WO2013013126A1 - Process and system for producing a fermentation product using a cone bottom fermenter - Google Patents

Abstract

A process and system for producing a fermentation product, such as ethanol, includes introducing biomass and a microorganism into a mixing tank, mixing the biomass and microorganism in the mixing tank to form a slurry, pumping the slurry to a cone bottom fermenter, and allowing the microorganism to ferment the biomass into a fermentation product. The cone bottom fermenter can have a cone-shaped bottom having an angle in a range from above 45 degrees to about 80 degrees.

Description

PROCESS AND SYSTEM FOR PRODUCING A FERMENTATION PRODUCT

USING A CONE BOTTOM FERMENTER

STATEMENT REGARDING FEDERALLY- SPONSORED

RESEARCH AND DEVELOPMENT

[0001] This invention was funded, in part, by the United States government under a grant with the Department of Energy, Award No. DE-FC36-08GO18103. The U.S.

Government has certain rights in this invention.

BACKGROUND OF TBI- INVENTION Field of the Invention

[0002] The present invention is directed to processes and systems for producing a fermentation product using a cone-bottom fermenter. In particular, the invention relates to an improved method of fermenting biomass in which a vessel configuration and a specialized microorganism are combined to obtain a controlled fermentation which reduces the need for costly physical agitation or mixing in the fermenter, for the production of fermentation products such as ethanol. Useful fermentation products other than ethanol can also be produced.

Background Art

[0003] Energy conversion, utilization, and access underlie many of the great challenges of our time, including those associated with sustainability, environmental quality, security, and poverty. New applications of emerging teclmologies are required to respond to these challenges. Biotechnology, one of the most powerful of the emerging technologies, can give rise to important new energy conversion processes. Biomass, which is available in abundance, can be used as a feedstock for production of fuels and chemicals, such as ethanol.

[0004] An obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful products. Ethanol, and other products, can be produced from a wide variety of lignocellulosic biomass feedstocks including agricultural plant wastes (e.g., corn stover, cereal straws, sugarcane, and sugarcane bagasse), plant wastes from industrial processes (e.g., sawdust, paper pulp), and consumer waste and energy crops grown specifically for fuel production, such as switchgrass. Cellulosic biomass is composed of cellulose, hemicellulose and lignin, with smaller amounts of proteins, lipids (fats, waxes and oils) and ash. Roughly, two-thirds of the dry mass of cellulosic materials are present as cellulose and hemicellulose. Lignin makes up the bulk of the remaining dry mass.

[0005] Lignocellulosic biomass is particularly well-suited for energy applications because of its large-scale availability, low cost, and environmentally benign production. In particular, many energy production and utilization cycles based on lignocellulosic biomass have near-zero greenhouse gas emissions on a life-cycle basis.

[0006] The production of ethanol, and other products, from biomass typically involves the breakdown or hydrolysis of lignocellulose-containing materials into disaccharides and, ultimately, monosaccharides. Biological processing of cellulosic biomass aims to extract fermentable sugars from the feedstock. The sugars in cellulose and hemicellulose are locked in complex carbohydrates called polysaccharides (long chains of monosaccharides or simple sugars). Separating these complex polymeric structures into fermentable sugars is essential to the efficient and economic production of cellulosic ethanol.

[0007] Many factors are involved in efficient bioprocessing, but the final concentration of product and good mixing are two of the most important. In order to achieve a high concentration of the fuel or chemical product, it is necessary to start with high concentrations of an initial starting material (e.g., biomass feedstock). With biomass fermentation, the use of high substrate concentrations creates problems for another key factor, mixing. Cellulosic biomass is highly fibrous, strong and water-absorbing, making it very difficult to mix at high concentrations. Mechanical mixing of cellulosic biomass requires a great deal of energy expense in the form of electricity to drive the impellers and is costly.

[0008] It would therefore be an advance in the art to minimize mechanical mixing/agitation in a fermenter so that high substrate concentrations can be used and fermentation occurs in a minimally agitated vessel. BRIEF SUMMARY OF THE INVENTION

[0009] In some embodiments, a process for producing a fermentation product comprises introducing biomass into a tank; introducing a microorganism into the tank; mixing the biomass and the microorganism in the tank to form a slurry; pumping the slurry from the tank to the cone bottom fermenter; allowing the microorganism to ferment the biomass in the cone bottom fermenter; and recovering one or more products of the fermentation, wherein the cone bottom fermenter has a cone-shaped bottom having an angle, measured with respect to a horizontal flat plane, in a range of from above 45 degrees to about 80 degrees.

[0010] In some embodiments, the biomass and microorganism are mixed in the tank with an agitator.

[0011] In some embodiments, a residence time of the slurry in the cone bottom fermenter is in a range of from about 30 minutes to about 10 hours.

[0012] In some embodiments, a solids content of the biomass in weight percentage is in a range of from about 5% to about 45%.

[0013] In some embodiments, the tank comprises a cone bottom tank.

[0014] In some embodiments, the tank is proportionally dimensioned from about l/5th to about 1/50th a size of the cone bottom fermenter.

[0015] In some embodiments, the slurry is pumped into a top portion of the cone bottom fermenter.

[0016] In some embodiments, a portion of the slurry exiting the cone-shaped bottom of the cone bottom fermenter is recycled to the tank.

[0017] In some embodiments, the recycled portion passes through a heat exchanger before entering the tank.

[0018] In some embodiments, the slurry passes through a heat exchanger before entering the cone bottom fermenter.

[0019] In some embodiments, the slurry comprises a solids content in a range of from about 5% to about 45% by weight.

[0020] In some embodiments, the angle is in a range of from about 60 degrees to about

80 degrees, with respect to a horizontal flat plane. [0021] In some embodiments, the angle is about 60 degrees, with respect to a horizontal flat plane.

[0022] In some embodiments, the biomass comprises lignocellulosic biomass.

[0023] In some embodiments, the lignocellulosic biomass comprises grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof.

[0024] In some embodiments, the microorganism is selected from the group consisting of yeast, bacteria, fungi, and combinations thereof.

[0025] In some embodiments, the microorganism is a recombinant microorganism.

[0026] In some embodiments, the microorganism produces ethanol.

[0027] In some embodiments, the microorganism comprises Saccharomyces cerevisiae,

Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, Schwanniomyces occidentalis, as well as bacteria, such as, Eschirichia coli, Thermoanaerobacteium saccharolyticum, and Clostridium thermocellum.

[0028] In some embodiments, the microorganism is a yeast.

[0029] In some embodiments, the microorganism is Saccharomyces cerevisiae.

[0030] In some embodiments, the microorganism is a xylose-utilizing microorganism, a glucose-utilizing microorganism, or both.

[0031] In some embodiments, the microorganism expresses one or more enzymes that break down cellulose in the biomass into sugars and ferments the sugars into ethanol. In some embodiments, the enzymes are heterologous to the microorganism.

[0032] In some embodiments, the fermenting of the biomass in the cone bottom fermenter occurs without the use of a mixing device in the cone bottom fermenter.

[0033] In some embodiments, the fermentation product comprises ethanol.

[0034] In some embodiments, the slurry further comprises one or more exogenous enzymes. [0035] In some embodiments, the slurry further comprises one or more catalysts that mimic enzymes.

[0036] In some embodiments, the biomass is pretreated.

[0037] In some embodiments, a system for producing a fermentation product is provided which comprises: a mixing tank configured to form a slurry comprising biomass and a microorganism; a cone bottom fermenter configured to receive the slurry, wherein the cone bottom fermenter has a cone-shaped bottom having an angle, measured with respect to a horizontal flat plane, in a range of from above 45 degrees to about 80 degrees; and a pump configured to move the slurry from the mixing tank to the cone bottom fermenter.

