RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/358,214, filed on June 24, 2010. The entire teaching of the above application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

There has been an increasing interest in the use of alternative feedstocks for the production of fuels and chemicals, rather than relying exclusively on fossil fuels. Fuels and chemicals are now produced through biotechnology using sugars (to produce ethanol and Butanol) and triacyl glycerides (to produce biodiesel). The term "biodiesel" is used for a variety of ester-based oxygenated fuels made from vegetable oils, fats, greases, or other sources of triglycerides. It is a nontoxic and biodegradable substitute and supplement for petroleum diesel. Even in blends as low as 20% biodiesel and 80% petroleum diesel (B20), biodiesel can substantially reduce the emission levels and toxicity of diesel exhaust.

Biodiesel can be used in any diesel engine, without the need for mechanical alterations, and is compatible with existing petroleum distribution infrastructure. Biodiesel processing involves the production of mono-alkyl esters of long chain fatty acids by reacting the source acid with a low molecular weight alcohol, such as methanol or ethanol. A traditional process for manufacturing fatty acid alkyl esters involves the transesterification of triacyl glycerides ("triglycerides" or "TAGs") using methanol, in the presence of an alkali catalyst. In addition to the desired fatty acid alkyl esters, this process produces an effluent stream comprising glycerol (glycerine), excess alcohol, water, alkyl esters and a mixture of mono-, di- and triglycerides resulting from the transesterification step. The rapid worldwide expansion of the production of biodiesel fuel since 2000 has been creating a rapidly growing supply of byproduct crude glycerol. Recently, the value of crude glycerol has decreased and it is anticipated that biodiesel producers may receive little or no value for this material. At one time there was a valuable market for glycerol, which assisted the economics of the biodiesel process as a whole. However, with the increase in global biodiesel production, the market price for crude glycerol has diminished.

Large quantities of crude glycerol are also produced in the oleochemicals industry, where fatty acids and fatty alcohols are produced and used for the production of soaps, detergents, chemical intermediates and other products.

The volume of glycerol that is produced by the oleochemicals industry and as a by product in the production of biodiesel is predicted to continue to increase because of the tremendous global growth in the production of biodiesel.

Crude glycerol has few direct uses due to the presence of a large percentage of impurities such as salts and other species, and its fuel value is marginal. However, crude glycerol has recently become an attractive carbon source for fermentation processes, in part because of its low cost. Not only is glycerol abundant and cheaper than sugar-based feedstocks, but its higher reduced state, compared to cellulosic sugars, promises to significantly increase the product yield of chemicals whose production from sugars is limited by the availability of reducing equivalents. Taking full advantage of the higher reduced state of carbon in glycerol requires the use of microaerobic or completely anaerobic fermentations. Various strains of bacteria and bioengineered organisms are known to be capable of growing anaerobically (fermentatively) on glycerol substrates (e.g. US Pat. Pub. No. 2009/0186392).

Glycerol does have drawbacks as a feed stock in a fermentative process. The crude glycerol by-product stream from a biodiesel plant is typically comprised of a mixture of glycerol, water, inorganic salts (catalyst residue), methanol, free fatty acids, unreacted mono-, di-, and triglycerides and methyl esters, as well as a variety of other organic non- glycerol components in varying quantities. In raw form, this crude glycerol has a high salt and free fatty acid content and substantial color (yellow to dark brown). Extensive removal ("refining") of these impurities prior to use is costly.

However, glycerol does have advantages over the more traditional feedstocks for conversion to ethanol such as grains, molasses, corn, sugarcane and the like. These traditional feedstocks have high solids contents and so these solids build up in the fermenter during continuous batch fed biofermentation. Consequently there is no commercial advantage to a process that significantly extends the fermentation time using traditional feedstocks. However, crude glycerol or crude glycerol that has been partially or fully desalted has a low solids content. Therefore, solids do not build up throughout the process at the rate that build up occurs with the use of traditional sugars.

An improved, efficient, cost effective means for using glycerol and other waste stream feedstocks in a bioconversion system such as anaerobic fermentation to create a higher value product is desirable. SUMMARY OF THE INVENTION

