Bacterial process for preparation of microbial storage lipids

Abstract

The present invention relates to a bacterial process for the production of microbial storage lipids, such as wax esters, triacylclycerides, oils and polyhydroxyalkanoates, from readily available feedstocks derived from biomass. The feedstocks are rich in volatile fatty acids and ammonium, and are generated from biomass via the process of acidogenic fermentation. This allows for the preparation of microbial storage lipids from cheap and readily available biomass sources, such as plant or agricultural waste, or sewage. In particular, the present invention is directed to a process in which the carbon-to-nitrogen ratio of the composition containing volatile fatty acids and ammonium can be effectively controlled so as to optimise the yield of microbial storage lipid production from this process. The present invention also relates to microbial storage lipids that are obtainable by this process, and an apparatus for carrying out the process.

Description

[0001] BACTERIAL PROCESS FOR PREPARATION OF MICROBIAL STORAGE LIPIDS

[0002] Related application

[0003] The present application claims priority from United Kingdom Patent Application No. GB 2405054.4, filed 9 April 2024, the entire contents of which are hereby incorporated by reference.

[0004] Field of the invention

[0005] The present invention relates to a bacterial process for the production of microbial storage lipids, such as wax esters, triacylclycerides, oils and polyhydroxyalkanoates, from readily available feedstocks derived from biomass. The feedstocks are generally rich in volatile fatty acids and ammonium, and can be generated from biomass via the process of acidogenic fermentation. This allows for the preparation of microbial storage lipids from cheap and readily available biomass sources, such as plant or agricultural waste, or sewage. In particular, the present invention is directed to a process in which the carbon-to-nitrogen ratio of the composition containing volatile fatty acids and ammonium can be effectively controlled so as to optimise the yield of microbial storage lipid production by the bacteria. In particular, the present invention is directed to processes in which the carbon-to-nitrogen ratio of the composition containing volatile fatty acids and ammonium can be effectively controlled using aluminosilicates (e.g. zeolites). The present invention also relates to microbial storage lipids that are obtainable by this process, and an apparatus for carrying out the process.

[0006] Background of the invention

[0007] Microbial storage lipids (MSLs) are a broad class of energy storage molecules accumulated by microbes when the limitation of essential nutrients, especially nitrogen, precludes the growth of biomass. MSLs may take various forms, including wax esters and triacylglycerides, oils and polyhydroxyalkanoates. They are of great economic relevance due to their similarity to many products conventionally derived from petrochemicals, for which they may provide renewable alternatives (Martin et al., Bioresour. TechnoL Rep. 2023, 22; Sabapathy et al., Bioresour. TechnoL, 2020, 306). The substitution of conventional petrochemicals and petrochemical-derived products with MSL alternatives offers myriad benefits. MSLs can be produced through microbial synthesis from biological carbon sources, meaning they have the potential to be carbon neutral. They are also typically biodegradable, such that they do not accumulate in the environment as do their conventional fossil-derived alternatives.

[0008] One particularly industrially-important class of MSLs are the polyhydroxyalkanoates (PHAs): polyesters of hydroxyalkanoic acid monomers which are accumulated by various microbial species as hydrophobic, intracellular granules (Alves et al., Ini. J. Environ. Sci. TechnoL 2023, 20). These are of interest for their similarity to many of the most widely used plastics, including low-density polyethylene, polypropylene (Dartiailh et al., Front. Bioeng. BiotechnoL, 2021, 8), and polystyrene. PHA polymers may be constituted from a range of around 150 monomers, yielding plastics with various and customizable chemical, thermal, and mechanical properties. The specific PHA composition will depend on the microbial species in question, the carbon sources available, and on the pathways through which these are metabolized (Dartiailh et al., Front. Bioeng. BiotechnoL, 2021, 8). Much work to date has focused on polyhydroxy butyrate (PHB), a short-chain PHA polymer consisting of four-carbon (3 -hydroxybutyric acid) monomers, and to its accumulation by Cupriavidus necator, a metabolically flexible bacterium capable of accumulating up to 80% of its cell weight as PHB (Zhang et al., Front. Bioeng. BiotechnoL, 2022, 10). PHB is highly crystalline and brittle, however, rendering it poorly suited for many industrial applications. The medium chain PHAs produced by species such as Pseudomonas putida, which occur mainly as copolymers of 6-14 carbon monomers, provide plastics far more amenable to industrial use (Dartiailh et al., Front. Bioeng. BiotechnoL, 2021, 8).

[0009] Despite the many benefits offered by PHAs over their conventional plastic counterparts, their deployment is hampered by their high cost. Substrates constitute a large fraction of this cost (Choi & Lee, Bioprocess Eng., 1997, 17), and considerable effort has gone into identifying cheaper carbon sources, such as from the waste streams and byproducts of existing industrial processes (Dalsasso et al., Process Biochem., 2019, 85; Passanha et al., Bioresour. TechnoL, 2013, 147; Penkhrue et al., PloS one, 2020, 15(3)). Among these alternative feedstocks, volatile fatty acids (VFAs) stand out for their extremely low cost, and P. putida has demonstrated an ability to accumulate PHA using VFA as a substrate (Dartiailh et al., Front. Bioeng. BiotechnoL, 2021, 8), as have other species (Jia et al., Bioresour. TechnoL, 2013, 140; Passanha et al., Bioresour. TechnoL, 2013, 147).

[0010] The attractiveness of VFAs is due to their ability to be derived from essentially any biomass source, including dedicated agricultural crops, food and agricultural waste, meat industry waste or sewage sludge, through the process of acidogenic fermentation. Acidogenic fermentation is the anaerobic decomposition of biomass to produce fatty acids. However, this process also degrades proteins present in the biomass, which releases the nitrogen therein as ammonium. If a high amount of nitrogen is present (i.e. there is a low carbon to nitrogen (C / N) ratio), it has been found that the VFA-processing microbes will prioritize growth over (e.g.) PHA production, and hence PHA yields are low.

[0011] Some previous studies have demonstrated that increased PHA production can be achieved if the C / N ratio of the biomass feedstock is high. Example studies include PHB production in a mixed microbial consortium grown on acetate (Zhou et al., Set. Total Environ., 2022, 811), PHB production in Bacillus megaterium grown on sucrose (Faccin et al., J. Chem. TechnoL Biotechnol., 2009, 84(12)), and production of PHB-co-PHV (a PHA copolymer) by Nostoc muscorum on a mixture of acetate, valerate and glucose (Angra et al., Microbial ecology, 2023, 85 (2)). However, in these studies, control of C / N ratios was achieved using independent carbon and nitrogen sources whose precise dosing allowed for control of this parameter. Thus these techniques are incompatible with production of PHAs from naturally occurring biomass sources containing VFAs. This use of pure compounds increases cost and is a major obstacle to the commercialization of PHAs. Bulk prices for acetate for example, the cheapest of the VFAs, stand around US $400-800 / tonne, and are over US $2000 / tonne for the longer C4 and C5 fatty acids.

[0012] Therefore, there is a need for a method for PHA production which is suitable for use with a wide range of biomass carbon sources, irrespective of their purity and chemical heterogeneousness, that are cheap and readily available feedstocks. Such a method also needs to be suitable for production of PHAs on a large scale.

[0013] Summary of the invention

[0014] Aluminosilicates (e.g. zeolites) are a broad class of microporous minerals containing aluminium, silicon and oxygen (i.e. oxides of both aluminium and silicon) with an inherent ability to participate in ion-exchange reactions. While the ions in question are usually metal cations, aluminosilicates may also reversibly bind ammonium (NH ). The present inventors have surprisingly found that through cation exchange, aluminosilicates may be used to deplete ammonium in acidogenic fermentate and increase the C / N ratio in a highly controlled fashion. This level of control allows for optimisation of the C / N ratio of the VFA-containing feedstock such that there is sufficient nitrogen to grow and maintain a sufficiently effective culture of bacteria whilst also prioritise MSL production over bacterial growth. The optimisation of the MSL production process in this manner allows for significantly improved yields of MSL production, with surprisingly up to c. 300% increase in MSL production observed compared to the absence of treatment of the VFA-containing composition with aluminosilicate.

[0015] Thus, the present inventors have found that acidogenic fermentation augmented with aluminosilicate treatment of the resulting VFA-rich composition unexpectedly allows combination of the cost benefits of heterogeneous feedstocks with the substrate homogeneity and exquisite control of reactor parameters afforded by pure- substrate systems. Acidogenic fermentation allows extraction of VFA from both the soluble and insoluble fractions of the biomass. It homogenizes all available carbon sources to a pool of VFA which is widely utilizable by many MSL-accumulating species. Moreover, the heterogeneous nitrogen sources in biomass, which may take the form of proteins, free amino acids, urea, nitrates, or other chemicals, are hydrolysed to ammonium ions.

[0016] Therefore, the presently described aluminosilicate process represents a low-cost solution to allow for direct bioproduction of MSLs in acidogenic fermentates. Given the wide range of carbon sources available for acidogenic fermentation, and the hundreds of known species able to accumulate MSLs on fatty acids, this process has wide-ranging implications for the deployment of cheap and renewable MSL alternatives to conventional industrial and commercial products.

[0017] In an embodiment, the present invention therefore provides a method for preparing one or more microbial storage lipids from biomass, said method comprising:

[0018] (a) subjecting the biomass to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium-containing compounds;

[0019] (b) treating said composition with an aluminosilicate such that the carbon-to- nitrogen ratio of the treated composition is 30: 1 or greater; and

[0020] (c) incubating the treated composition with microorganisms to produce one or more microbial storage lipids. In another embodiment, the present invention provides a method of producing one or more microbial storage lipids from a composition comprising volatile fatty acids and ammonium-containing compounds and optionally further comprising biomass, wherein the composition has a carbon-to-nitrogen ratio of 30: 1 or greater; the method comprising incubating the composition with microorganisms thereby producing one or more microbial storage lipids.

[0021] In another embodiment, the present invention provides a microbial storage lipid obtainable by the process of the invention.

[0022] Also provided is the use of an aluminosilicate to reduce the nitrogen content of a composition comprising volatile fatty acids and ammonium-containing compounds and optionally further comprising biomass, thereby forming a treated composition having a carbon-to-nitrogen ratio of 30: 1 or greater.

[0023] The invention also provides a system for carrying out such methods. Thus, in an embodiment, the present invention provides an apparatus which comprises:

[0024] (i) a composition comprising volatile fatty acids and ammonium-containing compounds, wherein the carbon-to-nitrogen ratio of the composition is 30:1 or greater;

[0025] (ii) an aluminosilicate; and

[0026] (iii) microorganisms.

[0027] Brief description of the Figures

[0028] Figure 1 summarises the three-step process of the present invention.

[0029] Figure 2 shows analysis of carbon and nitrogen composition of untreated and treated Opuntia fermentates with zeolite. A: Fatty acid composition of total VFA; from top centre proceeding clockwise: C2 = Acetate, C3 = Propionate, C4 = butyrate + Isobutyrate +, C5 = valerate + isovalerate, C6 = hexanoate. B: Total organic carbon (TOC) and Total organic acids (TOA). C: Total nitrogen (TN) and Total ammonium nitrogen (TAN). D: Carbon to nitrogen ratio (mass basis).

[0030] Figure 3 shows growth characteristics (OD600) of P. putida grown on acidogenic fermentates of varying C / N ratio. A-F correspond to C / N ratios of 10: 1, 20: 1, 40: 1, 60: 1, 80: 1, and 100: 1, respectively. Figure 4 shows 3 -hydroxydecanoic acid production over a parameter space of zeolite treatment vs. time. A: Titres of 3 -hydroxy decanoic acid (grams per litre of culture medium). B: Yields of 3 -hydroxy decanoic acid (% of cell dry weight).

[0031] Figure 5 shows a statistical comparison of 3HD titre and yield maxima across timepoints. Panels A and B show maximum titres and yields respectively. Comparisons of statistical significance of deviation of treatment groups from the untreated control performed using Dunnett’s Multiple Comparisons Test. Non-significant comparisons have been omitted.

[0032] Figure 6 shows the fate of input volatile solids (VS) through sequential acidogenic fermentation and P. putida bioproduction steps. Input volatile solids from Opuntia and anaerobic digestion (AD) sludge are fermented to yield 38.03% of their mass as volatile fatty acids. These fatty acids are fed to P. putida to yield 20% of their mass (7.7 % of input VS) as P. putida biomass. Of this P. putida biomass, 26.6% (or 2.05% of input VS) are recoverable as PHA bioplastic. All values quoted in percentages of initial VS (mass basis).

[0033] Figure 7 shows the rate of removal of ammonium from a medium by aluminosilicates (lines from top to bottom: 0% (w / v) zeolite; 6.25% (w / v) zeolite; 12.5% (w / v) zeolite; 25% (w / v) zeolite; 50% (w / v) zeolite; 75% (w / v) zeolite; 1000% (w / v) zeolite).

[0034] Figure 8 shows the relative decrease in VFA concentration and ammonium concentration during aluminosilicate treatment over time, and demonstrates that zeolites are selective for ammonium removal over VFA removal from fermentates.

[0035] Figure 9 shows the cumulative effect of the observations of Figures 7 and 8, which is an increase in C / N ratio over time (lines from top to bottom: 50% (w / v) zeolite; 25% (w / v) zeolite; 0% (w / v) zeolite).

[0036] Figure 10(a) is a process flow diagram which summarises the process of acidogenic fermentation. Figure 10(b) is a process diagram which summarises the example method of production of microbial lipids from acidogenic fermentate.

[0037] Figure 11 shows analysis of carbon and nitrogen composition of untreated and treated Opuntia fermentates. A: Fatty acid composition of total VFA C2 = Acetate, C3 = Propionate, C4 = Butyrate + Isobutyrate +, C5 = Valerate + Isovalerate, C6 = Hexanoate, B: Total organic carbon (TOC) and Total organic acids (TOA), C: Total nitrogen (TN) and Total ammonium nitrogen (TAN), D: Carbon to nitrogen ratio (mass basis). Figure 12 shows Growth characteristics (ODeoo) of P. putida grown on acidogenic fermentates of varying C:N ratio. A, B, C, D, E, and F, correspond to ratios of 12.5, 13.5, 17.5, 26, 43 and 93.5, respectively.

[0038] Figure 13 shows PHA production over a parameter space of zeolite treatment vs. time. A: Titres of PHA (milligrams per liter of culture medium), B: Yields of PHA (% of cell dry weight).

