US 2014O106420A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0106420 A1 RAZAV-SHRAZI et al. (43) Pub. Date: Apr. 17, 2014

(54) BOCONVERSION PROCESSESUSING cation No. 61/689,933, filed on Jun. 15, 2012, provi WATER-INSOLUBLE LIQUIDS sional application No. 61/689.935, filed on Jun. 15, 2012, provisional application No. 61/689,939, filed on (71) Applicant: Microvi Biotech, Inc., Hayward, CA Jun. 15, 2012, provisional application No. 61/689,940, (US) filed on Jun. 15, 2012, provisional application No. 61/689,943, filed on Jun. 15, 2012, provisional appli (72) Inventors: Fatemeh RAZAVI-SHIRAZI, cation No. 61/689,945, filed on Jun. 15, 2012, provi Hayward, CA (US); Mohammad Ali sional application No. 61/689,953, filed on Jun. 15, DORRI, Milpitas, CA (US); Ameen 2012, provisional application No. 61/849,725, filed on RAZAVI, Fremont, CA (US) Feb. 1, 2013, provisional application No. 61/850,631, filed on Feb. 20, 2013, provisional application No. (73) Assignee: Microvi Biotech, Inc., Hayward, CA 61/851,467, filed on Mar. 8, 2013, provisional appli (US) cation No. 61/852.451, filed on Mar. 15, 2013, provi sional application No. 61/689,921, filed on Jun. 15, (21) Appl. No.: 14/106,659 2012, provisional application No. 61/849,725, filed on Feb. 1, 2013. (22) Filed: Dec. 13, 2013 Publication Classification Related U.S. Application Data (51) Int. C. (63) Continuation-in-part of application No. 13/918,868, CI2P 7/06 (2006.01) filed on Jun. 14, 2013, Continuation-in-part of appli (52) U.S. C. cation No. PCT/US2013/046029, filed on Jun. 14, CPC ...... CI2P 7/06 (2013.01) 2013. USPC ...... 435/139; 435/132; 435/145; 435/161 (60) Provisional application No. 61/689.921, filed on Jun. 15, 2012, provisional application No. 61/689,922, (57) ABSTRACT filed on Jun. 15, 2012, provisional application No. Processes are disclosed for bioconversion processes in which 61/689,923, filed on Jun. 15, 2012, provisional appli a ME biocatalyst is surrounded by water-insoluble liquid cation No. 61/689,924, filed on Jun. 15, 2012, provi during the bioconversion to facilitate one or more of mass sional application No. 61/689.925, filed on Jun. 15, transfer of substrate to and bioproduct from the biocatalyst 2012, provisional application No. 61/689,929, filed on and the separation and recovery of bioproduct from the water Jun. 15, 2012, provisional application No. 61/689,930, insoluble liquid. The ME biocatalyst irreversibly retains filed on Jun. 15, 2012, provisional application No. microorganisms for the bioconversion and has, in its interior, 61/689,932, filed on Jun. 15, 2012, provisional appli an aqueous environment. Patent Application Publication Apr. 17, 2014 Sheet 1 of 2 US 2014/0106420 A1

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BOCONVERSION PROCESSESUSING the contact of an aqueous medium with the microorganisms. WATER-INSOLUBLE LIQUIDS The aqueous medium may be a continuous phase in which the microorganisms exist or may be an aqueous phase wetting the CROSS-REFERENCE TO RELATED microorganisms or Support on which the microorganisms APPLICATIONS reside. 0001. This is a continuation-in-part of U.S. patent appli 0006 The use of an aqueous medium can pose challenges. cation Ser. No. 13/918,868, filed on Jun. 14, 2013, which For instance, one or more Substrates or one or more metabolic claims priority to U.S. Provisional Patent Applications Nos. products may have little, if any solubility in water, thereby 61/689,921, filed on Jun. 15, 2012: 61/689,922, filed on Jun. presenting mass transfer challenges. Another challenge, is the 15, 2012: 61/689,923, filed on Jun. 15, 2012: 61/689,924, separation of the sought bioproduct from an aqueous filed on Jun. 15, 2012: 61/689,925, filed on Jun. 15, 2012: medium. Distillation is an often used process, which can be 61/689,929, filed on Jun. 15, 2012: 61/689,930, filed on Jun. energy intensive especially where the boiling points of the 15, 2012: 61/689,932, filed on Jun. 15, 2012: 61/689,933, sought bioproduct and water are proximate. With some oxy filed on Jun. 15, 2012: 61/689,935, filed on Jun. 15, 2012: genated organic compounds Such as alcohols, azeotropes can 61/689,939, filed on Jun. 15, 2012: 61/689,940, filed on Jun. form thereby presenting additional problems in the separation 15, 2012: 61/689,943, filed on Jun. 15, 2012: 61/689,945, of the sought bioproduct from water. filed on Jun. 15, 2012: 61/689,953, filed on Jun. 15, 2012: 0007 Some disclosed bioprocesses inherently or by 61/849,725, filed on Feb. 1, 2013: 61/850,631, filed on Feb. choice generate relatively dilute broths of the sought bioprod 20, 2013: 61/851,467, filed on Mar. 8, 2013; and 61/852,451, uct in water. Woods, et al., in U.S. Pat. No. 7,682,821 B2, filed on Mar. 15, 2013, and a continuation-in-part of Interna disclose a photobioreactor System in which Solar energy is tional Application No. PCT/US2013/046029, designating the used to provide a concentrated ethanol product that can be United States of America, filed on Jun. 14, 2013, which more economically distilled. The patentees state that photo claims priority to U.S. Provisional Patent Applications Nos. bioreactor systems experience various limitations for use in 61/689,921, filed Jun. 15, 2012, and 61/849,725, filed on Feb. massive scale industrial production of low-cost biofuels. One 1, 2013, all herein incorporated by reference in their entirety. such limitation is stated to be the evaporation of ethanol from the aqueous medium. The photobioreactor comprises a cham STATEMENT REGARDING FEDERALLY ber having a translucent or clear region to allow in Sunlight to SPONSORED RESEARCH ORDEVELOPMENT contact an aqueous fermentation medium in a lower part of the chamber wherein ethanol and water vapors can condense 0002. None on the upper part of the chamber. The condensate has an enhanced ethanol concentration thus enabling more eco TECHNICAL FIELD nomic recovery of an anhydrous ethanol product. 0003. This invention pertains to bioconversion processes 0008. In other instances, for example, fermentation pro in which the exteriors of biocatalysts comprising microorgan cesses to make butanols, including, but not limited to n-bu isms that are substantially irreversibly retained in the interior tanol and isobutanol, the butanol in the aqueous medium must of an open, porous, highly hydrophilic polymer, are Sur be maintained relatively dilute since the butanol is toxic or rounded by water-insoluble liquid. inhibitory toward the microorganisms. In general, the con centration ofbutanol is less than about 3 volume percent in the BACKGROUND aqueous medium. Due to the amount of water required to be 0004 Metabolic processes have long been proposed for removed, distillation is not the preferred recovery process. anabolic and catabolic bioconversions. Microorganisms of Workers have sought to modify microorganisms to increase various types have been proposed for these bioconversions tolerance. See, for instance, U.S. Patent Application Publica and include bacteria and archaea, both of which are prokary tion 2010/0105103 A1. Workers have also proposed extrac otes; fungi, and algae. Metabolic processes are used by tion of butanol from the aqueous medium to maintain the nature, and some have been adapted to use by man for mil butanol concentration below that which adversely affects the lennia for anabolic and catabolic bioconversions ranging microorganisms. Erdner-Tindall, et al., in U.S. Patent Appli from culturing yogurt and fermentation of Sugars to produce cation Publication No. 2010/0143993 A1 have proposed pro alcohol to treatment of water to remove contaminants. Meta cesses for producing alcohols using an ionic liquid as a sol bolic processes offer the potential for low energy consump vent to separate an alcohol product from a fermentation broth. tion, high efficiency bioconversions in relatively inexpensive They state that ionic liquids will generally have no measur processing equipment and thus may be and are often viable able vapor pressure, have a high solubility for alcohol prod alternatives to chemical synthesis and degradation methods. uct, and are immiscible with the aqueous fermentation broth. Often anabolic processes can use raw materials that are pre They further state that the ionic liquid has little to no toxicity ferred from a renewable or environmental standpoint but are to the microorganism. not desirable for chemical synthesis, e.g., the conversion of 0009 Aprior disclosure of extraction of some bioproducts carbon dioxide to biofuels and other bioproducts. Catabolic from aqueous media was made by Tedder in U.S. Pat. No. bioconversions can degrade Substrates and have long been 4.517,298. The patentee discloses a process for producing used for waste water treatment. Considerable interests existin simple aliphatic alcohols, most particularly ethanol, using an improving metabolic processes for industrial use and expand organic solvent containing an extractant for contact with ing the variety of metabolic process alternatives to chemical aqueous fermentation medium withdrawn from fermentation syntheses and degradations. unit. An alcohol-solvent extract phase and an aqueous phase 0005 Virtually all microorganisms require the presence of are formed, and the alcohol is separated from the alcohol water in liquid or vaporous form. Consequently, metabolic Solvent phase, e.g., by evaporation or distillation. The paten processes that have been proposed by prior workers involve tee contemplates returning the aqueous phase to the fermen US 2014/0106420 A1 Apr. 17, 2014 tation unit. The solvent system disclosed comprises a microorganisms can be achieved in a bioreactor without hydrophobic solvent, such as aliphatic hydrocarbon, and operational problems such as high viscosity media that occur eXtractant. with high, Suspended cell densities. Thus, high rates of bio 0010 Shirazi, et al., in the above-mentioned U.S. patent conversion per unit volume of bioreactor can be achieved. application Ser. No. 13/918,868 disclose biocatalysts having 0017. As the ME biocatalyst is surrounded by water-in a high tolerance to the presence of toxins. These biocatalysts soluble liquid, the processes of this invention are particularly comprise advantageous for bioconversion processes where the Sub 0011 i. a solid structure of hydrated hydrophilic poly strate has little solubility in water. This preferred aspect of the mer defining an interior structure having a plurality of invention is useful in the bioconversion of syngas and meth interconnected major cavities having a smallest dimen ane to oxygenated organic products. The Substrate can be sion of between about 5 to about 100 microns and an directly introduced into a bioreactor assembly containing the HEV of at least about 1000 and biocatalyst and water-insoluble liquid or can be contacted 0012 ii. a population of microorganisms capable of with the water-insoluble liquid, and a water-insoluble liquid converting Sugars to at least one organic product Sub ladened with substrate can be introduced into the bioreactor stantially irreversibly retained in the interior of the solid assembly for contact with the biocatalyst. The ME biocatalyst structure, said population of microorganisms being in a facilitates separation of the water-insoluble liquid phase and concentration of at least about 60 grams per liter based thus enables process flow design flexibility without incurring upon the volume defined by the exterior of the solid costs for, e.g., centrifugation to separate the biocatalysts. structure when fully hydrated wherein the microorgan 0018. Additionally, as the water-insoluble liquid is sub isms maintain their population Substantially stable. stantially anhydrous, bioproducts that can form azeotropes 0013 The microorganisms are believed to undergo phe with water can typically be more readily separated from the notypic alterations enabling, inter alia, enhanced tolerance. liquid medium. The wide variety of water-insoluble liquids The disclosed biocatalysts are particularly attractive for con that are available for the practice of this invention enables the tinuous processes for the bioconversion offermentable Sugars selection of a water-insoluble liquid that has appropriate to ethanol as the biocatalyst is substantially devoid of solids properties for the separation and recovery of bioproduct. For generation, and, being a solid, enables separation of the bio example, where the separation is effected by distillation, the catalyst from the fermentation broth. Additionally, the phe selection of the water-insoluble liquid can be based upon notypic alterations reduce the requirement of the microorgan boiling point. In some instances, such as where the bioprod ism for Sugars for metabolic Sustenance thereby enabling the uct is normally a gas, e.g., in a denitrification bioconversion, bioconversion of as much as about 99 percent of the ferment the bioproduct may pass from the water-insoluble liquid in able Sugars to bioproducts. Moreover, the biocatalyst has a the bioreactor assembly. In other instances, it may be desired long lifetime and competition with undesired microorganism to remove the water-insoluble liquid which contains the bio is substantially eliminated. For ease of reference, these bio product from the bioreactor assembly for recovery by any catalysts are herein referred to as ME biocatalysts. Suitable means, including, but not limited to, distillation, 0014 Improved processes are sought for bioconverting membrane separation, extraction, and crystallization. Substrate to bioproduct where the microorganisms need to be 0019. In its broad aspect, the processes for the bioconver retained in an aqueous medium. sion of a Substrate to a bioproduct comprise: 0020 a. providing in a bioreactor assembly a water SUMMARY insoluble liquid containing said Substrate containing 0015. In accordance with the processes of this invention, said Substrate; certain biocatalyst compositions (ME biocatalysts) that sub 0021 b. contacting said water-insoluble liquid in said stantially irreversibly retain microorganisms in their interior bioreactor assembly with an internally hydrated biocata are surrounded by water-insoluble liquid during bioconver lyst under metabolic conditions for a time sufficient to sion of substrate to bioproduct. The ME biocatalysts exhibit bioconvert at least a portion of said substrate to said enhanced tolerance, and thus the water-insoluble liquid can bioproduct wherein said water-insoluble liquid is be chosen over a wide range of materials without undue capable of receiving said bioproduct from the interior of adverse effect on the microorganisms. Hence, the water-in said biocatalyst, and in some instances, said water-in soluble liquid can be selected to facilitate one or more of mass soluble liquid is a solvent for said bioproduct, to provide transfer of substrate to and bioproduct from the biocatalyst a bioproduct-containing liquor, said contacting being and the separation and recovery of bioproduct from the water Substantially absent an aqueous phase external to said insoluble liquid. biocatalyst, wherein said biocatalyst comprises: 0016. The ME biocatalysts have a high Hydration Expan 0022 i. a solid structure of hydrated hydrophilic sion Volume (HEV) and are hydrated and thus physically polymer defining an interior structure having a plu protect the microorganisms from the external, water-in rality of interconnected major cavities having a small soluble liquid environment. The water-insoluble liquid also est dimension of from about 5 to about 100 microns can, in some instances, serve to provide a hostile environment and an HEV of at least about 1000 and to adventitious microorganisms. The microorganisms are 0023 ii. a population of microorganisms capable of retained in the interior of the biocatalysts and thus are not a converting said Substrate to said bioproduct, said source of debris that can foul the biocatalyst or the water population of microorganisms being Substantially insoluble liquid. Thus, the processes of this invention are irreversibly retained in the interior of the solid struc particularly attractive for continuous metabolic operations. ture, said population of microorganisms being in a Further, the biocatalysts can be moved without damage to the concentration of at least about 60 grams per liter microorganisms therein. Since the microorganisms are con based upon the volume defined by the exterior of the tained in the interior of the biocatalyst, high densities of solid structure when fully hydrated; and US 2014/0106420 A1 Apr. 17, 2014

