WO2000024918A1

WO2000024918A1 - Method for purifying a polyol product stream

Info

Publication number: WO2000024918A1
Application number: PCT/US1999/025130
Authority: WO
Inventors: Rod Fisher; Ashwin M. Madia
Original assignee: Cargill Inc
Current assignee: Cargill Inc
Priority date: 1998-10-27
Filing date: 1999-10-26
Publication date: 2000-05-04
Anticipated expiration: 2001-04-27
Also published as: AU1234400A

Abstract

A method for purifying a sugar polyol product stream is provided by the invention. The process facilitates the isolation and recovery of sugar polyol from a culture generated by polyol-producing microorganisms. The method employs a pretreatment operation which includes microbial cell separation in combination with the removal and/or deactivation of proteinaceous material. In one embodiment of the method, microbial cells are separated from the fermentation broth without disruption of the cells, thereby avoiding the release of substantial amounts of intracellular components, cell organelles and related components into the culture broth.

Description

METHOD FOR PURIFYING A POLYOL PRODUCT STREAM

Background of the Invention

Many commercially available sugar polyols are prepared by fermentation of organic substrates. Common substrates for fermentation includes carbon sources such as dextrose, sucrose, starch and related carbohydrates. The polyol is then recovered from the fermentation broth by any of numerous process operations which make use of techniques such as extraction, crystallization, precipitation, and chromatography.

Methods for polyol production by fermentation using different microorganism are well documented in the literature. The typically process involves the growth of the microorganism(s) in a nutrient medium under the specific fermentation parameters. The fermentation can be carried out in a batch, semi-batch, fed-batch, semi-fed-batch, semi-continuous or continuous mode.

The recovery, separation and/or purification processes for the polyols produced by the microorganisms generally involves a heat treatment step or analogous procedure after fermentation is complete to kill the microorganisms. The destruction of the microorganisms may be carried out either in the fermentation tank and/or in a separate vessel. The destruction of the microorganism(s) present in the fermentation broth at the end of the fermentation is generally accomplished by subjecting the fermentation broth to high(er) temperature under acidic or alkaline conditions for a period of time. The time requirement is usually longer at lower temperature and shorter at the higher temperature. The effectiveness and the extent of destruction of the microorganisms can be monitored by routine microbiological procedures. Appropriate combinations of fermentation broth pH, time and temperature can be chosen to accomplish the desired level of destruction of the microorganisms. In most commercial polyol fermentation processes, complete destruction of all microorganisms is desired and accomplished by selecting an appropriate combination of time, temperature and pH of the fermentation broth.

The process of killing microorganisms generally results in disruption of the microorganisms' cell wall and a resulting release of intracellular cell components into the fermentation broth. For example, the breakdown of cell wall and plasma membrane due to cell disruption can result in the release of cytoplasmic contents of the microorganism including the contents of endoplasmic reticulum, golgi apparatus, endosomes, exosomes, mitochondria, ribosomes, intracellular and cell membrane and cell wall bound enzymes. The cell contents typically include proteins, amino acids, minerals, salts and other cell components such as carbohydrates, lipids, nucleic acids, all of which are released into the fermentation broth. The presence of large amounts of such materials in the broth can make the separation and purification of desired fermentation products more difficult.

In addition, the conditions employed to kill the microorganisms not only results in the release of the contents of cell and cell wall into the fermentation broth but also can result in the dissolution of these materials in fermentation broth. The pH, temperature and time conditions generally involved in killing microorganisms favors dissolution of cell materials, including cell bound components. Similarly, the conditions employed for killing microorganisms can also result in the dissolution of other normally insoluble materials (if any) present in the fermentation broth. In common polyol production operations, once the microorganisms have been killed, the fermentation broth is typically subjected to a series of steps to separate, recover and purify the polyol product(s). The first step in this overall process is the removal by filtration of the killed microorganisms, together with cell wall, cell debris and other insoluble components and fragments which result from killing of microorganisms. Filtration and related processes (e.g., centrifugation) commonly employed to remove killed microorganisms from the fermentation broth are useful in removing the insoluble materials from the broth. Such processes do not, however, generally remove much of the soluble materials produced during fermentation and/or released in the fermentation broth due to the killing of microorganisms.

After removal of insoluble materials the normal separation, recovery and purification process (referred to herein as the "downstream recovery process") generally involves some combination of carbon treatment, chromatography, ion exchange, concentration, crystallization, and other types of standard unit operations to obtain the final purified polyol product(s). Where the liquid fraction obtained after removal of the insoluble materials contains significant levels of soluble proteinaceous material, typical downstream recovery processes may not be effective at reducing the amount of proteinaceous material to an adequate degree. The removal of proteins and related materials ("proteinaceous material") from polyols is generally necessary to achieve food grade product(s). This is true especially where the proteinaceous material may have allergenic properties. In the commercial production of polyols for use in food grade products, removal of proteins down to sub-ppm levels may be required. In a typical process, fermentation of carbohydrate to polyol is followed by pasteurization and cell separation, then a separation train (that usually includes one or more techniques such as ion exchange and carbon treatment), and then a final purification (that is usually crystallization). The separation train and final purification operations are collectively referred to herein as "downstream recovery operations." Because the separation train is generally not designed for removal of protein (the bulk impurities in polyol fermentations are typically sugars and other alcohols, as well as metallic salts), the proteins that pass into the separation train often are not effectively removed by those operations (and may make them less effective at removing other impurities as well) and pass on to the later stages of the purification. Consequently, conventional purification processes can produce sugar polyol product with an unacceptably high level of proteinaceous material.

