HK1125391B - Polymer extraction methods - Google Patents
Polymer extraction methods Download PDFInfo
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- HK1125391B HK1125391B HK09104103.7A HK09104103A HK1125391B HK 1125391 B HK1125391 B HK 1125391B HK 09104103 A HK09104103 A HK 09104103A HK 1125391 B HK1125391 B HK 1125391B
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Description
The present application is a divisional application of the Chinese invention application (title of the invention: method of extracting polymer; application date: 7/23/2003; application number: 03823836.5 (International application number: PCT/US 2003/023034)).
Technical Field
The present invention relates to a method for extracting a polymer.
Background
Polyhydroxyalkanoates ("PHAs") can be extracted from biomass having PHA-containing cells. Generally, the process involves mixing the biomass with a solvent for the PHA, followed by heating and stirring. This generally provides a system comprising two phases, one phase being a solution containing solvent and PHA, the other phase containing residual biomass having cells containing a reduced amount of PHA. Typically, the two phases are separated and the PHA is then removed from the solvent.
SUMMARY
The present invention relates generally to polymer extraction processes.
In one aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system to provide a residual biomass and a solution. The solvent system includes a solvent for the polymer and a precipitant for the polymer, and the solution includes the polymer, the solvent for the polymer, and the precipitant for the polymer. The method further includes applying a centrifugal force to the solution and the residual biomass to separate at least some of the solution from the residual biomass.
In another aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system to provide a residual biomass and a solution including a polymer and the solvent system, and separating at least some of the solution from the residual biomass. The method also includes adding a precipitant for the polymer to the solution to remove at least some of the polymer from the solvent system.
In another aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system to provide residual biomass and a solution including a polymer and the solvent system. The solution has a polymer concentration of at least about 2% and a viscosity of at most about 100 centipoise. The method also includes separating at least some of the solution from the residual biomass.
In one aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system to provide residual biomass and a solution. The solvent system includes a solvent for the polymer, and the solution includes the polymer and the solvent for the polymer. The boiling point of the solvent for the polymer may be higher than 100 ℃. The method also includes separating the polymer from the residual biomass.
In another aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a volume of a solvent system to provide a residual biomass and a solution including a polymer and a solvent for the polymer, and separating at least some of the solution from the residual biomass. The method further includes adding a volume of a precipitant for the polymer to the separated solution to remove at least some of the polymer from the solution. The volume of precipitant added is less than about 2 parts relative to the volume of solvent system.
In another aspect, the invention features a method of separating a polymer from biomass containing the polymer and biomass impurities. The method includes contacting the biomass with a precipitant for the polymer to remove at least some biomass impurities from the biomass containing the polymer and the biomass impurities, thereby providing a purified biomass containing the polymer. The method also includes contacting the purified biomass with a solvent system to provide a solution of residual biomass and a solvent including a polymer and a polymer.
In another aspect, the invention features a method of separating a polymer from biomass containing the polymer and biomass impurities. The method includes chemically pretreating the biomass to remove at least some biomass impurities from the biomass containing the biomass and impurities, thereby providing a purified biomass containing the polymer. Chemical treatment involves controlling pH, temperature, and contact time in the presence or absence of additional chemicals, such as surfactants, detergents, enzymes, or similar materials that can aid in the removal of biomass impurities. The method also includes contacting the purified biomass with a solvent system to provide a solution of residual biomass and a solvent including a polymer and a polymer.
In one aspect, the invention features a method of separating a polymer from biomass containing the polymer. The method includes contacting the biomass with a solvent system under countercurrent flow conditions.
In another aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system using a one-step process that forms a PHA phase and a residual biomass phase. The ratio of the volume of solvent system present in the PHA phase to the volume of solvent system contacted with the biomass is at least about 0.8.
In another aspect, the invention features a method of separating a polymer from a biomass containing the polymer. The method includes contacting the biomass with a solvent system using a one-step process that forms a PHA phase and a residual biomass phase. The ratio of the volume of solvent system present in the residual biomass phase to the volume of solvent system contacted with the biomass is at most about 0.2.
In particular embodiments, the method is capable of extracting polymers (e.g., PHA) from biomass in relatively high yields. In certain embodiments, relatively high yields of polymer (e.g., PHA) may be extracted from biomass without using multiple steps (e.g., using a single step process).
In certain embodiments, the method is capable of extracting relatively pure polymers (e.g., PHA).
In certain embodiments, the method enables the use of solvents and/or precipitants in a relatively efficient manner. For example, a relatively high proportion of the solvent and/or precipitant used in the process may be recovered (e.g., for reuse).
In certain embodiments, the method may have reduced environmental impact.
In particular embodiments, the process is capable of extracting polymer at relatively high space velocities (e.g., high throughput, with overall low residence time in the process equipment).
In certain embodiments, the process can result in relatively small amounts of undesirable reaction by-products (e.g., organic acids). This can, for example, reduce the likelihood of corrosion or other undesirable damage to the systems used in the present process, and/or extend the useful life of such systems.
In certain embodiments, the method can provide relatively high volume throughput (e.g., by using a single step process).
In certain embodiments, the method can provide relatively high solvent recovery.
In certain embodiments, the method can be performed using a single-step apparatus (e.g., a counter-current centrifugal contactor).
In certain embodiments, a relatively low viscosity residual biomass is formed (e.g., using countercurrent conditions), which may enhance subsequent processing, such as stripping residual solvent and concentrating solids content (solidcontents) (e.g., by evaporation, filtration, or drying).
The present invention specifically relates to the following aspects:
a method of separating a polymer from biomass containing the polymer, the method comprising:
contacting the biomass with a solvent system comprising a solvent for the polymer and a precipitant for the polymer to provide a solution of residual biomass and precipitant comprising the polymer, the solvent for the polymer, and the polymer; and
a centrifugal force is applied to the solution and the residual biomass to separate at least some of the solution from the residual biomass.
Item 2 the method of item 1, wherein the solvent for the polymer has a density of less than about 0.95 kg/liter.
The process of item 3, item 1, wherein the solvent for the polymer is non-halogenated.
The method of item 4, item 1, wherein the solvent of the polymer has a solubility in water of less than about 1%.
The method of item 5, item 1, wherein the solvent for the polymer is substantially non-hydrolyzable.
The process of clause 6, 1, wherein the solvent for the polymer has a logK value relative to water at 100 ℃ of at least about 1.5.
Item 7 the process of item 1, wherein the solvent has a boiling point above 100 ℃.
The method of item 8, item 1, wherein the solvent for the polymer is selected from the group consisting of ketones, esters, and alcohols.
Item 9 the method of item 1, wherein the solvent for the polymer is selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
The method of item 10, item 1, wherein the precipitant for the polymer dissolves less than about 0.2% of the polymer at room temperature.
The method of item 11, wherein the relative volatility of the solvent for the polymer and the precipitant for the polymer is at least about 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
The process of item 12, item 1, wherein the solvent for the polymer and the precipitant for the polymer do not form an azeotrope.
The process of item 13, item 1, wherein the precipitating agent comprises at least one alkane.
Item 14 the method of item 1, wherein the solution comprises up to about 25% by volume of the polymeric precipitant.
The method of item 15, item 1, wherein the polymer concentration of the solution is at least about 2%.
Item 16 the method of item 1, wherein the viscosity of the solution is up to about 100 centipoise.
The method of item 17, item 1, wherein the biomass containing the polymer is derived from a microorganism and has a polymer content of at least about 50 wt.%.
The method of item 18, wherein the biomass containing the polymer is plant derived and has a polymer content of less than about 50% by weight.
The method of item 19, item 1, wherein the biomass containing the polymer comprises cells containing the polymer.
The method of item 20, item 1, further comprising removing at least some of the polymer from the solution.
The method of item 20, further comprising extruding the removed polymer to dry and pelletize the polymer.
The method of item 22, item 20, wherein removing the polymer from the solution does not include exposing the solution to hot water.
The method of item 23, item 20, wherein removing the polymer from the solution comprises adding a second precipitant for the polymer to the solution.
The method of item 24, item 23, wherein the first and second precipitating agents for the polymer are the same.
The method of item 25, item 20, further comprising evaporating a portion of the solution before removing at least some of the polymer from the solution.
Item 26 the method of item 1, further comprising, after applying the centrifugal force to the solution, adding a volume of a second precipitant for the polymer to remove at least some of the polymer from the solution, wherein the volume of the second precipitant is less than about 2 parts relative to the volume of the solvent system.
The method of item 1, wherein the polymer comprises PHA.
A method of separating a polymer from a biomass containing the polymer, the method comprising:
contacting the biomass with a solvent system to provide residual biomass and a solution comprising a polymer and the solvent system;
adding a precipitant for the polymer to the solution; and
at least some of the solution is separated from the residual biomass after the addition of the precipitant for the polymer.
The method of item 29, item 28, wherein the solvent system comprises a solvent for the polymer, the solvent having a density of less than about 0.95 kilograms per liter.
The method of item 30, item 28, wherein the solvent system comprises a non-halogenated solvent for the polymer.
The method of item 31, item 28, wherein the solvent system comprises a solvent for the polymer, the solvent having a solubility in water of less than about 1%.
The method of item 32, item 28, wherein the solvent system comprises a solvent for the polymer, the solvent being substantially non-hydrolyzable.
The method of item 33, item 28, wherein the solvent system comprises a solvent for the polymer having a logK value relative to water at 100 ℃ of at least about 1.5.
The method of item 34, item 28, wherein the solvent system comprises a solvent for the polymer, the solvent having a boiling point greater than 100 ℃.
The method of item 35, item 28, wherein the solvent system comprises at least one solvent selected from the group consisting of ketones, esters, and alcohols.
The method of item 36, item 28, wherein the solvent system comprises at least one solvent selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
The method of item 37, item 28, wherein the solvent system comprises a precipitant for the polymer that dissolves less than about 0.2% of the polymer at room temperature.
The method of item 38, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the relative volatilities of the solvent for the polymer and the precipitant for the polymer are at least about 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
The method of item 39, item 28, wherein the solvent system comprises a solvent for the polymer and a precipitating agent for the polymer, and the solvent for the polymer and the precipitating agent for the polymer do not form an azeotrope.
