Methods for recovering PHAs from biomass

Abstract

A method for recovering polyhydroxyalkanoates from biomass is disclosed. According to the method, polynucleotide chains are cleaved by adding an endonuclease. A lysis agent is used to break the cell walls of the microbial cells and release the intracellular polyhydroxyalkanoates from the cells. Proteins are also degraded by adding a peptidase. The polyhydroxyalkanoates are then separated from the cell debris of the cells. According to the disclosure, the method is carried out without the use of organic solvents in the cleavage, lysis and degradation steps.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the earlier filing date of provisional application 63 / 235,853, filed August 23, 2021, the disclosure of which is incorporated by reference in its entirety.

[0002] The present disclosure relates to biodegradable polymer compositions. More specifically, the present disclosure relates to methods for recovering polyhydroxyalkanoates from microbial cell biomass. [Background technology]

[0003] Due to environmental concerns associated with traditional petroleum-based polymers, there is growing interest in alternative polymers that are bio-based and / or biodegradable. One class of polymers that are both bio-based and biodegradable are polyhydroxyalkanoates. Polyhydroxyalkanoates are typically produced by fermentation of bacterial or other microbial biomass in a bioreactor. Polyhydroxyalkanoates are synthesized by and accumulate in microbial cells. To recover the polyhydroxyalkanoates for use, the microbial cells must be lysed, and the polyhydroxyalkanoates must then be extracted from the lysed cell material.

[0004] Traditionally, organic solvents have been used in the polyhydroxyalkanoate extraction process. Although such solvents are successful in isolating the polyhydroxyalkanoate product, they then introduce additional challenges. For example, organic solvents may themselves raise health, safety, or environmental concerns. Thus, organic solvents may require special recovery and disposal methods. They may also cause product contamination.

[0005] It would therefore be desirable to provide an alternative method for the extraction of biomass-derived polyhydroxyalkanoates that avoids the use of hazardous organic solvents and is therefore more environmentally friendly and does not require special disposal measures. Summary of the Invention

[0006] In a first aspect, the present disclosure provides a method for recovering polyhydroxyalkanoates from a biomass of microbial cells that contains intracellular polyhydroxyalkanoates. According to one embodiment, the method includes enzymatically treating the microbial cells of the biomass by (a) cleaving polynucleotide chains from the microbial cells by adding an endonuclease to the biomass, (b) lysing the microbial cells in the biomass by adding a lysis agent to the biomass so that the cell walls of the microbial cells are disrupted and the intracellular polyhydroxyalkanoates are released from the microbial cells, and (c) degrading proteins from the microbial cells by adding a peptidase to the biomass. The polyhydroxyalkanoates are then separated from the cell debris of the microbial cells. Importantly, according to the present disclosure, the method is carried out without the use of organic solvents in the cleavage, lysis, and degradation steps.

[0007] According to certain embodiments, the endonuclease is preferably a deoxyribonuclease. In certain other embodiments, the endonuclease is preferably a ribonuclease.

[0008] In some embodiments, the lysing agent preferably comprises an agent selected from the group consisting of lysozyme, bromelain, papain, trypsin, and mixtures thereof. More preferably, the lysing agent comprises lysozyme. The lysing agent is preferably present in an amount of about 0.01 mg / L to about 8 g / L, preferably about 0.01 mg / L to about 2 g / L, based on the volume of biomass. Furthermore, in certain embodiments, the lysing agent preferably also comprises a non-ionic surfactant and a detergent.

[0009] In some cases, the dissolving step is preferably carried out at a temperature of about 20° C. to about 70° C. for a period of about 20 minutes to about 24 hours.

[0010] In certain embodiments, the peptidase preferably comprises a protease selected from the group consisting of serine proteases, neutral metalloproteinases, cysteine ​​proteases, and mixtures thereof. Also, according to certain embodiments, the proteolysis step is preferably carried out for a period of about 20 minutes to about 24 hours at a temperature of about 30° C. to about 65° C.

[0011] In certain embodiments, the microbial cells are preferably bacterial cells. More preferably, the microbial cells comprise at least one bacterial species selected from the group consisting of the genera Ralstonia, Bacillus, Escherchia, Cupriavidus, Alcaligenes, Wausteria, Aeromonas, and Pseudomonas. A particularly preferred species is E. coli.

