrobacter nicotinovorans, Paenarthrobacter ureafaciens, or any combination thereof for the degradation of one or more alkaloids on an alkaloid-contaminated object.

[0068] In a further embodiment, the bacterial extract is an enzyme derived from Pseudomonas putida S16, Paenarthrobacter nicotinovorans, Paenarthrobacter ureafaciens, or any combination thereof.

[0069] In yet a further embodiment, the enzyme is one or more of the group comprising or consisting of nicotine oxidoreductase (NicA2), L-6-hydroxynicotine oxidase, berberine bridge enzyme or berberine bridge enzyme-like polypeptides, and variants thereof.

[0070] In an embodiment, the one or more alkaloids are selected from the group consisting of: nicotine, nornicotine, anabasine, anatabine, myosime, cotinine, and pseudooxynicotine. Suitably, any combination of the following alkaloids may be degraded: nicotine, nornicotine, anabasine, anatabine, myosime, cotinine, and pseudooxynicotine.

[0071] In a further embodiment, the one or more alkaloids is nicotine. In other words, at least one alkaloid is nicotine.

[0072] In a further embodiment, the one or more alkaloids is nornicotine. In other words, at least one alkaloid is nornicotine.

[0073] In a further embodiment, the one or more alkaloids is anabasine. In other words, at least one alkaloid is anabasine.

[0074] In a further embodiment, the one or more alkaloids is anatabine. In other words, at least one alkaloid is anatabine.

[0075] In a further embodiment, the one or more alkaloids is myosime. In other words, at least one alkaloid is myosime.

[0076] In a further embodiment, the one or more alkaloids is cotinine. In other words, at least one alkaloid is cotinine.

[0077] In a further embodiment, the one or more alkaloids is pseudooxynicotine. In other words, at least one alkaloid is pseudooxynicotine.

[0078] The alkaloids to be degraded may be natural or synthetic alkaloids. In this context, “natural” alkaloids are those produced by a plants, bacteria, fungi, or animals from which they may have been subsequently extracted. “Synthetic” alkaloids are those that have been manufactured (i.e. man-made by synthesis) and may encompass semi-synthetic alkaloids, which are those that have been manufactured by modifying a natural compound. The natural compound that is modified to become a semi-synthetic alkaloid may be a natural alkaloid, “synthetic” and “semi-synthetic” alkaloids may comprise structural modifications of naturally occurring alkaloids.

[0079] In an embodiment, the one or more alkaloids are natural alkaloids. Suitably, at least one alkaloid may be a natural alkaloid. In an embodiment, the one or more alkaloids are synthetic alkaloids. Suitably, at least one alkaloid may be a synthetic alkaloid.

[0080] In an embodiment, the one or more alkaloids are semi-synthetic alkaloids. Suitably, at least one alkaloid may be a semi-synthetic alkaloid.

[0081] In an embodiment, the one or more alkaloids are natural alkaloids, synthetic alkaloids, semisynthetic alkaloids, or any combination thereof.

[0082] In an embodiment, the alkaloid-contaminated object is selected from the group consisting of: combustible aerosol provision systems, non-combustible aerosol provision systems, aerosol- free delivery systems, and consumable or non-consumable components thereof.

[0083] In a further embodiment, the alkaloid-contaminated object is a combustible aerosol provision system, or consumable or non-consumable component thereof.

[0084] In yet a further embodiment, the combustible aerosol provision system is selected from the group comprising cigarette, cigarillo, and cigar, wherein the tobacco component has been removed or consumed.

[0085] In yet a further embodiment, the consumable or non-consumable component of the combustible aerosol provision system is selected from the group comprising: filter, filter rod, filter segment, a spill, an aerosol-modifying agent release component such as a capsule, a thread, or a bead, or a paper such as a plug wrap, a tipping paper and a cigarette paper.

[0086] In a further embodiment, the alkaloid-contaminated object is a non-combustible aerosol provision system, or consumable or non-consumable component thereof.

[0087] In yet a further embodiment, the non-combustible aerosol provision system is selected from the group comprising an electronic cigarette, also known as a vaping device or electronic nicotine delivery system (END); and an aerosol-generating material heating system, also known as a heat-not-burn system.

[0088] In yet a further embodiment, the consumable or non-consumable component of the non- combustible aerosol provision system is selected from the group comprising: an aerosolgenerating material storage area, such as a vaping cartridge, an aerosol-generating material transfer component, an aerosol generator, an aerosol generation area, a housing, a wrapper, a filter and a mouthpiece.

[0089] In an embodiment, the alkaloid-contaminated object is a vaping cartridge.

[0090] In a further embodiment, the alkaloid-contaminated object is an aerosol-free delivery system, or consumable or non-consumable component thereof.

[0091] In yet a further embodiment, aerosol-free delivery system is selected from the group comprising lozenges, gums, patches and articles comprising inhalable powders.

[0092] In yet a further embodiment, the consumable or non-consumable component of the aerosol- free delivery system is selected from the group comprising: pouches, gums and wrappers.

[0093] Within the context of this disclosure, the alkaloid-contaminated object does not comprise the tobacco which may be present in any of the provision or delivery systems disclosed herein. However, the alkaloid-contaminated object may be any object that has come into contact with tobacco and thus unwanted alkaloids have transferred to the object and thus the object requires decontamination.

[0094] Method

[0095] In one aspect, there is provided a method of degrading one or more alkaloids on an alkaloid- contaminated object, wherein the method comprises: a) inoculating the alkaloid-contaminated object with alkaloid-degrading (e.g. nicotinedegrading) microorganisms, to decontaminate the object.

[0096] In one embodiment, the alkaloid-degrading microorganisms are pre-cultured with at least one alkaloid.

[0097] In one embodiment, the alkaloid-degrading microorganisms are nicotine-degrading microorganisms.

[0098] In one embodiment, the nicotine-degrading microorganisms are pre-cultured with nicotine.

[0099] The microorganisms e.g. bacteria may be pre-cultured with alkaloid (e.g. nicotine) to promote the alkaloid-degrading (e.g. nicotine-degrading) enzymatic pathways in these bacteria. Known pathways in nicotine-degrading bacteria are the pyridine pathway and the pyrrolidine pathway, with a hybrid pathway also described (Huang, et al. (2020). Front Microbiol, 11: 598207).

[0100] In one embodiment, prior to step (a) the alkaloid-contaminated object is suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloid-contaminated object provides the main carbon and nitrogen source when added to the liquid media.

[0101] In a further alternative embodiment, prior to step (a) the alkaloid-contaminated object is suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloids on the alkaloid-contaminated object are degraded.

[0102] In an alternative embodiment, prior to step (a) the alkaloid-contaminated object is suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloids on the alkaloid-contaminated object are preferentially degraded. Suitably, the alkaloids on the alkaloid-contaminated object may be preferentially used as a carbon and / or nitrogen source over any other carbon and / or nitrogen present.

[0103] “Substantially free from” means that the liquid media contains minimal amounts of the excluded matter, such that any source of the excluded matter that is added to the liquid media is preferentially degraded by the bacteria. For example, the liquid media disclosed herein contains minimal amounts of carbon and nitrogen, such that when the alkaloid- contaminated object is added to the liquid media, the bacteria use the alkaloids in the alkaloid-contaminated object as their sole source of carbon and nitrogen.

