HK1160179A - Enhanced ethanol fermentation using biodigestate - Google Patents

Abstract

Methods and systems for enhancing ethanol production using a suspending fluid are described The suspending fluid includes organic material that has at least partially been anaerobically digested and anaerobic microorganisms, and is substantially free of non-anaerobic microorganisms Also described are methods and systems for hydrolyzing a feedstock for fermentation that include hydrolyzing a feedstock suspension The feedstock suspension can include feedstock that includes complex sugars, and a suspending fluid, wherein the suspending fluid includes organic material that has at least partially been anaerobically digested and anaerobic microorganisms, and is substantially free of non-anaerobic microorganisms

Description

Enhanced ethanol fermentation using biodigestate

Background

Ethanol has a variety of commercial uses, for example, it can be used as a fuel or fuel additive for combustion. Ethanol (also known as bioethanol) may be produced by fermenting sugars contained in the feedstock. The fermentation may be performed by microorganisms such as yeast or bacteria, which convert sugars to ethanol by biochemical processes. The feedstock may comprise organic material, typically plant material, containing sugars. Examples of plant material that can be used as feedstock include plants that produce and store simple sugars (such as sugar cane and sugar beets), plants that produce and store starch (such as grains, e.g., corn and wheat), and other cellulose and/or hemicellulose-rich plant materials (such as agricultural or forestry residues, e.g., plant stalks and leaves).

Ethanol production by fermentation may require many materials in addition to feedstock and microorganisms. These materials may include fresh process water, which may be added to the feedstock to produce a feedstock suspension for microbial fermentation; and nutritional supplements, particularly nitrogen supplements (such as urea or ammonium compounds), which can provide the necessary nutrients to the microorganism undergoing fermentation. However, these materials are expensive and can be prohibitively high increasing the cost of ethanol production, which is one of the major obstacles in the failure of ethanol-based fuels to compete with gasoline economy. For example, the water consumption in a conventional ethanol plant is about 10gPM per million gallons of annual ethanol production. This means that in the near future, large amounts of fresh water will be consumed for large scale bioethanol production. However, to date, no significant research effort and associated action has been invested in alleviating this problem.

Feedstocks for ethanol fermentation may include complex sugars (polysaccharides), which are often difficult to ferment to ethanol by microorganisms. To assist in fermenting the complex sugars contained therein, the feedstock may be subjected to a hydrolysis reaction in which the complex sugars are converted to simpler sugars that can be more readily converted to ethanol by microorganisms. The hydrolysis process can also be expensive, in part, due to the need for materials such as fresh water and enzymes to perform the conversion.

In addition, conventional ethanol plants are also fairly criticized as lacking in energy efficiency. The greatest loss in energy efficiency typically comes from fossil fuels used in the distillation and drying of distillers grains (distillers grains), which are the wet residue in fermented beer after the ethanol produced is distilled.

Organic wastes such as municipal wastewater or livestock excrements can release greenhouse gases such as methane and carbon dioxide, and can be sources of air pollution, soil pollution and water pollution. Anaerobic biodigesters (bio-digesterters) can process organic waste through treatment with organisms, which can be obligate or facultative and/or archaea. These organisms can use biochemical reactions to convert organic materials into a variety of products. These products include gas mixtures commonly referred to as biogas and liquid and solid mixtures commonly referred to as biodigestate. Biodigestate is typically disposed of as waste.

Disclosure of Invention

The present invention provides methods and systems for enhancing ethanol production and obtaining value-added products from biodigestate, which is generally considered waste. The method and system of the present invention is based in part on the discovery that: biodigestate and its different fractions do not inhibit the activity of many of the enzymes required for a microorganism-based fermentation process for ethanol production and, therefore, can be used directly as a suspension fluid in a fermentation process without the addition of any fresh water or nutritional supplements. This not only provides useful use of biodigestate (generally considered waste), but also saves valuable resources such as fresh water and nutritional supplements. The methods and systems of the present invention are also based in part on the following surprising findings: biodigestate or certain fractions thereof provide for increased ethanol yields compared to using freshwater, thereby further increasing the cost-efficiency of ethanol production using microbial fermentation. While not wishing to be bound by any particular theory, it is possible that the observed increased ethanol production is due to the presence of certain nutrients and other organic matter that is deficient in fresh water, such as nutrients in water-insoluble matter (WIS) and AD effluents (effluent), which may contribute to the final yield of ethanol fermentation. It is also possible that the observed increased ethanol production is due to the presence of certain microorganisms in the anaerobic digestate that may synergistically enhance saccharification and fermentation of cereal bioethanol production.

The combination of AD technology and bioethanol production processes not only converts anaerobic digestion effluents into value-added products, but also helps the bioethanol industry to achieve a positive balance in energy consumption, bioethanol production, waste management and environmental protection to maximize its profit.

Accordingly, one aspect of the present invention provides a process for producing ethanol comprising: (1) adding a suspension fluid to the feedstock to produce a fermentation suspension, wherein the suspension fluid comprises organic material that has been at least partially anaerobically digested; (2) if desired, adjusting the pH of the fermentation suspension to a value at which (productive) fermentation can take place; and (3) fermenting the fermentation suspension to produce ethanol, wherein the suspension fluid is substantially free of fresh water (e.g., exogenously added) or nutrient supplements.

In certain embodiments, the method further comprises inoculating the fermentation suspension with a microorganism capable of fermenting the fermentation suspension to produce ethanol. For example, the microorganism may be a yeast or bacteria, or any other microorganism capable of fermentation to produce ethanol. Exemplary ethanol-producing microorganisms include Saccharomyces (Saccharomyces) and Zymomonas (Zymomonas), facultative anaerobic thermophilic strains (such as those described in WO/88/09379), and genetically engineered microorganisms using genetic engineering (that otherwise would not produce significant amounts of ethanol). See, e.g., engineered E.coli (E.coli) containing ADH and PDC enzymes from Zymomonas mobilis (Zymomonas mobilis), Ingram et al, "Genetic Engineering of Ethanol Production in Escherichia coli," appl.environ Microbil.53: 2420, 2425, 1987; genetically modified photosynthetic Cyanobacteria (Cyanobacteria), such as those described in U.S. patent No. 6,699,696; engineered Klebsiella oxytoca (Klebsiella oxytoca); and see generally, Dien et al, bacterial engineered for fuel ethanol production: current status applied microbiology and biotechnology, 63: page 258-.

Preferred ethanol-fermenting microorganisms are tolerant to high concentrations of ethanol (e.g., 10%, 15%, 20%, 25%, or 30%) in AD medium fermentation broth. Preferred ethanol fermenting microorganisms are also effective in decomposing non-starch cellulosic biomass, which can hydrolyze various non-cereal biomass and convert it to monosaccharide molecules for fermentation. Recombinant DNA technology can be used to genetically enhance the traits of these fermenting microorganisms that are favorable for ethanol fermentation.

In certain embodiments, the suspension fluid comprises, consists essentially of, or consists of anaerobic biodigestate or effluent thereof. Anaerobic biodigestate can be derived from the anaerobic digestion of organic material, including any organic waste material, such as organic material including animal offal, livestock manure, food processing waste, municipal wastewater, thin stillage (thin stillage), wine tanks, or other organic material.