Summary of the Invention The present invention relates to the purification of polyols produced via fermentation. The processes described herein, by applying an appropriate pretreatment to a polyol-containing fermentation broth, can remove potential interfering substances, such microbial cells, soluble and/or insoluble materials, present in the fermentation broth and enhance the efficacy of subsequent separation, recovery and purification steps to obtain polyol products free of undesired contaminating substances including the intracellular contents of microorganisms. The method provided by the application is able to economically handle large process stream volumes while providing a robust separation of cells and removal/deactivation of proteinaceous material from the main process stream. The present method can accommodate changing input streams (e.g., in terms of the impurity profile of the culture broth) while efficiently avoiding leakage of significant amounts of proteinaceous material forward into the downstream polyol recovery operation(s).

In particular, the present process can substantially reduce or eliminate the potential of carrying over potential allergenic compounds through downstream recovery operations into the final polyol products. The invention also concerns purification processes to produce a polyol product with acceptable levels of proteinaceous material. By limiting the amount of proteinaceous material in the polyol product to a very low level, the value of the polyol product for use in food products can be greatly enhanced. The process disclosed herein allows polyol- containing fermentation broths to be purified to provide polyol products in solution, liquid or crystalline form having low levels of proteinaceous material, typically no more than about 5 ppm, preferably no more than about 2 ppm and, more preferably, no more than about 1 ppm total protein. As employed herein, the term "proteinaceous material" refers to the total amount of proteins, protein degradation products, amino acids, and other protein derived components of the culture broth, such as lipoproteins, glycoproteins and related protein complexes. The present invention describes processes which include the separation and removal of cells and cell bound materials from a fermentation broth, e.g., by centrifugation and/or filtration. The cell separation may be conducted with or without the aid of flocculant, protein inactivating and/or denaturing agents or other appropriate material to flocculate and/or absorb microbial cells and/or particulate materials. The present processes also typically include the separation and removal of soluble and insoluble proteinaceous material, including proteins, glycoproteins and other proteinoic complexes and materials, from a fermentation broth. This may be accomplished with or without the use of flocculant, protein inactivating or denaturing agents or other appropriate material to flocculate and/or absorb microbial cells and/or particulate materials. Throughout this application the terms "fermentation broth" and "culture broth" are used synonymously.

The separation of cells and cell bound materials and removal proteinaceous material is advantageously carried out before the fermentation broth is subjected to the typical downstream polyol recovery operation. The downstream polyol recovery operation relates to the separation, recovery and purification of polyol(s) and generally involves one or more steps such as activated carbon treatment, chromatography, ion exchange, concentration, crystallization and/or other types of standard unit operations to obtain a final purified polyol product(s). The present processes also can allow the collection of live microbial cells for potential reuse for subsequent fermentation and/or other potential applications. Alternatively, if desired, the live cells can be killed in an operation separate from the polyol isolation and purification process by using appropriate pH, temperature and time combinations or other appropriate methods of cell disruption. In one embodiment of the present invention, microbial cells are separated from the fermentation broth without killing or disrupting the cells. This may be done on a continuous or semi-continuous basis or batchwise after completion of the fermentation process. Removal of microbial cells from the culture broth without killing or disrupting the cells, can avoid allowing substantial amounts of intracellular components, cell organnelles and related components being released into the culture broth. This also can essentially eliminates the dissolution of intracellular cell components including proteins, carbohydrates, lipids, nucleic acids and cell bound materials as well as the associated reaction products typically formed under the conditions required to destroy microbial cells. Separation of the microorganisms without the destruction of their cell walls can also avoid the formation of undesirable byproducts having potential undesired allergenic activity. Such allergenic byproducts may be carried over into subsequent separation, recovery and purification processes and can potentially contaminate the finished desired polyol product(s).

Brief Description of the Drawings

Figure 1 is a schematic diagram showing the recovery of polyol from a fermentation according to one embodiment of the invention employing a continuous fermentation process in combination with removal of proteinaceous material early in the polyol purification process.

Figure 2 is a schematic diagram showing the recovery of polyol from a fermentation according to another embodiment of the invention which includes a continuous fermentation process employing a filtration membrane located within the fermentation tank.

Figure 3 is a schematic diagram showing the recovery of polyol from a fermentation according to another embodiment of the invention which employs a hollow fiber fermentation unit.

