Method for producing polyhydroxyalkanoates from biomass

Abstract

A method for producing a polyhydroxyalkanoate from biomass is described herein. The method can include providing a biomass stream; treating the biomass stream in an anaerobic digestion reactor to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

Description

VTIP 25-021 (103418-001PCT)METHOD FOR PRODUCING POLYHYDROXYALKANOATES FROM BIOMASSCROSS REFERENCE TO RELATED APPLICATIONThis application claims priority to U.S. Provisional Patent Application No. 63 / 723,666, filed November 22, 2024, the contents of which are hereby incorporated by reference in their entirety.FEDERAL RESEARCH STATEMENTThis invention was made with government support under Grant no. 2023-79000-38973 awarded by the United States Department of Agriculture National Institute of Food and Agriculture. The government has certain rights in the invention.BACKGROUND

[0001] Polyhydroxyalkanoates (PHAs) are a family of bioplastics produced by a wide range of bacteria and archaea with mechanical and thermal properties similar to commercially available plastics such as polyethylene and polypropylene. The homopolymer poly (3- hydroxybutyrate) (PHB) is amongst the most produced PHAs. However, PHB is typically crystallized into large, dense, radially orientated lamellar spherulites, which make the material exceptionally crystalline and brittle for many commercial applications without the use of additives and additional processing. This can be addressed through copolymerization of 3- hydroxyvalyrate (HV), which forms poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Studies have shown that an increase in the HV fraction in PHBV leads to lower melting temperatures, improved ductility, enhanced elasticity, and higher biodegradation rate. As a result, the copolymer PHBV disrupts the high crystallinity of PHB, creating a more flexible and tough material with a wider temperature processing window. This makes PHBV a promising biopolymer for replacing petroleum-based plastics, offering superior thermal and mechanical properties that PHB and other PHAs do not possess.

[0002] It would therefore be advantageous to provide improved processes for production of PHAs. It would be particularly advantageous to provide a process capable of tuning the composition of the resulting PHA.SUMMARY

[0003] An aspect of the present disclosure is a method for producing a polyhydroxyalkanoate from biomass, the method comprising: providing a biomass stream;VTIP 25-021 (103418-001PCT) treating the biomass stream in an anaerobic digestion reactor comprising a microbial electrolysis cell to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

[0004] Another aspect is a polyhydroxyalkanoate made by the method, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprises greater than 10 to 20 mole percent of repeating units derived from 3 -hydroxy valerate.

[0005] Another aspect is an article comprising the polyhydroxyalkanoate.

[0006] The above described and other features are exemplified by the following figures and detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The following figures represent exemplary embodiments.

[0008] FIG. 1 shows volatile fatty acid (VFA) in terms of C3&C5 fractions and total VFA concentration according to an aspect.

[0009] FIG. 2 shows pH at the end of the cycle in food waste digestate with a microbial electrolysis cell (MEC) at 1.5 volts (V) and 0 V.

[0010] FIG. 3 shows hydroxybutyrate (HB) and hydroxyvalyrate (HV) fractions in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) according to an aspect.

[0011] FIG. 4 shows total organic carbon flow using food waste digestate with MEC at 1.5 V and O V.

[0012] FIG. 5 shows correlation of HV fractions in PHBV and C3&C5 VFA fractions in VFAs.

[0013] FIG. 6 shows VFA concentration and C3&C5 VFA fractions in semi-continuous arrested AD during 367-day operation.

[0014] FIG. 7 shows a schematic illustration of the sequencing batch reactor (SBR) setup for Haloferax mediterranei (HM)-based PHBV production from food waste digestate according to an aspect.

[0015] FIG. 8 shows cycle time and volume exchange ratio according to an aspect.

[0016] FIG. 9 shows organic loading rate (OLR) scheduled in SBR during 450 days of operation according to an aspect.

[0017] FIG. 10A shows the effect of dilution factors on HM growth in terms of optical density at 600 nanometers (nm) (ODeoonm), dry cell weight (DCW), and total organic carbonVTIP 25-021 (103418-001PCT)(TOC) consumption.

[0018] FIG. 10B shows PHBV production in terms of PHBV content and titer.

[0019] FIG. 11 shows HM growth profdes in terms of ODeoonm and DCW in SBR during 450 days of operation. Dashed line indicates the day when initial substrate concentration was lowered from 7 to 5 grams per liter (g / L) total organic carbon (TOC).

[0020] FIG. 12 shows HM growth profdes in terms of HM cyclic substrate utilization profdes in terms of TOC removal (ATOC) and substrate utilization efficiency (ATOC / initial TOC) in SBR during 450 days of operation. Dashed line indicates the day when initial substrate concentration was lowered from 7 to 5 g / L TOC.

[0021] FIG. 13A shows profdes of HB, HV and PHBV cellular contents in unit DCW, and HV fraction in PHBV during 450 days of operation.

[0022] FIG. 13B shows profdes of HB, HV and PHBV titer during 450 days of operation.

[0023] FIG. 13C shows profdes of PHBV yield during 450 days of operation.

[0024] FIG. 13D shows profdes of PHBV productivity in SBR during 450 days of operation.

[0025] FIG. 14A shows OLR effects on PHBV productivity.

[0026] FIG. 14B shows cycle time effects on substrate utilization (circle) and residual substrate (triangle) at 5 and 7 g / L initial TOC concentration.

[0027] FIG. 14C shows product inhibition in terms of ATOCx(l-a) on PHBV yield. ATOC - substrate utilization; a - volume exchange ratio.

[0028] FIG. 14D shows HRT effects on PHBV titer. Note: all data were expressed as the mean and standard error of multiple analyses conducted during the steady state.

[0029] FIG. 15 shows effects of the percent of maintenance energy expenditure (ASm / AS) on cellular polyhydroxyalkanoate (PHA) content and observed growth yield (Y obs).

[0030] FIG. 16 shows Effects of the percent of maintenance energy expenditure (ASm / AS) on cellular polyhydroxyalkanoate (PHA) content.

[0031] FIG. 17 shows effects of the percent of maintenance energy expenditure (ASm / AS) on PHA yield.

[0032] FIG. 18 shows effects of the ratio of substrate consumed for maintenance to the new cell growth (ASm / AX) on cellular p polyhydroxyalkanoate (PHA) content. R2= 0.99 between predicted and measured data.

[0033] FIG. 19 shows profdes of pH, optimal density at 600nm, and cell dry mass (determined as volatile suspended solids) during H. mediterranei growth of 216 hours.VTIP 25-021 (103418-001PCT)

[0034] FIG. 20 shows profiles of PHBV content during H. mediterranei growth.

[0035] FIG. 21 shows cell growth and cellular PHA content of H. mediterranei cultivated in 3 x diluted aAD-treated digestate supernatant without other treatment, with overliming treatment, and with pH 2 treatment.

[0036] FIG. 22 shows cell growth and cellular PHA content of H. mediterranei cultivated in raw crude glycerol with 3x and 6x dilution with and without pH 2 treatment.DETAILED DESCRIPTION

[0037] The present inventors have discovered improved processes for production of PHAs. In an advantageous feature, the processes described herein can use organic waste as a carbon source. The United States alone generates nearly 66 million tons of food waste annually, representing 24% of landfill waste. This landfilled food waste is a significant source of greenhouse gas emissions. By utilizing food waste for PHA production, these emissions can be mitigated, feedstock costs can be reduced, and additional revenue can be generated through tipping fees associated with waste disposal. Food waste needs to be hydrolyzed into smaller molecules such as monosaccharides and volatile fatty acids (VFAs) to be readily consumable by microbes such as halophilic microorganisms including Haloferax mediterranei (Hid). Halomonas boliviensis, Halomonas sp. Kid-1. Halomonas bluephagenesis TD01, Halomonas nitro reduce ns. Halomonas sp. O-l, Halomonas elongata, Halomonas halophila, Halomonas marina, Halomonas maura, Halomonas ventosae, Halomonas halodenitrificans , Halomonas halodeneurihalina, Halomonas salina, Halomonas sp. SF2003, Halomonas profundus, Halomonas campisalis, Halomonas hydrothermalis, Vibrio proteolyticus , Yangia sp. ND 199, Yangia sp. CCB-MM3, and Paracoccus sp. LL1. Arrested anaerobic digestion (aAD) is an useful technique for transforming food waste into VFAs such as acetic (C2), propionic (C3), butyric (C4), and valeric (C5) acids, which are important building blocks for PHAs.

[0038] The present inventors have unexpectedly discovered that inclusion of certain process parameters or method steps can provide an improved process for production of PHAs from halophilic microorganisms. Advantageously, the biologically-derived PHAs provided by the methods of the present disclosure can have desirable properties similar to their petrochemically-derived counterparts. In another advantageous feature, synthesis of the PHAs takes place intracellularly in halophilic microorganisms in high salinity conditions which can aid in reduction of contamination. In an aspect, a process for the production of PHAs from food waste can include incorporation of a microbial electrolysis cell (MEC) into arrested anaerobic digestion. In an aspect, a process for production of PHAs can include use of a sequencing batchVTIP 25-021 (103418-001PCT) reactor (SBR). The processes described herein can provide an advanced biotechnology that simultaneously addresses both plastic and food waste pollution issues. A significant improvement is therefore provided by the present disclosure.

[0039] Accordingly, an aspect of the present disclosure is a method for producing a polyhydroxyalkanoate from biomass. The term “polyhydroxyalkanoate” or “PHA” as used herein refers to polymers having the general structure according to Formula (I) or copolymers comprising repeating units derived from at least two different repeating units of Formula (I)wherein in Formula (I) is R is H, C1-12 alkyl, or C1-12 alkenyl, and m is an integer from 1 to 12, preferably 1 to 6, more preferably 1 to 4. Preferably, R is H, methyl, ethyl, propyl, or butyl. Exemplary PHAs can include PHB (poly-3 -hydroxy butyrate), PHBV (poly(hydroxybutyrate-co- hydroxyvalerate)), P4HB (poly(4-hydroxybutyrate), P3HB4HB (poly(3-hydroxybutyrate-co-4- hydroxybutyrate)), PHV (polyhydroxy valerate), and PHHX (polyhydroxyhexanoate).