[0039] Figure 14 shows statistical comparison of PHA titre and yield maxima across timepoints. Panels A, and B show maximum titres and yields respectively. Comparisons of statistical significance of deviation of treatment groups from the untreated control performed using Dunnett’s Multiple Comparisons Test.

[0040] Figure 15 shows compositional analysis of PHAs produced by P. putida on Opuntia fermentates of various C:N ratios at different timepoints. All data are shown as mean ± standard deviation of biological triplicates. Timepoints of 18, 24, 30, 36, 43, and 48 hours are shown in panels A, B, C, D, E, and F, respectively.

[0041] Figure 16 shows bulk analysis of carbon and nitrogen composition of untreated and zeolite treated molasses fermentates. A: Concentrations of Total organic carbon (TOC) and Total organic acids (TOA), B: Total nitrogen (TN) and Total ammonium nitrogen (TAN), D: Carbon to nitrogen ratio (mass basis).

[0042] Figure 17 shows C. necator growth, fatty acid consumption, and PHA accumulation on molasses digestates of various C:N ratios. A: C. necator growth as measured by ODeoo measurement from an initial ODeoo of 0.1, B: Consumption of VFA by C. necator at various medium C:N ratios, C: Titres of PHA accumulation by C. necator across time at various medium C:N ratios. All data are given as means ± standard deviations as calculated from biological triplicates.

[0043] Figure 18 shows statistical comparison of mean PHA titre and yield maxima across timepoints. Panels A, and B show maximum titres (g / L) and yields (gPHA / gcDw) observed, respectively. All data are given as the means ± standard deviations of biological triplicates. Pairwise tests of statistical difference between control (C:N 12) and treatment groups shown in figures were determined by Dunnett’s Multiple Comparisons Test.

[0044] Detailed description

[0045] Definitions As defined herein, the term “microbial storage lipids” refers to lipids (i.e. hydrophobic or amphiphilic small molecules that are typically capable of self-assembly in an aqueous environment into structures such as vesicles, multilamellar / unilamellar liposomes, or membranes) that can be accumulated and stored in microorganisms as a reserve of energy. Typically, a microbial storage lipid is a polyhydroxyalkanoate (PHA), a triacylglycerol, a wax ester or an oil. Most typically, a microbial storage lipid is a polyhydroxyalkanoate (PHA).

[0046] As defined herein, the term “polyhydroxyalkanoate” (“PHA”) refers to a type of polyester synthesised by microorganisms which comprises the following repeat unit: wherein each R may be the same or different and each R is independently an alkyl group, an alkenyl group or an alkynyl group and each x is an integer from 1 to 12. Typically, each R is a C1-20 alkyl group, a C1-20 alkenyl group or a C1-20 alkynyl group, preferably a C1-12 alkyl group, a C1-12 alkenyl group or a C1-12 alkynyl group, more preferably a Cns alkyl group, a Cns alkenyl group or a Cns alkynyl group and most preferably a C1-6 alkyl group, a C1-6 alkenyl group or a C1-6 alkynyl group. Preferably, each R is independently an alkyl group. Thus, typically each R is a C1-20 alkyl group, preferably a C1-12 alkyl group, more preferably a Cns alkyl group, and most preferably a C1-6 alkyl group. Typically, x is an integer from 1 to 8, preferably from 1 to 6, and more preferably from 1 to 4, and most preferably x is 1. A polyhydroxyalkanoate comprising a single type of monomer unit therefore typically has the following formula: wherein R and x are as defined as above, and n is typically from 100 to 1000.

[0047] The nature of the PHA species produced in the present invention is not particularly limited, and over 150 different monomer units can be used in nature by bacteria to produce PHAs. However, preferred PHA species in the present invention comprise one or more types of monomer repeat unit having from 3 to 26 carbon atoms, preferably from 3 to 14 carbon atoms, e.g. from 6 to 12 carbon atoms. The PHA species may be a homopolymer (i.e. comprising a single type of monomer repeat unit) or a copolymer (i.e. comprising two or more types of repeat unit). Particularly preferred examples of PHA species are poly(3- hydroxybutanoic acid), poly(3 -hydroxypentanoic acid), poly(3 -hydroxyhexanoic acid), poly(3-hydroxyheptanoic acid), poly(3-hydroxyoctanoic acid), poly(3-hydroxynonanoic acid), poly(3 -hydroxydecanoic acid), poly(3-hydroxyundecanoic acid), poly(3- hydroxydodecanoic acid), poly(3 -hydroxytetradecanoic acid), copolymers of two or more monomer units selected from 3 -hydroxybutanoic acid, 3 -hydroxypentanoic acid, 3- hydroxyhexanoic acid, 3 -hydroxyh eptanoic acid, 3 -hydroxy octanoic acid, 3- hydroxynonanoic acid, 3 -hydroxy decanoic acid, 3-hydroxyundecanoic acid, 3- hydroxydodecanoic acid and 3 -hydroxytetradecanoic acid, and a combination thereof. In some embodiments, the PHA product is a homopolymer. In other embodiments, the PHA product is a copolymer. A copolymer of PHA monomer units may comprise two different types of monomer unit. Alternatively, a copolymer of copolymer of PHA monomer units may comprise three different types of monomer unit. Alternatively, a copolymer of copolymer of PHA monomer units may comprise four or more different types of monomer unit, e.g. four, five, six, seven, eight or more types of monomer unit. A copolymer may be a random copolymer, an alternating copolymer, or a block copolymer.

[0048] In some embodiments, the PHA species of the present invention may be a “shortchain” PHA, that is to say a PHA which comprises a monomer repeat unit having from 3 to 5 carbon atoms. In other embodiments, the PHA species of the present invention may be a “medium-chain” PHA, that is to say a PHA which comprises a monomer repeat unit having from 6 to 14 carbon atoms. In some embodiments, the PHA species of the present invention may be a “long-chain” PHA, that is to say a PHA which comprises a monomer repeat unit having from 15 to 26 carbon atoms.

[0049] PHAs may be thermoplastic (i.e. become pliable or moldable at a certain elevated temperature and solidifies upon cooling) or elastomeric (i.e. a polymer with viscoelasticity and with weak intermolecular forces). Typically, the melting point of PHAs ranges from about 40°C to about 180°C. Typically, PHAs are UV-stable, in contrast to other bioplastics from polymers such as polylactic acid. The crystallinity of PHAs can vary from a few % to around 70%. The wide variety of physical properties of different PHAs makes them a useful class of materials for a variety of applications.

[0050] As used herein, the term “wax ester” is an ester of a fatty acid and a fatty alcohol of the formula R-(C=O)-O-R’, wherein R and R’ are independently selected from saturated or unsaturated hydrocarbon groups. Typically, R is a saturated or unsaturated hydrocarbon group comprising from 12 to 24 carbon atoms and preferably from 12 to 20 carbon atoms. Thus, preferably R is selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12-24 alkynyl group. More preferably, R is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group. Typically, R’ is a saturated or unsaturated hydrocarbon group comprising from 12 to 34 carbon atoms and preferably from 24 to 34 carbon atoms. Thus, preferably R’ is selected from a C12-34 alkyl group, a C12-34 alkenyl group and a C12-34 alkynyl group. More preferably, R’ is selected from a C24-34 alkyl group, a C24-34 alkenyl group and a C24-34 alkynyl group.

[0051] Typically, fully saturated wax esters (i.e. wax esters wherein both R and R’ are saturated hydrocarbon groups) have higher melting points and are solids at room temperature. Typically, unsaturated wax esters have lower melting points and are liquids at room temperature.

[0052] As used herein, the term “triacylglycerol” refers to an ester derived from glycerol and three fatty acids. A “triacylglycerol” may also be called a “triglyceride” and these terms are interchangeable. Thus, typically a triacylglycerol has the following formula: wherein R, R’ and R’ ’ are independently selected from saturated or unsaturated hydrocarbon groups. R, R’ and R” may be the same, but typically they are different. When R, R’ and R” are different, the triacylglycerol is typically referred to as a “mixed triglyceride”. Typically, R is a saturated or unsaturated hydrocarbon group comprising from 12 to 24 carbon atoms and preferably from 12 to 20 carbon atoms. Thus, preferably R is selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12-24 alkynyl group. More preferably, R is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group. Typically, R’ is a saturated or unsaturated hydrocarbon group comprising from 12 to 24 carbon atoms and preferably from 12 to 20 carbon atoms. Thus, preferably R’ is selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12-24 alkynyl group. More preferably, R’ is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12- 20 alkynyl group. Typically, R” is a saturated or unsaturated hydrocarbon group comprising from 12 to 24 carbon atoms and preferably from 12 to 20 carbon atoms. Thus, preferably R” is selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12-24 alkynyl group. More preferably, R” is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group.

[0053] As used herein, the term “oil” refers to a composition that is liquid at room temperature and comprises one or more triacylglycerol compounds, one or more fatty acids (typically a long-chain fatty acid) and / or one or more wax esters. The composition is nonpolar and hydrophobic in nature.

[0054] As used herein, the term “volatile fatty acid” refers to a short-chain fatty acid having from 2 to 6 carbon atoms. Typically, a volatile fatty acid has the formula R-COOH, wherein R is a C1-5 alkyl group. Thus, a volatile fatty acid is typically selected from acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2 -methylbutyric acid and hexanoic acid. A composition comprising a volatile fatty acid may comprise one or more different volatile fatty acids, e.g. one, two, three, four, five or more different types of fatty acid.

[0055] As used herein, the term “long chain fatty acid” refers to a fatty acid having 12 or more carbon atoms. Typically, a long chain fatty acid has the formula R-COOH, wherein R is a saturated or unsaturated hydrocarbon group having 11 or more carbon atoms. Preferably, R is a Cn-35 alkyl group, a Cn-35 alkenyl group or a Cn-35 alkynyl group, more preferably a Cn-21 alkyl group, a Cn-21 alkenyl group or a Cn-21 alkynyl group. Nonlimiting examples of long chain fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoeladic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.

[0056] As used herein, the term “ammonium” refers to the cation NH .

[0057] As used herein, the term “ammonium-containing compound” refers to a compound comprising ammonium. Examples include organic anions with an ammonium cation. Ammonium-containing compounds are in some embodiments derived from proteins, free amino acids, urea, or nitrates. In some embodiments an ammonium-containing compound is an ammonium salt such as ammonium acetate, ammonium butyrate, ammonium lactate, ammonium propionate and ammonium formate. Other ammonium-containing compounds include ammonium valerate, ammonium caproate, ammonium succinate, ammonium fumarate, ammonium malate, ammonium carbonate, ammonium bicarbonate, ammonium oxalate, ammonium citrate, ammonium tartrate, ammonium benzoate, ammonium amino acid salts; ammonium alaninate, ammonium glutamate, ammonium glyoxylate, ammonium isobutyrate, ammonium isovalerate and ammonium phenyl acetate. In some embodiments the term “ammonium-containing compound” refers to the free (e.g. solubilized) ammonium ion e.g. NH4+(aq).

[0058] As used herein, the term “alkyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix. Thus, the term “Ci-6 alkyl” refers to a linear saturated monovalent hydrocarbon radical of one to six carbon atoms or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms, e.g. methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- pentyl and n-hexyl.

[0059] As defined herein, the term “alkenyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond. Thus, the term “C2-6 alkenyl” refers to a linear saturated monovalent hydrocarbon radical of two to six carbon atoms having at least one double bond, or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms having at least one double bond, e.g. ethenyl, propenyl, 1,3-butadienyl, (CH3)2CH=C(CH3)2, CH3CH=CHCH(CH3)2, and the like.

[0060] As defined herein, the term “alkynyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one triple bond. Thus, the term “C2-6 alkynyl” refers to a linear saturated monovalent hydrocarbon radical of two to six carbon atoms having at least one double bond, or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms having at least one double bond, e.g. ethynyl, propynyl, 1,3-butadiynyl, (CH3)2C=CCH3, CH3C=CCH(CH3)2, and the like.

[0061] As used herein, the term “room temperature” refers to a temperature of from 20°C to 25°C.

[0062] As used herein, the term “biomass” refers to any energy source derived from biological material. Typically, the biomass which acts as the feedstock for the process of the present invention is selected from plant matter, agricultural waste, food waste, meat industry waste, sewage, human or animal effluent, waste from biofuel production, molasses, blood, cheese whey, pulp and paper mill effluents, and olive oil mill effluents. As used herein, the term “microorganism” refers to an organism of microscopic size, which may exist in its single-celled form or as a colony of cells. Preferably microorganisms used in the process of the present invention are bacteria.

[0063] As used herein, the term “bacteria” refers to unicellular organisms which have cell walls but lack organelles and an organised nucleus.

[0064] As used herein, the term “carbon-to-nitrogen ratio” (or “C / N ratio”) refers to the ratio by mass of total organic carbon in a given composition to mass of total nitrogen. Total organic carbon and total nitrogen can be determined spectrophotometrically, e.g. using a Hach Lange DR 2800 spectrophotometer (Hach UK, Manchester, UK). Carbon and nitrogen concentrations are calculated using HACH Lange analysis kits. Total Organic Carbon is calculated by the purging method with persulphate digestion (HACH kit LCK 386; obtainable from Hach UK, Manchester, UK; kit operated according to the manufacturer’s instructions; kit accords with standard EN1484). Nitrogen is calculated by the method of Koroleff digestion with photometric detection with 2.6 dimethylphenol (HACH kit LCK238; obtainable from Hach UK, Manchester, UK; kit operated according to the manufacturer’s instructions; kit accords with standard EN ISO 11905-1). Ratios are quoted on a mass basis (gC / gN).

[0065] As used herein, the term “reactor” refers to a substantially enclosed volume in which a chemical reaction takes place. The nature of the reactors used in the present process are not particularly limited, and can be of essentially any feasible shape or size, and made of any suitable material. The skilled person is equipped to select an appropriate reactor type for each particular process step. A reactor may be operated as a batch reactor, i.e. a reactor in which all reagents are added at an initial time point, and the reaction is allowed to proceed to completion without addition of any further reagents during the course of the reaction. Alternatively, a reactor may be operated as a continuous (flow) reactor, i.e. a reactor in which reagent is added either continuously or periodically (i.e. batchwise) over time, and product is continuously or periodically removed from the reactor as it is produced. The skilled person is able to select an appropriate mode of operation for a given reactor for a given process step.

[0066] Step (a): Acidogenic fermentation

[0067] The present invention is directed to a method for preparing one or more microbial storage lipids from biomass, said method comprising: (a) subjecting the biomass to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium-containing compounds;

[0068] (b) treating said composition with an aluminosilicate such that the carbon-to- nitrogen ratio of the treated composition is 30: 1 or greater; and

[0069] (c) incubating the treated composition with microorganisms to produce one or more microbial storage lipids.