Detailed Description of the Invention

The present invention relates to enhancements in the recovery of sugar polyol from a fermentation broth. The present method includes a combination of microbial cell separation together with the removal and/or deactivation of proteinaceous material. The present method typically includes an initial separation of at least a portion of the culture broth from the microorganisms to generate (i) a cell retentate stream, and (ii) a clarified polyol-containing stream. The clarified polyol-containing stream is then subjected to one or more steps to remove and/or deactivate proteinaceous material. As used herein, "removal of proteinaceous material" means that at least a portion of the proteinaceous material, e.g., at least about 50% and preferably at least about 75% of the proteinaceous material present in a given polyol- containing stream is taken out of the stream by the operation. Very often, the protein removal operation will be conducted so as to lower the protein content of the polyol-containing stream below a target level. The target level may be higher than the concentration of proteinaceous material considered allowable in the purified sugar polyol to ultimately be produced by the overall isolation/purification scheme. By substantial reducing the protein levels present prior to the initiation of downstream polyol recovery operation, i.e., in the pretreatment phase, it may be possible to enhance the efficacy of downstream polyol recovery operation at removing proteinaceous material as well as other impurities. As referred to herein, the term "sugar polyol" refers to polyhydric alcohols which can be derived from sugar based carbon sources by fermentation. Most common sugar polyols are sugar alcohols related to simple sugars by reduction. Monosaccharide sugar alcohols generally have the formula C_nO_nH_2n+2, where n is an integer from 3 to 6 and more commonly have the chemical formula HOCH₂- (CHOH)_m-CH₂OH, where m is an integer from 1 to 4. Common examples of sugar polyols include glycerol, tetritols, pentitols, hexitols as well as disaccharide alcohols such as maltitol, lactitol and isomalt. Examples of hexitols which can be produced and purified using the present method include allitol, altritol, mannitol, galactitol, glucitol, sorbitol, and iditol. The present method can also be employed in the production and purification of tetritols, such as threitol and erythritol, and pentitols, such as ribitol, arabinitol, adonitol, and xylitol. The term "sugar polyol," as employed in this application, may include a single polyol or may refer to mixtures of two or more polyols. Many microbial fermentation processes produce mixtures of two or more polyhydric alcohols in relative amounts which can vary depending on the particular fermentation conditions chosen. For example, fermentation of sugar, such as glucose (dextrose), sucrose, fructose and/or maltose, with the sugar tolerant fungus Moniliella tomentosa var. pollinis can produce mixtures of glycerol, erythritol, ribitol and/or arabitol.

A variety of microorganisms are capable of producing fermentation broths which include one or more sugar polyols. Such microorganisms are referred to collectively herein as polyol-producing microorganisms and include polyol- producing bacteria, polyol-producing fungi, and polyol-producing yeast. Fermentation of a carbon source with a polyol-producing microorganism may result in the production of other metabolites in addition to sugar polyol(s). For example, such fermentations may also produce ethanol, an organic acid such as lactic acid and/or more complex fermentation product(s). Preferably, the microorganism and fermentation conditions are chosen so that the major fermentation product(s), and more preferably at least about 90 wt.% of the fermentation product(s) is sugar polyol(s) in order to maximize the utilization of the fermentation equipment and the overall efficiency of the polyol production process. As indicated above, the process of the invention relates to methods for purifying a polyol-containing broth from a fermentation process. In this process, a carbon source is fermented to produce sugar polyol(s). As is well known, such fermentations may be conducted using fungi, yeast or other microorganisms capable of forming polyol(s) upon metabolizing a carbon source such as a carbohydrate or petroleum-based hydrocarbon. Fungi of the genus Moniliella can suitably be employed, for example. As to yeast, those such as the genus Candida may be employed. It is well within the purview of one ordinarily skilled in this art to select and use a suitable polyol-producing microorganisms from among the many know and available for this purpose. Other examples of polyol-producing microorganisms suitable for use in generating culture broths which may be purified according to the present methods include fungi from the genus Trichosporonoides or Auriobasidium and polyol-producing yeast from the genus T gonopsis or Yarrowia. The cultures employed in the present processes are typically generated by fermentation of polyol-producing microorganisms in a nutrient medium which includes about 200 to 600 g/L of a carbon source such as glucose. While the cultures normally contain about 40 to 60 g/L microbial cells, fermentations may be suitably conducted over a broader range of cell concentrations, e.g., at microbial cell concentrations of about 10 to 80 g/L. Under typical conditions, the cultures commonly contain about 100 to 300 g/L of sugar polyol after fermentation is complete.

The separation of microbial cells from culture broth may be carried out on a continuous basis or the separation may be conducted in a batchwise manner after completion of fermentation. When the separation is conducted in a continuous or semi-continuous manner, generally only a fraction (e.g., about 20 to about 60%) of the culture broth is separated from the cells. If, however, the fermentation in conducted as a batch process, it is generally advantageous to recover a large fraction of the culture broth free of cells and related insoluble cell debris. Where the overall process is aimed at producing a polyol product having very low levels of proteinaceous impurities, it may be preferred to separate culture broth from the microbial cells without prior destruction of the cells. This avoids the release of large amounts of contaminating materials into the culture broth. As alluded to above, the conditions which are required to destroy substantially all the microorganisms present also typically result in the dissolution of some of the cell debris as well as other previously insoluble material present in the fermentation broth. Although some relatively small amount of destruction and lysis of the microbial cells typically occurs during the fermentation process, the fermentation conditions are generally selected such that no more than about a small fraction polyol-producing microorganisms are destroyed. Preferably, the fermentation is carried out under conditions which minimize the destruction and lysis of microbial cells. This avoids the release of substantial amounts of intracellular cell components into the fermentation broth and reduces the load of impurities to be removed by the downstream polyol recovery process. As the cell separator, use may be made of a liquid cyclone to be used in ordinary solid-liquid separation and a solid-liquid separator such as a precipitation separating tank. In view of the size of common polyol-producing microorganisms, it is often preferable to use a separated plate type (De Laval type) or inclined type (super decant type) centrifugal precipitation device which can continuously separate the cells from the liquid by utilizing centrifugal forces of 1,000 G to 10,000 G.