[0040] The method comprises providing a biomass stream; treating the biomass stream in an anaerobic digestion reactor to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

[0041] The biomass is preferably an organic waste stream. Organic waste refers generally to biodegradable organic residues. Suitable organic waste includes, but is not limited to, food waste, agricultural residues, organic portion of municipal solid waste, industrial byproducts, and green waste. The production of bioplastics from organic wastes can also provide many benefits to the environment.

[0042] In an aspect, the biomass preferably comprises food waste. Food waste as a biomass carbon source has previously presented certain technical challenges. For example, food waste can have significant variation in composition, which makes it difficult to render suitable for use as a uniform substrate. Food waste can also comprise growth inhibitors, which can affect PHA production. The methods disclosed herein can overcome these and other technical challenges associated with the use of food waste, as will be further described herein.

[0043] In an aspect, the biomass can be an organic waste comprising glycerol. With the global expansion of biodiesel as a renewable energy source, waste glycerol, a major byproduct of transesterification, has become an abundant and underutilized carbon source. Crude glycerolVTIP 25-021 (103418-001PCT) typically contains impurities such as methanol, salts, and free fatty acids, rendering it unsuitable for many high-value applications without costly purification. However, certain extremophilic and halophilic microorganisms, such as Halofercix mediierranei. exhibit remarkable tolerance to these impurities and can efficiently metabolize crude glycerol into PHAs.

[0044] In some aspects, the glycerol waste can include long chain fatty acids, which can act as inhibitors. Thus when glycerol waste is used, the method can optionally further comprise adjusting the pH to precipitate the long chain fatty acid inhibitors from the glycerol waste. When pH adjustment is included, the glycerol waste can be neutralized prior to subsequent method steps (further discussed below). Advantageously, the low pH precipitation step can reduce the feedstock dilution factor (e.g., from greater than 20x to 5x or less). Such a method step is further described in the working examples below.

[0045] The biomass (e.g., agricultural waste, including food waste) can be prepared for use in the present method by homogenizing the biomass to obtain a feedstock. The homogenization can comprise chopping, blending, milling, and the like, or a combination thereof. The homogenized biomass can have a total solids content of, for example, 10 to 50%, or 10 to 40%, or 10 to 30%, or 15 to 25%, and a volatile solids content of, for example, 5 to 50 %, or 5 to 40%, or 5 to 30%, or 10 to 25%, or 15 to 25%. The homogenized biomass can be diluted with water to achieve a particular desired solids content.

[0046] In an aspect, the method can optionally comprise pre-treating the organic waste to remove growth inhibitor. The present inventors have found that growth inhibitors present in organic waste, specifically in food waste, can be removed at least partially by a shift in pH. For example, the method can comprise adjusting the pH of the food waste to 2 or less, adjusting the pH of the food waste to neutral (e.g., 6 to 8) to precipitate the growth inhibitors, and separating precipitated growth inhibitors from the food waste (e.g., by filtration, centrifugation, or other solid-liquid separation technique). In some aspects, the concentration of growth inhibitor can be reduced by up to 30% using this method.

[0047] The method comprises treating or decomposing the biomass (or the pre-treated biomass described above) using anaerobic digestion to provide a treated biomass stream. The treated biomass can be provided by anaerobic digestion in any suitable reactor. In some aspects, the treated biomass can be provided by anaerobic digestion in a batch reactor. In some aspects, the treated biomass can be provided by anaerobic digestion in a sequencing batch reactor. Preferably, the anaerobic digestion is arrested anaerobic digestion wherein volatile fatty acids are the main product. Microorganisms used for the anaerobic digestion can be, for example, hydrolytic and acidogenic microbes (e.g., bacteria). The anaerobic digestion can be carried outVTIP 25-021 (103418-001PCT) in the presence of methanogenic bacteria. In some aspects, the methanogenic bacteria can be mixed from the treated biomass (e.g., food waste) at a ratio of methanogenic bacteria to substrate of 1: 1 to 5: 1, or 1: 1 to 3: 1, or 1.5: 1 to 2.5: 1, or 1.8: 1 to 2.2: 1, wherein the ratio is based on volatile solid content. In some aspects, the anaerobic digestion is carried out at a temperature of 20 to 40°C, or 30 to 40°C. In some aspects, the anaerobic digestion is carried out at a pH suitable to support survival of the methanogenic bacteria. For example, in an aspect, the pH can be less than 4.5. In an aspect, an initial pH of less than 4.5 can be used, and after a predetermined period of time, the pH can be adjusted to 5 to 7.5, or 5 to 7, or 5 to 6. An exemplary anaerobic digestion process is further described in the working examples below.

[0048] In an aspect, the effluent from the anaerobic digestion reactor can be separated into a solid stream and a liquid stream. Separated solids can be discarded. The separation can provide a substantially solid-free solution (e.g., an aqueous solution) containing volatile fatty acids and other soluble nutrients. This separated liquid stream can also be referred to as the “treated biomass stream” herein. The treated biomass stream can be concentrated, if desired, to provide a treated biomass stream having a particular solids content.

[0049] The treated biomass stream comprises soluble nutrients derived from the biomass such as volatile fatty acids (VFAs), sugars, and the like. The term “volatile fatty acid” as used herein refers to fatty acids having six carbon atoms or less, for example formic acid, acetic acid, priopionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. In an aspect, the treated biomass stream comprises a mixture of volatile fatty acids, for example comprising two or more of 3 -hydroxybutyrate, 3 -hydroxypropionate, 4-hydroxybutyrate, 3-hydroxyvalerate, glycolic acid, 5 -hydroxypentanoic acid, and the like.

[0050] In an aspect, the treated biomass can comprise a volatile fatty acid composition comprising 30 to 50 weight percent acetic acids, 10 to 30 weight percent propionic acids, 20 to 40 weight percent butyric acids, and 1 to 20 weight percent valeric acids, wherein weight percent is based on the total weight of the volatile fatty acid composition.

[0051] In some aspects, it can be advantageous to obtain a treated stream having a particular distribution of volatile fatty acids. For example, in some aspects, it can be preferable to provide a treated biomass stream comprising greater than 35 weight percent, or greater than or equal to 40 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream.

[0052] The present inventors have unexpectedly found that incorporation of a microbial electrolysis cell can into the anaerobic digester can advantageously allow for tuning of the composition of the volatile fatty acids in the treated biomass. Accordingly, an aspect of theVTIP 25-021 (103418-001PCT) present disclosure includes a microbial electrolysis cell in an anaerobic digestion reactor for providing the treated biomass. In an aspect, the electrolysis cell can be operated at a voltage of greater than 1 volt, or 1 to 2 volts. As further shown herein, when the treated biomass stream is provided by the anaerobic digestion reactor comprising the electrolysis cell operated at a voltage of 1 to 2 volts, the treated biomass comprises a weight percent of C3 and C5 volatile fatty acids that is greater than a weight percentage of C3 and C5 volatile fatty acids in a comparative treated biomass stream provided by an anaerobic digestion reactor operated at zero voltage. Also as shown herein, when the treated biomass stream is provided by the anaerobic digestion reactor comprising the electrolysis cell operated at a voltage of 1 to 2 volts, the treated biomass can comprise a higher weight fraction of volatile fatty acids compared to a treated biomass not provided in the electrolysis cell (i.e., provided at a voltage of 0 volts).

[0053] In an aspect, when a microbial electrolysis cell is used, the treated biomass can comprise 0.2 to 0.4, preferably 0.3 grams per liter (g / L) of acetic acid, 0.4 to 0.6, preferably 0.5 g / L propionic acid, 1.6 to 1.8, preferably 1.7 g / L butyric acid, 0.7 to 0.9, preferably 0.8 g / L isovaleric acid, 0.3 to 0.5, and preferably 0.4 g / L valeric acid. In an aspect, when a microbial electrolysis cell is not used, the treated biomass can comprise 0.1 to 0.3, preferably 0.2 g / L acetic acid, 0.2 to 0.4, preferably 0.3 g / L propionic acid, 1.6 to 1.8, preferably 1.7 g / L butyric, 0.2 to 0.4, preferably 0.3 g / L isovaleric acid, and 0.1 to 0.3, preferably 0.2 g / L valeric acid.

[0054] The method further comprises contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly. Advantageously, halophilic microorganisms require a high salinity environment, and can grow at salt concentrations of, for example 5 to 30 weight percent. The high salt concentration can serve as natural protection from contamination by other microorganisms that may be present in the media or the biomass. Preferred halophilic microorganisms are capable of synthesizing and accumulating polyhydroxyalkanoate granules intracellularly using volatile fatty acids, and exemplary halophilic microorganisms can include Haloferax mediterranei (HM), Halomoncis boliviensis, Halomoncis sp. KM-1 , Halomonas bluephagenesis TD01, Halomonas nitroreducens , Halomonas sp. O-l, Halomonas elongata, Halomonas halophila, Halomonas marina, Halomonas maura, Halomonas ventosae, Halomonas halodenitrificans , Halomonas halodeneurihalina, Halomonas salina, Halomonas sp. SF2003, Halomonas profundus, Halomonas campisalis, Halomonas hydrothermalis, Vibrio proteolyticus , Yangia sp. ND 199, Yangia sp. CCB-MM3, and Paracoccus sp. LL1.

[0055] In an aspect, the halophilic microorganism can comprise Haloferax mediterranei, which can synthesize and accumulate polyhydroxyalkanoate granules intracellularly usingVTIP 25-021 (103418-001PCT) volatile fatty acids (e.g., derived from biomass or food waste) as a carbon source. In some aspects, a support medium for contacting the treated biomass stream with the halophilic microorganism can have a salt concentration of greater than 100 grams of salt per liter of support medium, or 100 to 300 grams of salt per liter of support medium, or 150 to 250 grams of salt per liter of support medium. In another advantageous feature, the salt can be recycled to further improve the sustainability and cost-effectiveness of the method.

[0056] The halophilic microorganism can be separated from the growth media after a sufficient time for PHA synthesis. Various separation techniques can be used. In an aspect, centrifugation can be used to separate the cellular biomass from the media. The PHA can then be extracted from the harvested cellular biomass.