[0012] In some cases, the method also includes an additional step of inactivating the microbial cells in the biomass prior to the enzyme treatment. This inactivation can be carried out by various means. For example, in one embodiment, the cells can be inactivated by exposing the cells to electromagnetic radiation. Alternatively, in another embodiment, the cells can be inactivated by exposing the cells to infrared energy in an amount sufficient to heat the biomass to a temperature of at least 50° C. for a period of at least 2 minutes.

[0013] In a third embodiment, the cells may be inactivated by injecting steam into the biomass. In yet another embodiment, the cells may be inactivated by either (a) adding one or more acids to the biomass in an amount sufficient to establish a biomass pH below about 6.0, or (b) adding one or more bases to the biomass in an amount sufficient to establish a biomass pH above about 8.0, or (c) high pressure (shear) homogenization of the biomass.

[0014] In some embodiments, the method also includes the additional step of bleaching the polyhydroxyalkanoates from the microbial cells by adding an oxidizing agent (preferably an oxidizing bleaching agent) to the biomass.

[0015] According to certain embodiments, the step of separating the polyhydroxyalkanoate from the cell debris is preferably carried out by filtration or centrifugation.

[0016] In certain embodiments, it is also preferred to add a surfactant to the biomass mixture before, during, or immediately after the proteolysis step. The surfactant can be ionic or non-ionic. In some cases, anionic surfactants may be preferred.

[0017] In some embodiments, the endonuclease is preferably added to the biomass prior to adding the lysis agent to the biomass, while in other embodiments, the endonuclease and the lysis agent are preferably added to the biomass simultaneously.

[0018] Also, in certain embodiments, the lysis agent is preferably added to the biomass prior to adding the peptidase to the biomass, while in other embodiments, the lysis agent and the peptidase are preferably added to the biomass simultaneously. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In a first aspect, the present disclosure provides a method for recovering polyhydroxyalkanoates from a biomass of microbial cells that contains intracellular polyhydroxyalkanoates.

[0020] Polyhydroxyalkanoates are a class of polymers that are both biologically derived and biodegradable and have attracted commercial interest in a variety of applications. In some cases, the polyhydroxyalkanoates recovered according to the present disclosure may be composed of homopolymers, such as polyhydroxybutyrate. In other cases, the polyhydroxyalkanoates are composed of copolymers, or even terpolymers, formed from different hydroxyalkanoate monomers.

[0021] For example, in some embodiments, the polyhydroxyalkanoate can be a copolymer having from about 75 to about 99.9 mole percent of monomeric repeat units of 3-hydroxybutyrate and from about 0.1 to about 25 mole percent of monomeric repeat units of a second hydroxyalkanoate having 5 to 12 carbon atoms, such as hydroxyhexanoate, hydroxyoctanoate, hydroxydecanoate, hydroxydodecanoate, or hydroxytetradecanoate. More preferably, the second hydroxyalkanoate is 3-hydroxyhexanoate.

[0022] In other embodiments, the polyhydroxyalkanoate can be a terpolymer having from about 75 to about 99.9 mole percent monomeric repeat units of 3-hydroxybutyrate, from about 0.1 to about 25 mole percent monomeric repeat units of 3-hydroxyhexanoate, and from about 0.1 to about 25 mole percent monomeric repeat units of a third 3-hydroxyalkanoate having from 5 to 12 carbon atoms.

[0023] Polyhydroxyalkanoates are typically produced by fermentation of microbial biomass in a bioreactor. In certain embodiments, the microbial cells are preferably bacterial cells. More preferably, the microorganism comprises at least one bacterial species selected from the group consisting of Ralstonia, Bacillus, Escherchia, Cupriavidus, Alcaligenes, Wausteria, Aeromonas, and Pseudomonas. A particularly preferred species is E. coli.