Alternatively, separation of the microorganisms from the culture broth may be carried out with a filtration membrane, such as a microfiltration membrane or a ultra filtration membrane. As a separating membrane of the membrane separator employed as the cell separator, it is generally preferable to use a microfiltration membrane having pores of not more than 1 μm in diameter or a ultra filtration membrane having a cut-off molecular weight of not less than 10,000 for the purpose of assuredly separating the cells. If the pore diameter is more than 1 μm, the pores of the filter can be prone to plugging by the microorganisms. Consequently, a permeation flux tends to lower in a short time. Similarly, if the fractionation molecular weight is not more then 10,000, the permeation flux can drop and advantageous separation can become more difficult. It is generally advantageous to operate the membrane separator in a cross-flow filtration mode (as opposed to dead- end filtration) as this makes the membrane less prone to buildup of cells and insoluble materials on the face of the membrane.

Proteinaceous material can be removed from the culture broth using any of a variety of well known techniques. To enhance its efficiency, the protein removal step is typically carried out immediately after microbial cell separation, i.e., on the clarified polyol-containing stream generated by the separation step. Common techniques for protein removal include froth flotation, flocculation, adsorption, absorption and combinations thereof.

In one embodiment of the invention, the amount of proteinaceous material in a polyol-containing process stream is substantially reduced by froth flotation. The froth flotation operation may be carried out either before or after separation of microbial cells from the culture broth. In a typical embodiment of the invention, however, in order to avoid the release of significant amounts of intracellular proteinaceous material into the broth, the culture broth is typically separated from the cells prior to conducting the froth flotation operation. Preferably, the cell separation is carried out without any prior step which kills the cells or disrupts their cell walls. The froth flotation is generally carried out by bubbling a gas, such as air, up through a liquid polyol-containing process stream, thereby causing proteinaceous material to separate out as a frothy solid phase on the top surface of the liquid. Commonly, the passing about 0.1 to about 0.5 volumes of gas bubbles per volume of liquid phase per minute through the liquid process stream will effectively separate substantial amounts of proteinaceous material from the liquid within a period of a few seconds to a few minutes. Very commonly, the froth filtration operation is conducted by passing air through the process stream for 1 to 2 minutes. One common apparatus for carrying out this operation is a bubble column. A bubble column typically includes a vertically oriented cylindrical tank with a porous ring at the bottom for introducing small bubbles of gas into the liquid phase. The bubble column normally also includes a mechanical skimmer for removing the froth from the upper surface of the polyol-containing liquid and an outlet valve located near the bottom of the column for drawing off the liquid phase after the froth filtration has been completed. Depending on the nature of the process stream and the proteinaceous which are present, it may be advantageous to add a flocculation agent to the liquid phase as the gas is being sparged up through it. The addition of flocculation agent, e.g., a synthetic polymer flocculating agent such as polyethyleneimine or an acrylamide polymer, can aid in the separation of proteinaceous material as froth during the sparging operation and can increase efficiency and rate of protein removal.

In instances where the froth filtration operation can efficiently remove substantial amounts of proteinaceous material in a relatively short period of time, e.g., within 10 to 15 seconds or less, the froth filtration operation may be carried out in a continuous manner. The polyol-containing process stream to be treated can be introduced in a continuous fashion into the bubble column through a inlet port located below but close to the upper surface of the liquid in the column. Air is typically introduced into the column through a porous ring ring near the bottom of the column. The protein-depleted liquid stream can then be drawn off through an outlet port near the bottom of the column. Thus, the bubble column operates in a "countercurrent" fashion, with air bubbles flowing up through and removing proteinaceous material as froth from the liquid polyol process stream as it passes down through the column.

As an alternative to froth flotation, proteinaceous material can be removed from a polyol-containing process stream via flocculation. The flocculation operation includes the addition of an agent to a polyol-containing process stream which results in the adsorption of proteinaceous material as well as causing the aggregation of suspended colloidal protein-containing particles into small clumps to facilitate their removal. Examples of flocculating agents which can used to reduce the levels of proteinaceous material in a liquid polyol-containing process stream include synthetic polymers such as polyethyleneimine, polypropylene, acrylamide polymers, dialkyldiallylammonium halide polymers and methacryloyloxyethyl trimethylammonium halide-acrylonitrile copolymers.

In another embodiment of the present method, the level of proteinaceous material in a liquid polyol-containing process stream can be reduced by adsorption/absorption. The adsorption/absorption operation is generally carried out on a polyol-containing process stream from which microbial cells have already been removed. Typical adsorbing agents which are suitable for removing proteins and related materials include sepharose, cellulose, lignin, lignocellulosic materials, polystyrene latex and bentonite. Suitable sepharose adsorbing agents include phenyl sepharose and alkyl sepharoses, such as octyl sepharose. Lignin and lignocellulosic materials adsorbing agents suitable for use in the present method include native as well as modified materials. Similarly native or modified cellulose adsorbing agents may be suitably used. Examples of modified celluloses which may be employed as a adsorbing agents in the present method include acid treated cellulose and DEAE- cellulose. Other examples of suitable adsorbing agents include silica gel, alumina, and zeolites. The adsorbent may be in immobilized form, e.g., in a bed or column or in the form of beads, or may simply be added to the liquid process stream and subsequently separated. In either, case the process of separating the resulting protein-depleted liquid stream from the adsorbent generally has the added benefit of removing residual insoluble particulate matter, thereby further clarifying the liquid. One suitable manner of carrying out the adsorption/absorption operation, is to add the adsorbing agent to a liquid polyol-containing process stream in a mixing tank. After contacting the liquid phase with the adsorbing agent for a period of time suitable for reducing the total amount of proteinaceous material to a desired level, the adsorbing agent can be physically separated from the liquid phase by conventional methods such as filtration or centrifugation.

