SOLUTION AND METHOD FOR THE OXIDATIVE LYSIS AND CONDITIONING OF POLYHYDROXALKANOATE PRODUCING CELLS

Abstract

The present specification generally relates to a method for the oxidative conditioning of a microbial cell wall, and its eventual lysis. In particular, the specification pertains to an oxidative lysis solution and methodology, wherein the selective lysis solution is capable of conditioning and lysing the cell wall of polyhydroxyalkanoate-producing microbes, while having a limited effect on the degradation of the polyhydroxyalkanoate (PHA) biopolymer solids contained therein, thereby allowing for the further high yield recovery, and purification of high molecular weight PHA biopolymer solids.

Claims

1. A method for decreasing the production of foam when adding an oxidizing agent to a cellular biomass, comprising: heating the cellular biomass to a temperature sufficient to deactivate any enzymes present in the cellular biomass that adversely interact with said oxidizing agent; and adding said oxidizing agent to said heated cellular biomass forming a heated suspension to release the cellular content of said cellular biomass.

2. The method of claim 1, wherein the cellular biomass is heated to a temperature in the range of 80° C.-130° C.

3. The method of claim 1, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (E.sub.O) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

4. The method of claim 3, wherein said oxidizing agent comprises hydrogen peroxide (H.sub.2O.sub.2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na.sub.2O.sub.2), sodium perborate (Na.sub.2H.sub.4B.sub.2O.sub.8), sodium percarbonate (Na.sub.2H.sub.3CO.sub.6), and sodium persulfate (Na.sub.2S.sub.2O.sub.8); chlorite, chlorate, metachloro perbenzoic acid (C.sub.7H.sub.5ClO.sub.3) perchlorate, performic acid (CH.sub.2O.sub.3), peracetic acid (CH.sub.3CO.sub.3H), perchlorate (ClO.sub.4), chlorine dioxide (ClO.sub.2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO.sub.3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

5. The method of claim 1, wherein said heated suspension of cellular biomass in contact with said oxidizing agent contains a final concentration of 0.1%-10% of said oxidizing agent.

6. The method of claim 1, wherein said cellular biomass comprises polyhydroxyalkanoate (PHA) containing microorganisms containing greater than 25% PHA by dry cell weight.

7. (canceled)

8. The method of claim 6, further comprising the step of separating and purifying said PHA from the cellular biomass and said cellular content.

9. The method of claim 8, wherein said PHA has a molecular weight of about 100 kDa-3,000 kDa and a purity greater than 95.5%.

10. A method for providing a source of polyhydroxyalkanoate (PHA) for further purification, comprising: heating a suspension of microorganisms containing PHA; exposing said heated suspension of PHA-containing microorganisms to an oxidizing agent; and maintaining said heated suspension of PHA-containing microorganisms in contact with said oxidizing agent for a period of time and at a temperature sufficient to condition and lyse said microorganisms thereby releasing PHA and non-polymer cell mass (NPCM).

11. The process of claim 10, wherein said heated suspension of PHA-containing microorganisms in contact with said oxidizing agent contains a final concentration of 0.1%-10% of said oxidizing agent.

12. (canceled)

13. The process of claim 11, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (E.sub.O) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

14. The process of claim 11, wherein said oxidizing agent comprises hydrogen peroxide (H.sub.2O.sub.2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na.sub.2O.sub.2), sodium perborate (Na.sub.2H.sub.4B.sub.2O.sub.8), sodium percarbonate (Na.sub.2H.sub.3CO.sub.6), and sodium persulfate (Na.sub.2S.sub.2O.sub.8); chlorite, chlorate, metachloro perbenzoic acid (C.sub.7H.sub.5ClO.sub.3) perchlorate, performic acid (CH.sub.2O.sub.3), peracetic acid (CH.sub.3CO.sub.3H), perchlorate (ClO.sub.4), chlorine dioxide (ClO.sub.2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO.sub.3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

15-16. (canceled)

17. The process of claim 10, wherein said temperature and time sufficient to condition and lyse said microorganisms and dissolve said NPCM is in the range of 80° C.-130° C. for greater than 1 hour.

