CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims the benefit of U.S. Provisional Application No. 61/898,158, filed on October 31, 2013, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[02] The invention generally relates to methods for recovering bioproducts from a solution, in particular absorption by a polymeric resin, release of the bioproducts from the polymer by a triggering agent or event, and optional recycling of the polymer resin.

BACKGROUND

[03] Purification of n-butanol from fermentation processes has a long history, nearly as long as the initial discovery of the Acetone -Butanol-Ethanol (ABE) process by Louis Pasteur in 1862. What have evolved over time are some alternative isolation and purification strategies. One that emerged as a lower energy process compared to distillation is a process that involves some kind of sparging or stripping using an inert carrier stream. This can be an inert gas such as carbon dioxide, or simply steam, in which case one takes advantage of the butanol-water azeotrope. This azeotrope can then phase separate into an organic phase, which can then be decanted away from the bottom water layer, which is returned for use as the sparging "solvent." In some cases, the sparging stream might be a gas at ambient temperature and then simple re-evaporation of the gas leaves behind the liquid butanol.

[04] Recently, in patent application WO 2007/149399 A2, four general methods of purifying butanol from fermentation broth were summarized as related to zso-butanol, although most of the methodology is applicable to both iso- and n-butanol. It should be noted that each of the butanol isomers, namely n-, iso-, sec-, and t-butanol, will behave somewhat differently in each of the recovery/purification strategies discussed below.

[05] The first method takes the organic layer containing butanol {e.g., the stripping method noted above), other products, and some water, and is further purified by fractional distillation. The first fractions are low boiling co-products like acetone, followed by azeotropic (butanol-water) removal of the water, and this is followed by collection of the purified butanol, typically at purity levels of >98%, depending on the fractionation column. The butanol-water azeotrope is recycled and must be distilled again, utilizing process time and energy. Although this process leads to very pure butanol, it is a very time and energy consuming process.

[06] In method two, the butanol can be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent. This method can require large amounts of a flammable co-solvent and can present difficulties with scale. Furthermore, handling of the liquid organic extractant presents environmental and health hazards as well as the requirement for efficient separation.

[07] In method three, a distillation in combination with adsorption can be used to sequester butanol from the fermentation medium. For this process the fermentation broth containing the butanol is sparged near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al,

"Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover," Report NREL/TP- 510-32438, National Renewable Energy Laboratory, June 2002). This can require large amounts of adsorbent since distilled butanol-water mixtures typically have water content 15-20 wt-%.

[08] A fourth approach is to combine a sparging process to first remove the butanol from the reactor and solids, followed by a pervaporation process. As above, the fermentation broth containing the butanol (-1.5-2 wt-%) is sparged from the bioreactor at approximately azeotropic composition (or some butanol saturated inert gas and/or hydrocarbon) and the remaining water is removed by pervaporation, for example, through a hydrophilic membrane (Guo et al. (2004) J. Membr. Sci. 245: 199-210). Typically pervaporation processes are difficult to operate at large scale and can be extremely costly on a commercial scale.

[09] A fifth approach, described in U.S. Patent No. 8,283,505, which is unique to iso- butanol, is removal of this bioproduct from the fermentation broth by application of reduced pressure in the fermentation vessel followed by removal of the iso-butanol. This approach is specific to zso-butanol and thus has severe limitations and application in the practice of bioproduct removal/recovery from dilute aqueous solutions. [10] Product toxicity in fermentations results in low titers and productivities, creating a barrier for commercialization. Low titers have significant influence on the economics of product recovery as well as on the hydraulic load. Reduced productivities increase the risk associated with upfront capital expenditure in building commercial scale production plants. Although in situ product removal strategies such as gas stripping and extractive liquid- liquid fermentations have been widely studied to improve the effective titer and

productivity in the case of ABE fermentations, a requirement of large amounts of biocompatible gas and liquids provides a barrier to process scalability due to low partition coefficients. In addition, losses associated with extractant separation and recycling due to emulsion formation warrant the use of additional unit operations such as centrifugation for effective and efficient product recovery. With these severe limitations, large-scale application is technically difficult, very energy intensive, and very costly from both an equipment and plant operations point of view.