As either an alternative or an adjunct to the protein removal step, after separation of microbial cells the present method may include an operation which results in the degradation and/or inactivation (e.g., through denaturation) of proteinaceous material still present in the clarified polyol-containing stream.

Depending on the nature of the impurity profile in a polyol-containing culture broth, it may be sufficient to simply inactivate the proteinaceous material present to achieve the desired overall properties of the ultimate purified polyol to be produced. Alternatively, a protein degradation/ deactivation step may be included in the pretreatment phase of processes employing a combination of microbial cell separation and protein removal. In instances where highly allergenic proteinaceous materials may be associated with a polyol-containing culture broth, it may be advantageous to subject the cell free fraction obtained from the culture to both protein removal and protein deactivation operations. Where the culture broth is suspected of containing allergenic proteinaceous material of moderate to high molecular weight, heat treatment of the clarified broth may be carried out at temperatures of at least about 80°C and more typically at about 90°C to about 120°C. While such conditions can result in some degree of chemical degradation of proteinaceous materials, a more important consequence is that most proteins are denatured upon being heated under such conditions. In addition to simple heating, the proteinaceous material may be degraded and/or denatured by heating the material at an acidic pH (e.g., about 2.0 to 5.0 and preferably about 3.5 to 4.5) for an appropriate amount of time. It is well within the skill of those in the art to choose appropriate pH, temperature and time conditions to achieve the desired degree of protein deactivation. Simply denaturing the proteinaceous materials which are present may be sufficient to decrease the allergenic content of a polyol- containing stream to a desired level. More commonly, the protein deactivation operation includes a protease treatment and, preferably an acid protease treatment of the polyol-containing stream. Typically, the clarified polyol-containing stream is treated with the protease at an acidic pH (e.g., pH 2.0 to 5.0) for a sufficient period of time to degrade at least about 50% and preferably at least about 90% of the proteinaceous material to lower molecular weight peptides and amino acids. The lower molecular weight peptides are preferably not capable of being precipitated by heat sterilization, an increase in salt content, or treatment with either 5% trichloroacetic acid or organic solvent. The protease treatment step is advantageously carried out at about 40°C to about 65 °C to enhance its efficiency. Figure 1 is a schematic diagram showing the pretreatment operations of one embodiment of the present invention. The microbial fermentation is carried out in a stirred fermentation tank 1 equipped with a mechanical stirrer 2 and a gas inlet 3 for introducing a gas, such as air, into the bottom of the tank. The gas is generally passed through a filter 10 to remove insoluble particulate matter prior to introduction into the fermentation broth. During the fermentation, a slurry of the cells is circulated through the cell separation unit 4 by means of pipes 5,7. The cell separation unit 4 preferably includes a microfiltration membrane which will remove intact microbial cells with high efficiency. If it is believed advantageous to the overall process, a ultrafiltration step can be included later in the pretreatment operation. The combination of an initial microfiltration together with a subsequent ultrafiltration operation can provide a highly clarified polyol-containing stream, while avoiding clogging the ultrafiltration membrane or suffering a loss in throughput do to restricted flow through the filtration membrane(s).

The process operation shown in Figure 1 allows the cell retentate fraction from the cell separation unit to be recycled back to fermentation tank 1 through conduit 5. The effluent stream ("clarified polyol-containing stream") exiting the cell separation unit is transferred to the protein removal unit 8. The removal may based on be any of a variety of common techniques, such a froth flotation, flocculation, and/or adsorption.

If, necessary or desired, a protein deactivation unit can be integrated into the system between the protein removal unit 8 and the downstream (polyol(s)) recovery operation(s) 9. The deactivation can be carried out by heating the effluent stream from the protein removal unit 8 to a temperature sufficient to denature proteins present in the clarified polyol-containing stream. The protein deactivation unit generally is capable of being heated to a temperature of at least about 60°C (and preferably about 90-110°C). The configuration of the fermentation tank 1 and cell separation unit 4 shown in Figure 1 allows the fermentation to be run in either a continuous or batch mode. If the fermentation is run in a continuous (or semi-continuous mode), the slurry of cells is circulated through the cell separation unit 4 in a continuous (or intermittent) fashion while the fermentation is occurring in the tank 1. Alternatively, the fermentation may be conducted as a batch operation. When run in this mode, the slurry of cells and culture broth in the tank 1 is circulated through the cell separation unit 4 after fermentation is completed. If desired, all or a portion of the cell retentate may be left in the tank 1 to be used as an inoculant for a subsequent fermentation batch. If the microbial cells in the retentate are to be discarded, the cells can be destroyed by heating at a temperature of about 80°C to 120°C. The microorganisms can be killed in the fermentation tank 1 or the cells may be removed from the system via the bleed line 6 and solid-liquid separator and transferred to another tank (not shown) before being destroyed.

Bleed line 6 leads to a solid-liquid separator (e.g., a second microfiltration membrane unit). The bleed line allows the removal of cells during the course of a continuous fermentation so that the amount of microbial cells in the fermentation tank 1 can be maintained within desired limits. Microbial fermentations which produce sugar polyol(s) are typically run under conditions where the concentration of microbial cells in the fermentor is maintained at about 10 to about 80 g/L and, preferably, about 40 to about 60 g/L.