The present invention provides methods and apparatus for the highly efficient, cost effective production of desired chemical products by anaerobic fermentation. In a preferred embodiment, the method is a process for anaerobic fermentation of glycerol to produce ethanol using a biofermentation process that couples a fermentation zone to a vacuum assisted purification zone and ultimately a product recovery zone. The process is characterized in that vacuum assisted evaporation is used to produce a product enriched vapor from which the product may be efficiently isolated and collected wherein the vacuum assisted evaporation is carried out in a zone that is separate from the fermenter. The process is also characterized in that it uses microorganisms that are heat tolerant thereby allowing the entire process to be run at higher temperatures. This enables the recycling of microorganisms to the fermenter for renewed fermentation even after ethanol is extracted from the fermentation liquid by evaporation under a vacuum at elevated temperatures. Using the process of the invention, ethanol is produced with high efficiency from a low cost and abundant crude feedstock. The invention also provides highly efficient batch- fed processing for the production of ethanol from glycerol over longer periods of time as compared to traditional ethanol fermentation processes.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1 is a schematic flow chart representing an embodiment of the invention. FIG. 2 is a schematic flow chart representing a variation of the embodiment of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the invention are particularly useful when low value and/or highly impure feedstocks (referred to herein as "disadvantaged feedstocks") are used as the substrate for the fermentation process. Feedstocks that may be used in accordance with the present invention and apparatus include gums, mono-, di- or tri-glycerides, fatty acids and crude oil extracts from animals or plants. Preferred feedstocks include those that are waste products from other industries and particularly the oleochemical and biofuels industries. Particularly preferred feedstocks are those that have low solids content such as glycerol or glycerol/sugar mixtures. The use of low solids feedstocks avoids the build up of solids in the fermenter and does not limit the time and volume of feedstocks that can be used per fermentation as compared to the classic high solids feedstocks for fermentation to produce ethanol such as feedstocks associated with sugarcane and grains such as corn. Chemical products produced by the methods and apparatus of the invention include but are not limited to alcohols (e.g ethanol, 1,4 butanediol, isopropanol, butanol and 1,2 propanediol), acetate, succinate, gamma-butyrolactone, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, butyrate, mevalonate, ethanolamine, propionate and isoprene.

In one embodiment, the method is a process for anaerobic fermentation of a feedstock to produce a product by combining a fermentation zone with a vacuum assisted purification zone and a product recovery zone collectively referred to herein as a

"biorefmery system". In one embodiment, the method of the invention comprises: 1) combining a feedstock with a heat-tolerant microorganism capable of producing the desired product from the feedstock in a fermenter under conditions suitable for the bioconversion of feedstock to product; 2) removing at least a portion of the fermented liquid comprising the product from the fermenter to the vacuum assisted purification zone; 3) subjecting the fermentation liquid to vacuum evaporation at elevated temperatures in the vacuum assisted purification zone thereby vaporizing at least a portion of the product contained in the fermentation liquid to form a product-rich vapor wherein the elevated temperatures are tolerated by the heat-tolerant microorganisms present in the fermentation broth; and 4) optionally separating and collecting the product from the product-rich vapor. In one preferred embodiment, the fermentation broth removed from the fermenter in step 2 is degassed prior to vacuum evaporation of step 3.

In one preferred embodiment, fermentation broth (including microorganisms) remaining in the fermentation liquid after depletion of ethanol via vacuum evaporation in step 3 are recycled back to the fermenter for renewed fermentation. In one embodiment, water and other soluble nutrients are recycled back to the fermenter after the isolating and collecting of step 4. In one embodiment the vacuum assisted purification zone includes removal of carbon dioxide and hydrogen gasses from the fermentation liquid prior vaporizing ethanol in step 3. In one embodiment, solids or other toxic non- volatile by products do not accumulate in the fermentation broth in the fermenter. In one embodiment ethanol does not accumulate in the fermentation broth of the fermenter and cause inhibition of the production of ethanol by the microorganisms. In one embodiment the process of converting glycerin to ethanol and recovering ethanol proceeds continuously for at least 3 days and preferably for at least 6 days without renewal of the entire contents of the fermenter. In one embodiment the process is a batch-fed process that continuously converts glycerol to ethanol over a period of at least about 3 days and preferably at least about 6 days.

In one embodiment, the method of invention comprises the steps of:

a) fermenting a feedstock in a fermenter with a heat tolerant microorganism capable of producing the product from the feedstock under conditions suitable for the

bioconversion of the feedstock thereby forming a fermented liquid;

b) removing at least a portion of the fermented liquid comprising the product from the fermenter and conducting the fermentation liquid to a vacuum assisted purification zone wherein the vacuum assisted purification zone includes at least one degassing apparatus and at least one vacuum evaporation apparatus;

c) degassing the fermented liquid to at least partially remove by-product gasses from the fermentation liquid in the degassing apparatus prior to the fermentation liquid entering the vacuum evaporation apparatus;

d) vaporizing at least a portion of the product contained in the fermentation liquid in the vacuum evaporation apparatus operated under vacuum and at elevated temperatures wherein the elevated temperatures are tolerated by the heat tolerant microorganisms present in the fermentation broth; and wherein the vapor produced in the vacuum evaporator is product-enriched vapor. Optionally, the method further comprises the step of separating and recovering product from the product-enriched vapor.

In one preferred embodiment the invention provides a method of converting glycerol to ethanol by anaerobic fermentation comprising the steps of:

a) fermenting glycerol in a fermenter with a heat tolerant microorganism capable of producing ethanol from glycerol under conditions suitable for the bioconversion of glycerol to ethanol thereby forming a fermented liquid;

b) removing at least a portion of the fermented liquid comprising ethanol from the fermenter and conducting the fermentation liquid to a vacuum assisted purification zone wherein the vacuum assisted purification zone includes at least one degassing apparatus and at least one vacuum evaporation apparatus;

c) degassing the fermented liquid to at least partially remove by-product gasses from the fermentation liquid in the degassing apparatus prior to the fermentation liquid entering the vacuum evaporation apparatus;

d) vaporizing at least a portion of the ethanol contained in the fermentation liquid in the vacuum evaporation apparatus operated under vacuum and at elevated temperatures wherein the elevated temperatures are tolerated by the heat tolerant microorganisms present in the fermentation broth; and wherein the vapor produced in the vacuum evaporator is ethanol-enriched vapor. Optionally the method further comprises the step of separating and recovering ethanol from the ethanol-enriched vapor.