In certain embodiments, the suspension fluid comprises, consists essentially of, or consists of biodigestate as a whole. In other embodiments, the suspension fluid comprises, consists essentially of, or consists of fractionated (fractionated) anaerobic biodigestate. The fractionated anaerobic biodigestate can be a liquid fraction produced by removing substantially all solids from the anaerobic biodigestate, such as by centrifugation. In certain embodiments, the supernatant obtained by the centrifugation process functions best in ethanol fermentation when a certain amount of suspended solids are present in the supernatant. Thus, in certain embodiments, the supernatant is produced by centrifuging the AD effluent at 200g, 400g, 600g, 800g, 1000g, 1500g, 2000g, 2500g, 3000g, 3500g, 4000g, 5000g, 6000g, 7500g, or 10,000 g.

Alternatively, the liquid fraction may be produced by passing the anaerobic biodigestate through a screw press (e.g., a "FAN" brand screw press) or other similar device.

Preferably, the AD digestate is from a "healthy" batch of anaerobic digestion, as biogas production is optimal (contrast drops to near zero) in said healthy batch.

In certain embodiments, an amount of urea is added to the AD effluent to increase the yield.

AD may be used fresh, or may be stored for a period of time, such as 12 hours, 1, 2, 3, 5, 7, 10, 2 weeks, 1 month, etc.

In certain embodiments, the liquid fraction comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (preferably 3-9%) solids.

In certain embodiments, the liquid fraction may also be fortified with nutrients recovered from the anaerobic biodigestate.

In certain embodiments, the fractionated anaerobic biodigestate is an ultrafiltration concentrate or ultrafiltration permeate produced from a liquid fraction of the anaerobic biodigestate, wherein the liquid fraction is produced by removing substantially all solids from the anaerobic biodigestate.

In certain embodiments, the pH of the fermentation suspension is adjusted to less than 6.0 (e.g., 4.0-5.0) for optimal enzymatic catalysis.

In certain embodiments, the method further comprises distilling the fermented beer to collect ethanol without prior removal of solids from the beer.

In certain embodiments, the feedstock is high starch wheat, corn, or other high starch crops.

In certain embodiments, the high starch wheat, corn or other high starch crop is at least partially converted to simple sugars in the suspending fluid.

In certain embodiments, the conversion comprises (in no particular order, and without limitation to repetition) mechanical milling, steam heating, reaction with an acid, liquefaction using an alpha-amylase, and/or saccharification using a glucoamylase.

In certain embodiments, the pH is controlled within an optimal range required for a wheat or crop conversion reaction.

In certain embodiments, about 75% of the suspension fluid is added prior to liquefaction and about 25% of the suspension fluid is added prior to saccharification after liquefaction.

In certain embodiments, the high starch wheat, corn, or other crop is present in an amount up to about 28% (w/v), or up to 36% (w/v), of the suspending fluid.

In certain embodiments, the method further comprises adding cellulase, xylanase and/or acid proteolytic enzyme to the suspension fluid.

In certain embodiments, the method further comprises incubating the fermentation mixture at about 30-50 ℃ (inclusive) for about 24, 36, 48, or 72 hours.

In certain embodiments, wet distillers grains (wet distillers grains) obtained from ethanol distillation are fed to livestock animals (e.g., swine, poultry, fish, or cattle) as feed, optionally with fortified nutritional elements, or used as fertilizer with enhanced nutritional value (e.g., nitrogen enhancement).

In certain embodiments, the suspension fluid is substantially free of non-anaerobic microorganisms.

In certain embodiments, the pH of the suspension fluid is adjusted to a value that is substantially incompatible with the growth of non-anaerobic microorganisms.

In certain embodiments, the pH of the suspension fluid is adjusted to a value for optimal growth of the fermenting microorganisms.

In certain embodiments, the nutritional supplement is a nitrogen supplement.

In certain embodiments, the production of ethanol is increased or enhanced as compared to an otherwise identical process using fresh water in place of the suspending fluid. Preferably, ethanol production is increased by 5-15% or 7-10% when about 20-36% or 22-28% wheat is used.

Another method of the present invention provides a method for hydrolyzing a feedstock, wherein the feedstock comprises a polysaccharide, and wherein the hydrolyzed feedstock produces more ethanol upon fermentation than prior to hydrolysis, the method comprising: (1) adding a suspension fluid to the feedstock to produce a feedstock suspension, wherein the suspension fluid comprises organic material that has been at least partially anaerobically digested; and (2) hydrolyzing the feed suspension such that at least a portion of the polysaccharides are converted to simple sugars, wherein the suspension fluid is substantially free of (exogenously added) fresh water or nutritional supplements.

In certain embodiments, the hydrolysis step comprises (in no particular order, and without limitation to repetition) mechanical milling, steam heating, reaction with an acid, liquefaction using an alpha-amylase, and/or saccharification using a glucoamylase.

Unless specifically excluded or clearly unsuitable or inapplicable, it is contemplated that all embodiments of the invention described herein may be combined with any other embodiment, including those described in different aspects of the invention.

Drawings

The above and other advantages of the present invention will become more apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout, and in which:

FIG. 1 is a flow chart 100 illustrating an exemplary method for increasing ethanol production comprising steps 102, 104, and 106, according to one embodiment of the invention.

FIG. 2 is a schematic diagram illustrating an exemplary system 200 for increasing ethanol production, according to one embodiment of the invention. The system 200 may include a bio-digester 202 in which organic waste 204 is anaerobically bio-digested to produce bio-digestate and biogas. At least a portion of biodigestate 206 is passed to hydrolysis unit 214 for mixing with the feedstock to produce a suspension. The hydrolysis may be performed using enzymes 208 and/or acids 210 and/or heat 212 (e.g., in the form of steam, etc.). The resulting hydrolyzed feedstock suspension 218 is then fermented to produce ethanol 224. Alternatively, at least a portion of biodigestate 216 may be transferred directly to a fermentor (fermenter) 220 and mixed with feedstock 218. Feedstock 222 may also be added to produce ethanol.

FIG. 3 is a flow chart 300 illustrating an exemplary method for hydrolyzing a feedstock including steps 302, 304, and 306, according to one embodiment of the invention.

FIG. 4 shows the change in specific gravity and potential ethanol content (% by volume) for different fermentation groups at 22 ℃ for up to 14 days. Description of the drawings: group (2): tap H₂O: tap water, UF-per: ultrafiltration (UF) permeate, UF-con: ultrafiltration (UF) concentrate, S: granulated sugar, SY: super Tubor yeast. Specific gravity (S.G.) was measured on days 0, 4,7, 11, and 14 of fermentation. The potential ethanol content was calculated based on the Oechsle scale.

Figure 5 is a comparison of Anaerobic Digestate (AD) catalyzed by two-step enzyme and wheat conversion in tap water based on glucose content (grams per gram of dry wheat).

Figure 6 shows the glucose yield after two steps of enzymatic conversion versus different contents of wheat in AD and water of FAN separation.

Figure 7 shows two procedures used in wheat transformation.

Figure 8 shows ethanol yield for Simultaneous Saccharification and Fermentation (SSF) versus AD with and without BG and water.

Fig. 9 shows the dose-dependent ethanol yield in SSF of an anaerobic digest of FAN isolation (FSD) versus different amounts of dry wheat.