[0038] In some embodiments, the angle is in a range of from about 60 degrees to about

80 degrees, with respect to a horizontal flat plane.

[0039] In some embodiments, the angle is about 60 degrees, with respect to a horizontal flat plane.

[0040] In some embodiments, the cone bottom fermenter further comprises a cylindrical portion above the cone-shaped bottom and wherein a ratio of a height of the cone-shaped bottom to the cylindrical portion is in a range of from about 0.2 to about 50.

[0041J In some embodiments, the mixing tank comprises a cone bottom tank.

[0042] In some embodiments, the mixing tank is proportionally dimensioned in a range of from about l/5th to about l/50th a size of the cone bottom fermenter.

[0043] In some embodiments, the cone bottom fermenter further comprises an inlet nozzle arranged near a top portion of the cone bottom fermenter at an angle, measured with respect to a horizontal flat plane, in a range of from about 0 degrees to about 40 degrees for introducing the slurry into the cone bottom fermenter.

[0044] In some embodiments, the angle of the inlet nozzle is about 0 degrees.

[0045] In some embodiments, the system is configured for the microorganism to ferment the biomass in the cone bottom fermenter without the use of a mixing device in the cone bottom fermenter.

[0046] In some embodiments, the system further comprises a heat exchanger through which the slurry passes before entering the cone bottom fermenter.

[0047] In some embodiments, the system further comprises a second pump configured to recycle a portion of the slurry exiting the cone-shaped bottom of the cone bottom fermenter to the mixing tank< [0048] In some embodiments, the system further comprises a second heat exchanger through which the recycled portion passes before entering the mixing tank.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.

[0050] FIG. 1 schematically illustrates an exemplary method and system of the present invention for producing a fermentation product.

[0051] FIG. 2 schematically illustrates an alternative exemplary method and system of the present invention for production of a fermentation product.

[0052] FIG. 3 illustrates the fermentation time course in a 2L agitated flat bottom laboratory fermenter.

[0053] FIG. 4 illustrates the fermentation time course in a 1,000 gallon cone bottom fermenter without the use of an agitator.

[0054] FIG. 5 illustrates the fermentation time course in a 1,000 gallon flat bottom fermenter with the use of an agitator.

[0055] FIG. 6 illustrates the fermentation time course in a 5,000 gallon cone bottom fermenter without the use of an agitator.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

[0057] In order to further define this invention, the following terms and definitions are herein provided. [0058J As used herein, the terms "comprises," "comprising," "includes," "including,"

"has," "having," "contains" or "containing," or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0059] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0060] The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

[0061] As used herein, the term "about" when modifying a quantity or numerical value means within 10% of the numerical value, alternatively within 5% of the numerical value. Whether or not modified by the term "about", the claims include equivalents to the quantities.

[0062] "Biomass" as used herein means biological material made by the growth of living organisms.

[0063] "Fermentation" or "fermentation process" as used herein means any process comprising a fermentation step.

[0064] "Fermentation product" as used herein means a product of a fermentation process.

[0065] "Fermenter" as used herein means a vessel in which fermentation occurs.

[0066] "Mixing tank" as used herein means a vessel wherein mixing takes place with use of mixing device, such as, for example, an agitator. [0067] The present invention provides processes and systems for producing a fermentation product. in some embodiments, biomass and a microorganism are introduced into a tank and mixed in the tank to form a slurry. The slurry is pumped to a cone bottom fermenter where the microorganism is allowed to grow and ferment the biomass into a fermentation product, such as, for example, ethanol. The cone bottom fermentei can have a cone-shaped bottom having an angle in a range from above 45 degrees to about 80 degrees.

[0068] The processes and systems of the present invention will be described with reference to the Figures. Figure 1 illustrates an exemplary process and system 100 for producing a fermentation product. A feed stream of biomass 102 can be fed into a mixing tank 106. Biomass can be classified into three main categories: sugar, starch, and cellulose-containing plants. In some embodiments biomass stream 102 can include sugars, including glucose and xylose, starch, cellulose-containing plants, or combinations thereof.

[0069] Cellulose-containing plants and waste products are the most abundant forms of biomass and are referred to as lignocellulosic biomass because they contain cellulose (e.g., 10% to 80%), hemicellulose (e.g., 2% to 40%) and lignin (e.g., 5% to 45%). In some embodiments, biomass stream 102 includes lignocellulosic biomass.

[0070] The terms "hemicellulose," "hemicellulosic portions," and "hemicellulosic fractions" mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan, mamian, glucomannan, and galactoglucomannan, inter alia), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan), and proteoglycans (e.g., arabinogalactan-protein, extensin, and proline-rich proteins).

[0071] In some embodiments, lignocellulosic biomass can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, mixed prairie grasses, or a combination thereof; sugar-processing residues, such as but not limited to energy cane, sugar cane bagasse; agricultural wastes, such as but not limited to cobs and corn fiber, rice straw, rice hulls, barley straw, cereal straw, wheat straw, canola straw, oat straw, oat hulls, beet pulp, palm residue, and; stover, such as but not limited to soybean stover, corn stover; and forestry wastes, such as but not limited to recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch), softwood, newsprint or any combination thereof.

[0072] Paper sludge is also a viable feedstock for ethanol production and can be another type of lignocellulosic biomass. Paper sludge is solid residue arising from pulping and paper-making, and is typically removed from process wastewater in a primary clarifier. The size range of the substrate material varies widely and depends upon the type of substrate material used as well as the requirements and needs of a given process.

[0073] in some embodiments, the lignocellulosic biomass may be prepared in such a way as to permit ease of handling in conveyors, hoppers and the like. In the case of wood, the chips obtained from commercial chippers are suitable; in the case of straw it is sometimes desirable to chop the stalks into uniform pieces about 0.5 to about 3 inches in length, and about 3 to about 5 mm in width. Depending on the intended degree of pretreatment, the size of the substrate particles prior to pretreatment may range from less than a millimeter to inches in length.

[0074] Cellulose molecules are linear, unbranched and can have polymerization ranges from 500 to 20,000 and have a strong tendency to form inter- and intra-molecular hydrogen bonds. Bundles of cellulose molecules are thus aggregated together to form microfibrils in which highly ordered (crystalline) regions alternate with less ordered (amorphous) regions. Microfibrils make fibrils and finally cellulose fibers. As a consequence of its fibrous structure and strong hydrogen bonds, cellulose has a very high tensile strength and is insoluble in most solvents.

[0075] Lignocellulosic biomass can undergo pre-treatment to enhance susceptibility to hydrolysis. The degradation of lignocellulosic biomass is primarily governed by its structural features because cellulose possesses a highly ordered structure and the lignin surrounding cellulose forms a physical barrier.

[0076] Pretreatment of lignocellulosic biomass can reduce the lignin content, reduce the order of the cellulose and increase surface area. In some embodiments, pretreatment can increase conversion yield during fermentation. Pretreatment methods can be physical, chemical, physicochemical and/or biological, depending on the mode of action. The various pretreatment methods that have been used to increase cellulose digestibility include, for example, ball-milling treatment, two-roll milling treatment, hammer milling treatment, colloid milling treatment, high pressure treatment, radiation treatment, pyrolysis, catalytic treatment, acid treatment, alkaline treatment, organic solvent treatment, steam treatment, heat treatment, low-pH treatment, steam explosion treatment, pulping treatment, white rot fungi treatment, steam explosion and ammonia fiber explosion, continuous hydrolysis treatment, and combinations thereof. Pretreatment can be a single-stage pretreatment or a multiple-stage pretreatment (such as, two-stage pretreatment).