002.4 c. separating said bioproduct from said bioprod 0031. The use of the terms “a” and “an is intended to uct-containing liquor. include one or more of the element described. Lists of exem 0025. The processes of this invention may be batch, semi plary elements are intended to include combinations of one or batch, or continuous. Often continuous processes are pre more of the element described. The term “may as used herein ferred. Especially in continuous processes, preferably at least means that the use of the element is optional and is not portion of the bioproduct-containing liquor is withdrawn intended to provide any implication regarding operability. from the bioreactor assembly and bioproduct is separated 0032. As used herein, the term “normally gaseous compo from the withdrawn bioproduct-containing liquor to provide nent’ means the component is a gas under normal conditions a regenerated liquor. The regenerated liquor may be directly of 0° C. and 101 kPa absolute. passed to the bioreactor assembly, or Substrate may be Sup 0033) Adhering to the solid structure of the biocatalyst plied to the least a portion of the regenerated liquor to provide means that the microorganism is located in cavities in the a feed liquor, and the feed liquor is introduced into the biore interior of the biocatalyst and is substantially irreversibly actor assembly as at least a portion of the water-insoluble retained therein although extraordinary conditions and treat liquid containing said Substrate. ments (i.e., not normal bioconversion conditions for biocon 0026. In another preferred aspect of the invention, the ME version using the microorganism) might be able in some biocatalyst, which has been used for the bioconversion of instances to cause the microorganism to exit the biocatalyst. Substrate to bioproduct, is rehydrated. In some instances, the Adhering includes Surface attachment to the polymer forming biocatalyst will not lose any appreciable amount of water the walls of the porous matrices as well as where the micro during use. In Such situations, the rehydration may serve to organism are retained microorganisms that are proximate to a replenish one or more nutrients. The rehydration may occur polymeric Surface, e.g., within about 10 or about 20 microns, by ceasing the contact between the biocatalyst and water but not directly contacting the Surface. Adhering thus insoluble liquid, separating the biocatalyst and water-in includes physical and electrostatic adherence. In some soluble liquid and then contacting the biocatalyst with an instances, the polymer used to make the biocatalyst may aqueous medium for the hydration. The rehydration may become embedded in the extracellular polymeric substance occur in the bioreactor assembly, e.g., by cycling a vessel around a cell or even in or on the cell wall of the microorgan containing the biocatalyst between an external environment 1S of the water-insoluble liquid and the aqueous medium for the 0034 Bioconversion activity is the rate of consumption of hydration. Alternatively, especially in continuous processes, Substrate per hour per gram of microorganism. Where an a portion of the biocatalyst can continuously or intermittently increase or decrease in bioconversion activity is referenced be withdrawn from the bioreactor assembly and any water herein, Such increase or decrease is ascertained under similar immiscible liquid removed therefrom. This biocatalyst can bioconversion conditions including concentration of Sub then be contacted with the aqueous medium for hydration. strate and product in the aqueous medium. Bioconversion Preferably, the rehydration provides a biocatalyst that is fully activity to bioproduct is the rate of production of the bioprod hydrated. After hydration, the aqueous medium and biocata uct per hour per gram of microorganism. lyst are separated, and the rehydrated biocatalyst can be rein 0035 Biofilm means an aggregate of microorganisms troduced into the bioreactor assembly. The aqueous medium embedded within an extracellular polymeric substance (EPS) for hydration of the biocatalyst preferably contains nutrients, generally composed of polysaccharides, and may contain including micronutrients, sufficient to maintain the microor other components such as one or more of proteins, extracel ganism population in the biocatalyst. Since the aqueous lular DNA and the polymer used to make the biocatalyst. The medium, including the nutrients, are within the interior of the thickness of a biofilm is determined by the size of the aggre ME biocatalyst and the exterior of the biocatalyst is sur gate contained within a continuous EPS structure, but a con rounded with the water-immiscible liquid, environment can tinuous EPS structure does not include fibrils that may extend be maintained for extended periods of time in the interior of between separated biofilms. In some instances, the biofilm the biocatalyst that Supports the microorganism population. extends in a random, three dimensional manner, and the BRIEF DESCRIPTION OF THE DRAWINGS thickness is determined as the maximum, straight line dis tance between the distal ends. A thin biofilm is a biofilm 0027 FIG. 1 is a schematic depiction of an apparatus that which does not exceed about 10 microns in any given direc can be used in the processes of this invention in which a tion. gaseous Substrate is passed into a bioreactor assembly in 0036 Bioproduct means a product of a bioconversion which it is converted to an anabolic bioproduct. which may be an anabolic product or a catabolic product and 0028 FIG. 2 is a schematic depiction of an apparatus that includes, but is not limited to, primary and secondary metabo can be used in the processes of this invention in which water lites. Bioproducts include, but are not limited to, sought insoluble liquid is passed into an absorber for contact with metabolites, co-products, and by-products, and the metabo Substrate, and once ladened with Substrate, is passed into a lites may be final products or intermediate products or a bioreactor assembly for bioconversion of the substrate. product which has no utility. DETAILED DESCRIPTION 0037. A bioreactor assembly is an assembly of one or 0029 All patents, published patent applications and more vessels suitable to contain water-insoluble liquid and articles referenced herein are hereby incorporated by refer ME biocatalyst and can contain associated equipment Such as ence in their entirety. injectors, recycle loops, agitators, and the like. 0038 Capable of receiving bioproduct from a biocatalyst DEFINITIONS means the bioproduct is able to pass from the interior of the 0030. As used herein, the following terms have the mean biocatalyst to the water-insoluble liquid. The bioproduct may ings set forth below unless otherwise stated or clear from the or may not dissolve in the water-insoluble liquid. For context of their use. instance, a bioproduct may pass into the water-insoluble liq US 2014/0106420 A1 Apr. 17, 2014

uid as a separate phase, which may be gaseous, liquid or Solid. microorganisms contained therein) wherein fluid can enter Preferably, the bioproduct is at least partially soluble in the and exit the major cavities from and to the exterior of the water-insoluble liquid. matrix. The porous matrix may contain larger and Smaller 0039. An exo-network is a community of spaced-apart channels or cavities than the major cavities, and may contain microorganisms that can be in the form of individual cells or channels and cavities not open to the exterior of the matrix. biofilms that are interconnected by extracellular polymeric The major cavities, that is, open, interconnected regions of Substance in the form of strands. The spacing between the between about 5 or about 10 to about 70 or about 100 microns microorganisms or biofilms in the exo-network is sufficient to in the Smallest dimension (excluding any microorganism con enable the passage of nutrients and Substrates there between tained therein), have nominal major dimensions of less than and is often at least about 0.25, say, at least about 0.5, micron about 300, preferably less than about 200, microns, and some and may be as large as about 5 or about 10 microns or more. times a smallest dimension of at least about 10 microns. The 0040 Exterior skin is an exterior layer of polymer on the term open, porous thus refers to the existence of channels or biocatalyst that is less open than the major channels in the cavities that are interconnected by openings therebetween. interior structure of the biocatalyst. A biocatalyst may or may 0049 Metabolic conditions include conditions of tem not have a skin. Where a skin is present, it may or may not perature, pressure, oxygenation, pH, and nutrients (including have surface pores. Where no surface pores are present, fluids micronutrients) and additives required or desired for the diffuse through the skin. Where pores are present, they often microorganisms in the biocatalyst. Nutrients and additives have an average diameter of between about 1 to about 10 include growth promoters, buffers, antibiotics, vitamins, min microns. erals, nitrogen sources, and Sulfur sources and carbon Sources 0041 Free liquid phase on the surface of a biocatalyst where not otherwise provided. means the presence of liquid beyond that required for incipi 0050. Oxygenated organic product means a product con ent wetness, or filling the pores or capillaries. Often the pres taining one or more oxygenated organic compounds having 2 ence of free liquid results on the Surface results in a glistening to 100, and frequently 2 to 50, carbons and at least one moiety appearance whereas the absence of free liquid on the Surface selected from the group consisting of hydroxyl, carbonyl, results in a dull appearance. ether and carboxyl. 0042 Fully hydrated means that a biocatalyst is immersed 0051 Permeable means that a component can enter or exit in water at about 25° C. until no further expansion of the the major cavities from or to the exterior of the biocatalyst. superficial volume of the biocatalyst is perceived. 0.052 Quiescent means that the aqueous medium in a bio 0043. The “Hydration Expansion Volume” (HEV) for a catalyst is still; however, flows of nutrients and substrates and biocatalyst is determined by hydrating the biocatalyst in bioproducts can occur through the aqueous medium via dif water at about 25°C. until the volume of the biocatalyst has fusion and capillary flow. stabilized and measuring the superficial volume of the bio 0053 Population of microorganisms refers to the number catalyst (V), removing the biocatalyst from water and of microorganisms in a given Volume and includes Substan removing excess water from the exterior, but without drying, tially pure cultures and mixed cultures. and immersing the biocatalyst in ethanol at about 25°C. for a 0054) A phenotypic change or alternation or phenotypic time sufficient that the volume of the biocatalyst has stabi shift is a change in a microorganism's traits or characteristics lized and then measuring the superficial volume of the bio from environmental factors and is thus different from a catalyst (V). change in the genetic make-up of the microorganism. 0044) The HEV in volume percent is calculated as the 0055 Retained solids means that solids are retained in the amount of IV/Vx100%. To assure dehydration with the interior of the biocatalyst. The solids may be retained by any ethanol, either a large Volume ratio of ethanol to biocatalyst is Suitable mechanism including, but not limited to, restrained used or successive immersions of the biocatalyst in fresh by not being able to pass through pores in the skin of a ethanol are used. The ethanol is initially anhydrous ethanol. biocatalyst, by being captured in a biofilm or a polysaccharide 0045 Irreversibly retained and substantially irreversibly structure formed by microorganisms, by being retained in the retained mean that the microorganism is adhering to poly polymeric structure of the biocatalyst, or by being sterically meric structures defining open, porous cavities. Irreversibly entangled within the structure of the biocatalyst or the micro retained microorganism does not include microorganisms organisms. located on the exterior Surface of a biocatalyst. Microorgan 0056 Smallest dimension means the maximum dimen ism is irreversibly retained even if the biocatalyst has exterior sion of the shortest of the maximum dimensions defining the pores of sufficient size to permit egress of the microorganism. length, width and height of a major cavity. Usually a prepon 0046 Highly hydrophilic polymers are polymers to which derance of the major cavities in a matrix are substantially water is attracted, i.e., are hydroscopic. Often the polymers width and height symmetrical. Hence the Smallest dimension exhibit, when cast as a film, a water contact angle of less than can be approximated by the maximum width of a cavity about 60°, and sometimes less than about 45°, and in some observed in a two dimensional cross section, e.g., by optical instances less than about 10, as measured by the sessile drop or electronic microscopy. method using a 5 microliter drop of pure distilled water. 0057. A solubilized precursor for the polymer is a mono 0047. Highly hydrated means that the volume of the bio mer or prepolymer or the polymer itself that is dissolved or catalyst (excluding the Volume of the microorganisms) is at dispersed such that Solids cannot be seen by the naked eye and least about 90 percent water. is stable. For instance, a solid can be highly hydrated and be 0.048. A matrix is an open, porous, polymeric structure and Suspended in an aqueous medium even though the Solid is not is an article of manufacture having an interconnected plural dissolved. ity of channels or cavities (herein “major cavities') defined by 0.058 Sorption means any physical or chemical attraction polymeric structures, said cavities being between about 5 to and can be adsorption or absorption and may be relatively about 100 microns in the Smallest dimension (excluding any weak, e.g., about 10 kilojoules per mole or a chemical inter US 2014/0106420 A1 Apr. 17, 2014 action with a sorbent. Preferably the sorptive attraction by the in this patent application. Principles and details of standard sorbent is greater than that between water and the substrate, separation and purification steps are known in the art, for but not so great that undue energy is required to desorb the example in “BioSeparations Science and Engineering. Roger substrate. Frequently the sorptive strength is between about G. Harrison et al., Oxford University Press (2003), and Mem 10 to about 70, say, about 15 and about 60, kilojoules per brane Separations in the Recovery of Biofuels and Biochemi mole. A sorbent is a Solid having sorptive capacity for at least cals—An Update Review, Stephen A. Leeper, pp. 99-194, in one substrate. Separation and Purification Technology, Norman N. Li and 0059 A stable population of microorganisms means that Joseph M. Calo, Eds. Marcel Dekker (1992). the population of microorganisms does not decrease by more 0065. A water-insoluble liquid comprises one or more than about 50 percent nor increase by more than about 400 components, and wateris Substantially insoluble in the liquid, percent. often less than about 0.2, preferably less than about 0.05, 0060 Substrates are carbon sources, electron donors, elec volume percent water is soluble in the water-insoluble liquid tronacceptors and other chemicals that can be metabolized by at about 25°C. The water-insoluble liquid is also substantially a microorganism, which chemicals, may or may not provide insoluble in water, often less than about 0.2, preferably less Sustaining value to the microorganisms. than about 0.05, volume percent of the water-insoluble liquid 0061 Sugar means carbohydrates having 5 to 12 carbon is soluble in water at about 25°C. atoms and includes, but is not limited to, D-glyceraldehyde, 0066. The wet weight or wet mass of cells is the mass of L-glyceraldehyde, D-erythrose, L-erythrose, D-threose, cells from which free water has been removed, i.e., are at the L-threose, D-ribose, L-ribose, D-lyxose, L-lyxose, D-allose, point of incipient wetness. All references to mass of cells are L-allose, D-altrose, L-altrose 2-keto-3-deoxy D-gluconate calculated on the basis of the wet mass of the cells. (KDG), D-mannitol, guluronate, mannuronate, mannitol, 0067 References to organic acids herein shall be deemed lyxose, Xylitol, D-glucose, L-glucose, D-mannose, L-man to include corresponding salts and esters. nose, D-idose, L-idose, D-galactose, L-galactose, D-xylose, 0068 References to biocatalyst dimensions and volumes L-Xylose, D-arabinose, L-arabinose, D-talose, L-talose, glu herein are of fully hydrated biocatalyst unless otherwise curonate, galacturonate, rhamnose, fructooligosaccharide stated or clear from the context. (FOS), galactooligosaccharide (GOS), inulin, mannan oli gosaccharide (MOS), oligoalginate, mannuronate, gulur Process Discussion onate, alpha-keto acid, or 4-deoxy-L-erythro-hexoselulose 0069. This invention can be used for a wide range of ana uronate (DEHU). bolic and catabolic bioconversion processes. Substrates may 0062 Typical Bioreactor Systems are those operated on a be one or more of normally a gas, liquid or Solid. The Sub continuous, semi-continuous or batch mode of operation and strates preferably are capable of being dissolved in the water include bioreactor assemblies Such as, but are not limited to, insoluble liquid for contact with the ME biocatalyst although ponds (in the case of photosynthetic processes), bubble col the processes can find advantageous application where the umn reactors, stirred reactors, packed bed reactors, trickle substrate has little, if any, solubility in the water-insoluble bed reactors, fluidized bed reactors, plug flow (tubular) reac liquid but can be transported by the liquid to the biocatalysts, tors, and membrane (biofilm) reactors. In conducting photo e.g., as a dispersion. The bioproduct is soluble in the water synthetic bioconversions, the reactors may be designed to insoluble liquid to facilitate its removal from the ME biocata permit the transfer of photo energy. The biocatalyst may be lyst. freely mobile in the water-insoluble liquid or fixed, e.g., to a 0070 The substrate can be introduced into the water-in structure in the reactor vessel, or may itself provide a fixed soluble liquid by any suitable technique. For instance, the structure. More than one reactor vessel may be used in a substrate can be directly introduced into the bioreactor bioreactor assembly. For instance, reactor vessels may be in assembly. Alternatively, the substrate may first be admixed parallel or in sequential flow series. with water-insoluble liquid to form a solution, aerosol or 0063 Typical Mesophilic Conditions are metabolic con colloidal mixture and then introduced into the bioreactor ditions that include a temperature in the range of between assembly. In some embodiments, the water-insoluble liquid about 0° C. to about 50° C. or more depending upon the may contact a gas or liquid containing Substrate to selectively temperature tolerance of the microorganism, most frequently, remove Substrate. Any suitable apparatus may be used for the about 5° C. or about 10° C. to about 40°C. or about 45° C.; a contacting, including but not limited to, scrubbers, spray tow pressure in the ranges from about 70 to about 500, say, about ers, and liquid-liquid extraction columns. Where the water 90 to about 300, kPa absolute due to equipment configura immiscible liquid is admixed with the substrate prior to intro tions although higher and lower pressures could find applica duction into the bioreactor assembly, it is possible to effect the bility; and a pH in the range of between about 3 and about 9. mixing under conditions different than those in the bioreactor The Typical Mesophilic Conditions can be aerobic or anaero assembly since microorganisms are not present. Thus, condi bic. tions of temperature and pressure can be used that facilitate 0064. Typical Separation Techniques for chemical prod the mixing and, where Substrate is removed from a gas or ucts include phase separation for gaseous chemical products, liquid, facilitate the removal. the use of a still, a distillation column, liquid/liquid phase (0071. Substrates can be natural or xenobiotic substances separation, gas stripping, flow-through centrifuge, Karr col in an organism (plant or animal) or can be obtained from other umn for liquid-liquid extraction, mixer-settler, or expanded Sources. Hence, Substrates include, but are not limited to, bed adsorption. Separation and purification steps may pro those that can be, or can be derived from, plant, animal or ceed by any of a number of approaches combining various fossil fuel sources, or can be produced by a chemical or methodologies, which may include centrifugation, filtration, industrial process. The processes of this invention can also be reduced pressure evaporation, liquid/liquid phase separation, applicable to water Supply or waste water clean-up operations membranes, distillation, and/or other methodologies recited where the substrate is one or more contaminants. The bio US 2014/0106420 A1 Apr. 17, 2014 catalysts generate metabolites as a result of anabolic or cata atoms including but not limited to, nitrogen, Sulfur, oxygen, bolic activity and the metabolites may be primary or second and phosphorus atoms. Examples of organic products as end ary metabolites. The processes of this invention can be used to products from metabolic processes are those listed in United produce any type of anabolic or catabolic metabolite. States published patent application no. 2010/0279354 A1, 0072 Examples of substrate that may be contained in a gas especially as set forth in paragraphs 0129 through O149. See phase include, but are not limited to, hydrogen, carbon mon also, United States published patent application no. 2011/ oxide, carbon dioxide, nitrogen oxides, ammonia, hydrogen 0165639 A1. Other bioproducts include p-toluate, terephtha Sulfide, Sulfur oxides, carbon disulfide, phosphine, carbonyls late, terephthalic acid, aniline, putrescine, cyclohexanone, (such as phosgene and carbonyl Sulfide), halocarbons (such as adipate, hexamethylenediamine (HMDA), 6-aminocaproic carbon tetrachloride and tetrafluoromethane), sulfur com acid, malate, acrylate, apidipic acid, methacrylic acid, 3-hy pounds (such as mercaptains and thioethers), Volatile organic droxypropionic acid (3HP), Succinate, butadiene, propylene, compounds (such as lower alkanes, lower alkenes, lower caprolactam, fatty alcohols, fatty acids, glycerates, acrylic alkynes, aromatic organic compounds, alkanols, phenols, tet acid, acrylate esters, methacrylic acid, methacrylic acids, rahydrofurans, aldehydes, ketones, ethers, epoxides and halo fucoidan, muconate, iodine, chlorophyll, carotenoid, cal containing organic compounds. cium, magnesium, iron, sodium, potassium, and phosphate. 0073 Substrates such as sugars and oligosaccharides can The bioproduct may be a chemical that provides a biological find application in the processes of this invention where dis activity with respect to a plant or animal or human. The persed in the water-insoluble liquid. Lipids may also find biological activity can be one or more of a number of different utility as substrates. Other substrates include, but are not activities such as antiviral, antibiotic, depressant, stimulant, limited to, aliphatic and aromatic molecules, often having growth promoters, hormone, insulin, reproductive, attractant, from, e.g., about 1 to about 44 carbon atoms which may repellant, biocide, and the like. Examples of antibiotics contain hetero atoms, e.g., oxygen, Sulfur, phosphorus, and include, but are not limited to, aminoglycosides (e.g., amika nitrogen, and which may be substituted, e.g., with acyl, halo cin, gentamicin, kanamycin, neomycin, netilmicin, tobramy gen, hydroxyl, amine, amide, thiol, nitro, or phosphine cin, paromomycin); ansamycins (e.g., geldanamycin, herbi groups. mycin); carbacephem (loracarbef); carbapenems (e.g., 0.074. In some instances, the gases containing Substrate ertapenem, doripenem, imipenem/cilastatin, meropenem); may also contain components that may be adverse to the cephalosporins (first generation, e.g., cefadroxil, cefazolin, microorganism. Although often the biocatalyst exhibits cefalotin, cefalexin); cephalosporins (second generation, enhance the resistance to such toxins, it may be desired to e.g., cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime); pretreat the gases to reduce the concentration of Such toxins. cephalosporins (third generation, e.g., cefixime, cefdinir, The pretreatment may comprise any Suitable unit operation cefditoren, cefoperaZone, cefotaxime, cefpodoxime, ceftazi including, but not limited to, Sorption, chemical reaction, dime, ceftibuten, ceftizoxime, ceftriaxone); cephalosporins membrane separation, ultrafiltration, and metabolic treat (fourth generation, e.g., cefepime); cephalosporins (fifth gen ment. eration, e.g., ceftobiprole); glycopeptides (e.g., teicoplanin, 0075. In some instances, the two or more substrates Vancomycin, telavancin); lincosamides (e.g., clindamycin, present may be able to be bioconverted by a single species of lincomycin); macrollides (e.g., azithromycin, clarithromycin, microorganism contained in the biocatalyst. For example, dirithromycin, erythromycin, roXithromycin, troleandomy microorganisms have been proposed that are capable of con cin, tellithromycin spectinomycin); monobactams (e.g., aztre Verting hydrogen and carbon dioxide to ethanol as well as onam): nitrofurans (e.g., furazolidone, nitrofurantoin); peni converting carbon monoxide to ethanol. In a bioreactor cillins (e.g., amoxicillin, ampicillin, azlocillin, carbenicillin, assembly more than one type of microorganism can be used, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methi say, by using different biocatalysts, each retaining a different cillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacil microorganism, or by including more than one microorgan lin, temocillin, ticarcillin); penicillin combinations (e.g., ism in a biocatalyst. The different microorganisms may be amoxicillin/clavulanate, amplicillin/Sulbactam, piperacillin/ used to metabolize different substrates or one may bioconvert taZobactam, ticarcillin/clavulanate); polypeptides (e.g., baci the bioproduct of one microorganism to a further bioproduct. tracin, colistin, polymyxin B); quinolones (e.g., ciprofloxa 0076 Bioproducts may be degradation products espe cin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, cially where contaminants are being removed from a fluid moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trova Such as for water Supply or waste water treatment. Such floxacin, grepafloxacin, sparfloxacin, temafloxacin); Sulfona degradation bioproducts include, but are not limited to, car mides (e.g., mafenide; Sulfonamidochrysoidine, Sulfaceta bon dioxide, carbon monoxide, hydrogen, carbonyl sulfide, mide, Sulfadiazine, silver Sulfadiazine, Sulfamethizole, hydrogen Sulfide, water, and salts such as carbonate, bicar Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisox bonate, Sulfide, Sulfite, Sulfate, phosphate, phosphite, chlo azole, trimethoprim, trimethoprim-Sulfamethoxazole (Co ride, bromide, iodide, and borate salts of ammonium, or trimoxazole) (TMP-SMX); tetracyclines (e.g., demeclocy group 1 to 16 (IUPAC) metals such as Sodium, potassium, cline, doxycycline, minocycline, Oxytetracycline, manganese, magnesium, calcium, barium, iron, copper, tetracycline); drugs against mycobacteria (e.g., clofazimine, cobalt, tin, selenium, radium, uranium, bismuth, cadmium, dapsone, capreomycin, cycloserine, ethambutol, ethiona mercury, molybdenum and tungsten. mide, isoniazid, pyrazinamide, rifampin, rifabutin, rifapen 0077 Bioproducts may be one or more of aliphatic com tine, Streptomycin) and others (e.g., arsphenamine, chloram pounds and aromatic compounds including but not limited to phenicol, fosfomycin, fusidic acid, lineZolid, metronidazole, hydrocarbons of up to about 44 or about 50 carbons, and mupirocin, platensimycin, 1 uinupristin/dalfopristin, rifaxi hydrocarbons substituted with one or more of hydroxyl, acyl, min, thiamphenicol, tinidazole). carboxyl, amine, amide, halo, nitro, Sulfonyl, and phosphino 0078 Preferably an anabolic bioproduct is at least one of moieties, and hydrocarbons containing one or more hetero an oxygenated organic compound and hydrocarbon of up to US 2014/0106420 A1 Apr. 17, 2014