The invention is useful for improving the efficiency and productivity of any biofermentation process using a feedstock, particularly a low solids feedstock that may include high levels of impurities and/or from which product is otherwise difficult to produce by fermentation and/or isolate from fermentation liquid. The invention ultimately lowers costs for isolating product from fermented liquid because it provides for recycling and reuse of expensive reagents and allows for longer periods of operation of fermentation and product recovery than prior art systems.

The invention may be integrated, retrofitted or otherwise conducted in any facility wherein the feedstock is generated as a byproduct. For example, the invention may be integrated, retrofitted or otherwise conducted in a biodiesel production plant. The invention may also be integrated, retrofitted or otherwise conducted in any facility that produces the product by other fermentation processes. For example, when the product is ethanol, the invention produces ethanol, the invention may also be integrated, retrofitted or otherwise conducted in any facility that produces bio fuels.

In the application, unless specifically stated otherwise, the following abbreviations and definitions apply.

The "biorefmery system" refers to the entire process and apparatus for

bioconversion of a feedstock by anaerobic fermentation to a desired product and final collection of the product in substantially purified form. In accordance with the present invention, the biorefmery system includes at least the fermentation zone, the vacuum assisted purification zone and the product recovery zone.

In accordance with the invention a single "run" is the continuous production of product via batch-fed fermentation which begins when fresh feedstock, microorganisms and nutrients are added to the fermenter and which ends upon the emptying of the fermenter of its contents and shut down of the biorefmery system for optionally cleaning, sterilization and preparation for a new run. In accordance with the invention, a single run can last for at least 3 days and preferably at least 6 days.

As used herein "glycerol" refers to glycerol that may be crude, treated or refined. Crude glycerol may contain up to 90% of impurities such as inorganic salts e.g. chloride, sulfate phosphate organic salts and others, organic compounds (e.g. fatty acids, fatty esters, protein residues, methanol, acids bases or combinations thereof). Treated glycerol includes any glycerol that has undergone at least one treatment process such as a desalting process. Refined glycerol is glycerol that has been treated such that less than 10% impurity remains.

As used herein "feedstock" refers to the material that serves as a substrate for the bioconversion of the material to a desired product by the biocatalyst. The feedstock is combined with the biocatalyst preferably in a fermenter under conditions suitable for bioconversion of the feedstock to the desired product by the microorganisms.

The "fermentation zone" also referred to herein as "fermentation" is the process and apparatus in which a biocatalyst is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the biocatalyst converts raw materials, such as a feedstock, into products. Any feedstock that contains a fermentable carbon source is suitable for the present invention. The fermentation zone generally comprises a fermenter vessel comprising feedstock, biocatalyst(s), nutrients for the biocatalyst and water.

The "biocatalyst" may be any microorganism capable of converting a selected feedstock to a desired product. The biocatalyst can be a whole microorganism, one or more isolated enzymes or any combination thereof. For the purposes of this application,

"microorganism" includes one or more eukaryotes or prokaryotes and includes bacteria, yeast or cells from an insect, animal or plant or tissues therefrom. Further aspects of the biocatalyst are discussed below.

"Conditions suitable for suitable for bioconversion of a feedstock" refers to the material and methods for maintenance and growth of microbial cultures that support the biochemical pathways that are necessary for the microorganism to convert a specific feedstock to a specific product. Such materials and methods are well known in the art of microbiology and biofermentation. For example, anaerobic fermentation of glycerol in Paenibacillus macerans is described in Gupta et al., Applied and Environmental

Microbiology, (2009) 75: 5871-5883, and the anaerobic fermentation of glycerol in E. coli is described in U.S. Pat. Pub. No. 2009/0186392. Consideration must be given to appropriate media, pH, temperature and requirements for anaerobic conditions depending on the specific requirements of the microorganism to support bioconversion of the given feedstock. "Media" generally refers to the liquid containing nutrients for culturing the microorganisms.

"Product" refers to the biocatalytically produced product that is selectively removed from the fermentation liquid using a process herein described. This may be a compound that is produced by the biocatalyst or non-native genes may be genetically engineered into a microorganism for their functional expression in the biofermentation. Generally, the desired product is ethanol.

"Byproduct" refers to any by-product of the fermentation that would be desirable to selectively remove from the biofermentation system to eliminate for example, feedback inhibition and/or improve biocatalyst activity and/or improve or facilitate purification of the desired product and/or collect for further treatment or use. Such byproducts include byproduct gasses generated during the fermentation process including C0 ₂ and H ₂ .

"Fermentation liquid" also referred to herein as "fermentation broth", "beer", or "fermented liquid", is the liquid in which the fermentation and bioconversion of the feedstock to product takes place which generally takes place in the fermenter. The fermentation liquid may be removed from the fermenter for selective removal of the desired product or spent cell mass as described herein. In addition to product, the fermentation liquid may also comprise microorganisms, metabolic intermediates, nutrients, cofactors, water, and salts.