[0077] A further discussion of pretreatments can be found, for example, in U.S. Patent

Nos. 5,865,898, and 5,846,787 which are hereby incorporated by reference in their entirety. Exposure time, temperature, and pH are the additional metrics that govern the extent to which the cellulosic carbohydrate fractions cleaved during pretreatment are amenable to further enzymatic hydrolysis in subsequent biological conversion steps.

[0078] In some embodiments, pretreatment of biomass stream 102 can be carried out at severities of about 3, about 3.2, about 3.5, about 3.7, about 3.9, about 4, about 4.2, about 4.5, or about 4.6. In some embodiments, pretreatment of biomass stream 102 can be carried out at severities in a range of about 3 to about 4.6, about 3 to about 4.5, about 3 to about 4.2, about 3 to about 4, about 3 to about 3.9, about 3 to about 3.7, about 3 to about 3.5, about 3 to about 3.2, about 3.2 to about 4.6, about 3.2 to about 4.5, about 3.2 to about 4.2, about 3.2 to about 4, about 3.2 to about 3.9, about 3.2 to about 3.7, about 3.2 to about 3.5, about 3.5 to about 4.6, about 3.5 to about 4.5, about 3.5 to about 4.2 about 3.5 to about 4, about 3.5 to about 3.9, about 3.5 to about 3.7, about 3.7 to about 4.6, about 3.7 to about 4.5, about 3.7 to about 4.2 about 3.7 to about 4, about 3.7 to about 3.9, about 3.9 to about 4.6, about 3.9 to about 4.5, about 3.9 to about 4.2, about 3.9 to about 4, about 4 to about 4.6, about 4 to about 4.5, about 4 to about 4.2, about 4.2 to about 4.6, or about 4.2 to about 4.5. Severity defines the extent to which feedstock (e.g., biomass stream 102) is cooked in an aqueous medium as a function of time and temperature. The numerical value of severity, R₀, is given by the formula

R₀ = t exp[(T-100)/14.75] (1) wherein t is the time in minutes and T is the temperature in °C.

[0079] In some embodiments, the biomass can be pretreated in a first stage, for example a low severity treatment of about 3 to about 3.8 to separate hemicellulose and cellulose. In some embodiments the low severity treatment can be followed by a washing step to separate solubilized hemicellulose from cellulose. The hemicellulose can be fed to mixing tank 106 as part of biomass stream 102 and can be broken down into 5-carbon sugars, such as xylose, through the catalytic action of xylanases which may be added as an exogenous enzyme or produced by a CBP microorganism. In some embodiments, the cellulose separated from the hemicellulose in the first stage can be pretreated in a second stage, for example a high severity treatment in the range of about 3.8 to about 4.6. The cellulose can be fed to mixing tank 106 as part of biomass stream 102 and can be broken down into 6-carbon sugars, such as glucose. In some embodiments, the biomass can be pretreated in a single stage at low severity conditions to obtain hemicellulose, which can be broken down into 5-carbon sugars. In some embodiments, the biomass can be treated in a single state at high severity conditions to obtain cellulose, which can be broken down into 6-carbon sugars. In some embodiments, pretreatment temperatures can exceed 215 °C, thereby providing an aseptic or sterile feedstock.

In some embodiments, the pretreated biomass can be utilized at the same rate at which it is generated. However, if the pretreated substrate is stored for a period after pretreatment before it is fermented, the pretreated substrate can be processed to maintain aseptic or sterile conditions. In some embodiments, aseptic conditioning is obtained by hot washing an aqueous suspension of the substrate at a high temperature, for example at 190 °F, followed by the addition of an acid, for example phosphoric acid, to bring the aqueous suspension of the substrate (i.e., slurry) to a low pH, for example a pH of 2. The aseptic conditioning can reduce introduction of bacteria when pretreated substrate is stored at the room temperature. In some embodiments, sterile conditioning is obtained by holding a slurry of the pretreated substrate for at least about 15 minutes at about 121 °C. In some embodiments, sterile conditioning is obtained by holding a slurry of the pretreated substrate for about 3 minutes at about 134 °C. In some embodiments, the sterile conditioning is obtained by an autoclave that commonly uses steam heated to about 121 - 134 °C.

A feed stream 104 of fermentation microorganism can also be fed into mixing tank 106. In some embodiments, fermentation microorganisms can be selected from bacteria, fungi, yeast, or combinations thereof. In some embodiments, the microorganism can be a recombinant microorganism. In some embodiments, useful recombinant microorganisms can include Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, Schwanniomyces occidentalis, as well as bacteria, such as, Eschirichia coli, Thermoanaerobacteium saccharolyticum, and Clostridium thermocellum. In some embodiments, the recombinant microorganism is a consolidated bioprocessing (CBP) enabling microorganism that performs hydrolysis and fermentation concurrently. In some embodiments, a combination of exogenous helper enzymes and fermentative CBP microorganisms can be used.

[0082] CBP is a process configuration that can perform the following transformations in a single step: (1) the production of saccharolytic enzymes (e.g., amylases, cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose) and uronic acids; and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). In some embodiments, the microorganism is a xylose-utilizing microorganism (e.g., it ferments xylose). In some embodiments, the microorganism is a glucose-utilizing microorganism (e.g., it ferments glucose). In some embodiments, the microorganism is capable of fermenting glucose and xylose to ethanol, and has demonstrated substrate range so that it may utilize other sugars or organic components to produce ethanol. In some embodiments, the microorganism expresses one or more enzymes that break down cellulose in the biomass to sugars (e.g., hydrolyze cellulose) and ferments the sugars into a fermentation product, such as, for example, ethanol, organic acids, propanols, butanols, butanediols, polyols, and/or other fermentation products.

[0083] A major cost in producing ethanol from cellulose is the cost of added enzymes needed to break down the cellulose to glucose. CBP microorganisms secrete cellulases needed to provide sugar, as well as a pathway to convert the sugar into ethanol. The use of CBP microorganisms thus reduces the cost of ethanol production and helps achieve an efficient and economic production of ethanol.

[0084] The use of CBP fermentation also allows for an extended ethanol production in which fermentable substrate is released at a rate determined by the metabolism of the yeast. This is unlike a corn or sugar fermentation, or traditional cellulose hydrolysis followed by fermentation, in which glucose is present in large metabolic excess at the beginning of the fermentation, and where the fermentation system must be designed to control a biologically uncontrolled system, i.e., rapid initial fermentation of the glucose to ethanol and concomitant release of heat. Thus, in some embodiments, the CBP technology places the control point at a microbial (microscopic) level, and enables the use of simple vessel design consisting of a cone bottom and external mixing tank where slow recirculation of broth through the two vessels during the fermentation process is sufficient to control the fermentation and allow sustained ethanol production.

[0085] Further discussion of CBP and CBP-enabling microorganisms can be found, for example, in commonly-owned U.S. Appl. No. 61/351 , 165, filed June 3, 2010; U.S. Appl. No. 61/420,142, filed December 6, 2010; International Appl. No. PCT/US2011/039192, filed June 3, 201 1, published as International Pub. No. WO 2011/153516; U.S. Appl. No. 61/331 ,657, filed May 5, 2010; U.S. Appl. No. 61/351,133, filed June 3, 201 1 ; International Appl. No. PCT/US201 1/035416, filed May 5, 201 1, published as International Pub. No. WO 201 1/140386; U.S. Appl. No. 61/472,085, filed April 5, 201 1 ; International Appl. No. PCT/US2012/032443; U.S. Appl. No. 61/565,261, filed November 30, 201 1 ; and International Pub. No. WO 2010/060056, each of which is hereby incorporated by reference in its entirety.