FIG. 10 shows the use of AD or H₂Ethanol yield in SSF for the two-step addition procedure of O. Description of the drawings: 1/4 volumes of H were added₂O or FSD, and adding G-ZYME480 (modified Pre-saccharification and saccharification enzyme mixture, from GENENCOR)Roche Wser, NY) and OPTIMASH^TMBG (beta glucanase/xylanase complex from GENENCORRochester, NY) was incubated at 55 ℃ for a further 30 minutes. W36 or W28: 130 or 100ml FSD or H₂36 g in O or 28g wheat. H₂O, W28 was used as a control. Each group n is 4.

FIG. 11 shows Total Solids (TS) and Volatile Solids (VS) in the samples after fermentation.

FIG. 12 is total nitrogen in the solids after different sets of fermentations.

FIG. 13 shows the glucose yield of FSD catalyzed with OPTIMASH XL and ethanol cellulase (Accellerase).

FIG. 14 shows the use of OPTIMASH^TMXL (high concentration cellulase/xylanase Complex from GENENCORRochester, NY) and ethanol cellulase. *: and (5) counting the significance.

FIG. 15 shows the use/non-use of FERMAGEN^TM(Low pH protease from GENENCORRochester, NY) of FSD and H₂Ethanol yield of O-wheat mixture.

FIG. 16 shows FSD/wheat and H having the same weight after fermentation₂Ethanol yield of O mixture. *: and (5) counting the significance.

FIG. 17 shows the nutrient value of wet distillers' grains (WDG) in fermentation using anaerobic digestateThe value is obtained. "AD alone" represents the nutritional value of the anaerobic digestate alone prior to fermentation; "AD/wo centrif" represents the nutritional value of whole AD (without centrifugation) fermented with wheat; "ADS, nnn rpm" represents the nutritional value of AD centrifuged at different speeds (respectively "nnn rpm") fermented with wheat; ' H₂O control "represents the nutritional value of wheat fermented in water; and "dry wheat" represents the nutritional value of unfermented milled whole wheat. "P-F" stands for "after fermentation". The bars from left to right for each group are the values of crude (crud) protein, crude fiber, fat and ash, respectively.

Figures 18 and 19 show the results of an analysis of the different nutrient elements required for animal feed, which are present in different pastes (mash) or WDG. In FIGS. 18 and 19, the bars from left to right for each group are H₂Values for O control, ADS (1000rpm), ADS (4000rpm), ADS (6000rpm), AD alone, dried wheat and AD/wo centrif.

Figure 20 shows calculated animal feed values for different ADS (AD supernatant) batches compared to fresh water alone. "TD" means "total digestible nutrients"; "NF" is a "non-fiber carbohydrate"; "DE" is "digestible energy"; "GE" is "Total energy"; and "ME" is "metabolizable energy". The bars from left to right of each group are respectively H₂Values for control 0, ADS (1000rpm), ADS (4000rpm), ADS (6000rpm), AD alone, dried wheat and AD/wocent.

Detailed Description

As noted above, it is desirable to reduce or eliminate the use of fresh process water and/or nutrient supplements (particularly nitrogen supplements) during fermentation. Thus, according to the present invention, a suspension fluid may be added to the feedstock to produce a fermentation suspension. The suspension fluid may have a liquid content sufficient to suspend the feedstock, thereby reducing and in some embodiments substantially eliminating the need for fresh process water. In certain embodiments, the suspending fluid contains no more than 20%, 10%, 5%, 2%, 1%, or is substantially free of exogenously added fresh water and/or commercially available nutritional supplements.

The suspension fluid may contain solid materials therein, including organic materials that have been at least partially anaerobically digested. These solid materials contain nitrogen, and in some embodiments, a nutritional supplement may not be required.

The suspension fluid may also contain one or more types of anaerobic microorganisms. In certain preferred embodiments, the suspension fluid is substantially free of non-anaerobic microorganisms, which is advantageous because aerobic microorganisms can interfere with the fermentation process (e.g., by consuming the feedstock).

In some embodiments, the suspension fluid may be a biodigestate produced by anaerobic biological digestion of organic waste. The organic waste can be, and typically is, a mixture of waste organic materials having relatively low commercial value. Organic waste can include by-products from various industries including agriculture, food processing, animal and plant processing, and livestock. Examples of organic waste include, but are not limited to, livestock manure, animal carcasses and offal, plant material, wastewater, sewage, food processing, and any combination thereof. Organic waste may also include waste from humans, such as sewage and wastewater, waste food, plant or animal material, and the like.

In certain embodiments, the suspension fluid may be fractionated from the anaerobic biodigestate, thereby allowing selected fractions to be used in the present methods.

For example, in certain embodiments, fractionated anaerobic biodigestate is a liquid fraction produced by removing substantially all solids (e.g., greater than 91%, 93%, 95%, 97%, 99%, or close to 100%) from anaerobic biodigestate. This can be done, for example, by passing the anaerobic biodigestate through a FAN screw press or other equivalent mechanical device. The liquid fraction obtained from this process can be used directly in the present invention.

In certain embodiments, the liquid fraction contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (e.g., 3-9%) solids.

In certain embodiments, such liquid fractions may also be further enhanced by nutrients recovered from anaerobic biodigestate. Such nutrients (including nitrogen or phosphate nutrients) can be obtained (e.g., isolated, purified, or enriched) from the liquid fraction of the anaerobic digest using methods known in the art.

In other embodiments, the fractionated anaerobic biodigestate can be an ultrafiltration concentrate (UFC) or ultrafiltration permeate (UFP) produced from a liquid fraction of the anaerobic biodigestate, wherein the liquid fraction is produced by removing at least a portion or substantially all of the solids from the anaerobic biodigestate.

Anaerobic bio-digesters can be used to convert useful products from organic waste or to extract useful products from organic waste. The anaerobic bio-digester may comprise a closed container, which may be a barrel or vessel (vessel) or tank (houseing), in which anaerobic bio-digestion of organic waste takes place. Anaerobic bio-digesters are typically closed to prevent contact with air or other atmospheric or local contaminants. Many anaerobic bio-digestion apparatuses and systems are known (e.g., plug-flow, multi-tank, vertical-tank, fully mixed and covered lagoon digesters), any of which may be suitable for the purposes of the present invention.

In certain embodiments, the anaerobic bio-digester is an integrated system described in co-pending U.S. s.N.12/004,927, entitled "INTEGRATED BIO-differentiation FACILITY", filed on 21.12.2007. The entire contents of the co-pending' 927 application are incorporated herein by reference.

Anaerobic biological digestion of organic waste is carried out by anaerobic organisms, which, as mentioned above, can thus produce biogas and biodigestate (also referred to as anaerobic digestion effluent). Biogas typically comprises a mixture of the gases methane, carbon dioxide and nitrogen (which may be in the form of ammonia), but may also contain significant amounts of hydrogen, sulfides, siloxanes, oxygen and air particles, and is itself a useful product that can be combusted to produce energy.

Anaerobic biological digestion of organic material can produce biodigestate in addition to biogas. Biodigestate can be a mixture of materials and can include organic material that is not digested by anaerobic organisms, byproducts of anaerobic biological digestion released by organisms, and the organisms themselves. For example, biodigestate can include carbohydrates, nutrients (such as nitrogen compounds and phosphates), gaseous organics, wild-type yeast, and large volumes of wastewater. In some embodiments, the solids content may be about 5-9 wt% or about 5-6 wt%. The biodigestate is sufficiently digested such that it is substantially free of non-anaerobic organisms that can be eliminated by consumption of anaerobic organisms, conditions of anaerobic biological digestion (which can include predetermined temperature and pH settings based on optimal survival conditions for anaerobic organisms, in addition to the substantial absence of oxygen), or a combination thereof.