18-20. (canceled)

21. The process of claim 10, wherein said polyhydroxyalkanoates (PHAs) is selected from the group consisting of poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO, or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), or other short- or medium-chain length, saturated or unsaturated PHAs; or polylactic acid (PLA); or their copolymers or any combinations thereof.

22. The process of claim 10, wherein the suspension of microorganisms containing greater than 25% PHA by dry cell weight.

23. An aqueous extraction process of polymers from a culture of microorganisms, comprising: contacting a suspension of microorganisms with an oxidizing agent wherein said oxidizing agent has a final concentration of approximately 0.5%-6.0%; maintaining said suspension of microorganisms in contact with said oxidizing agent at a temperature greater than about 87° C. for approximately 2.5 hours to lyse open said microorganisms freeing said polymer and dissolve said non-polymer cell mass.

24. The process of claim 23, wherein said freed polymer has a molecular weight of about 100 kDa-3,000 kDa.

25. The process of claim 23, wherein said oxidizing agent comprises a non-gaseous compound at room temperature having an oxidation-reduction potential (E.sub.O) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol.

26. The process of claim 25, wherein said oxidizing agent comprises hydrogen peroxide (H.sub.2O.sub.2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na.sub.2O.sub.2), sodium perborate (Na.sub.2H.sub.4B.sub.2O.sub.8), sodium percarbonate (Na.sub.2H.sub.3CO.sub.6), and sodium persulfate (Na.sub.2S.sub.2O.sub.8); chlorite, chlorate, metachloro perbenzoic acid (C.sub.7H.sub.5ClO.sub.3) perchlorate, performic acid (CH.sub.2O.sub.3), peracetic acid (CH.sub.3CO.sub.3H), perchlorate (ClO.sub.4), chlorine dioxide (ClO.sub.2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO.sub.3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

27-28. (canceled)

29. The lysis solution of claim 25, wherein said non-gaseous compound comprises a 30-35% hydrogen peroxide solution having a final concentration of 0.4-4% volume/volume.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is related to a selective lysis solution and process, proven to be industrially feasible, useful for the selective lysis or disruption of the cell walls of polyhydroxyalkanoates (PHA) producing microbes while preserving at maximum the original characteristics of the PHA biopolymer, with high yield and efficiency.

[0020] As used herein the term “PHA biopolymers” refers to poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO, or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), or other short- or medium-chain length, saturated or unsaturated PHAs; or polylactic acid (PLA); or their copolymers or any combinations thereof.

[0021] As used herein the term “fermentation” or “fermentation process” refers to any fermentation process or any process comprising a fermentation step. A fermentation process includes, without limitation, fermentation processes used to produce PHAs and are well known in the art. Examples of such can be found in U.S. Pat. Nos. 7,579,176 and 9,850,508 issued to Herrema, et al., all of which are incorporated herein by reference.

[0022] As used herein the term “fermentation media” or “fermentation medium” refers to the environment in which the fermentation is carried out and which includes the fermentation substrate, that is, the carbon source that is metabolized by the fermenting microorganism. The fermentation media, including fermentation substrate and other raw materials used in the fermentation process may be processed prior to or simultaneously with the fermentation process. Accordingly, the fermentation media can refer to the media before the fermenting microorganisms are added, as well as the media which comprises the fermenting microorganisms.

