BIOLOGICAL CONVERSION OF BIOMASS-DERIVED SUGARS TO VALUE ADDED CHEMICALS

A method of growing a microorganism by culturing the microorganism in a an aqueous solution of carbohydrates containing C6-sugar monomers or C5-sugar monomers, wherein the aqueous solution of carbohydrates is made by reacting biomass or a biomass-derived reactant with a solvent system including a lactone and water, and an acid catalyst. The reaction yields a product mixture containing water-soluble C6-sugar-containing oligomers, C6-sugar monomers, C5-sugar-containing oligomers, C5-sugar monomers, or any combination thereof. The product mixture is then partitioned or extracted to yield an aqueous layer containing the carbohydrates and a substantially immiscible organic layer containing the lactone. The aqueous layer is used for growing the microorganisms.

DETAILED DESCRIPTION

Abbreviations and Definitions

“Biomass” as used herein includes materials containing cellulose, hemicellulose, lignin, protein and carbohydrates such as starch and sugar. Common forms of biomass include trees, shrubs and grasses, corn and corn husks as well as municipal solid waste, waste paper and yard waste. Biomass high in starch, sugar or protein such as corn, grains, fruits and vegetables, is usually consumed as food. Conversely, biomass high in cellulose, hemicellulose and lignin is not readily digestible by humans and is primarily utilized for wood and paper products, fuel, or is discarded as waste. “Biomass” as used herein explicitly includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, vines, hard and soft woods. In addition, biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. “Biomass” includes virgin biomass and/or non-virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper, and yard waste. Municipal solid waste generally includes garbage, trash, rubbish, refuse and offal that is normally disposed of by the occupants of residential dwelling units and by business, industrial and commercial establishments, including but not limited to: paper and cardboard, plastics, food scraps, scrap wood, saw dust, and the like.

“Biomass-derived”=Compounds or compositions fabricated or purified from biomass.

Brønsted-Lowry Acid/Base=A Brønsted-Lowry acid is defined herein as any chemical species (atom, ion, molecule, compound, complex, etc.), without limitation, that can donate or transfer one or more protons to another chemical species. Mono-protic, diprotic, and triprotic acids are explicitly included within the definition. A Brønsted-Lowry base is defined herein as any chemical species that can accept a proton from another chemical species. Included among Brønsted-Lowry acids are mineral acids, organic acids, heteropolyacids, solid acid catalysts, zeolites, etc. as defined herein. Note that this list is exemplary, not exclusive. The shortened term “Brønsted” is also used synonymously with “Brønsted-Lowry.”

“Carbohydrate” is defined herein as a compound that consists only of carbon, hydrogen, and oxygen atoms, in any ratio.

“C5carbohydrate” refers to any carbohydrate, without limitation, that has five (5) carbon atoms. The definition includes pentose sugars of any description and stereoisomerism (e.g., D/L aldopentoses and D/L ketopentoses). C5carbohydrates include (by way of example and not limitation) arabinose, lyxose, ribose, ribulose, xylose, and xylulose.

“Cellulose” refers to a polysaccharide of glucose monomers ((C6H10O5)n); “cellulosic biomass” refers to biomass as described earlier that comprises cellulose, and/or consists essentially of cellulose, and/or consists entirely of cellulose. Lignocellulosic biomass refers to biomass comprising cellulose, hemicellulose, and lignin. Lignocellulosic biomass comprises xylose, as does hemicellulose. For the experiments described below, dried corn stover was obtained through the Great Lakes Bioenergy Research Center, Madison, Wis., USA. Dried maple wood was obtained from Mascoma corporation, Hanover, N.H.

“Growth” of a microorganism (e.g., a unicellular prokaryote or eurkaryote) denotes the division of one microorganism into two identical daughter cells. Both daughter cells from the division do not necessarily survive. However, if on average more than one (1) of the daughter cells survives, the population of cells undergoes exponential growth. Measuring the exponential growth curve in batch culture can accomplished via several well-known methods, such as individual cell counting via microscopy or flow cytometry, colony counting, turbidity, optical density, nutrient uptake, etc. Growth in batch culture can be modeled with four different phases: lag phase, exponential or log phase, stationary phase, and death phase (D). During “lag phase” the microorganisms adapt themselves to growth conditions. It is the period where the individual organisms are maturing and not yet able to divide. The “exponential phase” (sometimes also called the “logarithmic” or “log” phase) is a period characterized by cell doubling. The number of new organisms appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate such that both the number of cells and the rate of population increase doubles with each consecutive time period. The actual rate of growth depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. Exponential growth cannot continue indefinitely because the medium is soon depleted of nutrients and enriched with wastes. In the “stationary phase” the growth rate slows as a result of nutrient depletion and accumulation of toxic products. This phase is reached as the microorganisms begin to exhaust the resources that are available to them. In the “death phase,” the organisms run out of nutrients and die. Spore-forming species undergo sporulation in this phase.