In some embodiments, the amount of each component in the biodigestate can be adjusted. For example, the amount of time an organism is exposed to organic material can be varied to vary the amount of undigested organic material and anaerobic biological digestion byproducts.

In some embodiments, biodigestate can be delivered to the ethanol feedstock without storage for suspension. This can be done, for example, by using a delivery tube. These embodiments are advantageous because they can reduce the risk of contamination of the biodigestate by non-anaerobic organisms.

As mentioned above, the fermentation suspension may already contain anaerobic organisms. Alternatively, an anaerobic microorganism suitable for ethanol production may be inoculated into the culture medium.

The fermentation suspension may additionally contain other microorganisms that may interfere with the fermentation by, for example, digesting the feedstock and/or digesting the organism undergoing fermentation. However, these organisms may be pH sensitive. Thus, in certain embodiments, the pH of the fermentation suspension may be adjusted such that the growth of interfering microorganisms is substantially inhibited. The inhibition can prevent the interfering microorganisms of this type from destroying/inhibiting the fermentation of the feedstock to ethanol. In some embodiments, the inhibition can be by killing interfering microorganisms. In some embodiments, the pH may be adjusted to below 6.0. In certain embodiments, the pH may be adjusted to fall within the range of 4.0-5.0.

The fermentation suspension may be fermented to produce ethanol under conditions (pH, temperature, etc.) at which ethanol production may occur. The process of the present invention can be advantageous because the suspension fluid used reduces or eliminates the need for fresh process water, nutrient make-up, or both. The method of the present invention may also be advantageous because ethanol production may be increased due to the presence of fermentable materials in the suspension fluid (but lack thereof in fresh water).

In certain embodiments, the post-fermentation beer can be distilled directly to collect ethanol without prior removal of solids from the beer. This further reduces the cost of operating an ethanol plant according to the invention.

Wet Distillers Grain (WDG) is the remainder of the feedstock wheat added to the ethanol process after distillation is complete. Most of the starch in wheat is converted to ethanol by microorganisms, while the proteins and any lipids are not used. These remaining portions of the grain are valuable and palatable as cattle feed.

Thus, in certain embodiments, the methods of the present invention propose building a comprehensive ethanol plant near an animal farm, where it is not necessary to be forced to use large amounts of energy to dry wet distillers grains for long-term storage as many ethanol plants. Furthermore, it would not be necessary to use large quantities of fuel to transport the stillage over long distances to remote markets or farms. Alternatively, the stillage may be sent to a nearby farm and consumed in wet form by farm animals (e.g., cattle). This configuration/combination not only provides major energy savings for ethanol plants, but also reduces the amount of fresh drinking water consumed by cattle.

In certain embodiments, the suspending fluid is added to the feedstock in multiple steps (e.g., two steps). For example, in the first step, about 75% of the suspension fluid is added to the feedstock (e.g., high starch wheat) prior to the liquefaction step using alpha-amylase. The remaining 25% can be added after liquefaction but before saccharification with glucoamylase.

The amount of feedstock used may also be optimized. In certain preferred embodiments, the amount of high starch wheat added to the suspending fluid is up to about 28% (w/v).

Systems designed for performing the methods of the present invention may include anaerobic bio-digesters, wherein the organic waste material produced in the anaerobic bio-digesters may be subjected to anaerobic bio-digestion to produce biodigestate and biogas as described above.

As noted above, the feedstock may contain complex sugars, such as polysaccharides, cellulose, or hemicellulose, which may generally be hydrolyzed by specific chemical agents to produce more readily digestible sugars. In certain embodiments, at least a portion of the biodigestate is conveyed as biodigestate to a hydrolysis unit where it is mixed with feedstock to produce a feedstock suspension. Because biodigestate contains materials that can be hydrolyzed (e.g., cellulose or hemicellulose), more sugar can be produced in the hydrolysis than in the use of fresh water to produce the feed suspension. In some embodiments, hydrolysis may be performed by using one or more enzymes (e.g., alpha-amylase, glucoamylase, cellulase, xylanase, and/or acid proteolytic enzyme). In some embodiments, hydrolysis may also be performed by using an acid. In some embodiments, the hydrolysis may be performed by using heat in the form of steam. The hydrolyzed feedstock suspension may have the following results: it contains more simple sugars that can be fermented to produce ethanol.

In certain embodiments, the suspension fluid is substantially free of exogenously added fresh water or nutritional supplements.

At least a portion of the biodigestate can be transferred to a fermentor. Within the fermentor, the biodigestate or fractions thereof can be mixed with feedstock and ethanol produced after fermentation.

The present invention also provides an exemplary method for hydrolyzing feedstock according to one embodiment of the present invention.

For example, a suspension fluid comprising organic material that has been at least partially anaerobically digested, and preferably comprising one or more anaerobic microorganisms suitable for ethanol production, substantially free of non-anaerobic microorganisms can be added to a feedstock (e.g., corn or wheat, preferably high starch wheat) to produce a feedstock suspension.

As described above, the feedstock may be hydrolyzed. In the above embodiments, without wishing to be limited to a particular sequence or repetition of steps, one or more mechanical milling or grinding of the feedstock may be performed, one or more enzymes may be added, and the feedstock may be heated (preferably by steam). All of these steps can be carried out in the suspension fluid of the present invention, preferably without any fresh water and/or nutritional supplements added exogenously. The feedstock suspension is hydrolyzed such that at least a portion of the polysaccharides therein are converted to simple sugars, which can then be fermented to produce ethanol. Without wishing to be bound by any particular theory, the suspending fluid contains certain complex polysaccharides, such as cellulose or hemicellulose, which can be digested by added enzymes to produce simple sugars.

While certain preferred illustrative embodiments of the invention have been described above, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the invention. It is intended that the appended claims cover all such changes and modifications as fall within the true spirit and scope of this present invention.

Examples

Having generally described the invention, applicants provide the following exemplary embodiments to facilitate an understanding of some aspects of the invention that are generally described. These specific examples, which include embodiments only intended to illustrate certain aspects and embodiments of the invention, are not intended to limit the invention in any way. However, certain general principles described in the examples may be applied generally to other aspects or embodiments of the invention.

The examples described herein below show that integration of bioethanol plants and farms and IMUS (integrated fecal use system) technology is an excellent way to share infrastructure and use by-products on site. This integration increases the value of the fertilizer in the form of electricity and heat, which is magnified through the use of ethanol plants. This value also translates into a significant reduction in the cost of use of the ethanol plant and helps to allow small ethanol plants to coexist with large farms in a balanced feed/byproduct relationship.