"Vacuum assisted purification zone" refers to process and apparatus whereby a portion of the fermentation liquid containing the desired product is removed from the fermenter and product recovery is initiated using at least one evaporator operated separately (from the fermenter or any other apparatus) under vacuum at elevated temperatures sufficient to vaporize the desired product from the remainder of the fermentation liquid. The vacuum assisted recovery system also preferably includes an apparatus (e.g. a purge column) for removing volatile gas by-products from the

fermentation liquid prior to separation of the desired product from the fermentation broth by flash evaporation in an evaporator. The system may also include means for recycling components of the fermentation broth such as cells and water back to the fermenter at one or more stages of the vacuum assisted product recovery process.

"Product recovery zone" refers to a process which includes apparatus for collecting (e.g. via further distillation) and isolating product from the vacuum assisted purification zone or any other zone which provides fermentation broth containing product. The product recovery system may also include means for recycling components of the fermentation broth such as water and nutrients back to the fermenter after isolation and collection of the desired product.

As used herein "ATCC" refers to the American Type Culture Collection

International Depository located at 10801 University Blvd., Manassas VA 20110-1109. The "ATCC No" is the accession number to cultures on deposit with the ATCC.

The present invention is adaptable to a variety of bio fermentation methodologies, especially those suitable for large-scale industrial processes. The invention may be practiced using batch, fed-batch, or continuous processes, but is preferably practiced in fed-batch mode and can be practiced with a wide variety of fermenter vessels. The present process preferably uses a fed-batch method of biofermentation. Fed-batch biofermentation processes comprise a typical batch system with the exception that the substrate is added in increments as the biofermentation progresses. The biocatalyst may be whole microorganisms or in the form of isolated enzyme catalysts. Whole microbial cells can be used as biocatalyst without any pretreatment or alternatively cells may be treated, for example, to improve the rate of diffusion of materials into and out of the cells.

Optionally, the microorganisms are cultured and grown in a separate "seed" bioreactor and are added to the primary fermenter upon initiation of the biofermentation cycle for recovering ethanol. Microorganisms may be grown anaerobically or aerobically, as appropriate, in this preproduction stage of the fermentation in the seed fermenter and the primary fermenter. One advantage of growing the microorganism in a separate bioreactor is that the need to sterilize the fermenter between runs may be eliminated.

Preferred microorganisms that are suitable as biocatalysts for use in the present invention include those that are capable of converting glycerol to ethanol and that are heat tolerant. The optimum fermentation temperature in the fermenter varies depending on the particular fermentation microorganisms employed, but a range of 25°C to 70°C is generally preferred. Higher fermentation temperatures are preferred, and thus the preferable microorganism for fermentation is heat resistant. Preferred organisms are able to withstand temperatures in excess of 40 °C or higher and produce the desired product. Organisms suitable for use in the conversion of glycerol to ethanol include, but are not limited to: wild-type and bioengineered microorganisms described in United States Patent Publication 2009/0186392 such as wild-type E. Coli K12 strains MG1655 (ATCC

700926), W3110 (ATCC 27325), MC4100 (ATCC 35695) and E. coli B (ATCC 11303), enteric bacteria Enterobacter cloacae subsp., cloacae NCDC 279-56 (ATCC 13047) and yeast Saccharaomyces cerevisiae. Other suitable organisms include strains such as wild type Paenibacillus macerans P. macerans) Northrup strain N234A (=LMG13285=N234A) available from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG, Gent, Belgium); and wild-type P. macerans strains B-394 (NRRL collection Peoria , IL) and ATCC 7068 (American Type Culture Collection, Manassas, VA), Bacillus coagulans, Geobacillus. stearothermophilus, and Geobacillus. thermoglucosidasius . Suitable B. coagulans, G. stearothermophilus and G. thermoglucosidasius strains are available from the Bacillus Genetic Stock Center at The Ohio State University.

Materials and methods suitable for maintenance and growth of microbial cultures are well known to those in the art of microbiology or biofermentation science art. Consideration must be given to appropriate media, pH, temperature, and requirements for anaerobic conditions, depending on the specific requirements of the microorganism.

Large-scale microbial growth may use a wide range of simple or complex carbohydrates, organic acids and alcohols, and saturated hydrocarbons as feedstocks. Suitable feedstocks may include, but are not limited to, monosaccharides (such as glucose and fructose), disaccharides (such as lactose or sucrose), oligosaccharides and

polysaccharides (such as starch or cellulose or mixtures thereof, or unpurified mixtures from renewable feedstocks (such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt)). The feedstocks may also be free fatty acids, monoclycerides, diglycerides, triglycerides, phospholipids or combinations thereof. The feedstocks may also be one-carbon substrates (such as carbon dioxide, methanol, or methane).

Preferred feedstocks include those that are by products from other industries and particularly the biochemicals and biofuels industries. Feedstocks that may be used in accordance with the present invention and apparatus include gums, fatty acids, crude oil extracts from animals or plants and algae. Particularly preferred feedstocks are those that are water soluble such as glycerol or glycerol/sugar mixtures. Optionally, the glycerol feedstock may be treated prior to its use as a feedstock. One such treatment includes a desalting treatment. In one preferred embodiment, the glycerol used as feedstock is obtained as a byproduct of biodiesel production and includes no more than 70% by weight impurities.