[0023] As used herein the term “fermenting microorganism” refers to any microorganism suitable for use in a desired fermentation process. Suitable fermenting microorganisms according to the invention are able to ferment, i.e., convert, methane, carbon dioxide, sugars, alkanes, vegetable oils, organic acids, and alcohols, directly or indirectly into the PHA. Sources from which PHA is extracted via the process of the present invention include single-cell organisms such as bacteria or fungi and higher organisms such as plants (herein collectively referred to as “biomass”). While such biomass could be genetically manipulated species, they are preferably wild-type organisms specifically selected for the production of a specific PHA of interest. Bacteria useful in the present invention include any bacteria which naturally produce PHA. To date, Cupriavidus necator (formerly known as Wautersia eutropha, Ralstonia eutropha and Alcaligenes eutrophus) is the most extensively studied microorganism for the cost-effective production of PHA. Numerous other strains such as Bacillus megaterium, Bacillus cereus SPV, Sinorhizobium meliloti, Azotobacter spp, Pseudomonas putida KT2440 and Metylobacterium spp, and Methylococcus spp are also gaining attention for PHA production. These bacteria can accumulate up to 30-90% of their weight as PHB under limiting nitrogen substrate and in the presence of an abundant source of carbon such as but not limited to methane, carbon dioxide, sugars, alkanes, vegetable oils, organic acids, and alcohols. For further examples of such bacteria the following articles and patents are incorporated herein by reference—NOVEL BIODEGRADABLE MICROBIAL POLYMERS, E. A. Dawes, ed., NATO ASI Series, Series E: Applied Sciences—Vol. 186, Kluwer Academic Publishers (1990); Herrema, et. al., (U.S. Pat. No. 7,579,176); Shiotani, et. al., (U.S. Pat. No. 5,292,860,); and, Peoples, et. al., (U.S. Pat. No. 5,250,430).

[0024] As used herein the term “oxidizing agents” refers to compounds that are non-gaseous at room temperature having an oxidation-reduction potential (E.sub.O) of more than 1.4 volts, a pH of more than 3 and less than 11, and a molar mass of less than 250 g/mol. Examples of such oxidizing agents includes but is not limited to hydrogen peroxide (H.sub.2O.sub.2) and other inorganic peroxides, such as but not limited to sodium peroxide (Na.sub.2O.sub.2), sodium perborate (Na.sub.2H.sub.4B.sub.2O.sub.8), sodium percarbonate (Na.sub.2H.sub.3CO.sub.6), and sodium persulfate (Na.sub.2S.sub.2O.sub.8); chlorite, chlorate, metachloro perbenzoic acid (C.sub.7H.sub.5C.sub.1O.sub.3) perchlorate, performic acid (CH.sub.2O.sub.3), peracetic acid (CH.sub.3CO.sub.3H), perchlorate (ClO.sub.4), chlorine dioxide (ClO.sub.2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO.sub.3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate.

[0025] The general conditions of growth and fermentation are well known in the art For carrying out the process according to the invention, at least part of the fermentation solution or the water is first removed from the fermented, aqueous cell suspension. Examples of separation processes which can be employed here are decanting, centrifugation, spray drying, evaporation, and filtration of the biomass from the fermentation medium. It is preferred to optionally remove part of the fermentation medium from the cell mass by centrifugation, preferably with the aid of a separator so that the resulting biomass slurry contains a final biomass concentration of approximately 5-10 percent, 10-16 percent, 16-20 percent, 20-25 percent, or 25-35 percent solids, or preferable targeted percentages therein.

[0026] One of the advantages of the process according to the invention is that there is no need to pretreat the biomass slurry by breaking it up or drying it. However, it is also possible to employ pretreated cell material in the process according to the invention. Surprisingly, as discussed below, it was discovered that when a heated biomass is suspended in a solution of an oxidizing agent such as 5-50% hydrogen peroxide having a final concentration of 0.01-30.0 percent volume/volume the cell wall of PHA-producing microorganisms having an overall PHA concentration of greater than 30% can be selectively disrupted with little damage to the PHA biopolymer. Applicant theorizes, without wishing to be bound thereto, that as the PHA concentration increases within a PHA-producing microorganisms the cell wall becomes both strained and more vulnerable to lysis. In addition, it is believed that the enzymes present in the biomass are deactivated as a result of the heating; consequently, when the oxidizing agent is added to the slurry, the native enzymes do not immediately decompose the oxidizing agent into harmless components. Many oxidizing agents when exposed to microorganisms will increase the permeability of the cell wall by disrupting sulphydryl (—SH) and sulphur (—S) bonds creating “holes” in the cell wall that typically do not result in lysis. However, when PHA-producing cells are engorged with PHA, and the oxidizing agent is not readily decomposed, the cells are lysed according to the present invention. The inventors theorize that the cell wall becomes strained as a result of the PHA content and as “holes” appear, as a result of the oxidizing agent, the cell wall begins to fail and spill out the contents of the cell. The discovery by the present inventors that the PHA-producing cells could be oxidized by the oxidizing agents disclosed herein at the concentrations used was surprising, as it is a finding quite contradictory to the well known principle that cell walls are not vulnerable to lysis when exposed to the oxidizing agents disclosed herein at the concentrations used. Thus, the method of this invention displays a number of notable advantages in comparison to the prior art. The PHA-producing cells are able to be disrupted while preserving the maximum original characteristics of the PHA biopolymer located inside the cell.