[0057] Thus, the method further comprises extracting the intracellularly accumulated polyhydroxyalkanoate from the halophilic microorganism. In an aspect, the specific selection of Haloferax mediterranei can be advantageous for the extraction step. In an aspect, the polyhydroxyalkanoate can be extracted by addition of salt-free water to enable cell self-lysis due to the change in osmotic pressure. The released polyhydroxyalkanoate can be recovered by isolation, for example using any suitable solid-liquid separation technique such as centrifugation.

[0058] In some aspects, the intracellularly accumulated polyhydroxyalkanoate can be extracted using a high pressure homogenization process. In an aspect, the high pressure homogenization can comprise subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to a pressure of at least 100 bar, for example to a pressure of 100 to 1000 bar. In some aspects, increasing the pressure can increase the yield or purity of the isolated polyhydroxyalkanoate. For example, the pressure can be 200 to 1000 bar, or 300 to 1000 bar, or 400 to 1000 bar, or 500 to 1000 bar, or 600 to 1000 bar, or 700 to 1000 bar or 800 to 1000 bar, or 900 to 1000 bar. The released polyhydroxyalkanoate can be recovered by isolation, for example using any suitable solid-liquid separation technique such as centrifugation.

[0059] In some aspects, the produced polyhydroxyalkanoate comprises poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3 -hydroxybutyrate), poly(3-hydroxyvalerate), or a combination thereof. In a specific aspect, the produced polyhydroxyalkanoate can comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 80 to 95 mole percent of repeating units derived from 3 -hydroxybutyrate and 5 to 20 mole percent of repeating units derived from 3 -hydroxy valerate. As discussed above, it has been discovered that the volatile fatty acid composition can be tuned. Accordingly, by tuning the composition of the volatile fatty acids present in the treated biomass, it can be possible to vary the composition of the resultingVTIP 25-021 (103418-001PCT) polyhydroxyalkanoate composition. For example, in an aspect, the polyhydroxyalkanoate can comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and the poly(3-hydroxybutyrate-co-3- hydroxyvalerate) can comprise 80 to 90 mole percent of repeating units derived from 3- hydroxybutyrate and 10 to 20 mole percent of repeating units derived from 3 -hydroxy valerate. In some aspects, the 3 -hydroxy valerate content can be 12 to 20 mole percent, or 15 to 20 mole percent.

[0060] The method described herein can provide the desired polyhydroxyalkanoate in high yields and at high purity. For example, the polyhydroxyalkanoate can obtained in a yield of greater than 65 weight percent, or greater than 70 weight percent, wherein weight percent is based on grams of polyhydroxyalkanoate per grams of the halophilic microorganism dry mass. In some aspects, the isolated polyhydroxyalkanoate can have a purity of at least 65%, or at least 80%, or at least 85%. In an aspect, when present residual impurities present in the isolated polyhydroxyalkanoate can comprise cell wall debris, proteins, or a combination thereof.

[0061] In a specific aspect, the method according to the present disclosure can comprise pre-treating a biomass stream comprising food waste to remove growth inhibitor and provide a pre-treated biomass stream; treating the pre-treated biomass stream in an anaerobic digestion reactor to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with the halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

[0062] In a specific aspect, the method according to the present disclosure can comprise providing a biomass stream, treating the biomass stream in an anaerobic digestion reactor to provide a treated biomass stream comprising volatile fatty acids, wherein the anaerobic digestion reactor is a sequencing batch reactor; contacting the treated biomass stream with the halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

[0063] In an aspect, the sequencing batch reactor can be operated under conditions that induce mild substrate inhibition, thereby increasing maintenance energy expenditure in the halophilic microorganism. In an aspect, the maintenance energy expenditure is characterized by a substrate allocation ratio (ASm / AS) can be 0.4 to 0.5, or 0.43 to 0.49, or 0.45 to 0.47, or approximately. 0.46. In some aspects, the sequencing batch reactor can be operated with a volume exchange ratio of 0.3 to 0.7, or 0.4 to 0.6, or 0.45 to 0.55, or approximately 0.5 to achieve optimal dilution of the treated biomass stream. Advantageously, the intracellular polyhydroxyalkanoate content of the halophilic microorganism can be increased in response to elevated maintenance energy expenditure. For example, in some aspects, the intracellularVTIP 25-021 (103418-001PCT) polyhydroxyalkanoate content can reach a maximum of approximately 60% by weight of total cell organic carbon.

[0064] In some aspects, the method can further comprise monitoring the lag phase duration of the halophilic microorganism as an indicator of environmental stress and maintenance energy demand.

[0065] In another specific embodiment, the method according to the present disclosure can comprise pre-treating a biomass stream comprising food waste to remove growth inhibitor and provide a pre-treated biomass stream; treating the pre-treated biomass stream in the anaerobic digestion reactor comprising the microbial electrolysis cell at a voltage of 1 to 2 volts to provide a treated biomass stream, wherein the treated biomass stream comprises greater than 35 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with the halophilic microorganism in a support medium having a salt concentration of greater than 100 grams of salt per liter of support medium to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism, wherein the extracting comprises contacting the halophilic microorganism comprising the polyhydroxyalkanoate with water; or subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to high pressure homogenization at a pressure of at least 100 bar.

[0066] A polyhydroxyalkanoate made by the methods described herein represents another aspect of the present disclosure. In an aspect, the polyhydroxyalkanoate can comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3- hydroxyvalerate) comprises greater than 10 mole percent, or greater than 15 mole percent, or greater than or equal to 20 mole percent of repeating units derived from 3 -hydroxy valerate. In an aspect, the polyhydroxyalkanoate can comprise poly(3-hydroxybutyrate-co-3- hydroxyvalerate) comprising 80 to 95 mole percent of repeating units derived from 3- hydroxybutyrate and 5 to 20 mole percent of repeating units derived from 3-hydroxyvalerate.

[0067] The polyhydroxyalkanoate made by the methods described herein can be suitable for formation of various articles, for example fdm, sheets, or packaging materials.

[0068] This disclosure is further illustrated by the following examples, which are nonlimiting.EXAMPLES

[0069] Food waste was digested using arrested anaerobic digestion (aAD) according to the following procedure. A stainless-steel fermenter (Ss Brite Tank, CM Brewing Technologies,VTIP 25-021 (103418-001PCT)Inc., USA) with a total volume of 37.8 L and a working volume of 30 L was operated in a semi- continuous mode as an anaerobic digester with cycle time of 3 days for 367 days at organic loading rates (OLR) of 1.5, 2.5, 3.5, 5, and 7.5 g VS / L-day during different phases. At the end of each cycle, 15 L of digestate was discharged, followed by adding 15 L mixture of food waste and deionized (DI) water according to the desired OLR, which gives a hydraulic retention time (HRT) of 6 days. A pump (Jabsco 31801-0115 Pump, Xylem, USA) connected to a programmed timer was utilized to provide mixing through recirculating digestate from the bottom to the top of the digester at an interval of eight times a day. The reactor was operated at mesophilic temperature (35 ± 0.5°C) maintained using an external heating control and internal radiant heating pipes (Chronical, CM Brewing Technologies, Inc., USA and Electric Mini-Tank Water Heater, Bosch Inc., USA). In order to start up the aAD, the anaerobic inoculum with an average total solid (TS) and volatile solids (VS) content of 0.60% and 0.46%, respectively, was collected from an existing inoculum and blended with food waste at a 2: 1 inoculum-to-substrate ratio based on VS concentration. The pH of the aAD was not adjusted until the 34th day after startup. The pH was firstly adjusted to 5.5 using potassium hydroxide at the beginning of each cycle from Day 34 to 164 and then to pH 7.0 from day 167 to 367.

[0070] The food waste used as a substrate for the aAD was prepared according to a recipe based on food waste characterization by the US Department of Agriculture. The food waste was manually chopped into small pieces and blended as a paste using the electrical blender and stored at -20°C until use. The prepared food waste had average TS of 20.4% and VS of 19.3%. In order to control the OUR at desired levels, food waste was diluted with DI water accordingly prior to feeding.

[0071] A microbial electrolysis cell was incorporated into the arrested anaerobic digestion vessel. Six units of 500 E serum bottles were operated as aAD reactors. An electrode made of a carbon fiber brush and a stainless-steel mesh was placed inside of each bottle with a butyl rubber sheet cut to fit inside the cap to hold the brush in the center of the bottle. A voltage (1.5 V) between the brush and stainless-steel mesh was applied in three bottles as a triplicate to determine the effect of MEC on aAD. Zero voltage was applied in the other three bottles as a triplicate control. Each bottle aAD reactor was operated at an OLR of 1.5 g VS / L-day and 35 °C. All aAD bottles were inoculated with sludge collected from the aforementioned 30 L aAD. After inoculation, the bottle aAD reactors were equilibrated for 17 days with no feeding to enable establishment of a biofilm on the electrode surface. After this equilibration period, these reactors were fed semi-continuously for 38 days at a 6-day HRT. The pH of all bottle aAD reactor was adjusted to 7 during each feed cycle. These reactors were run for a total of 63 days.VTIP 25-021 (103418-001PCT)

[0072] Microorganism and culture conditions were as follows. HM strain (ATCC 33500, American Type Culture Collection, USA) was purchased from ATCC and activated in ATCC medium: 1176 Halobacterium medium containing 156g / L NaCl, 5g / L yeast extract, Ig / L glucose, 13g / L MgCl2«6H2O, 20g / L MgSO4«7H2O, Ig / L CaCl2«2H2O, 4g / L KC1, 0.2g / L NaHCCh, and 0.5g / LNaBr. The pH was adjusted to 7.0 using 1 N NaOH or 1 N H2SC>4. Sterilization was provided through autoclaving at 121 °C for 20 mins. After 48-hour cultivation at 37 °C and 150 rpm, the cell density measured in terms of optical density (ODeoonm) reached 0.5. The activated culture was distributed into 30 units of 5 ml sterile tubes with 15% glycerol (v / v) for long term storage at -80 °C until use.

