WO1991018000A1

WO1991018000A1 - Isolation of oligosaccharides from biomass

Info

Publication number: WO1991018000A1
Application number: PCT/US1991/003550
Authority: WO
Inventors: Kaliprasanna Dhara; Wolfgang G. Glasser; Wiliam E. Kaar; James K. Palmer; Nancy C. Rauschenberg
Original assignee: Center For Innovative Technology
Priority date: 1990-05-22
Filing date: 1991-05-22
Publication date: 1991-11-28
Also published as: AU7953491A

Abstract

A process for isolating substantially pure oligosaccharide from an aqueous solution of oligosaccharide such as the aqueous extract of biomass by purifying the oligosaccharide in solution, preferably by bleaching, and then separating the oligosaccharide from associated aqueous extract components, preferably by ultrafiltration. The process may be advantageously utilized to produce macromolecular polysaccharides with a well defined molecular weight distribution and with known characteristics, such as water or alkaline solubility.

Description

ISOLATION OF OLIGOSACCHARIDES FROM BIOMASS

This application is a continuation-in-part of application serial number 07/526,597 filed May 22, 1990.

Field of the Invention

The present invention relates to a process for isolating oligosaccharides from aqueous solutions of oligosaccharides such as those which can be obtained from biomass.

Background Of The Invention

Aqueous solutions of oligosaccharides exist in many forms. The term ^,lbiomass" generally describes a collection or mass of organic material or residue of organic material. Sources of biomass include wood chips or sawdust, straw, sugar cane bagasse, corn stalks and other harvesting and agricultural residues. The treatment of biomass with steam at pressures between 200 and 400 psig, or with organic solvents at lower pressure, has gained commercial importance for its ability to render biomass separable or fractionable into constitutive chemical components. Terms such as ^■■steam- explosion", "autohydrolysis", "organosolv pulping" all describe types of technologies utilized to treat biomass. As generally understood by those of ordinary skill in the art, aqueous solutions of oligosaccharides may also be obtained by alkaline extraction, mild acid hydrolysis or other methods of biomass fractionation. The present invention generally relates to these technologies. When biomass from any source is treated with steam at elevated temperatures and pressures, a portion of the biomass becomes extractable by water. Similarly, treatment of biomass with organic solvents at elevated temperatures and pressures, or with aqueous alkali, results in the extraction of part of the biomass by the reaction medium. The solutions obtained from any of these fractionation regimes have concentrations of between approximately 1% and 20%, depending on the process conditions, and are comprised in substantial part by oligosaccharides. The water-soluble biomass fraction and other generally known aqueous solutions of oligosaccharides represent the source of material that is described in this invention as "oligosaccharide".

In addition to oligosaccharides and polysaccharides, the aqueous extract of biomass contains numerous other components. The other components in the aqueous extract of biomass include low molecular weight saccharides (monosaccharides, disaccharides, trisaccharides) and saccharide degradation products; fats and fatty acids; starches; proteins; low molecular weight lignins and tannins; lignins; plant pigments and other extractives.

It would be advantageous to be able to isolate the oligosaccharide in pure form from the mixtures of components found in aqueous extracts of biomass or aqueous solutions of oligosaccharides.

It would also be advantageous to produce a macromolecular oligosaccharide or polysaccharide, as distinguished from a monosaccharide, with a well defined molecular weight distribution and known characteristics, such as water or alkaline solubility, from mixtures of components from aqueous extracts of biomass or mixtures of aqueous solutions of oligosaccharides.

Summary of the Invention

We have discovered a process for isolating pure oligosaccharide from aqueous extracts of biomass. This oligosaccharide is generally comprised of 4-0- methylglucuronoxylan, although the exact composition of the isolated oligosaccharide is dependent on the original biomass resource extracted. The oligosaccharides isolated according to the process of the present invention include but are not limited to those having a xylose content between 45 and 80% of dry solids, with an average chain length in excess of five xylose units and molecular weights of at least 600 daltons, preferably between 1000 and 100,000 daltons. Preferably, a target product of the process of the present invention is a macromolecular oligosaccharide or polysaccharide, as distinguished from a monosaccharide, with a well defined molecular weight distribution and known characteristics such as water or alkaline solubility.

The oligosaccharides isolated by the process of the present invention have significant potential for use in numerous applications. The oligosaccharides isolated by the process of the present invention may be utilized as food or feed. They also have potential for use in pharmaceuticals, polymeric segments for bio-degradable plastics, and in separation technology.