“Heteropolyacid”=A class of solid-phase acids exemplified by such species as H4SiW12O40, H3PW12O40, H6P2W18O62, H3+xPMo12−xVxO40and the like. Heteropolyacids are solid-phase acids having a well-defined local structure, the most common of which is the tungsten-based Keggin structure. The Keggin unit comprises a central PO4tetrahedron, surrounded by 12 WO6octahedra. The standard unit has a net (−3) charge, and thus requires three cations to satisfy electroneutrality. If the cations are protons, the material functions as a Brønsted acid. The acidity of these compounds (as well as other physical characteristics) can be “tuned” by substituting different metals in place of tungsten in the Keggin structure. See, for example, Bardin et al. (1998) “Acidity of Keggin-Type Heteropolycompounds Evaluated by Catalytic Probe Reactions, Sorption Microcalorimetry and Density Functional Quantum Chemical Calculations,”J. of Physical Chemistry B,102:10817-10825.

“Homogeneous catalyst”=A catalyst that exists in the same phase (solid, liquid, or gas) as the reactants under reaction conditions.

“Heterogeneous catalyst”=A catalyst that exists in a different phase than the reactants under reaction conditions.

“Lactone” as used herein refers to an unsubstituted or substituted cyclic ester, having a single oxygen heteroatom in the ring, and having from four to six total atoms in the ring—i.e., beta, gamma, and delta lactones, derived from any corresponding C4 to C1-6carboxylic acid. Thus, as used herein, the term “lactone” explicitly includes (without limitation) unsubstituted and substituted beta- and gamma-butyrolactone and beta-, gamma-, and delta-valerolactones to beta-, gamma, and delta-hexadecalactones. Some lactones are miscible in water, such as GVL; other lactones have more limited solubility in water. Those lactones that can dissolve at least about 1 wt % water, and more preferably at least about 5 wt % (or more) of water (up to miscible) are suitable for use in the process described herein. Gamma- and delta-lactones are preferred. Gamma-valerolactone is most preferred.

Microorganism=all prokaryotic and eukaryotic unicellular (and sometimes multicellular) life forms and viruses, including wild-type and genetically engineered strains thereof. Thus, the term “microorganism” explicitly includes fungi, protista, archaea, and bacteria of any and all description, as well as virus, both wild-types and genetically engineered variants, whether now known or discovered or developed in the future.

Lewis Acid/Base=A Lewis acid is defined herein as any chemical species that is an electron-pair acceptor, i.e., any chemical species that is capable of receiving an electron pair, without limitation. A Lewis base is defined herein as any chemical species that is an electron-pair donor, that is, any chemical species that is capable of donating an electron pair, without limitation.

The Lewis acid (also referred to as the Lewis acid catalyst) may be any Lewis acid based on transition metals, lanthanoid metals, and metals from Group 4, 5, 13, 14 and 15 of the periodic table of the elements, including boron, aluminum, gallium, indium, titanium, zirconium, tin, vanadium, arsenic, antimony, bismuth, lanthanum, dysprosium, and ytterbium. One skilled in the art will recognize that some elements are better suited in the practice of the method. Illustrative examples include AlCl3, (alkyl)AlCl2, (C2H5)2AlCl, (C2H5)3Al2Cl3, BF3, SnCl4and TiCl4.

The Group 4, 5 and 14 Lewis acids generally are designated by the formula MX4; wherein M is Group 4, 5, or 14 metal, and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a pseudohalogen. Non-limiting examples include titanium tetrachloride, titanium tetrabromide, vanadium tetrachloride, tin tetrachloride and zirconium tetrachloride. The Group 4, 5, or 14 Lewis acids may also contain more than one type of halogen. Non-limiting examples include titanium bromide trichloride, titanium dibromide dichloride, vanadium bromide trichloride, and tin chloride trifluoride.

Group 4, 5 and 14 Lewis acids useful in the method may also have the general formula MRnX4−n; wherein M is Group 4, 5, or 14 metal; wherein R is a monovalent hydrocarbon radical selected from the group consisting of C1to C12alkyl, aryl, arylalkyl, alkylaryl and cycloalkyl radicals; wherein n is an integer from 0 to 4; and wherein X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include benzyltitanium trichloride, dibenzyltitanium dichloride, benzylzirconium trichloride, dibenzylzirconium dibromide, methyltitanium trichloride, dimethyltitanium difluoride, dimethyltin dichloride and phenylvanadium trichloride.

Group 4, 5 and 14 Lewis acids useful in method may also have the general formula M(RO)nR′mX(m+n); wherein M is Group 4, 5, or 14 metal; RO is a monovalent hydrocarboxy radical selected from the group consisting of C1to C30alkoxy, aryloxy, arylalkoxy, alkylaryloxy radicals; R′ is a monovalent hydrocarbon radical selected from the group consisting of C1to C12alkyl, aryl, arylalkyl, alkylaryl and cycloalkyl radicals; n is an integer from 0 to 4; m is an integer from 0 to 4 such that the sum of n and m is not more than 4; and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include methoxytitanium trichloride, n-butoxytitanium trichloride, di(isopropoxy)titanium dichloride, phenoxytitanium tribromide, phenylmethoxyzirconium trifluoride, methyl methoxytitanium dichloride, methyl methoxytin dichloride and benzyl isopropoxyvanadium dichloride.

Group 5 Lewis acids may also have the general formula MOX3; wherein M is a Group 5 metal; X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. A non-limiting example is vanadium oxytrichloride.

The Group 13 Lewis acids have the general formula MX3; wherein M is a Group 13 metal and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include aluminum trichloride, boron trifluoride, gallium trichloride, indium trifluoride, and the like.