The study was based at least in part on the following integrated analyses:

ethanol production to farm operations: wet distillers' grains and thin stillage

Ethanol production to IMUS process: low heat (< 50 ℃) and thin stillage

IMUS process to ethanol production: electric and thermal

IMUS process to ethanol production: digestion products

IMUS process to farm run: electric power

Farm run to IMUS process: fertilizer

The results of this study indicate that anaerobic digestate can be used to replace freshwater and fertilizer utilization for bioethanol production. Based on the data of this study, one can improve the economic viability of a bioenergy aggregation model by i.e. creating an ecological agricultural or bioengineered network where all waste streams or related by-products are used. Finally, the system can be used to convert the output to value added products such as beef, heat, bioethanol, biofertilizer, electricity in an environmentally friendly mannerAnd collectable food-grade CO₂。

Example 1 Anaerobic Digestate (AD) and its grade supporting fermentation

This example shows that anaerobic biodigestate (AD) can replace fresh water used for bioethanol production.

IMUS from Vegreville (Alberta, Canada) was collected^TMFour different isolates of AD for the exemplary plant included fresh Anaerobic Digestate (AD), FAN-separated digestate (FSD), and permeate of FSD by Ultrafiltration (UFP) and concentrate (UFC).

Specifically, FSD (FAN separated digestate) may be produced by using a screw press (e.g., FAN brand screw press) or other similar mechanical device to separate the digestate into two fractions-a liquid fraction and a solid fraction. The liquid fraction was FSD in this study. It contains about 5-7% total solids.

UFP/UFC may be produced by subjecting the FSD fraction to ultrafiltration. The permeate (UFP) is a relatively clear liquid (mostly water). The remainder of the concentration, where it passed through the ultrafiltration system, was designated UFC.

For small scale laboratory production, as when used in this example, UFP and UFC fractions were produced by using a laboratory system without lime before the ultrafiltration system. In a typical operation, one unit of FAN separated liquid digest produces about 80% permeate and 20% concentrate.

Three experimental experiments were performed, showing that:

(1) effect of AD on yeast fermentation of granulated sugar (food grade),

(2) the ability of AD to ferment granulated sugar in the absence of yeast, and

(3) ethanol yield compared to tap water collected in the laboratory.

Specifically, granulated sugar was dissolved in AD (pH 8.1) and tap water (pH 5.5) to a concentration of about 28g/dl, respectively, and the pH was adjusted to 5.4 with 12N HCl. The fermentation was carried out in a 3.5 liter fermentor in a volume of 1.0 liter for 14 to 24 days. The fermentation process was observed daily by measuring the change in specific gravity of the mixture using a hydrometer. The potential ethanol content (% by volume) was calculated using the olechs scale (see, e.g., en.

The Oechsle scale is a hydrometer scale that measures the density of a wine liquor, which is an indication of the ripening and sugar content of the grapes used in the wine brewing process. It is named as Ferdinand Oechsle and is widely used in the wine brewing industry in Germany, Switzerland and Lusenberg. In the Oechsle scale, one degree of Oechsle (° Oe) corresponds to one gram difference between the mass of 1 liter of grape liquid at 20 ℃ and 1 kilogram (mass of 1 liter of water). For example, 1084 grams of mass of grape juice per liter is 84 ° Oe. The difference in mass between equal volumes of liquid glucose and water is almost entirely due to the dissolved sugars in the liquid glucose. Since alcohol in wine is produced by sugar fermentation, the Oechsle scale is used to predict the maximum alcohol content possible for a finished wine.

The selected samples were sent to a Quality Control (QC) laboratory for Alberta Centre for Toxicology (ACFT, University of Calgary) and ethanol analysis was performed using a gas chromatograph (GC, HP6890) and a Flame Ionization Detector (FID).

The results show that AD does not have a significant inhibitory effect on yeast-promoted fermentation for ethanol production compared to the use of tap water. The potential ethanol yield in different AD was about 13-16.7%, and the potential ethanol yield in the water control was-18% (fig. 4). When different AD isolates with the same sugar concentration were fermented, different ethanol contents were detected, the highest in the UPC (13.7g/dL) and the lowest in the UFP (10.2g/dL) during 24 days of fermentation (table 1).

As a negative control, when water and sugar were mixed without addition of yeast (0.3g/dL), almost no ethanol was produced under fermentation conditions up to 24 days. However, in the yeast-free UFC and sugar mixture, 8.0g/dL of ethanol was produced, indicating that some components of the UFC may contribute to fermentation. Furthermore, ethanol content was much lower (1.5g/dL) in the yeast-free UFP/sugar mixture. The results show that some anaerobic microorganisms in the yeast-free UFP/sugar mixture contribute to the fermentation during this process.

Single-step distillation experiments also showed that UFP and UFC beer can be distilled to produce clear ethanol at concentrations of 70-71g/dL (table 1).

TABLE 1 ethanol concentrations determined by GC and FID for different fermentation groups up to 24 days at 22 ℃

Description of the drawings: fy: a yeast; BP: a boiling point; and (2) DS: distillation; ethanol (g/dL) was measured by GC, and ethanol (%) was measured by a hydrometer.

In summary, this example shows that: (1) anaerobic digestate can be used as a water substitute for bioethanol fermentation; (2) when total solids in AD increased (UFC > UFP), ethanol concentration also increased; and (3) post-fermentation beer from AD is distillable to produce clear ethanol without prior removal of solids from the mixture.

Example 2 transformation of wheat in AD and tap Water

This example shows that AD does not inhibit alpha-amylase and glucoamylase during the conversion of wheat to glucose. This example also provides a comparison between the conversion rates of tap water and AD when they are used as culture medium.

The conversion from wheat or other crops to starch and then to glucose is a critical step in bioethanol production, as the amount of glucose will be directly related to the ethanol content in beer. Typically, the average conversion rate of wheat to glucose in the bioethanol industry is about 56%.

The two most important enzymes in the conversion process are alpha-amylase and glucoamylase. The first enzyme catalyzes the conversion of wheat to starch and the second enzyme catalyzes the conversion of starch to glucose. Two commercial invertases-from GenencorAlpha-amylase of inc (Spezyme XTRA) and glucoamylase (G-ZYME)^TM480 ethanol) was used in a two-step transformation experiment. D-glucose analysis is suitable for assessing the conversion of AD and wheat in water.

Specifically, wheat (soft white wheat-Andrew) ground using a hammer mill was obtained from Highmark Renewables Research. Unscreened wheat was prepared at different concentrations in AD and tap water. For the different treatment groups, final concentrations were 70, 140, 175 and 280 grams wheat per 1 liter of medium. 12 experiments were set up in 1.0 liter of medium using a 2.0 liter beaker.

After the dose and reaction time were optimized, the first step of liquefaction by Spezyme XTRA was carried out at 85 ℃ for 60 minutes at pH 5.0-6.0 by G-ZYME^TM480 the second step of saccharification is carried out at 60 ℃ for 30 minutes at a pH of 4.0-4.5. Samples were taken before and after the addition of both enzymes and centrifuged at 4,750rpm for 15 minutes. Collecting the supernatant, and using H₂And O, diluting. The glucose concentration in the supernatant was determined by glucose assay using a glucose assay kit (Sigma GAHK20-1KT) or YSI instrument with specified standard values. Total carbohydrates in AD were also analyzed to determine whether carbohydrates were present as substrates for conversion.

The results show that there was no significant difference in glucose yield during the wheat conversion process by both enzymes in AD and tap water (figure 5). The efficiency of wheat conversion reaches the average wheat conversion rate (-56%). When different alpha-amylases and glucoamylases from different manufacturers (Novozyme Inc) were tested, it appeared that there was no significant difference between the conversion efficiency of the enzymes Genencor and Novozyme with respect to the yield of glucose (data not shown).