[0104] In one embodiment, substantially free from may be any concentration less than 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% w / v of carbon and / or nitrogen.

[0105] In one embodiment, substantially free from may be any concentration less than 15% w / v of carbon and / or nitrogen.

[0106] In one embodiment, substantially free from may be any concentration less than 14% w / v of carbon and / or nitrogen.

[0107] In one embodiment, substantially free from may be any concentration less than 13% w / v of carbon and / or nitrogen. In one embodiment, substantially free from may be any concentration less than 12% w / v of carbon and / or nitrogen.

[0108] In one embodiment, substantially free from may be any concentration less than 11% w / v of carbon and / or nitrogen.

[0109] In one embodiment, substantially free from may be any concentration less than 10% w / v of carbon and / or nitrogen.

[0110] In one embodiment, substantially free from may be a concentration of 0% w / v of carbon and / or nitrogen.

[0111] It is important that the liquid media is substantially free from carbon and nitrogen so that the bacteria use the alkaloids present in the alkaloid-contaminated object as their carbon and nitrogen source, thus degrading the alkaloids in the alkaloid-contaminated object. The liquid media may contain carbon or nitrogen from sources other than the alkaloid-contaminated object, such as yeast extract, providing some minimal nitrogen for the bacterial culture to establish before it begins degrading the alkaloids in the alkaloid-contaminated object.

[0112] In a further embodiment, the liquid media is adjusted to between pH 6.0 and pH 8.0.

[0113] In a further embodiment, the liquid media is adjusted to between pH 6.1 and pH 7.9.

[0114] In a further embodiment, the liquid media is adjusted to between pH 6.2 and pH 7.8.

[0115] In a further embodiment, the liquid media is adjusted to between pH 6.3 and pH 7.7.

[0116] In a further embodiment, the liquid media is adjusted to between pH 6.4 and pH 7.6.

[0117] In a further embodiment, the liquid media is adjusted to between pH 6.4 and pH 7.5.

[0118] In a further embodiment, the liquid media is adjusted to between pH 6.5 and pH 7.5.

[0119] In a further embodiment, the liquid media is adjusted to between pH 6.6 and pH 7.4.

[0120] In a further embodiment, the liquid media is adjusted to between pH 6.7 and pH 7.3. In a further embodiment, the liquid media is adjusted to between pH 6.8 and pH 7.2.

[0121] In a further embodiment, the liquid media is adjusted to between pH 6.9 and pH 7.1.

[0122] In a further embodiment, the liquid media is adjusted to pH 7.0.

[0123] In an embodiment, the method further comprises: b) incubating the inoculated alkaloid-contaminated object until it is decontaminated.

[0124] An alkaloid-contaminated object is considered to be decontaminated when the level of alkaloids are below the level required to safely dispose of or recycle the object. The safe level of alkaloids for disposal or recycling may be determined by the law or by-laws which govern the location in which the alkaloid-contaminated object is being processed. Therefore, the safe level of alkaloid may differ from region to region.

[0125] In one embodiment, the one or more alkaloids are reduced to a level suitable for safe disposal.

[0126] “Suitable for safe disposal” is considered to be the level of alkaloids at which the alkaloids will cause minimal harm to the environment or people. This level may not be a complete elimination of the alkaloids and local regulations for what is considered suitable for safe disposal may differ between locations or jurisdictions. The parameters of the method, such as temperature, cell density and incubation time, can be tailored to alter the final concentration of the alkaloids, such that they result in a level which is considered “suitable for safe disposal”.

[0127] In an embodiment, the level of nicotine is reduced to less than or equal to 0.25 % w / w in the alkaloid contaminated object.

[0128] Due to their water solubility, some alkaloids may dissolve into the liquid media. Therefore, it is important that the liquid media is also decontaminated.

[0129] In an embodiment, the level of nicotine is reduced to less than or equal to 0.12 % w / v in the liquid media.

[0130] The skilled person will understand that the percentage reduction of alkaloid required to reach a safe level for disposal or recycling is dependent on both the legally mandated maximum alkaloid level and the starting concentration of the alkaloids on the alkaloid-contaminated object. The skilled person will also note that it may not be required to reduce the level of all alkaloids on an alkaloid-contaminated object to meet the legal requirements for safe disposal or recycling. Therefore, the reduction of one or more of the alkaloids on the alkaloid- contaminated object may meet the legal requirements for safe disposal or recycling.

[0131] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 30 minutes.

[0132] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 40 minutes.

[0133] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 50 minutes.

[0134] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 1 hour.

[0135] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 1 hour 15 minutes.

[0136] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 1 hour 30 minutes.

[0137] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 1 hour 45 minutes.

[0138] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 2 hours.

[0139] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 3 hours.

[0140] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 4 hours. In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 5 hours.

[0141] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 6 hours.

[0142] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 8 hours.

[0143] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 10 hours.

[0144] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 15 hours.

[0145] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 24 hours.

[0146] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 36 hours.

[0147] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 48 hours.

[0148] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 72 hours.

[0149] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 96 hours.

[0150] In one embodiment, the inoculated alkaloid-contaminated object is incubated for a minimum of 120 hours.

[0151] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 30 minutes and about 168 hours. In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 30 minutes and about 120 hours.

[0152] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 30 minutes and about 24 hours.

[0153] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 1 hour and about 168 hours.

[0154] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 1 hour and about 120 hours.

[0155] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 1 hour and about 24 hours.

[0156] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 2 hours and about 168 hours.

[0157] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 2 hours and about 120 hours.

[0158] In one embodiment, the inoculated alkaloid-contaminated object is incubated for between about 2 hours and about 24 hours.

[0159] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 15°C and 45°C.

[0160] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 15°C and 40 °C.

[0161] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 15°C and 39 °C.

[0162] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 15°C and 38 °C. In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 15°C and 37 °C.

[0163] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 16°C and 37 °C.

[0164] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 17°C and 37 °C.

[0165] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 18°C and 37 °C.

[0166] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 19°C and 37 °C.

[0167] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 20°C and 37 °C.

[0168] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 21°C and 37 °C.

[0169] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 22°C and 37 °C.

[0170] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 23°C and 37 °C.

[0171] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 24°C and 36 °C.

[0172] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 25°C and 35 °C.

[0173] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 26°C and 34 °C. In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 27°C and 33 °C.

[0174] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 28°C and 32 °C.

[0175] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at between 29°C and 31 °C.

[0176] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at a minimum of 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20°C, 21 °C, 22 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C.

[0177] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at a maximum of 45 °C , 44 °C , 43 °C , 42 °C , 41 °C, 40 °C, 39 °C, 38 °C, 37 °C, 36 °C, 35 °C, 34 °C, 33 °C, 32 °C, 31 °C, 30 °C, 29 °C, 28 °C, 27 °C, 26 °C, 25 °C, 24 °C, 23 °C, 22 °C, 21 °C, 20 °C, 19 °C, 18 °C, 17 °C, 16 °C or 15 °C.

[0178] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 15 °C,

[0179] 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C,

[0180] 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C or 45 °C.

[0181] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 26 °C.

[0182] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 27 °C.

[0183] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 28 °C.

[0184] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 29 °C.

[0185] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 30 °C.

[0186] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 31 °C.

[0187] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 32 °C. In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 33 °C.

[0188] In a further embodiment, the inoculated alkaloid-contaminated object is incubated at 34 °C.