[0086] In some embodiments, additional materials can be fed into mixing tank 106, such as, for example, media, water, inoculum, exogenous enzymes (i.e., enzymes that are not provided by the CBP microorganism), and/or one or more catalysts that mimic enzymes. In some embodiments, exogenous enzymes are added to mixing tank 106 to aid in hydrolysis of the biomass. In other embodiments, the microorganism expresses the enzymes, either endogenously and/or heterologously. Suitable exogenous enzymes can include, for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, β- glucosidases, xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases, mannases, glactases, pectinases, glucuronidases, amylases, a-amylases, β-amylases, glucoamylases, a-glucosidases, isoamylases, proteases, esterases, nucleases, and mixtures thereof.

[0087] Biomass feed stream 102 and microorganism feed stream 104, as well as any additional materials fed to mixing tank 106, are mixed/agitated in mixing tank 106 to form a slurry. In some embodiments, a particle size of the biomass in the slurry ranges from about 10 microns to greater than about 3,000 microns. In some embodiments, a particle size of the biomass in the slurry is about 10 microns, about 100 microns, about 500 microns, about 1,000 microns, about 2,000 microns, about 3,000 microns, and/or mixtures thereof. In some embodiments, a size of the microorganism ranges from about 2 microns to about 20 microns. During mixing/agitation, enzymes expressed by the microorganism and/or exogenously added enzymes hydrolyze the biomass.

[0088] In some embodiments, mixing of biomass feed stream 102 and microorganism feed stream 104 can be accomplished through the use of conventional agitators. Agitators typically include an electric motor, a gearbox for speed reduction, a shaft for transmitting rotational motion to various portions of a vessel, and impellers. The impellers can induce axial flow, radial flow, or combinations thereof in the fluid which they contact.

[0089] In some embodiments, mixing of biomass feed stream 102 and microorganism feed stream 104 occurs as a result of rotational motion of liquid in mixing tank 106. In some embodiments, the rotational motion can be caused by liquid (e.g., feed streams 102 and 104) flowing into mixing tank 106 in an off-center direction, on a path which does not intersect with a centerline of mixing tank 106. In some embodiments, mixing in mixing tank 106 can occur as a result of the rotational motion without the use of a mixing device (e.g., an agitator). In some embodiments, mixing in mixing tank 106 can occur as a result of a combination of the rotational motion and the use of a mixing device.

[0090] In some embodiments, mixing tank 106 can be made from conventional materials, such as, for example, stainless steel, fiber reinforced polymer, metal matrix composite, ceramic matrix composite, thermoplastic matrix composite, metal, epoxy lined carbon steel, plastic lined carbon steel, plastic, fiberglass, glass lined carbon steel, metal alloys based on iron, nonferrous metals in pure or alloy forms, plastic lined concrete, and/or metal lined concrete.

[0091] In some embodiments, a pipe leading to the fermenter 110 is sufficiently large to allow for the mixing of the biomass feed stream 102 and the microorganism feed stream 104 in the pipe so that no mixing tank 106 is needed. In some embodiments, the pipe has a diameter in a range of about 3 inches to about 2 feet.

[0092] Slurry stream 108 exits mixing tank 106 and enters fermenter 110. In some embodiments, slurry stream 108 can have a solids content, as a percentage of weight, of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%), or about 45%. In some embodiments, slurry stream 108 can have a solids content, as a percentage of weight, in a range from about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 45%, about 30% to about 45%, about 30% to about 35%, 35% to about 45%, about 35% to about 40%, or about 40% to about 45%.

[0093] In some embodiments, pump 1 12 moves slurry stream 108 to fermenter 1 10.

Pump 1 12 can be, for example, a centrifugal-type pump or a reciprocating-type pump. In some embodiments, the hold-up volume is approximately 5 gallons and the head against which pump 1 12 must move slurry stream 108 is within a range from less than about 15 feet to as much as about 1 10 feet of liquid. In some embodiments, the flow rate through pump 1 12 can be within a range bounded by 0.1 turnovers per hour of fermenter 1 10 and about 5 turnovers per hour. In some embodiments, the flow rate through pump 112 can be within a range of from about 100 gallons/minute to about 9,000 gallons/minute. In some embodiments, the flow rate of the pump, and therefore the pump size, depends on the turn over rate (the reciprocal of residence time) and fermentation volume, and can be adjusted accordingly for optimization of the fermentation process.

[0094] In some embodiments, liquid slurry stream 108 can pass through a heat exchanger

114 prior to entering fermenter 110 to control the temperature of slurry stream 108, and thereby control the temperature inside fermenter 1 10. As the microorganism hydrolyzes the biomass, heat is generated, thereby raising the temperature of slurry stream 108. The microorganism can become inactivated if the temperature of slurry stream 108 rises too high. Thus, heat exchanger 1 14 can cool slurry stream 108 to maintain the temperature in an active temperature range of the microorganism. In some embodiments, heat exchanger 1 14 is positioned just before the inlet to fermenter 1 10. In some embodiments, heat exchanger 1 14 is designed to control the temperature of fermenter 1 10 within a range of +/- 2 °C based on the optimal temperature needed for the microorganism used. In some embodiments, heat exchanger 1 14 controls the temperature at about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C. In some embodiments, heat exchanger 1 14 controls the temperature in a range of from about 20°C to about 60°C, about 20°C to about 55°C, about 20°C to 50°C, about 20°C to about 45°C, about 20°C to about 40°C, about 20°C to about 35°C, about 20°C to about 30°C, about 20°C to about 25°C, 25°C to about 60°C, about 25°C to about 55°C, about 25°C to 50°C, about 25°C to about 45°C, about 25°C to about 40°C, about 25°C to about 35°C, about 25°C to about 30°C, 30°C to about 60°C, about 30°C to about 55°C, about 30°C to 50°C, about 30°C to about 45°C, about 30°C to about 40°C, about 30°C to about 35°C, 35°C to about 60°C, about 35°C to about 55°C, about 35°C to 50°C, about 35°C to about 45°C, about 35°C to about 40°C, 40°C to about 60°C, about 40°C to about 55°C, about 40°C to 50°C, about 40°C to about 45°C, about 45°C to about 60°C, about 45°C to about 50°C, about 50°C to about 60°C, about 50°C to about 55°C, or about 55°C to about 60°C.

[0095] In some embodiments, heat exchanger 1 14 can be, for example, any of the following types: shell and tube, tube in tube, finned tube, scraped-surface tube, wide-gap plate and frame, spiral, and/or combinations thereof.

[0096] Inside fermenter 1 10, fermentation of the biomass into a fermentation product, such as, for example, ethanol occurs. A fermentation process includes, without limitation, fermentation processes used to produce alcohols, organic acids, ketones, amino acids, gases, antibiotics, enzymes, vitamins and hormones. Fermentation processes also include fermentation processes used in the consumable alcohol industry, dairy industry, leather industry and tobacco industry. The product of the fermentation process is referred to herein as a fermentation product.