[0073] The activated culture was used as the inoculum for the PHBV fermentation using food waste digestate. Specifically, a 2% (v / v) activated culture was inoculated into a 500 m shake flask containing 100 m of food waste digestate supplemented with 156g / L NaCl, 5g / L yeast extract, 13g / L MgCl2«6H2O, 20g / L MgSO4*7H2O, Ig / L CaCl2«2H2O, 4g / L KC1, 0.2g / L NaHCOv and 0.5g / LNaBr. NH4C1 was added to control C / N ratio at 15. The medium was adjusted to pH 7.0 and sterilized by 0.22 pm-filtration to prevent VFA evaporation from food waste digestate that occurs with autoclaving. The HM culture was incubated at 37 °C and 150 rpm for 120 hours until the growth reached stationary phase.

[0074] VFA concentration and composition were determined using a gas chromatography (GC, Model 7890, Agilent Technologies, USA) equipped with a flame ionization detector (FID) and a capillary GC column (NUKOU -15m x 0.53 mm x 0.50 pm, Supelco, USA). GC was operated under the following conditions: 75 °C initial column temperature of with a ramp rate of 4 °C / min to 120 °C, 200 °C injection temperature, and 250 °C detector temperature. Carrier gas was nitrogen with a flow rate of 40 mU / min, and flame gases were hydrogen and air. Fermented food waste was centrifuge at 8,000 xg for 30 min; the supernatant was acidified with 0.1% (v / v) phosphoric acid and filtered with 0.22 um membrane for VFA analysis.

[0075] ODeoonm was monitored by a microplate reader (Synergy™ Hl, BioTek Instruments, Inc., USA). Nitrogen was determined using the persulfate digestion method by testing kits (TNT828, Hach Company, USA) following Hach procedures with a spectrophotometer (DR3900, Hach Company, USA). Dry cell weight (DCW) was determined as the volatile suspended solids (VSS) of cell broth and followed the standard VSS measurement method. Briefly, samples were centrifuged at 8,000 xg for 20 min, and the pellet was then transferred to a ceramic crucible. Samples were dried to constant weight, weighed and then all organic matter burnt at 500°C for 4 h in a furnace. The DCW was calculated as the differenceVTIP 25-021 (103418-001PCT) between the dry weight (salts plus organic matter) and the burnt sample (salts only).

[0076] Total organic carbon (TOC) was measured by a TOC analyzer (TOC-LCSN, Shimadzu, USA) for liquid sample analysis. This TOC analyzer was also equipped with a solid sample combustion unit (SSM-5000A, Shimadzu, USA) for solid sample analysis. Uiquid samples were fdtered with 0.45 pm syringe fdters and diluted with DI water. Solid samples were dried at 105°C till a constant weight before being loaded into the TOC analyzer. Potassium hydrogen phthalate standards and sodium carbonate standards were used to establish total carbon (TC) and inorganic carbon (IC) calibration curves, respectively.

[0077] PHBV extraction and quantification were performed according to the following procedure. Briefly, 40-45 ml sample was collected and centrifuged at 8,000 xg for 10 min. The cell pellet was washed with 4% sodium hypochlorite and DI water. The washed pellets were freeze-dried in a freeze dryer at -50 °C and 0.12 mBar (FreeZone 2.5 Uiter Freeze Dry System, Uabconco, USA) till a constant weight is reached. Then, a mixture of 25 mg of freeze-dried cells, 2 ml chloroform, and 2 ml acidified methanol (containing 3% sulfuric acid) were incubated at 105 °C for 120 mins. The samples were then allowed to cool to the room temperature. The samples were then mixed with 1 ml of DI water and vortexed. The resultant solution was centrifuged at 5,000 xg for 15 mins and the chloroform layer at the bottom of tube was then collected. This chloroform layer was passed through 0.22 pm filter and finally transferred to the GC vials. The samples were analyzed using a GC (Model 8890, Agilent, USA) equipped with FID. The Agilent HP-5 column (30m x 320pm x 0.25pm) was used with nitrogen as carrier gas. Specifically, inlet temperature and pressure: 200 °C and 13 psi; total flowrate: 86 mU / min; split ratio: 30: 1; oven temperature: initial 80°C for 4 min; ramping from 80 to 160 °C, with a rate of 6 °C / min. FID temperature was 275 °C, with 30 mU / min of H2 flow, 390 mU / min of air flow, and 25 mU / min of nitrogen. PHBV (CAS Number: 80181313, Sigma-Aldrich, USA) with 8 mol % HV and 92 mol % HB were sued as the standards and corrected using methyl benzoate (CAS Number: 93583, Sigma-Aldrich, USA) an internal standard.

[0078] Electro-fermentation has been reported to be capable of enhancing the production of C3&C5 VFAs. Although the exact mechanism still remains to be confirmed, this enhancement is presumably attributed to the buffering effect of MEC against pH drop during aAD, which causes either the microbial community shift towards the ones that tend to produce higher levels of C3 or C5 acids, or a change of the redox environment energetically favor the metabolism producing these acids. This approach was also experimented in the present examples. The results in FIG. 1 revealed that aAD equipped with MEC being operated at 1.5V produced digestate with VFAs consisting of 44%, 39%, 49% C3&C5 VFAs in triplicate bottleVTIP 25-021 (103418-001PCT) reactors, respectively. These consistent results double the fraction of C3&C5 VFAs (23%, 19%, 20%) in VFAs produced in the control aAD equipped with MEC operated at 0V. The pH in aAD with and without MEC were 6 ± 0.02 and 5.5 ± 0.01 (FIG. 2), respectively, indicating MEC resulted in higher pH, which may be the root cause of higher C3&C5 VFA production. It should be pointed out that the level of total VFAs (C2-C5 VFAs) remained the same at 4g / L in both aAD (FIG. 1), indicating that MEC only altered the VFA composition but not the VFA yield. Subsequently, the two digestates were fed to HM for PHBV production. Results in FIG. 3 revealed that the PHBV produced from the digestate treated by MEC contained 20 mol% HV. In contrast, PHBV produced from the digestate without MEC treatment only contained 7 mol% HV. This threefold difference in HV fraction in PHBV once again evidenced that the C3&C5 VFAs directly produced from food waste aAD facilitated by MEC can be taken advantage for significantly increasing the HV fraction in PHBV. Again, there was no obvious difference in cell growth and PHBV yield in terms of carbon flow in FIG. 4, with only HV and HB contents varying. This confirms that cell growth and PHBV yield depend only on TOC loading, while HV and HB fractions depend on VFA composition. Importantly, this experiment also confirmed that the HV fraction in PHBV produced by HM can be indirectly enhanced by MEC during the aAD. The present inventors also found that applying 1.5 V through MEC on aAD process was able to not only shift the VFA profile but also improve the overall VFA yield.

[0079] Although producing PHBV with HV fraction > 10 mol% is promising when food waste digestate is used as a substrate for HM, the bioplastic industry prefers to use PHBV with HV fraction > 20 mol% because PHBV with 20 mol% HV can exhibit 5 -fold greater toughness and flexibility than PHBV with 10 mol% HV. Comparing PHBV with 20 mol % HV to that with 10 mol% HV, the former exhibited a lower melting temperature at 168.5 °C compared to the latter at 174.5 °C, and a high decomposition temperature at 103.2 °C compared to the latter at 75.6 °C. Thereby, PHBV with 20 mol % HV was rated as the best polymer for injection molding purposes. According to FIG. 5, in order for HMto produce PHBV with HV fraction > 20 mol%, C3 and / or C5 fraction in VFAs has to be > 45%. Unexpectedly, the triplicate MEC-facilitated aAD experiment performed on the same food waste under the loading rate of 1.5g VS / L-day and pH of 7 showed 100% reproducibility in boosting C3 and C5 VFA fractions in VFAs to the range > 45% (FIG. 6). The results found herein strongly indicate that MEC can be utilized as an effective approach to boost HV fractions in PHBV produced by / / AT fed with food waste digestate.

[0080] Increasing the fractions of propionic acid and valeric acid in food waste digestate has therefore been shown to be effective in enhancing the fraction of 3 -hydroxy valerate inVTIP 25-021 (103418-001PCT) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Haloferax mediterranei, with a strong linear correlation. Furthermore, microbial electrolysis cell-facilitated arrested anaerobic digestion was proven to be capable of improving both total volatile fatty acid production and the proportion of propionic acid and valeric acid.

[0081] The present inventors have also discovered that additional advantages arise from the use of a sequencing batch reactor (SBR) for Haloferax mediterranei (HM) to produce poly(3-hydroxybutyrate-co-3 -hydroxy valerate) (PHBV). While the conversion of food waste to PHBV by HM using food waste digestate has been demonstrated in single-batch reactors, such reactors are unsuitable for large-scale industrial applications due to the inefficiencies associated with batch changeover. In contrast, continuous or semi-continuous flow reactors, such as sequencing batch reactors (SBRs), are more desired for industrial-scale application. SBRs combine the advantages of single batch and continuous flow reactors while minimizing their respective limitations. The high initial substrate concentration in each cycle of the SBR enables high bioreaction rates that a continuous flow bioreactor does not offer. Meanwhile, the sequencing nature of the SBR avoids the downtime that limits the productivity of single-batch reactors. Traditionally, continuous or semi -continuous flow reactors are generally unsuitable for pure culture fermentation due to the high risk of contamination. However, the halophilic nature of the HM may offer an opportunity to get around this problem. In addition, because only a portion of the cells and salts are discharged at the end of each SBR cycle, the remaining cells and salts can be automatically recycled as the inoculum and cultivation materials for the subsequent cycle of fermentation, which are bound to reducing the operational costs. Besides, the volume exchange ratio of SBRs also provides an automatic dilution of the influent digestate. Hence, SBRs may not only increase PHBV productivity and conserve salts for HM cultures but also mitigate the accumulation of inhibitors, a common issue in fed-batch reactors that has also been explored for HM fermentation of food waste for bioplastic production.

[0082] The food waste mixture used for the following examples was obtained according to the procedure already described. The food waste had an average total solid content of 20.4% and a volatile solids content of 19.3%.