about 100, often up to about 50, carbonatoms. Most preferred pH, and nutrients (including micronutrients) and additives oxygenated organic product includes methanol, ethanol, ace required or desired for the microorganisms. The hydration of tic acid, n-propanol, i-propanol, propionic acid, n-butanol, the interior of the ME biocatalysts provides the environment i-butanol, butyric acid, acetone, and methyl ethyl ketone. for the microorganisms and thus will define conditions such 0079. Examples of anabolic or catabolic processes suit as pH, oxidation reduction potential and nutrients. As the able to be practiced by the processes of this invention include, biocatalysts are surrounded by water-insoluble liquid, water but are not limited to: is retained in the interior of the ME biocatalysts. Due to the 0080 Syngas, i.e., gas containing carbon monoxide and microenvironments and phenotypic alterations associated optionally hydrogen, for conversion to oxygenated with the ME biocatalysts, often a broader range of metabolic organic product and hydrocarbons. In typical prior art conditions can be effectively used, and a broader range of processes for the conversion of syngas to oxygenated conditions in the interior of the ME biocatalyst tolerated than organic product, a limiting factor on productivity is the those Suitable for planktonic microorganisms. mass transfer of carbon monoxide and hydrogen from 0087. The metabolic conditions used are those suitable for the gas phase into the liquid phase of the aqueous the microorganisms and the bioconversion. As stated above, medium. By using the processes of this invention for the useful range of metabolic conditions are typically broader syngas bioconversion, mass transfer can be enhanced. than those for planktonic bioconversion systems. In general, I0081 Carbon dioxide-containing gases for conversion a microorganism can fall into the categories of psychrophile to oxygenated organic product and hydrocarbons. The (optimal growth at -10°C. to 25°C.), a mesophile (optimal anabolic conversion may be effected by algae, cyano growth at 20-50° C.), a thermophile (optimal growth 45° C. to bacteria, or other photo activated microorganisms, e.g., 80° C.), or a hyperthermophile (optimal growth at 80° C. to to produce alcohols, biodiesel, and like. Other biocon 100° C.). Many microorganisms are mesophiles and Typical version processes using carbon dioxide to produce bio Mesophilic Conditions are typically preferred. products include those to make organic acids and esters I0088 Any suitable bioreactor assembly may be used and diacids and diesters such as Succinic acid and lactic including Typical Bioreactor Systems. The bioreactor may or acid. may not be sterilized prior to introducing the aqueous I0082 Combustion gases, e.g., from the disposal of solid medium. Due to the use of biocatalysts containing significant wastes or generation of energy, where the Substrate com populations of microorganisms, bioreactors can have a rapid prises contaminants sought to be removed from the start-up time. gases such as oxygenated halides, sulfoxy moieties, I0089. The bioreactor assembly contains water-insoluble nitrogen oxides, heavy metal compounds and the like. liquid and ME biocatalyst. The relative amounts of the liquid I0083 Industrial process waste gases containing, for and biocatalyst can fall within a broad range, especially since instance, Volatile organic compounds; solvents such as the microorganisms are contained within the interior of the chlorine containing solvents, ketones, aldehydes, per ME biocatalyst. Preferably sufficient water-insoluble liquid oxygenates, and the like; ammonia or volatile amines; is present to maintain the exterior Surfaces of the biocatalyst mercaptains and other Sulfur containing compounds; Wet. nitrogen oxides; and the like. The industrial process 0090 The bioconversion processes may be optimized to waste gases may be air-based. Such as exhaust from achieve one or more objectives. For instance, the processes painting operations, or maybe devoid of air Such as may be designed to provide high conversions of Substrate to purge or waste gases. The ability to Subject these Sub bioproduct or may be designed to balance capital and energy strates to catabolic degradation can often eliminate the costs against conversion to bioproduct. As the biocatalysts are necessity forathermal oxidation unit operation resulting highly hydrated, generally their density is close to that of in both capital and energy savings as often natural gas or water. In some instances where the metabolic processes gen other fuel is required to maintain temperature for the erate a gas, the gas can accumulate in the biocatalyst to thermal oxidation unit. increase buoyancy. This accumulated gas can reduce the I0084 Natural gas (including, but not limited to, gas energy consumption for a fluid bed operation and can facili recovered by underground fracturing processes, i.e., frac tate the use of other bioreactor designs such as jet loop biore gas) wherein the Substrate for catabolic processing may actOrS. be one or more of oxygenates, such as nitrogen oxides, 0091. The bioproduct may be recovered from the aqueous Sulfur oxides; perchlorates; Sulfides, ammonia; mercap medium in any Suitable manner including the Typical Sepa tans; and the like or wherein the substrate for anabolic ration Techniques. processing may be methane and other light hydrocar 0092. The duration of contact between the water-insoluble bons. liquid and the ME biocatalyst in the bioreactor assembly is I0085 Carbohydrate, including, but not limited to cellu Sufficient to achieve a sought conversion of Substrate to bio lose, hemicellulose, starches, and Sugars for bioconver product. Both mass transfer and biocatalytic rates are factors sion to oxygenated organic product and hydrocarbons. in determining the duration of contact. I0086. The bioconversion processes using the ME biocata (0093. Hydration of the ME biocatalysts may be achieved lysts may be conducted in any Suitable manner employing by any Suitable procedure. Typically for continuous processes metabolic conditions sufficient for the biocatalyst to convert a portion of the ME biocatalyst is continuously or intermit the substrate to the sought bioproduct. In the processes of this tently withdrawn for rehydration and supply of nutrients (in invention, certain metabolic conditions are defined by the cluding micronutrients). Where, for instance, the Substrate is Surrounding water-insoluble liquid and others are defined by not a carbon Source for the microorganisms, suitable carbon the environment in the interior of the ME biocatalyst. Meta Source is included in the nutrients. The processes can be bolic conditions broadly include conditions of temperature, conducted with all carbon requirements being provided in the pressure, oxygenation (oxidation reduction potential (ORP)). aqueous medium and introduced during hydration or on a US 2014/0106420 A1 Apr. 17, 2014