The method of item 40, item 28, wherein the solvent system comprises a precipitant for the polymer, the precipitant comprising an alkane.
The method of item 41 item 28, wherein the polymer concentration of the solution is at least about 2%.
Item 42 the method of item 28, wherein the viscosity of the solution is up to about 100 centipoise.
The method of item 43, wherein the biomass containing the polymer is derived from a microorganism and has a polymer content of at least about 50% by weight.
The method of item 44, wherein the biomass containing the polymer is plant derived and has a polymer content of less than about 50% by weight.
The method of item 45, item 28, wherein the biomass containing the polymer comprises cells containing the polymer.
The method of item 46. item 28, further comprising removing at least a portion of the polymer from the solution, wherein removing the polymer does not include exposing the solution to hot water.
Item 47 the method of item 28, wherein separating at least some of the solution from the residual biomass comprises applying a centrifugal force to the solution and the residual biomass.
The method of item 48, item 28, further comprising evaporating a portion of the solution to remove at least some of the polymer from the solvent system prior to adding the precipitant for the polymer to the solution.
Item 49 the method of item 28, further comprising, after separating, adding a volume of a second precipitant for the polymer to remove at least some of the polymer from the solution, wherein the volume of the second precipitant is less than about 2 parts relative to the volume of the solvent system.
The method of item 28, further comprising: removing at least a portion of the polymer from the solution; and
the removed polymer was extruded to dry and pelletize the polymer.
The method of item 51. item 28, wherein the polymer comprises PHA.
A method of separating a polymer from a biomass containing the polymer, the method comprising: contacting the biomass with a solvent system to provide residual biomass and a solution comprising a polymer and the solvent system, the solution having a polymer concentration of at least about 2% and a viscosity of at most about 100 centipoise; and
separating at least some of the solution from the residual biomass.
The method of item 53, item 52, wherein the solvent system comprises a solvent for the polymer, the solvent having a density of less than about 0.95 kilograms per liter.
The method of item 54 item 52, wherein the solvent system comprises a non-halogenated solvent for the polymer.
The method of item 55, wherein the solvent system comprises a solvent for the polymer, the solvent having a solubility in water of less than about 1%.
The method of item 56, item 52, wherein the solvent system comprises a solvent for the polymer, the solvent being substantially non-hydrolyzable.
The method of item 57, item 52, wherein the solvent system comprises a solvent for the polymer having a LogK value relative to water at 100 ℃ of at least about 1.5.
The method of item 58, item 52, wherein the solvent system comprises a solvent for the polymer, the solvent having a boiling point greater than 100 ℃.
The method of item 59, item 52, wherein the solvent system comprises at least one solvent selected from the group consisting of ketones, esters, and alcohols.
The method of item 52, wherein the solvent system comprises at least one solvent selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
The method of item 61 item 52, wherein the solvent system comprises a precipitant for the polymer that dissolves less than about 0.2% of the polymer at room temperature.
Item 62 the method of item 52, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the relative volatilities of the solvent for the polymer and the precipitant for the polymer are at least about 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
Item 63 the method of item 52, wherein the solvent system comprises a solvent for the polymer and a precipitating agent for the polymer, and the solvent for the polymer and the precipitating agent for the polymer do not form an azeotrope.
The method of item 64, item 52, wherein the solvent system comprises a precipitant for the polymer, the precipitant comprising an alkane.
Item 65 the method of item 52, wherein the biomass containing the polymer is derived from a microorganism and has a polymer content of at least about 50 weight percent.
The method of item 66, wherein the biomass containing the polymer is plant-derived and has a polymer content of less than about 50% by weight.
The method of item 67, item 52, wherein the biomass comprising the polymer comprises cells comprising the polymer.
The method of item 68, item 52, further comprising removing at least some of the polymer from the solution.
The method of item 69, item 68, further comprising extruding the removed polymer to dry and pelletize the polymer.
The method of item 68, wherein removing the polymer from the solution does not include exposing the solution to hot water.
The method of item 71, item 68, wherein removing the polymer from the solution comprises adding a precipitant for the polymer to the solution.
The method of item 68, item 72, further comprising evaporating a portion of the solution before removing at least some of the polymer from the solution.
Item 73 the method of item 52, wherein separating at least some of the solution from the residual biomass comprises applying a centrifugal force to the solution and the residual biomass.
The method of item 74, item 52, wherein the polymer comprises a PHA.
A method of separating a polymer from biomass containing the polymer and biomass impurities, the method comprising:
contacting the biomass with at least one alkane to remove at least some biomass impurities from the biomass containing the polymer and biomass impurities, thereby providing a purified biomass containing the polymer; and
the purified biomass is contacted with a solvent system to provide a solution of residual biomass and a solvent comprising a polymer and a polymer.
Item 76 the method of item 75, further comprising separating at least some of the solution from the residual biomass, and adding a precipitant for the polymer to the solution to remove at least some of the polymer from the solvent system.
The method of item 77, item 75, wherein the solvent system comprises a solvent for the polymer, the solvent having a density of less than about 0.95 kilograms per liter.
The method of item 78, item 75, wherein the solvent system comprises a non-halogenated solvent for the polymer.
The method of item 75, wherein the solvent system comprises a solvent for the polymer, the solvent having a solubility in water of less than about 1%.
The method of item 80, item 75, wherein the solvent system comprises a solvent for the polymer, the solvent being substantially non-hydrolyzable.
The method of item 81, item 75, wherein the solvent system comprises a solvent for the polymer having a logK value relative to water at 100 ℃ of at least about 1.5.
The method of item 82, item 75, wherein the solvent system comprises a solvent for the polymer, the solvent having a boiling point greater than 100 ℃.
The method of item 83, item 75, wherein the solvent system comprises at least one solvent selected from the group consisting of ketones, esters, and alcohols.
Item 84 the method of item 75, wherein the solvent system comprises at least one solvent selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
The method of item 85, item 75, wherein the solvent system comprises a precipitant for the polymer that dissolves less than about 0.2% of the polymer at room temperature.
Item 86 the method of item 75, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the relative volatilities of the solvent for the polymer and the precipitant for the polymer are at least about 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
The method of item 87, item 75, wherein the solvent system comprises a solvent for the polymer and a precipitating agent for the polymer, and the solvent for the polymer and the precipitating agent for the polymer do not form an azeotrope.
The method of item 88, item 75, wherein the alkane is hexane, heptane, or isoalkane.
The method of item 89, item 75, wherein the polymer concentration of the solution is at least about 2%.
Item 90 the method of item 75, wherein the viscosity of the solution is up to about 100 centipoise.
Item 91 the method of item 75, wherein the biomass containing the polymer is derived from a microorganism and has a polymer content of at least about 50 weight percent.
The method of item 92, item 75, wherein the biomass containing the polymer is plant-derived and has a polymer content of less than about 50 wt.%.
The method of item 93, item 75, wherein the biomass containing the polymer comprises cells containing the polymer.
Item 94 the method of item 75, comprising removing at least a portion of the polymer from the solution, wherein removing the polymer does not comprise exposing the solution to hot water.
Item 95 the method of item 75, wherein separating at least some of the solution from the residual biomass comprises applying a centrifugal force to the solution and the residual biomass.
The method of item 96, item 75, further comprising evaporating a portion of the solution to remove at least some of the polymer from the solvent system prior to adding the precipitant for the polymer to the solution.
The method of item 97, item 75, further comprising: removing at least a portion of the polymer from the solution; and
the removed polymer was extruded to dry and pelletize the polymer.
The method of item 75, item 98, wherein the polymer comprises a PHA.
A method of separating a polymer from a biomass containing the polymer, the method comprising:
the biomass and solvent system are contacted under countercurrent flow conditions.
The method of item 99, further comprising forming a PHA phase and a residual biomass phase, wherein a ratio of a volume of the solvent system present in the PHA phase to a volume of the solvent system contacted with the biomass is at least about 0.8.
Item 101 the method of item 99, further comprising forming a PHA phase and a residual biomass phase, wherein a ratio of a volume of the solvent system present in the residual biomass phase to a volume of the solvent system contacted with the biomass is at most about 0.2.
The method of item 102 item 99, wherein the method is a single step method.
The method of item 103, item 99, wherein the method is a multi-step method.
The method of item 104 item 99, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
A method of separating a polymer from a biomass containing the polymer, the method comprising:
contacting the biomass and the solvent system using a one-step process that forms a PHA phase and a residual biomass phase, wherein the ratio of the volume of the solvent system present in the PHA phase to the volume of the solvent system contacted with the biomass is at least about 0.8.
Item 106 the method of item 105, wherein a ratio of a volume of the solvent system present in the residual biomass phase to a volume of the solvent system contacted with the biomass is at most about 0.2.
Item 107. a method of separating a polymer from a biomass containing the polymer, the method comprising:
contacting the biomass with a solvent system using a one-step process that forms a PHA phase and a residual biomass phase, wherein a ratio of a volume of the solvent system present in the residual biomass phase to a volume of the solvent system contacted with the biomass is at most about 0.2.
Item 108 the method of item 1, wherein the solvent system is contacted with the biomass under countercurrent flow conditions.
The method of item 109, item 108, wherein the method is a single step method.
The method of item 110, item 108, wherein the method is a multi-step method.
Item 111 the method of item 108, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
The method of item 112, item 28, wherein the solvent system is contacted with the biomass under countercurrent flow conditions.
The method of item 113, item 112, wherein the method is a single step method.
The method of item 114, item 112, wherein the method is a multi-step method.
The method of item 115, item 112, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
The method of item 116, item 52, wherein the solvent system is contacted with the biomass under countercurrent flow conditions.
The method of item 117 item 116, wherein the method is a single step method.
The method of item 118, item 116, wherein the method is a multi-step method.
The method of item 119, item 116, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
The method of item 120 item 75, wherein the at least one alkane is contacted with the biomass under countercurrent flow conditions.
The process of item 121 item 120, wherein the process is a one-step process.
The method of item 122, item 120, wherein the method is a multi-step method.