[0027] The solids content of the biomass slurry, which preferably contains water, is then adjusted to approximately, and preferably, 10-30 g/L, 30-50 g/L, 50-70 g/L, 70-200 g/L, or 200-500 g/L total solids and the cells are treated to inactivate metabolic and enzymatic activity to prevent degradation of both (i) the PHA biopolymer and (ii) the oxidizing agent. The preferred method to inactivate metabolic and enzymatic activity of the cells is to hold the biomass slurry at a temperature in the range of 50-80° C., 80-95° C., 95-99° C., or overlapping ranges therein, including for various time durations, including 1-5, 10, 30, 60, 120, 240, 480, and 960 minutes, including overlapping durations therein. This temperature assures that the cells are metabolically and enzymatically inactive without lysing the cells. Other methods known to selectively kill cells without lysis are described in Lawlis, Jr., et. al., (U.S. Pat. No. 5,378,621) describing the use adding about 1 to 2% by weight of acetic acid. Other methods may include the addition of detergents or caustic chemicals as is well known in the art.

[0028] The inactivated cells are then selectively lysed by mixing in an oxidizing agent, and water which is stirred at temperatures greater than 40° C., 50° C., 60° C., 70° C., 72° C., 74° C., 77° C., 83° C., 85° C., 89° C., 92° C., 95° C., or 99° C. and preferably great than 60° C., 73° C., 87°, or 94° C. for a period of time sufficient to lyse the inactive cells which is typically 0.5-24 hours. The mixture is preferably stirred with the aid of mixers, for example with the aid of static mixers. The oxidizing agent is added to achieve a final concentration of 0.5-30.0% volume/volume. The oxidizing agents useful in the present invention are preferably, non-gaseous at room temperature, have an oxidation-reduction potential (E.sub.O) of more than 1.4 volts, a pH between 3 and 11, and a molar mass of less than 250 g/mol. The oxidizing agent may be for example one or more types each selected from organic and inorganic peroxides, such as but not limited to sodium peroxide (Na.sub.2O.sub.2), hydrogen peroxide (H.sub.2O.sub.2), sodium perborate (Na.sub.2H.sub.4B.sub.2O.sub.8), sodium percarbonate (Na.sub.2H.sub.3CO.sub.6), and sodium persulfate (Na.sub.2S.sub.2O.sub.8); chlorite, chlorate, metachloro perbenzoic acid (C.sub.7H.sub.5ClO.sub.3) perchlorate, performic acid (CH.sub.2O.sub.3), peracetic acid (CH.sub.3CO.sub.3H), perchlorate (ClO.sub.4), chlorine dioxide (ClO.sub.2) and other analogous halogen compounds; permanganate compounds such as potassium permanganate; sodium perborate; potassium nitrate (KNO.sub.3), sodium bismuthate; and cerium (IV) compounds such as ceric ammonium nitrate and ceric sulfate. It is preferred, however, to use hydrogen peroxide either as such or as a compound which produces hydrogen peroxide in situ or acts as an equivalent thereof, suitably a percarboxylic acid, for example peracetic acid, a perborate or a percarbonate. It is preferred however to introduce a 25-45 percent, and more preferably 30-40 percent and ideally 30-35 percent hydrogen peroxide to achieve a final concentration of 0.5 (0.5 to 3) percent volume/volume (0.1N H.sub.2O.sub.2).