[11] Heady and Frankiewicz disclosed the use of granular activated carbon to extract n- butanol from an active fermentation process (U.S. Patent No. 4,520,104). In this process use of granular activated carbon avoided interference with the fermentation process and provided enhanced growth of the bacteria and an overall increase in sugar consumption for the ABE fermentation. Recovery (i.e., "desorbing" or desorption) of the n-butanol was disclosed as a process involving exposure of the butanol soaked granular carbon to organic vapors such as acetone. This requires a complicated process and/or isolation of the granular carbon for recovery of the butanol. U.S. Patent No. 4,520,104 does not disclose a recovery efficiency of the butanol from the fermentation process nor capabilities to produce pure n- butanol.

[12] Qureshi et al. (2005) Bioprocess Biosyst. Eng. 27:215-222 later described the use of polymeric materials for a low energy pathway providing n-butanol separation from an ABE fermentation process. These authors disclosed that polymeric materials can be used to adsorb n-butanol from the fermentation broth and then release the butanol by thermally heating the polymeric material. This process requires significant heating of the polymer to desorb the n-butanol from the polymer host. Furthermore, the partition coefficient of the n- butanol within the polymeric material is quite low and would require large and

unreasonable amounts of polymer to effectively increase fermentation titer and n-butanol recovery. [13] More recently, MIT workers (Nielson & Prather (2009) Biotech. Bioeng.

102(3):811-821) explored use of a polymeric material to adsorb n-butanol from

fermentation broth. This resulted in higher than expected overall concentration of the n- butanol in the fermentation medium {e.g., 22 g/L) and as for Qureshi et al, discussed above, the n-butanol was released by thermal heating and/or the application of reduced pressure. Both the Qureshi and Nielsen methods disclose recovery of n-butanol from a fermentation medium, requiring a separate, energy consuming step, i.e., releasing butanol from the polymer by application of external heating. Although the energy consumed is less than with traditional distillation methods, there remains a significant "energy penalty" for the thermally induced release of the n-butanol from the polymeric material.

[14] There is a need for a material, process, and method that can provide isolation of bioproducts such as butanol from a fermentation broth and that is: 1) very low in energy consumption, 2) permits an increase in overall fermentation batch bioproduct {e.g., butanol) titer and productivity, 3) provides bioproduct {e.g., butanol) isolation without application of solvents, heat, or vacuum, and/or 4) provides an efficient recycling of the polymeric material over many cycles of use. A method that provides any of these advantages would be an improvement over the currently available methods in the art.

BRIEF SUMMARY OF THE INVENTION

[15] A method is provided that involves the use of solid extractants, such as porous polymeric materials (resins) for in situ absorption and/or adsorption of bioproducts from dilute aqueous solutions. Simultaneous fermentation and removal of bioproducts {e.g., butanol) through absorption into the polymeric material can result in increased fermentation titer and productivity. For example, in some embodiments, if a 20% loading of a solid extractant that possesses a partition coefficient (Ka) of 6 is provided, then an overall titer and productivity of 30 g/L and 1.2 g/L/h, respectively, may be obtained {see, e.g., FIG 1 and FIG 2). This is illustrated schematically in the reactor diagram displayed in FIG 3. At the end of the fermentation, the polymeric material may be treated with a chemical trigger {e.g., low or high pH solution), which induces a release of the bioproduct {e.g., butanol) without the need for thermal heating (FIG 4). Thus, a highly efficient and low energy method for bioproduct {e.g., butanol) isolation is disclosed herein. In addition, the ease of separation {e.g. , using a simple mesh or other filtration method) and regeneration of the solid extractant using this simple chemical trigger has significant operational advantage over liquid and solid extractants that lack a low-energy mechanism to trigger release of the bioproduct. The optional use of anti-fouling coatings on the polymeric materials used in the methods of the invention provides additional utility to the process in certain embodiments, such as embodiments in which the polymeric material is used to extract bioproducts in situ during a microbial fermentation process. In some embodiments, an anti-fouling coating may reduce or eliminate microbial {e.g., bacterial) growth or adherence on the polymeric material, including but not limited to formation of a biofilm. In some embodiments, an enzyme coating may be included to convert absorbed and/or adsorbed bioproducts to other products of interest.

[16] In one aspect, a method is provided for isolating at least one bioproduct from an aqueous solution, including: contacting an aqueous solution that comprises the bioproduct with at least one polymeric material {e.g., at least one organic polymer resin), wherein the bioproduct is absorbed by said polymeric material; and contacting said polymeric material with a trigger solution comprising at least one chemical reagent, thereby releasing said bioproduct from said polymeric material. In some embodiments, the polymeric material is separated from the aqueous solution prior to contacting with the trigger solution.

[17] In some embodiments, contacting the polymeric material with the trigger solution results in physical and/or chemical change in the polymeric material, thereby effecting release of the bioproduct from the polymer.