The process of the present invention includes a purification/fractionation step and preferably a color removal step. The color removal step is preferably performed by bleaching utilizing hydrogen peroxide and the purification/fractionation step is preferably performed, on the basis of molecular parameters, by ultrafiltration. The desired oligosaccharide is obtained in dry form utilizing any suitable technology including freeze-drying and spray-drying. Optionally the process may include one or more of the following steps: ion exchange, for the purpose of desalination; and/or concentration utilizing rotation evaporation or other method. The process of the present invention may be performed on any aqueous solution containing oligosaccharides.

More particularly, according to a process of the present invention, biomass is treated with steam at a pressure between 200 and 400 psig, or with organic solvents, aqueous alkali, or weak acid at lower pressure, in the manner known to those of ordinary skill of the art, to separate the aqueous extractable components from the biomass. The oligosaccharides are then isolated from the aqueous extract biomass components by the steps of: bleaching the water soluble biomass component at a pH between about 8 and 11; concentrating and liquid/liquid extracting of the bleached solution with water, utilizing ultrafiltration technology to remove salts and other low molecular weight components; fractionating of the oligosaccharides utilizing ultrafiltration technology; and

■ recovering the oligosaccharide solids. Optionally, an ion- exchange step can be performed to facilitate complete desalination of the oligosaccharide.

An advantage of the present invention is that it allows the oligosaccharides to be isolated in substantially pure form from other aqueous extractable biomass components.

Another advantage of the present invention is that it may be utilized to produce macromolecular polysaccharides with well defined molecular weight distributions and known characteristics, such as water or alkaline solubility.

An advantage of the oligosaccharides produced by the process of the present invention is that the oligosaccharides may be utilized as food, feed or polymer components.

Another advantage of the oligosaccharides produced by the process of the present invention is that the oligosaccharides may be utilized in pharmaceuticals and separation technology.

Other details and advantages of the present invention will become apparent in the following more detailed description.

Brief Description of the Drawings

Figure 1 is a diagram of a representative

* _ ultrafiltration/di.af.ltrati.on process as used in the present invention. Figure 2 is a flow diagram of a process for isolating oligosaccharides from an aqueous extract of biomass according to the present invention.

Figure 3 is a flow diagram of a process for isolating oligosaccharides from an aqueous extract of biomass according to one embodiment of the present invention.

Figure 4 is a plot of the molecular weight distribution of oligosaccharides isolated by a process of the present invention, determined by aqueous gel permeation chromatography using polyethylene glycol molecular weight standards.

Detailed Description of the Invention

The present invention provides a novel and advantageous process for isolating oligosaccharides from aqueous solutions of biomass. The oligosaccharides isolated from the aqueous extracts of biomass according to the present invention include but are not limited to those having a xylose content between 45 and 80% of the dry solids, with an average chain length in excess of 5 xylose units and a molecular weight greater than 600 daltons and preferably greater than 1000 daltons.

According to the present invention the technique for isolating the oligosaccharides includes purification/fractionation from associated components on the basis of molecular parameters such as molecular weight and preferably includes decolorization prior to purification/fractionation. More particularly, the process of the present invention may include bleaching the aqueous extract solution with hydrogen peroxide, at a pH between 8 and

11; concentrating the resulting solution using an ultrafiltration process; eliminating salts and low molecular weight components via liquid/liquid extraction of the solution with water using an ultrafiltration process; and recovering the oligosaccharide solids using spray-drying, freeze-drying or non-solvent precipitation. Optionally, the bleached solution may be passed through a cation exchange resin to effect the desalination, or additional ultrafiltration steps can be utilized to fractionate the oligosaccharides into more closely defined molecular weight ranges.

Preferably, the decolorization procedure includes treating the aqueous extract solution with hydrogen peroxide

(H₂0₂) in an amount, by weight, of from 50 to 100% of the lignin content of the aqueous biomass solution, at conditions wherein the solution has a pH of above approximately 9, in the present of a chelating agent, preferably sodium ethylene diamine tetraacetate (Na-EDTA) but also sodium silicate or other qualified chelating agents, at a temperature of approximately 50°C.

Preferably, before the purification/fractionation step of the present process, the decolorized solution is cooled and the pH of the solution is reduced using a mineral acid such a hydrochloric acid (HCI) .

The purification/fractionation procedure preferably includes ultrafiltration of the bleached solution.

Ultrafiltration is a technology that fractionates mixtures of molecules on the basis of molecular size using membranes with specific porosities. Molecules that are of a sufficiently small molecular size so as to pass through a particular membrane are carried through the membrane in the "permeate" stream, whereas molecules that are retained by the membrane are isolated in the "retentate" stream. By utilizing membranes of different porosities, it is possible to fractionate a mixture of molecules to narrower distributions while at the same time concentrating the retentate streams.