When the wheat concentration in the mixture was increased (up to 28g/dL in these experiments), the yield of glucose increased accordingly whether the wheat in water or AD was converted (figure 6). In FSD, the total carbohydrate content in AD is 4.11 g/dL. After centrifugation, the supernatant of FSD contained only 0.12g/dL (2.9% of original) total carbohydrate.

In summary, no inhibition of both convertases by AD was observed during the conversion of wheat to glucose, as long as the pH was controlled within the optimal range required for the reaction. A dose-dependent increase in glucose content was achieved when the amount of wheat was increased up to 28g/dL in AD-and water-wheat mixtures. The conversion efficiency of the enzyme was higher in the low concentration wheat-medium mixture, but the difference was not significant.

As expected, a small amount of total carbohydrate is present in AD, but cannot be broken down by invertase. Carbohydrates are more likely to be in a non-soluble form and are considered to be cellulose or hemicellulose (rather than starch-based polysaccharides).

Example 3 ethanol yield from Simultaneous Saccharification and Fermentation (SSF) Using AD and tap Water

A Simultaneous Saccharification and Fermentation (SSF) study was conducted to evaluate the production of wheat-based bioethanol in AD and water. Since AD has no negative impact on glucose conversion from wheat and direct yeast fermentation of sugars, ethanol yield in beer after fermentation represents the impact of AD on the fermentation process.

This example provides a direct comparison between the final beer ethanol content of SSF using AD-wheat mix and water-wheat mix. This example also optimizes the process of lab-scale SSF and studies which components of nutrients, carbohydrates, proteases, microorganisms, or AD are helpful for the improvement of ethanol production.

28 grams or 36 grams in water containing 100ml or 130ml AD (FSD and UFP) respectivelyIn a 250ml flask of dry wheat, the SSF experiment was set up. Testing of beta 3-glucanase/xylanase mixtures (OPTIMASH)^TMBG from GenencorRochester, NY) for analysis of non-starch carbohydrates in wheat and/or AD in addition to the two standard invertases used in example 2. Liquefaction was carried out at 85 ℃ for 1.0 hour as described in example 2 above. Then adding G-ZYME at 60 deg.C during saccharification^TM480 (from GenencorRochester, NY) and BG for 30 minutes. Ethanol fermentation was started by pouring Super yeast X-press powder (AG grade for bioethanol) into distilled water at 34 ℃ for 20 minutes, and then adding an aliquot to the flask together with the yeast nutrients.

SSF fermentation was set to last 48 hours in a water bath at 32 ℃. Three SSF experiments were performed. The first experiment was intended to test the effect of both AD and BG on the final ethanol yield; the second experiment was used to test the dose-dependent ethanol yield in 100ml FSD containing 12, 20 and 28 grams of dry wheat and BG; and a third experiment to test the effect of two additions of AD or water (total liquid volume of 3/4 for liquefaction and 1/4 prior to saccharification after liquefaction) on ethanol yield (fig. 7).

The samples were centrifuged at 4,750rpm for 15 minutes before being sent to ACFT for ethanol analysis. 50ml of post-fermentation mixture from each group was retained for analysis of Total Solids (TS), Volatile Solids (VS) and total nitrogen (TKN) in the biological waste laboratory.

Somewhat surprisingly, in the SSF-1 experiment, the highest ethanol content was obtained in FSDs with BG (9.57. + -. 0.5g/dL) and without BG (9.20. + -. 0.17g/dL), which was higher (p < 0.05 and < 0.01, t-test) than the ethanol content in water with and without BG (8.25. + -. 0.07 and 8.36. + -. 0.15g/dL), respectively. When FSD is used instead of water, the ethanol content is 10-16% higher. There was no difference in ethanol yield between paired groups with and without BG (fig. 8). The increase in ethanol content in AD-wheat fermentation appears to be due to AD instead of β -glucanase/xylanase catalysis.

A dose-dependent increase in ethanol content was observed in SSF-2 experiments. A good linear relationship of ethanol production was observed as dry wheat increased from 12 grams to 28 grams in 100ml FSD (figure 9). It is estimated that 0.3 grams of additional ethanol is produced per gram of additional dry wheat in this range.

In SSF-3 experiments, AD or H was added₂The ethanol yield in the two-step procedure of O was compared to the ethanol yield in the one-step procedure. Interestingly, the ethanol yield was increased in all two-step procedures compared to the one-step procedure, regardless of whether FSD or water was added after the liquefaction stage. With similar final wheat concentration in the fermentation mixture (28 g/dL), the highest ethanol content (8.93. + -. 0.07) was observed in the FSD/FSD mixture, the second being in H₂In O (8.50. + -. 0.21) and then in H₂O/H₂O (8.21. + -. 0.22 g/dL). wheat-H₂The ethanol content in the O-mix control was achieved by a one-step procedure only (-7.9 g/dL) (FIG. 10).

With FSD/FSD mixture and H in a two-step procedure₂O/H₂The ethanol content increased by 0.72g/dL compared to the O-mixture (Table 2). The results show that different procedures of conversion appear to affect the final ethanol yield.

TABLE 2 statistical analysis of ethanol yields (p-value) for different groups (significance level p < 0.05)

The Total Solids (TS) and Volatile Solids (VS) in the samples after fermentation are summarized in fig. 11. TS, VS (as% TS) in FSD/FSD were 14.8% respectively with the same amount of wheat in the fermentation mixture、76.76％，H₂O/H₂Group O contained 8.69% and 92.86%, respectively. The total nitrogen content in the solid after fermentation was 0.87. + -. 0.007 g/g TS in FSD/FSD and in H₂O/H₂Group O had a TS of 0.51. + -. 0.016 g/g (FIG. 12).

Considering wheat/FSD mixture and wheat/H₂Total solids difference between O mixtures, total nitrogen ratio wheat/H in solids after fermentation₂wheat/FSD was much higher in group O, indicating that the fermentation process was healthy and improved by using AD.

In summary, using the FSD-wheat mixture, single step SSF can increase the ethanol content of the fermented sample by 10-16%. The beta-glucanase/xylanase mix had no significant effect on the final ethanol yield, indicating that a limited amount of non-starch carbohydrate substrate specific for the enzyme mix could be obtained from the AD effluent. Adding AD or H during SSF, especially in FSD/FSD group₂The two-step procedure of O results in an increase in ethanol yield over the use of the one-step procedure. This indicates that: (1) the wheat content of the mixture may be further increased in excess of 28g/dL during the liquefaction step; and (2) some microorganisms, biomolecules (e.g., proteolytic enzymes) and nutrients in crude AD contribute to yeast fermentation.

Example 4 improvement of ethanol yield Using combination of enzymes

It was observed that small amounts of carbohydrates were present in AD, but these carbohydrates may not be catalyzed by amylases, glucoamylases and glucanase/xylanases. This example shows that these carbohydrates in AD can be broken down by a combination of different enzymes that are used to increase bioethanol production. This example also provides an analysis of what these carbohydrates are in AD, how much their effect on ethanol production. This example also provides evidence that it is indicated in AD-and H₂The use of proteases in the conversion and fermentation of O-mixtures can increase the yield of ethanol.