The Group 13 Lewis acids useful in method may also have the general formula: MRnX3−nwherein M is a Group 13 metal; R is a monovalent hydrocarbon radical selected from the group consisting of C1to C12alkyl, aryl, arylalkyl, alkylaryl and cycloalkyl radicals; and n is an number from 0 to 3; and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include ethylaluminum dichloride, methylaluminum dichloride, benzylaluminum dichloride, isobutylgallium dichloride, diethylaluminum chloride, dimethylaluminum chloride, ethylaluminum sesquichloride, methylaluminum sesquichloride, trimethylaluminum and triethylaluminum.

Group 13 Lewis acids useful in this disclosure may also have the general formula M(RO)nR′mX3−(m+n); wherein M is a Group 13 metal; RO is a monovalent hydrocarboxy radical selected from the group consisting of C1to C30alkoxy, aryloxy, arylalkoxy, alkylaryloxy radicals; R′ is a monovalent hydrocarbon radical selected from the group consisting of C1to C12alkyl, aryl, arylalkyl, alkylaryl and cycloalkyl radicals; n is a number from 0 to 3; m is an number from 0 to 3 such that the sum of n and m is not more than 3; and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include methoxyaluminum dichloride, ethoxyaluminum dichloride, 2,6-di-tert-butylphenoxyaluminum dichloride, methoxy methylaluminum chloride, 2,6-di-tert-butylphenoxy methylaluminum chloride, isopropoxygallium dichloride and phenoxy methylindium fluoride.

Group 13 Lewis acids useful in this disclosure may also have the general formula M(RC(O)O)nR′mX3−(m+n); wherein M is a Group 13 metal; RC(O)O is a monovalent hydrocarbacyl radical selected from the group consisting of C2to C30alkacyloxy, arylacyloxy, arylalkylacyloxy, alkylarylacyloxy radicals; R′ is a monovalent hydrocarbon radical selected from the group consisting of C1to C12alkyl, aryl, arylalkyl, alkylaryl and cycloalkyl radicals; n is a number from 0 to 3 and m is a number from 0 to 3 such that the sum of n and m is not more than 3; and X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine. X may also be a psuedohalogen. Non-limiting examples include acetoxyaluminum dichloride, benzoyloxyaluminum dibromide, benzoyloxygallium difluoride, methyl acetoxyaluminum chloride, and isopropoyloxyindium trichloride.

The most preferred Lewis acids for use in the method are metal halides generally and more specifically transition metal halides, lanthanoid metal halides, and Group 5, 13, and 14 metal halides. Preferred among the metal halides are metal chlorides. Preferred transition metal chlorides include, but are not limited to, TiCl4, VCl3. and the like. Preferred Group 13 and 14 metal halides and chlorides include, but are not limited to, BF3, AlCl3, SnCl4, InCl3, and GaCl3. Preferred lanthanoid chlorides include, but are not limited to, LaCl3, DyCl3and YbCl3.

The terms “solid acid” and “solid acid catalyst” are used synonymously herein and can comprise one or more solid acid materials. The solid acid catalyst can be used independently or alternatively can be utilized in combination with one or more mineral acid or other types of catalysts. Exemplary solid acid catalysts which can be utilized include, but are not limited to, heteropolyacids, acid resin-type catalysts, mesoporous silicas, acid clays, sulfated zirconia, molecular sieve materials, zeolites, and acidic material on a thermo-stable support. Where an acidic material is provided on a thermo-stable support, the thermo-stable support can include for example, one or more of silica, tin oxide, niobia, zirconia, titania, carbon, alpha-alumina, and the like. The oxides themselves (e.g., ZrO2, SnO2, TiO2, etc.) which may optionally be doped with additional acid groups such as SO42−or SO3H may also be used as solid acid catalysts.

Further examples of solid acid catalysts include strongly acidic ion exchangers such as cross-linked polystyrene containing sulfonic acid groups. For example, the Amberlyst®-brand resins are functionalized styrene-divinylbenzene copolymers with different surface properties and porosities. (These types of resins are designated herein as “Amb” resins, followed by a numeric identifier of the specific sub-type of resin where appropriate.) The functional group is generally of the sulfonic acid type. The Amberlyst®-brand resins are supplied as gellular or macro-reticular spherical beads. (Amberlyst® is a registered trademark of the Dow Chemical Co.) Similarly, Nafion®-brand resins are sulfonated tetrafluoroethylene-based fluoropolymer-copolymers which are solid acid catalysts. Nafion® is a registered trademark of E.I. du Pont de Nemours & Co.)

Solid catalysts can be in any shape or form now known or developed in the future, such as, but not limited to, granules, powder, beads, pills, pellets, flakes, cylinders, spheres, or other shapes.

Zeolites may also be used as solid acid catalysts. Of these, H-type zeolites are generally preferred, for example zeolites in the mordenite group or fine-pored zeolites such as zeolites X, Y and L, e.g., mordenite, erionite, chabazite, or faujasite. Also suitable are ultrastable zeolites in the faujasite group which have been dealuminated.

The term “solute” is broadly defined herein to include any non-reactive salt (such as NaCl, NaBr, and any other inorganic or organic salts) or other non-reactive organic or inorganic solutes that drive the formation of an aqueous layer and a substantially immiscible organic layer containing the lactone when the solute is added to the product mixture after reaction. Sodium salts are preferred. Sodium chloride is also preferred. Alternatively, the product mixture may be extracted with a solvent such as supercritical CO2or an alkylphenol, which has the same effect of partitioning (or extracting) the water-soluble products into an aqueous product stream comprising sugars and a small concentration of GVL. Such solvents are included in the term “solute.”