[0024] In other embodiments, the microorganism is selected from the group consisting of Rhodococcus spp., Bacillus spp., Cupriavidus spp., Aeromonas spp., Pseudomonas spp., Ralstonia spp., Alcaligenes spp., Wausteria spp., Azotobacter spp., Halococcus spp., Halorubrum spp., Halopiger spp., Haloarcula spp., Halomonas spp., Haloferax spp., Halostagnicola spp., Haloterrigena, Halobiforma spp., Halobacter spp., The present invention relates to a method for treating bacterial infections comprising the steps of: treating bacterial infections with at least one of the following bacteria: Acinetobacterium species, Natrinema species, Natronobacterium species, Natronococcus species, Halogranum species, Burkholderia species, Thiococcus species, Sinorhizobium species, Methylobacterium species, Zobellella species, Clostridium species, Salmonella species, Chloroflexus species, Shimwellia species, Klebsiella species, Azohydromonas species, and Vibrio species.

[0025] Polyhydroxyalkanoates are synthesized by and accumulate in microbial cells, and therefore, to recover the polyhydroxyalkanoates for use, the microbial cells must be lysed and the polyhydroxyalkanoates subsequently extracted from the lysed cell material.

[0026] In some embodiments of the present disclosure, the method will include an initial step of inactivating the microbial cells in the biomass prior to any other recovery steps. Inactivation of the biomass cells can be carried out by a variety of means.

[0027] For example, biomass cells can be inactivated by exposing the cells to electromagnetic radiation (such as gamma rays, X-rays, ultraviolet rays, or infrared rays) for a time sufficient to inactivate the cells. Typically, this will range from about 10 seconds to about 30 minutes. In some cases, biomass cells can be inactivated by exposing the cells to infrared energy in an amount sufficient to heat the biomass to a temperature of at least 50° C. for a time of at least 2 minutes.

[0028] In an alternative embodiment, the cells may be inactivated by injecting steam into the biomass. In yet another embodiment, the cells may be inactivated by either (a) adding one or more acids to the biomass in an amount sufficient to establish a biomass pH below about 6.0, or (b) adding one or more bases to the biomass in an amount sufficient to establish a biomass pH above about 8.0, or (c) high pressure (shear) homogenization of the biomass.

[0029] After any cell inactivation step, the method includes enzymatically treating the microbial cells of the biomass, which enzymatic treatment includes at least three parts: (a) cleaving polynucleotide chains derived from the microbial cells by adding an endonuclease to the biomass, (b) lysing the microbial cells in the biomass by adding a lysis agent to the biomass such that the cell walls of the microbial cells are disrupted and intracellular polyhydroxyalkanoates are released from the microbial cells, and (c) degrading proteins derived from the microbial cells by adding a peptidase to the biomass.

[0030] In general, it is believed that the three aforementioned enzymatic processing parts (i.e., polynucleotide strand cleavage, cell lysis, and protein degradation) may be performed in any order according to the methods of the present disclosure. Indeed, in some cases, two or even all three of these enzymatic processing parts may be performed simultaneously.

[0031] For example, according to certain embodiments of the present disclosure, the endonuclease may be added to the biomass prior to adding the lysis agent to the biomass, while in other embodiments, the endonuclease and the lysis agent may be added to the biomass simultaneously.

[0032] Also, according to certain embodiments of the present disclosure, the lysis agent may be added to the biomass prior to adding the peptidase to the biomass, while in other embodiments, the lysis agent and the peptidase may be added to the biomass simultaneously.

[0033] In certain embodiments of the present disclosure, endonuclease addition may be performed first, followed by lysis agent addition, then peptidase addition.

[0034] Again, the enzymatic treatment of the method involves cleaving polynucleotide chains derived from microbial cells by adding an endonuclease to the biomass.

[0035] The inventors have found that in conventional methods for recovering polyhydroxyalkanoates, the viscosity of the biomass mixture increases dramatically as substantially complete cellular polynucleotide chains are released from the cells. This high viscosity makes it more difficult to recover the polyhydroxyalkanoates. However, the inventors have found that by adding an endonuclease at this point in the process to cleave the polynucleotide chains into smaller fragments, the viscosity of the biomass mixture remains reduced, which may facilitate the recovery of the polyhydroxyalkanoates.