Item 123 the process of item 120, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
The method of item 99, wherein the at least one alkane is contacted with the biomass under countercurrent flow conditions.
The process of item 125 item 124, wherein the process is a one-step process.
The method of item 126, item 124, wherein the method is a multi-step method.
The method of item 127 item 124, wherein the countercurrent conditions comprise a pressure of at least about 65 psig.
Furthermore, the features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Drawings
FIG. 1 is a flow diagram of an embodiment of a method of extracting PHA from biomass having PHA-containing cells;
FIG. 2 is a partial flow diagram of an embodiment of a method of extracting PHA from biomass having PHA-containing cells; and
FIG. 3 is a graph showing the viscosity and polymer content of example III.
Detailed Description
Fig. 1 is a flow diagram of an embodiment of a method of extracting PHA from biomass having PHA-containing cells. A slurry (slurry) containing biomass and water is provided. A solvent system is added to the slurry to form a mixture containing the slurry and the solvent system. The mixture is stirred (e.g., agitated) to provide a combination comprising two phases. One phase is formed from a solution containing PHA and a solvent system and trace amounts of biomass ("PHA phase"). The second phase is formed by the residual biomass of the cells with reduced polymer content, water and the solvent system leaving a portion ("residual biomass phase"). The two phases contained in the combination are separated using a suitable device that utilizes centrifugal force to facilitate separation (e.g., disk centrifuge, bowl centrifuge, decanter centrifuge, hydrocyclones, countercurrent centrifugal contactors). Optionally, one or more solvents may be added to the device that utilizes centrifugal force to facilitate separation. A precipitating agent for PHA is added to the PHA phase to form a mixture comprising the PHA phase and the precipitating agent. The mixture is stirred (e.g., agitated) to form a composition comprising precipitated PHA, the solvent system, and the precipitating agent. In certain embodiments, the solvent system and the precipitating agent are miscible, which results in a combination (precipitated PHA, solvent system, and precipitating agent) having two phases (e.g., one phase containing precipitated PHA, and one phase containing solvent system and precipitating agent). The combination (precipitated PHA, solvent system, and precipitating agent) is separated (e.g., by filtration or using centrifugal force) to remove the separated extracted PHA.
The process of fig. 1 may be referred to as a single-step process. Generally, a single step process is a process that uses only one centrifugation step in the separation of a polymer (e.g., PHA) from biomass. Generally, a multi-step process refers to a process that uses more than one centrifugation step in the separation of a polymer (e.g., PHA) from biomass (see other description below). For example, the residual biomass formed in the process of fig. 1 can be processed and ultimately centrifuged, thereby producing a two-step process (see, e.g., fig. 2 and the description below).
In certain embodiments, the process results in a relatively high yield of PHA. For example, in certain embodiments, the ratio of the dry weight of extracted PHA to the dry weight of PHA initially contained in the biomass is at least about 0.9 (e.g., at least about 0.95, at least about 0.97, at least about 0.98). In certain embodiments, relatively high yields of PHA can be obtained without the use of a multi-step process (e.g., using a single-step process).
In particular embodiments, the process can be carried out using relatively large amounts of solvent transferred to the PHA phase. For example, in certain embodiments, the ratio of the volume of solvent recovered in the PHA phase to the volume of solvent contacted with the biomass is at least about 0.8 (e.g., 0.85, at least about 0.9, at least about 0.95, at least about 0.98, at least about 0.99). In certain embodiments, a relatively large amount of solvent can be transferred to the PHA phase using, for example, countercurrent flow conditions during the separation of a polymer (e.g., PHA) from the biomass.
In particular embodiments, the process may be carried out using relatively small amounts of solvent transferred to the residual biomass phase. For example, in certain embodiments, the ratio of the volume of solvent recovered in the residual biomass phase to the volume of solvent contacting the biomass is at most about 0.2 (e.g., at most about 0.15, at most about 0.1, at most about 0.05, at most about 0.02, at most about 0.01). In certain embodiments, a relatively small amount of solvent may be transferred to the residual biomass phase during the separation of a polymer (e.g., PHA) from the biomass using, for example, countercurrent flow conditions.
The slurry may be provided in any desired manner. Typically, the slurry is provided by forming a fermentation broth (broth) containing water and biomass, and removing a portion of the water from the fermentation broth. The water may be removed, for example, by filtration (e.g., microfiltration, membrane filtration) and/or by decantation and/or by using centrifugal force. In particular embodiments, biomass impurities (e.g., cell wall and cell membrane impurities) may be removed during the process of providing the slurry. These impurities may include proteins, lipids (e.g., triglycerides, phospholipids, and lipoproteins), and lipopolysaccharides.
The PHA content of the biomass (e.g., the PHA content of the dry biomass, including its polymer content, in weight%) can be varied as desired. As an example, in embodiments where the biomass is derived from a microorganism (microbial origin), the PHA content of the biomass can be at least about 50 wt.% (e.g., at least about 60 wt.%, at least about 70 wt.%, at least about 80 wt.%). As another example, in embodiments in which the biomass is plant-derived, the PHA content of the biomass can be less than about 50 wt% (e.g., less than about 40 wt%, less than about 30 wt%, less than about 20 wt%).
In certain embodiments, the solids content of the slurry (e.g., dry biomass, including its PHA content, weight relative to the total wet weight of the slurry) is from about 25 wt% to about 40 wt% (e.g., from about 25 wt% to about 35 wt%).
Biomass may be formed from one or more of a variety of entities (entities). Such entities include, for example, PHA-producing microbial strains (e.g., Alcaligenes eutrophus) (renamed Alcaligenes eutropha)Alcaligenes latus, Azotobacter, Aeromonas, Comamonas, Pseudomonas, genetically engineered organisms for PHA production (e.g., Pseudomonas, Ralstonia, Escherichia coli, Klebsiella), PHA-producing yeast and PHA-producing plant systems. Such entities are disclosed, for example, in Lee, Biotechnology& Bioengitieering 49: 1-14 (1996); braunegg et al (1998), J.Biotechnology65: 127-161; madison and Huisman, 1999; and Snell and Peoples2002, Metabolic Engineering 4: 29-40, which are incorporated herein by reference.
In embodiments where the biomass contains microbial cells, the size of the microbial cells contained within the biomass may also vary as desired. Typically, the microbial cells (e.g., bacterial cells) have at least one dimension that is at least about 0.2 microns (e.g., at least about 0.5 microns, at least about 1 micron, at least about 2 microns, at least about 3 microns, at least about 4 microns, at least about 5 microns) in size. In certain embodiments, the use of relatively large microbial cells (e.g., relatively large bacterial cells) in the biomass is advantageous because it can facilitate separation of the biomass to form a biomass slurry.
Typically, PHAs are formed by polymerizing (e.g., enzymatically polymerizing) one or more monomer units. Examples of the monomer unit include, for example, 3-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate, 3-hydroxydecanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, 3-hydroxyhexadecanoate, 3-hydroxyoctadecanoate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate and 6-hydroxyhexanoate.
In certain embodiments, the PHA has at least one chemical formula of-OCR1R2(CR3R4)nMonomeric units of CO-. n is 0 or an integer (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.). R1、R2、R3And R4Each is a hydrogen atom, a saturated hydrocarbon group, an unsaturated hydrocarbon group, a substituted group (e.g., a substituted hydrocarbon group), or an unsubstituted group (e.g., an unsubstituted hydrocarbon group). Examples of substituted groups include halogen-substituted groups (e.g., halogen-substituted hydrocarbyl groups), hydroxy-substituted groups (e.g., hydroxy-substituted hydrocarbyl groups), halogen groups, nitrogen-substituted groups (e.g., nitrogen-substituted hydrocarbyl groups), and oxygen-substituted groups (e.g., oxygen-substituted hydrocarbyl groups). Substituted groups include, for example, substituted saturated hydrocarbon groups and substituted unsaturated hydrocarbon groups. R1And R2、R3And R4Each of which is the same or different. R2And R1、R3AndR4 are each the same or different. R3And R1、R2And R4Each of which is the same or different. R4And R1、R2And R3Each of which is the same or different.
In certain embodiments, the PHA is a copolymer comprising two or more different monomer units. Examples of such copolymers include poly-3-hydroxybutyrate-co-3-hydroxypropionate, poly-3-hydroxybutyrate-co-3-hydroxyvalerate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, poly-3-hydroxybutyrate-co-4-hydroxybutyrate, poly-3-hydroxybutyrate-co-4-hydroxyvalerate, poly-3-hydroxybutyrate-co-6-hydroxyhexanoate, poly-3-hydroxybutyrate-co-3-hydroxyheptanoate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate, poly-3-hydroxybutyrate-co-3-hydroxydecanoate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, poly-3-hydroxybutyrate-co-4-hydroxyoctanoate, poly-3-hydroxybutyrate, Poly-3-hydroxybutyrate-co-3-hydroxydodecanoate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate, poly-3-hydroxydecanoate-co-3-hydroxyoctanoate, and poly-3-hydroxybutyrate-co-3-hydroxyoctadecanoate.
In a particular embodiment, the PHA is a homopolymer. Examples of the copolymer include poly-4-hydroxybutyrate, poly-3-hydroxypropionate, poly-3-hydroxybutyrate, poly-3-hydroxyhexanoate, poly-3-hydroxyheptanoate, poly-3-hydroxyoctanoate, poly-3-hydroxydecanoate and poly-3-hydroxydodecanoate.
The PHA has a polystyrene equivalent weight average molecular weight of at least about 500 (e.g., at least about 10,000, at least about 50,000) and/or less than about 2,000,000 (e.g., less than about 1,000,000, less than about 800,000).
In general, the amount of solvent system added to the slurry can vary as desired. In particular embodiments, an amount of solvent system is added to the slurry such that, after centrifugation, the PHA phase has a PHA solids content of less than about 10 wt.% (e.g., less than about 8 wt.%, less than about 6 wt.%, less than about 5 wt.%, less than about 4 wt.%, less than about 3 wt.%).