Ultrafiltration is also an efficient desalination technique, as it is used in the process of the present invention. An important stage in the ultrafiltration procedure is the process of diafiltration. Diafiltration is essentially a liquid/liquid extraction of the retentate stream using either fresh water or another buffer. To effect diafiltration of the retentate, the fresh water or other buffer, is added to the retentate stream at the same rate that the permeate stream is being removed from the retentate stream. The objective of diafiltration is to completely wash the retentate stream of any lower molecular weight compounds such as salts. The membrane used during the diafiltration stage defines the

"molecular weight cut-off" of the materials that are removed in the process of diafiltration. Figure 1 diagrams a typical ultrafiltration/diafiltration process.

Figure 2 shows a possible flow diagram for a process of the present invention. Referring to Figure 2, an alkaline extract (pH 12) from biomass comprising oligosaccharides and other material at a concentration of 8% (weight/volume) is treated with HCI to pH 11. Next, Na-EDTA is added in a quantity of 0.1% (weight/volume of total solution) and the solution is allowed to mix thoroughly for one hour.

At this point, ultraviolet spectroscopy is performed on a sample of the solution to calculate the approximate mass of lignin in the solution. Alternatively, the approximate mass of lignin in the solution may be determined in other manners known to those of ordinary skill in the art.

Next, hydrogen peroxide solution (50% weight/weight) is added in a quantity such that the added mass of hydrogen peroxide is equal to the calculated mass of lignin in the aqueous extract solution.

The solution is then slowly heated to 50°C. , at which temperature the solution is held isothermally for one hour.

As necessary during the bleaching state, 50% (w/w) sodium hydroxide solution is added to maintain the pH of the reaction mixture above pH 9. The solution is then allowed to cool, at which time the pH is adjusted to pH 2 with HCI. The solution is then concentrated using an ultrafiltration apparatus comprised of 3000 molecular weight cut-off (MWCO) membrane cartridges (such as those manufactured by the Amicon Division,

W.R. Grace and Company, Beverly, Massachusetts) to approximately one-fifth of the original volume. The concentrate is then diafiltered using a volume of fresh water equal to at least three times, but no more than ten times, the volume of concentrate. During the ultrafiltration stages, the temperature of the process solution is maintained at 30°C with a suitable temperature control device capable of both heating and refrigeration. At the conclusion of diafiltration, the retentate is further concentrated before spray-drying to yield to the desired oligosaccharide product. Alternatively, the xylan-rich oligosaccharide may be obtained by freeze-drying, non-solvent precipitation, or other suitable techniques.

Figure 3 shows a possible flow diagram for a process of the present invention, that includes the optional ion-exchange step. Referring to Figure 3, steam-exploded biomass is extracted with water using a fiber centrifuge or other appropriate devices capable of separating the unsolubilzed biomass from the aqueous extract. The pH of the extract is then adjusted to approximately pH 11 with sodium hydroxide, or another suitable base such as potassium hydroxide, and Na-EDTA is added in a quantity of 0.1% (w/v) . The mixture is then allowed to stir for one hour. Next, hydrogen peroxide solution (50%, w/w) is added in a quantity such that the added mass of hydrogen peroxide is equal to the calculated mass of lignin in the aqueous extract solution and the reaction mixture is allowed to slowly heat to 50°C; sodium hydroxide is added during the bleaching reaction to maintain the pH above pH 9. The bleached solution is then allowed to cool before adjusting the pH to pH 2 with 10% HCI solution. The neutralized solution is then concentrated using an ultrafiltration apparatus comprised of 3000 molecular weight cut-off (MWCO) membrane cartridges (such as those manufactured by the Amicon Division, W.R. Grace and Company, Beverly, Massachusetts) to approximately one-fifth of the original volume. The concentrate is then diafiltered using a volume of fresh water equal to at least three times, but no more than ten times, the volume of concentrate. During the ultrafiltration stages, the temperature of the process solution is maintained at 30°C with a suitable temperature control device capable of both heating and refrigeration. At the conclusion of diafiltration, the retentate is passed through an ion-exchange column that is packed with a cation- exchange resin in the hydrogen form, such as Amberlite IR-120 (manufactured and sold by The Dow Chemical Company, Midland, Michigan) in order to assure complete regeneration of the carboxylic acid groups present in the oligosaccharide. After the ion-exchange process has been completed, the solution is returned to the ultrafiltration unit for final diafiltration and concentration. The oligosaccharide is then recovered in powder form using a spray-dryer.