[18] In some embodiments, the polymeric material includes an organic polymer resin that includes at least one Bronsted acid functional group. In some embodiments, the polymeric material includes an organic polymer resin that contains at least one Bronsted base functional group. In some embodiments, the polymeric material is crosslinked.

[19] In some embodiments, the aqueous bioproduct-containing solution is a fermentation broth. In some embodiments, the contacting of the fermentation broth with the polymeric material is conducted during a microbial fermentation in which the bioproduct is produced. In some embodiments, the bioproduct production titer is higher in the presence of the polymeric material than in the absence of the polymeric material. In some embodiments, the microbial fermentation comprises batch, fed-batch, continuous suspended or

immobilized, or extractive fermentation. In some embodiments, the polymeric material may be present throughout the fermentation or added at a particular stage, for example, pre- stationary or stationary phase. In some embodiments, the microbial fermentation includes a mixed culture fermentation. In some embodiments, the fermentation includes co-cultures, mixotrophic, heterotrophic, chemotrophic, and/or mixed species cultures. In some embodiments, the microbial fermentation is conducted in a packed bed or fluidized bed bioreactor. In certain non-limiting embodiments, fermentation may be conducted as described in U.S. Patent Nos. 8,460,906 or 8,497, 105.

[20] In some embodiments, contacting of the fermentation broth with the polymeric material is conducted in a high cell density seed reactor that supplies inoculum for a larger scale fermentation.

[21] In some embodiments, the bioproduct(s) isolated by the method include(s) at least one solvent. In some embodiments, the at least one solvent includes butanol, selected from n-butanol, zso-butanol, sec -butanol, tert-butanol, and any combination thereof.

[22] In some embodiments, at least one co-product is produced during fermentation and absorbed by said polymeric material or in an additional polymeric material(s) for isolation of said co-product(s). For example, in an embodiment of a method for isolation of fermentatively produced butanol, co-product(s) may be acetone and/or ethanol. In some embodiments, co-product(s) may include vitamin(s), amino acid(s), and/or organic acid(s).

[23] A bioproduct isolated by the methods herein may be in the form of a gas, solid, or liquid.

[24] In some embodiments, the aqueous solution is contacted with the polymeric material for 30 sec to 6 h.

[25] In some embodiments, the trigger solution includes a pH of 0 to 14. In some embodiment, the trigger solution includes a pH of -1 to 14. In other embodiments, the trigger solution includes a pH of 7 to 14. In further embodiments, the trigger solution includes a pH of -1 to 7.

[26] In some embodiments, the polymeric material may be in the form of beads. In some embodiments, the polymeric material may be in the form of mixed beads (e.g., beads of different polymeric materials) to recover multiple products or a combination of a product and another chemical, e.g., co-product. In some embodiments, the polymeric material may be in the form of circular, rod, conical, or other morphologies or shapes, suitable for the application of use.

[27] In some embodiments, the polymeric material has a partition coefficient with respect to the bioproduct (e.g., the bioproduct has a partition coefficient between the polymeric material and the aqueous medium) of about 1.5 to about 10, about 1 to about 10, about 1 to about 100, about 10 to about 50, or about 50 to about 100 (e.g., favoring absorption of the bioproduct in the polymeric material over the aqueous medium by a factor of between about 1.5 to about 10, on an equal-weight basis) prior to addition of the trigger solution. In some embodiments, the polymeric material has a partition coefficient with respect to the bioproduct (e.g., the bioproduct has a corresponding partition coefficient between the polymeric material and the aqueous medium) of about 0.001 to about 1000, about 0.1 to about 1 , about 0.01 to about 1 , about 0.01 to about 0.5, or about 0.5 to about 1 after addition of and in the presence of the the trigger solution.

[28] In some embodiments, the bioproduct is released from the polymeric material into a second solution (e.g., aqueous solution), wherein the method further includes isolating the bioproduct from the second solution. For example, the bioproduct may be released into an aqueous or organic medium or solution, for example, from which the bioproduct may be further separated, for example, via distillation, centrifugation, or another separation method.

BRIEF DESCRIPTION OF THE DRAWINGS

[29] FIG. 1 Theoretical prediction of the effective titer (aqueous + organic) as a function of extractant (polymeric material) loading with an effective partition coefficient, IQ= 6.

[30] FIG. 2 Theoretical prediction of the effective productivity as a function of extractant (polymeric material) loading with an effective partition coefficient, IQ =6.