[0029] It may be desirable to introduce the oxidizing agent continuously or intermittently during the process rather than introducing the whole amount at the beginning thereby minimizing the losses of oxidizing agent due to thermal decomposition.

[0030] After the cell walls have been adequately disrupted the suspension mat undergoe a solid/liquid separation to obtain solids comprising exposed PHA bioploymer (e.g., as granules), proteins, peptides, amino acids and other cell residues (referred to herein as “NPCM”) and an aqueous phase. While it is preferred to separate out the aqueous/solid phases by filtration or gravity separation, other separation processes which can be employed here are decanting and centrifugation of the NPCM containing the PHA biopolymer from the aqueous phase. The recovery and purification of the PHA from the NPCM can then be performed by a variety of different extraction methodologies, whether a solvent or aqueous extraction methodology is used one skilled in the art will appreciate that the initial step of lysing the cells is so gentle that PHA degradation is avoided, thus resulting in product having average molecular weight of PHAs typically range from about 10 kDa-3,000 kDa, and more preferably in the range of 200 kDa-1,000 kDa and can account for up to approximately 90%, 95%, or 99% of the initial polymer available within the starting biomass. Purity of PHA from the oxidized lipid degradation products can be increased further through volatilization, as well as aqueous or solvent washing steps.

[0031] It should be noted that the process for extracting PHA biopolymer may comprise different steps, and also as having varied designs, which are not explicitly mentioned. Examples of such are one or more separation steps, concentration, stirring, controlling temperature and/or controlling pH, etc. Moreover, the design of the equipment used may vary, and the present invention, as according to the claims, should be seen as embodying different forms of equipment.

[0032] The present invention will become more clear from consideration of the following examples which are set forth to further illustrate the principles of the invention and are not intended, in any way, to be limitative thereof.

EXAMPLES

Example 1

[0033] A culture of Cupriavidus, Methylocystis, Methylsinus, Methylococcus, Halomonas, Zobellella, Pseudomonas, Bacillus, and/or Chromobacterium strains was grown in batch culture in an aqueous medium on a carbon source comprising methane to give a culture containing 25-200 g/l of cells containing 30-90% of a 3-polyhydroxybutyrate.

[0034] These PHA containing cells were then heated to 60° C.-95° C. and a hydrogen peroxide solution was added to a final concentration of 0.5-15% v/v. The mixture is then stirred for an additional 1-24 hours with maintenance of the same temperature. At the end of this time the solution undergoes a solid/liquid extraction and the PHA biopolymers solids were recovered by filtration, washed and dried.

[0035] Analysis of the polymer product for impurities indicated 0.5%. The polymer product was thus considered to be 99.5% pure poly-3-hydroxybutyrate.

Example 2

[0036] A culture of Cupriavidus, Methylocystis, Methylsinus, Methylococcus, Halomonas, Zobellella, Pseudomonas, Bacillus, and/or Chromobacterium strains was grown in batch culture in an aqueous medium on a carbon source comprising methane to give a culture containing 25-200 g/l of cells containing 30-90% of a 3-polyhydroxybutyrate.

[0037] These PHA containing cells were then heated 60° C.-95° C. A peracetic acid solution is added to a final pH of 3. The mixture is then stirred for an additional 1-24 hours with maintenance of the same temperature. At the end of this time the solution undergoes a solid/liquid extraction and the PHA biopolymers solids were recovered by gravity separation, washed and dried.

[0038] Having disclosed several embodiments, it will be recognized by those of skill in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosed embodiments. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

[0039] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0040] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a process” includes a plurality of such processes and reference to “the dielectric material” includes reference to one or more dielectric materials and equivalents thereof known to those skilled in the art, and so forth.

[0041] Also, the words “comprise,” “comprising,” “include,” “including,” and “includes” when used in this specification and in the following claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, acts, or groups.