Figure 2 schematically depicts another system for carrying out the present method. Like the system shown in Figure 1, the fermentation can be operated in a continuous, semi-continuous or batch mode using this system. The fermentation apparatus shown in Figure 2 includes a filtration membrane 20 arranged inside the fermentation tank 1. The membrane 20 is shown in a tubular type configuration in Figure 2. As will be evident to those skilled in the art, filtration membranes arranged in other configurations within the tank 1 will also be suitable for use in separating microbial cells from the culture broth. In a typical embodiment of the invention, filtration membrane 20 is a microfiltration membrane having an average pore size of no more than about 1 micron. The fermentation is conducted so that the major portion of the tank 23 contains the microbial cells together with the fermentation broth while the volume 22 defined by the filtration membrane 20 contains a cell-free, clarified liquid (the "clarified polyol-containing stream"). This cell-free, clarified liquid can be withdrawn on a continuous or intermittent basis and pumped through line 21 to the protein removal unit 8. If desired, the transfer of the clarified polyol containing broth to the protein removal unit can be facilitated by sealing the upper face of the tank 1 and pressurizing the liquid surface with a gas such as air, nitrogen or CO₂.

Another system which may be employed in the present method is shown schematically in Figure 3. The system includes a hollow fiber fermenter 30 in which the microbial cells are located outside the hollow fibers in a shell. The nutrient medium containing a carbon source such as a sugar is pumped through the hollow fibers. Nutrients and soluble fermentation products are allowed to diffuse freely through the microporous walls of the fibers while the microorganisms are unable to pass through the walls. The nutrient medium is introduced into a recycle vessel 31 and from there is pumped in a loop through the hollow fiber fermenter 30 via lines 32,33. The circulating fermentation broth is typically filtered to remove particulate matter using a microporous prefilter 34. The circulating fermentation broth can be diverted (completely or as a fraction of the recirculating stream) through line 35 to protein removal unit 8 on either a continuous or intermittent basis. As with the embodiments shown in Figures 1 and 2, the protein removal unit 8 can be replaced with a protein deactivation unit if substantial removal of proteins is not required and it is only necessary to deactivate proteinaceous material present in the fermentation broth.

The present method includes a pretreatment purification scheme which can enhance the efficacy of conventional downstream recovery operations typically employed in the separation, recovery and purification of a sugar polyol. The pretreatment purification scheme reduces the load of proteinaceous and other impurities to be removed by the downstream recovery operations. Typical downstream polyol recovery operations include at least one of ultrafiltration, activated carbon treatment, ion exchange chromatography, evaporation, and crystallization. A typical downstream recovery operation involves sequential treatment of a polyol-containing process stream via ion exchange chromatography, activated carbon treatment, evaporative concentration and crystallization. In other commercial processes The ion exchange chromatography demineralizes (removes salts from) the process stream and can also remove substantial amounts of other impurities such as oligo- and polysaccharides as well as colored impurities. Activated carbon treatment is normally employed to further reduce the level of colored materials present in the stream. While the evaporative concentration step is used primarily to raise the concentration of polyol in the process stream to a saturating concentration which will permit crystallization, the evaporation process can also remove volatile impurities from the polyol-containing process. Crystallization, which by its nature can provide a highly purified material, is generally the final step in the purification process. The demineralization of a sugar polyol-containing process normally includes the sequential passage of the process stream through a series of ion exchange resins, such as described in U.S. Patent 4,906,569, the disclosure of which is herein incorporated by reference. In a conventional demineralization scheme, the polyol- containing process stream is softened by passage through cation-exchange resins. The first resin in the series is typically a strongly acidic cation exchange resin, e.g., an alkali metal- (e.g., Na⁺) or ammonium-type strongly acidic sulfonate cation exchange resin. Passage of the process stream through a column of this type of

4-9 strong cation exchange resin is effective in exchanging "hard cations," such as Ca

4-9 and/or Mg ions, with an alkali metal cation or ammonium cation. The effluent from the strong cation exchange resin column is then passed through a weakly acidic cation exchange resin, e.g., a Na-type weakly acidic carboxylate cation exchange resin. Contacting the process stream with this weakly acidic resin is typically

4-9 4-9 employed to further reduce the level of divalent cations, such as Ca and Mg ions. The softened process stream can then be passed through a separation column packed with a cation exchange resin, preferably a strongly acidic cation exchange resin such as a Na-type sulfonate resin. This column allows salts, colored materials and high molecular weight polysaccharides to pass through most rapidly. Oligosaccharides and disaccharides are eluted next followed by polyol(s) in the later fractions. Thus, in addition to further desalting the polyol-containing process stream, this third ion exchange column can remove substantial amounts of colored materials, polysaccharides, oligosaccharides and disaccharides.

The polyol-containing process stream may optionally then be further treated with ion exchange resins to desalt and further decolorize the process stream, such as described in European Patent Application 525,659. This may be carried out by passing the softened process stream through a combination of H-type acidic cation exchange resin and OH-type basic anion exchange resin. For example, the softened process stream may be sequentially passed through (i) an H-type strongly acidic sulfonate cation-exchange resin, (ii) an OH-type weakly basic anion exchange resin and (iii) a "mixed bed column" packed with a mixture of H-type strongly acidic cation exchange resin and OH-type strongly basic anion exchange resin.