The solvent system includes a solvent for the one or more PHAs, and may optionally include a precipitant for the one or more PHAs. Without intending to be bound by theory, it is believed that the precipitation agent including the PHA in the solvent system can reduce the viscosity of the solution containing the polymer and the solvent system and/or enhance the selectivity in the process of extracting the desired PHA.
In general, a solvent for a given polymer is capable of dissolving the polymer to form a solution that is substantially uniform at the molecular or ionic scale level. Generally, the precipitating agent for a given polymer is capable of inducing precipitation of the polymer and/or weakening the solvent power of the polymer's solvent.
The choice of solvent and/or precipitating agent will generally depend on the given PHA to be purified. Without wishing to be bound by theory, it is believed that the appropriate solvent for a given polymer may be selected by substantially matching the appropriate solvation parameters (e.g., dispersion force, hydrogen bonding force, and/or polarity) for the given polymer and solvent. These solvation Parameters are disclosed, for example, in Hansen, solublity Parameters-A users's Handbook, CRC Press, NY, NY (2000).
In particular embodiments where the PHA is a poly-3-hydroxybutyrate copolymer (e.g., poly-3-hydroxybutyrate-co-3-hydroxypropionate, poly-3-hydroxybutyrate-co-3-hydroxyvalerate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, and/or poly-3-hydroxybutyrate-co-4-hydroxybutyrate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate), wherein a majority of the monomer units are 3-hydroxybutyrate (e.g., at least about 50% of the monomer units are 3-hydroxybutyrate, at least about 60% of the monomer units are 3-hydroxybutyrate), the solvent may be selected from ketones, esters and/or alcohols having at least 4 carbon atoms, and the precipitating agent may be selected from alkanes, methanol and ethanol.
In some embodiments where the PHA is a poly-3-hydroxyoctanoate, the solvent may be selected from ketones, esters, alcohols having at least 4 carbon atoms, or alkanes (e.g., hexane).
In general, the ketones may be cyclic or acyclic, straight or branched, and/or substituted or unsubstituted. Examples of acyclic and cyclic ketones include methyl isobutyl ketone ("MIBK"), 3-methyl-2-pentanone (butyl methyl ketone), 4-methyl-2-pentanone (methyl isobutyl ketone), 3-methyl-2-butanone (methyl isopropyl ketone), 2-pentanone (methyl n-propyl ketone), diisobutyl ketone, 2-hexanone (methyl n-butyl ketone), 3-pentanone (diethyl ketone), 2-methyl-3-heptanone (butyl isopropyl ketone), 3-heptanone (ethyl n-butyl ketone), 2-octanone (methyl n-hexyl ketone), 5-methyl-3-heptanone (ethyl amyl ketone), 5-methyl-2-hexanone (methyl isoamyl ketone), heptanone (amyl methyl ketone), cyclopentanone, Cyclohexanone.
Generally, esters may be cyclic or acyclic, straight or branched, and/or substituted or unsubstituted. Non-cyclic and cyclic esters include ethyl acetate, propyl acetate, butyl acetate, pentyl acetate, butyl isobutyrate, methyl n-butyrate, butyl propionate, butyl butyrate, methyl valerate, ethyl valerate, methyl hexanoate, ethyl butyrate, ethyl acetate, gamma-butyrolactone, gamma-valerolactone.
Typically, the alcohol having at least 4 carbon atoms may be cyclic or acyclic, linear or branched, and/or substituted or unsubstituted. Examples of such cyclic and acyclic alcohols include methyl-1-butanol, ethyl-1-butanol, 3-methyl-1-butanol (pentanol), 2-methyl-1-pentanol, 2-methyl-2-butanol (pentanol), 3-methyl-2-pentanol (methyl isobutyl carbinol), methyl-2-pentanol, 4-methyl-2-pentanol, butanol, pentanol, hexanol, heptanol, cyclohexanol, methyl-cyclohexanol, and fusel oil (a mixture of higher alcohols, which are typically by-products of alcohol distillation, and often predominantly pentanol (methyl butanol)).
In general, the paraffins may be cyclic or acyclic, straight-chain or branched, and/or substituted or unsubstituted. In certain embodiments, the alkane comprises a linear alkane and has 5 or more carbon atoms (e.g., heptane, hexane, octane, nonane, and dodecane). In particular embodiments, the alkane comprises an isoalkane (e.g., methyl heptane, methyl octane, dimethyl heptane). In particular embodiments, Soltrol can be used100 (mixture of isoparaffins from C9 to C11, available from Chevron Phillips Chemical Company, Houston, TX).
In general, the amount of solvent present in the solvent system can vary as desired. In certain embodiments, the solvent system has at least about 5 parts (e.g., at least about 10 parts, at least about 15 parts) solvent per part PHA and/or less than about 50 parts (e.g., less than about 30 parts, less than about 25 parts) solvent per part PHA.
In certain embodiments, the solvent of the PHA is non-halogenated. The use of non-halogenated solvents may be advantageous because it may reduce the negative impact of the solvent on the environment, reduce health risks associated with the use of the solvent, and/or reduce costs associated with storing, handling, and/or disposing of the solvent.
In particular embodiments, the solvent for the PHA can have a relatively low density. For example, the density of the solvent for the PHA can be less than about 0.95 kg/liter (e.g., less than about 0.9 kg/liter, less than about 0.8 kg/liter, less than about 0.7 kg/liter) at 20 ℃. Without wishing to be bound by theory, it is believed that the use of a relatively low density solvent can increase the quality of the PHA phase separated from the residual biomass phase.
In certain embodiments, the solvent for the PHA has a relatively low solubility in water. For example, the solubility of the solvent for PHA in water at 20 ℃ is less than about 1% (e.g., less than about 0.5%, less than about 0.2%). Solvents having a relatively low solubility in water are preferred because such solvents are not readily miscible with water. This can increase the ease of providing two separate phases during the process, thereby reducing cost and/or process complexity.
In certain embodiments, the solvent for the PHA is substantially non-hydrolyzable. For example, the solvent is at most as hydrolyzable as ethyl acetate. The use of a substantially non-hydrolyzable solvent can reduce the likelihood of the formation of undesirable by-products (e.g., chemically reactive species such as organic acids). This can reduce the amount and/or rate of corrosion of portions of the system (e.g., piping) in which PHA extraction is performed.
In certain embodiments, the solvent of the PHA is relatively easy to strip from the water. For example, a solvent has a logK value relative to water of at least about 1.5 (e.g., at least about 1.8, at least about 2, at least about 2.2) at 100 deg.C, as determined according to Hwang et al Ind. Eng. chem. Res., Vol.31, No.7, pp.1753-1767(1992), which is incorporated herein by reference. The use of a solvent that is easily stripped from water may be desirable because such a solvent is easily recovered and recycled compared to other solvents that are not easily stripped from water.
In a particular embodiment, the solvent for the PHA has a boiling point higher than 100 ℃.
In particular embodiments, suitable solvents are non-halogenated, have relatively low (e.g., lower than ethyl acetate) water solubility and relatively low reactivity in view of hydrolysis and/or in view of reactivity with the polymer.
In certain embodiments, the solubility of the PHA in the precipitating agent is less than about 0.2% (e.g., less than about 0.1%) of the PHA at 20 ℃.
In certain embodiments, a relatively small volume of precipitant is added to the PHA phase relative to the volume of solvent system added to the slurry, e.g., the ratio of the volume of precipitant added to the PHA phase to the volume of solvent system added to the slurry is less than about 0.2 (e.g., less than about 0.1, less than about 0.07, less than about 0.05).
In embodiments in which the solvent system comprises a solvent for one or more PHAs and a precipitating agent for one or more PHAs, the relative volatilities of the solvent and the precipitating agent may be at least about 2 (e.g., at least about 3, at least about 4) at atmospheric pressure at equimolar bubble points of the solvent and the precipitating agent.
In certain embodiments, wherein the solvent system comprises one or more solvents for the PHA and one or more precipitating agents for the PHA, the solvent and the precipitating agents do not form an azeotrope. The use of solvents and precipitants that do not form azeotropes may be desirable because it may be easier to separate and recover the solvents and precipitants for reuse relative to the azeotrope-forming solvents and precipitants.
In certain embodiments, wherein the solvent system comprises a solvent for PHA and a precipitant for PHA, the solution formed from PHA and the solvent system comprises less than about 25% (e.g., less than about 20%, less than about 15%, less than about 10%) by volume of the precipitant.
Typically, the mixture containing the solvent system and the slurry is heated to enhance the interaction of the solvent system and the PHA, thereby allowing the PHA to be removed from the biomass.
Generally, the temperature of the solvent system and slurry mixture during agitation can be varied as desired. In certain embodiments, the temperature is less than about 160 ℃ (e.g., less than about 125 ℃, less than about 95 ℃, less than about 65 ℃) and/or at least about 20 ℃. In particular embodiments, the temperature is from room temperature to about 95 ℃ (e.g., from about 40 ℃ to about 80 ℃, from about 60 ℃ to about 70 ℃). In particular embodiments, the pressure may be adjusted to greater than atmospheric pressure to facilitate extraction at elevated temperatures (e.g., greater than 1 atmosphere, up to 20 atmospheres).
In general, the shear force at which the solvent system and slurry mixture are agitated can be varied as desired. In a particular embodiment, the solvent system and slurry mixture are stirred by agitation in order to reduce the dissolution time. In certain embodiments, to aid in dissolution, a high shear rotary mixer and agitator (e.g., a flat blade rotary mixer, such as a 6 straight blade disc turbine) can be used, with tip speeds of, for example, about 5 meters/second or higher (e.g., up to about 10 meters/second). In certain embodiments, a high speed disperser with a small shaped blade (tip speed), for example, about 10 meters/second or more (e.g., about 15 meters/second or more, about 20 meters/second to about 25 meters/second), typically with a blade with a small shaped blade or serrated edge, may be used to produce high shear at increased tip speeds. In a particular embodiment, a rotor/stator system is used that produces relatively high shear (e.g., at tip speeds up to about 50 meters per second) in the gap between the high speed rotors that rotate within the slotted stator. The shape of the rotor and stator can generally be varied to suit a particular application, and many designs are commercially available.