[31] FIG. 3 Flow diagram of application of polymeric beads for in- situ extraction of butanol followed by its recovery using a pH trigger.

[32] FIG. 4 Block diagram showing use of a pH trigger for butanol recovery from polymeric beads, trigger solution is an aqueous Bronsted acid solution.

[33] FIG. 5 Block diagram showing the use and recycle of the polymeric resin absorbant for recovery of a bioproduct.

DETAILED DESCRIPTION

[34] Methods and systems are provided herein for low energy recovery of one or more bioproduct(s) from a solution, such as a fermentation broth, including absorption with polymeric resin, release of the bioproduct(s) from the resin by a triggering agent or event, and optional recycling of one or both of the polymeric absorbent and the solution (e.g. , fermentation broth) from which the bioproduct(s) have been removed. In some embodiments, absorption of bioproduct(s) by the polymeric resin during fermentative bioproduct production is accompanied by an increase in fermentative bioproduct production, e.g., an increase in bioproduct titer, yield, and/or productivity.

[35] In the methods and systems disclosed herein, bioproduct(s) produced using at least one chemical or biochemical conversion technique may be recovered from a solution {e.g., an aqueous solution, such as a fermentation broth) with at least one polymeric material, with release of the bioproduct(s) controlled by activation of a trigger. In particular, the bioproduct(s) are first absorbed within the polymeric matrix and then upon use of a specific trigger event are released {e.g., at high concentration), with application of no or

substantially no thermal energy, thus representing an extremely chemically and energy efficient method for isolation and recovery of bioproducts from mixtures which are often very complex. In some embodiments, the trigger event includes a change (i.e., increase or decrease) in pH. In some embodiments, the methods disclosed herein can be used to effect butanol {e.g., n-, iso, sec-, or t-butanol, or a combination thereof) extraction from a fermentation medium using a polymeric resin {e.g. , polymeric resin beads) followed by recovery of the butanol from the resin using a pH trigger, providing an energy efficient means of recovering the butanol product.

[36] "Bioproduct" refers to any substance of interest produced biologically, i.e., via a metabolic pathway, by a microorganism, e.g., in a microbial fermentation process.

Bioproducts include, but are not limited to solvents {e.g., butanol, acetone, ethanol), biomolecules {e.g., proteins {e.g., enzymes, polysaccharides), organic acids or their salts {e.g., formate, acetate, butyrate, propionate, succinate), alcohols {e.g., methanol, propanol, isopropanol, hexanol, n-butanol, 2-butanol, z ^' so-butanol, t-butanol), fatty acids, aldehydes, lipids, long chain organic molecules (for example, for use in surfactant production), vitamins, and sugar alcohols {e.g., xylitol).

[37] "Fermenting" or "fermentation" refers to the biological conversion of a carbon source into one or more bioproducts {e.g. , acetone, butanol, ethanol) by a microorganism. Fermentation may be aerobic or anaerobic. Anaerobic fermentation takes place in a medium or atmosphere that is free or substantially free of molecular oxygen.

[38] "Titer" refers to amount of a substance produced by a microorganism per unit volume in a microbial fermentation process. For example, titer of a bioproduct may be expressed as grams of the bioproduct produced per liter of solution. [39] "Yield" refers to amount of a product produced from a feed material (for example, sugar) relative to the total amount that of the substance that would be produced if all of the feed substance were converted to product. For example, yield of a bioproduct may be expressed as % of the bioproduct produced relative to a theoretical yield if 100% of the feed substance (for example, sugar) were converted to the bioproduct.

[40] "Productivity" refers to the amount of a substance produced by a microorganism per unit volume per unit time in a microbial fermentation process. For example, bioproduct productivity may be expressed as grams of the bioproduct produced per liter of solution per hour.

[41] "ABE fermentation" refers to production of acetone, butanol, and/or ethanol by a fermenting microorganism.

[42] The term "culturing" refers to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or solid medium.

[43] "Solvent" refers to a liquid or gas that is capable of dissolving a solid or another liquid or gas. Nonlimiting examples of solvents produced by microorganisms include butanol, acetone, and ethanol.

[44] Use of a solid extractant such as a polymeric resin for bioproduct recovery has several operational advantages for an industrial process. First the solid material may be selected to provide ease of separation, for example, by using a simple screen or filtration method. Secondly, polymeric materials may be selected to have low toxicity and ease of handling by the operators. Thirdly, provided a polymer with the desired physical properties (e.g. , as disclosed herein) is used, full regeneration and recycling of the solid extractant is possible and this lowers cost and provides an environmentally sound approach to bioproduct isolation. The methods disclosed herein provide for isolation of a bioproduct from a solution, such as an aqueous solution (e.g., fermentation broth), with low energy input and with optional recycling (re-use) of the solid extractant for further bioproduct extraction. In some embodiments, the solid extractant may be re -used at least once to 1000 times or more.