[0189] In a further embodiment, the inoculated alkaloid-contaminated object is shaken whilst it is incubated.

[0190] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 100 rpm to 450 rpm.

[0191] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 150 rpm to 400 rpm.

[0192] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 150 rpm to 350 rpm.

[0193] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 150 rpm to 300 rpm.

[0194] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 150 rpm to 250 rpm.

[0195] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at between 150 rpm to 200 rpm.

[0196] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at 150 rpm.

[0197] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at 160 rpm.

[0198] In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at 170 rpm. In yet a further embodiment, the inoculated alkaloid-contaminated object is shaken at 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, or 250 rpm.

[0199] In an embodiment, the method further comprises: c) drying the decontaminated object.

[0200] In an embodiment, the method further comprises: d) either

[0201] (i) further processing the decontaminated object for subsequent use; or

[0202] (ii) disposing of the decontaminated object.

[0203] The skilled person will understand that “subsequent use” may include recycling the decontaminated object.

[0204] In an embodiment, the initial nicotine-degrading microorganism cell density in step (a) is between about 2.0 x 106CFU / ml and about 1.0 x 109CFU / ml.

[0205] In an embodiment, the initial nicotine-degrading microorganism cell density in step (a) is between about 2.9 x 106CFU / ml and about 9.4 x 108CFU / ml.

[0206] In an embodiment, the initial nicotine-degrading microorganism cell density in step (a) is a minimum of about 2.9 x 106CFU / ml.

[0207] In a further embodiment, the nicotine-degrading microorganism is Pseudomonas putida S16.

[0208] In an embodiment, the initial nicotine-degrading microorganism cell density in step (a) is a minimum of about 9.3 x 108CFU / ml.

[0209] In a further embodiment, the nicotine-degrading microorganism is Paenarthrobacter nicotinovorans.

[0210] In an embodiment, the initial nicotine-degrading microorganism cell density in step (a) is a minimum of about 8.3 x 107CFU / ml.

[0211] In a further embodiment, the nicotine-degrading microorganism is Paenarthrobacter ureafaciens. In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 2.7 mg / L, wherein the one or more alkaloid is nicotine.

[0212] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 2.5 mg / L, wherein the one or more alkaloid is nicotine.

[0213] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 2.0 mg / L, wherein the one or more alkaloid is nicotine.

[0214] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 1.5 mg / L, wherein the one or more alkaloid is nicotine.

[0215] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 1.0 mg / L, wherein the one or more alkaloid is nicotine.

[0216] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.5 mg / L, wherein the one or more alkaloid is nicotine.

[0217] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 100 pg / L, wherein the one or more alkaloid is nicotine.

[0218] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 50 pg / L / L, wherein the one or more alkaloid is nicotine.

[0219] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 30 pg / L / L, wherein the one or more alkaloid is nicotine.

[0220] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 10 pg / L / L, wherein the one or more alkaloid is nicotine.

[0221] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 3.0 pg / L, wherein the one or more alkaloid is nicotine.

[0222] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 1.0 pg / L, wherein the one or more alkaloid is nicotine. In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.5 pg / L, wherein the one or more alkaloid is nicotine.

[0223] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.4 pg / L, wherein the one or more alkaloid is nicotine.

[0224] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.3 pg / L, wherein the one or more alkaloid is nicotine.

[0225] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.2 pg / L, wherein the one or more alkaloid is nicotine.

[0226] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 0.1 pg / L, wherein the one or more alkaloid is nicotine.

[0227] In an embodiment, the concentration of the one or more alkaloid is reduced to a final concentration equal to or less than 50 ng / L, wherein the one or more alkaloid is nicotine.

[0228] In an embodiment, the concentration of the one or more alkaloid is reduced to an undetectable level.

[0229] The skilled person will understand that the final concentration of the one or more alkaloid may relate to the total alkaloid concentration remaining on the alkaloid-contaminated object, or it may relate to the level of one or more alkaloids remaining on the alkaloid-contaminated object, such as the level of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more alkaloids.

[0230] In an embodiment, the one or more nicotine-degrading microorganisms are one or more nicotine-degrading bacteria.

[0231] In a further embodiment, the nicotine-degrading bacteria degrade nicotine through the pyridine pathway or the pyrrolidine pathway.

[0232] In yet a further embodiment, the nicotine-degrading bacteria is one or more of Pseudomonas putida S16, Paenarthrobacter nicotinovorans, and Paenarthrobacter ureafaciens. In a further embodiment, the nicotine-degrading bacteria degrade nicotine through the pyrrolidine pathway.

[0233] In yet a further embodiment, the nicotine-degrading bacteria is Pseudomonas putida S16.

[0234] In an embodiment, the one or more alkaloids are selected from the group consisting of: nicotine, nornicotine, anabasine, anatabine, myosmine, cotinine, and pseudooxynicotine.

[0235] In a further embodiment, the one or more alkaloids is nicotine. In other words, at least one alkaloid is nicotine.

[0236] In a further embodiment, the one or more alkaloids is nornicotine. In other words, at least one alkaloid is nornicotine.

[0237] In a further embodiment, the one or more alkaloids is anabasine. In other words, at least one alkaloid is anabasine.

[0238] In a further embodiment, the one or more alkaloids is anatabine. In other words, at least one alkaloid is anatabine.

[0239] In a further embodiment, the one or more alkaloids is myosmine. In other words, at least one alkaloid is myosmine.

[0240] In a further embodiment, the one or more alkaloids is cotinine. In other words, at least one alkaloid is cotinine.

[0241] In a further embodiment, the one or more alkaloids is pseudooxynicotine. In other words, at least one alkaloid is pseudooxynicotine.

[0242] The alkaloids to be degraded may be natural or synthetic alkaloids. In this context, “natural” alkaloids are those produced by a plants, bacteria, fungi, or animals from which they may have been subsequently extracted. “Synthetic” alkaloids are those that have been manufactured (i.e. man-made by synthesis) and may encompass semi-synthetic alkaloids, which are those that have been manufactured by modifying a natural compound. The natural compound that is modified to become a semi-synthetic alkaloid may be a natural alkaloid, “synthetic” and “semi-synthetic” alkaloids may comprise structural modifications of naturally occurring alkaloids. In an embodiment, the one or more alkaloids are natural alkaloids. Suitably, at least one alkaloid is a natural alkaloid.

[0243] In an embodiment, the one or more alkaloids are synthetic alkaloids. Suitably, at least one alkaloid is a synthetic alkaloid.

[0244] In an embodiment, the one or more alkaloids are semi-synthetic alkaloids. Suitably, at least one alkaloid is a semi-synthetic alkaloid.

[0245] In an embodiment, the one or more alkaloids are natural alkaloids, synthetic alkaloids, semisynthetic alkaloids, or any combination thereof.

[0246] In an embodiment, the alkaloid-contaminated object is selected from the group consisting of: combustible aerosol provision systems, non-combustible aerosol provision systems, aerosol- free delivery systems, and consumable or non-consumable components thereof.

[0247] In a further embodiment, the alkaloid-contaminated object is a combustible aerosol provision system, or consumable or non-consumable component thereof.

[0248] In yet a further embodiment, the combustible aerosol provision system is selected from the group comprising cigarette, cigarillo and cigar, wherein the tobacco component has been removed or consumed.