Typically, the solvent system and slurry mixture are agitated until the centrifuged sample of the mixture has a PHA phase of the desired PHA solid content. In certain embodiments, the solvent system and slurry mixture are stirred for less than about 3 hours (e.g., less than about 2 hours) and/or at least about 1 minute (e.g., at least about 10 minutes, at least about 30 minutes).
In particular embodiments, the PHA phase contains less than about 0.5 wt% (e.g., less than about 0.25 wt%, less than about 0.1 wt%) biomass relative to the amount of PHA dissolved in the PHA phase.
In certain embodiments, the biomass phase contains less than about 25 wt.% (e.g., less than about 20 wt.%, less than about 15 wt.%) of solvent (which was originally present in the solvent system) and/or at least about 1 wt.% (e.g., at least about 5 wt.%, at least about 10 wt.%) of solvent (which was originally present in the solvent system).
In certain embodiments, the PHA phase has a relatively low viscosity. For example, the viscosity of the phase may be less than about 100 centipoise (e.g., less than about 75 centipoise, less than about 50 centipoise, less than about 40 centipoise, less than about 30 centipoise). Without wishing to be bound by theory, it is believed that preparing the PHA phase to have a relatively low viscosity may result in relatively good separation of the PHA phase from the residual biomass phase. In particular, it is believed that the rate of phase separation during centrifugation is inversely proportional to the viscosity of the PHA phase, so that for a given centrifugation time, reducing the viscosity of the PHA phase results in improved phase separation relative to a particular system in which the PHA phase has a higher viscosity.
In particular embodiments, the PHA phase has a relatively high polymer concentration. For example, the polymer concentration of the PHA phase can be at least about 2% (e.g., at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%).
Various types of devices utilizing centrifugal force may be used. By way of example, centrifugation is performed in certain embodiments using a disc centrifuge (disc stack) (e.g., model SC-6 from Westfalia Separator US, Inc., located in Northvale, NJ). In a particular embodiment, centrifugation is performed using a decanter (e.g., model CA-220, Westfalia Separator US, Inc. from Northvale, NJ). In some embodiments, a hydrocyclone may be used.
In particular embodiments, a counter-current centrifugal contactor (e.g., a Podbielniak centrifugal contactor, a Luwesta centrifugal contactor, a Taylor-Couette centrifugal contactor) may be used. Typically, a counter-current centrifugal contactor is used by contacting two (or possibly more) liquid streams with each other. One stream (solvent stream) begins as a relatively solvent-rich stream. The other stream (biomass stream) begins as a relatively PHA-rich stream. The two streams are contacted under countercurrent conditions such that the solvent-rich fraction is contacted with the PHA-poor biomass stream fraction (to increase, e.g., optimize, PHA recovery from the biomass stream) and/or the PHA-rich fraction is contacted with the PHA-rich solvent stream fraction (to increase, e.g., optimize, the concentration of PHA in the solvent stream). In certain embodiments, this is achieved by reversing the flow of the solvent stream with respect to the flow of the biomass stream (counter-flow conditions). Counter-current centrifugal contactors are available, for example, from B & P Process Equipment (Saginaw, MI) and Quadronics. Examples of commercially available counter-current centrifugal contactors include Podbielniak A-1 counter-current centrifugal contactor (B & PProcess Equipment) and Podbielniak B-10 counter-current centrifugal contactor (B & P ProcessEquipment).
In general, the conditions (e.g., force, time) used for centrifugation can be varied as desired.
In certain embodiments using a disk centrifuge, the centrifugation can be performed using at least about 5,000RCF (relative centrifugal force) (e.g., at least about 6,000RCF, at least about 7,000RCF, at least about 8,000RCF) and/or less than about 15,000RCF (e.g., less than about 12,000RCF, less than about 10,000 RCF). In particular embodiments using a decanter, the centrifugation can be performed using at least about 1,000RCF (e.g., at least about 1,500RCF, at least about 2,000RCF, at least about 2,500RCF) and/or less than about 5,000RCF (e.g., less than about 4,000RCF, less than about 3,500 RCF). In particular embodiments using a counter-current centrifugal contactor, the centrifugation can be performed using at least about 1,000RCF (e.g., at least about 1,500RCF, at least about 2,000RCF, at least about 2,500RCF) and/or less than about 5,000RCF (e.g., less than about 4,000RCF, less than about 3,500 RCF).
In certain embodiments using a disk centrifuge, the centrifugation can be performed for less than about 1 hour (e.g., less than about 30 minutes, less than about 10 minutes, less than about 5 minutes, less than about 1 minute) and/or at least about 10 seconds (e.g., at least about 20 seconds, at least about 30 seconds). In certain embodiments using a decanter, centrifugation can be performed for less than about 1 hour (e.g., less than about 30 minutes, less than about 10 minutes, less than about 5 minutes, less than about 1 minute) and/or at least about 10 seconds (e.g., at least about 20 seconds, at least about 30 seconds). In particular embodiments using a counter-current centrifugal contactor, centrifugation can be performed for less than about 1 hour (e.g., less than about 30 minutes, less than about 10 minutes, less than about 5 minutes, less than about 1 minute) and/or at least about 10 seconds (e.g., at least about 20 seconds, at least about 30 seconds).
After centrifugation, a precipitating agent for PHA is added to the separated PHA phase to form a mixture. In embodiments where the solvent system contains one or more precipitating agents for the PHA, the precipitating agent added to the separated PHA phase may be the same as or different from the precipitating agent contained in the solvent system.
In general, the precipitating agent added to the separated PHA phase may be varied as desired. In certain embodiments, the amount of precipitating agent added to the isolated PHA is at least about 0.1 parts by volume (e.g., at least about 0.25 parts, at least about 0.5 parts) of precipitating agent relative to the volume of solvent in the PHA phase and/or less than about 2 parts by volume (e.g., less than about 1.5 parts, less than about 1 part, less than about 0.75 parts) of precipitating agent relative to the volume of solvent in the PHA phase.
The PHA phase/precipitant mixture is agitated to enhance the interaction of the PHA with the PHA precipitant. This causes the PHA to precipitate out of the mixture, resulting in a combination formed by: precipitated PHA and a mixture comprising a solvent system and added PHA precipitant. Typically, the PHA phase/precipitant mixture is stirred at room temperature, although other temperatures can be used if desired. In certain embodiments, the PHA phase/precipitant mixture is mixed using a high shear device, such as a high shear rotary mixer (e.g., a 6 straight blade disc turbine), a high speed disperser, and a rotor/stator high shear in-line (in-line) or in-tank (in-tank) mixer. The shear rate is determined by the tip speed of the various devices and can vary, for example, from about 5 meters/second to about 50 meters/second (e.g., from about 10 meters/second to about 25 meters/second). Without wishing to be bound by theory, it is believed that high shear mixing under specific conditions may improve the quality of the precipitated polymer.
The precipitated PHA is then separated from the residual liquid (e.g., solvent system and precipitant). This separation can be carried out, for example, by filtration or centrifugation (e.g., using a basket centrifuge, using a vacuum belt filter).
Typically, the precipitated PHA is then washed to help remove unwanted impurities, such as residual solvent and/or precipitant. In certain embodiments, the polymer may be washed with a solvent (e.g., a relatively fresh solvent), such as a mixture of PHA solvent and PHA precipitant (e.g., in any ratio between 0-100%). The composition used for washing is generally selected to reduce (e.g., minimize) re-dissolution of the polymer and/or enhance (e.g., maximize) removal of impurities. In particular embodiments, the appropriate ratio may depend on the particular polymer composition and/or may be determined by standard testing (wash efficiency). In certain embodiments, such washing steps may be conducted at elevated temperatures and appropriate residence times to further facilitate washing and removal of impurities.
Typically, the washed precipitated PHA is dried (e.g., at a temperature of about 40 ℃ to about 100 ℃). Drying may be performed under vacuum (e.g., to help facilitate recovery of residual solvent). In certain embodiments, it may be desirable to directly extrude the precipitated polymer while still containing solvent, for example, a devolatilizing extruder. Such extrusion may be carried out, for example, at a temperature near the melting point of the polymer, and the solvent may be recovered directly from the extruder. Optionally, water may be injected under pressure into the vented extruder (e.g., to generate steam in situ to facilitate efficient stripping and removal of trace amounts of residual solvent). Gas stream (e.g. air, CO)2Or steam) may optionally be injected into the extruder (e.g., to facilitate solvent removal). Extrusion can combine drying and product-forming operations (e.g., pelletizing) into a single unit while saving capital and process operating costs.
The residual liquid (solvent system and precipitant) can be further processed so that the components of the liquid (solvent and/or precipitant) can be reused. For example, the liquid may be distilled to separate the solvent and the precipitant. In certain embodiments, the separated solvent and/or precipitant can be reused in the processes described above (e.g., as a solvent in a solvent system, as a precipitant added to the PHA phase). In certain embodiments, the separated solvent and/or precipitant (e.g., as a solvent in a solvent system, as a precipitant added to the PHA phase) can be reused in the process described in fig. 2 (described below).
In particular embodiments, the process (or portions of the process) may be performed in a continuous and/or in-line manner. By way of example, the process may include an in-line rotor/stator dissolution process of PHA, and/or an in-line rotor/stator precipitation process of PHA, and/or an in-line vented extruder (e.g., Werner and Pfleiderer ZSK extruder, supplied by Coperion Corporation of Ramsey, NJ) for solvent removal and formation of PHA solids (e.g., pellets).
In some embodiments, the process uses a solvent in a relatively efficient manner. For example, at least about 90 vol% (e.g., at least about 95 vol%, at least about 97 vol%, at least about 98 vol%) of the solvent originally used in the solvent is recovered for reuse.
In certain embodiments, the process uses a precipitating agent in a relatively efficient manner. For example, at least about 90 volume percent (e.g., at least about 95 volume percent, at least about 97 volume percent, at least about 98 volume percent) of the total amount of the initially used precipitating agent and the precipitating agent added to the PHA phase in the recovery solvent is recycled for reuse.