All references to singular characteristics or limitations shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

The processes described herein can be run in batch mode, semi-continuous mode, and/or continuous mode, all of which are explicitly included herein.

The methods described and claimed herein can comprise, consist of, or consist essentially of the essential elements and limitations of the disclosed methods, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in synthetic organic chemistry.

The Process:

The process yields an aqueous solution of carbohydrates, selectively and with minimal degradation of carbohydrate monomers. The resulting aqueous solution can then be used as a carbon source in the manufacture of growth media for microorganisms. The process to generate the aqueous solution of carbohydrates comprises reacting biomass or a biomass-derived reactant with a solvent system comprising (i) an organic solvent selected from the group consisting of beta-, gamma-, and delta-lactones, and combinations thereof, and (ii) at least about 1 wt % water. The solvent system also includes an acid catalyst. The reaction is conducted for a time and under conditions to yield a product mixture wherein at least a portion of water-insoluble glucose-containing polymers or oligomers, or water-insoluble xylose-containing polymers or oligomers, if present in the biomass or biomass-derived reactant, are converted to water-soluble glucose oligomers, glucose monomers, xylose oligomers, xylose monomers, or any combination thereof. To the product mixture is added a non-reactive solute in an amount sufficient to cause partitioning of the product mixture into an aqueous layer and a substantially immiscible organic layer. The product carbohydrates are contained in the aqueous layer. The lactone is present in the organic layer and can be recycled. The aqueous product stream (which comprises water, carbohydrates, and a small concentration of GVL) is then either diluted with water or with minimal medium to yield a growth medium for microorganisms. Optionally, the GVL may be removed from the aqueous product stream. This can be done by any suitable means, such as by distillation, extraction, and the like.

InFIGS. 1A and 1B, fraction carbohydrate concentration results as a function of solvent volume flowed through the reactor depicted inFIG. 4are shown for reaction solvents containing 80 wt % GVL to 20 wt % water mixtures (80/20 GVL,FIG. 1A) and 90 wt % GVL to 10 wt % water (90/10 GVL,FIG. 1B), respectively. (See Examples for full experimental details.) Both solutions contained 5 mM H2SO4(˜0.05 wt %). In both cases, C5 (xylose and xylo-oligomer) and C6 (glucose and gluco-oligomer) fraction concentrations reach maxima between about 160° C. and about 200° C. and then decrease at higher temperatures. Equivalent results obtained with water as a solvent show a significantly different behavior, with glucose concentration continuously increasing with increasing temperature up to 493 K and potentially beyond. SeeFIG. 1C. This result indicates that GVL promotes cellulose deconstruction and hydrolysis with most cellulose hydrolysis occurring below about 210° C. Interestingly, when ethanol is used in the place of GVL, a maximum in C6 concentration was also observed. SeeFIG. 1D. However, overall C6 concentrations were lower when using 80/20 GVL-to-ethanol, suggesting that ethanol acts as an inhibitor rather than as a promoter for cellulose deconstruction.FIG. 1Eis a histogram of the cumulative results using the various solvent systems for comparison purposes.

Unlike water or water ethanol mixtures, lactone/water mixtures leave almost no solids in the reactor. The various fractions in GVL/water contain water-insoluble solids, which precipitate when the fraction is diluted with water and which must, according to the mass balance, be comprised mostly of solubilized lignin. The highest concentrations of water-insoluble solids always occurred in the first fraction and are almost absent after the first 10 fractions (seeFIGS. 5A and 5B). The total mass of water-insoluble solids corresponded to 95% and 84% of the original lignin mass for extraction with 80/20 wt % and 90/10 wt % GVL-to-water, respectively.

Quantitatively, the lactone/water/dilute acid process leads to an increase in overall C5 sugar recovery by 5-20 percentage points and, notably, to a 2 to 4-fold increase (200% to 400% increase) in C6 sugar recovery. SeeFIG. 1F. Also notable is that yields for both C5 sugars and C6 sugars are almost identical for maple wood as compared to corn stover. Again, seeFIG. 1F. This indicates that carbohydrate recovery is independent of the type of substrate used. Furthermore, due to the fractionation concentration profile, the C6 and C5 sugars are better resolved when using the lactone/water solvent system. Despite the increased amount of sugars associated with the GVL/water mixtures, over 80% of both extracted C5 and C6 sugars can be recovered in separate solvent fractions, as contrasted to 75% for water and 65% for ethanol. (These percentages were calculated as illustrated inFIGS. 6A through 6D; see the Examples.) The capability to separate the C5 sugars from the C6 sugars enables using the C5 and C6 fractions in separate and distinct upgrading processes. In addition, given the carbohydrate extraction profile associated with lactone/water systems (i.e., most sugars have been extracted once temperatures around 483-493 K have been reached), carbohydrate concentrations can be increased by decreasing the temperature ramp time without significantly decreasing carbohydrate yields. This is due to reducing the solvent volume flowed through biomass. As shown inFIG. 1E, soluble carbohydrate yields decrease by less than 4% when the temperature ramp time is shortened from 2 hr to 1 hr. Shortening the temperature ramp time to 30 min decreases soluble carbohydrate yield by less than 10% as compared to a 2 hour ramp time.