[0249] In yet a further embodiment, the consumable or non-consumable component of the combustible aerosol provision system is selected from the group comprising: filter, filter rod, filter segment, a spill, an aerosol-modifying agent release component such as a capsule, a thread, or a bead, or a paper such as a plug wrap, a tipping paper and a cigarette paper.

[0250] In a further embodiment, the alkaloid-contaminated object is a non-combustible aerosol provision system, or consumable or non-consumable component thereof.

[0251] In yet a further embodiment, the non-combustible aerosol provision system is selected from the group comprising an electronic cigarette, also known as a vaping device or electronic nicotine delivery system (END); and an aerosol-generating material heating system, also known as a heat-not-burn system. In yet a further embodiment, the consumable or non-consumable component of the noncombustible aerosol provision system is selected from the group comprising: an aerosolgenerating material storage area, such as a vaping cartridge, an aerosol-generating material transfer component, an aerosol generator, an aerosol generation area, a housing, a wrapper, a filter and a mouthpiece.

[0252] In an embodiment, the alkaloid-contaminated object is a vaping cartridge.

[0253] In a further embodiment, the alkaloid-contaminated object is an aerosol-free delivery system, or consumable or non-consumable component thereof.

[0254] In yet a further embodiment, aerosol-free delivery system is selected from the group comprising lozenges, gums, patches and articles comprising inhalable powders.

[0255] In yet a further embodiment, the consumable or non-consumable component of the aerosol- free delivery system is selected from the group comprising: pouches, gums and wrappers.

[0256] Within the context of this disclosure, the alkaloid-contaminated object does not comprise the tobacco which may be present in any of the provision or delivery systems disclosed herein. However, the alkaloid-contaminated object may be any object that has come into contact with tobacco and thus unwanted alkaloids have transferred to the object and thus the object requires decontamination.

[0257] The various aspects and / or embodiments described herein are presented only to assist in understanding and teaching the claimed features. These aspects and / or embodiments are provided as a representative sample of aspects and / or embodiments only, and are not exhaustive and / or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and / or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

[0258] The invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0259] Example 1 : Degradation of nicotine on waste vaping cartridges

[0260] Pre-culturing of the nicotine-degrading bacteria

[0261] Pre-cultures were grown as the following time schedule:

[0262] Day 0: From glycerol stocks, the different strains were streaked in Inorganic Salt (IS) + 4g / L Nicotine Agar plates. Petri plates were sealed with parafilm and incubated at 30C. After 3 days single colonies should appear in the media.

[0263] Day 3: in the morning, one single colony per strain was then selected and inoculated into 3 ml IS+ Nic 4g / L Nicotine. 4g / L Liguid Media. Inoculums were incubated at 190 rpm shaking and 30C until the afternoon. In the afternoon of the same day the 3 ml of inoculated media in the morning were used to inoculate 22 ml of fresh IS+Nic Media 4g / L Nicotine (total of 25 mL). Inoculums were incubated at 190 rpm shaking and 30C (50 ml centrifuge tube covered by aluminium film) overnight.

[0264] Day 4: in the morning, 25 ml of each strain culture was used to inoculate 100 ml of fresh media IS+Nic 4g / L Nicotine (Total of 125 ml). Inoculums were incubated at 190 rpm shaking and 30C (50 ml centrifuge tube covered by aluminium film) overnight.

[0265] Day5: in the morning, x4 30 ml of 125 ml previous culture was used to inoculate x4 270 ml of fresh media IS+Nic 4g / L Nicotine of every strain culture.

[0266] Day 6: in the morning, all cultures were placed in 50 ml centrifuge tubes and centrifuges at 3750 rpm shaking for 15 minutes at 21 °C. Supernatant was discarded, and cell pellets were resuspended in Na3PO4 0.05M pH 7.0. Cultures were resuspended to a final O.D 600 nm of 1.0. Cultures were then used for inoculation and treatment of vaping consumables.

[0267] O.D 1 eguates to 2.9 x 106CFU / ml for Pseudomonas putida S16. (-culture media

[0268] The media was comprised of the components listed in table 1, excluding nicotine. The final pH of the resultant media was pH 4.2 and was not adjusted. The prepared media was then sterilised by autoclaving (cold media). The nicotine was filtered by syringe filters and the desired amount was measured using a sterile microcentrifuge tube under sterile conditions, by measuring weight on a balance. Following autoclaving of the media, the filtered and weighed nicotine was transferred to the sterile media using a micropipette to take all the liquid. The final concentration of the nicotine in the media was 4 g / L.

[0269] Following addition of the nicotine to the media, the pH was corrected to pH 7.0 using filter sterilised NaOH 1 M and HCI 1 M.

[0270] Control media was prepared in the same way as above, excluding the addition of nicotine.

[0271] Table 1 : Components of the Inorganic Salt Media with Nicotine

[0272] Degradation of nicotine on used vapinq cartridges

[0273] Vapina Cartridges Preparation

[0274] Around 301 ePods vaping cartridges were collected by the Take Back Scheme at British American Tobacco, Southampton site. Several different e-liquid formulas were present in the collected cartridges. Each cartridge contained a small amount of e-liquid.

[0275] -Collected ePoD cartridges: x75 Golden Tobacco 18 mg / ml x77 Peppermint Tobacco 18 mg / ml x138 Creamy Tobacco 18mg / ml x11 Tequila Sunrise 18 mg / ml

[0276] To guarantee less variation of liquid amount between different replicates, cartridges were emptied by centrifugation, collecting all remaining e-liquids as a mix. Cartridges were centrifuged at 37,500 rpm shaking for 15 minutes with their basal part facing down in 50 ml centrifuge tubes, with a total of three cartridges per tube. Small traces of e-liquid were still present inside the cartridge chambers.

[0277] A total of 65.025 ml of e-liquid mix was recovered from 301 ePoDs. An estimated equivalent of 0.2167 ml / ePoD was recovered.

[0278] Vapin consumables treatment:

[0279] All cartridges were mixed in a big polyethylene bag and randomly picked for their treatment with microbial cultures.

[0280] • Bacteria culture (Pseudomonas putida S16 resuspended culture)

[0281] • Mock (NasPC resuspension buffer without bacteria)

[0282] Different concentrations of cartridges were tested for each of the treatments.

[0283] • 12 cartridges in 150 ml resuspended culture

[0284] • 6 cartridges in 150 ml resuspended culture

[0285] • 3 cartridges in 150 ml resuspended culture

[0286] Vaping cartridges were treated by incubation with 150 ml of resuspended bacteria at O.D600 nm 1.0 in 500 ml autoclaving bottles different containers for 48 hours. A total of 0.2167 ml / cartridge was included at the beginning of the incubation along with the cartridges into the liquid resuspended culture used for each treatment. All treatments were incubated for 48h at 30°C and 150 rpm shaking.

[0287] Sampling

[0288] Samples were collected several times during incubation and after incubation to measure nicotine biodegradation in the liquid solution (Oh, 2h, 4h, 6h, 24h and 48h after inoculation). 3 ml of liquid was sampled at each time point and stored at -80°C until it was analysed. Analysis was carried out by filtering with 0.2 um filters and performing HPTLC (High- performance thin-layer chromatography) for the detection of nicotine traces.

[0289] 2 hours after inoculation with the nicotine-degrading bacteria, 96% degradation of the nicotine on the vaping cartridges had occurred.

[0290] Results are shown in Figures 1-3.