In addition to an appropriate feedstock, biofermentation media contains suitable minerals, salts, cofactors, buffers, and other components, known to those skilled in the art. These supplements must be suitable for the growth of the biocatalyst and promote the biochemical pathway necessary to produce the biofermentation product.

In one embodiment bioconversion of feedstocks to product by the microorganisms takes about 24-72 hours. The withdrawal rate of the fermentation liquid from the fermenter to the downstream processes in the system depends on the particular arrangement of the overall biorefmery system and is driven, in part, by the desired concentration of product in the fermentation broth contained in the fermenter. Preferably product is removed from the fermentation broth to preserve the metabolic processes of the microorganisms by keeping product and other metabolites below a concentration that substantially inhibits or eliminates their activity. The vacuum assisted purification zone is a system that can receive fermentation liquid from the fermenter and initiate separation of desired product from the fermentation liquid. In a preferred embodiment, the vacuum assisted purification zone comprises at least one vacuum evaporator that is operatively connected to the fermenter and the process can further comprise circulating the fermentation liquid from the vacuum evaporator back to the fermenter. The system is preferably configured such that only the vacuum evaporator is operated under vacuum and the vacuum is isolated to the evaporator and preferably does not affect the fermenter or the other apparatus that may optionally be included in the vacuum assisted purification zone. The vacuum evaporator can be operated at a temperature which is at or near the boiling point of the volatile product in the fermented liquid when placed under a vacuum in order to sufficiently transfer the desired product to the vapor phase which is discharged from the evaporator to the product recovery zone.

In one embodiment, fermentation is conducted between about 40°C and 45°C. A portion of a fermentation liquid which may include microorganisms, nutrients and other components contained in the fermenter is removed from the fermenter and directed into the vacuum assisted purification zone to the vacuum evaporator. The temperature of the fermentation broth may be increased to about 45°C to 65°C prior to introduction into the vacuum evaporator by a heat exchanger in the line between the fermenter and the flash evaporator. Alternatively, the temperature of the portion of the fermentation broth is brought to the desired value after it is introduced in the vacuum evaporator. The heat source may be integral to the vacuum evaporator or may be external to the vacuum evaporator such as a steam source. Preferably, microorganisms are used that are viable and productive at these temperatures such that they can be recycled back to the fermenter for further fermentation.

Optionally, after the vacuum evaporation step, the product-depleted remaining portion of the fermentation broth can be conducted from the vacuum evaporator back to the fermenter.

In a preferred embodiment, before entering the vacuum evaporator, the

fermentation liquid may undergo one or more degassing steps for the removal of byproduct gasses such as carbon dioxide and hydrogen gas (e.g. during the fermentation of glycerol to ethanol) prior to the vaporization of the desired product in the vacuum evaporator. Degassing technology is well known in the fermentation art and is commonly used in a variety of processes based on fermentation technology including the commercial production ethanol bio fuels in the U.S. Such degassing may be conducted, for example, in a purge column that is operatively connected to the fermenter in line prior to the vacuum evaporator. The fermentation liquid passes from the fermenter through the purge column for degassing of dissolved by-product gasses contained in the fermentation liquid.

Alternatively, a flash evaporator may be used prior to the vacuum evaporation step under conditions that will vaporize the by-product gasses such as carbon dioxide and hydrogen gas without vaporizing the desired product such as ethanol. A heat exchanger may be used to heat the fermentation liquid prior to entry into the flash evaporator and/or the pressure can be dropped to a desired low vacuum sufficient enough to vaporize the by-product gasses in the flash evaporator. The by-product gasses which may contain trace amounts of the desired product may be discharged to an additional apparatus such as a scrubber for further separation of the desired product from the by-product gasses and be recirculated back to the line carrying the fermentation broth from the evaporator or purge column to the vacuum evaporator. The fermentation liquid which has been depleted of by-product gasses is then conducted to the vacuum evaporator for vaporization of the desired product.

In one embodiment, the vacuum assisted purification zone includes apparatus directly connected to the fermenter for collecting by-product gasses (and any trace amounts of product gas) generated by fermentation that collects as overhead vapor in the fermenter. This apparatus may include a scrubber column which separates trace product gas and water vapor generated in the fermenter for subsequent return to the vacuum assisted purification zone for further separation of the desired product. The byproduct gasses may be discharged from the scrubber column to atmosphere or collection for additional treatment.

The particular configuration of the vacuum assisted product recovery system and its specific integration with the fermentation zone and product recovery zone may take many forms. The vacuum assisted purification zone may receive all or only a portion of the fermentation liquid that passes to the product recovery zone for recovery of the product. The main goal of the vacuum assisted purification zone is to provide vapor or condensed vapor preferably having concentration of about 15-20 wt% product to the product recovery zone for an efficient separation and isolation of product having a concentration of at least about 90 wt% product. Preferably the vacuum assisted purification zone will also employ a circulation loop to circulate product-depleted fermentation liquid back to the fermenter for renewed fermentation and formation of product. To operate, the vacuum assisted purification zone generally requires only a heat addition equal to the latent heat of vaporization of the by-product gasses or the product. Since the evaporators require relatively low temperature heat inputs, particularly at low pressures, there are numerous opportunities for efficient heat inputs and heat exchanges such as steam.