[0083] The arrested anaerobic digester and digestate characterization was as follows. A stainless-steel fermenter (Ss Brite Tank, CM Brewing Technologies Inc., Tustin, CA, USA) with a total volume of 37.8 L and a working volume of 30 L was operated in a semi -continuous mode as an arrested anaerobic digester with a hydraulic retention time of 12 days for 367 days at an organic loading rate (OLR) of 2.5 g VS / L-day. At the end of each cycle, 15 L of digestate was discharged, followed by adding 15 L mixture of food waste and deionized (DI) water to reachVTIP 25-021 (103418-001PCT) the desired OLR. A pump (Jabsco 31801-0115 Pump, Xylem Inc, Washington, DC, USA) connected to a programmed timer was utilized to provide mixing through recirculating digestate from the bottom to the top of the digester at an interval of eight times a day. The digester was operated at mesophilic temperature (35 ± 0.5 °C) maintained using an external heating control and internal radiant heating pipes (Chronical, CM Brewing Technologies Inc., Tustin, CA, USA and Electric Mini-Tank Water Heater, Bosch Inc., Farmington Hills, MI, USA). To start up the arrested anaerobic digester, the anaerobic inoculum with an average total solid (TS) and volatile solids (VS) content of 0.60 % and 0.46 %, respectively, was collected and blended with food waste at a 2: 1 inoculum-to-substrate ratio based on VS concentration. The pH of the arrested anaerobic digester was not adjusted until the 34th day after startup to reduce the pH below the level suitable for methanogenic bacteria to survival (< pH 4.5). After this initial 34-day stabilization period, the pH was adjusted to 5.5 using potassium hydroxide at the beginning of each cycle. Referring to Table 1, the food waste digestate discharged from arrested anaerobic digester was primarily composed of 7.57 g / L volatile fatty acids (VFAs) with a composition of 1 % acetic acids, 21 % propionic acids, 27 % butyric acids, and 11 % valeric acids, which gives total organic carbon (TOC) of 14 g / E, total nitrogen (TN) of 1. 16 g / L, and total phosphorous (TP) of 0.11 g / L.

[0084] Referring to FIG. 7, a 500 mL glass bottle was operated with 400 mL working volume in SBR mode. Briefly, each cycle of the SBR consists of three phases, namely feeding, reaction, and discharging. The feeding and discharging took only 1 min each while the rest of the cycle time was dedicated to reaction. The discharging tubing was inserted to a given height above which the volume of the mixed liquor to the total working volume of the SBR is equivalent to the volume exchange ratio designed for the operation. The aeration was provided by injecting air to the bottom of the SBR at a flow rate of 100 ml / min. In order to reduce the mixed liquor loss to aeration, the air line passed through a humidifier to compensate water evaporation losses during aeration. To start up the SBR, the inoculum was added into SBR at a volumetric ratio of 10 % (v / v). The operating temperatures were set at 37 °C for the SBR and 4 °C for the feed reservoir.

[0085] Referring to FIG. 8, the entire 450 days of the experiment can be divided into two phases with either cycle time or volume exchange ratio actively controlled as a variable. Phase I started on day 0 and ended on day 148, during which the cycle times were varied from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and finally to 12 days, while the SBR volume exchange ratio was fixed at 0.5. Phase II started on day 149 and ended on day 450, during which the volume exchange ratios were actively changed from 0.3 to 0.35, 0.4, 0.45, and finally 0.5 while the cycle time wasVTIP 25-021 (103418-001PCT) passively controlled based on the HM growth curve measured by ODeoonm, i.e., each SBR cycle proceeded until HM reached their growth plateau. Throughout most of the SBR cycles, the initial substrate concentration was kept at the same level of 7 g / L TOC, as determined by the minimum dilution factor required when using the food waste digestate as a substrate. It should be pointed out that the initial substrate concentration was intentionally lowered to 5 g / L TOC from day 407 to 450 to understand the biodegradability of the residual TOC. Because of the fixed initial substrate concentration of 7 and 5 g / L TOC, the OLR of the SBR can be calculated with SBR cycle times and volume exchange ratios, as shown in FIG. 9. Basically, the OLR monotonously decreased from 7 to 3.5, 2.3, 1.4, 1.17, 0.88, 0.78, 0.70, 0.64, and then to 0.58 g TOC / L-day during the phase I study and from 1.4 to 1, 0.88, 0.63, and finally 0.58 g TOC / L-day during the phase II study (FIG. 9).

[0086] HM strain for the following experiments was purchased from ATCC (ATCC 33500, American Type Culture Collection, Manassas, VA, USA) and activated in ATCC medium (1176 Halobacterium medium ) which contains 156g / L NaCl, 5g / L yeast extract, Ig / L glucose, 13g / L MgCl2-6H2O, 20g / L MgSO4-7H2O, Ig / L CaCl2-2H2O, 4g / L KC1, 0.2g / L NaHCCh, and 0.5g / LNaBr. pH was adjusted to 7.0 using 1 N NaOH / 1 N H2SO4. Sterilization was done by autoclaving at 121 °C for 20 min. After 48-hour cultivation at 37 °C, the cell density measured at optical density (ODeoonm) reached 0.5. The activated culture was distributed into sterile tubes containing 15 % glycerol (v / v) for long-term storage at -80 °C. This activated culture was used as the inoculum for the SBR startup. The feed of SBR is the supernatant of food waste digestate collected by centrifuge at 15,000 x g for 20 mins, and supplemented with 156 g / L NaCl, 5g / L yeast extract, 13 g / L MgCl2«6H2O, 20 g / L MgSO4«7H2O, 1 g / L CaCl2«2H2O, 4 g / L KC1, 0.2 g / L NaHCCh, and 0.5 g / LNaBr.

[0087] First, a minimum dilution factor was examined for using food waste digestate as a substrate. To determine the minimum dilution factor of the food waste digestate that can be used for HM fermentation, a gradient of dilution factors of 1, 2, 3, 3.5, 4.5, and 9, corresponding to TOC concentrations of 14, 7, 4.7, 4, 3, and 1.6 g / L, was tested. Experimental results in FIG 10 revealed that the HM growth, TOC consumption, and PHBV production were ail inversely related to the dilution factor, with the exception of a dilution factor of 1 (i.e., no dilution). Hie fact that there was neither H rowth nor TOC consumption and PHBV production when food waste digestate was directly fed to HM without dilution just confirmed the presence of inhibitory compounds in the raw food waste digestate (FIG. 10). Notably, across ail tested conditions, a significant reduction of approximately 70% in TOC was observed, indicating efficient utilization of carbon sources by HM. Based on these findings, a dilution factor of 2, corresponding to anVTIP 25-021 (103418-001PCT) initial TOC concentration of 7 g / L, was regarded as an optimum dilution factor because it resulted in the highest PHBV titer of 2.1 1 g / L. At this dilution, the PHBV content reached approximately 70%, including 6% HV content. These results confirmed that the optimal dilution factor for HA growth and PHBV production should be 2, which was in turn selected as a default condition in subsequent SBR experiments.

[0088] FIG. 11 shows cell concentration measured in ODeoonm and DCW. There was no observed cell growth during the first 12 days when cycle time was only one day because the retention time was too short for HM cells to replenish themselves. Both ODeoonm and DCW profiles quickly ramped up from day 13 to day 66, and then stabilized around 5.5 and 2.5 g / L, respectively, for the rest of the 384 days of fermentation, indicating very stable long-term HM growth in unsterilized environments regardless of the cycle time and volume exchange ratio variations. The greatest ODeoonm and DCW values were obtained at the lowest volume exchange ratio, namely 0.3, between days 346 and 450. This is expected because the lower the volume exchange ratio was, the more HM cells were retained in SBR at the end of a given cycle and then transferred to the next cycle. The good linear correlation (R2= 0.99) between ODeoonm and DCW suggests that ODeoonm can be used as an accurate indicator of DCW for cycle time determination.

[0089] According to FIG. 12, the trend of the substrate utilization profile was in line with that of the HM cell growth profiles, indicating the cell growth was limited by TOC utilization. It should be pointed out that only 70 % TOC in food waste digestate can be maximumly utilized by HM when the initial substate concertation was 7 g / L TOC, even with cycle times as long as 12 days, leaving a residual 2 g / L TOC at the end of each cycle. To understand whether this residual TOC was biodegradable, the initial TOC level was intentionally reduced from 7 to 5 g / L on day 407. The results in FIG. 12 showed a TOC removal efficiency of up to 90%, indicating that the residual TOC was actually readily biodegradable by HM. As for the reason why HM cells refrained from utilizing it, it might have to do with the product inhibition which is presumably proportional to the extent of TOC utilization. This product inhibition could also explain why DCW did not exceed 3 g / L throughout the 450-day experiment (FIG. 11).

[0090] Traditionally, the fermentation performance can be measured by the productivity, yield, or titer of the final products. Additionally, the HV fraction in PHBV plays an important role in determining the product properties of PHBV, which directly influences its market value. For these reasons, the values of these performance indicating parameters along the 450 days of SBR operation were plotted in FIG. 13. As can be seen in FIG. 13, cellular PHBV content in HM ramped up along with cell growth from 44 to 68 % (g PHBV / g DCW) during the first 61VTIP 25-021 (103418-001PCT) days of the HM fermentation. For the rest of the fermentation period, cellular PHBV content remained almost constant, around 65 % (g PHBV / g DCW), which aligns with the typical cellular PHBV levels in HM cells reported in the literature. Therefore, one may conclude that cellular PHBV content in HM can remain fairly stable during long-term fermentation of food waste digestate and is insensitive to the range of cycle times and volume exchange ratios tested in this study. Notably, the HV cellular content stabilized at approximately 10% (g PHBV / g DCW), resulting in a steady HV fraction of 15% in PHBV (mol HV / mol PHBV) throughout the 450 days of the experiment (FIG. 13). This HV fraction is critical because it aligns with the threshold required for the properties of PHBV to meet the commercial application standard. Specifically, an HV fraction of at least 10 mol% in PHBV is reported to be necessary for the material to achieve the flexibility and toughness essential for practical uses, such as for packaging and agricultural films. The titer of PHBV followed the same trend as cellular PHBV content, as titer can be calculated with HM cell concentration, which also remained stable throughout the experiment (FIG. 13). In contrast, PHBV yield tended to decrease in the course of the fermentation duration (FIG. 13), which can be attributed to the product inhibition incurred by the greater fractions of SBR volume being retained without discharge. As for the PHBV productivity, its profile in FIG. 13 looks very similar to that of the OLR.