[0291] Surprisingly, nicotine biodegradation occurs in a significant way within the first two hours of incubation after inoculation. This means this method could be useful for a quick application and biodegradation of nicotine in vaping consumables leftovers, being a decontamination process prior to the recycling process.

[0292] Pending results

[0293] After 24 hours of incubation, random cartridges will be picked from tanks and once dried, will be washed off and nicotine content residues measured (nicotine content: pg / g). The selected cartridges will be dried at 30°C for 3 hours before analysing. Using 3 ml Extraction Buffer the dried cartridges will be incubated for 30 minutes spinning in a centrifuge tube noire for 30 minutes at room temperature.

[0294] After that period, extraction buffer will be collected and filtered into 96 well plates for its analysis.

[0295] Example 2: Nicotine biodegradation quantification

[0296] This protocol was performed to confirm viability of nicotine biodegradation in deionized water (DI) as a resuspending buffer and quantifying total reduction of nicotine for a total of 48h incubation after inoculation by Liquid Chromatography Mass Spectrophotometry (LCMS).

[0297] Pre-cultures

[0298] Pseudomonas putida S16 strain pre-cultures were grown in the same media and following same timeline as sown in Example 1. However, in this case at Day 6 cell pellets were resuspended in DI Water as resuspension buffer instead of NasPCU 0.05 M pH 7.0. Vaping Cartridges Preparation

[0299] A total of 68 vaping cartridges were collected by Take Back Scheme at BAT Southampton site. In this case all cartridges contained same e-liquid formula; chilled mint 18 mg / ml.

[0300] Each cartridge contained a small amount of e-liquid.

[0301] To guarantee less variation of liquid amount between different replicates, cartridges were emptied by centrifugation, collecting all remaining e-liquids as a mix. In this case, the cartridges were guillotined in half and centrifuged at 37500 rpm shaking for 15 minutes with their middle section facing down in 50 ml centrifuge tubes, with a total of two cartridges per tube.

[0302] Chambers were completely emptied of e-liquid.

[0303] A total of 15.35 ml of e-liquid was collected from guillotined cartridges. Therefore, an estimated equivalent of 0.225 ml / ePoD was recovered.

[0304] Vaping consumables treatment:

[0305] All guillotined cartridges were mixed in a big polyethylene bag and randomly picked for treatment with microbial cultures. Two different treatments were tested:

[0306] • Bacteria culture (Pseudomonas putida S16 resuspended culture)

[0307] • Mock (DI Water resuspension buffer without bacteria)

[0308] Both treatments were tested at a specific concentration:

[0309] • 2 cartridges in 50 ml resuspended culture (equivalent to 6 cartridges in 150 ml)

[0310] Vaping cartridges were treated by incubation with 50 ml of resuspended bacteria at O.D600 nm 1.0 (2.9 x 106CFU / ml of Pseudomonas putida S16) in 150 ml conical flasks or the mock treatment for 48 hours. A total of 0.225 ml per cartridge (x2) was included at the beginning of the incubation along with the cartridges into the liquid resuspended culture used for each treatment. All treatments were incubated for 48h at 30 °C and 150 rpm shaking. Sampling:

[0311] Samples were collected at several time points during incubation and after incubation to measure nicotine biodegradation in the liquid solution (Oh, 2h, 4h, 6h, 24h and 48h after inoculation). 3 ml of liquid was sampled at each time and stored at -80°C until analysis. The samples were analysed by filtering with 0.2 urn filters followed by HPTLC (High-performance thin-layer chromatography) for the detection of nicotine traces.

[0312] Results:

[0313] Massive nicotine reduction of nicotine content within 2 hours

[0314] About 94% reduction of nicotine content compared to the initial content was observed after 2 hours of treatment with resuspended Pseduomonas putida S16 in DI Water. Once this amount was reached nicotine levels did not drop below an average of 10 ug / mL, even after 48 hours. Nicotine average content in the liquid solution was originally at Oh 187 ug / mL. Nicotine content dropped massively down to an average of 11.01 ug / mL in two hours. Minimal nicotine content registered was achieved at 6h at a concentration of 9.34 ug / mL.

[0315] An exponential XY decay was then simulated to understand what level of nicotine should be expected in a period of 2h after inoculation. According to the simulated correlation in about 17 minutes nicotine content is expected to drop by 50% of the initial content and in about 35 minutes nicotine content would be expected to drop by 75% of the initial content.

[0316] Additionally, according to the correlation, nicotine content would drop by 88% of initial nicotine content. Table 2: Nicotine content in liquid incubation solution containing Pseudomonas putida S16 and vaping consumables at several times.

[0317] Results are shown in Figure 4 and Figure 5.

[0318] Example 3: Alternative method

[0319] An alternative approach for the decontamination of alkaloid contaminated objects is to preculture the nicotine-degrading bacteria in Luria-Bertani (LB) media (a general microbial media for growing bacteria) and then freeze dry the pre-cultured bacteria.

[0320] Prior to use, the freeze dried bacteria may be re-suspended in a nicotine containing liquid media. Once the initial nicotine content of the liquid media is low, alkaloid contaminated objects may be added to the nicotine-degrading bacterial culture for decontamination. The conditions of the decontamination step may be the same as in Example 1.

[0321] Example 4: Scale up of the method

[0322] Pre-cultures of the nicotine-degrading bacteria are prepared in advance grown in nicotine-containing liquid cultures. Once the pre-cultures reach a high density (close to the stationary phase of the culture) freeze dried bacteria are prepared from this.

[0323] Freeze dried bacteria is dehydrated bacteria in a powder form. Thus, making transportation and handling much easier.

[0324] This composition of freeze-dried bacteria is then added to a tank or bioreactors containing water and the alkaloid-contaminated objects to be treated and re-suspended to a desired concentration. The inoculated alkaloid-contaminated objects are then allowed to incubate for a specific time. Once the culture has been incubated and stablished, the alkaloid-contaminated objects are removed to continue the recycling process. Once the alkaloid-contaminated objects are removed, more alkaloid-contaminated objects could be added into the tank with bacteria culture on it, resulting in a continuous system.

[0325] An alternative approach for the scale-up of the method is to include inoculums of the nicotine-degrading bacteria into the washing step performed by recycling companies. For example, bacteria re-suspended in water could be used for washing contaminated material during the recycling process, i.e. during wet shredding. Example 5: Degradation of alkaloids by nicotine-degrading bacteria

[0326] The following example demonstrates the ability of three different strains of nicotinedegrading bacteria to degrade a variety of different alkaloids from a tobacco extract.

[0327] Pre-culturing bacteria

[0328] Three different bacteria strains were selected and used in this experiment. These microorganisms were chosen due their ability to degrade nicotine trough different metabolic pathways. Therefore, these bacteria could use nicotine as the sole carbon and nitrogen source.

[0329] • Pseudomonas putida S16 (BAA 2546) (pyrrolidine pathway)

[0330] • Paenarthrobacter nicotinovorans (DSM420) (pyridine pathway)

[0331] • Paenarthrobacte ureafaciens (DSM419) (pyridine pathway)

[0332] From glycerol stocks, the different strains were streaked in Inorganic Salt (IS) + 4g / L Nicotine Agar plates. Petri plates were sealed with parafilm and incubated at 30°C. After 3 days single colonies appeared in the media.