The product recovery zone may comprise any apparatus suitable for separation and collection of the products of fermentation. Any apparatus suitable for distillation of a desired product is contemplated including rectifiers, vacuum distillers, chromatography, phase separators, pervaporation, perstraction and combinations thereof.

In a preferred embodiment, the product recovery zone comprises at least one rectifier that is capable of receiving the product-enriched vapor generated in vacuum evaporator for further separation into a product that comprises at least 90 wt% product. Alternatively, the product-enriched vapor generated in the vacuum evaporator may be condensed using an optional condensation apparatus prior to entering the rectifier or other separation equipment. The product recovery zone may also include additional distillation apparatus for receiving fermentation broth that is conducted directly to the distillation apparatus and bypasses the vacuum assisted purification zone. The product recovery zone also includes means for recycling water and other components now depleted of product between the distillation equipment contained in the product recovery zone and/or to the vacuum assisted purification zone and/or ultimately back to the fermenter for further use.

It is understood that during any individual step of the process wherein a vapor stream is created that may comprise the desired product, by products, water or other inert gasses, such vapor streams may be collected individually or in combination and processed in downstream equipment to recover and purify the desired product.

FIG.1 is a schematic depicting one embodiment of the bioconversion system of the invention exemplifying the bioconversion of glycerol to ethanol. Depiction of the invention in schematic form does not limit the invention to the detail of the components shown therein which are provided for purposes of exemplification and not limitation.

A fermenter 1 is provided comprising an aqueous solution of glycerol feedstock and a microorganism biocatalyst capable of converting glycerol to ethanol along with the nutrients necessary for the biocatalyst to conduct bioconversion of glycerol to ethanol. Optionally, the microorganisms are cultured and grown in a separate bioreactor which may or may not be operatively connected to the fermenter, and are added to fermenter 1 upon initiation of the biofermentation cycle for recovering ethanol.

Optionally, the glycerol feedstock may be treated prior to its use as a feedstock in fermenter 1. Such treatment apparatus for the feedstock may or may not be operatively connected to the fermenter. One such treatment includes a desalting treatment. In one preferred embodiment, the glycerol used as feedstock is obtained as a byproduct of biodiesel production and includes no more than 70 wt% impurities. The fermenter 1 may comprise an optional salt purge 140 which functions to maintain a salt concentration in the fermentation below levels that inhibit either ethanol production or cell growth, whichever is lower.

Ethanol and carbon dioxide as well as hydrogen gas are produced by the fermentation process in fermenter 1. The gas generated as overhead vapor in fermenter 1 is discharged through a line 2 to a scrubber column 3. A portion of the fermented liquid which has been fermented to a desired ethanol concentration is conducted from the fermenter 1 through line 4 to a purge column 5. The scrubber column 3 functions to remove hydrogen and carbon dioxide gasses to an optional collection system or to atmosphere. Trace ethanol and water are discharged from the scrubber column through 6. Line 6 joins with the stream of degassed fermentation liquid leaving the purge column via line 7. The purge column 5 functions to degas the fermented liquid by removing byproduct gasses such as hydrogen and carbon dioxide gasses which are optionally conducted via line 9 to the scrubber column 3 for further treatment.

The partially degassed fermentation liquid leaving the purge column 5 via line 7 joins the ethanol and water stream from line 6 and is directed through an optional heat exchanger 8 to a vacuum evaporator 10. Heat for heat exchanger 8 may be provided by steam via 21. The heat exchanger 8 is used to raise the temperature of the fermentation liquid to a temperature suitable for vaporization of the product in the vacuum evaporator 10. Alternatively, the vacuum evaporator 10 may have its own heat source or be supplied with an external heat source (e.g. steam) with which to heat the fermentation liquid to the desired temperature. The evaporator is operated at below atmospheric pressure, such as at vacuum pressures and elevated temperatures. Preferred temperatures for operating the evaporator range from about 40°C to 65°C. The vacuum in the evaporator 10 may be created by a vacuum source 12 such as a vacuum pump or compressor. Optionally, the remaining fermentation liquid from which the ethanol has been substantially removed as ethanol vapor and which comprises mainly cells, medium and water is discharged from the bottom of the evaporator 10 and is conducted back to the fermenter 1 by line 20 for renewed fermentation.

Line 40 carries the overhead ethanol-containing vapor from the evaporator 10 to the rectifier column 14. The ethanol vapor preferably has an ethanol concentration of 15-20 wt%. Optionally equipment such as a condenser (not shown) in line 40 may be provided to condense the ethanol vapor leaving the evaporator 10 and entering the rectifier 14.

Preferably, the ethanol vapor is not condensed prior to entering the rectifier column

14. In the rectifier column, ethanol vapor is separated and product comprising at least 90 wt% ethanol is subsequently collected through line 100. Optionally, product collected through line 100 may be subsequently condensed using standard means (not shown).

Water used in the rectifier column is discharged via 60 to an optional stripper column 16. A first portion of the water treated in the stripper column 16 is recycled back to the rectifier column via line 70 and a second portion of the water treated in the stripper column 16 is recycled back to the fermenter via line 80.