Two species from Genencor incMixture of commercial cellulases: cellulase/xylanase (OPTIMASH)^TMXL) and ACCELLERASE1000^TM. In further experiments (results not shown) the enzymes of Novizyme were at least as good, if not better.

The evaluation of the conversion of non-starch carbohydrates in FSD was performed by glucose analysis (as described in example 2) and by ethanol enhancement using SSF with a modified procedure (as in example 3). In wheat-free samples containing 100ml of FSD or H₂O in a 250ml flask, set up the conversion test. Different doses of enzyme were added to the liquid and incubated at appropriate temperatures and times according to the manufacturer's instructions. Alpha-amylase and glucoamylase are then added for liquefaction and saccharification. The concentration of glucose was measured using a YSI instrument. The SSF experiments were performed in 250ml flasks containing 28 grams of Dried Wheat (DW) in 100ml of FSD or water. Mixing OPTIMASH^TMXL (0.01-0.1 ml/flask) and ACCELLERASE1000^TM(0.05-2.0 ml/flask) was added to a mixture containing alpha-amylase (Spezyme XTRA, 150. mu.l) using G-ZYME^TM480 (100. mu.l) were incubated at 50 ℃ for 24 hours before the saccharification step. SSF fermentation was carried out in a water bath at 32 ℃ for 48 hours. To test proteases (e.g.acid proteolytic enzymes, FERMGEN)^TM) In G-ZYME^TMAdding FERMAGEN after 480 and before adding yeast^TM(20 and 100. mu.l/flask). In ACFT, the ethanol content is measured by GC with FID.

The results show that a dose-dependent increase in glucose content using the two cellulase mixtures was observed in FSD, but not in wheat-free water. The highest yield of glucose was at ACCELLERASE1000^TM400 μ L (0.56g/L), then in OPTIMASH^TMXL 40. mu.l (0.45g/L, FIG. 13). After addition of both enzymes, at H₂Little glucose was detected in O (data not shown). Since both enzymes specifically catalyze the substrate of lignocellulosic biomass, increased glucose content is indicated in the fermentation, although not significantly compared to increased ethanol content after fermentationLignocellulosic biomass is present in AD. When mixed at 50 ℃ with H, FSD-wheat mixture for SSF₂When two enzymes were added to the O-wheat mixture for an extended period of time (24 hours), two enzymes were used (for OPTIMASH)^TMXL and ACCELLERASE1000^TM28% and 18%, respectively) increase in ethanol content in FSD as compared to H without similar doses of enzyme₂O increased significantly (p < 0.01, FIG. 14). No dose-dependent increase in ethanol yield was observed between the low and high doses, indicating that only a limited amount of lignocellulosic biomass was present in the FSD. Additional acid proteolytic enzyme (FERRGEN) in the mixture^TM) Slightly improves the ethanol content in the beer after fermentation. And does not contain FERMG^TMContains 20. mu.L of FERGMEN per flask compared with FSD of^TMThe FSD of (3) shows a 6% increase in ethanol content. However, when compared with a composition containing the same amount of FERGMEN^TMH of (A) to (B)₂The increase in ethanol content in FSD-wheat was 17% when compared to the O-wheat mixture (figure 15).

The FSD used in this experiment contained 5-7% total solids. The use of the same volume of water and FSD mixed with the same amount of wheat will result in a final volume difference in the fermented beer. To normalize the final yield of ethanol, the volume difference of beer between the two mixtures was analyzed. In the FSD-wheat mixture the ratio is H₂A5% smaller volume of beer was observed in the O-wheat mixture. The volume correction factor for the final yield of ethanol was 0.95 when the same volume of FSD was used instead of water. Using FSD-and H of exactly the same weight₂O-mixture, we found: with H having a final volume of 100ml₂The ethanol yield of FSD wheat mixture with a final volume of 95ml was increased by-15% compared to O-wheat mixture (figure 16).

In summary, hydrolysis was carried out at 50 ℃ for a long incubation time (24 h) by a modified liquefaction procedure, by adding cellulase OPTIMASH separately^TMXL and ACCELLERASE1000, ethanol yield increased by about 28% or 18%, respectively. Both enzymes catalyze lignocellulosic biomass present in AD, which has an effect on the final yield of ethanol. Acid proteolytic enzyme for FSD-SmallEffect ratio of fermentation of wheat mixture on H₂The extent of the effect of the fermentation of the O mixture was small, indicating that some protease was already present in the FSD-wheat mixture and contributing to the fermentation. These experiments also provide evidence that: AD itself plays a major role in the final ethanol yield by aiding enzymatic hydrolysis of wheat and improving fermentation by its microorganisms, proteases and nutrients. When FSD is used as the culture medium, a volume correction factor of 0.95 is used to normalize the final yield of ethanol. When this correction factor is taken into account, the final yield of ethanol in the FSD-wheat mixture from the different fermentation experiments is 5-11% in experiment 3 and 13-23% in example 4.

The results of these examples, taken together, show that:

(1) anaerobic Digestate (AD) has no inhibitory effect on various conversion/hydrolytic enzymes and yeast-facilitated fermentation processes;

(2) a dose-dependent increase in glucose conversion is obtained when the wheat content in the anaerobic digest is increased to about 28% (w/v) or even about 36% (w/v);

(3) as the total solids in the different isolates of anaerobic digestate increase, the ethanol content in the fermented beer increases;

(4) simultaneous Saccharification and Fermentation (SSF) increases the ethanol content of the fermented beer by 5-11%;

(5) by adding cellulase enzyme mixture and incubating at 30-50 deg.C (inclusive) for extended catalytic time (24 hours), the ethanol content is increased to 13-23%;

(6) small amounts of non-starch carbohydrates (e.g., lignocellulosic biomass) are present in anaerobic digests;

(7) the two-step procedure for adding anaerobic digestate increases the yield of ethanol over the one-step procedure;

(8) the fermented beer is distillable to produce clear ethanol without prior removal of solids;

(9) the increased nitrogen content in the fermented solids can facilitate the utilization of the distillers' grains as fertilizer; and

(10) the synergistic effect of microorganisms, proteases and nitrogen in the anaerobic digestate on fermentation plays a major role in the enhancement of ethanol.

Example 5 animal feed or Fertilizer analysis

The "mash" or vinasse-like material and wheat, optionally with fortified nutrients in the digesta after fermentation, can be used in animal feed (e.g., swine, poultry, fish, and cattle). The material can also be used as a fertilizer. This experiment shows that the "mash" has a comparable feed value to a conventional wet distillers' grains (WDG) obtained using fresh water alone. This experiment shows that the mash has an improved nutritional value compared to anaerobic digestate alone.

As shown in fig. 17, 18, and 19, "AD alone" represents the nutritional value of anaerobic digestate alone prior to fermentation; "AD/wo centrif" represents the nutritional value of total AD fermented with wheat ("P-F" means "after fermentation"); "ADS nnn rpm" represents the nutritional value of AD fermented with wheat centrifuged at different speeds, and "H₂O control "represents the nutritional value of wheat fermented in water.

