Patent Application
20250092428
Anaerobic digestion is used to convert soluble products into biogas which can be used to generate fuels, chemicals, fibers, and energy. To be economically viable, and reduce the amount of unconverted biomass waste, an adequate conversion percentage is desirable. A major problem arises in the digestion of lignocellulosic waste. Lignin is generally considered not available to anaerobic digestion and is largely not converted. The use of multiple anaerobic digesters to convert biomass into biogas has been suggested but none of these digestion systems are designed specifically to digest lignin, and with these systems most lignocellulose remains undigestible.
Lignocellulosic biomass is a relatively inexpensive, renewable, and abundant material. However, without some kind of chemical or mechanical processing of lignocellulosic biomass, anaerobic digestion typically converts only one-third of the carbon in the lignocellulosic biomass into biogas. In addition, the biogas is typically only 60% methane. Anaerobic digestion by microorganisms is effective on hemicellulose-side chains, but in lignocellulosic biomass that contains cellulose, long glucose chains, these chains are only slowly digested. Further, lignin, a complex polyphenol that can be abundant in plant biomass, is resistant or toxic to many microorganisms.
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The following describes two successive anaerobic digestion environments that are used to treat lignocellulosic biomass;
(1) a high-temperature biological anaerobic digestion environment by thermophilic anaerobic microorganisms and pasteurization of non-thermophilic anaerobes or mesophilic anaerobes. This digestion is described here as an anaerobic secretome bioreaction (ASB) environment.
(2) an anaerobic digestion (AD) environment with non-thermophilic or mesophilic anaerobes. An example anaerobic digestion system according to principles discussed herein includes an ASB environment and an AD environment. The system treats biomass for the production of biogas and includes structure adapted to receive a feed containing biomass. The ASB environment contains thermophilic anaerobic microorganisms that can only exist at thermophilic ASB temperature conditions, but thrive and digest lignocellulose. The thermophilic anaerobic microbes metabolize and solubilize the biomass through hydrolysis, acidogenesis, and acetogenesis to produce products accessible for digestion. These products include several products, but typically comprise one or more of acetic acid and lactic acid. As more fully described below, the ASB environment can be adjusted to favor acetic acid production over lactic acid production, or vice versa. This in turn determines the amount of CO2 in the biogas product. Accordingly, an essentially CO2 free biogas, or a biogas with a predetermined CO2 content can be produced.
The ASB environment is of a thermophilic temperature to support growth of the thermophilic anaerobic organisms. The thermophilic anaerobic microorganisms thrive at temperatures sufficiently high to damage or kill non-thermophilic or mesophilic microorganisms. The ASB temperatures accordingly pasteurize the biomass of non-thermophilic microbes.
A benefit of the ASB temperature, in addition to providing optimization for thermophiles and the advantage of pasteurization, is that the growth and pasteurization occurs at a faster rate than would be achieved at a non-thermophilic temperature.
Because of the pasteurization, the ASB environment is essentially free of non-thermophilic or mesophilic (NT) microbes. NT microbes include those commonly used in conventional or prior-art treatments and may occur naturally in biomass materials. NT microbes may also include common pathogens in lignocellulosic waste streams, such as manure. NT microbes do not thrive, or usually even survive, thermophilic temperatures. Specific examples of NT microbes include E. coli, Salmonella typhimurium, Salmonella Dublin, Campylobacter spp., Listeria monocytogenes, Yersinia enterocolitica, Cryptosporidium parvum, Giardia lamblia, Enterococcus, fecal coliform, and enterobacteria. Other examples are anticipated.
Many prior-art processes produce waste streams that are contaminated with pathogens that were present in the original biomass feed prior to treatment. This creates a disposal problem, as the waste stream may be a pathogenic biohazard that cannot be used for other purposes, such as soil remediation for food-crops. The pasteurization temperatures of the ASB eliminate this problem
Structure is provided to remove from the ASB environment, ASB treated biomass comprising solubilized biomass, liquid effluent, and solid effluent from the ASB environment, and introduce it to the anaerobic digestion (AD) environment. This structure may also include structure, such as coolers or heat exchangers, to cool the ASB effluent to a temperature suitable for the AD environment. The heat removed from the ASB effluent may be recycled, as described below, for example to heat future biomass feed for the ASB.