[0070] This process is summarised in the flow diagram of Fig. 1.

[0071] In some embodiments the method comprises:

[0072] (a) subjecting the biomass to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium;

[0073] (b) treating said composition with an aluminosilicate such that the carbon-to- nitrogen ratio of the treated composition is 30: 1 or greater; and

[0074] (c) incubating the treated composition with microorganisms to produce one or more microbial storage lipids.

[0075] Thus, the biomass is first subjected to acidogenic fermentation to produce a composition that is rich in volatile fatty acids and which also typically contains high levels of ammonium ions. The carbon-to-nitrogen ratio (C / N ratio) of this composition is unpredictable, highly dependent on the specific conditions under which acidogenic fermentation is carried out, and is difficult to control.

[0076] Acidogenic fermentation is the initial phase of anaerobic digestion of organic compounds to methane and carbon dioxide by microorganisms. This process is summarised in the flow diagram of Fig. 10(a). Typically, acidogenic fermentation is carried out in the presence of anaerobic bacteria, i.e. bacteria that do not require molecular oxygen for growth and which can thrive in low-oxygen environments. In acidogenic fermentation, soluble organic compounds present in biomass, such as sugars (hydrolysis products of carbohydrates), fatty acids (hydrolysis products of lipids) and amino acids (hydrolysis products of proteins) are fermented into organic acids, primarily volatile fatty acids (VFAs) which contain from 2 to 6 carbon atoms. Ammonium is also produced as a significant by-product of acidogenic fermentation due to the breakdown of amino acids and / or proteins in the biomass. Other by-products of acidogenic fermentation which may also be present in the resultant composition include phosphates, trace elements, alcohols and longer-chain fatty acids (e.g. lactic acid, succinic acid) but these are typically present in lower amounts and may be subjected to further reaction to produce VFAs. Hydrogen may also be generated as a by-product of acidogenic fermentation.

[0077] The biomass source for the acidogenic fermentation step is not particularly limited. This is a significant advantage of the present process, as it means that cheap, readily available biomass sources can be used. Often, these biomass sources are waste streams, and so the present process also enables recycling of waste to ultimately produce commercially useful products. Thus, the biomass source of the present process may, for example, be selected from plant matter, agricultural waste, food waste, meat industry waste, sewage, human or animal effluent, waste from biofuel production, molasses, blood digestate, cheese whey, pulp and paper mill effluents, and olive oil mill effluents. In some embodiments the biomass is comminuted prior to the acidogenic fermentation step; e.g. by grinding or milling.

[0078] In some embodiments the biomass has a volatile solids content of from about 40% to about 100% of total solids (VS / TS), e.g. from about 60% to about 95%, e.g. from about 70% to about 90% VS / TS.

[0079] The acidogenic fermentation step can be carried out under any suitable conditions for fermentation of biomass by microorganisms. These conditions are well-known to the skilled person. Typically, the acidogenic fermentation is carried out under anaerobic conditions. Thus, typically, the acidogenic fermentation comprises treatment of the biomass with anaerobic bacteria. Alternatively, the acidogenic fermentation comprises treatment of the biomass with aerobic bacteria. Typically, the acidogenic fermentation comprises treatment of the biomass with a mixed microbial culture, that is to say a culture of bacteria which contains more than one different species of bacteria. Typically, the mixed microbial culture is an aerobic or a micro-aerobic culture.

[0080] Typically, the composition comprising the biomass for fermentation may contain said biomass at from about 5 to about 100 gVS / L (volatile solids / L), such as from about 10 to about 50 gVS / L, e.g. from about 20 to about 40 gVS / L, e.g. about 30 gVS / L.

[0081] Acidogenic fermentation typically comprises the distinct steps of (1) hydrolysis, (2) acidogenesis (also referred to as fermentation) and (3) acetogenesis. These functions are typically performed by distinct microbial subpopulations within the mixed microbial culture that may perform one or more of these functions. Hydrolysis is the breakdown of organic macromolecules (e.g. carbohydrates, lipids, proteins and nucleic acids) to their monomeric constituents (e.g. saccharides, fatty acids, amino acids and nucleotides). Acidogenesis is the process by which these monomeric constituents are converted to VFAs. Acetogenesis is the process by which many of the VFAs in this resultant mixture are then further converted to acetic acid, producing a VFA mixture that is enriched in acetic acid (i.e. a C2 VFA).

[0082] Microorganisms for fermentation of the biomass may in some embodiments be provided from byproducts of waste treatment. Examples include anaerobic digestion (AD) sludge. Anaerobic digestion (AD) is widely used at industrial scale in the treatment of municipal food waste, sewage sludge, and agricultural residues and accordingly AD sludge is a widely available reagent for use in the disclosed methods. In some embodiments the most abundant microorganisms in AD sludge are of genus Clostridium. In some embodiments when microbes for fermentation are provided in the form of AD sludge, such sludge may be used in the composition comprising the biomass for fermentation at from about 1 to about 20 g / L, such as from about 2 to about 10 g / L, e.g. from about 3 to about 7 g / L, e.g. about 5 g / L.

[0083] In some embodiments the microbes used in acidogenic hydrolysis and / or acidogenesis steps are bacteria. The bacteria may comprise a single genus and / or species of bacteria; or may comprise a plurality of genii and / or species of bacteria. Examples of different genii of microbes which are typically involved in hydrolysis and / or acidogenesis steps include the bacterial genii Acetivibrio, Aminobacterium, Aminomonas, Anaeromusa, Anaerosphaera, Bacillus, Bacteroides, Bifidobacterium, Butyrivibrio, Caldanaerobacter, Caldicellulosiruptor, Campylobacter, Cellulomonas, Clostridium, Devosia, Espiroquetas, Eubacterium, Fervidobacterium, Birobacter, Fusobacterium, Gelria, Gracilibacter, Halocella, Lactobacillus, Paludibacter, Peptococcus, Peptoniphilus, Proteiniborus, Pseudomonas, Psychrobacter, Ralstonia, Ruminoclostridium, Ruminococcus, Selenomonas, Shewanella, Spotanaerobacter, Strptococcus, Streptomyces, Thermanaerovibtio, Thermomonas, Thermomonospora, Thermotoga, Treponema and Trichococcus and the fungus genii Aspergillus, Humicola, Penicillium and Trichoderma. Examples of particular species of microbes which are typically involved in hydrolysis and / or fermentation steps include the bacteria Pseudomonas mendocina, Bacillus halodurans, Clostridium hastiforme, Gracilibacter thermotolerans and Thermomonas haemolytica and the fungus Trichoderma reesei. Examples of different genii of microbes which are typically involved in acetogenesis include the bacterial genii Acetobacterium, Clostridium, Desulfotignum, Eubacterium, Holophaga, Moorella, Ruminococcus, Sporomusa, Thermoanaerobacter and Treponema. Examples of particular species of microbes which are typically involved in acetogenesis include the bacteria Moorella thermoacetica, Desulfotignum phosphitoxidans and Holophaga foetida.

[0084] Typically, the acidogenic fermentation step is carried out at a temperature of from 0°C to 75°C, preferably from 15°C to 60°C, more preferably from 25°C to 50°C and yet more preferably from 30°C to 40°C; e.g. at about 37 °C. Typically, the acidogenic fermentation step is carried out for at least 1 hour, preferably at least 2 hours, more preferably at least 4 hours, still more preferably at least 8 hours, even more preferably at least 12 hours, e.g. at least 1 day, at least 2 days, or at least 5 days. Typically, the acidogenic fermentation step is carried out for no more than 20 days, preferably no more than 16 days, more preferably no more than 12 days, and most preferably no more than 10 days. Thus, preferably the acidogenic fermentation step is carried out at a temperature of from 0°C to 75°C for a period of from 1 hour to 20 days, more preferably at a temperature of from 15°C to 60°C for a period of from 4 hours to 16 days, still more preferably at a temperature of from 25°C to 50°C for a period of from 2 days to 12 days, and most preferably at a temperature of from 30°C to 40°C for a period of from 5 days to 10 days. Typically, the acidogenic fermentation step is carried out under agitation. Alternatively, however, the acidogenic fermentation step is carried out in the absence of agitation.

[0085] Typically, the acidogenic fermentation step is carried out in a reactor operating in continuous mode, i.e. a reactor in which biomass is continuously being added to the reactor and in which the VFA- and ammonium-rich composition is continuously being removed from the reactor. Continuous mode operation of the process typically leads to higher yields of VFA than batch mode operation of the process. Without wishing to be bound by any particular theory, this is believed to arise from the better acclimatisation of the bacterial cultures to the biomass feedstock when the reactor operates in continuous mode.

[0086] The composition which results from the acidogenic fermentation step is rich in VFA and also comprises ammonium. Typically, therefore, the composition which results from the acidogenic fermentation step comprises VFAs having from 2 to 6 carbon atoms. Preferably, the VFAs are selected from acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, hexanoic acid and a combination thereof. The composition may comprise a single type of VFA. More typically, however, the composition comprises two or more types of VFA, e.g. two, three, four, five, six or more types of VFA. Typically, the VFA that is present in the highest concentration in the composition is acetic acid. Alternatively, however, the VFA that is present in the highest concentration in the composition is another VFA, e.g. propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-m ethylbutyric acid or hexanoic acid.

[0087] The C / N ratio of the composition which results from the acidogenic fermentation step may vary widely and may sometimes be difficult to control. Typically, however, a significant amount of ammonium is present due to the presence of proteins and amino acids in the biomass feedstock. Thus, typically, the C / N ratio of the composition is from 1:20 to 25: 1, more typically from 1 :5 to 20: 1 and most typically from 1 : 1 to 15: 1, e.g. from 2: 1 to 12: 1 or from 5: 1 to 10: 1.

[0088] Step (b): Treatment with aluminosilicate

[0089] The VFA- and ammonium-rich composition which is the product of the acidogenic fermentation step is subsequently treated with an aluminosilicate material. The present inventors have discovered that this treatment step provides exquisite control over the C / N ratio of the composition such that this ratio can be optimised for the subsequent production of microbial storage lipids (MSLs). Without wishing to be bound by any particular theory, it is believed that the aluminosilicate material sequesters ammonium ions from the VFA- and ammonium-rich composition via an ion exchange process. This reduces the concentration of the ammonium ions in the composition, which decreases the total amount of nitrogen and hence increases the C / N ratio of the composition.

[0090] Thus, the present process involves contacting the VFA- and ammonium-containing composition that is the product of the acidogenic fermentation step with an aluminosilicate. The nature of the aluminosilicate material useful for this purpose is not particularly limited. Typically, the aluminosilicate material may be selected from a sodium aluminosilicate, a potassium aluminosilicate, a calcium aluminosilicate, a magnesium aluminosilicate and a combination thereof.

[0091] The aluminosilicate material may be a zeolite. A zeolite is a class of aluminosilicate material containing several microporous, crystalline aluminosilicates. Zeolites typically have the general formula Mn+x / n[(A102)'x(Si02)y] ZH2O where Mn+is H+or one or more metal ions where n represents the charge on the one or more metal ions, and x, y and z are integers. Typically, the one or more metals M in Mn+i / nare one or more metal ions selected from sodium, potassium, calcium or magnesium. When M is sodium or potassium, n is 1. When M is calcium or magnesium, n is 2. The Si / Al ratio is variable, which provides a means to tune the properties of the zeolite. Zeolites with a Si / Al ratios higher than about 3 are classified as high-silica zeolites, which tend to be more hydrophobic. Typically, x is an integer from 1 to 20. Typically, y is an integer from 1 to 30. Typically, z is an integer from 1 to 40. Zeolites have microporous structures with a typical diameter of 0.3 -0.8 nm. The framework is formed by linking of aluminium and silicon atoms by oxides. This linking leads to a 3-dimensional network of Si-O-Al, Si-O- Si, and A1-0-A1 linkages. The aluminium centres are negatively charged, which requires an accompanying cation. These cations are hydrated during the formation of the materials. The hydrated cations interrupt the otherwise dense network of Si-O- Al, Si-O-Si, and Al-O- A1 linkage, leading to regular water-filled cavities. Because of the porosity of the zeolite, the water can exit the material through channels. As the zeolite framework is rigid, the loss of water does not result in collapse of the cavities and channels. This underpins the ability of zeolites to function as catalysts. Zeolites possess high physical and chemical stability due to the large covalent bonding contribution. They have excellent hydrophobicity and are suited for adsorption of bulky, hydrophobic molecules such as hydrocarbons.

[0092] When the aluminosilicate material is a zeolite, preferably the aluminosilicate is selected from analcime, chabazite, clinoptilolite, heulandite, natrolite, phillipsite, stilbite, amicite, barrerite, brewsterite, cowlesite, Cu Y zeolite, dachiardite-K, edingtonite, erionite, faujasite, ferrierits, garronite-Ca, gismondine, gmelinite, gonnardite, harmotome, hsianghualite, laumontite, levyne, mesolite, mordenite, nabesite, paulingite, pollucite, scolecite, SSZ-13, stellerite, thomsonite, ultramarine, wairakite, yugawaralite, ZSM-5 and a combination thereof.

[0093] The aluminosilicate may alternatively be a bentonite clay. Bentonite is an absorbent swelling clay comprising mostly montmorillonite (a type of smectite) which can either be Na-montmorillonite or Ca-montmorillonite. The montmorillonite making up bentonite is an aluminium phyllosilicate mineral whose crystal structure is described as low-charge TOT (tetrahedra-octahedra-tetrahedra). This means that a crystal of montmorillonite consists of layers, each of which is made up of two T sheets bonded to either side of an O sheet. The T sheets are so called because each aluminium or silicon ion in the sheet is surrounded by four oxygen ions arranged as a tetrahedron. The O sheets are so called because each aluminium ion is surrounded by six oxygen or hydroxyl ions arranged as an octahedron. The complete TOT layer has a weak negative electrical charge, and this is neutralized by calcium or sodium cations that bind adjacent layers together, with a distance between layers of about 1 nanometre. Because the negative charge is weak, only a fraction of the possible cation sites on the surface of a TOT layer actually contain calcium or sodium. Water molecules can easily infiltrate between sheets and fill the remaining sites. This accounts for the swelling property of montmorillonite and other smectite clay minerals.

[0094] When the aluminosilicate material is a bentonite clay, preferably the aluminosilicate is selected from sodium bentonite, calcium bentonite, potash bentonite and illite.