[45] In some embodiments, the methods disclosed herein may be conveniently carried out by contacting a polymeric resin with a dilute solution (e.g., an aqueous solution, such as a fermentation broth) containing at least one bioproduct of interest, for example, at a concentration of about 0.01 % (w/w) to about 15 % (w/w). In some embodiments, the at least one bioproduct includes at least one solvent, for example, butanol (e.g., n-, iso-, sec-, or t-butanol, or a combination thereof), acetone, ethanol, or a combination thereof. The bioproduct(s) in the solution may be absorbed within the polymeric resin and adsorbed on the surface of the resin. In some embodiments, the polymeric resin contacts a fermentation medium in situ {i.e., during fermentative bioproduct production), absorbing and adsorbing the bioproduct(s) as the fermentation proceeds.

[46] The bioproduct(s) of interest is (are) desorbed from the polymeric matrix {i.e. , released) via a "triggering" agent or event. Typically, a chemical release trigger is used. In some embodiments, the release trigger does not include or substantially does not include thermal energy {e.g. , the release trigger does not include thermal energy as the sole release mechanism).

[47] In some embodiments, the release trigger may include a change in pH {i.e., increase or decrease in pH).

[48] In other embodiments, the release trigger may include an oxidation or reduction event. For example, at least one redox active organic or inorganic moiety may be attached to the solid extractant. The oxidation or reduction of the redox moiety induces a bioproduct-affinity change and thus releases {i.e., effects desorption of) the bioproduct. In some embodiments, the complementary process (oxidation or reduction) versus the process that was used for bioproduct release may be used to regenerate the extractant, which may be optionally reused for extraction of further bioproduct. An exemplary, non-limiting embodiment of oxidation-induced bioproduct desportion, and extractant regeneration, is provided below. In other embodiments, reduction-induced bioproduct desorption and oxidation-induced extractant re eneration may be used.

butanol absorped in butanol debsorption from

extractant-resin extractant-resin

reduction reagent In this illustrative example, P stands for the polymeric material that supports the redox active moiety, and phenothiazine us used as the redox active chemical moiety.

[49] In other embodiments, the release trigger may include the use of Lewis acids or Lewis bases capable of a reversible reaction with the extractant resin. For example, a Lewis acid or Lewis base may form a complex with the extractant resin, which may induce a change in ionic character of the extractant resin. This change in ionic character may induce release of the bioproduct. Nonlimiting examples of such Lewis acid and base reactions include use of heteroatoms such as nitrogen and phosphorus for creating Lewis base sites of action or boron and aluminum for creating Lewis acid sites of interaction. In some embodiments, removal of the "triggering" Lewis acid (or base) is accomplished by treatment with a stronger corresponding Lewis base (or acid) than the Lewis base (or acid) site incorporated into the extractant resin, and/or repeated treatment of the "triggered" material with excess Lewis base (or acid), even if not stronger, until the "triggering" Lewis acid (or base) is removed from the "triggered" extractant resin.

[50] In some embodiments, the trigger is a gas phase trigger, for example, a gas that has better absorption and/or adsorption onto the polymeric material than the bioproduct. In some embodiments, the gas is a hydrophobic gas, for example, butane or hexane. In some embodiments, the gas may be applied under high pressure to displace liquid bioproduct from the polymeric material.

[51] In some embodiments, the polymeric resin is separated from the solution (e.g., aqueous solution, such as fermentation broth), for example, by physical means (e.g., filtration, decantation, or the like), prior to desorption of the bioproduct(s) with the trigger.

[52] In other embodiments, the trigger can be added to the solution (e.g. , fermentation broth) that contains the polymeric matrix, and the bioproduct is released into the solution. The released bioproduct along with bioproduct that was not physically absorbed by the polymeric resin, if any, can be isolated using methods known to those skilled in the art of bioproduct isolation and purification, such as sparging or other techniques.

[53] Polymeric resins suitable for use in the methods disclosed herein have an affinity for at least one bioproduct of interest that is present in the solution (e.g., dilute aqueous solution, such as fermentation broth). "Affinity" of a polymeric matrix for a bioproduct herein may be characterized by the partition coefficient between the aqueous solution and polymeric resin for the bioproduct (e.g., partition coefficient of the bioproduct between the polymeric resin and the aqueous solution).