[0333] One single colony per strain was then selected and inoculated into 10 ml IS+ Nicotine 4g / L Liquid Media. Inoculums were incubated at 235 rpm and 30°C (50 ml centrifuge covered by aluminium film) for 24 h.

[0334] Cell growth was confirmed prior to further use of the inoculums. (-culture media

[0335] The media was comprised of the components listed in table 1 (of Example 1), excluding nicotine. The final pH of the resultant media was pH 4.2 and was not adjusted. The prepared media was then sterilised by autoclaving.

[0336] The nicotine was filtered by syringe filters and the desired amount was measured using a sterile microcentrifuge tube under sterile conditions, by measuring weight on a balance. Following autoclaving of the media, the filtered and weighed nicotine was transferred to the sterile media using a micropipette to take all the liquid. The final concentration of the nicotine in the media was 4 g / L.

[0337] Following addition of the nicotine to the media, the pH was corrected to pH 7.0 using filter sterilised NaOH 1 M and HCI 1 M.

[0338] Control media was prepared in the same way as above, excluding the addition of nicotine.

[0339] Degrading alkaloids in tobacco extract

[0340] Tobacco Extract preparation

[0341] Tobacco extracts were made by mixing 5 grams of cured Tobacco, Burley TN90 Tobacco-K Control Upper Leaves with RO water up to a volume of 245ml. Once well mixed, the solution was filtered 2 times with laboratory paper filter to discard suspended particles in the resultant extract. Once filtered, pH was adjusted to 7.0 using NaOH 1 M. After this, the volume was adjusted in a measuring cylinder using RO water up to 250 ml. The final mix was then filter sterilised using vacuum filtering (0.2 uM filter) and stored at 5 °C.

[0342] Inoculation and sampling

[0343] The inoculums were prepared as outlined in above.

[0344] After the inoculums were ready and cell growth was confirmed, 15 ml of Tobacco Extract was inoculated with 1 ml of the initial inoculum. Initial concentrations of cultures were Pseudomonas putida S16 9.77 x 105CFU / ml; Paenarthrobacter nicotinovorans 5.24 x 108; Paenarthrobacter ureafaciens 2.08 x 107.

[0345] Samples were taken for each replicate at 0 h, 24h, 48h and 120h. pH was measured using 0.5 ml of the sample, 0.5 ml was centrifuged and re-suspended in MgCh 10 mM to measure O.D 600nm and 0.5 ml of sample was frozen to -80C for posterior nicotine content analysis by HPLC- LCMS.

[0346] Results are shown in Figures 6-11. Example 6: Assessing Bacteria resilience by Disk Diffusion Susceptibility Tests

[0347] The aim of this experiment was to assess the potential effect of commercial e-liquid dilutions on bacteria survival. Before this experiment was performed it was not known whether bacteria would survive in e-liquids and biodegrade alkaloids such as nicotine through metabolic processes.

[0348] Dilutions of different e-liquids (with different flavour blocks and nicotine content) were prepared. Dilutions were pipetted into test nylon disks used for Inhibition Zone Tests. As a positive control, diluted hydrogen peroxide was used.

[0349] The Inhibition Zone Area (mm) was measured.

[0350] Material and Methods

[0351] Dilutions of different e-liquids commercial formulas were prepared in deionized water diluting in the following ratios: 1 :1 , 1 :5, 1 :25, 1 :50, 1 :100. Different e-liquids dilutions were made for Golden Tobacco, Mint, Tobacco Marvel, Menthol and Blueberry Ice e-liquid formulas. The pH of the e-liquid dilutions was not adjusted. In parallel a dilution of H2O2 at 5% in deionized waster was prepared to be used as positive control.

[0352] The 1 :100 dilutions were analysed by Liquid Chromatography Mass Spectrophotometry (LCMS) to quantify the nicotine content of the different e-liquid formulas along with other additional formulas available for the test (3 replicates per e-liquid).

[0353] Pre-culturing media inorganic salt media was prepared in advance dissolving 0.025 g of FeSO4, 2g of KH2PO4, 5 of KCI, MgSO4 0.25 g and 0.1 g of yeast extract into 1 litre of deionized water. After being autoclaved, 4 g of nicotine was added per litre, adjusting pH of the solution to 7.0 using HCI 1 M and NaOH 1M. Agar media was prepared by adding 15 g of selected agar per litre. Once autoclaved agar media was heated in a microwave to a liquid form. When the temperature was about 50°C, 4 grams of nicotine per litre were added. Media was poured before solidifying into 90mm petri plates.

[0354] Pre-cultures were prepared in advance growing three different microbial strains separately: Pseudomonas putida S16, Paenarthrobacter nicotinovorans and Paenarthrobacter ureafaciens. On day one, glycerol stocks were used for inoculating agar inorganic salt (Nicotine 4 mg / m I) (pH 7.0). Plates were incubated for 56h at 30°C in the dark. After this, single colonies were picked and used to inoculate 3 ml of liquid inorganic salt (nicotine 4 mg / ml) (pH 7.0) for each of the strains and incubated overnight 30°C and shaking at 200 rpm. The next day, liquid cultures were scaled up by inoculating these 3 ml into a falcon tube with 22ml of liquid inorganic salt (nicotine 4 mg / ml) for incubation overnight until the next day at 30°C and shaking at 200 rpm. After this, the total of 25 ml of every culture was centrifuged in falcon tubes at 3,750 rpm at room temperature for 15 minutes. Supernatant was discarded and cell pellet was washed by pouring 30 ml of x1 PBS (phosphate buffered saline) in each falcon and centrifuged again at 3,750 rpm at room temperature for 15 minutes. After this, the supernatant was discarded, and cell pellet was resuspended for each of the cultures in PBS to 1 .0 OD600 nm. A volume of 50 pL of the adjusted resuspended bacteria was poured into 90 mm plates and extended using sterile loops to prepare a consistent bacterial lawn and let to be absorbed for 5 min.

[0355] After the initial inoculum was absorbed by the agar, six disks of 6 mm were place into agar inorganic salt (nicotine 4 mg / ml) (pH 7.0) media within an equal distance between each other. A volume of 10 pL of every e-liquid dilution was placed into each disk for each of the e-liquids.

[0356] Once inoculated and samples were loaded into the tests disks plates, plates were incubated for 48h upside down. After that time, the inhibition zones for each of the treatments were measured using Image J Software analysis.

[0357] Results

[0358] The tested e-liquids contained the same amount of nicotine as expected (labelled commercial e-liquid formulas) as shown in Figure 12:

[0359] Golden Tobacco e-liquid contained an average of 16.87 mg / ml;

[0360] Mint e-liquid contained an average of 16.52 mg / ml;

[0361] Tobacco Marvel e-liquid contained an average of 29.34 mg / ml; Menthol e-liquid contained an average of 18.17 mg / ml;

[0362] Blueberry Ice e-liquid contained an average of 32.23 mg / ml.

[0363] The different dilutions of Golden Tobacco e-liquid (16.87 mg / ml nicotine) had no significant effect on any of the three tested strains compared to the positive control, assuming a minimum of 6 mm (disk length) for each of the treatments. No differences were found between the three strains when exposed to the same dilutions.

[0364] In a similar manner, the different dilutions of Mint e-liquid (16.52 mg / ml nicotine) had no significant effect on any of the three tested strains compared to the positive control. Again, no differences between the three strains when exposed to the same dilutions were observed.