[0095] The aluminosilicate may alternatively be a kaolnite clay. Kaolnite is a clay mineral with the chemical composition A12Si2Os(OH)4. It is a layered silicate mineral, with one tetrahedral sheet of silica (Si O4) linked through oxygen atoms to one octahedral sheet of alumina (AlOe). Kaolnite is a 1 : 1 or TO (tetrahedra-octahedra) clay mineral, because its crystals consist of stacked TO layers. The two sheets in each layer are strongly bonded together via shared oxygen ions, while layers are bonded via hydrogen bonding between oxygen on the outer face of the T sheet of one layer and hydroxyl on the outer face of the O sheet of the next layer. A kaolinite layer has no net electrical charge and so there are no large cations (such as calcium, sodium, or potassium) between layers as with most other clay minerals.

[0096] Preferably, the aluminosilicate material is a zeolite.

[0097] In some embodiments the aluminosilicate is treated with acid or alkali prior to use in the disclosed methods. Suitable acids include sulfuric acid, hydrochloric acid, nitric acid, and acetic acid. Suitable alkalis (bases) include sodium hydroxide.

[0098] After treatment with an aluminosilicate material, the C / N ratio of the VFA- and ammonium-containing composition is reduced to the desired level. That is to say that the C / N ratio of the composition after treatment with aluminosilicate is about 30: 1 or greater. Preferably, the C / N ratio of the composition after treatment with aluminosilicate is from about 30: 1 to about 500: 1, e.g. about 35: 1 to about 350: 1, more preferably from 40: 1 to 200: 1, still more preferably from 40: 1 to 120: 1, yet more preferably from 40: 1 to 100: 1, even more preferably from 40: 1 to 90: 1, still more preferably from 40: 1 to 80: 1, e.g. from 40: 1 to 70: 1. In some embodiments the C / N ratio of the composition after treatment with aluminosilicate is from about 40: 1 to about 60: 1 or from about 40: 1 to about 50: 1, such as about 41 : 1, about 42 : 1 , about 43: 1, about 44 : 1 , about 45: 1, about 46: 1, about 47: 1, about 48: 1, or about 49: 1. In some embodiments the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 40: 1 to 500: 1. In some embodiments the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 50: 1 to 100: 1, and preferably from 60: 1 to 80: 1. In some embodiments the C / N ratio of the composition after treatment with aluminosilicate is 30: 1 to 500: 1, more preferably from 40: 1 to 200: 1, still more preferably from 50: 1 to 120: 1, yet more preferably from 52: 1 to 100: 1, even more preferably from 55: 1 to 90: 1, still more preferably from 58: 1 to 85: 1, and most preferably from 60: 1 to 80: 1. In some embodiments the C / N ratio of the composition after treatment with aluminosilicate is from about 40: 1 to about 80: 1 such as from about 40: 1 to about 70: 1; e.g. from about 60: 1 to about 80: 1 or from about 60: 1 to about 70: 1.

[0099] The specific optimum C / N ratio may vary slightly depending upon the particular microorganism to be used in the subsequent MSL-producing reaction (discussed further below) and the target MSL to be produced, but typically the C / N ratio will fall within the ranges set out above.

[0100] The VFA- and ammonium-containing composition is treated under conditions that are suitable for adjusting the C / N ratio to the desired level. The skilled person is able to adapt the reaction conditions in order to obtain the required C / N level. In particular, the skilled person is able to adjust the time for which the VFA- and ammonium-containing composition is contacted with aluminosilicate material, the temperature under which the process is carried out, and the ratio of VFA- and ammonium-containing composition to aluminosilicate.

[0101] Typically, however, the VFA- and ammonium-containing composition is treated by incubation with aluminosilicate at a temperature of from 0°C to 100°C, preferably from 10°C to 70°C, more preferably from 20°C to 50°C, and still more preferably from 25°C to 40°C, e.g. about 30°C or about 35°C. Typically, the VFA- and ammonium-containing composition is treated by incubation with aluminosilicate for from 15 minutes to 8 hours, preferably from 30 minutes to 4 hours, more preferably from 1 to 3 hours, and more preferably for about 2 hours. Typically, the VFA- and ammonium-containing composition is treated by incubation with from 25 to 100% (w / v) aluminosilicate, preferably from 35 to 95% (w / v) aluminosilicate, and more preferably from 50 to 90% (w / v) aluminosilicate. In some embodiments the VFA- and ammonium-containing composition is treated by incubation with from about 50 to about 60% (w / v) aluminosilicate. Thus, typically the VFA- and ammonium-containing composition is treated by incubation with aluminosilicate at a temperature of from 0°C to 100°C for from 15 minutes to 8 hours. Preferably, the VFA- and ammonium-containing composition is treated by incubation with aluminosilicate at a temperature of from 10°C to 70°C for from 30 minutes to 4 hours. More preferably, the VFA- and ammonium-containing composition is treated by incubation with aluminosilicate at a temperature of from 20°C to 50°C for from 1 to 3 hours. Typically the VFA- and ammonium-containing composition is treated by incubation with from 25 to 100% (w / v) aluminosilicate at a temperature of from 0°C to 100°C for from 15 minutes to 8 hours. Preferably, the VFA- and ammonium-containing composition is treated by incubation with 35 to 95% (w / v) aluminosilicate at a temperature of from 10°C to 70°C for from 30 minutes to 4 hours. More preferably, the VFA- and ammonium-containing composition is treated by incubation with from 50 to 90% (w / v) aluminosilicate at a temperature of from 20°C to 50°C for from 1 to 3 hours. In some embodiments, the VFA- and ammonium-containing composition is treated by incubation with from 50 to 60% (w / v) aluminosilicate at a temperature of from 25°C to 40°C for about 2 hours. Typically, the contacting step with aluminosilicate is carried out under agitation. Alternatively, however, the contacting step with aluminosilicate may be carried out without agitation (e.g. by passing the VFA- and ammonium-containing composition through a column comprising the aluminosilicate material as a stationary bed).

[0102] Steps (a) and (b) of the present process may be carried out sequentially or simultaneously. Thus, in an embodiment, steps (a) and (b) are carried out sequentially. In this embodiment, the biomass is subjected to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium-containing compounds, and then the acidogenic fermentation reaction is terminated before the said composition is treated with an aluminosilicate such that the C / N ratio of the treated composition is 30: 1 or greater. In some embodiments the biomass is subjected to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium, and then the acidogenic fermentation reaction is terminated before the said composition is treated with an aluminosilicate such that the C / N ratio of the treated composition is 30: 1 or greater. A non-limiting example of a possible reactor setup for this embodiment is where the acidogenic fermentation is carried out within a contained reactor, and the VFA- and ammonium-containing composition is removed from this reactor and fed into a column comprising a stationary aluminosilicate bed. In an alternative embodiment, steps (a) and (b) are carried out simultaneously. In this embodiment, the biomass is subjected to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium-containing compounds in the presence of an aluminosilicate, which simultaneously modulates the C / N ratio of the VFA- and ammonium-containing composition such that the final C / N ratio is 30: 1 or greater. Thus, in some embodiments the biomass is subjected to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium in the presence of an aluminosilicate, which simultaneously modulates the C / N ratio of the VFA- and ammonium-containing composition such that the final C / N ratio is 30: 1 or greater. A non-limiting example of a possible reactor setup for this embodiment is where the acidogenic fermentation is carried out within a contained reactor comprising both the microorganisms necessary to carry out the acidogenic fermentation step and the aluminosilicate material.

[0103] Following modulation of the C / N ratio of the VFA- and ammonium-containing material such that it is 30: 1 or greater (herein referred to as the “treated composition”), optionally the pH of the treated composition is adjusted. Typically in this embodiment, the pH of the treated composition is adjusted to 9 or less, preferably from 5 to 9, more preferably from 6 to 8, e.g. about 7. pH modulation can be effected by any means known to the skilled person. Typically pH modulation is effected by the addition of a buffering component. A “buffering component”, as defined herein, refers to any chemical entity, which when dissolved in solution, enables said solution to resist changes in its pH following the subsequent addition of either an acid or a base. The nature of the buffering component that can be employed is not particularly limited. Suitable buffering components are well-known to the skilled person. Examples of suitable buffering components include, but are not limited to, phosphates, sulfates, citrates and acetates. The buffer may be a salt of a monovalent cation, such as sodium, potassium or ammonium salts. The concentration of buffer salts used is typically from about 0.01 M to about 0.5M, such as from about 0.05 M to about 0.25 M; e.g. about 0.1 M.

[0104] Following modulation of the C / N ratio of the VFA- and ammonium-containing material such that it is 30: 1 or greater, typically the zeolite is removed from the treated composition. The method of removal is not especially limited, but typically removal is effected by filtration or centrifugation. Optionally, the treated composition may be clarified. Optionally, the treated composition may be sterilised. Step (c): Treatment with MSL-producing microorganisms

[0105] The treated composition (i.e. the composition comprising VFA and ammonium which has undergone treatment with an aluminosilicate such that the C / N ratio of the composition is 30: 1 or greater) is incubated with microorganisms to produce one or more microbial storage lipids.

[0106] The microorganisms employed in this step may be any microorganisms which produce microbial storage lipids from volatile fatty acids (“MSL-producing microorganisms”). Typically, the microorganisms are selected from one or more bacteria, one or more archaea and one or more fungi. Preferably the microorganisms are selected from one or more bacteria and one or more yeasts. A yeast is a type of fungus. Preferably, a single type of microorganism (i.e. a homogeneous microorganism culture) is employed in this step. Alternatively, a mixed culture of microorganisms is employed in this step. However, preferably, this step is not carried out in the presence of a mixed culture of microorganisms. The microorganisms may be wild-type microorganisms or they may be microorganisms which contain one or more genetic mutations.

[0107] In some embodiments a microorganism for use in the production of one or more microbial storage lipids as described herein is a bacterium. In some embodiments a microorganism for use in the production of one or more microbial storage lipids as described herein is a yeast.

[0108] In some embodiments microorganisms for use in the production of one or more microbial storage lipids as described herein are bacteria of family Pseudomonadaceae. Burkholderiaceae. Vibrionaceae. Moraxellaceae and / or Bacillaceae. In some embodiments microorganisms for use in the production of one or more microbial storage lipids as described herein are bacteria of genus Pseudomonas, Cupriavidus, Vibrio, Acinetobacter and / or Bacillus. In some embodiments microorganisms for use in the production of one or more microbial storage lipids as described herein are yeasts of family Dipodascaceae . In some embodiments microorganisms for use in the production of one or more microbial storage lipids as described herein are yeasts of genus Yarrowia.

[0109] In some embodiments microorganisms for use in the production of one or more microbial storage lipids as described herein are selected from: Pseudomonas putida, Cupriavidus necator, Vibrio natriegens, Acinetobacter baylyi, Bacillus megaterium and Yarrowia lipolytica. Particularly preferred microorganisms for use in this step are: Pseudomonas putida, Cupriavidus necator, Vibrio natriegens, Vibrio natriegens, Bacillus megaterium and Yarrowia lipolytica. Pseudomonas putida is a bacterial species that is capable of producing PHAs (specifically medium-chain PHAs, i.e. PHAs which typically contain from 6 to 14 carbon atoms in each monomer unit) from VFAs. Cupriavidus necator is a bacterial species that is capable of producing PHAs (specifically short-chain PHAs, i.e. PHAs which typically contain from 3 to 5 carbon atoms in each monomer unit) from VFAs. Vibrio natriegens is a bacterial species that is capable of producing PHAs (specifically short-chain PHAs) from VFAs. Acinetobacter baylyi is a bacterial species that is capable of producing wax esters from VFAs. Bacillus megaterium is a bacterial species that is capable of producing PHAs from VFAs. Yarrowia lipolytica is a yeast species that is capable of producing triacylglycerides from VFAs.

[0110] Thus, in an embodiment, the treated composition is incubated with Pseudomonas putida and the one or more microbial storage lipids produced is one or more polyhydroxyalkanoates (PHAs). In an alternative embodiment, the treated composition is incubated with Cupriavidus necator and the one or more microbial storage lipids produced is one or more polyhydroxyalkanoates (PHAs). In an alternative embodiment, the treated composition is incubated with Vibrio natriegens and the one or more microbial storage lipids produced is one or more polyhydroxyalkanoates (PHAs). In an alternative embodiment, the treated composition is incubated with Acinetobacter baylyi and the one or more microbial storage lipids produced is one or more wax esters. In an alternative embodiment, the treated composition is incubated with Bacillus megaterium and the one or more microbial storage lipids produced is one or more polyhydroxyalkanoates (PHAs). In an alternative embodiment, the treated composition is incubated with Yarrowia lipolytica and the one or more microbial storage lipids produced is one or more triacylglycerides.

[0111] The skilled person is able to modify the conditions under which step (c) is carried out in order to maximise the yield of MSL product, depending on factors such as the particular microorganism employed and the concentration of microorganism culture in the reaction mixture. In particular, the skilled person is able to select an appropriate time and temperature under which to carry out the reaction to product MSL product from VFA.

[0112] As regards time, without wishing to be bound by any particular theory, it is believed that there is an optimum time period to obtain a maximum yield of MSL. If the reaction period is too short, not all of the VFA will be converted to MSL by the microorganism. However, if the reaction period is too long, the microorganisms may begin to digest the MSL products due to a lack of alternative energy sources in the reaction medium for growth of the microorganisms (as the alternative energy sources are depleted over time). The skilled person is able to readily identify an optimum time period for a particular reaction setup. Typically, however, the treated composition is incubated with MSL-producing microorganisms for from 1 to 240 hours, preferably from 2 to 120 hours, more preferably from 4 to 96 hours, still more preferably from 12 to 72 hours, e.g. from 24 to 60 hours, from 30 to 48 hours, or from 36 to 44 hours.

[0113] As regards temperature, the skilled person is able to readily identify an optimum temperature at which any given MSL-producing microorganism can operate. Typically, however, the treated composition is incubated with MSL-producing microorganisms at a temperature of from 10°C to 70°C, preferably from 20°C to 50°C, and more preferably from 25°C to 45°C, e.g. from 30°C to 40°C or about 35°C.

[0114] As regards growth stage, the skilled person is readily identify an optimum growth stage at which any given MSL-producing microorganism can operate. For example, in some embodiments the MSL-producing microorganism is one or more bacterial species and the treated composition is incubated with MSL-producing microorganisms having an ODeoo (i.e. an optical density measured at a wavelength of 600 nm in 1 cm light path) of from about 0.1 to about 1, such as from about 0.2 to about 0.7, e.g. about 0.3 to about 0.5.