A typical oligosaccharide molecular weight distribution by gel permeation chromatography in water, of a water soluble oligosaccharide, isolated according to the process of the present invention, is illustrated in Figure 4. Column calibration using suitable water-soluble molecular weight standards permits the determination of the molecular weight standards permits the determination of the molecular weight distribution of the isolated oligosaccharide. As shown in Figure 4, a water soluble oligosaccharide isolated by the process of the present invention preferably has a minimum molecular weight greater than 1000 daltons and a maximum molecular weight greater that 10,000 daltons.

The advantages of the present invention will be further illustration by the following examples.

EXAMPLE 1

A water-soluble fraction from steam-exploded yellow poplar wood (Liriodendron tulipifera) having a concentration of approximately 30% molasses solids (solids on solution) is neutralized to pH 7.1 with one molar aqueous sodium bicarbonate (1M aq. NaHC0₃) solution. This neutralized solution is treated with an amount of aqueous hydrogen peroxide solution corresponding to approximately one half of the molasses solids. The resulting mixture is heated to 55 C for approximately one hour. The mixture in then cooled, treated with Mn0₂ to destroy excess H₂0₂, and filtered. The filtrate is subsequently passed through a column containing an Amberlite IR-250 H+ cation exchange resin suspended in water. This regenerates carboxyl functional groups from sodium carboxylate ions. The ion exchange solution, which has a pH of approximately 2.5 to 3, is then subjected to membrane filtration in a stirred cell filtration unit (manufactured and sold by Amicon) equipped with a membrane with nominal molecular weight cut-off of 1000. Pressure is adjusted to 50 psi with nitrogen. Membrane filtration is continued and pH of filtrate is monitored intermittently. When the pH of the membrane permeate has reached 5.5 to 6, the filtration is discontinued, and the retentate is reclaimed. 200 grams of a pure, water-soluble oligosaccharide fraction is recovered from the aqueous retentate by freeze drying. Its composition is given in Table I below:

Table I

MONOSACCHARIDE YIELD (in % of solids)

Rhamnose 0.56

Xylose 62.51

Mannose 4.84

Glucose 9.46

Galactose 0.05

Total Monosaccharides 77.32

Unaccounted Uronic Acids, Acetic Acid, etc. 22.68

EXAMPLE 2

The same procedure as in Example 1 is utilized except that the water soluble oligosaccharide is recovered from the aqueous retentate by solvent precipitation. The retentate, after concentration by membrane filtration to approximately 30% solids, is slowly added to an agitated volume of a xylan non-solvent, which in this instance is ethanol. The precipitate formed is collected by filtration or decantation to yield a water soluble oligosaccharide. EXAMPLE 3

The same procedure as in Example 1 is utilized except that the water soluble oligosaccharide is recovered from the aqueous retentate solution by spray drying.

EXAMPLE 4

A water-soluble fraction from steam-exploded yellow poplar wood (Liriodendron tulipifera) having a concentration of approximately 30-40% molasses solids (solids on solution) is subjected to continuous membrane filtration using an Amicon spiral membrane cartridge continuous filtration apparatus which permits prefiltration prior to bleaching. A 25% solids retentate fraction is then neutralized to approximately pH 7.1 with 1M aq. NaHC0₃ solution and treated with an amount of aqueous hydrogen peroxide solution corresponding to approximately one half of the molasses solids. The resulting mixture is heated to 55 C for approximately one hour. The mixture is then cooled, treated with Mn0₂ to destroy excess H₂0₂, and filtered. The filtrate is subsequently passed through a column containing an Amberlite IR-250 H⁺ cation exchange resin suspended in water. This regenerates carboxyl functional groups from sodium carboxylate ions. The ion exchanged solution, which has a pH of approximately 2.5 to 3, is then subjected to membrane filtration in a stirred cell filtration unit (manufactured and sold by Amicon Division, W.R. Grace and Company, Beverly, Massachusetts) equipped with a membrane with nominal molecular weight cut-off of 1000. Pressure is adjusted to 50 psi with nitrogen. Membrane filtration is continued and pH of filtrate is monitored intermittently. When the pH of the membrane permeate has reached 5.5 to 6, the filtration is discontinued, and the retentate is reclaimed. 200 grams of a pure, water-soluble oligosaccharide fraction is recovered from the aqueous retentate by freeze drying.