[54] A partition coefficient can be defined in general terms by the following equation:

Partition Coefficient = fe ^of Bioproduct in Resin] [g of Solution]

X

[g of Bioproduct in Solution] [g of Resin]

[55] For example, given an equal weight of an illustrative polymeric resin and aqueous solution, where the polymeric resin has a partition coefficient of 10: 1 for a bioproduct of interest, it follows that the resin would have ten times the mass of bioproduct absorbed versus the mass of bioproduct that remains in solution at equilibrium. In some

embodiments, polymeric resins useful in the methods disclosed herein have partition coefficients of at least about 1.5 for the bioproduct of interest versus the bioproduct- containing solution (e.g., fermentation broth). Partition coefficients of about 4 or greater between the resin and the bioproduct-containing solution are a preferred embodiment of the present methods. In some embodiments, a polymeric resin may exhibit different partition coefficients with respect to absorption of different bioproducts of interest. In some embodiments, the trigger that releases the bioproduct from the polymer may alter the partition coefficient. The change in partition coefficient may be different for each absorbed bioproduct when two or more bioproducts are absorbed in the polymeric material.

[56] The base polymeric material can be formed using one of many polymerization methods known to those skilled in the art of preparing polymeric resins. In some embodiments, polymers that can be used in the present methods herein include, but are not limited to, at least one polymer prepared by chemically altering a polystyrene, polyacrylate, polyamide, polyester, poly-p-phenylene, polyvinyl alcohol, or copolymers thereof. The chemical modification of the base polymer structure (e.g. , a polystyrene) may be selected so as to enhance the partition coefficient (i.e., affinity) for the bioproduct. This modification of the polymer structure can be done post polymerization or may be done during the manufacture of the resin by using one or more comonomers that alter (i.e., tailor) the affinity of the resin to the targeted bioproduct. Other organic resins known to those experienced in the art of polymer synthesis may be used provided they are not soluble in a solution in which the bioproduct of interest is soluble (e.g. , water or fermentation medium) and provide sufficient affinity for at least one bioproduct of concern. [57] In some embodiments, the polymeric resin may be cross-linked, for example, at levels of about 0.1% to about 10% (mol-%) to afford a mechanically robust material.

Modified polystyrene resins with 1 mol-% cross-linking (i.e., from copolymerization of divinylbenzene) represent just one example. Other examples are the cross-linked polyacrylamides that are often used as column chromatographic supports. Cross-linking provides mechanical strength and water insolubility to an organic resin, hence,

advantageously facilitating separation from an aqueous solution or stream. Furthermore, cross-linked polymeric resins provide a micro- and/or macroporous environment that favors absorption of the bioproduct from the aqueous solution.

[58] In some embodiments, a polymeric resin that is suitable for use in the methods disclosed herein may be constructed from monomers and/or chemically modified so that it possesses 1) a high affinity for at least one bioproduct in the dilute solution, and 2) at least one chemical functional group that can serve as a Bronsted acid or base functional group. The absorbed bioproduct may be released from the resin by adding one or more triggering agent(s) which changes the pH of the solution that contains the resin with absorbed bioproduct(s), thus altering the partition coefficient of the resin relative to the solution against which it is equilibrated, and reducing the relative affinity of the resin for at least one of the bioproduct(s). In some embodiments, the polymeric resin contains a Bronsted acid functional group with a pK _a in the range of about -5 to about +14. In some embodiments, the resin contains a Bronsted base with a conjugate acid pKa in the range of about -5 to about +14. In some embodiments, the polymeric resin contains at least about 0.1 mequiv of Bronsted acid or base per gram of resin. In some embodiments, the polymeric resin contains no more than about 1000 mequiv Bronsted acid or base per gram. An exemplary range of Bronsted sites is from about 1 to about 100 mEq/g, for example, about 3 to about 20 mEq/g.

[59] Illustrative, non-limiting examples of polymers containing Bronsted acid or base sites suitable for use in the methods herein include:

Bronsted acid Bronsted base [60] To tailor the affinity for at least one bioproduct of interest, the polymeric resin structure may be formulated to provide a balanced and favorable chemical interaction (e.g., balance of functional groups formulated or optimized to interact with the target product(s) of interest). In broad terms this can be described in terms of the partition coefficient. More specifically, the bioproduct should have an affinity to reside within the polymeric microstructure rather than in the solution. For example, for a partition coefficient of greater than 1.5, placing 10 g of polymeric resin in contact with a water solution (10 mL) containing 10 g of a target bioproduct should result in more than 6 g of bioproduct being absorbed in the polymer resin. In some embodiments of this example, at least about 6 g but less than about 10 g of the bioproduct is absorbed by the polymeric resin. In some embodiments, the direct measure for absorption in the polymeric resin is an equal and corresponding decrease in concentration (e.g., titer) of the bioproduct observed in the solution. In some embodiments, the polymeric resin has a partition coefficient greater than about 1.5, for example, about 4 to about 15.