[0115] Thus, typically, the treated composition is incubated with MSL-producing microorganisms for from 1 to 240 hours at a temperature of from 10°C to 70°C. Preferably, the treated composition is incubated with MSL-producing microorganisms for from 2 to 120 hours at a temperature of from 20°C to 50°C. More preferably, the treated composition is incubated with MSL-producing microorganisms for from 4 to 96 hours at a temperature of from 25°C to 45°C. Still more preferably, the treated composition is incubated with MSL-producing microorganisms for from 12 to 72 hours at a temperature of from 30°C to 40°C.

[0116] The MSL-producing microorganisms may or may not have been pre-cultured in the presence of an ammonium-containing composition prior to incubation with the treated composition.

[0117] Thus, in one embodiment, in step (c) the treated composition is incubated in a reactor with MSL-producing microorganisms which have not previously been cultured in the presence of an ammonium-containing composition. In this embodiment, typically the C / N ratio of the treated composition is from 30: 1 to 100: 1, preferably from 40: 1 to 90: 1, such as from 40: 1 to 80: 1, 40: 1 to 70: 1, 50: 1 to 80: 1, or 60: 1 to 80: 1. Typically the C / N ratio of the treated composition is from 40: 1 to 100: 1, preferably from 50: 1 to 90: 1, and more preferably from 60: 1 to 80: 1. The optimum C / N ratio in this embodiment reflects the fact that the MSL-producing microorganisms have not previously been cultured in the presence of an ammonium-containing composition and therefore (without wishing to be bound by any particular theory) it is believed that the microorganisms require a certain amount of ammonium to be present in the treated composition in order that the microorganisms have access to a sufficient level of nitrogen to grow and maintain an effective culture size throughout the course of the reaction to produce MSL from VFA.

[0118] In an alternative embodiment, however, in step (c) the treated composition is incubated in a reactor with MSL-producing microorganisms which have previously been cultured in the presence of an ammonium-containing composition. In this embodiment, typically the C / N ratio of the treated composition is from 60: 1 to 150: 1, preferably from 70: 1 to 120: 1, and more preferably from 80: 1 to 100: 1. Thus, the optimum C / N ratio in this embodiment is higher, because the MSL-producing microorganisms have already established a culture within an ammonium-containing medium and therefore (without wishing to be bound by any particular theory) it is believed that the microorganisms do not require as much ammonium to be present in the treated composition in order that the microorganisms can maintain an effective culture size throughout the course of the reaction to produce MSL from VFA. When the MSL-producing microorganisms are pre-treated with an ammonium-containing composition, the C / N ratio of that ammonium-containing composition is not particularly limited. However, typically the C / N ratio of that ammonium-containing composition is from 1 : 10 to 25: 1, preferably from 1 : 1 to 20: 1, and more preferably from 2: 1 to 15: 1, e.g. from 5: 1 to 10: 1.

[0119] The microbial storage lipids produced in step (c) can be any microbial storage lipids produced by microorganisms. Typically, the microbial storage lipids are selected from one or more polyhydroxyalkanoates (PHAs), one or more triacyl glycerols, one or more wax esters and one or more oils.

[0120] In an embodiment, the microbial storage lipids are one or more PHAs. Preferably, the one or more PHAs independently comprise a repeat unit of the following formula: wherein each R is the same or different and each R is independently a Ci-2oalkyl group, and each x is an integer from 1 to 12. Preferably each R is a C1-12 alkyl group, more preferably a C1-8 alkyl group and most preferably a C1-6 alkyl group. Preferably, x is an integer from 1 to 8, preferably from 1 to 6, and more preferably from 1 to 4. Thus, preferably, the one or more PHAs independently have the following formula: wherein R and x are as defined as above, and n is from 100 to 1000. Preferably, the one or more PHAs comprise a monomer repeat unit having from 6 to 12. More preferably, the one or more PHAs are selected from poly(3 -hydroxyhexanoic acid), poly(3- hydroxyheptanoic acid), poly(3-hydroxyoctanoic acid), poly(3-hydroxynonanoic acid), poly(3 -hydroxydecanoic acid), poly(3-hydroxyundecanoic acid), poly(3- hydroxydodecanoic acid) and a combination thereof.

[0121] In an embodiment, the microbial storage lipids are one or more wax esters. Preferably, the one or more wax esters independently have the formula R-(C=O)-O-R’, wherein R and R’ are independently selected from saturated or unsaturated hydrocarbon groups. Typically, R is selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12- 24 alkynyl group. More preferably, R is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group. Typically, R’ is selected from a C12-34 alkyl group, a C12- 34 alkenyl group and a C12-34 alkynyl group. More preferably, R’ is selected from a C24-34 alkyl group, a C24-34 alkenyl group and a C24-34 alkynyl group.

[0122] In an embodiment, the microbial storage lipids are one or more triacylglycerides. Preferably, the one or more triacylglycerides independently have the formula: wherein R, R’ and R” are independently selected from a C12-24 alkyl group, a C12-24 alkenyl group and a C12-24 alkynyl group. R, R’ and R” may be the same, but typically they are different. More preferably, R is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group. More preferably, R’ is selected from a C12-24 alkyl group, a C12- 24 alkenyl group and a C12-24 alkynyl group. More preferably, R” is selected from a C12-20 alkyl group, a C12-20 alkenyl group and a C12-20 alkynyl group.

[0123] In an embodiment, the microbial storage lipids are one or more oils. Preferably, the one ore more oils comprise one or more triacylglycerol compounds and / or one or more wax esters as defined herein.

[0124] Following production of MSLs in a process comprising steps (a) to (c) as described above, the desired MSL product may be subjected to further purification steps to obtain a product of high purity. Such purification steps are well-known in the art and may include, inter alia, stationary bed chromatography, simulated moving bed (SMB) chromatography, high-performance liquid chromatography, distillation, vacuum distillation, filtration and combinations thereof.

[0125] Further aspects

[0126] Further provided is the use of an aluminosilicate to reduce the nitrogen content of a composition comprising volatile fatty acids and ammonium-containing compounds (e.g. ammonium) and optionally further comprising biomass, thereby forming a treated composition having a carbon-to-nitrogen ratio of 30: 1 or greater. Also provided is a method comprising treating a composition comprising volatile fatty acids and ammonium- containing compounds (e.g. ammonium) and optionally further comprising biomass with an aluminosilicate, thereby reducing the nitrogen content of the composition so that the carbon-to-nitrogen ratio of the composition is 30: 1 or greater. In some embodiments the treated composition is for producing one or more microbial storage lipids via treatment (e.g. by incubation) of said composition with one or more microorganisms. The aluminosilicate is typically an aluminosilicate as disclosed herein. The composition is typically a composition as disclosed herein. The treated composition thereby produced typically has a carbon-to-nitrogen ratio as disclosed herein (e.g. a carbon-to-nitrogen ratio of from about 30: 1 to about 500: 1, e.g. about 35: 1 to about 350: 1, more preferably from 40: 1 to 200: 1, still more preferably from 40: 1 to 120: 1, yet more preferably from 40: 1 to 100: 1, even more preferably from 40: 1 to 90: 1, still more preferably from 40: 1 to 80: 1, e.g. from 40: 1 to 70: 1). In some embodiments the use is for reducing the nitrogen content of the composition for the bacterial production of microbial storage lipids from the treated composition. Also provided is a method of producing one or more microbial storage lipids from a composition comprising volatile fatty acids and ammonium-containing compounds (e.g. ammonium) and optionally further comprising biomass, wherein the composition has a carbon-to-nitrogen ratio of 30: 1 or greater; the method comprising incubating the composition with microorganisms thereby producing one or more microbial storage lipids. In some embodiments the composition is typically a composition as disclosed herein. In some embodiments the composition is a composition that has been treated with an aluminosilicate as described herein. The composition typically has a carbon-to-nitrogen ratio as disclosed herein (e.g. a carbon-to-nitrogen ratio of from about 30: 1 to about 500: 1, e.g. about 35: 1 to about 350: 1, more preferably from 40: 1 to 200: 1, still more preferably from 40: 1 to 120: 1, yet more preferably from 40: 1 to 100: 1, even more preferably from 40: 1 to 90: 1, still more preferably from 40: 1 to 80: 1, e.g. from 40: 1 to 70: 1). In some embodiments the microorganisms are as described herein. In some embodiments the composition is treated with said microorganisms as described herein.

[0127] Apparatus

[0128] The present invention also provides an apparatus which comprises:

[0129] (i) a composition comprising volatile fatty acids and ammonium-containing compounds, wherein the carbon-to-nitrogen ratio of the composition is 30:1 or greater;

[0130] (ii) an aluminosilicate; and

[0131] (iii) microorganisms.

[0132] Preferred features of the process as described above are also applicable to the present apparatus.

[0133] In some embodiments the apparatus comprises:

[0134] (i) a composition comprising volatile fatty acids and ammonium, wherein the carbon-to-nitrogen ratio of the composition is 30:1 or greater;

[0135] (ii) an aluminosilicate; and

[0136] (iii) microorganisms.

[0137] In some embodiments the VFA- and ammonium-containing composition has a C / N ratio of from 30: 1 to about 500: 1, e.g. about 35: 1 to about 350: 1, more preferably from 40: 1 to 200: 1, still more preferably from 40: 1 to 120: 1, yet more preferably from 40: 1 to 100: 1, even more preferably from 40: 1 to 90: 1, still more preferably from 40: 1 to 80: 1, e.g. from 40: 1 to 70: 1. Sometimes the VFA- and ammonium-containing composition has a C / N ratio of from 30: 1 to 500: 1, more preferably from 40: 1 to 200: 1, still more preferably from 50: 1 to 120: 1, yet more preferably from 52: 1 to 100: 1, even more preferably from 55: 1 to 90: 1, still more preferably from 58: 1 to 85: 1, and most preferably from 60: 1 to 80: 1.

[0138] Thus, the VFA- and ammonium-containing composition typically has a pH of 9 or less, preferably from 5 to 9, more preferably from 6 to 8, e.g. about 7.

[0139] Thus, the aluminosilicate is preferably selected from a sodium aluminosilicate, a potassium aluminosilicate, a calcium aluminosilicate, a magnesium aluminosilicate and a combination thereof. The aluminosilicate may be selected from a zeolite, a bentonite clay and a kaolinite clay. If the aluminosilicate is a zeolite, preferably the aluminosilicate is selected from analcime, chabazite, clinoptilolite, heulandite, natrolite, phillipsite, stilbite, amicite, barrerite, brewsterite, cowlesite, Cu Y zeolite, dachiardite-K, edingtonite, erionite, faujasite, ferrierits, garronite-Ca, gismondine, gmelinite, gonnardite, harmotome, hsianghualite, laumontite, levyne, mesolite, mordenite, nabesite, paulingite, pollucite, scolecite, SSZ-13, stellerite, thomsonite, ultramarine, wairakite, yugawaralite, ZSM-5 and a combination thereof. If the aluminosilicate is a bentonite clay, preferably the aluminosilicate is selected from sodium bentonite, calcium bentonite, potash bentonite and illite. Preferably, the aluminosilicate is a zeolite.

[0140] Thus, the microorganisms are typically any microorganisms which produce microbial storage lipids from volatile fatty acids (“MSL-producing microorganisms”). Preferably, the microorganisms are selected from one or more bacteria, one or more archaea and one or more fungi. More preferably the microorganisms are selected from one or more bacteria and one or more yeasts. Preferably, a single type of microorganism (i.e. a homogeneous microorganism culture) is present in the apparatus. Alternatively, a mixed culture of microorganisms is present in the apparatus. However, preferably, the apparatus does not comprise a mixed culture of microorganisms. Particularly preferred microorganisms for use in the apparatus: Pseudomonas pulida. Cupriavidus necalor. Vibrio natriegens, Vibrio nalriegens. Bacillus megaterium and Yarrowia lipolytica.

[0141] The publications, patent publications and other patent documents cited herein are entirely incorporated by reference. Herein, any reference to a term in the singular also encompasses its plural. Where the term “comprising”, “comprise” or “comprises” is used, said term may substituted by “consisting of’, “consist of’ or “consists of’ respectively, or by “consisting essentially of’, “consist essentially of’ or “consists essentially of’ respectively. Any reference to a numerical range or single numerical value also includes values that are about that range or single value. Unless otherwise indicated, any % value is based on the relative weight of the component or components in question.

[0142] Examples

[0143] The following are Examples that illustrate the present invention. However, these Examples are in no way intended to limit the scope of the invention.

[0144] EXAMPLE A

[0145] For ease of reference, the exemplified process described below is summarised by way of a process diagram in Fig. 10(b).

[0146] Example 1 - Preparation of materials

[0147] 1. Materials

[0148] Fresh Opuntia ficus indica (OFI) cladodes (biomass feedstock) were obtained from Pepe Aromas Lda. (Santa Justa, Portugal). These were oven dried at 80°C and ground in a blender (Waring USA) and screened through a 1 mm sieve to ensure consistent particle size.

[0149] Anaerobic digester (AD) sludge was obtained from an industrial AD facility processing municipal food waste (Severn Trent Green Energy, Cassington, UK).

[0150] All experimental reagents and VFA standards were purchased from Sigma-Aldrich (Merck Life Science, United Kingdom). The 3 -hydroxydecanoic acid standard was obtained from Manchester Organics Ltd. (Runcorn, United Kingdom).

[0151] Natural clinoptilolite zeolite was purchased from Finest Filters (Finest Aquatics Ltd., United Kingdom).

[0152] 2. Preparation of sodium-zeolite

[0153] Zeolite was equilibrated 1 : 1 (w / v) in 1 M NaCl solution overnight to saturate binding sites with Na+and then serially washed in deionized water (4x) to remove excess NaCl.

[0154] Zeolite was oven dried at 80°C before use in subsequent experiments.

[0155] 3. Preparation ofVFA-rich fermentate

[0156] VFA-rich fermentate was prepared by acidogenic fermentation of OFI biomass feedstock with AD sludge inoculum. Fermentations were performed with 2 L working volumes and concentration of 30 gVS / L OFI and 5 g / L AD sludge. The liquid phase consisted of a medium solution as per the recommendations of Angelidaki and Sanders (Rev. Environ. Sci. BiotechnoL, 2004, 3), with the omission of sodium sulfide.