[0036] In some cases, the endonuclease can be a deoxyribonuclease (such as an endodeoxyribonuclease, an exodeoxyribonuclease, or a micrococcal nuclease). Alternatively, the endonuclease can be a ribonuclease (such as an endoribonuclease or an exoribonuclease). Additionally, in some embodiments, multiple deoxyribonucleases, or multiple ribonucleases, or a combination of deoxyribonucleases and ribonucleases can be used in the cleavage step.

[0037] Typically, the cleavage step is carried out by mixing the endonuclease(s) with the cell biomass in an aqueous mixture without any organic solvent. The endonuclease(s) are typically added in an amount of about 0.001 micrograms / gram to about 5 milligrams / gram based on the weight of the biomass being treated.

[0038] Water may also be added to the mixture to adjust the total solids content in the mixture. Generally, the total solids content is about 1 to about 30 weight percent, preferably about 5 to about 25 weight percent, and more preferably about 10 to about 15 weight percent.

[0039] Generally, the biomass and endonuclease(s) are mixed and stirred at a temperature of about 30° C. to about 60° C. for a period of about 20 minutes to about 24 hours, during which time the endonuclease(s) enzymatically cleave the polynucleotide chains derived from the microbial cells. Conveniently, this step is carried out in the same bioreactor in which the biomass was originally fermented, or it can be carried out in another suitable reactor or mixing vessel.

[0040] The enzymatic treatment of the present disclosure also includes a lysis step, during which a lysis agent is added to the biomass to disrupt the cell walls of the microbial cells and liberate intracellular polyhydroxyalkanoates from the microbial cells.

[0041] In some embodiments, the lysis agent comprises an N-acetyl-muramidglycan hydrolase. Preferably, the lysis agent comprises a muramidase (such as lysozyme, amidase, glucosaminidase, lytic transglycosylase, and peptidoglycan hydrolase). In other embodiments, the lysis agent may be bromelain, papain, and / or trypsin. More preferably, the lysis agent is an agent selected from the group consisting of lysozyme, bromelain, papain, trypsin, and mixtures thereof, in an amount of about 0.01 mg / L to about 8 g / L based on the volume of biomass. Even more preferably, the lysis agent is lysozyme. Also, the amount of the lysis agent is more preferably about 0.01 g / L to about 2 g / L based on the volume of biomass.

[0042] In some cases, the lysis agent may also include a non-ionic surfactant and / or a detergent to enhance cell membrane disintegration. For example, the lysis agent may include a non-ionic surfactant (such as a polyethylene glycol and / or polypropylene glycol polymer or a polyacid), typically in an amount of about 0.01 weight percent to about 2 weight percent based on the weight of the biomass. The lysis agent may include a detergent (such as a polysorbate (e.g., Tween 20 or Tween 80), Triton, an ethoxylated alcohol (e.g., Tergitol), etc.), typically in an amount of about 0.01 weight percent to about 5 weight percent based on the weight of the biomass.

[0043] Generally, lysis is carried out by mixing the lysing agent with the cellular biomass in an aqueous mixture without any organic solvent. The biomass and lysing agent are typically mixed and stirred at a temperature of about 20° C. to about 70° C. for a period of about 20 minutes to about 24 hours, preferably about 2 to 4 hours. Preferably, the temperature is about 30° C. to about 65° C., more preferably about 37° C. to about 60° C. During this time, the lysing agent acts to enzymatically disrupt the cell walls of the microbial cells and liberate intracellular polyhydroxyalkanoates from the microbial cells.

[0044] As with the cutting step, this step can be carried out in the same bioreactor in which the biomass was originally fermented, or in another suitable reactor or mixing vessel.

[0045] The enzymatic treatment of the present disclosure also includes a proteolytic step during which peptidases are added to the biomass to break down proteins from the microbial cells.

[0046] Similar to the polynucleotide chains discussed above, the inventors have found that the release of large protein structures from biomass cells can also significantly increase the viscosity of the biomass mixture, making recovery of polyhydroxyalkanoates more difficult. The inventors have found that the addition of peptidases to break down cellular proteins into smaller fragments can maintain a reduced viscosity of the biomass mixture, thereby facilitating recovery of polyhydroxyalkanoates.