[0091] The dependency of PHBV productivity on the OLR of the influent is plotted in FIG. 14A with all the data collected from FIGs. 8, 9 and 13. The linear correlation between PHBV productivity and OLR is expected because the more feedstock (food waste digestate) loaded into the unit volume of the SBR per time, the faster the final bioproduct (PHBV) production will be resulted in. The value of the highest OLR, namely 7 g TOC / L-day, was excluded from this plot because of the lack of HM cell growth under one-day cycle time . In other words, the one-day cycle time was too short to allow HMto reproduce themselves to compensate for the cell loss in the discharge.

[0092] A plot of TOC utilization against cycle time in FIG. 14B indicates that a maximum of only 5 g / L TOC can be consumed by HM cells in the SBR, regardless of the cycle time, despite the fact that around 2 g / L TOC remained at the end of each SBR cycle. FIG. 12 and FIG. 14B confirm that this residual TOC was actually readily biodegradable, as evidenced by the lower residual TOC observed when a lower initial TOC concentration was used, even though the substrate utilization (ATOC) remained constant around 5 g / L. Therefore, the formation of inhibitors as a result of the TOC utilization might have limited further TOC utilization. To test this hypothesis, this study assumed that the concentration of inhibitors is proportional to TOC utilization and is also accounted for the dilution by the SBR volumeVTIP 25-021 (103418-001PCT) exchange ratio. Specifically, newly added food waste digestate may dilute inhibitors formed during the previous cycle of HM fermentation. Consequently, the concentration of product inhibitors can be hypothesized to be proportional to the value of ATOCx(l-a), where a represents the volume exchange ratio. A plot of PHBV yield against ATOCx(l-a) in FIG. 14C exhibited a linear correlation between the two parameters, indicating the product inhibition was responsible for the declining PHBV yield defined as g APHBV / g ATOC in FIG. 14C. It is broadly accepted that when more maintenance energy has to be spent on coping with the inhibition, less carbon becomes available for PHBV production. Therefore, minimizing product inhibitory effects is essential in HM fermentation of food waste. Examining the term ATOC’ / ( I - a), it is not difficult to see that the concentration of product inhibitors should be inversely related to the volume exchange ratio. Therefore, a large volume exchange ratio, e.g., 0.5, is preferred for HM fermentation of food waste digestate to mitigate product inhibition.

[0093] According to FIG. 14D, PHBV titer was very low at the beginning of the SBR operation because the cycle time used during the time was too short to allow substantial cell growth and subsequent PHBV formation. On the contrary, it is well understood that a prolonged cultivation time may force microbial cells to consume their own carbon storage, namely PHBV, to obtain the maintenance energy to survive. These bipolar effects can also be observed in the plot of PHBV titer against HRT which is calculated as the ratio of SBR cycle time to volume exchange ratio in FIG. 14D. The highest PHBV titer can be achieved only with the intermediate HRT values, such as 16 days. Too short or too long HRT is expected to result in low PHBV titer, which is critically important for reducing the cost in downstream PHBV processing.

[0094] Accordingly, the present examples show that stable PHBV fermentation from food waste digestate by HM can be achieved in SBR for the low contamination risk offered by the high salinity environment of HM culture. It was also found that a relatively high OLR, e.g., 3.5 g / L-day, should be employed to achieve a high PHBV productivity, and the cycle time of SBR should be no less than 5 days for the maximum substrate utilization. As for the PHBV titer, HRT that has factored in both the cycle time and volume exchange ratio becomes a decisive factor and should be around 16 days. It was further found that product inhibition is responsible for the PHBV yield decrease and should be avoided by keeping SBR volume exchange ratio no less than 0.5.

[0095] The present inventors further evaluated the role of maintenance energy expenditure in promoting polyhydroxyalkanoate (PHA) production by Halofercix mediterranei using food waste digestate as a substrate. It was found that under mild inhibitory conditions, caused by either substrate or product inhibition, H. mediterranei diverted a greater proportion ofVTIP 25-021 (103418-001PCT) substrate energy toward maintenance functions, which in turn enhanced cellular PHA accumulation (see, FIG. 15 and FIG. 16). A theoretical model was developed and validated to predict PHA content based on the ratio of substrate allocated for maintenance energy relative to cell growth. Experimental results demonstrated that maximum PHA yield occurred when approximately 46% of the substrate was consumed for maintenance energy (FIG. 17), indicating an optimal stress level for PHA biosynthesis. The model showed strong predictive accuracy (R2= 0.99) up to a cellular PHA content of 60%, beyond which intracellular space limitations constrained further accumulation (FIG. 18). These findings support the use of controlled stress conditions to optimize PHA production and reduce downstream processing costs, offering a practical and economically viable approach for bioplastic synthesis from waste-derived feedstocks.

[0096] The present inventors have further evaluated the use of glycerol waste as a substrate for forming PHAs by Haloferax mediterranei . Briefly, Haloferax mediterranei ATCC 33500 was used for converting glycerol waste into PHBV through fermentation but formulated with industrial grade chemicals. Instead of glucose, the medium was supplemented with 6.9 mL / L waste glycerol as a carbon source for fermentation. The fermentation was conducted at 37°C with agitation at 200 rpm for 216 hours without pH adjustment. During fermentation, the cell growth was monitored by taking 1 m of the fermentation broth samples at different time intervals and measuring their optical cell density (OD600). The cell dry mass (CDM) was determined as volatile suspended solids (VSS). For VSS measurement, 50 ml of the fermentation broth was centrifuged at 8,000 xg for 20 min. The resulting cell pellet was dried to a constant weight at 105°C in a convection oven. The dried pellet was then combusted at 550 °C for 4 hours in a muffle furnace. The VSS was calculated based on the weight difference before and after the muffle furnace combustion. After fermentation, the whole broth was harvested and stored in 4°C until the downstream processing to separate PHA.

[0097] To extract PHA from the H. mediterranei cells, a high-pressure homogenizer (PandaPLUS 2000, GEA, Frankfurt, Germany) with a processing volumetric flow rate of 9 L per hours was used. The fermentation broth was passed through the high-pressure homogenizer at different pressures (50 to 1000 bar) to break cell walls. The homogenized suspension was then centrifuged at 29,000 xg for 60 min to separate PHA granules (pellet) from the cell wall debris and other cell components (supernatant). The collected PHA granules were then washed once with deionized water, centrifuged at 29,000 xg for 30 min, and then freeze-dried using a freeze dryer (FreeZone 2.5 Liter Freeze Dry System, Labconco, Kansas City, MO, USA) to obtain PHBV powder.VTIP 25-021 (103418-001PCT)

[0098] The purity and monomer composition of the extracted PHBV and intracellular PHBV content were analyzed using gas chromatography equipped with a flame ionization detector (GC-FID, Agilent 8890, Agilent Technologies Inc., Santa Clara, CA, USA). To quantify the total intracellular PHBV content in the H. mediterranei cells, the cells were freeze- dried using freeze dryer at -50°C and 0.12 mBar (FreeZone 2.5 Liter Freeze Dry System, Labconco, Kansas City, MO, USA) until a constant weight was achieved. Then 25 mg of freeze- dried cells were mixed with 2 mL methanol acidified with 3% (v / v) sulfuric acid and 2 mL of chloroform in a pressure tube. The mixture was incubated in an oven at 105 °C for 4 hours for PHA methanolysis where the PHBV was depolymerized into its monomeric components. After the methanolysis, 1 mL of deionized water was added into the mixture for phase separation. The chloroform layer containing PHBV was sampled, filtered using a 0.2 pm filter, and analyzed by GC-FID equipped with a capillary column (30 m, ID: 0.32 mm, film thickness: 0.25 pm, Agilent HP-5 HP19091J-413). A sample of 1 pL was injected into the GC with inlet temperature and FID temperatures set at 200°C and 275°C, respectively. The oven temperature was programed from 80°C (4 min hold time) to 160°C at a heating rate of 6°C / min. Nitrogen was used as a carrier gas with a flow rate of 2.7 mL / min. To measure the purity of the extracted PHBV powder, the same procedures were followed, substituting the freeze-dried PHBV powders obtained from previous section for H. mediterranei cells.

[0099] Functional groups of the PHBV powder were identified by Fourier transform infrared (FTIR) spectroscopy. Spectral data were collected by an FTIR spectrometer equipped with an attenuated total reflectance (ATR) accessory (Thermo Fisher Scientific iS50, Thermo Fisher Scientific company, Waltham, MA, USA). The freeze-dried PHBV sample was directly placed in the ATR-FTIR cell. The FTIR spectrum was recorded from 500 to 4000 cm1with resolution of 1 cm'1with 32 scans.

[0100] To evaluate the feasibility of using waste glycerol as carbon source for PHBV production by H. mediterranei, 40-L fermentations were conducted under nonsterile conditions without pH control for 9 days (FIG. 19). The pH of the fermentation broth started at 7, quickly dropped to 5.7 within the first 48 hours, bounced back to 6.7 after 72 hours, and remained constant (at 6.7) thereafter. When glycerol serves as the carbon source, it is first metabolized into dihydroxyacetone phosphate (DHAP), which is then converted to glyceraldehyde -3- phosphate (G3P). G3P is further processed into pyruvate, which is converted to Acetyl-CoA and subsequently utilized for energy production and PHA biosynthesis. During the conversion of pyruvate to Acetyl-CoA, pyruvate is decarboxylated, releasing CO2 and hydrogen ions (H+), contributing to the initial pH drop. However, as the fermentation progresses, CO2 is graduallyVTIP 25-021 (103418-001PCT) removed from the liquid phase through agitation and aeration, leading to a decrease in carbonic acid concentration and stabilizing the pH. Additionally, the use of ammonium salt as nitrogen sources influences late-stage metabolism of / / . mediterranei . As the cell growth slows and nitrogen demand declines, H. mediterranei may release ammonia, which neutralize the acidity of medium. Despite the pH fluctuations during the fermentation process, both cell growth and PHBV production proceeded successfully (FIG. 19 and 20), indicating that pH control is not essential for pilot-scale PHBV production using waste glycerol.