[0157] Methanogenesis was inhibited by addition of 10 mM 2-bromoethane sulfonate. The headspace gas was not purged to remove excess oxygen. The headspace was kept anaerobic by means of a fermentation airlock, which permitted egress of surplus biogas, while isolating the system from ambient oxygen. Fermentations were incubated at 37°C with agitation at 125 rpm for 12 days, whereupon fermentation was arrested by freezing the cultures at -20°C.

[0158] Example 2 - Preparation of fermentate growth medium

[0159] The OFI fermentate was initially clarified by centrifugation at 2000 xg for 10 minutes and the supernatant collected. This was divided into two separate batches, of which one underwent zeolite treatment. The ‘treated’ fermentate was treated by incubation with 60% (w / v) of sodium-zeolite for two hours at 30 °C, with agitation at 125 rpm.

[0160] The pH of both treated and untreated fermentates was then adjusted to 7.0, and the two batches were autoclaved to induce precipitation. These batches were clarified by centrifugation at 12,000 xg for 15 minutes, the supernatant collected, and the pH again adjusted to 7.0. These were again clarified by centrifugation (12,000 xg for 15 minutes) to remove precipitates arising from pH adjustment.

[0161] These batches (treated and untreated) were then chemically characterized for their organic acid, total carbon, and total nitrogen composition (see Example 4 below), and the C / N ratio of the two media was thereby determined.

[0162] Microbial growth medium was prepared by diluting the clarified digestates 1 :3 with deionized water. For subsequent bioproduction experiments, the untreated fermentate provided the negative control. Various C / N ratios (i.e. the treatment groups) were obtained by blending the untreated and treated batches at different ratios, the highest C / N treatment group was provided by pure treated medium. 50 mL volumes of these media were aliquoted into 250 ml Erlenmeyer flasks, sealed with foil and autoclaved to achieve sterility.

[0163] As demonstrated by Fig. 2C, nearly all the total nitrogen detected in Opuntia digestate (i.e. in a medium prepared by the method above) could be accounted for as ammonium. This homogeneous nitrogen source is then in a form amenable to removal by zeolite, as per the requirements of the downstream bioproduction process. The relationship between ammonium removal and zeolite concentration is demonstrated in Fig. 7.

[0164] Example 3 - Pseudomonas putida growth time-courses

[0165] Cultures of Pseudomonas putida KT2440 were prepared in triplicate, with individual colonies taken to represent independent biological replicates. These colonies were used to inoculate initial precultures of 5 mL lysogeny broth, which were incubated overnight. Secondary and tertiary 50 mL precultures were prepared in 250 mL Erlenmeyer flasks, the former consisting of 50% lysogeny broth, 25% OFI fermentate and 25% deionized water, and the latter of 25% OFI fermentate in 75% deionized water. All precultures were incubated at 37°C, 125 rpm for ~12 hours.

[0166] The pellets from these final precultures were obtained by centrifugation at 12,000 rpm for 10 minutes, flushed with sterile PBS and used to inoculate the experimental flasks to an initial OD600 of 0.1. These were incubated at 37°C and 125 rpm.

[0167] P. putida cultures were sampled at 6-hour intervals and the OD600 determined. Once the culture OD600 reached 0.5, additional 5 mL samples were taken to measure PHA production. The pellet from these was obtained by centrifugation at 12,000 xg for 10 minutes, was resuspended in deionized water and again pelleted by centrifugation at 15,500 xg. Pellets were frozen at 80°C and lyophilized to allow determination of cell dry weights and to provide a dry pellet from which PHA could be extracted and quantified. Example 4 - Analytical Methods

[0168] The bulk characteristics of the OFI substrate and AD sludge inoculum were determined by standard methods (APHA, 1998 Standard Methods for the Examination of Water and Wastewater, American Water Works Association and Water Environment Federation), deviating from these only in that the oven temperature for total solids determination was set to 80°C. Determination of the cellulose, hemicellulose, and lignin content of the OFI substrate was performed by van Soest analysis (van Soest et al., J. Dairy Science, 1991, 74).

[0169] Liquid samples of both bulk fermentates and P. putida growth media were clarified by centrifugation at 15,500 xg and the supernatant collected prior to analysis. For VFA analysis clarified liquid samples were acidified by 1 : 1 dilution with 20% formic acid in 1.5 mL GC vials. VFAs quantified by gas chromatography with flame-ionization detection (GC-FID) using a ZB-FFAP column, with helium providing the carrier gas. Acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids were detected and quantified against standards; these values were summed to calculate total VFA (TVFA). Characterisation of bulk fermentate parameters for preparation of growth media was done using kit-based spectrophotometric assays from Hach Lange Ltd. (Manchester, United Kingdom) as follows: organic acids were quantified using the LCK365 kit, total organic carbon with the LCK386 kit, and total nitrogen with the LCK238 kit. C / N ratios of media were calculated as the ratio of TOC / TN as determined from these measures and are quoted here on a weight basis.

[0170] Example 5 - Extraction and quantification of PHA monomer 3 -hydroxydecanoic acid Quantification of PHA from lyophilized cell pellets was performed according to the protocol of Jia et al. (Bioresour. TechnoL, 2013, 140), with some minor alterations, wherein PHA undergoes methanolysis to obtain methylated monomers which are quantified by GC-FID detection. Lyophilized cell pellets were resuspended in 1 mL acidified methanol (9: 1 methanol: sulfuric acid v / v) and 2 mL of chloroform containing 10 mg / mL benzoic acid as an internal standard. These were incubated in sealed vials at 105°C for 2 hours and then cooled by incubation on ice. Once cool, 1 mL of ice-cold deionized water was added, the vial sealed and vortexed for 1 minute to mix. The vials were sat at ambient temperature to allow phase separation (~1 hour). The organic (chloroform) phase was collected and pipetted into 1.5 mL GC vials to which sodium sulphate had been added (as a desiccant). The methylated 3 -hydroxyalkanoate monomers were detected by GC-FID using a ZB-FFAP column with helium providing the carrier gas. Relative production levels of PHA were determined by quantification of the 3- hydroxydecanoic acid monomer, determined by Dartiailh et al. (Front. Bioeng.

[0171] BiotechnoL, 2021, 8) to be the most abundant PHA monomer produced in P. putida when short-chain VFA provides the carbon source.

[0172] Example 6 - Results

[0173] 1. Characterisation of initial biomass and AD sludge inoculum

[0174] Opuntia ficus indica biomass was found to have a volatile solids content of 83.51% of total solids (VS / TS), consistent with literature values for other related species such as Opuntia fragilis, Kalanchoe daigremonliana. and Ananas comosus (pineapple) (Lueangwattanapong et al.. Bioresour. TechnoL, 2020, 297). Lignin values were relatively low, at just 8.73%, consistent with values for Opuntia fragilis and Kalanchoe daigremontiana (Lueangwattanapong et al. , Bioresour. TechnoL, 2020, 297). AD sludge had a VS / TS of 60.30%, which is also in close agreement with literature values (Lueangwattanapong et al., Bioresour. TechnoL, 2020, 297; Tenci et al., Bioresour.

[0175] Technol. Reports, 2023, 21). It also was found to have a low initial VFA concentration (0.66 g / L), consistent with expectation for sludge from a methanogenic AD facility in which VFA is rapidly converted to methane.

[0176] Table 1: Characterisation parameters for initial biomass and AD sludge inoculum

[0177] 2. Characterisation of bulk VFA-rich fermentates, with and without zeolite treatment

[0178] The following results display controlled, effective and selective removal of ammonium from bulk fermentate over VFAs.

[0179] The bulk fermentate had a TVFA concentration of 13.31 ± 0.05 g / L. Given an initial concentration of 35 gVS / L of volatile solids (30 gVS of Opuntia, and 5 g / L AD sludge), this indicates a yield of 38.03% ± 0.15% VFA on a VS basis. The produced VFA was primarily acetate (C2) (50.1%) and butyrate (C4) (28.9%), with smaller, but still significant fractions of C3, C5, and C6 fatty acids detected (see Fig. 2A).

[0180] The effect of zeolite treatment on both carbon and nitrogen sources in the fermentate is shown in Fig. 2B-D. Zeolite treatment was not found to affect the abundance of either total organic carbon or of total organic acids in the fermentate (see Fig. 2B). Conversely both total nitrogen and total ammonium nitrogen were found to be heavily depleted (see Fig. 2C). The cumulative effect of this specific depletion of nitrogen in the fermentate can be seen in Fig. 2D, in which the C / N ratio of the fermentate shifts from 9.98 to 96.35 (mass basis). This lower value is rounded to 10 and higher value is rounded to 100 throughout the remainder of this discussion.

[0181] Zeolite treatment of the Opuntia fermentate produced a considerable decrease in total and ammonium nitrogen concentrations (see Fig. 2C). Zeolite demonstrated specificity for ammonium as concentrations of VFA were equal between treated and untreated conditions. This retention of VFAs is relevant as it suggests zeolite imposes no measurable loss in VFA yields which would need to be accounted for. The contrast between change in VFA concentration and ammonium concentration is shown in Fig. 8. Ammonium removal plateaued when all available binding sites were occupied. Different zeolite loadings were tested to increase the C / N ratio, with a maximum ratio of 100: 1 achieved, corresponding to 92% ammonium removal in a 4-hour time period (see Fig. 9).

[0182] P. putida demonstrated robust growth on the diluted fermentate medium at all C / N ratios tested (i.e., from 10: 1 to 100: 1). A reduced lag phase was observed in zeolite-treated media (in which nitrogen was reduced) in a dose dependent manner: the longest lag phases were seen in untreated media (C / N of 10: 1) and the shortest were seen in the most heavily treated media (C / N of 100: 1). The inverse of this effect was seen in growth rates, with the highest maximal growth rates seen at C / N 10: 1 and the slowest growth rates in C / N 100: 1, which likely represents the increased capacity for biomass accumulation afforded by the ammonium present in these samples.

[0183] 3. Effect of zeolite treatment on P. putida polyhydroxyalkanoate production on fermentate The following results show that zeolite treatment can provide a C / N ratio which greatly increases yield of PHA. The production is optimised at a C / N ratio of 60: 1-80: 1, with a 3 -fold increase in both titre and cell content of PHA at these ratios compared to untreated control fermentate.

[0184] Polyhydroxyalkanoate production, as measured through the abundance of the 3- hydroxydecanoate monomer, is shown in Fig. 4. PHA titres (see Fig. 4A) and yields (see Fig. 4B) were found to be highly responsive to both C / N ratio and to incubation time.

[0185] Maximal PHA titres (179.3 mg / L) were seen at a C / N ratio of 80: 1 at 36 hours, which corresponds to the maximal OD600 seen for this culture in Fig. 3E. This treatment and timepoint was also found to produce the maximal observed titres of PHA when measured as a percentage of cell dry weight (27.1%) suggesting this point represents a global optimum. Titres at C / N 60: 1 at 36 hours were comparable to those at C / N 80: 1, though yields were lower, suggesting a relatively higher allocation of carbon to biomass at this timepoint, likely due to the presence of more nitrogen, which facilitated biomass formation. Similarly, comparable yields were seen at C / N of 80: 1 at both 30 and 36 hours, however as absolute titres were maximal at 36 hours, the additional 6 hours of incubation allowed for additional PHA accumulation without an adverse effect on yields.

[0186] The various conditions achieved their maximal VFA titres and yields at different timepoints. For C / N ratios of 10: 1, 20: 1, 40: 1, 60: 1, 80: 1 and 100: 1 maximal titres were seen at 30, 24, 24, 36, 36, and 43 hours, respectively, while maximal yields were seen at 30, 24, 24, 30, 36, and 36 hours, respectively. These titre and yield maxima are shown in Fig. 5 A and 5B, respectively. Yield and titre maxima for each of the treatment groups were analysed for the statistical significance of their deviation from the untreated control group by Dunnett’s Multiple Comparisons Test. Both yields and titres deviated significantly from the control at C / N 60: 1 and 80: 1 (p-values for all four relevant comparisons <0.0001). Maximal titres at a C / N of 40: 1 had a p-value of 0.019. All other pairwise comparisons yielded nonsignificant results. The results of the experiments are summarised in Table 2 below.

[0187] Table 2: Summary of observed PHA titre and yield maxima at various C / N ratios.

[0188] A full accounting of the allocation of input volatile solids through the two-step acidogenic and PHA-accumulating stages of the process for the C / N 80: 1 condition is displayed graphically in Fig. 6.

[0189] EXAMPLE B

[0190] Materials and Methods

[0191] Materials

[0192] Fresh Opuntia ficus indica (OFI) cladodes were obtained from Pepe Aromas Lda. (Azaruja, Portugal). These were oven dried at 80 °C, ground in a blender (Waring, USA) and screened through a 1 mm sieve to ensure consistent particle size. Anaerobic digestion sludge was obtained from an industrial AD facility processing municipal food waste (Severn Trent Green Energy, Cassington, UK).

[0193] All experimental reagents were purchased from Sigma- Aldrich (Merck Life Science, United Kingdom). The 3-hydroxyalkanoic acid standards used for quantification of PHA monomers were obtained from Sigma-Aldrich, Manchester Organics Ltd. (Runcorn, United Kingdom), and Tokyo Chemical Industry Europe N.V. (Zwijndrecht, Belgium). Natural clinoptilolite zeolite was purchased from Finest Filters (Finest Aquatics Ltd., United Kingdom). Preparation of sodium-zeolite

[0194] Zeolite was equilibrated 1 : 1 (w:v) in IM NaCl solution overnight to saturate binding sites with Na+and then serially washed in excess deionized water (4x) to remove excess NaCl. Zeolite was oven dried at 80 °C before use in subsequent experiments. Preparation of VFA-rich fermentate

[0195] VFA-rich fermentate was prepared by acidogenic fermentation of OFI biomass substrate with AD sludge inoculum. Fermentations were performed with 2 L working volumes and concentration of 30 gVS / L OFI and 5 gVS / L AD sludge. The liquid phase consisted of a medium solution prepared as per the recommendations of Angelidaki and Sanders (2004), with the omission of sodium sulphide. Methanogenesis was inhibited by addition of 10 mM 2-bromoethane sulfonate. The headspace gas was not purged to remove excess oxygen. The headspace was kept anaerobic by means of a fermentation airlock, which permitted egress of surplus biogas, while isolating the system from ambient oxygen. Fermentations were incubated at 37 °C with agitation at 125 rpm for 12 days, whereupon fermentation was arrested by freezing the cultures at -20 °C.

[0196] Preparation of fermentate growth medium

[0197] The OFI fermentate was initially clarified by centrifugation at 2000 xg for 10 minutes and the supernatant collected. This was divided into two separate batches, of which one underwent zeolite treatment. The ‘treated’ fermentate was treated by incubation with 60% (w / v) of sodium-zeolite for two hours at 30 °C, with agitation at 125 rpm.