The data presented inFIGS. 1A through 1Fshow that the lactone/water/dilute acid system promotes cellulose and hemicellulose hydrolysis as contrasted to traditional solvents such as water and water/ethanol mixtures. An important feature of GVL/water solvent systems is that the aqueous phase can be separated from the GVL by addition of salt16. InFIGS. 2A,2B, and2C, carbohydrate recovery in the aqueous phase is shown as a function of NaCl content. (NaCl is exemplary. Any non-reactive salt or solute that drives partitioning of the product mixture into an aqueous layer and an organic layer that contains most of the lactone may be used.) Using simulated carbohydrate feeds in GVL/water mixtures, the data show that there was a tradeoff between obtaining higher aqueous concentrations of carbohydrates at low levels of NaCl addition (e.g., 4% aq) and increased recovery of carbohydrates in the aqueous phase at higher salt levels (10-12% aq). See the Examples for full details. This tradeoff is illustrated inFIGS. 2A,2B, and2C for feeds derived from corn stover. When recovering 40-50% of the sugars in the aqueous phase, the glucose and xylose concentrations can be increased by about 10- to 20-fold for 80/20 GVL/water and 90/10 GVL/water, respectively. The highest carbohydrate concentrations (over 110 g/L) are within range of the highest concentrations obtained without any drying from pretreatment and enzymatic hydrolysis, which range between 150 and 200 g/L depending on the substrate and enzyme loading17,18. When 80-90% and 60% of the sugars are recovered in the aqueous phase from 80/20 GVL/water and 90/10 GVL/water, carbohydrate concentration drops to 25 g/L and 60 g/L, respectively. SeeFIG. 2Afor the 80/20 data andFIG. 2Bfor the 90/10 data. Furthermore, concentrations can easily be increased by removing the first and last fractions from the flow-through process which contained very few sugars. As shown inFIG. 2C, while conserving the fractions that still contain 97% of both C5 and C6 sugars, carbohydrate concentrations can be increased from 25 g/L to 33 g/L simply by discarding low concentration fractions in the 80/20 GVL fractionation product. This 33 g/L solution was used in carbohydrate upgrading experiments to monomers and furans, discussed below.

As discussed earlier, carbohydrate concentrations can also be increased by shortening the temperature ramp time. As shown inFIG. 2C, using 80/20 GVL and a 12% aq NaCl content, carbohydrate concentrations increase to 48 g/L and 72 g/L with a 1 hr and 30 min temperature ramp respectively, with recovery efficiencies of around 90% and 80%.

Recovery was slightly reduced in biomass derived feeds compared to simulated feeds composed of monosaccharides. SeeFIGS. 7A and 7B, and the Examples. This was mainly due to oligomers partitioning more readily in the organic phase than monomers. In addition, NaCl proved to be less soluble in biomass-derived feeds with saturation in 90/10 GVL occurring between about 10 and 12% aq, while NaCl saturation occurred at about 16% aq in the simulated feeds.

As shown inFIGS. 2A,2B, and2C, the carbohydrates recovered in the aqueous phase are a mixture of monomers and oligomers. For biological upgrading, monomers are preferable starting products, especially compared to oligomers with a degree of polymerization greater than two. However, because most of the acid catalyst is recovered in the separated aqueous phase (when using homogeneous acid catalysts such as mineral acids), it offers an acidic aqueous environment that is well suited to oligomer depolymerization19. The data inFIG. 3Ashow that by heating the unmodified aqueous phase separated from the 80/20 GVL product stream, over 95% of total C5 sugars and C6 sugars can be recovered in the form of monomers after treatment at about 140° C. for 100 min. These monosacchamides are ideal substrates for biological upgrading.

Carbohydrates can also be upgraded to useful fuels and chemicals through the furan platform20-22. Xylose and glucose can undergo dehydration to furfural and 5-hydroxy-methyl-furfural (5-HMF), respectively. Both of these species are unstable at high temperatures in acidic environments and benefit from the continuous extraction in an organic phase such as 2-sec-butyl-phenol (SBP) during reaction23. Furthermore, selectivity to 5-HMF from glucose is greatly improved after glucose is isomerized to fructose, which can be catalyzed with a Lewis acid such as AlCl323. Phase modifiers such as NaCl, further promote 5-HMF production by modifying their partition coefficient to the organic phase21-23.

InFIG. 3B, furfural and 5-HMF yields are shown as a function of reaction time at 170° C. from the soluble carbohydrates (monomers and oligomers) recovered from the 80/20 GVL separated aqueous phase. This phase was used unmodified except for the addition of AlCl3and the presence of the SBP organic phase. Yields above 60 and 70% for 5-HMF and furfural, respectively are almost identical to those obtained from glucose and xylose despite the presence of oligomers and other biomass by-products23,24.

Because of the moderate conditions used to produce these sugars and the demonstrated versatility of these biomass-derived carbohydrates (suitable for both biological and chemical upgrading), this present method can be used in any number of biofuel and bioproduct production processes that use glucose, xylose or water-soluble oligomers comprising glucose and/or xylose as reactant or reagent.