EXAMPLE V

Comminuted yellow poplar, 5 kilograms, is extracted with 60 liters of 4% (w/v) sodium hydroxide solution for 24 hours at ambient temperature. The fiber is separated from the extract using a fiber centrifuge. The extract, pH 13, is neutralized to pH 11 with 10% HCI solution, at which time 60 grams of sodium ethylene diamine tetra-acetate is added; the mixture is allowed to stir for one hour. Next, 100 grams of 50% hydrogen peroxide solution is added and the reaction mixture is slowly heated to 50°C. Once the temperature of 50°C has been reached, the solution is maintained at 50°C for one hour before allowing the solution to cool. The solution is stirred continuously for the next four hours. Then, 10% HCI solution is added to adjust the pH to pH 2. At this time, the solution is transferred to an Amicon DC-30P semi-continuous ultrafiltration unit that is configured with four Amicon S-10 3000 molecular weight cut-off cartridges. The solution is concentrated to 20 liters, then diafiltered using 100 liters of fresh water. At the completion of diafiltration, the solution is further concentrated to 10 liters. The temperature during the ultrafiltration process is maintained at 30°C using a Polyscience (7800 Merrimac Avenue, Miles, IL

60648) heating/cooling recirculator. The oligosaccharide, 500 grams, is then recovered in powder form by spray-drying using a Yamato (33 Corporate Drive, Orangeburg, NY 10962) sprayer- dryer. The carbohydrate composition of the isolated oligosaccharide is 63.6% xylan and 1.55% glucan.

Claims

WHAT IS CLAIMED IS :

1. A process for isolating oligosaccharides comprising the steps of: a) treating a biomass, having oligosaccharide and polysaccharide components that are extractable by aqueous solutions to separate the extractable oligosaccharide and polysaccharide from the remainder of the biomass; b) bleaching the aqueous extract biomass component obtained in (a) , at a pH between about 8 and about 11; c) purifying, fractionating, and concentrating the bleached solution; d) recovering the oligosaccharide from the bleached extracted biomass components.

2. The process of claim 1 wherein the bleaching comprises adding sodium ethylene diamine tetra-acetate to the aqueous extract biomass component and adjusting the pH to pH 11.

3. The process of claim 2 wherein the bleaching further comprises adding an H₂0₂ solution to the aqueous extract biomass component.

4. The process of claim 3 wherein the bleaching further comprises adjusting the pH of the bleached solution to pH 2 with 20% HCI solution.

5. The process of claim 1 wherein the bleached solution is processed by ultrafiltration to remove salts and low molecular weight components.

6. The process of claim 5 wherein the ultrafiltered solution is passed through a cation exchange resin in hydrogen form to complete desalination.

7. The process of claim 5 wherein the ultrafiltered solution is concentrated using ultrafiltration.

8. The process of claim 1 wherein the recovering of the oligosaccharide solids further comprises spray-drying.

9. The process of claim 1 wherein the recovery of the oligosaccharide solids further comprises freeze-drying.

10. The process of claim 1 wherein the recovery of the oligosaccharide solids further comprises non-solvent precipitation followed by filtration.

11. A process for isolating oligosaccharides comprising the steps of: a) treating a biomass, having oligosaccharide and polysaccharide components that are extractable by aqueous solutions, to separate the extractable oligosaccharide and polysaccharide components from the insoluble biomass component; b) adjusting the pH of the aqueous extract obtained in (a) , using suitable acid or base, as required, to pH 11; c) adding Na-EDTA, or other chelating agent, such as sodium silicate, to the pH adjusted aqueous extract so as to stabilize peroxide; d) bleaching the pH adjusted aqueous extract with a solution of H₂0₂ in H₂0; e) heating the aqueous extract, Na-EDTA, H₂0₂ in H₂0 solution to about 50 "C for about one hour; f) cooling the bleached aqueous extract solution; g) adjusting the pH of the bleached aqueous extract solution to pH 2 using 10% HCI, or other suitable acidic solution; h) concentrating the pH adjusted bleached solution using ultrafiltration technology, using 3000 MWCO membrane cartridges of other suitable membrane system; i) diafiltering the concentrated, pH adjusted bleached solution (retentate) with fresh water, using ultrafiltration technology with a suitable membrane system, to complete the removal of salts and low molecular weight components from the retentate; k) further concentrating the diafiltered retentate using ultrafiltration technology with a suitable membrane system; 1) spray-drying the concentrate to recover the oligosaccharide in powder form.

12. A substantially pure oligosaccharide, having known molecular weight and with desirable characteristics such as water or alkaline solubility, isolated from an aqueous extract of biomass.

13. A substantially pure water soluble oligosaccharide comprising a xylose content of between 45 and 80% and a molecular weight above 1000 daltons.