carbon Source deficient basis where a polysaccharide is atoms; organosilicon liquids such as polydimethylsiloxane: included in the ME biocatalyst. Where operating in a carbon hydrophobic organophosphates and phosphonates, e.g., con Source deficiency, the aqueous medium often provides at least taining about 10 to about 30 carbonatoms; organopolysulfide about 50, frequently at least about 75, say, about 80 to less liquids; and water-insoluble ionic liquids. U.S. Patent Appli than about 100, mass percent on a carbon basis of the carbon cation Publication No. 2010/0.143993 discloses various ionic nutrient. The ME biocatalysts often retain nutrients (includ liquids. This patent publication states that representative ing micronutrients) and thus the demand for nutrients is that examples of typical ionic liquids are described in sources less than that for planktonic microorganisms. The rehydration such as J. Chem. Tech. Biotechnol. 68:351-356 (1997); can also, in some instances, serve to remove from the interior Chem. Ind., 68:249-263 (1996); J. Phys. Condensed Matter, of the ME biocatalyst water soluble metabolites and micro 5: (supp34B):B99-B106 (1993); Chemical and Engineering organism waste. The frequency of rehydration will depend News, Mar. 30, 1998, 32-37; J. Mater. Chem., 8:2627-2636 upon, interalia, the nature of the microorganism and biocon (1998); Chem. Rev. 99:2071-2084 (1999); and US Patent version and upon the design of the ME biocatalyst. Application Publication 2004/0133058. The publication fur 0094 Water for the aqueous medium for hydration may be ther states that many ionic liquids are formed by reacting a provided from any suitable source including, but not limited nitrogen-containing heterocyclic ring, preferably a het to, tap water, demineralized water, distilled water, and pro eroaromatic ring, with an alkylating agent (for example, an cess or waste water streams. As stated above, this aqueous alkyl halide) to form a quaternary ammonium salt, and per medium can contain nutrients and additives such as co-me forming ion exchange or other Suitable reactions with various tabolites, potentiators, enhancers, inducers growth promot Lewis acids or their conjugate bases to form the ionic liquid. ers, buffers, antibiotics, vitamins, minerals, nitrogen Sources, Examples of Suitable heteroaromatic rings include Substi and Sulfur sources as is known in the art. If desired, an anti tuted pyridines, imidazole, Substituted imidazole, pyrrole and foam agent may be used in the aqueous medium. In some substituted pyrroles. These rings can be alkylated with virtu instances, where additives are desired or required for the ally any straight, branched or cyclic Co alkyl group, but metabolic process, the ME biocatalysts exhibit at least preferably, the alkyl groups are C groups, since groups equivalent bioconversion activity at a lesser concentration of larger than this may produce low melting Solids rather than Such additives as compared to a planktonic, free-Suspension ionic liquids. Various triarylphosphines, thioethers and cyclic system, all else being Substantially the same. and non-cyclic quaternary ammonium salts may also been 0095. A wide variety of water-insoluble liquids can be used for this purpose. Counterions that may be used include used in the processes of this invention. The water-insoluble chloroaluminate, bromoaluminate, gallium chloride, tet liquids may comprise one component or may be a mixture of rafluoroborate, tetrachloroborate, hexafluorophosphate, two or more components, which preferably are miscible. For nitrate, trifluoromethane Sulfonate, methylsulfonate, p-tolu instance, where a substrate or bioproduct has limited solubil enesulfonate, hexafluoroantimonate, hexafluoroarsenate, tet ity in one component, another component may be provided to rachloroaluminate, tetrabromoaluminate, perchlorate, co-solubilize the substrate or bioproduct. The exterior of the hydroxide anion, copper dichloride anion, iron trichloride ME biocatalyst in the bioreactor assembly during the biocon anion, Zinc trichloride anion, as well as various lanthanum, version process is Substantially anhydrous and absent a sepa potassium, lithium, nickel, cobalt, manganese, and other rate water phase. Often less than about 2, say, less than about metal-containing anions. 1.5, and preferably less than about 0.5, volume percent of the (0098 ME Biocatalyst total liquid to the exterior of the ME biocatalyst is a separate water phase. Accordingly, preferred processes of this inven A. ME Biocatalyst Overview tion involve unit operations to remove water from substrate (0099. The ME biocatalysts used in the processes of this and from the exterior of the ME biocatalyst after hydration invention have a polymeric structure (matrix) defining inter prior to introduction into the bioreactor assembly. connected major cavities, i.e., are open, porous matrices, in 0096. The selection of the water-insoluble liquid is gener which the microorganism is retained in the interior of the ally based upon its ability to dissolve the bioproduct, its matrices. It is believed that the microorganisms and their ability to solubilize the substrate, and its compatibility with communities, inter alia, regulate their population. Also, in the unit operations, if any, for the recovery of the bioproduct conjunction with the sensed nature of the microenvironment from the water-insoluble liquid. Although some water-in in the matrices, it is believed that the microorganisms estab soluble liquids can be deleterious to the ME biocatalyst, e.g., lish a spatial relationship among the members of the commu by dissolving the polymer of the biocatalyst, the enhanced nity. tolerance of the microorganisms retained in the ME biocata 0100. The microorganisms that are retained in the matri lyst enables the use of all but the most toxic substances in the ces often have the ability to form an exo-network. The quies water-insoluble liquids. In some instances where the separa cent nature of the cavities facilitate forming and then main tion of the bioproduct from the water-insoluble liquid is taining any formed exo-network. A discernable exo-network effected by evaporation or distillation, the preferred water is not believed essential to achieving phenotypicalterations in insoluble liquids have a normal boiling point at least about the microorganism population Such as population modulation 20°C. higher or lower than that of the bioproduct. and metabolic shift. Where an exo-network develops, often 0097 Examples of water-insoluble liquids include, but are Strands of EPS interconnect proximate microorganisms and not limited to, aliphatic and aromatic hydrocarbons that are connect microorganisms to the Surface and form the exo liquid under the conditions of the process Such as alkanes and network. In some instances, the microorganisms form thin halo-substituted alkanes of about 5 to about 20 carbons, and biofilms and these thin biofilms are encompassed in the exo aromatic; alkyl-substituted aromatics and halo Substituted network. The biocatalysts have a substantial absence of bio aromatic and alkyl-substituted aromatics of about 6 to about films in their interiors that are larger than thin biofilms. 24 carbon atoms; alkanols of about 8 to about 24 carbon Hence, any biofilms that may ultimately form in the biocata US 2014/0106420 A1 Apr. 17, 2014 lysts are relatively thin, e.g., up to about 10, and preferably up establishing defenses against toxins. For instance, the bio to about 2 or about 5, microns in thickness, and stable in size. catalysts Survive the addition of toxins such as ethanol and Thus, each thin biofilm is often only a few cells and is con Sodium hypochlorite and the original bioconversion activity nected in an exo-network. is quickly recovered thus indicating the Survival of essentially 0101 Communication among the microorganisms is the entire community. believed to occur through emitting chemical agents, includ 0107. In summary, due to the microenvironments in the ing, but not limited to, autoinducers, and communication ME biocatalyst, communication among the microorganisms includes communications for community behavior and for and the phenotypic alterations undergone by the microorgan signaling. Often, the preparation of the biocatalysts used in isms, the biocatalysts provide a number of process-related the processes of this invention can result in a population of advantages including, but not limited to, microorganisms being initially located in the interior of the 0.108 no solid debris being generated, biocatalyst that is substantially that which would exist at the 0.109 the potential for high densities of microorganism steady-state level. At these densities of microorganisms in the in a bioreactor, biocatalysts, community communications are facilitated 0110 stable population of microorganisms and bioac which are believed to commence during the formation of the tivity over extended periods of time, biocatalysts, and phenotypic shifts occur to enable the meta 0.111 metabolic shift of microorganisms towards pro bolic retention and modulate the population of microorgan duction rather than growth and carbon flow shift, 1SS. 0112 ability of microorganisms to undergo essential 0102) Another phenotypic alteration occurring in the bio stasis for extended durations, catalysts, which is believed to be a result of this communica 0113 ability to quickly respond to changes in substrate tion, is a metabolic shift, i.e., the metabolic functions of the rate of Supply and concentration, community towards reproduction are diminished and the 0114 attenuation of diauxie, sought bioconversion continues. The population of microor 0115 enhanced control and modulation of pH and ganisms in the biocatalyst may tend to have an old average redox balances in the microenvironment of the biocata age due to this shift in the metabolic activity. Older microor lyst, ganisms also tend to provide a more robust and Sustainable 0116 greater tolerance to substrate, bioproduct and performance as compared to younger cells as the older cells contaminants, have adapted to the operating conditions. 0.117 ability to bioconvert substrate at ultralow concen 0103 Additional benefits of this communication can bean trations, increase in community-level strength or fitness exhibited by 0118 ability to use slower growing and less robust the community in warding off adventitious microorganisms microorganisms and increased resistance to competi and maintaining strain-type uniformity. In some instances, tiveness, the microorganisms during use of the biocatalyst may 0119 enhanced microorganism strain purity capabili undergo natural selection to cause the strain-type in the com ties, munity to become heartier or provide another benefit for the 0120 ability to be subjected to in situ antimicrobial Survival of the community of microorganisms. In some treatment, instances, the communication among the microorganisms 0121 ability to quickly start a bioreactor since the den may permit the population of microorganisms to exhibit mul sity of microorganism required at full operation is con ticellularity or multicellular-like behaviors. Thus, the popu tained in the biocatalyst, lation of microorganisms in a biocatalyst of this invention 0.122 ability to contact biocatalyst with gas phase sub may have microorganisms adapting to different circum strate, and stances but yet working in unison for the benefit of the com 0123 ease of separation of bioproduct from biocatalyst munity. thereby facilitating continuous operations. 0104. In some instances the porous matrix may provide 0.124. If desired, the ME biocatalysts may be treated to modulation of the Substrate and nutrients to the microorgan enhance the formation of the exo-network, and if desired, thin isms to effect to optimize metabolic pathways involving Sub biofilms, prior to use in the metabolic process. However, strates that are available, and these pathways may or may not performance of the porous matrices is not generally depen be the primarily used pathways where ample Substrate and dent upon the extent of exo-network formation, and often other nutrients are available. Accordingly, microorganisms in bioconversion activities remain relatively unchanged the biocatalysts may exhibit enhanced bioactivity for a pri between the time before the microorganisms have attached to marily used pathway or metabolic activity that is normally the polymeric structure and the time when extensive exo repressed. network structures have been generated. 0105. It is also believed that the microenvironments may promote genetic exchange or horizontal gene transfer. Con B. Physical Description of the Porous Matrices jugation or bacterial mating may also be facilitated, including 0.125. The ME biocatalysts used in the processes of this the transfer of plasmids and chromosomal elements. More invention comprise a matrix having open, porous interior over, where microorganisms lyse, strands of DNA and RNA structure with microorganism irreversibly retained in at least in the microenvironments are more readily accessible to be the major cavities of the matrix. taken up by microorganisms in these microenvironments. 0.126 The matrices may be a self-supporting structure or These phenomena can enhance the functional abilities of the may be placed on or in a preformed structure Such as a film, microorganisms. fiber or hollow fiber, or shaped article. The preformed struc 0106 The biocatalysts exhibit an increased tolerance to ture may be constructed of any suitable material including, toxins. In some instances, communications among microor but not limited to, metal, ceramic, polymer, glass, wood, ganisms and the exo-network may facilitate the population composite material, natural fiber, stone, and carbon. Where US 2014/0106420 A1 Apr. 17, 2014

self-supporting, the matrices are often in the form of sheets, rior of the biocatalyst. These contaminating microorganisms cylinders, plural lobal structures such as trilobal extrudates, are often subject to removal under even low physical forces hollow fibers, or beads which may be spherical, oblong, or such as by the flow of fluid around the biocatalysts. Thus, the free-form. The matrices, whether self-supporting or placed fouling of the biocatalyst can be substantially eliminated or on or in a preformed structure, preferably have a thickness or mitigated by washing or by fluid flows during use. axial dimension of less than about 5, preferably less than 0.132. Where present, the skin typically has pores of an about 2, say, between about 0.01 to about 1, centimeters. average diameter of between about 1 and about 10, preferably 0127. The porous matrices may have an isotropic or, pref about 2 to about 7, microns in average diameter. The pores erably, an anisotropic structure with the exteriorportion of the may comprise about 1 to about 30, say, about 2 to about 20, cross section having the densest structure. The major cavities, percent of the external Surface area. The external skin, in even if an anisotropic structure exists, may be relatively uni addition to providing a barrier to entry of adventitious micro form in size throughout the interior of the matrix or the size of organisms into the interior of the biocatalyst, is preferably the major cavities, and their frequency, may vary over the relatively smooth to reduce the adhesion of microorganisms cross-section of the biocatalyst. to the external side of the skin through physical forces such as 0128. The ME biocatalyst has major cavities, that is, open, fluid flow and contact with other solid surfaces. Often, the interconnected regions of between about 5 or about 10 to skin is Substantially devoid of anomalies, other than pores, about 70 or about 100 microns in the smallest dimension greater than about 2 or about 3 microns. Where a skin is (excluding any microorganisms contained therein). For the present, its thickness is usually less than about 50, say, purposes of ascertaining dimensions, the dimensions of the between about 1 to about 25, microns. It should be understood microorganisms includes any mass in the exo-network. In that the thickness of the skin can be difficult to discern where many instances, the major cavities have nominal major the porous matrix has an anisotropic structure with the dens dimensions of less than about 300, preferably less than about est structure being at the exterior of the matrix. 200, microns, and sometimes a smallest dimension of at least I0133) A high density of microorganisms can exist at about 10 microns. Often the biocatalyst contains smaller steady-state operation within the ME biocatalysts. The com channels and cavities which are in open communication with bination of the flow channels and the high permeability of the the major cavities. Frequently the Smaller channels have a polymeric structure defining the channels enable viable maximum cross-sectional diameter of between about 0.5 to microorganism population throughout the matrix, albeit with about 20, e.g., about 1 to about 5 or about 10, microns. The a plurality of unique microenvironments and nano-environ cumulative volume of major cavities, excluding the Volume ments. In some instances the cell density based upon the occupied by microorganisms and mass associated with the volume of the matrices is preferably at least about 100 grams microorganisms, to the Volume of the biocatalyst is generally per liter, preferably at least about 150 or about 200, and often in the range of about 40 or about 50 to about 70 or about 99. between about 250 and about 750, grams per liter. Volume percent. In many instances, the major cavities consti tute less than about 70 percent of the volume of the fully Polysaccharide-Containing ME Biocatalysts catalyst with the remainder constituting the Smaller channels I0134. By incorporating polysaccharide in the interior of and pores. The volume fraction of the biocatalyst that consti the ME biocatalyst, the viability of the microorganism popu tute the major cavities can be estimated from its cross-section. lation can be maintained. Typically polysaccharides are not The cross section may be observed via any suitable micro usable by most microorganisms. Often, the polysaccharide is Scopic technique, e.g., Scanning electron microscopy and provided in an amount of at least about 0.1, say, at least about high powered optical microscopy. The total pore Volume for 0.2 to about 100, gram per gram of cells retained in the the matrices can be estimated from the Volumetric measure biocatalyst, and sometimes the biocatalyst contains between ment of the matrices and the amount and density of polymer, about 25 and about 500 grams of polysaccharide per liter of and any other Solids used to make the matrices. volume of fully hydrated biocatalyst. The polysaccharide 0129. The ME biocatalyst is characterized by having high particles used in preparing the biocatalysts preferably have a internal Surface areas, often in excess of at least about 1 and major dimension of less than about 50, preferably less than Sometimes at least about 10, square meter per gram. In some about 20, often between about 0.1 to about 5, microns. The instances, the volume of water that can be held by a fully Solid polysaccharide particles are preferably granular and hydrated biocatalyst (excluding the volume of the microor often have an aspect ratio of minimum cross-sectional dimen ganisms) is in the range of about 90 to about 99 or more, sion to maximum cross sectional dimension of between about percent. Preferably, the biocatalyst exhibits a Hydration 1:10 to about 1:1, say about 1:2 to about 1:1. Expansion Volume (HEV) of at least about 1000, frequently I0135. Due to the ability of the polysaccharide to maintain at least about 5000, preferably at least about 20,000, and the viability of the microorganisms in the biocatalyst, the sometimes between 50,000 to about 200,000, percent. storage, handling and processes for use of the biocatalyst can 0130. Usually, the type of polymer selected and the void be facilitated. For instance, the biocatalysts can be used in Volume percent of the matrices are such that the matrices have bioconversion processes which are operated in a carbon defi adequate strength to enable handling, storage and use in a cient manner. In metabolic processes where carbon Source is bioconversion process. added to maintain the microorganisms and not used in the 0131 The porous matrices may or may not have an exte sought bioconversion of Substrate to bioproduct, Such as in rior skin. Preferably, the matrices have an exterior skin to the catabolysis of nitrate, nitrite, and perchlorate anions and assist in modulating the influx and efflux of components to the metabolic reduction of metalates, the polysaccharide may and from the interior channels of the porous matrix. Also, serve as the sole source of carbon and thereby eliminate the since the skin is highly hydrophilic, and additional benefit is necessity of adding carbon Source, or it may reduce the obtained as contaminating or adventitious microorganisms amount of carbon Source added, i.e., permit carbon deficient have difficulties in establishing a strong biofilm on the exte operation. An advantage is that the bioprocesses can be oper US 2014/0106420 A1 Apr. 17, 2014