[0097] In some embodiments, slurry stream 108 can contain no exogenous enzyme, or enzyme can be added to give slurry concentrations in a range of from about 0.05 grams/liter to about 5 grams/liter within the fermenter. In some embodiments, when a stream exiting fermenter 1 10 can be recycled back to fermenter 1 10 or mixing tank 106, a concentration of the microorganism in fermenter 1 10 can be up to about 100 grams/liter. In some embodiments, fermenter 1 10 can be a cone bottom fermenter that has a cone-shaped bottom portion 1 16 and a cylindrical-shaped top portion 1 1 8. In some embodiments, cone-shaped bottom portion 1 16 can be an accentric cone shape. In other embodiments, cone-shaped bottom portion 116 can be a concentric cone shape. In some embodiments, the slurry is shear thinning, meaning that slurry is viscous, but will flow upon being mixed. The angle of cone-shaped bottom portion 1 16 is important in order to ensure that the slurry will flow down the slope of cone-shaped bottom portion 116 and not just remain stuck to the slope of cone-shaped bottom portion 116. If the slurry does not flow down the slope of cone-shaped bottom portion 1 16, then fermenter 1 10 becomes difficult to clean and outlets of fermenter 1 10 located in cone-shaped bottom portion 116 can become clogged. In some embodiments, in order to avoid the problems discussed above, cone-shaped bottom portion 1 16 has an angle a of about 45 degrees, about 50 degrees, about 55 degrees, about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, or about 80 degrees. In some embodiments, in order to avoid the problems discussed above, cone-shaped bottom portion 1 16 has an angle a in a range from above 45 degrees to about 80 degrees, above 45 degrees to about 75 degrees, above 45 degrees to about 70 degrees, above 45 degrees to about 65 degrees, above 45 degrees to about 60 degrees, above 45 degrees to about 55 degrees, above 45 degrees to about 50 degrees, about 50 degrees to about 80 degrees, about 50 degrees to about 75 degrees, about 50 degrees to about 70 degrees, about 50 degrees to about 65 degrees, about 50 degrees to about 60 degrees, about 50 degrees to about 55 degrees, about 55 degrees to about 80 degrees, about 55 degrees to about 75 degrees, about 55 degrees to about 70 degrees, about 55 degrees to about 65 degrees, about 55 degrees to about 60 degrees, about 60 degrees to about 80 degrees, about 60 degrees to about 75 degrees, about 60 degrees to about 70 degrees, about 60 degrees to about 65 degrees, about 65 degrees to about 80 degrees, about 65 degrees to about 75 degrees, about 65 degrees to about 70 degrees, about 65 degrees to about 80 degrees, about 65 degrees to about 75 degrees, about 65 degrees to about 70 degrees, about 70 degrees to about 80 degrees, about 70 degrees to about 75 degrees, or about 75 degrees to about 80 degrees. Angle a can be measured between the surface of cone-shaped bottom portion 1 16 and a horizontal flat plane, as shown in FIG. 1, [0099] In some embodiments, a ratio of the height of cone-shaped bottom portion 1 16 to the height of cylindrical-shape top portion 1 18 can be in a range of from about 0.2 to about 50, about 0.2 to about 40, about 0.2 to about 30, about 0.2 to about 20, about 0.2 to about 10, about 0.2 to about 5, about 0.2 to about 1, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 10, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 50, about 30 to about 40, or about 40 to about 50.

[0100] In some embodiments, fermenter 110 can be made from stainless steel, fiber reinforced polymer, metal matrix composite, ceramic matrix composite, thermoplastic matrix composite, metal, epoxy lined carbon steel, plastic lined carbon steel, plastic, and/or fiberglass.

[0101] In some embodiments, fermenter 1 10 is sized to have a volume of about 1,000 gallons, about 5,000 gallons, about 10,000 gallons, about 50,000 gallons, about 100,000 gallons, about 200,000 gallons, about 300,000 gallons, about 400,000 gallons, about 500,000 gallons, about 750,000 gallons, or about 1,000,000 gallons. In some embodiments, fermenter 1 10 has a volume from about 1 ,000 gallons to about 1,000,000 gallons, about 1 ,000 gallons to about 750,000 gallons, about 1,000 gallons to about 500,000 gallons, about 1,000 gallons to about 400,000 gallons, about 1,000 gallons to about 300,000 gallons, about 1 ,000 gallons to about 200,000 gallons, about 1,000 gallons to about 100,000 gallons, about 1,000 gallons to about 50,000 gallons, about 1 ,000 gallons to about 10,000 gallons, about 1,000 to about 5,000 gallons, about 5,000 gallons to about 1,000,000 gallons, about 5,000 gallons to about 750,000 gallons, about 5,000 gallons to about 500,000 gallons, about 5,000 gallons to about 400,000 gallons, about 5,000 gallons to about 300,000 gallons, about 5,000 gallons to about 200,000 gallons, about 5,000 gallons to about 100,000 gallons, about 5,000 gallons to about 50,000 gallons, about 5,000 gallons to about 10,000 gallons, about 10,000 gallons to about 1,000,000 gallons, about 10,000 gallons to about 750,000 gallons, about 10,000 gallons to about 500,000 gallons, about 10,000 gallons to about 400,000 gallons, about 10,000 gallons to about 300,000 gallons, about 10,000 gallons to about 200,000 gallons, about 10,000 gallons to about 100,000 gallons, about 10,000 gallons to about 50,000 gallons, about 50,000 gallons to about 1,000,000 gallons, about 50,000 gallons to about 750,000 gallons, about 50,000 gallons to about 500,000 gallons, about 50,000 gallons to about 400,000 gallons, about 50,000 gallons to about 300,000 gallons, about 50,000 gallons to about 200,000 gallons, about 50,000 gallons to about 100,000 gallons, about 100,000 gallons to about 1 ,000.000 gallons, about 100,000 gallons to about 750,000 gallons, about 100,000 gallons to about 500,000 gallons, about 100,000 gallons to about 400,000 gallons, about 100,000 gallons to about 300,000 gallons, about 100,000 gallons to about 200,000 gallons, about 200,000 gallons to about 1,000,000 gallons, about 200,000 gallons to about 750,000 gallons, about 200,000 gallons to about 500,000 gallons about 200,000 gallons to about 400,000 gallons, about 200,000 gallons to about 300,000 gallons, about 300,000 gallons to about 1,000,000 gallons, about 300,000 gallons to about 750,000 gallons, about 300,000 gallons to about 500,000 gallons, about 300,000 gallons to about 400,000 gallons, about 400,000 gallons to about 1,000,000 gallons, about 400,000 gallons to about 750,000 gallons, about 400,000 gallons to about 500,000 gallons, about 500,000 gallons to about 1 ,000,000 gallons, about 500,000 gallons to about 750,000 gallons, or about 750,000 gallons to about 1 ,000,000 gallons.

In some embodiments, mixing tank 106 is smaller than fermenter 1 10. The larger the amount of biomass and microorganisms to be mixed, the more energy that is required to produce a slurry. Initially, the viscosity of the slurry peaks when hydrolysis of the biomass starts and then decreases over time. Thus, the larger the amount of biomass being hydrolyzed the larger the viscosity of the slurry during the initial phases of the fermentation until sufficient hydrolysis is achieved so that a noticeable decrease in viscosity occurs. The larger the viscosity of the slurry, the more energy that is needed to run an agitator and the larger the agitator needed to mix/agitate the slurry. Even though the viscosity of the slurry decreases over time, the agitator needs to be designed for the maximal energy input needed to mix the slurry during the peak viscosity time period. Thus, mixing with a mixing device (e.g., an agitator) and fermenting in a single vessel can be too expensive in terms of capital expenditure (e.g., the cost of the mixing device) and the energy expenditure (e.g., the amount of energy needed to run the mixing device). Performing the mixing of biomass feed stream 102 and microorganism feed stream 104 in batches in a mixing tank 106 that is smaller than fermenter 110 eliminates the need to have a mixing device in fermenter 1 10. Thus, in some embodiments, fermenter 1 10 does not have a mixing device (e.g, an agitator). In some embodiments fermenter 1 10 does not have any internal moving parts. As a result of mixing in a mixing tank 106 that is smaller than fermenter 1 10, larger quantities of biomass slurry can be fermented in fermenter 1 10 without the associated higher cost of mixing with a mixing device and fermenting the larger fermenter 110. Thus, the combination of mixing tank 106 and fermenter 1 10 results in a reduction in net greenhouse gases during the fermentation process and minimizes the carbon footprint of the fermentation process. In addition, the larger the amount of biomass and microorganisms being mixed the larger the amount of heat that is generated. Heat exchanger 1 14 must be designed to accommodate the peak heat load. Thus, the combination of mixing tank 106 and fermenter 110 reduces the amount of peak heat load generated, thereby reducing the cost of heat exchanger 1 14 and providing further reduction in the net greenhouse gases and carbon footprint of the fermentation process.