Optionally, after running the process continually for a period of days or weeks, the run can be shut down by emptying the entire contents of the fermenter 1 through line 90 to a cell/solids recovery unit 18 such as a centrifuge which discharges the cell mass and other solids through line 110 for optional collection and use as a fertilizer or other suitable use. The fermentation broth minus the cells and containing residual ethanol continues via line 111 to a distilling column 22 which further separates ethanol from the fermentation broth. The distillate having a concentration of ethanol of about 10-20 wt% ethanol exits the distillation column via line 120 and enters the rectifier 14. The remaining portion of the fermentation broth that is not distilled is discharged via line 130 and is recirculated back to the fermenter 1 by line 80.

Optionally, at the end of the run the contents of the fermenter 1 can be processed through the vacuum evaporator 10, which is operated at an elevated temperature compared to the production phase. This may inactivate the microorganisms but strips the remaining volatile product from the broth. The treated broth can then be processed through the cell/solids recovery unit. Upon conclusion of the run, the entire apparatus is cleaned and prepared for reuse. Optionally, the fermenter does not require sterilization between each run as the cell expansion of the microorganism culture does not take place in the fermenter where the bioconversion is taking place and contamination is less of an issue given the high temperature and anaerobic conditions in the fermenter.

FIG. 2 is a flow schematic of another exemplary embodiment of this invention wherein the purge column 5 of FIG. 1 is replaced with a flash evaporator 29. All similar elements in FIG. 2 have the same numbering as FIG.1.

In FIG. 2, a portion of the fermented liquid which has been fermented to a desired ethanol concentration is conducted from the fermenter 1 through line 4 to a flash evaporator 29. The flash evaporator 29 is operated below atmospheric pressure but is preferably not operated under strong vacuum. Optionally, prior to entering the flash evaporator 29, the fermentation liquid may enter a heat exchanger (not shown) to raise the temperature of the fermentation liquid such that the appropriate gasses vaporize in the flash evaporator 29. Alternatively, the flash evaporator itself may have its own heat source or be supplied with an external heat source (e.g. steam) with which to heat the fermentation liquid to the desired temperature. Alternatively, the temperature at which the fermentation process is conducted in the fermenter may be high enough such that the fermentation liquid entering the flash evaporator is already hot enough to vaporize the byproduct gasses at reduced pressure in the flash evaporator. The flash evaporator 29 functions to degas the fermented liquid by vaporizing at least a portion of the byproduct gasses such as hydrogen and carbon dioxide gasses which are optionally conducted via line 9 to the scrubber column 3 for further treatment as described with regard to FIG. 1.

The partially degassed fermentation liquid leaving the flash evaporator 29 via line 7 joins the ethanol and water stream from line 6 and is directed through an optional heat exchanger 8 to a vacuum evaporator 10. The remaining process as shown in FIG. 2 is as described with regard to FIG. 1.

Examples

Example 1

This example illustrates the production and recovery of ethanol from glycerol using the biorefmery system of the invention. P. macerans, strain LMG 13285 is sourced from the Belgian Coordinated

Collections of Microorganisms and is capable of converting glycerol to ethanol in high yields. This strain was maintained on plates containing Luria-Bertani (LB) medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar per liter). Liquid Nutrient Broth Yeast Extract (NBYE, 8 g/1 Difco nutrient broth, 5 g/1 Difco yeast extract and 5 g/1 NaCl) supplemented with 40 g/1 glycerol was used for overnight culture and fermentation. CaC0 ₃ (10 g/1) was added to all shake-flask cultures for pH control. Chemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma-Aldrich Co. (St Louis, MO).

Cell culture optical density was measured at 600 nm (OD ₆ oo) using a Biochrom Libra S22 spectrophotometer (Biochrom Ltd.). 10 μΐ of 10 M HC1 was used to remove

CaC03 residuals when measuring OD. Ethanol, glycerol, and organic acid concentrations were determined using a Shimadzu LC-20AD HPLC equipped with a UV-monitor (210 nm) and refractive index detector (RID). Products were separated using an Aminex HPX- 87H column (Bio-Rad Laboratories) with 2.5 mM H ₂ SO ₄ as the mobile phase (0.6 ml min ^" 55°C).

The fermentation seed culture was prepared as follows: the cultures (stored as glycerol stocks at -80°C) were streaked onto LB plates and incubated overnight at 42°C. Single colonies growing on these plates were used to inoculate 40 ml (NBYE medium as described above) seed cultures in 250 ml baffled flasks grown aerobically at 42°C and 175 rpm overnight. These cultures were used to provide an inoculum for a 5 1 fermenter.

Anaerobic fermentations containing NBYE medium as described above were conducted in a 5-L fermentation system (Biostat A ⁺ from Sartorius Stedim North America Inc., Bohemia, NY) with the temperature (42°C), pH (6.0) and agitation (200 rpm) controlled using manufacturer's software package (Sartorius Stedim North America Inc., Bohemia, NY). pH was controlled by adding 5 M NaOH. After 22 h, the fermenter was connected to the vacuum assisted purification zone for ethanol extraction.