ated such that the effluent has essentially no COD. The bio and microorganisms but including both the hydrophilic poly catalysts also have enhanced abilities to tolerate disruptions merand the particulates). More than one solid sorbent may be in Substrate presence and be able to quickly regain biocon used in a biocatalyst. Preferably the solid sorbent is relatively version activity. Also, the biocatalysts can be remotely manu uniformly dispersed throughout the interior of the biocatalyst factured and shipped to the location of use without undue although the solid sorbent may have a varying distribution deleterious effect on the bioconversion activity of the biocata within the biocatalyst. Where the distribution varies, the lyst. The biocatalysts may be able enter a state of essential regions with the higher concentration of solid sorbent often Stasis for extended durations of time in the absence of Sup are found toward the surface of the biocatalyst. plying Substrate and other nutrients to the microbial compos 0.138. Where a particulate sorbent is used, the sorbent ites even where excursions in the desired storage conditions comprises an organic or inorganic material having the sought Such as temperature occur. The bioactivity can be quickly Sorptive capacity. Examples of Solid sorbents include, with regained in a bioreactor even after extended episodic occur out limitation, polymeric materials, especially with polar rences of shutdown, feedstock disruption, or feedstock Vari moieties, carbon (including but not limited to activated car ability. The biocatalysts can be packaged and shipped in bon), silica (including but not limited to fumed silica), sili sealed barrels, tanks, and the like. The polysaccharide may be cates, clays, molecular sieves, and the like. The molecular from any Suitable source including, but not limited to, cellu sieves include, but are not limited to Zeolites and synthetic losic polysaccharides or starches. Polysaccharides are carbo crystalline structures containing oxides and phosphates of hydrates characterized by repeating units linked together by one or more of silicon, aluminum, titanium, copper, cobalt, glycosidic bonds and are Substantially insoluble in water. Vanadium, titanium, chromium, iron, nickel, and the like. The Polysaccharides may be homopolysaccharides or het Sorptive properties may comprise one or more of physical or eropolysaccharides and typically have a degree of polymer chemical or quasi-chemical sorption on the Surface of the ization of between about 200 to about 15,000 or more, pref Solid sorbent. Thus, Surface area and structure may influence erably from about 200 to about 5000. The preferred the sorptive properties of some solid sorbents. Frequently the polysaccharides are those in which about 10, more preferably, Solid sorbents are porous and thus provide high Surface area at least about 20, percent of the repeating units are amylose and physical sorptive capabilities. Often the pores in the solid (D-glucose units). Most preferably the polysaccharide has at sorbents are in the range of about 0.3 to about 2 nanometers in least about 20, more preferably, at least about 30, percent of effective diameter. the repeating units being amylose. The polysaccharides may 0.139. The solid sorbent may be incorporated into the poly or may not be functionalized, e.g., with acetate, Sulfate, phos meric structure in any convenient manner, preferably during phate, pyruvyl cyclic acetal, and the like, but such function the preparation of the biocatalyst. alization should not render the polysaccharide water soluble at temperatures below about 50° C. A preferred class of Phosphorescent ME Biocatalysts polysaccharides is starches. 0140. Another preferred aspect of the invention pertains to 0.136 Sources of polysaccharides include naturally occur biocatalysts containing phosphorescent material and photo ring and synthetic (e.g., polydextrose) polysaccharides. Vari synthetic microorganisms, i.e., microorganisms that uses ous plant based materials providing polysaccharides include light energy in a metabolic process. Preferably the microor but are not limited to woody plant materials providing cellu ganism is an algae, most preferably a microalgae, or cyano lose and hemicellulose, and wheat, barley, potato, Sweet bacteria. potato, tapioca, corn, maize, cassaya, milo, rye and brans 0.141. The bioactivity of photosynthetic microorganisms typically providing starches. can be enhanced to produce expressed bioproduct using broad-based light Source Such as Sunlight. In accordance with Solid Sorbent-Containing Biocatalysts the invention, the photosynthetic microorganisms are irre 0.137 The ME biocatalysts may contain a solid sorbent. versibly retained in biocatalysts in which the interior of the The solid sorbent may be the hydrophilic polymer forming biocatalyst contains phosphorescent material capable of the structure or may be a particulate, i.e., a distinct Solid shifting UV light to light having a wavelength of between structure regardless of shape) contained in the Solid structure. about 400 to about 800, preferably from about 450 to about The sorbent may be any suitable solid sorbent for the sub 650, nm and is capable of exhibiting persistence, with the strate or nutrients or other chemical influencing the sought emission of the light often lasting for at least about 5 seconds. metabolic activity Such as, but not limited to, co-metabolites, A phosphorescent material is a material that has the ability to inducers, and promoters or for components that may be be excited by electromagnetic radiation into an excited State, adverse to the microorganisms such as, and not in limitation, but the stored energy is released gradually. Emissions from toxins, phages, bioproducts and by-products. The Solid Sor phosphorescent materials have persistence, that is, emissions bent is typically an adsorbent where the Sorption occurs on the from Such materials can last for seconds, minutes or even surface of the sorbent. The particulate solid sorbents are pref hours after the excitation Source is removed. A luminescent erably nano materials having a major dimension less than material is a material capable of emitting electromagnetic about 5 microns, preferably, between about 5 nanometers to radiation after being excited into an excited State. Persistence about 3 microns. Where the solid sorbent is composed of is the time it takes, after discontinuing irradiation, for photo polymer, the Solid structure may be essentially entirely com luminescent emissions emanating from a photoluminescent posed of the polymer or may be a block copolymer or poly object to decrease to the threshold detectability. meric mixture constituting between about 5 and about 90 0142. The persistence of the radiation enables the micro mass percent of the solid structure (excluding water). Where organisms to be cycled in and out of a region of the culture the solid sorbent is a separate particulate in the biocatalyst, liquid exposed to the light source and still be productive. With the biocatalyst may comprise between about 5 to about 90 longer persistence durations, the photosynthetic microorgan mass percent of the mass of the biocatalyst (excluding water isms can continue photo-bioconversion in the absence of or US 2014/0106420 A1 Apr. 17, 2014 reduction in light intensity. The ability of the biocatalysts to 0147 High intensity and high persistence silicates have maintain photosynthetic activity over extended periods of been disclosed in U.S. Pat. No. 5,839,718, such as Sr.BaO. time, often at least about 30 days, and in some instances for at Mg.MO.SiGe:Eu:Ln wherein M is beryllium, zinc or cad least about one year, the cost of the phosphorescent materials mium and Ln is chosen from the group consisting of the rare is well offset by the increased production, reduced footprint earth materials, the group 3A elements, Scandium, titanium, of the bioreactor, and facilitated bioproduct recover). Vanadium, chromium, manganese, yttrium, Zirconium, nio 0143. The biocatalyst, being highly hydrated is a signifi bium, molybdenum, hafnium, tantalum, tungsten, indium, cant distributor of light radiation to photosynthetic microor thallium, phosphorous, arsenic, antimony, bismuth, tin, and ganisms retained in the interior of the biocatalyst and also lead. Particularly useful are dysprosium, neodymium, thu serves to protect the microorganism from photorespiration. lium, tin, indium, and bismuth. X in these compounds is at The solid debris in the culture liquid (an aqueous solution least one halide atom. comprising nutrients for metabolic processes) can be materi 0.148. Other phosphorescent materials include alkaline ally reduced, if not essentially eliminated, due to the micro earth aluminates of the formula MO. Al-O.B.O.R wherein organisms being irreversibly retained in the biocatalyst. Thus M is a combination of more than one alkaline earth metal the turbidity is reduced and a given light intensity can thus be (strontium, calcium or barium or combinations thereof) andR found at a greater depth in the culture liquid. These advan is a combination of Eu" activator, and at least one trivalent tages provided by the ME biocatalysts can be realized in any rare earth material co-activator, (e.g. lanthanum, cerium, photosynthetic process regardless of whether or not a phos praseodymium, neodymium, Samarium, gadolinium, ter phorescent material is used. bium, dysprosium, holmium, erbium, thulium, ytterbium, 0144. Examples of phosphorescent materials include, but lutetium), bismuth or manganese. Examples of Such phos are not limited to, phosphorescent materials are metal Sulfide phors can be found in U.S. Pat. No. 5,885.483. Alkaline earth phosphors such as ZnCdS:Cu: Al, ZnCdS:Ag: Al, ZnS:Ag:Al, aluminates of the type MAl-O, which are described in U.S. ZnS:Cu:Al as described in U.S. Pat. No. 3,595,804 and metal Pat. No. 5,424,006, may also find application as may phos sulfides that are co-activated with rare earth elements such as phorescent materials comprising a donor system and an those describe in U.S. Pat. No. 3,957,678. Phosphors that are acceptor system such as described in U.S. Pat. No. 6,953,536 higher in luminous intensity and longer in luminous persis B2. tence than the metal Sulfide pigments include compositions 0149. As can be appreciated, many other phosphors can comprising a host material that is generally an alkaline earth find application. See, for instance, Yen and Weber, Inorganic aluminate, or an alkaline earth silicate. The host materials Phosphors: Compositions, Preparation and Optical Proper generally comprise Europium as an activator and often com ties, CRC Press, 2004. prise one or more co-activators such as elements of the Lan 0150. The phosphorescent material may be a discrete par thanide series (e.g. lanthanum, cerium, praseodymium, ticle or may be a particle having a coating to facilitate incor neodymium, Samarium, gadolinium, terbium, dysprosium, poration and retention in the polymer forming the matrix. The holmium, erbium, thulium, ytterbium, and lutetium), tin, particles may be of any suitable shape. Generally the maxi manganese, yttrium, or bismuth. Examples of such phosphors mum dimension of the of the particles is less than about 1 are described in U.S. Pat. No. 5,424,006. millimeter, preferably less than about 0.1 millimeter. The 0145 High emission intensity and persistence phospho particles may be nanoparticles. rescent materials can be alkaline earth aluminate oxides hav 0151. The persistence time exhibited by the phosphores ing the formula MOAlO:Eu", R" wherein m is a number cent materials can range from a short duration, e.g., about 5 to ranging from about 1.6 to about 2.2, M is an alkaline earth about 10 seconds, to as much as about 10 or about 20 hours or metal (strontium, calcium or barium), Eu" is an activator, more and will be dependent upon the phosphorescent mate and R is one or more trivalent rare earth materials of the rial used. Preferred phosphorescent materials exhibit a per lanthanide series (e.g. lanthanum, cerium, praseodymium, sistence of at least about one minute. The intensity of the neodymium, Samarium, gadolinium, terbium, dysprosium, emitted radiation from the polymer of the matrices will, in holmium, erbium, thulium, ytterbium, lutetium), yttrium or part, depend upon the concentration of the phosphorescent bismuth co-activators. Examples of Such phosphors are material in the polymer and the nature of the phosphorescent described in U.S. Pat. No. 6,117.362. Phosphorescent mate material. Typically the phosphorescent material is provided rials also include alkaline earth aluminate oxides having the in an amount of at least about 0.1, say, between 0.2 and about formula M. Al-O.2xEu", 2yR'" wherein k=1-2x-2y, x is a 5 or about 10, mass percent of the polymer (non-hydrated) in number ranging from about 0.0001 to about 0.05, y is a the biocatalyst. One or more phosphorescent materials may number ranging from about X to 3X, M is an alkaline earth be used in the biocatalyst. Where more than one phosphores metal (strontium, calcium or barium), Eu" is an activator, cent material are used, the combination may be selected to and R is one or more trivalent rare earth materials (e.g. lan provide one or more of wave shifting from different light thanum, cerium, praseodymium, neodymium, Samarium, wavelengths contained in the band width of the radiation gadolinium, terbium, dysprosium, holmium, erbium, thu Source and providing differing persistence times. In preferred lium, ytterbium, lutetium), yttrium or bismuth co-activators. embodiments the phosphorescent materials are in the form of See U.S. Pat. No. 6,267,911B1. nanoparticles, e.g., having a major dimension of between 0146 Phosphorescent materials also include those in about 10 nm to about 10 Jum. In some instances, it may be which a portion of the Al" in the host matrix is replaced with desired to coat the phosphorescent materials with a compati divalent ions such as Mg" or Zn" and those in which the bilizing agent to facilitate incorporation of the phosphores alkaline earth metalion (M2) is replaced with a monovalent cent material within the polymer. Compatibilizing agents alkali metal ion such as Li", Na', K", Cs" or Rb" such as include, but are not limited to, molecules having one or more described in U.S. Pat. Nos. 6,117,362 and 6,267,911B1. of hydroxyl, thiol, silyl, carboxyl, or phosphoryl groups. US 2014/0106420 A1 Apr. 17, 2014