FIG. 2 is a flow diagram showing an embodiment of the second step of a two-step process that can be used to increase the efficiency of PHA extraction by extracting at least a portion of the PHA present in the residual biomass phase (FIG. 1). As shown in fig. 2, a solvent system is added to the biomass phase to provide a mixture containing the biomass phase and the solvent system. The mixture is agitated (e.g., using the conditions described above with respect to the agitation of the slurry and solvent system mixture) to provide a combination comprising a PHA phase (containing primarily the solvent system and PHA) and a biomass phase (containing primarily the biomass, water, and entrained solvent system). The PHA phase and the biomass phase are separated using centrifugation (e.g., using the conditions described above with respect to centrifuging the PHA phase and the biomass phase). The PHA phase may be treated as described above (e.g., by adding a precipitating agent for the PHA, stirring, separating, washing, drying), or the PHA phase may be added to the slurry and solvent mixture described above. The components of the solvent system (e.g., solvent and/or precipitant) can be stripped from the residual biomass phase using standard techniques. The residual solvent contained in the biomass can be recovered by various means, such as stripping in a suitable column, desolventizer dryer (e.g., desolventizer toaster) conventionally used to recover residual solvent from soy flour after extraction of oil, or direct drying and recovery of solvent (e.g., vacuum dryer, fluidized bed dryer with inert gas recycle and solvent condensation). In certain embodiments, the solvent-containing biomass can be co-dried with a compatible animal feed (e.g., gluten feed, distiller's dried grain, oilseed meal) in a dryer that is appropriately set up to process and recover and/or safely eliminate (e.g., adsorb or incinerate) residual solvent. In the overall process in fig. 2, the first step is shown in fig. 1 and the second step is shown in fig. 2. In certain embodiments, the residual biomass may be used as a nutrient for fermentation (e.g., ethanol fermentation using yeast), optionally after removal of residual solvent as described above. In certain embodiments, the biomass may be hydrolyzed (e.g., by exposure to acidic conditions at elevated temperatures, treatment with proteases, lytic enzymes) to improve its nutrient profile for fermentation.
While certain methods of extracting PHA from biomass have been described, other embodiments are possible.
As an example, dry biomass may be used. In certain embodiments, the dry biomass may be combined with water to provide a slurry.
As another example, a precipitating agent for PHA can be added to the slurry prior to addition of the solvent system. In certain embodiments, the amount of precipitant added is at least about 0.5 volume (e.g., about 0.5 volume to about 2 volume) of the slurry.
The addition of a precipitating agent prior to the addition of the solvent system can result in the formation of relatively pure, isolated, extracted PHA (e.g., at least about 99% pure, at least about 99.5% pure, at least about 99.9% pure). Polymer purity can be determined by Gas Chromatography (GC) analysis after butanolation of polymer samples under acidic conditions to form the butyl esters of PHA monomer units, and the butyl esters of lipid and phospholipid fatty acid residues (e.g., using a Hewlett Packard 5890 Series II GC equipped with a Supelco 24044 SBP-1 column with a 0.25 micron membrane at 30m x0.32mm ID). The chromatographic response is calibrated and normalized and quantified using suitable standards of fatty acids and hydroxy acids (e.g., palmitic, stearic, oleic, linoleic, and 3-hydroxybutyric acids). This can be used to quantify the polymer content as well as the impurity content. The inorganic impurities can be quantified by ashing.
Without wishing to be bound by theory, it is believed that adding a precipitant for PHA to the slurry prior to adding the solvent system can help remove biomass impurities present in the biomass (e.g., phospholipids, neutral lipids, lipoproteins). This may be particularly advantageous if the PHA solids content in the biomass is relatively high (e.g., at least about 65% PHA solids content, at least about 75%).
As another example, the biomass and/or slurry may be chemically pretreated, for example, under relatively mild caustic conditions (e.g., a pH of about 8.5 to 10, about 8.5 to about 9, about 9 to about 9.5, about 9.5 to about 10), followed by neutralization prior to addition of the solvent system. This can result in the formation of relatively pure, isolated, extracted PHA (e.g., at least about 99% pure, at least about 99.5% pure). Caustic conditions may be achieved using one or more relatively basic materials, for example, potassium hydroxide, sodium hydroxide, and/or ammonium hydroxide.
As another example, the temperature may be increased during the chemical pretreatment step (e.g., at any temperature between room temperature and about 95 ℃) and other chemicals, such as surfactants, detergents, and/or enzymes, may be added to further facilitate the formation of relatively pure, isolated, extracted PHA (e.g., at least about 99% pure, at least about 99.5% pure).
Without wishing to be bound by theory, it is believed that chemical treatment (e.g., relatively mild caustic treatment) of the slurry prior to addition of the solvent system can help remove biomass impurities present in the biomass (e.g., lipids, phospholipids, lipoproteins). This can be particularly advantageous when the PHA solids content of the biomass is relatively high (e.g., at least about 65% PHA solids content, at least about 75%) PHA solids content.
As another example, the method can include concentrating (e.g., evaporating) the PHA phase after separating the PHA phase from the residual biomass phase but before adding the precipitating agent of the PHA to the PHA phase. This can reduce the volume of the solution, thereby reducing the precipitant.
As a further example, the process can be carried out in certain embodiments without adding a precipitating agent for the PHA to the PHA phase.
Also, the solvent system can be formed and then contacted with the biomass, or the biomass can be contacted with less than all of the components of the solvent system followed by the addition of the remainder of the solvent system (e.g., sequential addition or all at once). For example, in embodiments where the solvent system comprises a solvent for the PHA and a precipitating agent for the PHA, the slurry may be contacted with the solvent followed by addition of the precipitating agent, or vice versa. Alternatively, the solvent and the precipitating agent can be combined to form a solvent system, followed by contacting the biomass.
Furthermore, while extraction of a single PHA from biomass has been described, the process can be used to extract multiple PHAs (e.g., 2, 3, 4, 5, 6) from biomass. Such processes may involve the use of various solvents, precipitants, and/or solvent systems.
Furthermore, while a solvent system has been described containing a single solvent for PHA and optionally a single precipitant for PHA, multiple solvents for PHA (e.g., 2, 3, 4, 5, 6) and/or multiple precipitants for PHA (e.g., 2, 3, 4, 5, 6) may be used.
As another example, in certain embodiments, the slurry/solvent system mixture may be stirred without heating. Alternatively, the slurry/solvent system mixture may be stirred under pressure and heat.
As a further example, the method can include distilling the formed (e.g., distilled) solvent system/precipitant mixture to separate the components (e.g., solvent for PHA, precipitant for PHA) so that one or more of the components can be reused.
The following examples are illustrative and not intended to limit the invention. In these examples, the chemicals were from Aldrich Chemical Co.Inc. (Milwaukee, Wis.) and the overhead stirrer (overhead stirrer) was Ika-werke eurostat power control-visc overhead stirrer (Ika Work inc., Wilmington, NC), and the centrifuge is a Sorvall RC 5B plus centrifuge.
Example I
A batch of e.coli biomass slurry containing 70% polymer (dry basis) of a composition of polyhydroxybutyrate-co-4-hydroxybutyrate with 25% 4-hydroxybutyrate was divided into three and treated as follows:
a spray drying and collecting 30 grams of dry biomass.
b. Spray-dried, 30 grams of biomass was collected and rewetted to 100g with Deionized (DI) water.
c. An unmodified 100 grams of the initial slurry containing 30 grams of dry biomass.
Each batch was extracted with 400ml of butyl acetate for 2 hours at room temperature with overhead stirring at 500 rpm. The resulting slurry was centrifuged at 5000g for 20 minutes and the PHA phase recovered. The PHA content was determined by precipitation of the PHA from the PHA phase using hexane as a precipitating agent, followed by drying overnight under a vacuum of 1mm Hg at a temperature of 40 ℃. The recovered polymer showed 32% dissolution of the starting polymer (scheme 1), 43% dissolution of the starting polymer (scheme 2) and more than 97% dissolution of the starting polymer (scheme 3).
Example II
In a side-by-side experiment, broth from an E.coli fermentation broth containing cells with a one-dimensional size greater than 2 microns was compared with an Alcaligenes sp (Ralstoniaeutropa) containing cells with a maximum size of 0.5 microns. In an Eppendorf 5415C microcentrifuge, the time to obtain a clear supernatant using a 1.5mL centrifuge tube filled with 1mL broth at 12000rpm was determined. In the case of E.coli broth, a clear supernatant was obtained at a centrifugation time of less than 1 minute, whereas Alcaligenes required more than 5 minutes for similar clarity.
Example III
A polymer solution containing 5 wt.% polymer (expressed relative to the total solution weight) of polyhydroxybutyrate-co-4-hydroxybutyrate containing 25% 4-hydroxybutyrate was prepared by dissolving an e.coli biomass slurry containing 70% polymer (dry basis) in butyl acetate (Aldrich Chemical co.inc., Milwaukee, WI) using the procedure of example Ic. The viscosity of the resulting solution was measured to be 365 centipoise (cP) using a Brookfield LVF Viscometer (Brookfield Engineering Laboratories Inc., Stoughton, Mass.). For solutions with a viscosity of less than 100cP, a number 1 rotary rod (spindle) was used, and for solutions with a viscosity of more than 100cP, a number 2 rotary rod was used. The solution was further diluted with additional butyl acetate to 4% and 3% polymer by total weight of the solution. The resulting viscosities were found to be 150cP and 40cP, respectively.
Some of the above prepared 5% polymer solutions in butyl acetate were then diluted with hexane (Aldrich Chemical co.inc., Milwaukee, WI) to prepare 4.5 wt%, 4.3 wt%, 4.1 wt%, and 3.9 wt% solutions. The viscosity of these solutions was measured as described above and determined to be 215cP, 37.5cP, 5cP and 27.5cP, respectively.
The viscosity of a 5% polymer solution in butyl acetate diluted with additional butyl acetate (PHA solvent) compared to the viscosity diluted with hexane (PHA precipitant at room temperature) is depicted in figure 3. Dilution with a precipitant has a non-linear and desirable effect on reducing viscosity. The viscosity increase observed for the polymer diluted with hexane to 3.9 wt% in solution was consistent with precipitation of the polymer from solution at the level of hexane addition.