[0198] The pH of both treated and untreated fermentates was then adjusted to 7.0, and the two batches were autoclaved to induce precipitation. These batches were clarified by centrifugation at 15,500 xg for 15 minutes, the supernatant collected, and the pH again adjusted to 7.0. These were again clarified by centrifugation (15,500 xg for 15 minutes) to remove precipitates arising from pH adjustment. These batches (treated and untreated) were then chemically characterized for their organic acid, total carbon, and total nitrogen composition, and the C:N ratio of the two media was thereby determined.

[0199] Microbial growth medium was prepared by diluting the clarified digestates 1 :3 with deionized water. For subsequent bioproduction experiments, the untreated fermentate provided the negative control. Various C:N ratios (i.e., the treatment groups) were obtained by blending the untreated and treated batches at different ratios, the highest C / N treatment group was provided by pure treated medium. Given a VFA concentration of ~11.5 g / L in the fermentate, this dilution method gave a medium concentration of <3 g / L. 50 mL volumes of these media were aliquoted into 250 ml Erlenmeyer flasks, sealed with foil and autoclaved to achieve sterility.

[0200] Pseudomonas putida growth time-courses

[0201] Cultures of Pseudomonas putida KT2440 were prepared in triplicate, with individual colonies taken to represent independent biological replicates. These colonies were used to inoculate initial precultures of 5 mL lysogeny broth, which were incubated overnight. Secondary and tertiary 50 mL precultures were prepared in 250 mL Erlenmeyer flasks, the former consisting of 50% lysogeny broth, 25% OFI fermentate and 25% deionized water, and the latter of 25% OFI fermentate in 75% deionized water. All precultures were incubated at 37 °C, 125 rpm for ~12 hours. The pellets from these final precultures were obtained by centrifugation at 15,500 xg for 10 minutes, flushed with sterile PBS and used to inoculate the experimental flasks to an initial ODeoo of 0.1. These cultures were incubated at 37 °C and 125 rpm.

[0202] P. putida cultures were sampled at 6-hour intervals and the ODeoo determined. Once the culture ODeoo reached 0.5, additional 5 mL samples were taken to measure PHA production. The pellet from these was obtained by centrifugation at 12,000 xg for 10 minutes, was resuspended in deionized water and again pelleted by centrifugation at 15,500 xg. Pellets were frozen at 80 °C and lyophilized to allow determination of cell dry weights and to obtain a dry pellet from which PHA could be extracted and quantified. Analytical methods

[0203] The bulk characteristics of the OFI substrate and AD sludge inoculum were determined by standard methods (APHA, 1998), deviating from these only in that the oven temperature for total solids determination was set to 80 °C. Determination of the cellulose, hemicellulose, and lignin content of the OFI substrate was performed by van Soest analysis (van Soest et al., J. Dairy Science, 1991, 74).

[0204] Liquid samples of both bulk fermentates and P. putida growth media were clarified by centrifugation at 15,500 xg and the supernatant collected prior to analysis. For VFA analysis clarified liquid samples were acidified by 1 : 1 dilution with 20% formic acid in 1.5 mL GC vials. VFA was quantified by gas chromatography with flame-ionization detection (GC-FID) using a ZB-FFAP column, with helium providing the carrier gas. Acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids were detected and quantified against standards, these values were summed to calculate total VFA (TVFA). Characterisation of bulk fermentate parameters for preparation of growth media was done using kit-based spectrophotometric assays from Hach Lange Ltd. (Manchester, United Kingdom) as follows: Organic acids were quantified using the LCK365 kit, total organic carbon with the LCK386 kit, and total nitrogen with the LCK238 kit. C:N ratios of media were calculated as the ratio of TOC:TN as determined from these measures and are quoted on a mass basis. Extraction and quanti fication of PHA

[0205] Quantification of PHA from lyophilized cell pellets was performed according to the protocol of Jia et al. (2013), with some minor alterations, wherein PHA undergoes methanolysis to obtain methylated monomers which are then quantified by GC-FID detection. Lyophilized cell pellets were resuspended in 1 mL acidified methanol (9: 1 methanol: sulfuric acid v / v) and 2 mL of chloroform containing 10 mg / mL benzoic acid as an internal standard. These were incubated in sealed vials at 105 °C for 2 hours and then cooled by incubation on ice. Once cool, 1 mL of ice-cold deionized water was added, and the vial sealed and vortexed for 1 minute to mix. The vials were sat at ambient temperature to allow phase separation (~1 hour). The organic (chloroform) phase was collected and pipetted into 1.5 mL GC vials to which sodium sulphate had been added (as a desiccant). The methylated monomers were detected by GC-FID using a 30 m x 0.25 mm x 0.25 pm fused-silica ZB-FFAP capillary column with helium providing the carrier gas and quantified against standards which had been subjected to the same methanolysis protocol. Total PHA was determined as the sum of the monomers (these being 3 -hydroxybutyrate, 3- hydroxyval erate, 3 -hydroxyhexanoate, 3 -hydroxyh eptanoate, 3-hydroxyoctanoate, 3- hydroxynonanoate, 3-hydroxydecanoate, 3-hydroxyundecanoate, 3hydroxydodecanoate, 3- hydroxytetradecanoate) .

[0206] Results

[0207] Characterisation of AF substrate and feedstock

[0208] Initial biomass and AD sludge inoculum were characterised and found to be as described in Example A. Characterisation o f bulk VFA-rich fermentate s, with and without zeolite treatment

[0209] The bulk fermentate had a TVFA concentration of 13.31 ±0.05 g / L. Given an initial concentration of 35 gVS / L of volatile solids (30 gVS of Opuntia, and 5 g / L AD sludge), this indicates a yield of 38.03 ±0.15 % VFA on a VS basis. The produced VFA was primarily acetate (C2) (50.1%) and butyrate (C4) (28.9%), with smaller, but still significant fractions of C3, 5, and 6 fatty acids detected (Fig. 11 A).

[0210] The effect of zeolite treatment on both carbon and nitrogen sources in the fermentate is shown in Fig. 11B-D. Zeolite treatment was not found to affect the abundance of either total organic carbon or of total organic acids in the fermentate, as confirmed by Student’s t-test (Fig. 1 IB). Conversely both total nitrogen and total ammonium nitrogen were found to be heavily depleted (Fig. 11C). The cumulative effect of this specific depletion of nitrogen in the fermentate can be seen in Fig. 1 ID, in which the C:N ratio of the fermentate shifts from 12.5 to 93.5 (mass basis). Growth characteristics ofP. putida on fermentate s

[0211] P. putida demonstrated robust growth on the diluted fermentate medium at all C:N ratios tested (i.e., from 12.5 to 93.5) (Fig. 12). A reduced lag phase was observed in zeolite-treated media (in which nitrogen was reduced) in a dose dependent manner: the longest lag phases were seen in untreated media (C:N of 12.5) and the shortest were seen in the most heavily treated media (C:N of 93.5). The inverse of this effect was seen in growth rates, with the highest maximal growth rates seen at C:N 10 and the slowest growth rates in C:N 93.5, which likely represents the increased capacity for biomass accumulation afforded by the ammonium present in these samples.

[0212] Effect of zeolite treatment on P. putida polyhydroxyalkanoate production on fermentate

[0213] Polyhydroxyalkanoate production, is shown in Fig. 13. PHA titres (Fig. 13A) and yields (Fig. 13B) were found to be highly responsive to both C:N ratio and to incubation time.

[0214] Maximal PHA titres (270 ±20 mg / L) were seen at a C:N ratio of 43 at 36 hours (Fig. 13 A), which corresponds to the maximal ODeoo seen for this culture (Fig 12). This treatment and timepoint was also found to produce the maximal observed titres of PHA when measured as a percentage of cell dry weight (40 ±7%) suggesting this point represents a global optimum for PHA production under these conditions (Fig. 13B). Across conditions, PHA yields and titres were seen to decrease after achieving their respective maxima, which likely represents the consumption of PHA as a carbon source by P. putida, once free carbon in the medium was exhausted, beyond which point biomass also began to decrease as seen in Fig. 12.

[0215] The various conditions achieved their maximal VFA titres and yields at different timepoints. For C / N ratios of 12.5, 13.5, 17.5, 26, 43 and 93.5 maximal titres were seen at 30, 24, 24, 36, 36, and 43 hours, respectively, while maximal yields were seen at 30, 24, 24, 36, 36, and 30 hours. These titre and yield maxima are shown in Fig. 14A and B. Mean yield and titre maxima for each of the treatment groups were analysed for the statistical significance of their deviation from the untreated control group by Dunnett’s Multiple Comparisons Test. Both yields and titres deviated significantly from the control at C:N ratios of 26 and above. The data in Fig. 14, including the p-values of pairwise comparisons between zeolite treated conditions and the negative control, are summarised in the following Table 3.

[0216] Table 3: Summary of observed PHA titre and yield maxima at various C:N ratios. p-values of pairwise comparisons between treatment groups and the negative control are provided, n.s. = nonsignificant.

[0217] Zeolite treatment of Opuntia fermentate produced a considerable decrease in total and ammonium nitrogen concentrations (Fig. 11) Zeolite demonstrated specificity for ammonium as concentrations of VFA were equal between treated and untreated conditions.

[0218] The synergy of acidogenic fermentation augmented with zeolite purification combines the benefits of heterogeneous feedstocks with substrate homogeneity and exquisite control of reactor parameters. Acidogenic fermentation allows production of VFA from both the soluble and insoluble fractions of biomass. It homogenizes all available carbon sources to a pool of VFA which is widely utilizable by many PHA accumulating species. Moreover, the heterogeneous nitrogen sources in biomass, which may take the form of proteins, free amino acids, urea, nitrates, or other chemicals, are hydrolysed to ammonium ions. This is evident in Fig. 11 where nearly all the total nitrogen detected in Opuntia digestate could be accounted for as ammonium. This homogeneous nitrogen source is then in a form amenable to removal by zeolite. The ability to modify the fermentate to the needs of any specific downstream process, whether a pure or mixed culture, is a specific advantage of the disclosed methods. EXAMPLE C

[0219] Composition of P. putida PHAs across the time / C:N parameter space

[0220] The compositions of PHAs produced at each timepoint in Example B are provided in Fig. 15. Under most conditions and timepoints PHAs were found to be rich in the CIO monomer 3 -hydroxy decanoic acid, which typically represented a majority of the polymer (by mass). Considerable quantities of the C6 monomer 3-hydroxyhexanoic acid were also observed. This monomer was particularly abundant in early timepoints, and in the C:N 93.5 condition, though PHAs broadly showed a trend of reducing C6 content with increased incubation time in all conditions.

[0221] Considerable variation in PHA composition was observed between treatment conditions and timepoints though this variation was mainly limited to the relative abundance of the C6 and CIO monomers. Whilst the most abundant PHA was a CIO-rich polymer with smaller percentages of other monomers under many conditions, at early timepoints and in the C:N 93.5 condition, the C6 monomer was found to dominate. Without being bound by theory, this may reflect a preferential consumption of hexanoate at early timepoints by P. putida.

[0222] EXAMPLE D

[0223] The underlying mechanism of nitrogen limitation driving PHA accumulation is not specific to Pseudomonas. As a PHA “hyperaccumulator” C. necator is capable of accumulating up to and even above 80% of its CDW as PHA. C. necator produces short chain PHAs, containing mostly the 3 -hydroxybutyrate monomer, and has primarily been investigated as a producer of the homopolymer PHB.

[0224] Materials and Methods

[0225] Materials

[0226] Sugarcane molasses was purchased from Harvest Foods (Alexandria, Egypt). AD sludge, which provided the inoculum for acidogenic fermentations, was obtained from Severn Trent Green Power (Cassington, UK) and stored anaerobically before use. Preparation of Zeolites

[0227] Zeolites used in these experiments were graded through a riddle to ensure a particle size of 1-3 mm and equilibrated by overnight incubation with 1 M NaCl at ambient temperature (21 °C). Equilibrated zeolites were flushed of surplus NaCl by washing in excess deionised water and oven dried at 80 °C before use.

[0228] Preparation offermentate

[0229] Bulk acidogenic fermentations were performed in triplicate with a 1.5 L working volume. The respective substrate and inocula were added to final concentrations of approximately 30 and 5 gVS / L, respectively. Molasses was added on a COD basis to a concentration of 43 gCOD / L.

[0230] Trace elements and micronutrients were provided by a medium solution, prepared according to Angelidaki and Sanders (Rev. Environ. Sci. BiotechnoL, 2004, 3), with the exclusion of sodium sulphide. The methanogenesis inhibitor bromoethane sulfonate was added to a final concentration of 10 mM and the medium pH adjusted to 8.0. Fermentations were incubated under anaerobic conditions at 37°C for 12 days with constant agitation at 125 rpm. Use of a fermentation airlock allowed relief of biogas pressure while maintaining fermentations under anaerobic conditions.

[0231] Zeolite treatment of acidogenic fermentates

[0232] Fermentates were zeolite treated with 50-60% (w / v) sodium-equilibrated zeolite for two hours, with shaking incubation at 125 rpm at 30 °C to obtain a treated (high C:N) fermentate.

[0233] Preparation of fermentate-based microbial growth media for C. necator

[0234] All bulk fermentates underwent clarification, pH-adjustment, and autoclaving. All batches were subjected to chemical characterisation for their organic acid, total carbon, SCOD, total ammonium nitrogen, and total nitrogen compositions via Hach methods and their C:N ratios determined. Treated (high C:N) and untreated (low C:N) fermentates were back blended to achieve intermediate C:N ratios.

[0235] Microbial growth medium was prepared by diluting clarified fermentates with deionised water to achieve VFA concentrations consistent with the VFA tolerances of C. necator determined by initial growth experiments. These concentrations equated to <1 g / L VFA for C. necator.

[0236] A high culture volume was used for C. necator experiments to compensate for the low culture density: 200 mL of media was added to 1 L flasks and these were sealed and autoclaved.

[0237] Culture conditions C. necator Cultures of C. necator Hl 6 were prepared in triplicate, with individual colonies, grown on LB A, taken to represent independent biological replicates. These colonies were used to inoculate initial precultures of 5 mL lysogeny broth, which were incubated overnight. Secondary and tertiary 50 mL precultures were prepared in 250 mL Erlenmeyer flasks. Fermentate concentrations in each were reduced to 5%, with the difference made up by deionized water. The volume of the final preculture was also increased to 200 mL.