C. Methods for Making ME Biocatalysts include Solution polymerization, slurry polymerization (char 0152 The components, including microorganisms, used acterized by having two or more initial phases), and Solidifi to make the ME biocatalysts and the process conditions used cation by cooling or removal of Solvent. for the preparation of the ME biocatalysts are not critical to 0157. The biocatalysts may be formed in situ in the liquid the broad aspects of this invention and may vary widely as is medium by Subjecting the medium to solidification condi well understood in the art once understanding the principles tions (such as cooling or evaporation) or adding a component described above. In any event, the components and process to cause a polymerization or cross-linking or agglomeration conditions for making the biocatalysts with the irreversibly, of Solids to occur to form a solid structure Such as a catalyst, metabolically retained microorganisms should not adversely cross-linking agent or coagulating agent. Alternatively, the affect the microorganisms. liquid medium may be extruded into a solution containing a 0153. The ME biocatalysts may be prepared from a liquid Solidification agent Such as a catalyst, cross-linking or coagul medium containing the microorganism and solubilized pre lating agent or coated onto a Substrate and then the composite cursor for the hydrophilic polymer which may be one or more subjected to conditions to form the solid biocatalyst. of a polymerizable or solidifiable component or a solid that is 0158 Polymeric materials used to make the biocatalysts fusible or bondable to form the matrix. Aqueous media are may have an organic or inorganic backbone but have suffi most often used due to the compatibility of most microorgan cient hydrophilic moieties to provide a highly hydrophilic isms and enzymes with water. However, with microorgan polymer which when incorporated into the matrices exhibits isms that tolerate other liquids, such liquids can be used to Sufficient water sorption properties to provide the sought make all or a portion of the liquid medium. Examples of Such Hydration Expansion Volume of the biocatalyst. Polymeric other liquids include, but are not limited to liquid hydrocar materials are also intended to include high molecular weight bons, peroxygenated liquids, liquid carboxy-containing com Substances such as waxes (whether or not prepared by a pounds, and the like. Mixed liquid media can also be used to polymerization process), oligomers and the like so long as prepare the biocatalyst. The mixed media may comprise mis they form biocatalysts that remain solid under the conditions cible or immiscible liquid phases. For instance, the microor of the bioconversion process intended for their use and have ganism may be suspended in a dispersed, aqueous phase and sufficient hydrophilic properties that the Hydration Expan the polymerizable or solidifiable component may be con sion Volume can be achieved. As stated above, it is not essen tained in a continuous solvent phase. tial that polymeric materials become cross-linked or further 0154 The liquid medium used to prepare the biocatalyst polymerized in forming the polymeric matrix. may contain more than one type of microorganism, especially 0159. Examples of polymeric materials include where the microorganisms do not significantly compete for homopolymers and copolymers which may or may not be the same Substrate, and may contain one or more isolated cross-linked and include condensation and addition polymers enzymes or functional additives such as polysaccharide, Solid that provide high hydrophilicity and enable the Hydration sorbent and phosphorescent materials, as described above. Expansion Volumes to be obtained. The polymer may be a Preferably, the biocatalysts contain a single type of microor homopolymer or a copolymer, say, of a hydrophilic moiety ganism. The concentration of the microorganisms in the liq and a more hydrophobic moiety. The molecular weight and uid medium used to make the biocatalysts should at least be molecular weight distribution are preferably selected to pro about 60 grams per liter. As discussed above, the concentra vide the combination of hydrophilicity and strength as is tion of microorganisms should preferably approximate the known in the art. The polymers may be functionalized with sought density of microorganisms in the biocatalyst. The hydrophilic moieties to enhance hydrophilicity. Examples of relative amounts of microorganism and polymeric material in hydrophilic moieties include, but are not limited to hydroxyl, forming the biocatalyst can vary widely. The growth of the alkoxyl, acyl, carboxyl, amido, and oxyanions of one or more population of microorganisms post formation of the biocata of titanium, molybdenum, phosphorus, Sulfur and nitrogen lyst is contemplated as well as the potential for damage to Such as phosphates, phosphonates, Sulfates, Sulfonates, and Some of the population of microorganisms during the biocata nitrates, and the hydrophilic moieties may be further substi lyst-forming process. Nevertheless, higher microorganism tuted with hydrophilic moieties such as hydroxyalkoxides, concentrations are generally preferred, e.g., at least about 100 acetylacetonate, and the like. Typically the polymers contain grams per liter, preferably at least about 200, and often carbonyl and hydroxyl groups, especially at Some adjacent between about 250 to 750, grams per liter of the liquid hydrophilic moieties such as glycol moieties. In some medium used to make the biocatalysts. instances, the backbone of the polymer contains ether oxy 0155 Any suitable process may be used to solidify or gens to enhance hydrophilicity. In some instances, the atomic polymerize the polymeric material or to adhere or fuse par ratio of oxygen to carbon in the polymer is between about ticles to form the open, porous polymeric matrix with micro 0.3:1 to about 5:1. organism irreversibly retained therein. The conditions of suit 0160 Polymers which may find use in forming the matri able processes should not unduly adversely affect the ces include functionalized or non-functionalized polyacryla microorganism. As microorganisms differ in tolerance to mides, polyvinyl alcohols, polyetherketones, polyurethanes, temperatures, pressures and the presence of other chemicals, polycarbonates, polysulfones, polysulfides, polysilicones, Some matrix-forming processes may be more advantageous olefinic polymers such as polyethylene, polypropylene, for one type of microorganism than for another type of micro polybutadiene, rubbers, and polystyrene, nylons, polythylox organism. aZyoline, polyethylene glycol, polysaccharides Such as 0156 Preferably the polymeric matrix is formed from Sodium alginate, carrageenan, agar, hyaluronic acid, chon Solidification of a high molecular weight material, by poly droitin Sulfate, dextran, dextran Sulfate, heparin, heparin Sul merization or by cross-linking of prepolymer in manner that fate, heparin Sulfate, chitosan, gellangum, Xanthan gum, guar a population of microorganisms is provided in the interior of gum, water Soluble cellulose derivatives and carrageenan, and the biocatalyst as it is being formed. Exemplary processes proteins such as gelatin, collagen and albumin, which may be US 2014/0106420 A1 Apr. 17, 2014 polymers, prepolymers or oligomers, and polymers and 0162 Cross linking agents, accelerators, polymerization copolymers from the following monomers, oligomers and catalysts, and other polymerization additives may be prepolymers: monomethacrylates such as polyethylene gly employed such as triethanolamine, triethylamine, ethanola col monomethacrylate, polypropylene glycol mine, N-methyl diethanolamine, N,N-dimethyl benzy monomethacrylate, polypropylene glycol monomethacry lamine, dibenzyl amino, N-benzyl ethanolamine, N-isopro late, methoxydiethylene glycol methacrylate, methoxypoly pyl benzylamino, tetramethyl ethylenediamine, potassium ethylene glycol methacrylate, methacryloyloxyethyl hydro persulfate, tetramethyl ethylenediamine, lysine, ornithine, gen phthalate, methacryloyloxyethyl hydrogen Succinate, histidine, arginine, N-vinyl pyrrolidinone, 2-vinyl pyridine, 3-chloro-2-hydroxypropyl methacrylate, Stearyl methacry 1-vinyl imidazole, 9-vinyl carbazone, acrylic acid, and 2-al late, 2-hydroxy methacrylate, and ethyl methacrylate; lyl-2-methyl-1,3-cyclopentane dione. For polyvinyl alcohol monoacrylates Such as 2-hydroxyethyl acrylate, 2-hydrox polymers and copolymers, boric acid and phosphoric acid ypropyl acrylate, isobutyl acrylate, t-butyl acrylate, isooctyl may be used in the preparation of polymeric matrices. As acrylate, lauryl acrylate, Stearyl acrylate, isobornyl acrylate, stated above, the amount of cross-linking agent may need to cyclohexyl acrylate, methoxytriethylene glycol acrylate, be limited to assure that the matrices retain high hydrophilic 2-ethoxyethyl acrylate, tetrahydrofurfuryl acrylate, phenoxy ity and the ability to have a high Hydration Expansion Vol ethyl acrylate, nonylphenoxypolyethylene glycol acrylate, ume. The selection of the polymer and cross-linking agents nonylphenoxypolypropylene glycol acrylate, silicon-modi and other additives to make porous matrices having the physi fied acrylate, polypropylene glycol monoacrylate, phenoxy cal properties set forth above is within the level of the artisan ethyl acrylate, phenoxydiethylene glycol acrylate, phenoxy in the art of water soluble and highly hydrophilic polymer polyethylene glycol acrylate, methoxypolyethylene glycol synthesis. acrylate, acryloyloxyethyl hydrogen Succinate, and lauryl 0163 The ME biocatalysts may beformed in the presence acrylate; dimethacrylates Such as 1,3-butylene glycol of other additives which may serve to enhance structural dimethacrylate, 1,4-butanediol dimethacrylate, ethylene gly integrity or provide a beneficial activity for the microorgan col dimethacrylate, diethylene glycol dimethacrylate, trieth ism Such as attracting or sequestering components, providing ylene glycol dimethacrylate, polyethylene glycol nutrients, and the like. Additives can also be used to provide, dimethacrylate, butylene glycol dimethacrylate, hexanediol for instance, a suitable density to be suspended in the aqueous dimethacrylate, neopentylglycol dimethacrylate, polyprene medium rather than tending to float or sink in the broth. glycol dimethacrylate, 2-hydroxy-1,3-dimethacryloxypro Typical additives include, but are not limited to, starch, gly pane, 2.2-bis-4-methacryloxyethoxyphenylpropane, 3.2-bis cogen, cellulose, lignin, chitin, collagen, keratin, clay, alu 4-methacryloxydiethoxyphenylpropane, and 2.2-bis-4-meth mina, aluminosilicates, silica, aluminum phosphate, diato acryloxypolyethoxyphenylpropane; diacrylates Such as maceous earth, carbon, polymer, polysaccharide and the like. ethoxylated neopentylglycol diacrylate, polyethylene glycol These additives can be in the form of solids when the poly diacrylate, 1.6-hexanediol diacrylate, neopentylglycol dia meric matrices are formed, and if so, are often in the range of crylate, tripropylene glycol diacrylate, polypropylene glycol about 0.01 to 100 microns in major dimension. diacrylate, 2.2-bis-4-acryloxyethoxyphenylpropane, 2-hy 0164. If desired, where the biocatalyst contains microor droxy-1-acryloxy-3-methacryloxypropane; trimethacrylates ganisms, they may be subjected to stress as is known in the art. Such as trimethylolpropane trimethacrylate; triacrylates Such Stress may be one or more of starvation, chemical or physical as trimethylolpropane triacrylate, pentaerythritol triacrylate, conditions. Chemical stresses include toxins, antimicrobial trimethylolpropane EO-added triacrylate, glycerol PO-added agents, and inhibitory concentrations of compounds. Physical triacrylate, and ethoxylated trimethylolpropane triacrylate; stresses include light intensity, UV light, temperature, tetraacrylates Such as pentaerythritol tetraacrylate, ethoxy mechanical agitation, pressure or compression, and desicca lated pentaerythritol tetraacrylate, propoxylated pentaeryth tion or osmotic pressure. The stress may produce regulated ritol tetraacrylate, and ditrimethylolpropane tetraacrylate; biological reactions that protect the microorganisms from urethane acrylates Such as urethane acrylate, urethane dim shock and the stress may allow the hardier microorganisms to ethyl acrylate, and urethane trimethyl acrylate; amino-con survive while the weaker cells die. taining moieties Such as 2-aminoethyl acrylate, 2-aminoethyl methacrylate, aminoethyl methacrylate, dimethylaminoethyl Microorganism methacrylate, monomethylaminoethyl methacrylate, t-buty laminoethylmethacrylate, p-aminostyrene, o-aminostyrene, 0.165. The microorganism for a ME biocatalyst is one or 2-amino-4-vinyltoluene, dimethylaminoethyl acrylate, more microorganisms. In another aspect, the biocatalysts can diethylaminoethyl acrylate, piperidinoethyl ethyl acrylate, contain, in addition to the microorganisms, one or more extra piperidinoethyl methacrylate, morpholinoethyl acrylate, cellular enzymes, or isolated enzymes, in the interior of the morpholinoethyl methacrylate, 2-vinyl pyridine, 3-vinyl biocatalyst to cause a catalytic change to a component which pyridine, 2-ethyl-5-vinyl pyridine, dimethylaminopropyl may be substrate or other nutrients, or a bioproduct or by ethyl acrylate, dimethylaminopropylethyl methacrylate, product or co-product of the microorganisms, or may be a 2-vinyl pyrrolidone, 3-vinyl pyrrolidone, dimethylaminoet toxin, phage or the like. hyl vinyl ether, dimethylaminoethyl vinyl sulfide, diethy 0166 Examples of enzymes include, but are not limited to, laminoethyl vinyl ether, 2-pyrrolidinoethyl acrylate, 2-pyrro one or more of oxidorectases, transferases, hydrolases, lidinoethyl methacrylate, lyases, isomerases, and ligases. The enzymes may cause one or more metabolic conversions. For instance, an enzyme may and other monomers such as acrylamide, acrylic acid, and metabolize a component in the feed such that it can be bio dimethylacrylamide. converted, or more easily be bioconverted, by the microor 0161. Not all the above listed polymers will be useful by ganisms in the biocatalyst. An enzyme may be used to themselves, but may be required to be functionalized or used metabolize a metabolite of the microorganism either to pro to form a co-polymer with a highly hydrophilic polymer. vide a sought bioproduct. An enzyme may be used to metabo US 2014/0106420 A1 Apr. 17, 2014

lize a component in the feed or a co-metabolite from the 0170 The microorganisms used in the ME biocatalysts microorganism that may be adverse to the microorganism may be unicellular or may be multicellular that behaves as a into a metabolite that is less adverse to the microorganism. If single cell microorganism such as filamentous growth micro desired, two or more different enzymes can be used to effect organisms and budding growth microorganisms. Often the a series of metabolic conversions on a component in the feed cells of multicellular microorganisms have the capability to or a metabolite from the microorganism. exist singularly. The microorganisms can be of any type, 0167 Representative enzymes include, without limita including, but not limited to, those microorganisms that are tion: cellulase, cellobiohydrolase (e.g., CBHI, CBHII), alco aerobes, anaerobes, facultative anaerobes, heterotrophs, holdehydrogenase (A, B, and C), acetaldehyde dehydroge autotrophs, photoautotrophs, photoheterotrophs, chemoau nase, amylase, alpha amylase, glucoamylase, beta glucanase, totrophs, and/or chemoheterotrophs. The cellular activity, beta glucosidase, invertase, endoglucanase (e.g., EGI, EGII. including cell growth can be aerobic, microaerophilic, or EGIII), lactase, hemicellulase, pectinase, hydrogenase, pull anaerobic. The cells can be in any phase of growth, including lulanase, phytase, a hydrolase, a lipase, polysaccharase, ligni lag (or conduction), exponential, transition, stationary, death, nase, Accellerase(R) 1000, Accellerase(R) 1500, Accellerase(R) dormant, vegetative, sporulating, etc. The one or more micro DUET, Accellerase(R) TRIO, or Cellic CTec2 enzymes, phos organisms be a psychrophile (optimal growth at about -10°C. phoglucose isomerase, inositol-1-phosphate synthase, inosi to about 25° C.), a mesophile (optimal growth at about tol monophosphatase, myo-inositol dehydrogenase, myo 20-about 50° C.), a thermophile (optimal growth about 45° C. inosose-2-dehydratase, inositol 2-dehydrogenase, deoxy-D- to about 80° C.), or a hyperthermophile (optimal growth at gluconate isomerase, kinase, 5-dehydro-2- about 80°C. to about 100° C.). The one or more microorgan deoxygluconokinase, deoxyphophogluconate aldolase, isms can be a gram-negative or gram-positive bacterium. A 3-hydroxy acid dehydrogenase, isomerase, topoisomerase, bacterium can be a cocci (spherical), bacilli (rod-like), or dehydratase, monosaccharide dehydrogenase, aldolase, spirilla (spiral-shaped; e.g., vibrios or comma bacteria). The phosphatase, a protease, DNase, alginate lyase, laminarinase, microorganisms can be phenotypically and genotypically endoglucanase, L-butanediol dehydrogenase, acetoin reduc diverse. tase, 3-hydroxyacyl-CoA dehydrogenase, or cis-aconitate 0171 The microorganisms can be a wild-type (naturally decarboxylase. The enzymes include those described by Hei occurring) microorganism or a recombinant (including, but nzelman et al. (2009) PNAS 106:5610-5615, herein incorpo not limited to genetically engineered microorganisms) micro rated by reference in its entirety. organism. A recombinant microorganism can comprise one (0168 The enzymes may be bound to the precursor for the or more heterologous nucleic acid sequences (e.g., genes). hydrophilic polymer of the biocatalyst prior to the formation One or more genes can be introduced into a microorganism of the biocatalyst or may be introduced during the preparation used in the methods, compositions, or kits described herein, of the biocatalyst, e.g., by addition to the liquid medium for e.g., by homologous recombination. One or more genes can forming the biocatalyst. There are many methods that would be introduction into a microorganism with, e.g., a vector. The be known to one of skill in the art for providing enzymes or one or more microorganisms can comprise one or more vec fragments thereof, or nucleic acids, onto a solid Support. tors. A vector can be an autonomously replicating vector, i.e., Some examples of Such methods include, e.g., electrostatic a vector that exists as an extra-chromosomal entity, the rep droplet generation, electrochemical means, via adsorption, lication of which is independent of chromosomal replication, via covalent binding, via cross-linking, via a chemical reac e.g., a linear or closed circular plasmid, an extra-chromo tion or process. Various methods are described in Methods in Somal element, a mini-chromosome, or an artificial chromo Enzymology, Immobilized Enzymes and Cells, Part C. 1987. some. The vector can contain a means for self-replication. Academic Press. Edited by S. P. Colowick and N. O. Kaplan. The vector can, when introduced into a host cell, integrate Volume 136: Immobilization of Enzymes and Cells. 1997. into the genome of the host cell and replicate together with the Humana Press. Edited by G. F. Bickerstaff. Series: Methods one or more chromosomes into which it has been integrated. in Biotechnology, Edited by J. M. Walker; DiCosimo, R., Such a vector can comprise specific sequences that can allow McAuliffe, J., Poulose, A. J. Bohlmann, G. 2012. Industrial recombination into a particular, desired site of the host chro use of immobilized enzymes. Chem. Soc. Rev.; and Immobi mosome. A vector system can comprise a single vector or lized Enzymes: Methods and Applications. Wilhelm Tischer plasmid, two or more vectors or plasmids, which together and Frank Wedekind, Topics in Current Chemistry, Vol. 200. contain the total DNA to be introduced into the genome of the Page 95-126. host cell, or a transposon. The choice of the vector will typi 0169. Typically extracellular enzymes bond or adhere to cally depend on the compatibility of the vector with the host Solid Surfaces, such as the hydrophilic polymer, Solid addi cell into which the vector is to be introduced. The vector can tives, cell walls and extracellular polymeric Substance. include a reporter gene. Such as a green fluorescent protein Hence, the enzymes can be substantially irreversibly retained (GFP), which can be either fused in frame to one or more of in the interior of the biocatalyst. Due to the structure of the the encoded polypeptides, or expressed separately. The vector ME biocatalysts, the microorganisms and the enzymes can be can also include a selection marker Such as an antibiotic in close proximity and thus effective, cooperative bioconver resistance gene that can be used for selection of Suitable sions can be obtained. The association of the enzymes with transformants. Means of genetically manipulating organisms the interior surfaces of the biocatalyst typically increases the are described, e.g., Current Protocols in Molecular Biology, resistance of the enzyme or enzymes to denaturation due to last updated Jul. 25, 2011, Wiley, Print ISSN: 1934-3639. In changes intemperature, pH, or other factors related to thermal Some embodiments, one or more genes involved in byproduct or operational stability of the enzymes. Also, by being formation are deleted in a microorganism. In some embodi retained in the biocatalyst, the use of the enzyme in a biore ments, one or more genes involved in byproduct formation actor is facilitated and undesirable post-reactions can be miti are not deleted. Nucleic acid introduced into a microorganism gated. can be codon-optimized for the microorganism. A gene can US 2014/0106420 A1 Apr. 17, 2014