Crystallization may be carried out in either a batch system or a continuous system. In the latter case, the concentration of the polyol inclusive of the precipitated crystals is typically maintained within the range of about 30 to 80% by weight based on the total content in a crystallizer. When crystallization is carried out as a batch operation, the polyol-containing stream is typically concentrated to a concentration suited for crystallization (e.g., from about 30 to 80% by weight depending on the polyol being isolated) at an elevated temperature just prior to crystallization. During the concentration step, relatively volatile impurities, such as acetoin, may be removed from the liquid by evaporation. Concentration is usually carried out at a temperature of from 50° to 100°C under either atmospheric pressure or reduced pressure. Apparatus which are suitable for use in the concentration step include an ordinary multiple effect evaporator, a Kestoner evaporator, and a thin film type evaporator.

It has been believed basically preferable for increasing the crystallization efficiency that the polyol-containing liquid be concentrated as highly as possible. For example, from the fact that some polyols have a water solubility of about 3 times by weight at 80° C, the polyol-containing liquid is often concentrated to have a weight of polyol which is 2 to 3 times that of water prior to crystallization (i.e., 67 to 75 wt.% polyol and 25 to 33 wt.% water). Under such a concentration condition, however, the precipitated polyol crystals may tend to become agglomerated crystals. Agglomerated crystals can have poor mechanical strength and may be easily powdered and liable to cake. In addition, a slurry containing a large amount of the crystal is sometimes difficult to move through a pipeline. Taking the crystallizing efficiency and crystal state into consideration, it may be preferable to maintain a polyol concentration in the polyol-containing liquid in a crystallizer from about 0.4 to 1.5 times the weight of water, i.e., from about 30 to 60% by weight. Crystallization is usually performed by heating the polyol-containing liquid to a temperature of 50° C or higher, e.g., about 60° to 90°C, followed by gradual cooling to room temperature or lower, and typically no more than about 15°C for efficiency reasons. While taking time in cooling is preferable, the rate of cooling may be somewhat increased by addition of seed crystals without adversely affecting the crystal properties. The polyol seed crystals preferably have a relatively small particle size, e.g., from 50 to 100 μm. The time of addition of the seed crystals is important for stable production of polyol crystals. A suitable stage of addition varies depending on the polyol concentration and the like but is, in general, before precipitation of a crystal and at the time when the liquid temperature decreases to below about 40 to 45°C.

After a concentrate of a polyol-containing liquid is crystallized, the wet crystal formed is separated from the mother liquor, usually by centrifugation. The wet crystal is then washed with a sufficient amount of water (usually 0.2 to 1.5 times the weight of the crystal) to remove most impurities not present in crystalline form.

Because both the mother liquor and the washing are expected to contain a considerable amount of polyol, at least a part is generally recycled back into the polyol purification operation at any of the steps preceding crystallization. While this may dilute the polyol-containing liquid to be concentrated prior to crystallization, the overall efficiency of the recovery process is generally enhanced by recycling polyol in this manner.

The invention will be further described by reference to the following examples. These examples illustrate but do not limit the scope of the invention that has been set forth herein. Variation within the concepts of the invention will be apparent.

Example 1

A sugar polyol can be produced by a batch fermentation process. The resulting fermentation broth is centrifuged to remove biomass and insoluble proteins using an inclined type (super decant type) centrifugal precipitation device which can continuously separate the cells from the liquid by utilizing centrifugal forces of about 5,000 G. The resulting supernatant has a polyol content of about 200 g/L and is subjected to froth flotation to reduce the level of soluble proteinaceous material present. This is carried out by by passing air up through 5 liters of the supernatant in a bubble column at a rate of about 1.0 L/min for about one to two minutes. A polyethyleneimine flocculation agent is added to the supernatant to aid in separation of proteinaceous material during the sparging operation. The addition of the flocculation agent increases the speed and efficiency of the protein removal. The air sparging together with the flocculation agent causes proteinaceous material to rise to the upper surface of the supernatant and separate as froth. Once froth begins to collect on the upper surface of the filtrate, the froth is mechanically removed using a skimmer. The resulting protein-depleted polyol-containing stream is then removed from the bubble column and further processed to recover crystalline polyol according to the following downstream recovery operation.

The protein-depleted polyol-containing stream is demineralized and purified in part by sequential passage through ion-exchange columns packed with (i) a Na- type strongly acidic sulfonate cation-exchange resin, and (ii) a weakly basic anion- exchange resin, respectively. The softened process stream is then desalted and further purified by passage through a mixed bed column containing a mixture of an H-type strongly acidic cation-exchange resin, and a strongly basic OH-type anion- exchange resin. Following this ion exchange treatment, activated carbon powder (circa 2 g/L) is added to the demineralized solution. The resulting mixture is thoroughly stirred and filtered. The activated carbon filtrate is washed with water. The combined filtrate and washing are concentrated under vacuum at about 80°C to produce a concentrate containing about 50 wt.% polyol. The concentrated solution is slowly cooled to about 15°C and allowed to stand until a substantial amount of polyol crystals have formed. The crystals are recovered by filtration, washed with a minimal amount of water and air dried with the aid of suction.

Example 2

The fermentation apparatus used for the extractive fermentation process described below is shown diagrammatically in Figure 1. A fermentor having a capacity of 5 L working volume is connected to an cell filtration unit containing an ultrafiltration membrane (Millipore HVLP000C5) having a pore size of 0.45 μm and a filtration area of 0.46 m². A variable speed pump is used to circulate the fermentation broth from the fermentor and provided tangential flow across the membrane. The retentate from the cell filtration system is pumped back into the fermentor. The filtrate (clarified polyol-containing stream ) is pumped into one of two bubble columns, each with a working volume of about 3 L, equipped with water jackets for cooling and a ring air sparger near the bottom. Prior to use, each column is sterilized with methanol, and the methanol then eluted with sterile water. The columns exited into a single line which is connected to the appropriate first stage of the downstream polyol recovery operation. This line is equipped with an in-line pH probe, flow meter and sampling port in order to permit convenient monitoring of the effluent stream (protein-depleted polyol-containing stream) exiting the bubble column. Air is typically sparged up through the clarified polyol-containing stream in the bubble column at a rate of about 0.5 L/min for a sufficient time to reduce the total protein content, typically about 1 to 2 minutes.