[0238] C. necator cultures were incubated at 30 °C, 125 rpm for ~12 hours. The pellets from the final preculture condition were obtained by centrifugation at 12,000 xg for 10 minutes, flushed with sterile, pH 7.0 PBS and used to inoculate the experimental flasks to an initial ODeoo of 0.1.

[0239] Experimental cultures of C. necator were incubated at 30 °C with agitation at 125 rpm, sampled at regular intervals and the ODeoo determined. Once the culture ODeoo reached 0.25, additional samples of 10 mL were taken to measure PHA production. The pellet from these cultures was obtained by centrifugation at 12,000 xg for 10 minutes, resuspension in deionized water and re-centrifugation at 15,500 xg. Pellets were frozen at 80 °C and lyophilized to allow determination of cell dry weights and to provide a dry pellet from which PHA could be extracted and quantified.

[0240] Growth experiments

[0241] Growth experiments were conducted broadly along the lines of those with Pseudomonas in Example A and B, except that a 20x dilution of fermentate in deionised water was used rather than the 4x dilution that had been used for Pseudomonas. To compensate for this change, the working volume was increased, and experiments performed in a 200 mL working volume in IL conical flasks, and the sample volume taken at each timepoint for analysis increased to 10 mL of culture.

[0242] Analytical methods

[0243] Bulk media parameters: TOC, TOA, TN, NHC-N, were determined by Hach Lange analysis kits. SCOD was determined by Hach Lange LC1400 COD analysis kits, using a DR 2800 Hach-Lange spectrophotometer. Analysis of VFA concentrations was performed by standard GC-FID protocols and determined against standards.

[0244] Quantification of PHA from lyophilized cell pellets was performed according to the protocol in Example A (Jia et al. Bioresour. TechnoL, 2013, 140), with some minor alterations. Quantification of PHA was performed against standards, subjected to the same methanolysis and extraction protocol as the cell pellets. Total PHA levels were determined by quantification of the individual monomers, with total PHA taken as the sum of these numbers. The 3-hydroxyalkanoic acid standards used for quantification of PHA monomers were obtained from Sigma-Aldrich, Manchester Organics Ltd., and Tokyo Chemical Industry Europe N. V.

[0245] Results

[0246] Molasses fermentate was treated with zeolite as described in Example B. The bulk parameters of the untreated and treated fermentates are shown in Figure 16. On a gram- per-gram basis, total organic carbon and total organic acid concentrations were found to be roughly equivalent. The absolute concentration of organic acids was approximately equal to that in Opuntia fermentate.

[0247] C. necator grew well on the diluted fermentate medium, showing rapid growth upon inoculation, with little lag phase. Growth and PHA production in both the untreated control and low-zeolite treated conditions (C:N 12 and 31) peaked at an ODeoo of approximately 1.25, at 20 hours - corresponding to the point at which medium VFA was depleted (Fig. 17B), beyond which point they gradually declined. The maximum mean PHA titres observed in each condition in Fig. 17C are again shown in Fig. 18 A. In addition, Fig. 18B presents the maximum observed PHA yields in each of the three conditions in which PHA yield is calculated on a percentage basis against CDW.

[0248] PHA titres and yields in the control achieved maxima of 156 ±6 mg / L and 49 ±0.04 % CDW, respectively. Pairwise significance tests between means of treatment groups and the negative control found all comparisons to be statistically significant at at least the 5% level. Maxima in the lightly-treated condition (C:N 30.8) were 192 ±3 mg / L (p = 0.0002), and 56 ±0.02 % CDW (p = 0.0379), while in the heavily zeolite-treated condition (C:N 67) they were calculated as being as high as 247 ± 5 mg / L (p < 0.0001) and 94 ±0.02 % CDW (p < 0.0001), indicative of a significant increase in both titre and concentration of PHA in both treated conditions.

[0249] Bioproduction of PHA was demonstrated in C. necator, using a molasses fermentate as the carbon and nitrogen source. The fermentate was treated with zeolite to induce nitrogen limitation and several C / N ratios, corresponding to differing degrees of zeolite treatment were analysed. The molasses digestate used in this experiment was prepared using the standard protocol used in Example B for the preparation of an Opuntia fermentate. Molasses was selected as the substrate for this digestion in part because of its high solubility which was expected to lend it a high degree of anaerobic degradability.

[0250] C. necator showed rapid growth in all zeolite treatment conditions with growth curves even lacking the lag phase seen in P. putida experiments, which may have been facilitated by the presence of unfermented sugars, which could have supplemented the VFA as an additional carbon source.

[0251] As seen in Fig. 17C, titres in both zeolite-treated conditions exceed those in the negative control at all timepoints from t=21. An even greater bifurcation in titres occurs from t=32 after which point PHA titres are highest in the most heavily treated, C:N 67 condition. The improvement in PHA titres with zeolite treatment is most clearly shown in Fig. 18 A, which compares the PHA titre maxima observed for each treatment across all timepoints. Titre maxima of 156 ±6, 192 ±3, and 247 ±5, mg / L PHA were observed for the negative control, and the two zeolite-treatments of C:N 31 and C:N 67, respectively.

[0252] PHA yields (%CDW) are similarly higher in zeolite-treated conditions than in the negative control, with the C:N 67 condition in particular showing nearly double the yield of the negative control. Noise levels around these means were low, as indicated by their standard deviations, indicating very good agreement between the biological triplicates. This allowed pairwise tests to confirm the statistical significance of the differences between all treatment means and that of the control.

[0253] The highest production values were found in this experiment at the highest C:N ratio tested (C:N 67).

[0254] These results confirm the disclosed methods can be effectively used in the production of short-chain polyhydroxyalkanoates, suggesting this as a route for their production from low-cost carbon sources. The demonstrated short-chain PHA production using C. necator complements the medium-chain PHA production demonstrated in P. putida.

Claims

CLAIMS1. A method for preparing one or more microbial storage lipids from biomass, said method comprising:(a) subjecting the biomass to acidogenic fermentation to produce a composition comprising (i) volatile fatty acids and (ii) ammonium-containing compounds;(b) treating said composition with an aluminosilicate such that the carbon-to- nitrogen ratio of the treated composition is 30: 1 or greater; and(c) incubating the treated composition with microorganisms to produce one or more microbial storage lipids.

2. The method of claim 1, wherein the one or more microbial storage lipids is selected from one or more polyhydroxyalkanoates (PHAs), one or more wax esters, one or more triacyglycerides and one or more oils.

3. The method of claim 2, wherein the one or more polyhydroxyalkanoates comprise a monomer repeat unit having from 3 to 26 carbon atoms, preferably from 3 to 14 carbon atoms, and more preferably wherein the one or more polyhydroxyalkanoates are selected from poly(3 -hydroxybutanoic acid), poly(3 -hydroxypentanoic acid), poly(3 -hydroxyhexanoic acid), poly(3-hydroxyheptanoic acid), poly(3- hydroxyoctanoic acid), poly(3-hydroxynonanoic acid), poly(3 -hydroxy decanoic acid), poly(3-hydroxyundecanoic acid), poly(3-hydroxydodecanoic acid), poly(3- hydroxytetradecanoic acid), copolymers of two or more monomer units selected from 3 -hydroxybutanoic acid, 3-hydroxypentanoic acid, 3 -hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3 -hydroxy octanoic acid, 3-hydroxynonanoic acid, 3- hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid, and a combination thereof.

4. The method of any one of claims 1 to 3, wherein the biomass is selected from plant matter, agricultural waste, food waste, meat industry waste, sewage, human or animal effluent, waste from biofuel production, molasses, blood, cheese whey, pulp and paper mill effluents, and olive oil mill effluents.

5. The method of any one of claims 1 to 4, wherein in (a) the acidogenic fermentation is carried out by microorganisms, preferably a mixed microbial culture.

6. The method of any one of claims 1 to 5, wherein in (a) the composition has a carbon-to-nitrogen ratio of from 1:1 to 20: 1.

7. The method of any one of claims 1 to 6, wherein in (a) the composition comprises volatile fatty acids having from 2 to 6 carbon atoms, preferably selected from acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid and a combination thereof.

8. The method of any one of claims 1 to 7, wherein in (b) the aluminosilicate is:(i) selected from a sodium aluminosilicate, a potassium aluminosilicate, a calcium aluminosilicate, a magnesium aluminosilicate and a combination thereof; and / or(ii) selected from a zeolite, a bentonite clay and a kaolinite clay, preferably a zeolite; optionally wherein the zeolite is selected from analcime, chabazite, clinoptilolite, heulandite, natrolite, phillipsite, stilbite, amicite, barrerite, brewsterite, cowlesite, Cu Y zeolite, dachiardite-K, edingtonite, erionite, faujasite, ferrierits, garronite-Ca, gismondine, gmelinite, gonnardite, harmotome, hsianghualite, laumontite, levyne, mesolite, mordenite, nabesite, paulingite, pollucite, scolecite, SSZ-13, stellerite, thomsonite, ultramarine, wairakite, yugawaralite, ZSM-5 and a combination thereof; and optionally wherein the bentonite clay is selected from sodium bentonite, calcium bentonite, potash bentonite and illite.

9. The method of any one of claims 1 to 8, wherein in (b) the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 40:1 to 500:1.

10. The method of claim 9, wherein in (b) the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 40: 1 to 100: 1, and preferably from 40: 1 to 70: 1.

11. The method of any one of claims 1 to 10, wherein steps (a) and (b) are carried out sequentially.

12. The method of any one of claims 1 to 10, wherein steps (a) and (b) are carried out simultaneously.

13. The method of any one of claims 1 to 12, wherein the treated composition of (b) is adjusted to a pH of 9 or less, preferably from 5 to 9, and more preferably from 6 to 8, before being subjected to step (c).

14. The method of any one of claims 1 to 13, wherein in (c) the treated composition is incubated with microorganisms selected from bacteria, archaea and fungi, preferably bacteria or yeast, and optionally wherein the microorganisms are selected from Pseudomonas pulida. Cupriavidus necalor. Vibrio natriegens, Acinetobacter baylyi, Bacillus megaterium and Yarrowia lipolytica.

15. The method of claim 14, wherein in (c):(i) the treated composition is incubated with Pseudomonas putida and the one or more microbial storage lipids is one or more polyhydroxyalkanoates (PH As); or(ii) the treated composition is incubated with Cupriavidus necator and the one or more microbial storage lipids is one or more polyhydroxyalkanoates (PH As); or(iii) the treated composition is incubated with Vibrio natriegens and the one or more microbial storage lipids is one or more polyhydroxyalkanoates (PHAs); or(iv) the treated composition is incubated with Acinetobacter baylyi and the one or more microbial storage lipids is one or more wax esters; or(v) the treated composition is incubated with Bacillus megaterium and the one or more microbial storage lipids is one or more polyhydroxyalkanoates (PHAs); or(vi) the treated composition is incubated with Yarrowia lipolytica and the one or more microbial storage lipids is one or more triacylglycerides.

16. The method of any one of claims 1 to 15, wherein in (c) the treated composition is incubated in a reactor with microorganisms which have not previously been cultured in the presence of an ammonium-containing composition.

17. The method of claim 16, wherein in (b) the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 30: 1 to 100: 1, preferably from 40: 1 to 90: 1, and more preferably from 40: 1 to 70: 1.

18. The method of any one of claims 1 to 15, wherein in (c) the treated composition is incubated in a reactor with microorganisms which have previously been cultured in the presence of an ammonium-containing composition.

19. The method of claim 18, wherein in (b) the composition is treated with an aluminosilicate such that the carbon-to-nitrogen ratio of the treated composition is from 60: 1 to 150: 1, preferably from 70: 1 to 120: 1, and more preferably from 80: 1 to 100: 1.

20. The method of claim 18 or claim 19, wherein the bacteria have previously been cultured in the presence of an ammonium-containing composition having a carbon- to-nitrogen ratio of from 1 : 10 to 25: 1, preferably from 1 : 1 to 20: 1, and more preferably from 2: 1 to 10: 1.

21. The method of any one of claims 1 to 20, wherein in (c) the treated composition is incubated with microorganisms for from 24 to 60 hours, preferably from 30 to 48 hours, and preferably for from 36 to 44 hours.

22. The method of any one of claims 1 to 21, wherein in (c) the treated composition is incubated with microorganisms at from 30°C to 40°C.

23. A method of producing one or more microbial storage lipids from a composition comprising volatile fatty acids and ammonium-containing compounds and optionally further comprising biomass, wherein the composition has a carbon-to-nitrogen ratio of 30: 1 or greater; the method comprising incubating the composition with microorganisms thereby producing one or more microbial storage lipids; wherein preferably said composition is obtainable by treatment of a composition comprising volatile fatty acids, ammonium-containing compounds and optionally biomass with an aluminosilicate; optionally wherein the one or more microbial storage lipids; the composition; the carbon-to-nitrogen ratio; the microorganisms and / or the method are as defined in any one of claims 2 to 22.

24. A microbial storage lipid which is obtainable by the method of any of the preceding claims.

25. Use of an aluminosilicate to reduce the nitrogen content of a composition comprising volatile fatty acids and ammonium-containing compounds and optionally further comprising biomass, thereby forming a treated composition having a carbon-to- nitrogen ratio of 30: 1 or greater; wherein preferably said treated composition is for producing one or more microbial storage lipids by incubation of said composition with one or more microorganisms; optionally wherein the composition; the aluminosilicate; the carbon-to-nitrogen ratio; the microbial storage lipids; the microorganisms and / or the use are as defined in any one of claims 2 to 22.

26. An apparatus which comprises:(i) a composition comprising volatile fatty acids and ammonium-containing compounds, wherein the carbon-to-nitrogen ratio of the composition is 30:1 or greater;(ii) an aluminosilicate; and(iii) microorganisms.

27. An apparatus according to claim 26, wherein:(i) the composition comprising volatile fatty acids and ammonium-containing compounds has a carbon-to-nitrogen ratio from 40: 1 to 120: 1, preferably from 40: 1 to 100: 1, and more preferably from 40: 1 to 70: 1; and / or(ii) the aluminosilicate is selected from a sodium aluminosilicate, a potassium aluminosilicate, a calcium aluminosilicate, a magnesium aluminosilicate and a combination thereof; and / or(iii) the microorganisms are selected from bacteria and yeast, and are preferably selected from Pseudomonas pulida. Cupriavidus necalor. Vibrio natriegens, Acinetobacter baylyi, Bacillus megaterium and Yarrowia lipolytica.

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