be modified (e.g., mutated) to increase the activity of the agitate the ME biocatalyst in bioreactor assembly 104. Unre resulting gene product (e.g., enzyme). acted carbon dioxide exits bioreactor assembly 104 via line 0172. The selected microorganism to be used in a biocata 106. Some ethanol contained in the water-insoluble liquid lyst can be targeted to the sought activity. The biocatalysts may be stripped by carbon dioxide not dissolved in the water thus often contain Substantially pure strain types of microor insoluble liquid and will be contained in the carbon dioxide ganisms and, because of the targeting, enable high bioactivity exiting the bioreactor assembly via line 106. The unreacted to be achieved and provide a stable population of the micro carbon dioxide in line 106 may be recycled to bioreactor organism in the biocatalyst. assembly 104. A stream of water-insoluble liquid containing 0173 Representative microorganisms for making ME ethanol is withdrawn from bioreactor assembly 104 via line biocatalysts include, without limitation, those set forth in 108 and this stream contains entrained ME biocatalyst. United States published patent application nos. 2011/ 0177 Line 108 directs the ME biocatalyst-containing liq 0072714, especially paragraph 0122: 2010/0279354, espe uid to solid separator 110 where biocatalyst is separated from cially paragraphs 0083 through 0089; 2010/0185017, espe the water-insoluble liquid. The separated biocatalyst is cially paragraph 0046; 2009/0155873; especially paragraph passed via line 112 to rehydration tank 126. The water-in 0093; and 2006/0063217, especially paragraphs 0030 and soluble liquid is withdrawn from solid separator 110 and 0031, and those set forth in Appendix A hereto. passes the line 114 to distillation column 116. Since the 0.174 Photosynthetic microorganisms include bacteria, water-insoluble liquid has an essential absence of water, the algae, and molds having biocatalytic activity activated by distillation provides a Substantially anhydrous, ethanol prod light radiation. Examples of photosynthetic microorganisms uct which is withdrawn from distillation column 116 via line for higher oxygenated organic compound production include, 120. Lights such as dissolved carbon dioxide exit distillation but are not limited to alga Such as Bacillariophyceae strains, column 116 via line 118. Abottoms fraction containing dode Chlorophyceae, Cyanophyceae, Xanthophyceaei, Chruso canol and being substantially devoid of ethanol is withdrawn phyceae, Chlorella (e.g., Chlorella protothecoides), Crypth via line 122. ecodinium, Schizocytrium, Nannochloropsis, Ulkenia, 0.178 The bottoms fraction in line 122 and is passed to Dunaliella, Cyclotella, Navicula, Nitzschia, Cyclotella, liquid-liquid extraction unit 124. Extraction unit 124 serves to Phaeodactylum, and Thaustochytrids; yeasts such as Rhodot remove any water-soluble impurities in the dodecanol such as orula, Saccharomyces, and Apiotrichum Strains; and fungi esters and acids. The water for the extraction unit is supplied species such as the Mortierella strain. Genetically enhanced via line 130 and countercurrent contact occurs with spent photoautotrophic cyanobacteria, algae, and other photoau water being removed from extraction unit 124 via line 132. A totrophic organisms have been adapted to bioconvert carbo raffinate, which is the water-insoluble liquid, exits extraction hydrates internal to the microorganism directly to ethanol, unit 124 via line 134. butanol, pentanol and other higher alcohols and other biofu 0179 Returning now to the ME biocatalyst passed to rehy els. For example, genetically modified cyanobacteria having dration tank 126, line 136 Supplies an aqueous medium con constructs comprising DNA fragments encoding pyruvate taining replacement nutrients for the biocatalyst to rehydra decarboxylase (pdc) and alcohol dehydrogenase (adh) tion tank 126 for contact with the biocatalyst. This aqueous enzymes are described in U.S. Pat. No. 6,699,696. Cyanobac medium is provided at a lower portion of rehydration tank 126 teria are photosynthetic bacteria which use light, inorganic for countercurrent contact with the biocatalyst. The aqueous elements, water, and a carbon Source, generally carbon diox medium is withdrawn from an upper portion of rehydration ide, to metabolize and grow. The production of ethanol using tank 126 via line 128. The aqueous medium will contain some genetically engineered cyanobacteria has also been described ethanol which was retained in the biocatalyst separated from in PCT Published Patent Application WO 2007/084477. the water-insoluble liquid. This aqueous medium constitutes a portion of the water used in extraction unit 124. A portion of DRAWINGS the contained ethanol will be extracted from the aqueous (0175. The processes of the invention will be further phase in extraction unit 124 by the dodecanol. A slurry of described in connection with FIGS. 1 and 2. The figures omit biocatalyst exits rehydration tank 126 via line 138 and is minor equipment such as pumps, compressors, valves, instru passed to solids separator 140. In solids separator 140 the ments and other devices the placement of which and opera biocatalyst is separated from the aqueous medium. The aque tion thereof are well known to those practiced in chemical ous medium exits via line 142 and is passed to line 134 use as engineering. The figures also all omit ancillary unit opera the aqueous medium for extraction unit 124. tions. The apparatus depicted in FIG.2 may be used to obtain 0180. The biocatalyst from solid separator 140 is passed Substrate from gaseous streams or liquid streams. via line 144 to mixing tank 146 where it is contacted with the 0176 FIG. 1 is a schematic depiction of an apparatus raffinate in line 134 obtained from extraction unit 124. A generally designated as 100 Suitable for practicing the pro slurry of biocatalyst in the water-insoluble liquid is produced cesses of this invention. Feedstock containing Substrate, in mixing tank 146, and the slurry is passed via line 148 to which for purposes of discussion is carbon dioxide, is pro knockout pot 150. Water that passes to the exterior of the vided via line 102 to bioreactor assembly 104. As depicted biocatalyst will exist as a separate phase and will be collected bioreactor assembly 104 is a photosynthetic bioreactor pro in knockout pot 150 and removed via line 154. A slurry of vides for contact between the substrate and water-insoluble biocatalyst in water-insoluble liquid is directed to bioreactor liquid, which for purposes of discussion is dodecanol. Biore assembly 104 via line 152 from knockout pot 150. actor assembly 104 contains ME biocatalyst in the form of 0181. With reference to FIG. 2, apparatus 200 depicts the spheres of about 2 millimeters in diameter. The ME biocata use of an absorption column to recover Substrate from a lyst contains photosynthetic microorganism capable of bio gaseous stream. As shown, absorption column 202 receives a converting carbon dioxide to ethanol Such as a cyanobacteria. gaseous stream containing hydrogen, carbon monoxide and As shown, the upward flow of carbon dioxide searched to carbon dioxide from line 204. Absorption column 202 pro US 2014/0106420 A1 Apr. 17, 2014

vides for countercurrent contact with water-insoluble liquid, tions, Inc. The Polymer Solution is mixed with a mechanical which for purposes of discussion is toluene. Absorbed gases stirrer to assure uniform dispersion of the components in the exit absorption column 202 via line 206. The water-insoluble aqueous medium. Where necessary to solubilize the precur liquid is provided to absorption column 202 via line 208. The sor, the Polymer Solution can be heated as appropriate. In water-insoluble liquid containing Substrate, i.e., hydrogen, Some instances, a micronutrient solution is also added to the carbon monoxide and carbon dioxide, exits absorption col Polymer Solution. umn 202 via line 210, and is passed to bioreactor assembly 0187 Aliquots of each of the Culture Medium (or dense 212. phase from centrifugation) and Polymer Solution are 0182 Bioreactor assembly 212 contains ME biocatalyst admixed under mechanical stirring at about 30°C. to for contained therein microorganisms adapted to convert syngas a Precursor Solution. About 70 volume parts of Polymer to ethanol. Water-insoluble liquid laden with ethanol is con Solution are used per 100 parts of Precursor Solution. tinuously withdrawn from bioreactor assembly 212 via line The microorganism density is about 310 grams per liter. 214 and passed to distillation column 216. Distillation col 0188 The Precursor Solution is then extruded through a umn 216 provides an ethanol offtake via line 218. Lights, perforated plate having orifices of about 0.75 millimeter in Such as methane and nitrogenas well as any unreacted syngas diameter to form droplets of about 3 millimeters in diameter. exits distillation column 216 via line 220. Abottoms fraction The droplets fall into a gently stirred coagulating bath of an containing toluene is withdrawn from distillation column 216 aqueous boric acid solution having a pH of about 5. The the line 208 and returned to absorption column 202. biocatalyst is recovered from the coagulating bath and 0183. As shown, ME biocatalyst is withdrawn from biore washed with distilled water. The biocatalyst, after washing, is actor assembly 212 via line 222 and is passed to rehydration placed in a liquid medium containing micronutrients and the tank 224. As with FIG. 1, an aqueous medium containing substrate under suitable metabolic conditions for the micro nutrients for the microorganisms is Supplied to rehydration organisms. tank 224 by line 226. An aqueous stream is withdrawn from 0189 The biocatalyst is used to bioconvert glucose con rehydration tank 224 via line 228. The biocatalyst, having tained in an aqueous medium to ethanol. The aqueous been rehydrated, is passed via line 230 to bioreactor assembly medium contains a nutrient package. The aqueous medium is 212. then drained from the biocatalyst and the biocatalyst is at the 0184 While not particularly shown in FIG. 2, it is to be point of incipient wetness. understood that the various unit operations described in con nection with FIG. 1 can be used to assure that a separate Example 2 aqueous phase does not get formed external to the biocatalyst. 0190. About 100 milliliters of dodecanol are placed in a 0185. In an alternative deployment, the apparatus of FIG. glass bioreactor and heated to about 32° C. Then about 5 2 can be used to remove nitrogen oxides from flue gases. In grams of glucose (powder) are added to the dodecanol to Such situations, bioreactor assembly 212 contains denitrifica provide an initial glucose concentration of about 50 grams per tion microorganisms, and a nitrogen-containing gas may liter. Most of the glucose remains undissolved, and the liquid directly be evolved from the bioreactor assembly without the is gray and cloudy. About 20 grams of the biocatalyst of need for distillation column 216. Also, the aqueous medium Example 1 are added. No nutrients are added. The bioreactor provided to rehydration tank 224 will need to contain a carbon is sealed and placed in an incubator shaker with shaking at Source for the microorganisms to retain their viability. medium speed. The bioreactor is provided with a tube to permit carbon dioxide to escape and the tube is directed to EXAMPLES water in a trap to enable observation of carbon dioxide evo lution. Example 1 0191 After about 3 hours the liquid in the bioreactor 0186. A biocatalyst is prepared for the bioconversion of becomes clear and the evolution of carbon dioxide is evident Sugar to ethanol. The following procedure is used. The micro from the bubbles passing through the trap. After about 72 organisms (Saccharomyces cerevisiae ATCCR 9763TM) for hours, the bioreactor is removed from the incubator, and a the biocatalyst are grown under Suitable planktonic condi Small aliquot sample of the liquid is analyzed for glucose tions in an aqueous medium for the microorganisms includ content, which is determined to be present in an amount of ing the presence of nutrients and micronutrients. This less than about 1 gram per liter. A small aliquot sample of the medium is referred to herein as the “Culture Medium'. The liquid is analyzed by gas chromatography and is found to microorganisms used are as available and thus may be either contain about 25 grams per liter of ethanol. No acqueous phase substantially pure strains or mixed cultures. The cell density is observed in the dodecanol and the biocatalyst appears to in the Culture Medium is determined by optical density. If too retain its size, both evidencing that the biocatalyst maintains thick, the cell density is determined through filtration of sol the aqueous medium in it interior. ids and determining the mass of solids per unit volume. If the 0.192 It is believed that the dissipation of the cloudy solu cell density of the Culture Medium is below that sought to tion of glucose in the dodecanol is due to the generation of make the biocatalyst, the Culture Medium is centrifuged or ethanol during the initial three hour period. Hence, when filtered to provide a denser, cell-containing fraction. A sepa about 5 grams of glucose (powder) are added to the bioreactor rately prepared aqueous solution of Solubilized precursor is after taking samples for the analyses, a Substantially clear made (referred to herein as the “Polymer Solution'). The Solution is obtained. In a further confirmation, about 5 grams Polymer Solution contains 15.0 wt. percent of polyvinyl alco of the glucose powder are added to a mixture of 100 milliliters hol available as Mowial(R) 28-99 from Kuraray Co., Ltd. hav of dodecanol and 2.5 milliliters of anhydrous ethanol. A clear ing a degree of hydrolysis of 99.0-99.8 mol percent and a solution is observed. molecular weight of 145,000; 3.5 wt. percent of sodium algi 0193 The biocatalyst used in this example retains its abil nate available as NalginTM MV-120 from Ingredient Solu ity to bioconvert Sugars to ethanol. US 2014/0106420 A1 Apr. 17, 2014 18

APPENDIX A mobilis, Pseudomonas sp. DLC-P11, P. mendocina, P 0194 Representative microorganisms include, without chichhori, strain IST 103), Pseudomonas fluorescens, limitation, Acetobacter sp., Acetobacter aceti, Achromno Pseudomonas denitrificans, Pyrococcus, Pyrococcus friri bacter, Acidiphilium, Acidovorax delafieldi P4-1, Acineto Osus, Pyrococcus horikoshii, Ralstonia sp., Rhizobium, bacter sp. (A. calcoaceticus), Actinomnadura, Actino Rhizomucor miehei, Rhizomucorpusillus Lindt, Rhizopus, planes, Actinomycetes, Aeropyrum permix, Agrobacterium Rhizopus deleimar; Rhizopus japonicus, Rhizopus niveus, sp., Alcaligenes sp. (A. dentrificans), Alloiococcus Otitis, Rhizopus Oryzae, Rhizopus Oligosporus, Rhodococcus, (R. Ancylobacter aquaticus, Ananas comosus (M), Arthro erythropolis, R. rhodochirous NCIMB 13064). Salmonella, bacter sp. Arthrobacter sulfurous, Arthrobacter sp. (A. Saccharomyces sp., Saccharomyces cerevisiae, protophormiae), Aspergillus sp., Aspergillus niger, Schizochytriu sp., Sclerotina libertina, Serratia sp., Shi Aspergillus Oryze, Aspergillus melleus, Aspergillus pull gella, Sphingobacterium multivorum, Sphingobium (Sph verulentus, Aspergillus Saitoi, Aspergillus Soiea, Aspergil ingbium chlorophenolicum), Sphingomonas (S. vanoiku lus usamii, Bacillus alcalophilus, Bacillus amyloliquefa vae, S. sp. RW1), Streptococcus, Streptococcus ciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, thermophilus Y-1, Streptomyces, Streptomyces griseus, Bacillus cereus, Bacillus lentus, Bacillus licheniformis, Streptomyces lividans, Streptomyces murinus, Streptomy Bacillus macerans, Bacillus Stearothermophilus, Bacillus ces rubiginosus, Streptomyces violaceOruber; Streptover subtilis, Beijerinckia sp., Bifidobacterium, Brevibacterium ticillium mobaraense, Synechococcus sp., Synechocystis sp. HL4, Breitanomyces sp., Brevibacillus brevis, sp., Tetragenococcus, Thermus, Thiosphaera pantotropha, Burkholderia cepacia, Campylobacter jejuni. Candida sp., Trametes, Trametes versicolor, Trichoderma, Trichoderma Candida cylindracea, Candida rugosa, Carboxydother longibrachiatum, Trichoderma reesei, Trichoderma viride, mus (Carboxydothermus hydrogenoformans), Carica TrichospOron sp., TrichospOron penicillatum, Vibrio algi papaya (L), Cellulosimicrobium, Cephalosporium, Cha molyticus, Xanthomonas, Xanthobacter sp. (X. autotrophi etomium erraticum, Chaetonium gracile, Chlorella sp. cus GJ 10, X. flavus), yeast, Yarrow lipolytica, Zygosac Citrobacter, Clostridium sp., Clostridium butvricum, charomyces rouxii, Zvinomonas sp. Zymomonus mobilis, Clostridium acetobutyllicum, Clostridium kluyveri, Geobacter sulfurreducens, Geobacter lovlevi, Geobacter Clostridium carboxidivorans, Clostridium thermocellum, metallireducens, Bacteroides succinogens, Butyrivibrio Cornynebacterium sp. Strain m15, Corynebacterium fibrisolvens, Clostridium cellobioparum, Ruminococcus (glutamicum), Corynebacterium efficiens, Deinococcus albus, Ruminococcus flavefaciens, Eubacterium cellulo radiophilus, Dekkera. Dekkera bruxellensis, Escherichia solvens, Clostridium cellulosolvens, Clostridium cellulo coli, Enterobacter sp., Enterococcus, Enterococcus vorans, Clostridium thermocellum, Bacteroides cellulo faecium, Enterococcus gallinarium, Enterococcus faeca solvens, and Acetivibrio cellulolyticus Gliricidia sp., lis, Erwinia sp. Erwinia chrysanthemi, Gliconobacter, Albizia sp., or Parthenium sp. Cupriavidus basilensis, Gluconacetobacter sp., Hansenula sp., Haloarcula, Humi Cupriavidus campinensis, Cupriavidus gillardi, Cupriavi Cola insolens, Humicola insolens. Kitasatospora setae, dus laharsis, Cupriavidus metallidurans, Cupriavidus Klebsiella sp., Klebsiella Oxytoca, Klebsiella pneumonia, Oxalaticus, Cupriavidus pauculus, Cupriavidus pinatu Kluyveromyces sp., Kluyveromyces fragilis, Kluyveromny bonensis, Cupriavidus respiraculi, Cupriavidus taiwanen ces lactis, Kocuria, Lactlactis, Lactobacillus sp., Lactoba sis, Oligotropha carboxidovorans, Thiobacillus sp., Thio cillus fermentum, Lactobacillus sake, Lactococcus, Lacto bacillus denitrificans, Thiobacillus thioxidans, coccus lactis, Leuconostoc, Methylosinus trichosporum Thiobacillus ferrooxidans, Thiobacillus concretivorus, OB3b, Methylosporovibrio methanica 812, Methanothrix Acidithiobacillus albertensis, Acidithiobacillus caldus, sp. Methanosarcina sp., Methanomonas sp., Methylocys Acidithiobacillus cuprithermicus, Rhodopseudomonas, tis, Methanospirilium, Methanolobus Siciliae, Methanoge Rhodopseudomonas palustris, Rhodobacter sphaeroides, nium Organophilum, Methanobacerium sp., Methanobac Rhodopseudomonas capsulate, Rhodopseudomonas aci terium bryantii, Methanococcus sp., Methanomicrobium dophila, Rhodopseudomonas viridis, Desulfotomaculum, sp., Methanoplanus sp., Methanosphaera sp., Methanolo Desulfotomaculum acetoxidans, Desulfotomaculum kuz bus sp., Methanoculleus sp., Methanosaeta sp., Metha netsovii, Desulfotomaculum nigrificans, Desulfotomacu nopyrus sp., Methanocorpusculum sp., Methanosarcina, lum reducens, Desulfotomaculum carboxydivorans, Methylococcus sp., Methylomonas sp. Methylosinus sp. Methanosarcina barkeri, Methanosarcina acetivorans, Microbacterium imperiale, Micrococcus sp., Micrococcus Moorella thermoacetica, Carboxydothermus hydrogeno lysodeikticus, Microlunalus, Moorella (e.g., Moorella formans, Rhodospirillum rubrum, Acetobacterium woodii, (Clostridium) thermoacetica), Moraxella sp. (strain B), Butyribacterium methylotrophicum, Clostridium autoet Morganella, Mucorjavanicus, Mvcobacterium sp. Strain hanogenium, Clostridiumn liungdahli, Eubacterium limo GP1, Myrothecium, Neptunomonas naphthovorans, Nitro sum, Oxobacter pfennigii, Peptostreptococcus productus, bacter; Nitrosomonas (Nitrosomonas europea), Nitzchia Rhodopseudomonas palustris P4, Rubrivivax gelatinosus, sp., Nocardia sp., Pachysolen sp., Pantoea, Papaya carica, Citrobacter sp Y19, Methanosarcina acetivorans C2A, Pediococcus sp., Pediococcus halophilus, Penicillium, Methanosarcina barkeri, Desulfosporosinus Orientis, Des Penicillium Camemberti, Penicillium citrinum, Penicillium ulfovibrio desulfuricans, Desulfovibrio vulgaris, Moorella emersonii, Penicillium roqueforti, Penicillum lilactinum, thermoautotrophica, Carboxydibrachium pacificus, Car Penicillum multicolor, Phanerochaete chrysoporium, bOxydocella thermoautotrophica, Thermincola carboxy Pichia sp., Pichia stipitis, Paracoccus pantotrophus, Pleu diphila, Thermolithobacter carboxydivorans, Thermosi rotus Ostreatus, Propionibacterium sp., Proteus, FS carboxydivorans, Methanothermobacter Pseudomonas (P. pavonaceae, Pseudomonas ADP, P thermoautotrophicus, Desulfotomaczdum carboxydi Stutzeri, Pputida, Pseudomonas Strain PSI, P. cepacia G4, vorans, Desulfotomaculum kusnetsovii, Desulfotomacu P. medocina KR, P. picketti PK01, P. vesicularis, P. pauci lum nigrificans, Desulfotomaculum thermobenzoicum US 2014/0106420 A1 Apr. 17, 2014 19