[61] Using butanol as an example of a target bioproduct, polymeric resin structures may be tailored to possess a "butanol-philic" molecular framework. This means creating a balance of sites with hydrocarbon character (e.g., alkyl chains), dipolar functional groups (e.g., amides), and sites capable of hydrogen bonding (e.g., hydroxyl groups) that best attract the target bioproduct. Butanol has both a hydrogen bonding site and significant amount of non-polar structure (butyl carbon chain). This matching of chemical

functionality of the polymeric resin to the target bioproduct is what produces a favorable partition coefficient ( e.g., >1.5). The ultimate capacity of the polymeric resin to absorb the bioproduct is controlled by the affinity for the bioproduct and the ability of the polymer resin to accommodate the bioproduct. In some embodiments, polymeric resins used in the methods described herein can absorb as little as 0.01 and up to 1000 times their weight of the bioproduct.

[62] In some embodiments, the affinity for the bioproduct in the resin is controlled by the chemical architecture (e.g. organic functional groups) and ability to absorb the bioproduct is controlled by the cross-link density. For the latter, a lower cross-link density allows more swelling of the polymer resin and thus can lead to a higher level of absorption. Also related to the ability of the polymeric resin to absorb the bioproduct of interest is the relative pore size and volume. Polymeric resins with larger pore volumes may absorb more bioproduct, provided they possess the proper bioproduct affinity (e.g. , as provided by chemical structure and functional groups).

[63] An exemplary, non-limiting example of a tailored poly(acrylamide) is shown below where the polymeric material contains an easily controlled and tunable mixture of substituents, e.g., side-groups on the polymer backbone. For example, the ratio of a, b, and c (mole fraction) may be used to match the chemical character of the bioproduct(s) (e.g., hy drophobic/hy drophilic character) :

Hydrophilic Trigger Site

Hydrophobic Cross-Link site

[64] In some embodiments, the polymer backbone type (e.g., polyester, polyamide, or polystyrene) is used to control the hydrophilic/hydrophobic character of the resin. This allows for the use of a higher mole fraction of the "trigger" side-group and thus induces a more significant change in hydrophilic/hydrophobic character when the trigger mechanism is applied for release of the bioproduct. Use of the polymer backbone polarity provides a mechanism for tuning the hydrophobic/hydrophilic character of the triggered-reversible absorption-desorption ("T-RAD") polymer backbone and leads to high levels of trigger loading (i.e., large b, meaning large mole fraction of trigger); hence, this in turn affords a maximum change in hydrophilic/hydrophobic character for a. It is noteworthy to mention that for the diagram above describing the poly(acrylamide), the mole fractions totaled (i.e., a + b + c + d) equal 1 , by definition.

[65] For the poly(acrylamide) shown above, the butanol-filled resin may be triggered by exposure to high pH solution (e.g., pH >12). This creates phenoxides, which are in turn very hydrophilic and therefore much less, for example, butanol-philic. This causes the polymer to absorb water and then release or "eject" the absorbed butanol from the polymeric resin, which is then available for separation from the trigger solution (see, e.g., FIG 5). In some embodiments, the amount of trigger solution used can be from about 10% to about 1000% of the weight of the T-RAD polymer with smaller volumes preferred.

[66] In some embodiments, the trigger solution can include salts to assist in separating the bioproduct from the trigger solution. In some embodiments, about 1% to about 100% salt saturated trigger solutions, or >5% salt saturated solution is used. The salt solution can include one or more salts. The salt may be chosen based on cost and compatibility with vessels and materials used in the process described herein. In one embodiment, an aqueous trigger solution containing sodium sulfate and at least one trigger reagent is used. Many common salts may be used, including but not limited to sodium chloride, sodium carbonate, sodium bicarbonate, and salts containing pairing of lithium ions, potassium ions, calcium ions, magnesium ions in combination with at least one anion selected from a sulfate, bisulfite, halide, carbonate, bicarbonate, or phosphate.