[0047] In certain embodiments, the peptidase may comprise a protease selected from the group consisting of serine proteases, neutral metalloproteinases, cysteine ​​proteases, and mixtures thereof. Combinations of peptidases may also be used. Typically, the proteolysis step is carried out by mixing the peptidase(s) with the cell biomass in an aqueous mixture without any organic solvent. The peptidase(s) are typically added in an amount of about 0.01 weight percent to about 5 weight percent based on the weight of the biomass being treated.

[0048] In some cases, a surfactant is added to the biomass mixture during or immediately before or after the proteolysis step. The surfactant may be ionic or non-ionic. In some cases, anionic surfactants may be preferred. For example, suitable surfactants may include polyethylene glycol sorbitan monolaurate, polyethylene glycol sorbitan monooleate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan palmitate, secondary ethoxylated alcohol, polyethylene tert-octylphenyl ether, or sodium lauryl sulfate, typically in an amount of about 0.01% to about 10% weight percent based on the weight of the biomass.

[0049] Generally, the biomass and peptidase(s) are mixed and stirred at a temperature of about 30° C. to about 65° C., preferably about 50° C. to about 60° C., more preferably about 57° C. to about 58° C., for a period of about 20 minutes to about 24 hours, preferably about 2 to about 4 hours, during which time the peptidase(s) enzymatically cleave the polypeptide chains from the microbial cells. As with the previous step, this step can be carried out in the same bioreactor in which the biomass was originally fermented, or in another suitable reactor or mixing vessel.

[0050] After proteolysis, the polyhydroxyalkanoates are physically separated from the cell debris of the microbial cells. This separation can be carried out by various separation techniques. For example, separation can be carried out by filtration using a plate and frame filter press, or tangential flow filtration, or centrifugation.

[0051] Prior to physical separation, a surfactant, preferably a non-ionic surfactant, can optionally be added to the biomass mixture to promote the formation of micelles that aggregate the cell debris and facilitate separation of the polyhydroxyalkanoates from the remaining cell debris.

[0052] Optionally, the polyhydroxyalkanoates can be bleached with an oxidizing agent (such as sodium hypochlorite, hydrogen peroxide, peracetic acid, or ozone, more preferably hydrogen peroxide) to deodorize and whiten the polyhydroxyalkanoates. Typically, this step is carried out by mixing the oxidizing agent with the biomass after the proteolysis step and before the separation step.

[0053] The polyhydroxyalkanoates may also be optionally treated in a final washing step after filtration or other physical separation. The washing step may be carried out while the polyhydroxyalkanoates are still in the filter cake on the filter press or in the separation tank after filtration is complete. The washing mixture may include water alone and / or a mixture of water and an aliphatic alcohol, such as methanol, ethanol, or isopropanol.

[0054] The present disclosure is further illustrated by the following embodiments.

[0055] EMBODIMENT 1 1. A method for recovering polyhydroxyalkanoates from a biomass of microbial cells that contains intracellular polyhydroxyalkanoates, the method comprising: (a) cleaving polynucleotide chains derived from the microbial cells by adding an endonuclease to the biomass; (b) lysing the microbial cells in the biomass by adding a lysis agent to the biomass such that cell walls of the microbial cells are disrupted and the intracellular polyhydroxyalkanoates are released from the microbial cells; and (c) degrading proteins from the microbial cells by adding a peptidase to the biomass; enzymatically treating the microbial cells of the biomass by Separating the polyhydroxyalkanoate from the cell debris of the microbial cells; Including, The method, wherein the cleaving, dissolving, and decomposing steps are carried out without the use of organic solvents.

[0056] EMBODIMENT 2 2. The method of embodiment 1, wherein the endonuclease comprises a deoxyribonuclease.

[0057] EMBODIMENT 3 2. The method of embodiment 1, wherein the endonuclease comprises a ribonuclease.

[0058] EMBODIMENT 4 13. The method of any of the preceding embodiments, wherein the lysing agent comprises an agent selected from the group consisting of lysozyme, bromelain, papain, trypsin, and mixtures thereof, in an amount of about 0.01 mg / L to about 8 g / L based on the volume of the biomass.

[0059] EMBODIMENT 5 5. The method of embodiment 4, wherein the lysis agent further comprises a non-ionic surfactant and a detergent.