Subsp. thermosyntrophicumn, Syntrophobacter fumaroxi ligmatophyla, Eutreplia, Fallacia, Fischerella, Fragilaria, dans, Clostridium acidurici, Desulfovibrio africanus, C. Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, pasteurianum, C. pasteurianum DSM 525. Paenibacillus Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, polymyxa, Acanthoceras, Acanthococcus, Acaryochloris, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Gloeococcus, Gloeocysis, Gloeodendron, Gloeomonas, Actinocyclus, Actinotaenium, Amphichrysis, Amphi Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiod dinium, Amphikrikos, Amphipleura, Amphiprora, Amphi ictyon, Golenkinia, Golenkiniopsis, Gomontia, Gompho thrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, cymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonvos Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanoth tomum, Granulochloris, Granulocystopsis, Groenbladia, ece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Ascochloris, Asterionella, Asterococcus, Audouinella, Hafniomonas, Hallassia, Ilammatoidea, Hannaea, Hantz Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Schia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Hemidinium, Hemitoma, Heribaudiella, Hleleronastix, Blidingia, Botraiopsis, Botrydium, Botryococcus, Botry Ileterothrix, Hibberdia, Hildenbrandia, Hillea, Holope Osphaerella, Brachiomonas, Brachysira, Brachytrichia, dium, Homoeothrix, Hormanthonema, Hormotila, Hvalo Brebissonia, Bulbochaete, Bunilleria, Bumilleriopsis, brachion, Hyalocardiumn, Hvalodiscus, Hvalogonium, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Cart Hvalotheca, Hydrianum, Hydrococcus, Hydrocoleum, eria, Catena, Cavinula, Centritractus, Centronella, Cera Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, tium, Chaetoceros, Chaetochloris, Chaetomorpha, Cha Hvella, Hymenomonas, Isthmochloron, Johannesbaptis etonella, Chaetonema, Chaetopeltis, Chaetophora, lia, Juranviella, Karayevia, Kathablepharis, Katodinium, Chaetosphaeridium, Chamnaesiphon, Chara, Characio Kephyrion, Keralococcus, Kirchneriella, Klebsormidi chloris, Characiopsis, Characium, Charales, Chilomonas, unm, Kolbesia, Koliella, Komarekia, Korshikoviella, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Chlamydomnonas, Chlamydomonopsis, Chlamydomyxa, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlo Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomo rella, Chlorobotrys, Chlorobrachis, Chlorochytrium, nas, Mantoniella, Marssoniella, Martyana, Mastigoco Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogo leus, Gastogloia, Melosira, Merismopedia, Mesostigma, nium, Chlorolobion, Chloromonas, Chlorophysema, Chlo Mesotaenium, Micraclinium, Micrasterias, Microchaete, rophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chro Microcoleus, Microcystis, Microglena, Micromonas. mophyton, Chromulina, Chroococcidiopsis, Chroococcus, Microspora, Microthamnion, Mischococcus, Monochry Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, sis, Monodus, Mononastix, Monoraphidium, Monos Chrysapsis, Chrysidiastrum, Chrysocapsa, Chryso troma, Mougeotia, Mougeotiopsis, Mvochloris, Myrome capsella, Chrysochaete, Chysochromulina, Chrysococcus, Cia, Myxosarcina, Naegeliella, Nannochloris, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Nautococcus, Navicula, Neglectella, Neidium, Nephroc Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, lamys, Nephrocytium, Nephrodiella, Nephroselmis, Chrysophaerella, Chrysostephanosphaera, Clodophora, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nos Clastidium, Closteriopsis, Closterlum, Coccomyxa, Coc toc, Ochromonas, Oedogonium, Oligochaelophora, Ony coneis, Coelastrella, Coelastrumm, Coelosphaerium, chonema, Oocardium, Oocystis, Opephora, Ophiocytium, Coenochloris, Coenococcus, Coenocystis, Colacium, Orthoseira, Oscillatoria, Oxvneis, Pachycladella, Coleochaete, Collodictyon, Compsogonopsis, Compsopo Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pas gon, Conjugatophyta, Conochaete, Coronastrum, Cos cherina, Paulschulzia, Pediastrum, Pedinella, Pedinomo marium, Cosmioneis, Cosmocladium, Crateriportula, nas, Pedinopera, Pelagodictyon, Penium, Peranema, Peri Craticula, Crimalium, Crucigenia, Crucigeniella, Cryp diniopsis, Peridinium, Peronia, Petroneis, Phacotus, toaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyan Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Odictyon, Cyanonephron, Cyanophora, Cyanophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopel Cyanothece, Cyanothomonas, Cyclonexis, CycloStepha tis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnu nos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylin laria, Pitophora, Placoneis, Planctonema, Plank drospermum, Cylindrotheca. Cymatopleura, Cymbella, tosphaeria, Planothidium, Plectonema, Pleodorina, Cymbellonitzschia, Cystodinium Dactylococcopsis, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Debarya, Denticula, Dermatochrysis, Dermocarpa, Der Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, mocarpella, Desmatractum, Desmidium, Desmococcus, Podohedra, Polyblepharides, Polychaetophora, Polvedri Desmonema, Desmosiphon, Diacanthos, Diacronema, ella, Polvedriopsis, Polygoniochloris, Polyepidomonas, Diadesmis, Diatomna, Diatomella, Dicellula, Dichothrix, Polytaenia, Polytoma, Polytomella, Porphyridium, Poste Dichotomococcus, Dicranochaete, Dictyochloris, Dictyo riochromonas, Prasinochloris, Prasinocladus, Prasino coccus, Dictyosphaerium, Didymocystis, Didymogenes, phyta, Prasiola, Prochlorphyta, Prochlorothrix, Proto Didymosphenia, Dilabifilum, Dimorphococcus, Dino derma, Protosiphon, Provasoliella, Prvinesium, bryon, Dinococcus, Diplochloris, Diplomeis, DiploStau Psammodictyon, Psammothidium, Pseudanabaena, ron, Distrionella, Docidium, Draparnaldia, Dunaliella, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudo Dysmorphococcus, Ecballocystis, Elakatothri, Eller characium, Pseudococcomyxa, Pseudodictyosphaerium, beckia, Encyonema, Enteromorpha, Entocladia, Entomo Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, neis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Pseudosphaerocyslis, Pseudostaurastrumm, Pseudostau Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucoc rosira, Pseudotetrastrum, Pteromonas, Punciastruata, coneis, Eudorina, Euglena, Euglenophyla, Eunotia, Eus Pyramichlamys, Pyramimonas, Pyrrophyta, Ouadrichlo US 2014/0106420 A1 Apr. 17, 2014 20

ris, Ouadricoccus, Ouadrigula, Radiococcus, Radiofilum, rubrum, Rhodobacter capsulatus, and Rhodopseudomo Raphidiopsis, Raphidocelis, Raphidonema, Raphido nas palusris (purple non-sulfur bacteria). phyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclo It is claimed: nium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rho 1. A process for the bioconversion of substrate to bioprod palodia, Rivularia, Rosenvingiella, Rossithidium, Rowa, uct, the process comprising: Scenedesmus, Scherffelia, Schizochlamydella, a. providing in a bioreactor assembly a water-insoluble Schizochlamys, Schizomeris, Schizothrix, Schroederia, liquid comprising said Substrate; Scolioneis, Scotiella, Scotiellopsis, Scoutfieldia, Scy b. contacting said water-insoluble liquid in said bioreactor tonema, Selena strum, Selenochloris, Selaphora, Senior assembly with an internally hydrated biocatalyst under bis, Siderocelis, Diderocysiopsis, Dimonsenia, Siphonon metabolic conditions for a time sufficient to bioconvert at least a portion of said Substrate to said bioproduct ema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, wherein said water-insoluble liquid is capable of receiv Spermatozopsis, Sphaerellocystis, Sphaerellopsis, ing said bioproduct from the interior of said biocatalyst Sphaerodinium, Sphaeroplea, Sphaerozosma, Spinifero to provide a bioproduct-containing liquor, said contact monas, Spirogyra, Spirotaenia, Spirulina, Spondylo ing being Substantially absent an aqueous phase external morum, Spondylosium, Sporotetras, Spumella, Stauras to said biocatalyst, wherein said biocatalyst comprises: trun, Stauerodesmus, Stauroneis, Staurosira, i. a solid structure of hydrated hydrophilic polymer Staurosirella, Stenopterobia, Stephanocostis, Stephano defining an interior structure having a plurality of discus, Stephanoporos, Stephanosphaera, Stichococcus, interconnected major cavities having a smallest Siichogloea, Stigeoclonium, Stigonema, Stipitococcus, dimension of between about 5 to about 100 microns Stokesiella, Strombon monas, Stylochrysalis, Stylodinium, and an HEV of at least about 1000 and Styloyxis, Stylosphaeridium, Surirella, Sykidion, Sym ii. a population of microorganisms capable of converting ploca, Synechococcus, Synechocystis, Synedra, Syno said Substrate to said bioproduct, said population of chromonas, Synura, Tabellaria, Tabularia, Teilingia, Tem microorganisms being Substantially irreversibly nogametum, Tetmemorus, Tetrachlorella, Tetracvclus, retained in the interior of the solid structure, said Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tet population of microorganisms being in a concentra raspora, Tetrastrum, Thalassiosira, Thamniochaete, tion of at least about 60 grams per liter based upon the Thorakochloris, Thorea, Tolypella, Tolypothrix, Trach volume defined by the exterior of the solid structure elomonas, Trachydiscus, Trebouxia, Trentepholia, when fully hydrated wherein the microorganisms Treubaria, Tribonema, Trichodesmium, Trichodiscus, Tro chiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Uro maintain their population Substantially stable; and Solenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, c. separating said bioproduct from said bioproduct-con Wolvulina, Weslella, Woloszynskia, Xanthidium, Xantho taining liquor. phyta, Xenococcus, Zygnema, Zygnemopsis, Zygonium, 2. The process of claim 1, wherein substrate is continu Chlorollexus, Chloronema, Oscillochloris, Heliothrix, ously or intermittently fed to the bioreactor assembly. Herpetosiphon, Roseiflexus, Thermomicrobium, Chloro 3. The process of claim 1, wherein at least a portion of the bium, Clathrochloris, Prosthecochloris, Allochromatium, bioproduct-containing liquor is withdrawn from the bioreac Chromatium, Halochromatium, Isochromatium, Mar tor assembly and bioproduct is separated from the withdrawn ichromatium, Rhodovulum, Thermochromatium, Thio bioproduct-containing liquor. capsa, Thiorhodococcus, Thiocystis, Phaeospirillum. 4. The process of claim 1, wherein the bioproduct com Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, prises oxygenated organic compound. Rhodopseudomonas, Rhodothalassium, Rhodospirillum, 5. The process of claim 4, wherein the oxygenated organic Ovibrio, Roseospira, Nitrobacteraceae sp., Nitrobacter sp., compound comprises alcohol that forms an azeotrope with Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomo water, and the separation is by distillation in the absence of nas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus formation of the azeotrope. sp., Nitrosovibrio sp., Thiovulum sp., Thiobacillus sp., Thi 6. The process of claim 5, wherein the alcohol comprises 1 Omicrospira sp., Thiosphaera sp., Thermothrix sp., Hydro to 4 carbon atoms. genobacter sp., Siderococcus sp., Aquaspirillum sp. 7. The process of claim 4, wherein the oxygenated organic Methanobacterium sp., Methanobrevibacter sp., Methano compound comprises at least one of Succinic acid or ester thermus sp., Methanococcus sp., Methanomicrobium sp., thereof, lactic acid or ester thereof, diol of 2 to 6 carbons. Methanospirillum sp., Methanogenium sp., Methanosa 8. The process of claim 1, wherein the substrate comprises rcina sp., Methanolobus sp. Methanothrix sp., Methano a normally gaseous component. coccoides sp., Methanoplanus sp., Thermoproteus sp., 9. The process of claim 8, wherein the normally gaseous Pyrodictium sp., Sulfolobus sp., Acidianus sp., Bacillus component is at least one of oxygen; hydrocarbon of 1 to 4 subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ral carbons; chloromethane; chlorodifluoromethane; difluo stonia sp., Rhodococcus sp., Corynebacieria sp., Brevi romethane; dichlorodifluoromethane; 1,1,1,2-tetrafluoroet bacteria sp., Mycobacteria sp., oleaginous yeast, Arabi hane; 2-chloro-1,1,1,2-tetrafluoroethane; pentafluoroethane; dopsis thaliana, Panicum virgatum, Miscanthus giganteus, chloropentafluoromethane; nitric oxide: nitrous oxide; nitro Zea mays (plants). Botryococcus braunii, Chlamydomonas gen dioxide; carbon monoxide; carbon dioxide; hydrogen; reinhardtii and Dunaliela Salina (algae), Synechococcus sp hydrogen Sulfide; Sulfur dioxide; and ammonia. PCC 7002, Synechococcus sp. PCC 7942, Synechocystis 10. The process of claim 1 wherein: sp. PCC 6803, Thermosynechococcus elongatus BP-1 (cy a. at least a portion of the bioproduct-containing liquor is anobacteria), Chlorobium tepidum (green Sulfur bacteria), withdrawn from the bioreactor assembly and bioproduct Chloroflexus auranticusl, Chromatium tepidum and Chro is separated from the withdrawn bioproduct-containing matium vinosum (purple Sulfur bacteria), Rhodospirillum liquor to provide a regenerated liquor, US 2014/0106420 A1 Apr. 17, 2014 21

b. Substrate is supplied to at least a portion of the regener ated liquor to provide a feed liquor, and c. the feed liquor is introduced into the bioreactor assembly as at least a portion of the water-insoluble liquid con taining said Substrate. 11. The process of claim 1 further comprising: a. continuously or intermittently ceasing contact between at least a portion of the biocatalyst and the water-in soluble liquid; b. contacting the biocatalyst with an aqueous medium com prising nutrients for said microorganism to provide an internally rehydrated biocatalyst; c. removing excess aqueous medium from said internally rehydrated biocatalyst; and d. contacting the internally rehydrated biocatalyst with the water-insoluble liquid in said bioreactor assembly. 12. The process of claim 1, wherein the bioconversion is a photosynthetic conversion. 13. The process of claim 1, wherein the bioconversion is anabolic. 14. The process of claim 1, wherein the bioconversion is catabolic.