[0101] Cell growth and biomass accumulation were monitored using ODeoo and CDM measured as VSS. The OD600 demonstrated a steady cell growth, reaching 10.0±0.07 by the end of the 216-hour fermentation. Correspondingly, CDM increased consistently, reaching 5.7±0.97 g / L after 216-hour fermentation, indicating substantial biomass accumulation. The low variability in the replicated fermentation confirmed the reliability of waste glycerol as a suitable carbon source for H. mediterranei in the pilot-scale fermentations.

[0102] The PHBV produced by H. mediterranei is a copolymer of 3 -hydroxy butyrate (HB) and 3-hydroxyvalerate (HV). The accumulation profiles of PHBV indicate a steady increase in both HB and HV throughout the fermentation (FIG. 20). By the end of fermentation, the HB titer reached 2.0±0.09 g / L, whereas HV was produced in much smaller quantities at about 0.2±0.02 g / L. The total PHBV titer (including HB and HV) is about 2.2±0.11 g / L, with HB being the dominant monomer. Additionally, PHBV properties can be customized by adjusting the HV content, enabling precise tailoring for specific use. This versatility makes PHBV a more attractive choice for commercial applications where balancing strength, flexibility, and processability is crucial. Unlike most microorganisms that require HV precursors for PHBV synthesis, H. mediterranei can efficiently synthesize PHBV without the addition of HV precursors, thus greatly reducing production cost.

[0103] Overall, these findings from the pilot-scale fermentation demonstrate that / / . mediterranei is a robust PHA producer capable of efficiently utilizing waste glycerol for scalable PHBV production.

[0104] The present inventors have further found that removal of long chain fatty acids from crude glycerol, which act as inhibitors, use of glycerol for PHA synthesis can be improved. For example, conducting an acid precipitation at pH 2 was found to be effective, removing LCFAs and enabling a reduction in the required feedstock dilution from 23 x to 3x. This treatment improved PHA titer by 40% and reduced the need for external salt supplementation by 46%, leveraging the inherent salinity of the crude glycerol. In contrast, overliming and arrested anaerobic digestion (aAD) were ineffective in significantly mitigating inhibition. The results areVTIP 25-021 (103418-001PCT) shown in FIG. 21 and FIG. 22. The results confirmed that the dark, hydrophobic precipitate formed during acidification contained the inhibitory LCFAs, and its removal restored microbial growth and PHA production. These findings demonstrate that pH 2 treatment is a practical and scalable pretreatment method for enabling the valorization of crude glycerol into bioplastics, offering both economic and environmental benefits.

[0105] This disclosure further encompasses the following aspects.

[0106] Aspect 1: A method for producing a polyhydroxyalkanoate from biomass, the method comprising: providing a biomass stream; treating the biomass stream in an anaerobic digestion reactor comprising a microbial electrolysis cell to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism .

[0107] Aspect 2: The method of aspect 1, wherein the biomass comprises organic waste, preferably food waste.

[0108] Aspect 3: The method of aspect 2, further comprising pre-treating the organic waste to remove growth inhibitor.

[0109] Aspect 4: The method of aspect 3, wherein the pre-treating to remove growth inhibitor comprises adjusting the pH of the food waste to 2 or less, adjusting the pH of the organic waste to neutral to precipitate the growth inhibitors, and separating precipitated growth inhibitors from the organic waste.

[0110] Aspect 5: The method of any of aspects 1 to 4, wherein the treated biomass stream comprises greater than 35 weight percent, or greater than or equal to 40 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream.

[0111] Aspect 6: The method of any of aspects 1 to 5, wherein the electrolysis cell is operated at a voltage of greater than 1 volt, or 1 to 2 volts.

[0112] Aspect 7: The method of any of aspects 1 to 6, wherein the treated biomass stream provided by the anaerobic digestion reactor comprising the electrolysis cell operated at a voltage of 1 to 2 volts comprises a weight percent of C3 and C5 volatile fatty acids that is greater than a weight percentage of C3 and C5 volatile fatty acids in a comparative treated biomass stream provided by an anaerobic digestion reactor operated at zero voltage; or a total volatile fatty acid content that is greater than a total volatile fatty acid content in a comparative treated biomass stream provided by an anaerobic digestion reactor operated at zero voltage; or both.

[0113] Aspect 8: The method of any of aspects 1 to 7, wherein the halophilicVTIP 25-021 (103418-001PCT) microorganism is Haloferax mediterranei (HM). Halomonas boliviensis. Halomonas sp. KM-1. Halomoncis bluephagenesis TD01, Halomonas nitroreducens , Halomonas sp. 0-1, Halomonas elongata, Halomonas halophila, Halomonas marina, Halomonas maura, Halomonas ventosae, Halomonas halodenitrificans , Halomonas halodeneurihalina, Halomonas salina, Halomonas sp. SF2003, Halomonas profundus, Halomonas campisalis, Halomonas hydrothermalis, Vibrio proteolyticus , Yangia sp. ND 199, Yangia sp. CCB-MM3, and Paracoccus sp. LL1, preferably, Haloferax mediterranei (HM).

[0114] Aspect 9: The method of any of aspects 1 to 8, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3- hydroxybutyrate), poly(3-hydroxyvalerate), or a combination thereof; preferably, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 80 to 95 mole percent of repeating units derived from 3 -hydroxybutyrate and 5 to 20 mole percent of repeating units derived from 3 -hydroxy valerate.

[0115] Aspect 10: The method of any of aspects 1 to 9, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprises 80 to 90 mole percent of repeating units derived from 3 -hydroxybutyrate and 10 to 20 mole percent of repeating units derived from 3 -hydroxy valerate .

[0116] Aspect 11 : The method of any of aspects 1 to 10, wherein a support medium for contacting the treated biomass stream with the halophilic microorganism has a salt concentration of greater than 100 grams of salt per liter of support medium, or 100 to 300 grams of salt per liter of support medium, or 150 to 250 grams of salt per liter of support medium.

[0117] Aspect 12: The method of any of aspects 1 to 11, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises contacting with water.

[0118] Aspect 13: The method of any of aspects 1 to 12, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises high pressure homogenization .

[0119] Aspect 14: The method of aspect 13, wherein high pressure homogenization comprises subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to a pressure of at least 100 bar.

[0120] Aspect 15: The method of any of aspects 1 to 14, further comprising isolating the polyhydroxyalkanoate, and drying the polyhydroxyalkanoate.

[0121] Aspect 16: The method of any of aspects 1 to 15, wherein the polyhydroxyalkanoate is obtained in a yield of greater than 65 weight percent, wherein weightVTIP 25-021 (103418-001PCT) percent is based on grams of polyhydroxyalkanoate per grams of the halophilic microorganism dry mass.

[0122] Aspect 17: The method of aspect 1, comprising pre-treating a biomass stream comprising food waste to remove growth inhibitor and provide a pre-treated biomass stream; treating the pre-treated biomass stream in the anaerobic digestion reactor comprising the microbial electrolysis cell at a voltage of 1 to 2 volts to provide a treated biomass stream, wherein the treated biomass stream comprises greater than 35 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with Haloferax mediterranei in a support medium having a salt concentration of greater than 100 grams of salt per liter of support medium to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the Haloferax mediterranei, wherein the extracting comprises contacting the Haloferax mediterranei comprising the polyhydroxyalkanoate with water; or subjecting the Haloferax mediterranei comprising the polyhydroxyalkanoate to high pressure homogenization at a pressure of at least 100 bar.

[0123] Aspect 18: A method for producing a polyhydroxyalkanoate from biomass, the method comprising: providing a biomass stream; treating the biomass stream in an anaerobic digestion reactor comprising a sequencing batch reactor cell to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

[0124] Aspect 19: The method of aspect 18, wherein the biomass is derived from organic waste, preferably food waste.

[0125] Aspect 20: The method of aspect 18, wherein the biomass is derived from organic waste comprising glycerol.

[0126] Aspect 21 : The method of aspect 18 or 19, further comprising pre-treating the organic waste to remove growth inhibitor.

[0127] Aspect 22: The method of aspect 21, wherein the pre-treating to remove growth inhibitor comprises adjusting the pH of the organic waste to 2 or less, adjusting the pH of the organic waste to neutral to precipitate the growth inhibitors, and separating precipitated growth inhibitors from the organic waste.

[0128] Aspect 23: The method of any of aspect 18 to 22, wherein the treated biomass stream comprises greater than 35 weight percent, or greater than or equal to 40 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream.VTIP 25-021 (103418-001PCT)

[0129] Aspect 24: The method of any of aspects 18 to 23, wherein the halophilic microorganism is Haloferax mediterranei (HM), Halomoncis boliviensis, Halomoncis sp. KM-1 , Halomonas bluephagenesis TD01, Halomoncis nitroreducens , Halomonas sp. 0-1, Halomonas elongata, Halomonas halophila, Halomonas marina, Halomonas maura, Halomonas ventosae, Halomonas halodenitrificans , Halomonas halodeneurihalina, Halomonas salina, Halomonas sp. SF2003, Halomonas profundus, Halomonas campisalis, Halomonas hydrothermalis, Vibrio proteolyticus , Yangia sp. ND 199, Yangia sp. CCB-MM3, and Paracoccus sp. LL1, preferably, Haloferax mediterranei (HM).

[0130] Aspect 25: The method of any of aspects 18 to 24, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3- hydroxybutyrate), poly(3-hydroxyvalerate), or a combination thereof; preferably, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 80 to 95 mole percent of repeating units derived from 3 -hydroxybutyrate and 5 to 20 mole percent of repeating units derived from 3 -hydroxy valerate.