EXAMPLES

The following Examples are included solely to provide a more thorough disclosure of the method described and claimed herein. The Examples do not limit the scope of the claimed method in any fashion. The Examples provide the experimental methods by which the results presented in the figures were obtained.

A schematic of the flow-through reaction system used in the Examples is shown inFIG. 4. Corn stover was obtained through the Great Lakes Bioenergy Research Center (GLBRC, Madison, Wis.) and maple wood was obtained from Mascoma (Hanover, N.H.). Their compositions are given in Table 1.

Approximately 2.5 g of biomass were mixed with 5 g of silicon dioxide fused granules (Sigma-Aldrich, St. Louis, Mo.) and placed into the heated zone of the flow-through reactor between two beds of pure silicon dioxide granules separated by quartz wool plugs (Grace-Davison, Albany, Oreg.). SeeFIG. 4. The reactor's heated zone is fitted between two aluminum blocks placed within an insulated furnace (Applied Test Systems, Butler, Pa.). A type-K thermocouple (Omega Engineering, Inc. Stamford, Conn.) was placed at the reactor wall and was used to monitor and control the reactor temperature using a 16A series controller (Love Controls, a division of Dwyer Instruments, Inc., Michigan City, Ind.). Solvent was flowed through the system using an HPLC pump as shown at the top ofFIG. 4(Lab Alliance-brand Series I, Thermo-Fischer Scientific, Waltham, Mass.). Pressure was maintained constant at 300 psi by flowing helium (Airgas, West Chicago, Ill.) in the headspace of the liquid collector through a back-pressure regulator (1500 psi, 10.3 MPa, Tescom, a wholly owned subsidiary of Emerson Process Management, Elk River, Minn.). At the start of the reaction, dry biomass was heated to 423 K in the presence of helium using a 20 min ramp. The temperature was allowed to equilibrate between 423 and 433° C. for 3 min after which solvent was flowed through the biomass at a rate of 2 ml/min while a 2 hr temperature ramp was applied between 433 and 493 K. The resulting flow-through liquid was sampled approximately every 5 min by draining the liquid collector.

A given amount sodium chloride (Sigma-Aldrich) was added to the liquids resulting from flow-through experiments using GVL-H2O mixtures as a solvent in order to separate the aqueous phase. The resulting solutions were repeatedly shaken and sonicated in a sonication bath (FS28, Fisher-Scientific) until no solids were visible. The mixtures were then centrifuged at 4500 rpm for 4 min in a Sorvall ST16 centrifuge (ThermoFisher). The heavier aqueous phase was removed using a syringe and needle to measure its mass, after which both phases were analyzed.

Oligomer depolymerization reactions were carried out in thick-walled glass reactors (5 mm, Supelco, a subsidiary of Sigma-Aldrich, Bellefonte, Pa.) with a magnetic stirrer. Approximately 2.5 g of unmodified aqueous solutions resulting from the aqueous phase separation were placed in the reactors. The glass reactors were heated and stirred using an oil bath at 413 K placed an Isotemp digital stifling hotplate set at 800 rpm (Fisher Scientific). Reactors were stopped at specific reaction times by placing the reactors in an ice slurry.

Aqueous solutions (1.5 g each) resulting from the aqueous phase separation to which 100 mM of AlCl3(Sigma-Aldrich) was added, were contacted with 3 g of 2-sec-butyl-phenol (Alfa-Aesar, a Johnson Matthey Company, Ward Hill, Mass.) in a 10 ml thick-walled glass reactor (Grace Davison). To begin each reaction, the resulting mixture was placed in an oil bath heated with an Isotemp digital stifling hotplate (Fisher Scientific). The hotplate was used to stir a magnetic stir bar in the reactor at 1200 rpm. In the same fashion as in the monomer production experiments, reactors were cooled at specific reaction times by placing the them in an ice slurry.

Aqueous phase, GVL/water and ethanol/H2O were analyzed for glucose, xylose, 5-hydroxymethylfurfural (HMF) and furfural and after 10× dilution in water using a Waters 2695 HPLC system with a Bio-Rad Aminex HPX-87H column and a 5 mM H2SO4aqueous mobile phase flowing at 0.6 ml/min (Waters, Inc., Milford, Mass.; Bio-Rad Laboratories, Inc., Hercules, Calif.). The 2-sec-butyl-phenol phase was analyzed using a Waters 2695 HPLC system with a Zorbax SB-C18 5 μm column (Agilent, Santa Clara, Calif.) using 5 mM H2SO4as the aqueous phase with acetonitrile as the organic modifier. Both systems were equipped with a RI 2414 (refractive index) detector and a PDA 960 (photodiode array) detector (Waters). Sugars were measured using the RI detector while 5-HMF and furfural were measured using the PDA detector at 320 and 230 nm respectively. Oligomers were measured according to the procedure published by the National Renewable Energy Laboratory19using unstirred 10 ml thick-walled glass reactors (Grace-Davison) placed in an oil bath set to 121° C.

Water insoluble solids in GVL/water fractions were measured by diluting the solutions 10 times using water and filtering the resulting mixture using a 0.2 μm nylon filter (Millipore, Billerica, Mass.). The filter was dried overnight in a vacuum oven (Fisher-Scientific) set at 333 K and weighed for recovered solids.