[0103] In some embodiments, mixing tank 106 can also have a cone-shaped bottom portion and a cylindrical-shaped top portion. In some embodiments, the cone-shaped bottom portion can be an accentric cone shape. In other embodiments, the cone-shaped bottom portion 116 can be a concentric cone shape. In some embodiments, the dimensions of mixing tank 106 are proportional to the dimensions of fermenter 110. In some embodiments mixing tank 106 is proportionally dimensioned to be about l/5^th, about l/10^th, about l/20^th, about l/30^th, about l/40^th, or about l/50^th the size of fermenter 1 10. In some embodiments, mixing tank 106 is proportionally dimensions to be in a range of from about l/5^th to about l/50^th, about l/5^th to about l/40^th, about l/5^th to about l/30^th, about l/5^th to about l/20^th, about l/5^th to about l/10^th, about l/10^th to about l/50^th, about 1/10^th to about l/40^th, about 1/10^th to about l/30^th, about 1/10^th to about l/20^th, about l/20^th to about l/50^th, about l/20^th to about l/40^th, about l/20^th to about l/30^th, about l/30^th to about l/50^th, about l/30^th to about l/40^th, or about l/40^th to about l/50^th the size of fermenter 110.

[0104] Residence time of slurry stream 108 inside fermenter 1 10 is the amount of time slurry stream 108 spends insider fermenter 1 10 from the time it enters fermenter 1 10 until the time it leaves fermenter 1 10. The number of tank turn overs per hour can be calculated by taking the reciprocal of the residence time. In some embodiments, residence time is dependent upon the microorganism and/or the enzymes used. In some embodiments, the enzyme has the smallest diffusion constant of the water soluble contents of fermenter 1 10, which permits for a longer residence time. In some embodiments the diffusion constant of the enzyme can be in a range of from about 10^"6 cm²/sec about 10^"7 cm²/sec. In some embodiments, in order to make use of the potential maximum residence time provided by the enzyme having a small diffusion constant, the microorganism can be chosen based on its ability to stay active (e.g., to hydrolyze and/or ferment) for longer periods of time in a controlled environment. In some embodiments, the residence time (or the tank turn over rate, which is the reciprocal of residence time), depends on the pump size and fermentation volume, and can be adjusted accordingly for optimization of the fermentation process.

In some embodiments, a residence time of slurry stream 108 inside fermenter 1 10 can be about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. In some embodiments, a residence time of slurry stream 108 inside fermenter 1 10 can be in a range of from about 30 minutes to about 10 hours, about 30 minutes to about 9 hours, about 30 minutes to about 8 hours, about 30 minutes to about 7 hours, about 30 minutes to about 6 hours, about 30 minutes to about 5 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 10 hours, about 1 hour to about 9 hours, about 1 hour to about 8 hours, about 1 hour to about 7 hours, about 1 hour to about 6 hours, about 1 hour to about 5 hours, about 1 hour to about 4 hours, about 1 hour to about 3 hours, about 1 hour to about 2 hours, about 2 hours to about 10 hours, about 2 hours to about 9 hours, about 2 hours to about 8 hours, about 2 hours to about 7 hours, about 2 hours to about 6 hours, about 2 hours to about 5 hours, about 2 hours to about 4 hours, about 2 hours to about 3 hours, about 3 hours to about 10 hours, about 3 hours to about 9 hours, about 3 hours to about 8 hours, about 3 hours to about 7 hours, about 3 hours to about 6 hours, about 3 hours to about 5 hours, about 3 hours to about 4 hours, about 4 hours to about 10 hours, about 4 hours to about 9 hours, about 4 hours to about 8 hours, about 4 hours to about 7 hours, about 4 hours to about 6 hours, about 4 hours to about 5 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours, 5 hours to about 8 hours, about 5 hours to about 7 hours, about 5 hours to about 6 hours, about 6 hours to about 10 hours, about 6 hours to about 9 hours, about 6 hours to about 8 hours, about 6 hours to about 7 hours, about 7 hours to about 10 hours, about 7 hours to about 9 hours, about 7 hours to about 8 hours, about 8 hours to about 10 hours, about 8 hours to about 9 hours, or about 9 hours to about 10 hours.

[0106] In some embodiments, slurry stream 108 is pumped into a top portion of fermenter

1 10 (e.g., a top portion of cylindrical-shaped top portion 1 18). In some embodiments, fermenter 1 10 has one or more inlet nozzles through which slurry stream 108 enters fermenter 1 10. In some embodiments a plurality of inlet nozzles can be arranged in a horizontal plane of fermenter 110. In some embodiments, the inlet nozzle(s) can be arranged at an angle to move slurry stream 108 away for the walls of fermenter 1 10 and induce a circular mixing pattern in fermenter 110. In some embodiments, the angle of the inlet nozzle(s) can be measured between a surface of the inlet nozzle(s) and a horizontal flat plane. In some embodiments, the angle of the inlet nozzle(s) can be about 0 degrees, 10 degrees, about 20 degrees, about 30 degrees, or about 40 degrees. In some embodiments, the angle of the inlet nozzle(s) can be in a range of from about 0 degrees to about 40 degrees, about 0 degrees to about 30 degrees, about 0 degrees to about 20 degrees, about 0 degrees to about 10 degrees, about 10 degrees to about 40 degrees, about 10 degrees to about 30 degrees, about 10 degrees to about 20 degrees, about 20 degrees to about 40 degrees, about 20 degrees to about 30 degrees, or about 30 degrees to about 40 degrees.

[0107] Introduction of slurry stream 108 into fermenter 1 10 through a one or more inlet nozzles positioned at an angle establishes a swirling motion inside fermenter 1 10 that mixes the contents of fermenter 110. In addition during fermentation, the microorganisms generate carbon dioxide, and the carbon dioxide bubbles provide some mixing of the contents of fermenter 1 10. Thus, even without the use of a mixing device in fermenter 110, mixing occurs within fermenter 1 10.

[0108] In some embodiments, a stream 120 exits fermenter 1 10 containing the fermentation product, for example, ethanol, and the remainder of the slurry. The fermentation product can be recovered from stream 120 through conventional means (not shown) and the remainder of the contents of stream 120 (e.g., microorganism, unconverted biomass, unfermented sugars, etc.) can be recycled to mixing tank 106. In some embodiments, a pump 122 is used to move the recyclable contents of stream 120 back to mixing tank 106. In some embodiments, pump 122 can be the same as pump 1 12. In some embodiments, the recyclable contents of stream 120 can pass through a heat exchanger 124 before entering mixing tank 106. In some embodiments, heat exchanger 124 can be the same as heat exchanger 114. In some embodiments, the recyclable contents of stream 120 can include the carbon dioxide generated by the microorganism. In some embodiments, a rate of carbon dioxide generation is in a range of from less than about 0.05 volumes of gas per volume of fermentation broth per hour (vvh) to about 0.3 vvh. Carbon dioxide can dissolve in the fermentation broth and form carbonic acid, which can create foaming when the acid is released as carbon dioxide when slurry from fermenter 1 10 is pumped into mixing tank 106. The contents at the bottom of fermenter 1 10 are under a pressure in a range of from about 1 bar to about 3 bar as a result of the height of liquid (i.e., the liquid head) in fermenter 1 10, which is determined by the height of fermenter 1 10. In some embodiments, recycling carbon dioxide as carbonic acid back to mixing tank 106 prevents buildup of carbonic acid in fermenter 1 10 and advantageously permits degassing and defoaming to occur in mixing tank 106 in an incremental manner.