[67] In the methods described herein, addition of a triggering agent may effect a chemical and/or physical change in the polymeric structure or ionic character of the polymer that results in release of bound biomolecules. It is well known to those skilled in the art of polymer chemistry that changing the ionic character of a polymeric backbone induces a large change in hydrophilicity. Perhaps the best known example is poly(acrylic acid) which has a somewhat low affinity for water; however, the deprotonated form poly(sodium acrylate) is extremely hydrophilic, in certain cases labeled as superabsorbent and capable of retaining -1000 g of water per gram of polymer. The effect of pH changes on the hydrophilicity of poly(acrylate) copolymers is well known (see, e.g., Wang and Wang (2010) "Synthesis and swelling properties of pH-sensitive semi-IPN superabsorbent hydrogels based on sodium alginate poly(sodium acrylate) and polyvinylpyrrolidone" Carbohydrate Polymers 80(4): 1028-1036).

[68] The process described herein is applicable to the separation of many types of bioproducts from aqueous solutions, e.g. , bioproducts with many common organic functional groups. Typical examples include, but are not limited to: alcohols, diols, triols, ketones, aldehydes, esters, aromatics containing at least one of these functional groups, and combinations thereof. One illustrative example of a bioproduct containing combined functional groups in a single molecule is levulinic acid.

[69] One embodiment of the current process includes increasing a fermentation titer while providing simultaneously a chemically efficient and low energy path for isolation of at least one bioproduct from the dilute aqueous solution (e.g., the fermentation broth). [70] In some embodiments, an anti-fouling coating is included on at least a portion of the polymeric material. In some embodiments, such a coating may provide protection against fouling of the polymeric material in a microbial fermentation system, for example, protection against growth and/or adherence of microbes such as bacteria or fungi and/or adherence of components of the fermentation medium such as components of biomass hydrolysates. One nonlimiting example of an anti-fouling coating which may be employed is a polyethylene glycol based material, for example, a polyethylene glycol with an amine (e.g., oligo-amine) functional group at one end, for example, coupled to a surface such as a carboxylated surface (e.g., Blockmaster™CE510 (JSR Life Sciences, Sunnyvale, CA)).

[71] In other embodiment, the polymeric material may be coated with one or more enzyme(s) that catalyze specific reaction(s) of interest. Bioproduct(s) that are absorbed by the polymeric material, as described herein, may be converted to other product(s) of interest. One non-limiting example is a lipase enzyme. For example, lipase can catalyze conversion of butanol and acrylic acid to butyl acrylate.

[72] The following examples are intended to illustrate, but not limit, the invention.

EXAMPLES

Example 1

[73] n-Butanol (1.5 g, 98% purity) obtained by fermentation of sugar was added to 100 g of water. A sample was taken and the n-butanol titer measured. Next a 10 g sample of poly(vinylpyridine) (Sigma-Aldrich chemical Co., 2% cross-linked) was added to the aqueous n-butanol solution and liquid sample taken, and the n-butanol titer measured. These data showed that there was an almost instantaneous absorption of n-butanol by the polymeric material(see sample 1 and 2 below in Table 1), hence reducing the effective titer in the solution to 12.2 g/L. Further stirring for several hours did not afford any significant additional n-butanol uptake, demonstrating that the system had reached equilibrium. These data afford en experimentally determined partition coefficient of 2.5 for this particular polymeric material versus water. The polymer was collected on a glass-frit, quickly air dried, and then placed in a clean and new reaction flask. The filtrate was shown to have the same titer as the original solution in equilibrium with the polymer before the polymer was removed by filtration. The collected polymer was treated with 20 mL of a pH 2 solution prepared from adding concentrated nitric acid to water. This was the trigger solution for this particular polymer. The polymer was in contact with the trigger solution for 5 min and a liquid sample taken and the titer measured. The measured titer was 7.73 g/L, which corresponds to 50% recovery/release of the n-butanol absorbed in the polymer. This corresponds to the triggered polymer having a partition coefficient less than 2.5. The new partition coefficient may be a result of pyridinium salt formation that afforded an increase in the resin hydrophilicity and induced a decrease for the resin's butanol-philicity. Hence this experiment demonstrated a change in the partition coefficient as a result of the trigger solution.

Table 1

Experimental data gathered demonstrating TRAD using poly(vinylpyridine) resin and butanol extraction from water

[74] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention, which is delineated in the appended claims. Therefore, the description should not be construed as limiting the scope of the invention.

[75] All publications, patents, and patent applications cited herein are hereby

incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.