Two simulated feeds (80 wt % GVL to 20 wt % water and 90 wt % GVL to 10 wt % water) were constructed based on corn stover fractionation results. Given amounts of glucose and xylose, equivalent to the amounts of their equivalent monomer and oligomer concentrations in the solutions derived from corn stover were added to the respective solvent. The effect of increasing NaCl concentrations on the aqueous phase separation is shown inFIGS. 7A(80 wt % GVL/20 wt % water) and7B (90 wt % GVL/10 wt % water). The solution containing 80 wt % GVL showed salt saturation at between 16 and 20% aq (i.e., mass fraction with respect to the total amount water present) NaCl while the solution containing 90 wt % GVL showed saturation between 12 and 16% aq NaCl.

7. Growth Media Comprising Biomass-Derived Sugars and Growth of Microorganisms on the Resulting Media:

Overnight cultures ofE. coliwere started from single colonies picked into LB (5 mL) and incubated at 37° C. and 30° C. with 250 rpm shaking. The overnight cultures were used to inoculate test media (2 mL) to an OD600(optical density at 600 nm) of 0.01. These cultures were then incubated as described below. The final OD600of each culture was measured after 24 hours of incubation.

A single colony ofL. caseiwas picked and transferred to 5 ml of rich MRS broth (de Man, J. D.; Rogosa, M.; Sharpe, M. E. (1960). “A Medium for the Cultivation of Lactobacilli,”. J Appl Bact 23:130-135) and incubated at 37° C. and 250 rpm overnight. The overnight culture was used to inoculate test media (2 ml) to an OD600of 0.01. The test media were then incubated for 24 hours under the same conditions. The final OD600was taken at the end of the 24-hour incubation period.

For the aerobic test media growth experiments, an overnight culture was used to inoculate test media (2 mL) to an OD600of 0.01. The test cultures were then incubated for 40 hours under the same conditions. A final OD600was measured after the 40-hour incubation period.

For the anaerobic growth experiments and ethanol production studies, a single colony ofS. cerevisiaePE2 was transferred to 5 mL of YPD broth and grown anaerobically overnight at 30° C. and 250 rpm. The culture was used to inoculate 5 mL of the anaerobic media in 16×125 hungate tubes capped with rubber septa. These were then incubated to an OD600of 0.02. The hungate tubes were then sealed and sparged with N2for 3 minutes. The sparged tubes with inoculum were incubated at 30° C. and 200 rpm. In the ethanol production studies, OD600measurements were taken every 2 hours and 100 μL of solution was extracted via syringe for HPLC analysis. HPLC samples were filter sterilized into HPLC vials and stored at −80° C. until processed.

Test media forE. coliandP. putidawere made using MOPS minimal salts and either varying the weight percent of NaCl and GVL or varying dilutions of the aqueous product stream in water.L. caseitest media were made using Chemically Defined Media forL. caseiand yeast aerobic test media were made with Yeast Nitrogen Base (6.7 g/L; Difco, a wholly owned subsidiary of Becton Dickinson and Company, Franklin Lakes, N.J.). Yeast anaerobic media were made with Yeast Nitrogen Base and supplemented with Tween 80 (0.4 wt %) and amino acids (50 mg/L).

7.2 Growth of Microorganisms in Biomass-Derived Media:

The aqueous product stream described herein above (comprising sugars and GVL) was used to make growth media for the microorganisms described in Sec. 7.1. The raw aqueous product stream comprised a modest concentration of sugar, roughly 50 g/L. The aqueous product stream was used as the base for formulating media to grow various types of microorganisms. The first step was to determine what level of salt and/or GVL would be tolerated by any given type of microorganisms. Therefore, the raw aqueous product stream from the biomass processing was diluted into an appropriate laboratory minimal media for each organism. See Sec. 7.1. The microorganisms were then grown in increasing dilute solutions of the aqueous product stream. The results are shown inFIG. 8.

FIG. 8is a histogram depicting the growth ofE. coli(gram negative),L. casei(gram positive),S. cerevisiaePE2 (yeast),S. passalidarumNN245,S. stipitis7124, andS. stipitis6054 in various dilutions of model media comprising GVL, NaCl, glucose, and xylose dissolved in the stated ratios with laboratory minimal medium. As shown in the figure, at a dilution of 1:4, only minimal growth was observed. At a dilution of 1:8, all of the tested microorganisms exhibited some growth and some of the organisms displayed very robust growth—matching that of the minimal medium.FIG. 8also shows thatL. caseiandS. cerevisiaeare capable of growing in 1:6 dilutions whileE. coliand the other yeast strains tested require a 1:10 dilution for robust (>75% of baseline) growth.

The next step was to investigate the sensitivity of various microorganisms to salt concentration and GVL concentration. This was accomplished by measuring the growth ofE. coliand yeasts in minimal media supplemented with known concentrations of GVL or NaCl under aerobic conditions. The results are depicted inFIGS. 9 through 13.FIG. 9depicts the growth characteristics ofE. coliTY05 in response to increasing concentrations of GVL.FIG. 10depicts the growth characteristics ofS. cerevisiaein response to increasing concentrations of GVL.FIG. 11depicts the growth characteristics ofS. cerevisiaePE2 in response to increasing concentrations of NaCl.FIG. 12depicts the growth characteristics ofS. passalidarumNN245 in response to increasing NaCl concentration.FIG. 13depicts the growth characteristics ofE. colistrain MG1655 (ATCC 47076) in response to increasing concentrations of NaCl.