To determine whether the resulting wet distillers' grains-like mash is also a nutritional animal feed, the protein, crude fiber and fat content of the mash was compared to WDGs made from fresh water alone. Figure 17 shows that the paste obtained from fermentation using centrifuged Anaerobic Digest (ADS) has substantially the same quality compared to WGD obtained using fresh water fermentation. For example, crude protein increased from 13% (in dry wheat) to the fresh water control (H)₂O control) 45-50% and 43-47% after fermentation (ADS). Total digestible nutrients, non-fibrous carbohydrates and fats and H₂The WGD of the O control was comparable. In addition, the following essential metal elements and H for animal feed in the post-fermentation solids using ADS₂The comparison in the post-fermentation solids in the O control is also equivalent or improved, the metallic elementIncluding calcium, magnesium and zinc. In addition, mercury, lead or other undesirable elements are not present in the solids after fermentation. Thus, the resulting vinasse reaches the quality of the animal feed.

Figures 18 and 19 show the results of analyzing the different nutrient elements required in animal feed, which are present in different pastes or WDGs. The results show that different ADS batches contain slightly different concentrations of elements. Note that the concentration of the metallic element can be adjusted by simple centrifugation at different speeds. Pastes or WDGs with different contents of metallic elements can be fed directly to animals in specific growth phases to meet their physiological needs.

Figure 20 shows calculated animal feed values for different ADS batches compared to fresh water alone. The results show that the different ADS batches are at least as nutritious, if not more nutritious, than the water control alone.

It is clear that the use of AD instead of fresh water in an ethanol fermentation process is not only not detrimental to the fermentation process, but also may be expected to produce a wet distillers' grains-like mash that is more nutritious as a fertilizer than the digestate effluent without fermentation and the mash or WDG obtained using fresh water. Note that nitrogen values are not shown in fig. 17, but the percentage of crude protein per unit is increased by more than 60% compared to AD and dry wheat alone. And H₂All element contents in the post-fermentation mash with AD of wheat were increased compared to in the O control fermentation. However, the heavy metal content in the post-fermentation mash with wheat AD was reduced compared to AD alone without fermentation (fig. 20). This will make the fermented paste or WDG a better fertilizer than the digestate effluent.

Furthermore, depending on the wheat content used in the fermentation, the total volume of the mash is typically increased by about 30-50% compared to fresh water WDG. The net yield of the fermented product as fertilizer is remarkably increased. At the same time, ash in post-fermentation mash with AD of wheat was reduced by 50% (as dry matter, 30% to 15%) compared to AD alone (data not shown in the figure).

Reference to the literature

1.(S&T)²Consultants Inc. and Meyer Norris Penny LLP. Econoomic, finial, social analysis and public poles for bioethanol, Phase I report, 2004, 11/22.

Ethanol Short Course, North American Bioproducts Corporation, 11-15.2.2008, Schaumburg, Illinois.

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13.en.wikipedia dot org/wiki/Oechsle_scale。

All of these references are incorporated herein by reference.

Abbreviations herein

Claims (30)

1. A method of producing ethanol, comprising:

(1) adding a suspension fluid to the feedstock to produce a fermentation suspension, wherein the suspension fluid comprises organic material that has been at least partially anaerobically digested;

(2) if desired, adjusting the pH of the fermentation suspension to a value at which fermentation can take place; and

(3) fermenting the fermentation suspension to produce ethanol, wherein the suspension fluid is substantially free of (exogenously added) fresh water or nutrient supplement.

2. The method of claim 1, further comprising inoculating the fermentation suspension with a microorganism capable of fermenting the fermentation suspension to produce ethanol.

3. The method of claim 2, wherein the microorganism is a yeast.

4. The method of claim 1, wherein the suspension fluid comprises anaerobic biodigestate.

5. The method of claim 4 wherein the anaerobic biodigestate is obtained from anaerobic digestion of organic material.

6. The method of claim 5, wherein the organic material comprises animal sewage, livestock manure, food processing waste, municipal wastewater, thin stillage, wine troughs, or other organic material.

7. The method of claim 1, wherein the suspension liquid comprises fractionated anaerobic biodigestate.

8. The method of claim 7, wherein the fractionated anaerobic biodigestate is a liquid fraction produced by removing substantially all solids from the anaerobic biodigestate.

9. A method according to claim 8, wherein the liquid fraction is produced by passing the anaerobic biodigestate through a screw press or through centrifugation.

10. The method of claim 8, wherein the liquid fraction comprises about 3-9% solids.

11. The method of claim 8 wherein said liquid fraction is further fortified with nutrients recovered from said anaerobic biodigestate.

12. The method of claim 7, wherein the fractionated anaerobic biodigestate is an ultrafiltration concentrate or ultrafiltration permeate produced from a liquid fraction of the anaerobic biodigestate, wherein the liquid fraction is produced by removing substantially all solids from the anaerobic biodigestate.

13. The method of claim 1, wherein the pH of the fermentation suspension is adjusted to less than 6.0.

14. The method of claim 1, wherein the pH of the fermentation suspension is adjusted to 4.0-5.0.

15. The method of claim 1, further comprising distilling the fermented beer to collect ethanol without prior removal of solids from the beer.

16. The method of claim 1 wherein the feedstock is high starch wheat, corn or other high starch crops.

17. The method of claim 16, wherein the high starch wheat, corn or other high starch crop is at least partially converted to simple sugars in the suspending fluid.

18. The process of claim 16, wherein the converting comprises (in no particular order and without limitation to repetition) mechanical milling, steam heating, reacting with acid, liquefaction with alpha-amylase, and/or saccharification with glucoamylase.

19. The method of claim 17, wherein the pH is controlled within an optimal range required for the wheat or crop conversion reaction.

20. The method of claim 17, wherein about 75% of the suspension fluid is added prior to liquefaction and about 25% of the suspension fluid is added prior to saccharification after liquefaction.

21. The method of claim 16, wherein the amount of high starch wheat is up to about 28% (w/v) in the suspending fluid.

22. The method of claim 1, further comprising adding cellulase, xylanase and/or acid proteolytic enzyme to the suspension fluid.

23. The method of claim 22, further comprising incubating the fermentation mixture at 50 ℃ for about 24-72 hours.

24. The method according to claim 16, wherein the wet distillers grains from ethanol distillation are fed to livestock animals (such as pigs, poultry, fish or cattle) as feed or used as fertilizer.

25. The method of claim 1, wherein the suspension fluid is substantially free of non-anaerobic microorganisms.

26. The method of claim 1, wherein the pH of the suspension fluid is adjusted to a value for optimal growth of fermenting microorganisms.

27. The method of claim 1, wherein the nutritional supplement is a nitrogen supplement.

28. The method of claim 1, wherein the yield of ethanol is increased or enhanced as compared to an otherwise identical method using fresh water in place of the suspending fluid.

29. A method for hydrolyzing a feedstock, wherein the feedstock comprises polysaccharides, and wherein the hydrolyzed feedstock produces more ethanol upon fermentation than prior to hydrolysis, the method comprising:

(1) adding a suspension fluid to the feedstock to produce a feedstock suspension, wherein the suspension fluid comprises organic material that has been at least partially anaerobically digested; and

(2) hydrolyzing the feed suspension so that at least a portion of the polysaccharides are converted to simple sugars,

wherein the suspending fluid is substantially free of (e.g., exogenously added) fresh water or nutritional supplements.

30. The process of claim 29, wherein the hydrolyzing step comprises mechanical milling, steam heating, reaction with acid, liquefaction with alpha-amylase, and/or saccharification with glucoamylase, in no particular order and without limitation to repetition.