[0060] EMBODIMENT 6 The method according to any of the preceding embodiments, wherein the dissolving step is carried out for a time period of about 20 minutes to about 24 hours at a temperature of about 20° C. to about 70° C.

[0061] EMBODIMENT 7 13. The method of any of the preceding embodiments, wherein the peptidase comprises a protease selected from the group consisting of serine proteases, neutral metalloproteinases, cysteine ​​proteases, and mixtures thereof.

[0062] EMBODIMENT 8 The method according to any of the preceding embodiments, wherein the proteolysis step is carried out for a period of from about 20 minutes to about 24 hours at a temperature of from about 30° C. to about 65° C.

[0063] EMBODIMENT 9 The method of any of the preceding embodiments, wherein the microbial cells are bacterial cells.

[0064] EMBODIMENT 10 The method of any of the preceding embodiments, further comprising, prior to the cutting step, inactivating the microbial cells in the biomass by exposing the microbial cells to electromagnetic radiation.

[0065] EMBODIMENT 11 10. The method of any of the preceding claims, further comprising, prior to the cutting step, inactivating the cells in the biomass by exposing the cells to infrared energy in an amount sufficient to heat the biomass to a temperature of at least 50°C for a period of at least 2 minutes.

[0066] EMBODIMENT 12 10. The method according to any one of the preceding claims, further comprising, prior to the cutting step, inactivating the microbial cells in the biomass by injecting steam into the biomass.

[0067] EMBODIMENT 13 10. The method of any one of the preceding claims, further comprising the step of inactivating the microbial cells in the biomass by either (a) adding one or more acids to the biomass in an amount sufficient to establish a biomass pH of less than about 6.0, or (b) adding one or more bases to the biomass in an amount sufficient to establish a biomass pH of greater than about 8.0, prior to the cutting step.

[0068] EMBODIMENT 14 The method of any of the preceding embodiments, further comprising bleaching the polyhydroxyalkanoates from the microbial cells by adding an oxidizing agent to the biomass.

[0069] EMBODIMENT 15 The method of any of the preceding embodiments, wherein the separation of the polyhydroxyalkanoate from the cell debris is carried out by filtration or centrifugation.

[0070] EMBODIMENT 16 The method of any of the preceding embodiments, further comprising adding a surfactant to the biomass mixture before, during, or after the proteolysis step.

[0071] EMBODIMENT 17 13. The method of any of the preceding embodiments, wherein the endonuclease is added to the biomass prior to adding the lysis agent to the biomass.

[0072] EMBODIMENT 18 13. The method of any preceding embodiment, wherein the endonuclease and the lysing agent are added to the biomass simultaneously.

[0073] EMBODIMENT 19 13. The method of any preceding embodiment, wherein the lysis agent is added to the biomass prior to adding the peptidase to the biomass.

[0074] EMBODIMENT 20 4. The method of any preceding embodiment, wherein the lysing agent and the peptidase are added to the biomass simultaneously. EXAMPLES

[0075] The following non-limiting examples illustrate various additional aspects of the present invention. Unless otherwise specified, temperatures are in degrees Celsius and percentages are by weight based on the dry weight of the preparation.

[0076] Example 1 : In this example, a biomass consisting of an aqueous broth of E. coli cells from a fermenter was enzymatically treated to extract polyalkanoates from the biomass cells. The total volume of the treated biomass broth was about 100 mL and its dry solids content was about 15 weight percent.

[0077] The extraction process was carried out in a fermenter with gentle agitation at about 50 rpm and an initial temperature of about 37° C. In the first addition step, the following chemicals were added to the biomass broth: [Table 1]

[0078] The additives included two surfactants, an endonuclease (C-Lecta Denarase) to cleave polynucleotide chains, and a lysing agent (lysozyme) to break down cell walls in the biomass. After adding the chemicals, the biomass broth was stirred for 60 minutes while maintaining the temperature at about 37°C and the pH at about 6.2 to allow the endonuclease and lysozyme to break down the polynucleotide chains and cell walls, respectively.

