Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-05-12
2002-01-22
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S146000, C435S243000, C435S261000, C528S354000, C528S361000, C528S493000, C528S50200C
Reexamination Certificate
active
06340580
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention generally relates to methods for recovering polyhydroxyalkanoates (“PHAs”) from microbial or plant biomass. An improved understanding of the PHA biosynthetic pathways has allowed for the use of microbial organisms, both natural and recombinant, as well as plant cells, to produce significant quantities of PHA. However, difficulties remain in developing efficient and cost-effective recovery of the PHA at a useful levels of quality and purity from these biological source materials. Previous methods for isolating and purifying PHAs from biomass have included, for example, aqueous routes as well as organic solvent routes.
For example, U.S. Pat. No. 5,364,778 to Byrom discloses an aqueous method wherein biomass comprising PHA is maintained as an aqueous slurry in which the PHA is generally insoluble. The slurry is subjected to various treatments designed to digest, degrade, or otherwise make water-soluble the non-PHA biomass. This solubilized biomass then is removed from the slurry by centrifugation, filtration, or other means. Aqueous-based routes, however, generally have certain disadvantages, particularly which applied to large scale processing. Examples of these disadvantages include (a) effective purification is made more difficult because many impurities, including some surfactants useful for the solubilizing treatments, may be tightly adsorbed to the surface of the PHA particles; (b) many volumes (i.e. large quantities) of wash water may be required by the process, creating used wash water and its attendant disposal difficulties; (c) multiple solubilizing treatments may be required to obtain high purity PHA; (d) drying of the water-based PHA slurry may be time-consuming and costly; (e) PHA particles may cause extensive fouling of filtration membranes, centrifuges, and other process equipment; and (f) solubilizing treatments may require expensive reagents and lengthy process times and/or high temperatures to be effective.
Examples of organic solvent-based methods processes are disclosed in U.S. Pat. No. 4,101,533 to Lafferty et al. and U.S. Pat. No. 5,422,257 to Ohleyer. In these methods, an organic solvent for the PHA contained in a biomass is mixed with the biomass, resulting in the dissolution of the PHA. The organic solution comprising the PHA then is separated from the remaining insoluble biomass by filtration, centrifugation, or other means. The organic solution then is desolventized to recover the PHA. These organic solvent routes suffer disadvantages similar to the disadvantages associated with aqueous routes, including (a) a relatively large volume of solvent is required to completely extract the PHA from biomass; (b) biomass may need to dried prior to solvent extraction, which may be costly and time-consuming; and (c) solvents may co-extract impurities along with the PHA, such as lipids or other hydrophobic biological materials, necessitating further processing of the extract to obtain PHA of satisfactory purity. It would be advantageous to develop improved, more cost-effective processes for recovering PHA from PHA-containing biomass.
It is therefore an object of this invention to provide a method of recovering PHA from PHA-containing biomass using a process that is more simple, relatively faster, uses aqueous and/or organic solvents more efficiently, and possibly yields a more pure PHA product than conventional processes.
It is another object of the present invention to provide a method of recovering PHA from PHA-containing biomass using a process that can be employed economically in a commercial-scale production process.
SUMMARY OF THE INVENTION
A method is provided for isolating and purifying PHA from biomass which comprises PHA. The method includes the step of extracting PHA from the biomass using at least one solvent while simultaneously subjecting the biomass to a filtration process to remove cells. In a preferred embodiment of the method, biomass comprising PHA (for example an aqueous slurry of microbial cells obtained from a fermentation process) is directly extracted by diafiltration sing an organic solvent, to obtain PHA.
In a preferred diafiltration process, an aqueous slurry of microbial cells comprising PHA is recirculated through a filtration membrane, wherein the membrane has a pore size sufficiently small to reject individual cells or such aggregates of cells as may exist in the slurry. An outflow of liquid from the filtration membrane occurs under conditions where a pressure drop exists across the filtration membrane. As the liquid is progressively removed from the biomass slurry, an organic solvent, preferably a water-miscible solvent that also is a solvent for the PHA, is added to the biomass slurry. The solvent addition should be made at a rate which approximates the rate of liquid permeation through the filter in order to maintain the volume of the biomass slurry. As the concentration of organic solvent in the slurry increases, various impurities which are insoluble in water become dissolved in the solvent-water mixture and pass through the filter membrane. When the organic solvent concentration reaches a certain level, the PHA becomes soluble and flows through the filtration membrane. The filtrate comprising PHA then is desolventized to recover the polymer.
The method has the advantages that (a) it is not generally necessary to dry the biomass prior to solvent extraction; (b) it is readily possible to fractionate the PHA from other impurities to obtain relatively pure PHA in a single process, because the biomass is subjected to a gradient in solvent concentration; (c) the entire process of extracting and purifying PHA from biomass can be accomplished using a minimum of process stages and equipment; and (d) the method efficiently uses solvents, especially when the biomass slurry is relatively concentrated and when the diafiltration is conducted at a constant volume diafiltration. Furthermore, by using volatile organic solvents, it is relatively easy to desolventize the PHA solutions and to recover and reuse the solvent from the filtrates generated in the diafiltration process.
DETAILED DESCRIPTION OF THE INVENTION
A method has been developed for isolating and purifying polyhydroxyalkanoates (“PHAs”)from biomass comprising PHAs. The method includes the step of extracting PHA from the biomass using at least one solvent while simultaneously subjecting the biomass to a filtration process to remove cells.
1. The PHA-Containing Biomass
The biomass materials are derived from PHA-producing plants or PHA producing microorganisms.
Polymer Compositions
As used herein, “polyhydroxyalkanoate” and “PHA” refer to polymers that contain one or more units, for example between 10 and 100,000, and preferably between 100 and 30,000 units of the following formula I:
—OCR
1
R
2
(CR
3
R
4
)
n
CO—;
wherein n is an integer, for example between 1 and 15, and in a preferred embodiment, between 1 and 4; and
wherein R
1
,
2
, R
3
, and R
4
independently can be hydrocarbon radicals including long chain hydrocarbon radicals; halo- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substituted radicals; and/or hydrogen atoms.
As used herein, the formula —(CR
3
R
4
)
n
— is defined as including the following formulas:
—CR
3
R
4
—(where n=1);
—CR
3
R
4
CR
3′
R
4′
—(where n=2); and
—CR
3
R
4
CR
3′
R
4′
CR
3″
R
4″
—(where n=3);
wherein R
3
, R
4
, R
3″
, R
4″
, R
3″
, and R
4
″, can be independently hydrocarbon radicals including long chain hydrocarbon radicals; halo- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substituted radicals; and/or hydrogen atoms. Thus, formula I includes units derived from 2-hydroxyacids (n=0), 3-hydroxyacids (n=1),
4
-hydroxyacids (n=2), and 5-hydroxyacids (n=3), and 6-hydroxyacids (n=4).
These units may be the same in a homopolymer, or be more different units, as for exa
Holland & Knight LLP
Lilling Herbert J.
Metabolix Inc.
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