Example IV
In fed-batch fermentation, poly-3-hydroxybutyrate-co-4-hydroxybutyrate (30% 4-hydroxybutyrate on a molar basis) was prepared using recombinant E.coli with glucose as the primary carbon source. After fermentation is complete, the E.coli cells swell to greater than 2 microns in size in at least one dimension. Biomass accumulated 70% polymer (dry weight). The biomass is then harvested using centrifugation to produce wet biomass pellets substantially free of dissolved impurities.
100g of wet biomass pellets (48% dry solids) containing 70% (dry basis) of poly-3-hydroxybutyrate-co-4-hydroxybutyrate were charged with 500ml of ethyl acetate and stirred with an overhead stirrer at room temperature for 1 hour. The polymer composition was 30% 4-hydroxybutyrate (molar). After the viscosity increases to a point where further mixing is ineffective to mix the materials, mixing is stopped. A total of 350ml of slurry was collected and centrifuged at 5000g for a total of 20 minutes (Sorvall RC 5B plus centrifuge, Kendro laboratory products, Newtown CT). The theoretical amount of ethyl acetate that should be recovered from 350mL of slurry is 300mL, calculated on the basis of mass balance.
The PHA content of the PHA phase was about 5.3%. 220 ml of PHA phase was recovered by precipitation after centrifugation, representing about 73% by volume of the total recoverable ethyl acetate of the slurry before centrifugation.
Example V
The previous examples were repeated except that butyl acetate (Aldrich Chemical co.inc., Milwaukee, WI) was used instead of ethyl acetate. The polymer in solution was about 4.3%. An emulsion layer appeared at the interface after centrifugation. The 250 ml PHA phase was recovered by decantation after centrifugation, representing about 83% of the total recoverable butyl acetate present in the slurry before centrifugation.
Example VI
The previous examples were repeated except that MIBK (Aldrich Chemical co.inc., Milwaukee, WI) was used instead of butyl acetate. The polymer in solution was about 4.2%. 290 ml of PHA phase was recovered by decantation after centrifugation, representing about 97% of the total recoverable MIBK present in the slurry before centrifugation.
Example VII
100 grams of a wet E.coli biomass paste containing 28% dry solids, containing 35% (on a dry solids basis) of 75% poly-3-hydroxybutyrate-co-4-hydroxybutyrate of 4-hydroxybutyrate, was contacted with 200 grams of hexane (Aldrich Chemical Co. Inc., Milwaukee, Wis.) and stirred overhead (Ika) at room temperature-werke eurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) for 2 hours.
The hexane supernatant was separated by centrifugation at 3,500g for 20 minutes, and the solid pellets were recovered after decanting the hexane supernatant. Then, at room temperature, stirring was carried out with an overhead stirrer (Ika)-werke Eurostar powercontrol-visc overhead stirrer, Ika Work inc., Wilmington, NC) using 425 grams of MIBK (Aldrich Chemical co.inc., Milwaukee, WI) for 3 hours. The supernatant (solution of polymer in MIBK) was separated by centrifugation at 3,500g for 20 minutes and the polymer was precipitated by adding 355 grams of hexane. The precipitated polymer was recovered by filtration through a funnel using a grooved filter paper (VWR Scientific Products, West Chester, Pa.), and dried overnight at 45 ℃ in a Buchi rotary evaporator (rotavap) under a vacuum of 1mm Hg to yield 13 grams of dry polymer. The dried polymer was subjected to hot film pressing (hot film pressing) at 180 ℃. An appropriate amount of PHA (typically 0.5 grams) was placed between 2 PET sheets separated by spacers to form a film with a thickness of 100 microns. The film assembly (i.e., 2 sheets, shims and PHA) was placed between blocks (blocks) of a heated (180 ℃) Press (Carver hydralic Press Model #3912, Carver inc., Wabash, IN) and a 10 ton load was applied for 30 seconds. The film was then cooled between aluminum blocks and observed for color and clarity. This produced a substantially clear film that was substantially free of a smoky or unpleasant taste at an operating temperature of 180 ℃ during the press cycle.
Example VIII
The previous examples were repeated except that heptane (Aldrich Chemical co.inc., Milwaukee, WI) was used instead of hexane. The process produces a substantially clear film that is substantially free of a smoky or unpleasant taste.
Example IX
The previous examples were repeated except that Soltrol was used100(C9-C11A mixture of isoalkanes from Chevron Phillips Chemical Company, Houston, TX) instead of hexane. The process produces a substantially clear film that is substantially free of a smoky or unpleasant taste.
Example X
100 grams of a wet E.coli biomass paste having 28% dry solids containing 75% of 35% (on a dry solids basis) of poly 3-hydroxybutyrate-co 4-hydroxybutyrate of 4-hydroxybutyrate for 20 minutes was treated with an effective 0.02N NaOH (Aldrich Chemical Co.Inc., Milwaukee, Wis.) at 65 deg.C and rapidly cooled to room temperature over 5 minutes. The resulting slurry was neutralized to pH 7 using 85% phosphoric acid (Aldrich chemical co.inc., Milwaukee, WI), then centrifuged (3,500g) for 20 minutes and washed with 2 volumes of deionized water. The supernatant was discarded and 425 g of MIBK was used with overhead stirring at room temperature (Ika)-werke eurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) extracts the paste for 3 hours. The supernatant (solution of polymer in MIBK) was separated by centrifugation at 3,500g for 20 minutes and the polymer was precipitated by addition of 355 g hexane. The precipitated polymer was recovered by filtration and dried in a Buchi B-171 rotary evaporator (65 ℃ C. and 1mm Hg vacuum for 8 hours) to give 12 g of dry polymer. The dried polymer was subjected to hot film pressing at 180 ℃. This results in a film with only very slight discoloration/opaqueness.
Example XI
The previous example was repeated, but without the steps of treatment with NaOH at 65 ℃ for 20 minutes and rapid cooling. The PHA so recovered produced films with strong yellow discoloration and opacity during hot film pressing. There was also evidence of thermal degradation during the test, as evidenced by fuming during the film test pressure cycle (180 ℃ and 10 tons of pressure, 30 seconds).
Example XII
The following is an example of a single step process using a counter current centrifugal contactor.
11kg of biomass paste containing 26% of dry solids of E.coli was contacted with 38.6kg of methyl isobutyl ketone (4-methyl-2-pentanone or MIBK) at 30 ℃ for 3 hours in a dissolution tank equipped with a stirrer of a marine impeller (marine impeller) to maintain a uniform mixture. Biomass contained 71 wt% poly-3-hydroxybutyrate-co-4-hydroxybutyrate (22% molar concentration of 4-hydroxybutyrate) (dry basis). After 3 hours, the supernatant solution of MIBK and PHA obtained by centrifuging the sample from the dissolution tank contained 4.1 wt% PHA, indicating 91.2% dissolution.
The mixture of cell paste and MIBK was fed to an A-1 pilot scale Podbielniak extractor (B & P Process Equipment, Saginaw, MI) as a heavy liquid feed (heavy liquid in) (HLI) at a rate of 635 ml/min. At the same time, fresh MIBK was fed as light liquid feed (LLI) to perform counter current washing and extraction of the cell paste in a Podbielniak contactor. LLI was supplied at a rate of 175ml/min to maintain the feed ratio HLI: LLI at 3.6: 1. A total of 49.6kg of HLI and 12.8kg of LLI were fed in during 90 minutes. A total of 8.9kg of residual cell paste was collected as a heavy liquid take-off (HLO) and during a period of 90 minutes 53.6kg of PHA solution in MIBK was collected as a light liquid take-off (LLO). The LLO contained 3.75% PHA by weight in solution, as determined by drying a sample of the material. A total of 2.0kg PHA recovery was achieved in LLO, compared to 2.04kg PHA in the HLI cell paste feed (98.4% overall recovery).
Mass balance measurements showed that more than 98% of the total MIBK contained in the combined HLI and LLI was recovered in the clear PHA of MIBK solution (LLO). Laboratory centrifugation showed a very clear interface formed after 1 minute at 3000 g. Aggregates without any interface were also confirmed by LLO remaining clear for 90 min test time.
The improved PHA recovery (98.4%) of the Podbielniak extractor compared to the recovery obtained with a single step of dissolution (91.2%) demonstrates the effectiveness of countercurrent contact with fresh solvent to improve PHA recovery. The viscosity of the residual biomass paste is also significantly reduced by countercurrent contact, which is a result of the almost total removal of PHA.
Example XIII
The following is an example of extracting PHA using cyclohexanone.
90 grams of wet E.coli biomass paste having 28% dry solids containing 80% poly 3-hydroxybutyrate-co-4-hydroxybutyrate (PHA) at 12% 4-hydroxybutyrate (on a dry solids basis) was added to 400 grams of cyclohexanone (Aldrich Chemical Co., Inc., Milwaukee, Wis.) at 90 ℃. The solution was homogenized at 30,000rpm for 5 minutes using a hand-held homogenizer (Virtis, Gardiner, NY) equipped with a single-tank rotor-stator combination, followed by an overhead stirrer (Ika)-werkeeurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) for 30 minutes. During the solvent contacting step, the temperature was controlled at 90. + -. 5 ℃. The supernatant (solution of polymer in cyclohexanone) was then separated from the residual biomass paste pellet by decanting using a Sorvall RC 5B plus centrifuge (Kendro Laboratory Products, Newtown, CT) to centrifuge the biomass paste/cyclohexanone mixture at 3000g for 5 minutes.
The supernatant was then heated again to 80 ± 5 ℃ in a beaker and an equal volume of heptane (kept at room temperature) was added slowly to the solution over a period of 5 minutes while using an overhead stirrer (Ika)-werke eurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) intensive stirring to precipitate the polymer while maintaining the temperature between 70 ℃ and 80 ℃. The precipitated polymer was recovered by filtration through a funnel using a fluted filter paper (VWR Scientific Products, West Chester, PA) and air dried overnight in a chemical fume hood to yield 16 grams of white polymer particles (80% overall recovery).