Θ9] In some embodiments, fermenter 1 10 and all streams entering fermenter 1 10 are kept sterile in order to avoid contaminants.

10] In some embodiments, the pH of the contents of fermenter 1 10 (e.g., the fermenter broth) is maintained according to optimal ranges for the microorganism used. In some embodiments, the pH of the contents of fermenter 1 10 is maintained at about 4, about 4.5, about 5, about 5.5, or about 6, In some embodiments, the pH of the contents of fermenter 1 10 is maintained in a range of about 4 to about 6, about 4 to about 5.5, about 4 to about 5, about 4 to about 4,5, about 4.5 to about 6, about 4.5 to about 5.5, about 4.5 to about 5, about 5 to about 6, about 5 to about 5.5, or about 5.5 to about 6.

111 In an alternative exemplary process and system illustrated in FIG. 2, slurry stream

108 can enter fermenter 110 through an inlet nozzle located in a cone-shaped portion 116. In some embodiments, the recyclable contents of stream 120 exiting fermenter 1 10 can be split into a stream 126 that is recycled back to mixing tank 106 and a stream 128 that is recycled back to fermenter 1 10. In some embodiments, the split into streams 126 and 128 occurs after heat exchanger 124. In some embodiments, the split can occur before heat exchanger 124 and each stream 126 and 128 can pass through a separate heat exchanger. In some embodiments, stream 128 can enter a top portion of fermenter 1 10 (e.g., a top portion of cylindrical-shaped top portion 1 18). in some embodiments, stream 128 enters fermenter through an inlet nozzle positioned at an angle as discussed above. The flow of streams 128 and 108 into different portions of fermenter 1 10 can create a cross flow tha increases mixing in fermenter 110. In some embodiments, a cross flow can also be created by having stream 108 enter fermenter 1 10 through a top portion of fermenter 1 10 (as shown in FIG. 1) and having stream 128 enter through a bottom portion of fermenter 1 10 (not shown).

Examples

Example 1

The yeast strain Ml 873, described in International Appl. No.

PCT/US201 1 /039192 filed June 3, 201 1 , at an exogenous enzyme loading of 2.8 mg enzyme per g chips (dry basis), and a yeast inoculum level of 10% v/v (equivalent to 0.5 g/L) was tested. Data for calculation of the specific production rates of ethanol, C0₂ and heat, are summarized in Table 1. The data in Table 1 were based on runs carried out in a 2L, flat bottom, agitated fermenter with pretreated hardwood feedstock MS 887 (2 stage pretreated solids) for which the fermentation time course is shown in FIG. 3. There was 1L of fermentation broth in the 2L fermenter. FIG. 3 illustrates the concentration of ethanol, glycerol, lactic acid, and acetic acid over the time course of the fermentation.

Table 1.

CBP: Properties and Factors

(a) CBP Yeast Measured Characteristics

(b) Characteristic Dimensions and Diffusion Constants

l

(d) Summary of Static Head (Pressure) as a Function of Fermenter Volume

Example 2

The fermentation of Example 1 was run again in a 1,000 gallon cone bottom fermenter without the use of an agitator and in a 1,000 gallon traditional (i.e., flat bottom) fermenter with the use of an agitator. The 1,000 gallon cone bottom fermenter had an angle of 60 degrees as measured between the surface of the cone and a horizontal flat plane. FIG. 4 illustrates the concentration of ethanol and lactic acid produced over the time course of the fermentation in the 1 ,000 gallon cone bottom fermenter without the use of an agitator. The feed time was 48 hours. FIG. 5 illustrates the concentration of ethanol and lactic acid produced over the time course of the fermentation in the 1,000 gallon traditional fermenter with the use of an agitator. The feed time was 36 hours. [0114] Mixing achieved in a small scale fermenter, such as in Example 1 , is often not directly scalable to larger fermenters, due to the intensity that would require unrealistic power inputs at a large scale. Surprisingly, a comparison of the results in Example 1 shown in FIG. 3 to (a) results from the 1,000 gallon cone bottom fermenter (FIG. 4) and (b) results from the 1,000 gallon traditional (e.g., flat bottom) agitated fermenter (FIG. 5) show similar results between the laboratory agitated 2L fermenter and (a) the 1,000 gallon cone bottom fermenter and (b) the 1 ,000 gallon traditional agitated fermenter.

[0115] It was also surprising that the results achieved from the fermentation in the 1 ,000 gallon cone-bottom fermenter without agitation (FIG. 4) was essentially equivalent to the results achieved from the fermentation in the 1,000 gallon agitated traditional (i.e., flat bottom) fermenter (FIG. 5), when the different feeding times (48 hours for FIG. 4 and 36 hours for FIG. 5) were accounted for. The solids fed over the course of the fermentations was equivalent to 23% w/w pretreated wood/fermentation broth. These results surprisingly demonstrate that the agitation provided by the cone bottom fermenter without the use of an agitator provides adequate mixing for the obtaining a desired amount of fermentation product (e.g., ethanol). Since the fermentation in the cone bottom fermenter without the use of an agitator had little effect on the fermentation time course, scale-up to larger fermenters based on micro-scale properties of the CBP yeast and pretreated wood system can be accomplished.

Example 3

[0116] The yeast strain Ml 873, described in International Appl. No.

PCT/US201 1/039192 filed June 3, 2011 was tested in a series of runs carried out in a 1 ,000 gallon epoxy-lined cone bottom fermenter. The feedstock was hardwood pretreated in a single stage at a severity of 4.4 and washed. Enzymes were loaded at a chip basis of 3.5 to 3.8 mg protein per g of solid and a washed pretreated basis of about 5 mg protein per g of solid. Two fermentation runs were made at three tank turnovers (TTO) per hour. The fermentation conditions and ethanol titers are summarized in Table 2 below. Table 2: Ethanol Titers for Three Tank Turnovers per Hour in a 1,000 Gallon Cone

Bottom Fermenter

[0117] As can be seen in Table 2, when the fermentation runs were conducted at three tank turnovers per hour, the ethanol titer for the two runs were measured at 68 g/L. When normalized for lactic acid production (i.e., assuming only ethanol is produced and no contaminating bacteria are present) the ethanol titer for the two runs were estimated to be equivalent to 68 g/L and 70 g/L, with an average of 69 g/L.

[0118] Six fermentation runs were also made at one tank turnover (TTO) per hour. The fermentation conditions and ethanol titers are summarized in Table 3 below.

Table 3 : Ethanol Titers for One Tank Turnover per Hour in a 1 ,000 Gallon Cone Bottom

Fermenter

[0119] As can be seen in Table 3, when the fermentation runs were conducted at one tank turn over per hour, the ethanol titers ranged from 55 g/L to 64 g/L after 120 hours of fermentation, with an average ethanol titer of 59 g/L. When normalized for lactic acid production (i.e., assuming only ethanol is produced and no contaminating bacteria are present), the average ethanol titer was equivalent to 63 g/L. As shown in Table 3, two of the runs (R3-041 and R3-042) were carried out with increased mixing being using in a satellite tank prior to introduction to the fermenter (indicated as "Incr. Sat. Mixing").