The data inFIGS. 9-13suggest that GVL concentration has the stronger impact on cell growth as compared to NaCl. These tests are being repeated for yeast under anaerobic conditions. (Data not shown.) The data inFIGS. 9-13also show that growth media can be formulated using the biomass-derived sugars as obtained by the process described herein. Notably, bothE. coliand yeast displayed very significant growth at GVL concentrations of about 2% (wt/vol) and less. This is typically about the upper limit for the GVL concentration found in the raw aqueous product stream derived from biomass. Of course, for organisms more sensitive to GVL, the GVL may optionally be removed (or its concentration reduced) in the aqueous product stream that is incorporated into the medium.

7.3 Production of Fatty Acids from Biomass-Derived Sugars:

The possibility of producing small molecules via bioconversion of biomass-derived sugars using engineered microbes was then explored. This concept was tested with a strain ofE. coli(TY05) that has been engineered to produce lauric acid, a precursor to diesel fuel and many commodity chemicals. (Youngquist, J. T., R. M. Lennen, D. R. Ranatunga, W. H. Bothfeld, W. D. Marner, and B. F. Pfleger. 2012. “Kinetic modeling of free fatty acid production inEscherichia colibased on continuous cultivation of a plasmid free strain,” Biotechnol Bioeng 109:1518-1527)E. coliTY05 was grown in minimal media formulated with a 1:10 dilution of the biomass-derived aqueous product stream. The same strain was also grown in MOPS minimal media that was formulated to be phosphate-limiting (unlike traditional recipes which are often carbon-limiting). The results are depicted inFIG. 14.FIG. 14is a graph depicting the aerobic growth ofE. coliTY05 in laboratory minimal medium (♦) versus the GVL-containing medium derived from biomass as described herein (▪). The growth rate ofE. coliwas slightly slower in the GVL-biomass medium, but the GVL-biomass cultures reached a higher final OD600. This result indicates that the GVL-biomass medium contained additional nutrients (phosphate) as compared to the laboratory medium, which was phosphate limited.

Lauric acid (fully saturated, C12 carbon, i.e. dodecanoic acid) and myristic acid (fully saturated, C14 carbon chain, i.e., tetradecanoic acid) were extracted from samples of each culture after 24 hours of growth.FIGS. 15 and 16depict fatty acid yield fromE. coliTY05 using the growth medium containing biomass-derived sugars.FIG. 15depicts the results when theE. coliwere grown in minimal medium supplemented with a 1:10 dilution of biomass aqueous product stream.FIG. 16depicts the results when theE. coliwere grown in MOPS minimal medium supplemented with a 1:10 dilution of biomass aqueous product stream. (MOPS medium is available commercially from Teknova, Inc., Hollister, Calif. 95023.)FIG. 16also depicts the yield of lauric and myristic acids when theE. coliTY05 were grown in carbon-limited minimal medium and phosphate-limited minimal medium. In all instances fatty acid production was decreased in the GVL-biomass medium as compared to the controls, but significant amounts of the fatty acids were produced in the GVL-biomass cultures.

Similar results were found when the yields of fatty acid from sugars were plotted. SeeFIG. 17. InFIG. 17, the values for the growth ofE. coliTY05 (cell density at OD600; ) glucose concentration (♦), and xylose concentration (▪) are plotted simultaneously over time using the biomass-derived growth medium described herein.E. coliwas able to consume both the glucose and xylose found in the biomass, with glucose consumption occurring before xylose consumption.

7.4 Production of Ethanol from Biomass-Derived Sugars:

Ethanol is a major component of transportation fuels and a potential replacement for gasoline in the personal transportation sector. Thus, experiments were conducted to gauge the ability to use the GVL-biomass medium described herein to produce ethanol via yeast fermentation. A growth medium was made by combining a 1:6 ratio of the aqueous product stream containing biomass-derived GVL and carbohydrates with minimal medium suitable for yeast growth. Yeast (S. cerevisiaePE2) cultures were grown anaerobically for 30 hours as described above. The yeast cells consumed the glucose in the medium and converted it to ethanol at near quantitative yield. SeeFIG. 18. The figure is a graph depicting superimposed values for the growth ofS. cerevisiaePE2 (cell density at OD600; ), glucose concentration (♦), xylose concentration (), and ethanol concentration (▴) over time using the biomass-derived 1:6 growth medium described herein.

As shown inFIG. 18, the yeast were unable to consume the xylose (▪) in the medium. This can be corrected by engineering a pathway for the catabolism of xylose into the yeast. A number of such xylose-fermenting strains are known. See, for example, Lee S M, Jellison T, Alper H S (August 2012) “Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeastSaccharomyces cerevisiae,” Appl Environ Microbiol. 78(16):5708-16; Ha et al (2011) “EngineeredSaccharomyces cerevisiaecapable of simultaneous cellobiose and xylose fermentation,” PNAS 108(2): 504-509; Shao et al. (online 12 Dec. 2008) “DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways,” Nucleic Acids Res. 2009 February; 37(2): e16. This example clearly shows that the GVL-biomass medium described herein can be used to grow yeast and to use the cultures for the production of ethanol (or other desirable, value-added chemicals that can be formed via fermentation).

REFERENCES CITED