The resulting protein-depleted polyol-containing stream is then processed to recover crystalline polyol according to downstream recovery operation including ion exchange demineralization, activated carbon treatment, concentration and crystallization described in Example 1.

The invention has been described with reference to various specific and preferred embodiments and techniques. The invention is not to be construed, however, as limited to the specific embodiments disclosed in the specification. It should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

WE CLAIM:

1. A method for producing a purified polyol product stream from a culture broth, said process comprising: (a) providing a culture generated by polyol-producing microorganisms, wherein the culture contains culture broth including sugar polyol;

(b) separating the microorganisms from the culture; and

(c) removing proteinaceous material from the culture broth; wherein the removing step includes at least one of conducting a flocculating operation, conducting a froth flotation operation or adsorbing proteinaceous material with an adsorbing agent.

2. The method of claim 1 wherein the separating step comprises centrifuging the culture to generate: (i) a cell retentate stream; and

(ii) a polyol-containing supernatant.

3. The method of claim 1 wherein the separating step comprises filtering the culture to generate: (i) a cell retentate stream; and

(ii) a polyol-containing filtrate.

4. The method of claim 1 wherein the separating step comprises preferentially separating the microorganisms and the culture broth in the culture mixture from one another to generate:

(i) a cell retentate stream; and

(ii) a clarified polyol-containing stream.

5. The method of claim 4 wherein the removing step comprises conducting a flocculating operation on the clarified polyol-containing stream.

6. The method of claim 4 wherein the removing step comprises adsorbing proteinaceous material in the clarified polyol-containing stream with an adsorbing agent.

The method of claim 4 wherein the removing step comprises conducting a froth flotation operation on the clarified polyol-containing stream to generate: (i) a first proteinaceous byproduct stream; and (ii) a protein-depleted polyol-containing stream.

8. The method of claim 7 wherein the removing step comprises conducting the frothing operation with flocculating agent added to the clarified polyol- containing stream.

9. The method of claim 7 further comprising recovering polyol from the protein-depleted polyol-containing stream.

10. The method of claim 9 wherein the recovering step comprises at least one of activated carbon treatment, ion exchange chromatography, evaporative concentration, ultrafiltration, and crystallization.

11. The method of claim 7 further comprising conducting a protein deactivating operation on the protein-depleted polyol-containing stream.

12. The method of claim 11 wherein conducting a protein deactivating operation comprises heating the protein-depleted polyol-containing stream.

13. The method of claim 1 wherein the removing step comprises processing the culture broth to generate: (i) a second proteinaceous byproduct stream; and (ii) a second polyol-containing stream; and the separating step comprises preferentially separating the microorganisms and the culture broth in the second polyol-containing stream to generate:

(i) a second cell retentate stream; and (ii) a protein-depleted, clarified polyol-containing stream.

14. The method of claim 1 wherein the separating step is conducted without a prior step of disruption of the microorganisms.

15. A method for producing a purified polyol product stream from a culture broth, said process comprising:

(a) providing a culture generated by polyol-producing microorganisms, wherein the culture contains culture broth including sugar polyol; (b) preferentially separating the microorganisms and the culture broth in the culture from one another to generate:

(i) a cell retentate stream; and

(ii) a clarified polyol-containing stream; and (c) removing proteinaceous material from the clarified polyol-containing stream; wherein the removing step includes at least one of conducting a flocculating operation, conducting a froth flotation operation or adsorbing proteinaceous material with an adsorbing agent.

16. A process for producing a purified polyol product stream from a culture broth comprising:

(a) providing a culture generated by polyol-producing microorganisms, wherein the culture contains culture broth including sugar polyol;

(b) preferentially separating the microorganisms and the culture broth from one another to generate:

(i) a cell retentate stream; and

(ii) a clarified polyol-containing stream; and

(c) conducting a protein deactivating operation on the clarified polyol- containing stream.

17. The method of claim 16 wherein the protein deactivating operation comprises treating the clarified polyol-containing stream with protease.

18. The method of claim 16 wherein the protein deactivating operation comprises heating the clarified polyol-containing stream to at least about

80°C.

19. An apparatus for purifying a polyol product stream from a culture of polyol- producing microorganisms comprising: a cell separation unit; and a protein removal unit for removing proteinaceous material from a polyol-containing product stream.

20. A purified polyol product stream comprising: an output stream from a pretreatment operation for removal of microorganisms and depletion of proteinaceous material from a culture broth, said output stream comprising about 100 to about 300 g/L sugar polyol and no more than about 100 mg/L total protein.

21. The purified polyol product stream of claim 20, wherein the output stream is a fluid stream from a frothing operation.

22. The purified polyol product stream of claim 20, wherein the output stream is a fluid stream from a flocculation operation.

23. The purified polyol product stream of claim 20, wherein the output stream is a fluid stream from an adsorption process.

24. The purified polyol product stream of claim 20, wherein the sugar polyol includes tetritol, pentitol, hexitol, glycerol, disaccharide alcohol or a mixture thereof.