[0131] Aspect 26: The method of any of aspects 18 to 25, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprises 80 to 90 mole percent of repeating units derived from 3 -hydroxybutyrate and 10 to 20 mole percent of repeating units derived from 3 -hydroxyvalerate .

[0132] Aspect 27: The method of any of aspects 18 to 26, wherein a support medium for contacting the treated biomass stream with the halophilic microorganism has a salt concentration of greater than 100 grams of salt per liter of support medium, or 100 to 300 grams of salt per liter of support medium, or 150 to 250 grams of salt per liter of support medium.

[0133] Aspect 28: The method of any of aspects 18 to 27, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises contacting with water.

[0134] Aspect 29: The method of any of aspect 18 to 28, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises high pressure homogenization .

[0135] Aspect 30: The method of aspect 29, wherein high pressure homogenization comprises subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to a pressure of at least 100 bar.

[0136] Aspect 31: The method of any of aspects 18 to 30, further comprising isolating the polyhydroxyalkanoate, and drying the polyhydroxyalkanoate.

[0137] Aspect 32: The method of any of aspects 18 to 31, wherein theVTIP 25-021 (103418-001PCT) polyhydroxyalkanoate is obtained in a yield of greater than 65 weight percent, wherein weight percent is based on grams of polyhydroxyalkanoate per grams of halophilic microorganism dry mass.

[0138] Aspect 33: The method of any of aspects 18 to 32, wherein the sequencing batch reactor is operated under conditions effective to induce substrate inhibition and increase maintainence energy expenditure in the halophilic microorganism.

[0139] Aspect 34: The method of aspect 18, comprising pre-treating a biomass stream comprising organic waste to remove growth inhibitor and provide a pre-treated biomass stream; treating the pre-treated biomass stream in the anaerobic digestion reactor comprising the sequencing batch reactor to provide a treated biomass stream, wherein the treated biomass stream comprises greater than 35 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with the halophilic microorganism in a support medium having a salt concentration of greater than 100 grams of salt per liter of support medium to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism, wherein the extracting comprises contacting the halophilic microorganism comprising the polyhydroxyalkanoate with water; or subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to high pressure homogenization at a pressure of at least 100 bar.

[0140] Aspect 35: A polyhydroxyalkanoate made by the method of any of aspects 1 to 34, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3- hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprises greater than 10 to 20 mole percent of repeating units derived from 3 -hydroxy valerate.

[0141] Aspect 36: An article comprising the polyhydroxyalkanoate of aspect 35.

[0142] Aspect 37: The article of aspect 36, wherein the article is a fdm, a sheet, or a packaging material.

[0143] The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components, that are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles.

[0144] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. “Combinations” is inclusive of blends, mixtures,VTIP 25-021 (103418-001PCT) alloys, reaction products, and the like. The terms “first,” “second,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The terms “a” and “an” and “the” do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. “Or” means “and / or” unless clearly stated otherwise. Reference throughout the specification to “an aspect” means that a particular element described in connection with the aspect is included in at least one aspect described herein, and may or may not be present in other aspects. The term “combination thereof’ as used herein includes one or more of the listed elements, and is open, allowing the presence of one or more like elements not named. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various aspects.

[0145] Unless specified to the contrary herein, all test standards are the most recent standard in effect as of the filing date of this application, or, if priority is claimed, the filing date of the earliest priority application in which the test standard appears.

[0146] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this application belongs. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.

[0147] Compounds are described using standard nomenclature. For example, any position not substituted by any indicated group is understood to have its valency filled by a bond as indicated, or a hydrogen atom. A dash that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CHO is attached through carbon of the carbonyl group.

[0148] Unless substituents are otherwise specifically indicated, each of the foregoing groups can be unsubstituted or substituted, provided that the substitution does not significantly adversely affect synthesis, stability, or use of the compound. “Substituted” means that the compound, group, or atom is substituted with at least one (e.g., 1, 2, 3, or 4) substituents instead of hydrogen, where each substituent is independently nitro (-NO2), cyano (-CN), hydroxy (-OH), halogen, thiol (-SH), thiocyano (-SCN), C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, C1-9 alkoxy, C1-6 haloalkoxy, C3-12 cycloalkyl, C5-18 cycloalkenyl, C6-12 aryl, C7-13 arylalkylene (e.g., benzyl), C7-12 alkylarylene (e.g, toluyl), C4-12 heterocycloalkyl, C3-12 heteroaryl, C1-6 alkyl sulfonyl (-S(=O)2-alkyl), C6-12 arylsulfonyl (-S(=O)2-aryl), or tosyl (CH3C6H4SO2-), providedVTIP 25-021 (103418-001PCT) that the substituted atom’s normal valence is not exceeded, and that the substitution does not significantly adversely affect the manufacture, stability, or desired property of the compound. When a compound is substituted, the indicated number of carbon atoms is the total number of carbon atoms in the compound or group, including those of any substituents.

[0149] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents.

Claims

VTIP 25-021 (103418-001PCT)CLAIMS1. A method for producing a polyhydroxyalkanoate from biomass, the method comprising: providing a biomass stream; treating the biomass stream in an anaerobic digestion reactor comprising a microbial electrolysis cell to provide a treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with a halophilic microorganism to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism.

2. The method of claim 1, wherein the biomass is derived from organic waste, preferably food waste.

3. The method of claim 2, further comprising pre-treating the organic waste to remove growth inhibitor.

4. The method of claim 3, wherein the pre -treating to remove growth inhibitor comprises adjusting the pH of the organic waste to 2 or less, adjusting the pH of the organic waste to neutral to precipitate the growth inhibitors, and separating precipitated growth inhibitors from the organic waste.

5. The method of claim 1, wherein the treated biomass stream comprises greater than 35 weight percent, or greater than or equal to 40 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream.

6. The method of claim 1, wherein the electrolysis cell is operated at a voltage of greater than 1 volt, or 1 to 2 volts.

7. The method of claim 1, wherein the treated biomass stream provided by the anaerobic digestion reactor comprising the electrolysis cell is operated at a voltage of 1 to 2 volts comprises a weight percent of C3 and C5 volatile fatty acids that is greater than a weight percentage of C3 and C5 volatile fatty acids in a comparative treated biomass stream provided by an anaerobic digestion reactor operated at zero voltage; or a total volatile fatty acid content that is greater than a total volatile fatty acid content in aVTIP 25-021(103418-001PCT) comparative treated biomass stream provided by an anaerobic digestion reactor operated at zero voltage.

8. The method of claim 1, wherein the halophilic microorganism is Haloferax mediterranei (HM), Halomoncis boliviensis, Halomoncis sp. KM-1 , Halomonas bluephagenesis TD01, Halomonas nitroreducens, Halomonas sp. 0-1, Halomonas elongata, Halomonas halophila, Halomonas marina, Halomonas maura, Halomonas ventosae, Halomonas halodenitrificans, Halomonas halodeneurihalina, Halomonas salina, Halomonas sp. SF2003, Halomonas profundus, Halomonas campisalis, Halomonas hydrothermalis , Vibrio proteolyticus , Yangia sp. ND 199, Yangia sp. CCB-MM3, and Paracoccus sp. LL1, preferably, Haloferax mediterranei (HM).

9. The method of claim 1, wherein the polyhydroxyalkanoate comprises poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3 -hydroxybutyrate), poly(3-hydroxyvalerate), or a combination thereof; preferably, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3- hydroxyvalerate) comprising 80 to 95 mole percent of repeating units derived from 3- hydroxybutyrate and 5 to 20 mole percent of repeating units derived from 3-hydroxyvalerate.

10. The method of claim 1, wherein the polyhydroxyalkanoate comprises poly(3- hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3- hydroxyvalerate) comprises 80 to 90 mole percent of repeating units derived from 3- hydroxybutyrate and 10 to 20 mole percent of repeating units derived from 3-hydroxyvalerate.

11. The method of claim 1, wherein a support medium for contacting the treated biomass stream with the halophilic microorganism has a salt concentration of greater than 100 grams of salt per liter of support medium, or 100 to 300 grams of salt per liter of support medium, or 150 to 250 grams of salt per liter of support medium.

12. The method of claim 1, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises contacting with water.

13. The method of claim 1, wherein extracting the polyhydroxyalkanoate from the halophilic microorganism comprises high pressure homogenization.

14. The method of claim 13, wherein high pressure homogenization comprisesVTIP 25-021(103418-001PCT) subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to a pressure of at least 100 bar.

15. The method of claim 1, further comprising isolating the polyhydroxyalkanoate, and drying the polyhydroxy alkanoate.

16. The method of claim 1, wherein the polyhydroxyalkanoate is obtained in a yield of greater than 65 weight percent, wherein weight percent is based on grams of polyhydroxyalkanoate per grams of halophilic microorganism dry mass.

17. The method of claim 1, comprising pre-treating a biomass stream comprising organic waste to remove growth inhibitor and provide a pre-treated biomass stream; treating the pre-treated biomass stream in the anaerobic digestion reactor comprising the microbial electrolysis cell at a voltage of 1 to 2 volts to provide a treated biomass stream, wherein the treated biomass stream comprises greater than 35 weight percent of C3 and C5 volatile fatty acids, based on the total weight of the treated biomass stream comprising volatile fatty acids; contacting the treated biomass stream with the halophilic microorganism in a support medium having a salt concentration of greater than 100 grams of salt per liter of support medium to produce the polyhydroxyalkanoate intracellularly; and extracting the polyhydroxyalkanoate from the halophilic microorganism, wherein the extracting comprises contacting the halophilic microorganism comprising the polyhydroxyalkanoate with water; or subjecting the halophilic microorganism comprising the polyhydroxyalkanoate to high pressure homogenization at a pressure of at least 100 bar.

18. A polyhydroxyalkanoate made by the method of any of claims 1 to 17, wherein the polyhydroxyalkanoate comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and wherein the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprises greater than 10 to 20 mole percent of repeating units derived from 3 -hydroxy valerate.

19. An article comprising the polyhydroxyalkanoate of claim 18.VTIP 25-021(103418-001PCT)20. The article of claim 19, wherein the article is a film, a sheet, or a packaging material.

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