[0365] The different dilutions Tobacco Marvel (29.34 mg / ml nicotine) had no significant effect on any of the three tested strains compared to the positive control. Additionally, no differences were found between the three strains when exposed to the same dilutions. There were no significant effects of different dilutions of Blueberry Ice e-liquid (32.23 mg / ml nicotine) on any of the three tested strains, with no major difference between these strains.

[0366] The dilutions of Menthol (18.17 mg / ml nicotine) had no significant effect on any of the three tested strains compared to the positive control.

[0367] Based on the results, we conclude the three bacterial strains are minimally affected by different dilutions of the tested commercial e-liquids.

[0368] Example 7: Measuring nicotine biodegradation in E-liquids

[0369] Dilutions of different commercial e-liquids (with different flavour blocks and nicotine content) were prepared. Dilutions were inoculated in micro well plates with an initial inoculum for their incubation and monitoring over 27 hours.

[0370] Cell Growth (OD600nm) and Nicotine content (mg / ml) were measured.

[0371] Material and Methods

[0372] Dilutions of different e-liquids commercial formulas were prepared in inorganic salt media with no nicotine, diluting e-liquids in two ratios: 1 :8, 1 :16. The following e-liquids were diluted: Golden Tobacco e-liquid contained an average of 16.87 mg / ml; Mint e-liquid contained an average of 16.52 mg / ml; Tobacco Marvel e-liquid contained an average of 29.34 mg / ml. The pH of the dilutions was adjusted to pH 7.0 prior to inoculation using 1M HCL and 1 M NaOH. In parallel a dilution of H2O2 at 5% in Deionized water and Luria Broth liquid media (pH 7.0) were prepared to be used as positive controls.

[0373] Pre-cultures were prepared in advance growing three different microbial strains separately: Pseudomonas putida S16, Paenarthrobacter nicotinovorans and Paenarthrobacter ureafaciens. On day one, glycerol stocks were used for inoculating Luria Broth Agar plates (pH 7.0). Plates were incubated for 56h at 30°C in the dark. After this, single colonies were picked and used to inoculate 3 ml of liquid Luria Broth (pH 7.0) for each of the strains and incubated overnight 30°C and shaking at 200 rpm. Next day, liquid cultures were scaled up by inoculating these 3 ml into a falcon tube with 22ml of liquid Luria Broth for its incubation overnight until the next day at 30°C and shaking at 200 rpm. After this, the total of 25 ml of every culture was centrifuged in falcon tubes at 3,750 rpm at room temperature for 15 minutes. Supernatant was discarded and cell pellet was washed by pouring 30 ml of PBS in each falcon and centrifuged again at 3,750 rpm at room temperature for 15 minutes. After this, the supernatant was discarded, and cell pellet was resuspended for each of the cultures in PBS to 1 .0 ODeoo nm. A volume of 25 pL of each of the resuspended cultures were inoculated into 225 L of each of the diluted e-liquids for three replicates per treatment and dilution (n=3) in a sterile 96 well plate (300 pL wells). Mock was included and inoculated with plain PBS as reference to be used as a blank for absorbance at ODeoo nm.

[0374] Samples in a 96 well plate were incubated in a FLUOstar BMG LABTECH for 27h at 30°C in dark. During the incubation time, the absorbance at 600 nm of every sample was measured every 5 minutes. Liquid samples were taken for each replicate at 0 h, 4h, 6 h and 24h. Samples were frozen at -80°C for posterior nicotine content analysis by LCMS (3 replicates per strain).

[0375] Results

[0376] Figure 13 show 99% biodegradation of nicotine at 24h for both dilutions, starting to drop at 6h for Dilution 1 :16. Results show no nicotine biodegradation for P. nicotinovorans compared to the mock treatment. These results show a contrast when compared to cell growth, potentially indicating that the strain is growing and metabolising other compounds in the e-liquid mix.

[0377] Results show 99% biodegradation of nicotine at 24h for Mint Dilution 1 :16 when treated with P. putida S16. However, Mint Dilution 1 :8 has a significant lower drop in nicotine content when treated with the same strain. As previously described in Figure 11 , the other two strains are not biodegrading nicotine below significant levels compared to the mock treatment.

[0378] Results show 99% nicotine biodegradation in Tobacco Marvel Dilution 1 :16 when treated with P. putida S16.

[0379] Notably, a slight increase in nicotine content is observed in the “mock” over time, it is believed this is due to evaporation.

[0380] Example 8: Nicotine exposure biodegradation

[0381] Results from previous experiments show nicotine biodegradation from non-exposed to nicotine strains. We next assessed the potential effect of nicotine exposure to three tested nicotine to understand the degradation speed at early times in the biodegradation process to determine if nicotine presence is needed for the nicotine biodegradation at early times.

[0382] Nicotine-exposed and non-nicotine exposed pre-cultures were produced and we assessed their efficiency to degrade nicotine in Luria Broth containing nicotine 4 mg / ml, taking sample at early times from 0 h to 4 h.

[0383] The nicotine content mg / ml, ODeoonmwas measured. Material and Methods:

[0384] Pre-cultures were prepared in advance growing three different microbial strains separately: Pseudomonas putida S16, Paenarthrobacter nicotinovorans and Paenarthrobacter ureafaciens. On day one, glycerol stocks were used for inoculating Luria Broth Agar with 4 mg / ml nicotine (LB+) and without nicotine plates (LB-) (pH 7.0). Plates were incubated for 56h at 30°C in dark. After this, single colonies were picked and used to inoculate 3 ml of liquid Luria Broth (pH 7.0) LB + and LB - and incubated overnight 30°C and shaking at 200 rpm. Next day, liquid cultures would be scaled up by inoculating these 3 ml into a falcon tube with 22ml of liquid Luria Broth LB+ and LB - for its incubation overnight until the next day at 30°C and shaking at 200 rpm. After this, the total of 25 ml of every culture was centrifuged in falcon tubes at 3,750 rpm at room temperature for 15 minutes. Supernatant was discarded and cell pellet was washed by pouring 30 ml of x1 PBS (Phosphate Buffer) in each falcon and centrifuged again at 3,750 rpm at room temperature for 15 minutes. After this, the supernatant was discarded, and cell pellet was resuspended for each of the cultures in PBS to 1.0 ODeoo nm.

[0385] A volume of 25 pL of each of the resuspended cultures were inoculated into 225 pL of LB (4 mg / ml)+ (Nicotine 4three replicates per treatment and dilution (n=3) in a sterile 96 well plate (300 pL wells). 225 pL of 5% H2O2 was used as positive control, inoculated with the same volume of bacteria as other treatments. Mock was included and inoculated with plain PBS as reference to be used as a blank for absorbance at ODeoo nm.

[0386] Samples in a 96 well plate were incubated in a FLUOstar BMG LABTECH for 27h at 30°C in dark. During the incubation time, the absorbance at 600 nm of every sample was measured every 5 minutes. Liquid samples were taken for each replicate at 0 h, 1h, 2 h and 4h. Samples were frozen at -80°C for posterior nicotine content analysis by Liquid Chromatography Mass Spectrophotometer (3 replicates per strain).

[0387] Results - shown in Figures 14-19.