The film was prepared by placing approximately 0.5 grams of polymer between 2 PET sheets separated by a spacer to form a film having a thickness of 100 microns. The film assembly (i.e., 2 sheets, shims and PHA) was placed between blocks (blocks) of a heated (180 ℃) Press (Carver hydralic Press Model #3912, Carver inc., Wabash, IN) and a 10 ton load was applied for 30 seconds. The film was then cooled between aluminum blocks and observed for color and clarity. This produced a substantially clear film that was substantially free of a smoky or unpleasant taste at an operating temperature of 180 ℃ during the press cycle.
Example XIV
The following is an example of PHBH extraction.
Using genetically engineered alcaligenes strains (prepared as described in Kichise et al, (1999), int.j. biol. macromol.25: 69-77)) and Naylor in the fermentation process described in US patent No.5,871,980 using fructose and lauric acid as carbon sources, a wet cell paste (27 wt% biomass solids in water) of the alcaligenes genus (ralstoniae eutropha) was prepared, which paste contained approximately 65% poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBH) (on a dry biomass basis), consisting of 5-7% molar hydroxycaproate (Kichise et al, (1999), int.j.biol. macromol.25: 69-77). This biomass was added to a suitable amount of MIBK to give a 5% solution (w/w) of PHBH in solvent. At 30,000rpm, a hand-held homogenizer (Virtis, Gardiner, N.Y.) equipped with a single-slot rotor-stator combination was used) The solution was homogenized for 5 minutes and then an overhead stirrer (Ika) was used-werke Eurostar powercontrol-visc overhead stirrer, Ika Work inc., Wilmington, NC) for 30 minutes. During the solvent contacting step, the temperature was controlled at 80 ± 5 ℃. The resulting biomass/solvent mixture was then separated by centrifugation using a Sorvall RC 5B plus centrifuge (Kendro Laboratory Products, Newtown, CT). The biomass paste/cyclohexanone mixture was centrifuged at 3000g for 5 minutes to separate the supernatant (solution of polymer in cyclohexanone) from the residual biomass paste pellet by decantation.
The supernatant was added to a beaker and an equal volume of heptane was slowly added to the solution over a period of 5 minutes while using an overhead stirrer (Ika)-werke eurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) intensive stirring to precipitate the polymer. After drying overnight in a chemical fume hood, white polymer crystal powder was recovered.
Example XV
The following is an example of PHBX extraction.
Such as Matsusakai et al (1999, Biomacromolecules)1: 17-22), and grown on glucose, containing approximately 50% of poly 3-hydroxybutyrate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate (phbbxx) with a composition of 92% 3-hydroxybutyrate, 1% 3-hydroxyoctanoate, 3% 3-hydroxydecanoate, 3% 3-hydroxydodecanoate and 1% 3-hydroxydodecanoate (molar concentration), based on dry biomass. Adding biomass to a suitable amount of MIBK to obtain PHBX in solvent5% solution (w/w). The solution was homogenized at 30,000rpm for 5 minutes using a hand-held homogenizer (Virtis, Gardiner, NY) equipped with a single-tank rotor-stator combination, followed by an overhead stirrer (Ika)-werke eurostat power control-visc overhead stirrer, Ika Work inc., Wilmington, NC) for 30 minutes. During the solvent contacting step, the temperature was controlled at 80. + -. 5 ℃. The resulting biomass/solvent mixture was separated using a Sorvall RC 5B plus centrifuge (Kendro Laboratory Products, Newtown, CT). The biomass paste/cyclohexanone mixture was then centrifuged at 3000g for 5 minutes and the supernatant (solution of polymer in cyclohexanone) was separated from the residual biomass paste pellet by decantation.
The supernatant was then added to a beaker and an equal volume of heptane was slowly added to the solution over a period of 5 minutes while using an overhead stirrer (Ika)-werke Eurostar powercontrol-visc overhead stirrer, Ika Work inc., Wilmington, NC) intensive stirring to precipitate the polymer. After drying overnight in a chemical fume hood, white polymer crystal powder was recovered.
Other embodiments are within the claims.
Claims (44)
1. A method of separating a polymer from a biomass containing the polymer, the method comprising:
contacting the biomass with a solvent system comprising a solvent for the polymer to provide a solution of residual biomass and the solvent comprising the polymer and the polymer;
applying centrifugal force to the solution and the residual biomass to separate at least a portion of the solution from the residual biomass as a separated solution;
adding a precipitant for the polymer to the separated solution; and
wherein the polymer comprises a PHA, and the method separates biomass from the polymer.
2. The method of claim 1, wherein the solvent system comprises a solvent for the polymer, the solvent having a density of less than 0.95 kg/liter.
3. The method of claim 1, wherein the solvent system comprises a non-halogenated solvent for the polymer.
4. The method of claim 1, wherein the solvent system comprises a solvent for the polymer, the solvent having a solubility in water of less than 1%.
5. The method of claim 1, wherein the solvent system comprises a solvent for the polymer, the solvent being non-hydrolyzable.
6. The method of claim 1, wherein the solvent system comprises a solvent for the polymer having a logK value relative to water at 100 ℃ of at least 1.5.
7. The method of claim 1, wherein the solvent system comprises a solvent for the polymer, the solvent having a boiling point greater than 100 ℃.
8. The method of claim 1, wherein the solvent system comprises at least one solvent selected from the group consisting of ketones, esters, and alcohols.
9. The method of claim 1, wherein the solvent system comprises at least one solvent selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
10. The method of claim 1, wherein the solvent system comprises a precipitant for the polymer that dissolves less than 0.2% by weight of the polymer at room temperature.
11. The method of claim 1, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the relative volatilities of the solvent for the polymer and the precipitant for the polymer are at least 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
12. The method of claim 1, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the solvent for the polymer and the precipitant for the polymer do not form an azeotrope.
13. The method of claim 1, wherein the solvent system comprises a precipitant for the polymer, the precipitant comprising an alkane.
14. The method of claim 1, wherein the polymer concentration of the solution is at least 2 weight percent.
15. The method of claim 1, wherein the viscosity of the solution is at most 100 centipoise.
16. The method of claim 1 wherein the biomass containing the polymer is of microbial origin and has a polymer content of at least 50% by weight.
17. The method of claim 1, wherein the biomass containing the polymer is of plant origin and has a polymer content of less than 50% by weight.
18. The method of claim 1, wherein the biomass containing the polymer comprises cells containing the polymer.
19. The method of claim 1, further comprising removing at least a portion of the polymer from the separated solution, wherein removing the polymer does not include exposing the separated solution to hot water.
20. The method of claim 1, further comprising evaporating a portion of the solution to remove at least some of the polymer from the solvent system prior to adding the precipitant for the polymer to the solution.
21. The method of claim 1, further comprising, after isolating, adding a volume of a second precipitant for the polymer to remove at least some of the polymer from the solution, wherein the volume of the second precipitant is less than 2 parts relative to the volume of the solvent system.
22. The method of claim 1, further comprising: removing at least a portion of the polymer from the solution; and
the removed polymer was extruded to dry and pelletize the polymer.
23. A method of separating a polymer from a biomass containing the polymer, the method comprising: contacting the biomass with a solvent system to provide residual biomass and a solution comprising a polymer and the solvent system, the solution having a polymer concentration of at least 2 weight percent and a viscosity of at most 100 centipoise; and
separating at least some of the solution from the residual biomass;
wherein the solvent has a density of less than 0.95 kg/l.
24. The method of claim 23, wherein the solvent system comprises a non-halogenated solvent for the polymer.
25. The method of claim 23, wherein the solvent system comprises a solvent for the polymer, the solvent having a solubility in water of less than 1%.
26. The method of claim 23, wherein the solvent system comprises a solvent for the polymer, the solvent being non-hydrolyzable.
27. The method of claim 23, wherein the solvent system comprises a solvent for the polymer having a LogK value relative to water at 100 ℃ of at least 1.5.
28. The method of claim 23, wherein the solvent system comprises a solvent for the polymer, the solvent having a boiling point greater than 100 ℃.
29. The method of claim 23, wherein the solvent system comprises at least one solvent selected from the group consisting of ketones, esters, and alcohols.
30. The method of claim 23 wherein the solvent system comprises at least one solvent selected from the group consisting of MIBK, butyl acetate, cyclohexanone, and combinations thereof.
31. The method of claim 23, wherein the solvent system comprises a precipitant for the polymer that dissolves less than 0.2% by weight of the polymer at room temperature.
32. The method of claim 23, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the relative volatilities of the solvent for the polymer and the precipitant for the polymer are at least 2 at equimolar bubble points of the solvent for the polymer and the precipitant for the polymer.
33. The method of claim 23, wherein the solvent system comprises a solvent for the polymer and a precipitant for the polymer, and the solvent for the polymer and the precipitant for the polymer do not form an azeotrope.
34. The method of claim 23, wherein the solvent system comprises a precipitant for the polymer, the precipitant comprising an alkane.
35. The method of claim 23, wherein the biomass containing the polymer is of microbial origin and has a polymer content of at least 50% by weight.
36. The method of claim 23, wherein the biomass containing the polymer is of plant origin and has a polymer content of less than 50% by weight.
37. The method of claim 23, wherein the biomass containing the polymer comprises cells containing the polymer.
38. The method of claim 23, further comprising removing at least some of the polymer from the solution.
39. The method of claim 38, further comprising extruding the removed polymer to dry and pelletize the polymer.
40. The method of claim 38, wherein removing the polymer from the solution does not include exposing the solution to hot water.
41. The method of claim 38, wherein removing the polymer from the solution comprises adding a precipitant for the polymer to the solution.
42. The method of claim 38, further comprising evaporating a portion of the solution prior to removing at least some of the polymer from the solution.
43. The method of claim 23, wherein separating at least some of the solution from the residual biomass comprises applying a centrifugal force to the solution and the residual biomass.
44. The method of claim 23 wherein the polymer comprises PHA.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40149802P | 2002-08-06 | 2002-08-06 | |
| US60/401,498 | 2002-08-06 | ||
| US42896302P | 2002-11-25 | 2002-11-25 | |
| US60/428,963 | 2002-11-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1125391A1 HK1125391A1 (en) | 2009-08-07 |
| HK1125391B true HK1125391B (en) | 2012-09-21 |
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