The vacuum assisted purification zone comprises a 1000 ml round flask (flash vessel) connected via an angled distillation arm to a West condenser and a vacuum adapter with a distribution flask and four 10 ml receivers. Assembly was connected to a vacuum pump (GAST Manufacturing Corporation) via two cold traps to prevent the residual ethanol vapors from entering into the vacuum pump. After 22 h fermentation (at an ethanol concentration of 7 g/L), the continuous ethanol extraction process was started by pulling the ethanol rich broth from the fermenter to the flash vessel (10 kPa) using vacuum as the driving force and a needle valve to control the flow rate (0.5 1/h) and returning the ethanol- depleted medium to the fermenter using a peristaltic pump (Cole-Parmer). The flash vessel was heated using a heat mantle (380 W, Glass-Cole) to keep broth boiling (at 10 kPa) and to control the evaporation rate. A vacuum gauge (Ashcroft Digital, 0 - -30 in Hg) and a glass thermocouple were installed on the vacuum vessel. The vacuum assisted purification zone was connected to the fermenter for 8 h and extracted 13.2 g ethanol (extraction rate 0.41 g/l/h). Ethanol was produced in the fermenter during ethanol removal using the vacuum assisted purification zone (ethanol concentration did not decrease over time in the 5-L fermenter).

Example 2

P. macerans, strain LMG 13285 sourced from the Belgian Coordinated Collections of Microorganisms was maintained on plates containing Luria-Bertani (LB) medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar per liter). Liquid Nutrient Broth Yeast Extract (NBYE, 8 g/1 Difco nutrient broth, 5 g/1 Difco yeast extract and 5 g/1 NaCl) supplemented with 40 g/1 glycerol, was used for overnight cultures and fermentations unless otherwise specified. CaC0 ₃ (10 g/1) was added to all shake-flask cultures for pH control. Chemicals were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma- Aldrich Co. (St Louis, MO).

Cell culture optical density was measured at 600 nm (OD ₆₀ o) using a Biochrom Libra S22 spectrophotometer (Biochrom Ltd.). 10 μΐ of 10 M HC1 was used to remove CaC03 residuals when measuring OD. Ethanol, glycerol, and organic acid concentrations were determined using a Shimadzu LC-20AD HPLC equipped with a UV-monitor (210 nm) and refractive index detector (RID). Products were separated using an Aminex HPX- 87H column (Bio-Rad Laboratories) with 2.5 mM H ₂ SO ₄ as the mobile phase (0.6 ml min ^" 55°C).

Anaerobic fermentation contained NBYE medium as described above were conducted in 0.5 1 (working volume) fermentation system (Ward's Natural Science, Rochester, NY). The vessels were immersed in a water bath, whose temperature was maintained at 42°C. The pH was controlled at 6.0 with a Jenco 3671 pH Controller fitted with a Jenco 600p pH probe (Jenco, San Diego, CA). Base (5 M NaOH) for pH control was added by gravity flow using a pinch valve (Bio-Chem Inc., Boonton, NJ) connected to the pH controller. The stirrer speed was maintained at 350 rpm by using an IKA color squid magnetic stirrer (IKA Works, Inc.).

The fermentation seed culture was prepared as follows: the cultures (stored as glycerol stocks at -80°C) were streaked onto LB plates and incubated overnight at 42°C. Single colonies growing on these plates were used to inoculate 40 ml (NBYE medium as described above) seed cultures in 250 ml baffled flasks grown aerobically at 42°C and 175 rpm overnight. These cultures were used to provide inoculums in the 500 ml working volume.

The vacuum assisted purification zone comprises a 1000 ml round flask (flash vessel) connected via an angled distillation arm to a small West condenser and a vacuum adapter with a distribution flask and four 10 ml receivers. Assembly was connected to a vacuum pump (GAST Manufacturing Corporation) via a cold trap to prevent the residual ethanol vapors from entering into the vacuum pump. After 24 h fermentation (ethanol ~10 g/1) ethanol rich broth was transferred to the flash vessel using vacuum as the driving force and a needle valve to control the flow rate and was kept under vacuum (10 kPa) for 1 h. Vessel was heated using a heat mantle (380 W, Glass-Cole) to keep broth boiling and to control the evaporation rate. A vacuum gauge (Ashcroft Digital, 0-30 in Hg) and a glass thermocouple were installed on the vacuum vessel. Evaporated ethanol was collected using the West condenser. Ethanol free broth was then returned to the Ward's reactor using a peristaltic pump (Control Company) to continue fermentation under normal conditions. NBYE medium (40 ml) was added to the fermenter to compensate for the evaporated water-ethanol mixture. Glycerol was added to the media to bring the glycerol concentration back up to 35 g/1.

LMG 13285 produced 8.7 g/1 ethanol in 24 h fermentation in NBYE medium at

42°C and 101.3 kPa. Ethanol was extracted and collected in the vacuum assisted purification zone and broth containing 3.5 g/1 ethanol was returned to the reactor for more ethanol production. 2.6 g ethanol was removed from the broth in one step evaporation. Glycerol was fed to the reactor to bring the glycerol concentration back up to ~35 g/1. Total produced ethanol was 5 g in 72 h.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from the various embodiments may be combined in whole or in part in accordance with the invention.