[0388] For P. putida S16, bacterial cell growth for this strain is similar in both liquid medias, Luria Broth - and Luria Broth containing nicotine 4 mg / ml reaching about ODeoonm 1.0. Surprisingly, nicotine drops within 2 and 4h for LB + precultured strains. This means that for quick treatments it would be advisory to pre-culture this strain in nicotine containing media. However, we have probed in previous experiments that nicotine degradation occurs despite of the absence of nicotine in the pre-culturing media.

[0389] Similarly, we see similar cell growth for P. nicotinovorans. However, cell growth seems to be higher at the experiment (24h) in Luria Broth containing nicotine 4 mg / ml reaching about ODeoonm 1.0 and about 0.6 mg / ml. However, no significant decrease in the nicotine content occurs within 4 h for any of the treatments, indicating that this strain would be better for longer incubation times, as it has been proven do biodegrade nicotine and other minor alkaloids efficiently in previous experiments.

[0390] By last, show less cell growth for all liquids medias tested, being a significant difference between liquid media LB+ and LB - and being higher in this last liquid media at 24h. Additionally, no nicotine decrease within 4 h was observed for any of the treatments, indicating that this strain would be better for longer incubation times. Moreover, results from previous experiments show this strain in is less efficient that P. nicotinovorans when degrading alkaloids, being advisory to prioritise the use of P. nicotinovorans in first place, which also shows a higher cell growth in the tested medias.

[0391] The invention is further described by the following numbered paragraphs:

[0392] 1. Use of one or more nicotine-degrading microorganisms or an extract thereof for the degradation of one or more alkaloids on an alkaloid-contaminated object.

[0393] 2. A method of degrading one or more alkaloids on an alkaloid-contaminated object, wherein the method comprises: a) inoculating the alkaloid-contaminated object with nicotine-degrading microorganisms, to decontaminate the object.

[0394] 3. The method of paragraph 2, wherein the nicotine-degrading microorganisms are precultured with nicotine.

[0395] 4. The method of paragraph 2 or paragraph 3, wherein prior to step (a) the alkaloid- contaminated object is suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloid-contaminated object provides the main carbon and nitrogen source when added to the liquid media. 5. The method of any one of paragraphs 2-4, wherein the method further comprises: b) incubating the inoculated alkaloid-contaminated object until it is decontaminated.

[0396] 6. The method according to paragraph 5, wherein the inoculated alkaloid-contaminated object is incubated for a minimum of 30 minutes.

[0397] 7. The method according to paragraph 5 or paragraph 6, wherein inoculated alkaloid- contaminated object is incubated for between about 30 minutes and about 120 hours.

[0398] 8. The method of any one of paragraphs 2-7, wherein the method further comprises: c) drying the decontaminated object.

[0399] 9. The method of any one of paragraphs 2-8, wherein the method further comprises: d) either

[0400] (i) further processing the decontaminated object for subsequent use; or

[0401] (ii) disposing of the decontaminated object.

[0402] 10. The method according to any one of paragraphs 2-9, wherein the initial nicotinedegrading microorganism cell density in step (a) is between about 2.0 x 106CFU / ml and about 1.0 x 109CFU / ml.

[0403] 11. The method according to any one of paragraphs 2-10, wherein the concentration of the one or more alkaloid is reduced to a final concentration of equal to or less than 2.7 mg / L, wherein the one or more alkaloid is nicotine.

[0404] 12. The use of claim 1 or the method of any one of paragraphs 2-11 , wherein the one or more nicotine-degrading microorganisms are one or more nicotine-degrading bacteria.

[0405] 13. The use or method of paragraph 12, wherein the nicotine-degrading bacteria degrade nicotine through the pyrrolidine pathway.

[0406] 14. The use or method of paragraph 12 or paragraph 13, wherein at least one of the nicotine-degrading bacteria is Pseudomonas putida S16.

[0407] 15. The use of any one of paragraphs 1 or 12-14 or the method of any one of claims 2- 14, wherein the one or more alkaloids are selected from the group consisting of: nicotine, nornicotine, anabasine, anatabine, myosmine, cotinine and pseudooxynicotine. 16. The use or method of paragraph 15, wherein the one or more alkaloids is nicotine.

[0408] 17. The use of any one of paragraphs 1 or 12-16 or the method of any one of claims 2- 16, wherein the alkaloid-contaminated object is selected from the group consisting of: combustible aerosol provision systems, non-combustible aerosol provision systems, aerosol- free delivery systems, and consumable or non-consumable components thereof.

[0409] 18. The use or method of paragraph 17, wherein the alkaloid-contaminated object is a consumable or non-consumable component of a non-combustible aerosol provision system, wherein the component is a vaping cartridge.

Claims

CLAIMS1. Use of one or more nicotine-degrading microorganisms or an extract thereof for the degradation of one or more alkaloids on an alkaloid-contaminated object, wherein at least one of the nicotine-degrading microorganisms is Pseudomonas putida S16.

2. A method of degrading one or more alkaloids on an alkaloid-contaminated object, wherein the method comprises: a) inoculating the alkaloid-contaminated object with nicotine-degrading microorganisms, to decontaminate the object wherein at least one of the nicotinedegrading microorganisms is Pseudomonas putida S16.

3. The method of claim 2, wherein the nicotine-degrading microorganisms are precultured with nicotine.

4. The method of claim 2 or claim 3, wherein prior to step (a) the alkaloid-contaminated object is suspended in a liquid media substantially free from carbon and nitrogen, such that the alkaloid-contaminated object provides the main carbon and nitrogen source when added to the liquid media.

5. The method of any one of claims 2-4, wherein the method further comprises: b) incubating the inoculated alkaloid-contaminated object until it is decontaminated.

6. The method according to claim 5, wherein the inoculated alkaloid-contaminated object is incubated for a minimum of 30 minutes.

7. The method according to claim 5 or claim 6, wherein inoculated alkaloid- contaminated object is incubated for between about 30 minutes and about 120 hours.

8. The method of any one of claims 2-7, wherein the method further comprises: c) drying the decontaminated object.

9. The method of any one of claims 2-8, wherein the method further comprises: d) either(i) further processing the decontaminated object for subsequent use; or(ii) disposing of the decontaminated object.

10. The method according to any one of claims 2-9, wherein the initial nicotine-degrading microorganism cell density in step (a) is between about 2.0 x 106CFU / ml and about 1.0 x 109CFU / ml.11 . The method according to any one of claims 2-10, wherein the concentration of the one or more alkaloid is reduced to a final concentration of equal to or less than 2.7 mg / L, wherein the one or more alkaloid is nicotine.

12. The use of claim 1 or method of any one of claims 2-11 , wherein the nicotinedegrading bacteria degrade nicotine through the pyrrolidine pathway.

13. The use of any one of claim 1 or claim 12 or the method of any one of claims 2-12, wherein the one or more alkaloids are selected from the group consisting of: nicotine, nornicotine, anabasine, anatabine, myosmine, cotinine and pseudooxynicotine.

14. The use or method of claim 13, wherein the one or more alkaloids comprises nicotine.

15. The use of any one of claims 1 or 12-14 or the method of any one of claims 2-14, wherein the alkaloid-contaminated object is selected from the group consisting of: combustible aerosol provision systems, non-combustible aerosol provision systems, aerosol- free delivery systems, and consumable or non-consumable components thereof.

16. The use or method of claim 15, wherein the alkaloid-contaminated object is a consumable or non-consumable component of a non-combustible aerosol provision system, wherein the component is a vaping cartridge.

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