[0079] The temperature of the biomass mixture was then increased to about 60° C. and a second chemical addition was performed. In this second addition step, the following chemicals were added to the biomass broth: [Table 2]

[0080] After this addition, the biomass broth was stirred for an additional 60 minutes while maintaining the temperature at about 60° C. to allow the proteases to break down proteins from the cells of the biomass.

[0081] A third chemical addition was then made in which 3.69 grams of hydrogen peroxide was added to the biomass mixture. After this addition, the biomass broth was stirred for an additional 240 minutes while maintaining the temperature at about 58° C. to allow the hydrogen peroxide to bleach the mixture, thereby improving its color and odor.

[0082] Finally, the polyalkanoates were separated from the remainder of the biomass by filtration.

[0083] The foregoing description of the preferred embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments have been chosen and described in an effort to provide the best illustration of the principles of the invention and its practical application, thereby enabling others skilled in the art to utilize the invention in various embodiments, with various modifications made to suit the specific uses contemplated. All such modifications and variations are within the scope of the invention, as determined by the appended claims, when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Claims

1. 1. A method for recovering polyhydroxyalkanoates from a biomass of microbial cells containing intracellular polyhydroxyalkanoates, said method comprising: (a) adding an endonuclease to the biomass to cleave polynucleotide chains derived from the microbial cells at a temperature of about 30°C to about 60°C for a time period of about 20 minutes to about 24 hours; (b) lysing the microbial cells in the biomass by adding a lysing agent to the biomass so that the cell walls of the microbial cells are disrupted and the intracellular polyhydroxyalkanoates are released from the microbial cells; and (c) adding peptidase to the biomass to degrade proteins derived from the microbial cells; enzymatically treating the microbial cells of the biomass by Separating the polyhydroxyalkanoate from the cell debris of the microbial cells; Including, the dissolving step is carried out for a time period of 20 minutes to 24 hours at a temperature of 20°C to 70°C; the proteolysis step is carried out for a time period of 20 minutes to 24 hours at a temperature of 30°C to 65°C; the endonuclease is added to the biomass before adding the lysing agent to the biomass, and the lysing agent is added to the biomass before adding the peptidase to the biomass; and The method, wherein the cutting step, the dissolving step, and the decomposition step are carried out without using organic solvents.

2. The method of claim 1 , wherein the endonuclease comprises a deoxyribonuclease.

3. The method of claim 1 , wherein the endonuclease comprises a ribonuclease.

4. 10. The method of claim 1, wherein the lysing agent comprises an agent selected from the group consisting of lysozyme, bromelain, papain, trypsin, and mixtures thereof, in an amount of 0.01 mg / L to 8 g / L based on the volume of the biomass.

5. The method of claim 4 , wherein the lysing agent further comprises a non-ionic surfactant and a detergent.

6. 10. The method of claim 1, wherein the peptidase comprises a protease selected from the group consisting of serine proteases, neutral metalloproteinases, cysteine ​​proteases, and mixtures thereof.

7. The method of claim 1 , wherein the microbial cells are bacterial cells.

8. 10. The method of claim 1, further comprising, prior to said cutting step, inactivating the cells in said biomass by exposing said microbial cells to electromagnetic radiation.

9. 10. The method of claim 1, further comprising, prior to said cutting step, inactivating the cells in the biomass by exposing the microbial cells to infrared energy in an amount sufficient to heat the biomass to a temperature of at least 50°C for a period of at least 2 minutes.

10. 10. The method of claim 1, further comprising, prior to said cutting step, inactivating said microbial cells in said biomass by injecting steam into said biomass.

11. 10. The method of claim 1, further comprising, prior to said cutting step, inactivating the microbial cells in the biomass by either (a) adding one or more acids to the biomass in an amount sufficient to establish a biomass pH of less than 6.0, or (b) adding one or more bases to the biomass in an amount sufficient to establish a biomass pH of greater than 8.

0.

12. 10. The method of claim 1, further comprising bleaching the polyhydroxyalkanoate from the microbial cells by adding an oxidizing agent to the biomass.

13. 2. The method of claim 1, wherein the separation of the polyhydroxyalkanoate from the cell debris is carried out by filtration or centrifugation.

14. 10. The method of claim 1, further comprising adding a surfactant to the biomass mixture before, during, or after the proteolysis step.