[0120] The fermentations were conducted at three tank turnovers (TTO) per hour and at one tank turnover (TTO) per hour to determine the effect of the number of tank turnovers per hour on ethanol titer. A lower TTO has the advantage that less capital is expended for fermenter circulation pumps and piping. In contrast, a higher TTO enables enhanced removal of heat and faster feed and reagent addition rates, thus balancing out or buffering potential changes in the critical fermentation parameters of temperature and pH control. These runs were performed as part of an optimization process for determining the effect of TTO on ethanol titer.

Example 4

[0121] The yeast strain Ml 873, described in International Appl. No.

PCT/US201 1/039192 filed June 3, 2011 , at an exogenous enzyme loading of 2.8 to 3.1 mg enzyme per g chips (dry basis), and a yeast inoculum level of 10.9% v/v (equivalent to 0.26 g/L) was tested in a 5,000 gallon cone bottom fermenter without agitation. The 5,000 gallon cone bottom fermenter had an angle of 60 degrees as measured between the surface of the cone and a horizontal flat plane. Washed pretreated hardwood was provided as the feedstock using a 39 hour feeding schedule. FIG. 6 illustrates the concentration of ethanol, lactic acid, and acetic acid over the time course of the fermentation on the left side of the graph. Fig. 6 also illustrates the cumulative mass (on a dry basis) of washed pretreated total solids fed to the fermenter over the time course of the fermentation on the right side of the graph.

[0122] Data from the 1 ,000 gallon cone bottom fermentation run shown in Fig. 4 is superimposed onto Fig. 6 to show that the ethanol yield and rate of production for the 5,000 gallon cone bottom fermentation run is similar to the ethanol yield and rate of production for the 1,000 gallon cone bottom fermentation run. The similarity in the ethanol yield and rate of production in the 5,000 gallon cone bottom fermentation run and the 1,000 gallon cone bottom fermentation run indicate that scale-up to larger fermenters based on micro-scale properties of the CBP yeast and pretreated wood system can be accomplished.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A process for producing a fermentation product comprising:

introducing biomass into a tank;

introducing a microorganism into said tank;

mixing said biomass and said microorganism in said tank to form a slurry;

pumping said slurry from said tank to said cone bottom fermenter;

allowing said microorganism to ferment said biomass in said cone bottom fermenter; and recovering one or more products of said fermentation,

wherein said cone bottom fermenter has a cone-shaped bottom having an angle, measured with respect to a horizontal flat plane, in a range of from above 45 degrees to about 80 degrees.

2. The process of claim 1, wherein said biomass and microorganism are mixed in said tank with an agitator.

3. The process of any one of claims 1-2, wherein a residence time of said slurry in said cone bottom fermenter is in a range of from about 30 minutes to about 10 hours.

4. The process of any one of claims 1-3, wherein a solids content of said biomass in weight percentage is in a range of from about 5% to about 45%.

5. The process of any of claims 1 -4, wherein said tank comprises a cone bottom tank.

6. The process of any one of claims 1-5, wherein said tank is proportionally dimensioned from about l/5th to about l/50th a size of said cone bottom fermenter.

7. The process of any one of claims 1-6, wherein said slurry is pumped into a top portion of said cone bottom fermenter.

8. The process of any one of claims 1-7, wherein a portion of said slurry exiting said cone- shaped bottom of said cone bottom fermenter is recycled to said tank.

9. The process of claim 8, wherein said recycled portion passes through a heat exchanger before entering said tank.

10. The process of any one of claims 1-9, wherein said slurry passes through a heat exchanger before entering said cone bottom fermenter.

1 1. The process of any one of claims 1-10, wherein said slurry comprises a solids content in a range of from about 5% to about 45% by weight.

12. The process of any one of claims 1-11, wherein said angle is in a range of from about 60 degrees to about 80 degrees.

13. The process of any one of claims 1- 12, wherein said angle is about 60 degrees.

14. The process of any one of claims 1-13, wherein said biomass comprises lignocellulosic biomass.

15. The process of claim 14, wherein said lignocellulosic biomass is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof.

16. The process of any one of claims 1-15, wherein said microorganism is selected from the group consisting of yeast, bacteria, fungi, and combinations thereof.

17. The process of any one of claims 1-16, wherein said microorganism is a recombinant microorganism.

18. The process of any one of claims 1-17, wherein said microorganism produces ethanol.

19. The process of any one of claims 1-18, wherein said microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastor is, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, Schwanniomyces occidentalis, Eschirichia coli, Thermo anaerobacteium saccharolyticum, and Clostridium thermocellum.

20. The process of any one of claims 1-19, wherein said microorganism is a yeast.

21. The process of any one of claims 1-20, wherein said microorganism is Saccharomyces cerevisiae.

22. The process of any one of claims 1-21, wherein said microorganism is a xylose-utilizing microorganism.

23. The process of any one of claims 1-22, wherein said microorganism is a glucose-utilizing microorganism.

24. The process of any one of claims 1-23, wherein said microorganism expresses enzymes that break down cellulose in said biomass into sugars and ferments said sugars into ethanol.

25. The process of any one of claims 1-24, wherein the fermenting of said biomass in said cone bottom fermenter occurs without the use of a mixing device in said cone bottom fermenter.

26. The process of any one of claims 1-25, wherein the fermentation product comprises ethanol.

27. The process of any one of claims 1-26, wherein the slurry further comprises one or more exogenous enzymes.

28. The process of any one of claims 1-27, wherein the slurry further comprises one or more catalysts that mimic enzymes.

29. The process of any one of claims 1-28, wherein said biomass is pretreated.

30. A system for producing a fermentation product comprising:

a mixing tank configured to form a slurry comprising biomass and a microorganism; a cone bottom fermenter configured to receive said slurry, wherein said cone bottom fermenter has a cone-shaped bottom having an angle, measured with respect to a horizontal flat plane, in a range of from above 45 degrees to about 80 degrees; and

a pump configured to move said slurry from said mixing tank to said cone bottom fermenter.

31. The system of claim 30, wherein said angle is in a range of from about 60 degrees to about 80 degrees.

32. The system of any one of claims 30-31 , wherein said angle is about 60 degrees.

33. The system of any one of claims 30-32, wherein said cone bottom fermenter further comprises a cylindrical portion above said cone-shaped bottom and wherein a ratio of a height of said cone-shaped bottom to said cylindrical portion is in a range of from about 0.2 to about 50.

34. The system of any one of claims 30-33, wherein said mixing tank comprises a cone bottom tank.

35. The system of any one of claims 30-34, wherein said mixing tank is proportionally dimensioned in a range of from about l /5th to about 1/50th a size of said cone bottom fermenter.

36. The system of any one of claims 30-35, wherein said cone bottom fermenter comprises an inlet nozzle arranged near a top portion of said cone bottom fermenter at an angle, measured with respect to a horizontal flat plane, in a range of from about 0 degrees to about 40 degrees for introducing said slurry into said cone bottom fermenter.

37. The system of claim 36, wherein said angle of said inlet nozzle is about 0 degrees.

38. The system of any one of claims 30-37, wherein the system is configured for said microorganism to ferment said biomass in said cone bottom fermenter without the use of a mixing device in said cone bottom fermenter.

39. The system of any one of claims 30-38, further comprising a heat exchanger through which said slurry passes before entering said cone bottom fermenter.

40. The system of any one of claims 30-39, further comprising a second pump configured to recycle a portion of said slurry exiting said cone-shaped bottom of said cone bottom fermenter to said mixing tank.

41. The system of claim 40, further comprising a second heat exchanger through which said recycled portion passes before entering said mixing tank.