The AD environment is of a temperature to sustain and support growth of NT anaerobic microbes. After the NT anaerobic microbes are pasteurized in the ASB environment and thus become inactive in the AD feed, additional anaerobic microbes must be initially introduced or inoculated into the AD environment for the AD treatment. This may be accomplished by any suitable means, such as a direct, inoculation, or through a satellite reservoir described below. The AD environment is not thermophilic and does not sustain or support growth of thermophilic ASB microorganisms. The NT anaerobic microbes introduced to the AD environment digest the ASB treated biomass through methanogenesis to produce methane. Unlike prior-art anaerobic digestion processes, difficult or impossible to digest lignocellulosic materials in the biomass have been converted in the ASB to readily anaerobic digestible materials for the AD feed. In addition to methanogenesis in the AD, hydrolysis, acidogenesis, acetogenesis, and other processes may also occur.
Moreover, various contents may be introduced or recycled to the ASB environment by the AD environment. Furthermore, at least one satellite reservoir may supply contents to at least one of the ASB and AD environments according to principles described herein.
A heat recovery system may be used to recover and recycle heat from at least one environment. For example, at least one heat exchanger may be used to direct heat from at least one environment to at least one other environment. Purification treatments may also be used on contents produced by at least one environment. Also, contents may be recycled from at least one environment to at least one other environment.
The treatment processes according to principles described herein advantageously have lower energy costs compared to current treatment processes, render biomass significantly more available to anaerobic treatment, and introduce no chemical agents that are poisonous or inhibitory to anaerobic microorganisms. The two-part anaerobic digestion environments described herein are to treat biomass more completely and quickly without the use of mechanical and chemical pretreatment. It is acknowledged that processes are available that use anaerobic digestion by anaerobic microbes to solubilize biomass into materials that can be converted into biogas. The microbes may be readily available and often occur naturally with the biomass. A problem with these processes is that biomass materials often contain significant amounts of lignocellulosic materials that are essentially insoluble under anaerobic digestion.
An example of such a process is disclosed in U.S. Pat. No. 6,342,378 to Zhang, wherein digestion is modified to control volatile fatty acids which can inhibit the conversion to biogas. However, even with this improved digestion system, problems with materials that are unable to be solubilized or digested are significant, as noted in column 8, line 67 through column 9, line 13. Materials like rice straw are difficult to biodegrade. This is due, at least in part, to the plant fibers having lignin, cellulose, and hemi-cellulose, all of which are water-insoluble. Breakdown of these insoluble materials can be achieved to some degree by chemical hydrolysis or biodegradation. However, the breakdown is severely inhibited by lignin, cellulose, and hemi-cellulose which form barriers or seals around materials and which are considered non-biodegradable by conventional digestion.
Efforts have been made to improve anaerobic digestion of biomass by treating biomass with chemical or mechanical means beforehand, however, there are several drawbacks. Mechanical treatment systems, such as grinding and cutting, are generally energy intensive, uneconomical, and only modestly effective in improving conversion. Chemical treatment introduces components, such as acids or bases, to chemically react with biomass and make it more available to anaerobic digestion. These components, however, are often poisonous to anaerobic bacteria and are also only modestly effective. Strong acids or alkalies used to breakdown or damage lignin actually poison the anaerobic digestion. Any advantages from breaking down the biomass components by the chemicals are compromised by causing a less than optimal anaerobic environment in the subsequent anaerobic digestion tank. Chemical means can further create an expensive disposal problem for toxic wastes that result.
There are known microbes that in nature digest lignin, but none have been used in a biomass to biogas system, particularly a system that can be operated on an industrial scale. Because of the ASB treatment prior to the AD treatment, the AD treatment functions in a more efficient and controlled manner that is not achievable in conventional anaerobic treatments. In addition, the ASB treated biomass to the AD is pasteurized, free from problems associated with conventional anaerobic digesters where the composition of the methanogenic consortium cannot be controlled from contamination from unpasteurized feedstock. The ASB treatment thus allows customization and optimization of the AD anaerobic microorganisms, depending on, for example, the nature of the biomass, process conditions, and other variables.
In contrast to conventional anaerobic digestion, any of certain suitable thermophilic microorganisms that digest lignocellulosic materials can be used in the ASB to biologically treat biomass fed to the AD to make it significantly more available to anaerobic digestion. The ASB may occur in a suitable reactor or tank, or any other environment that produces an effluent comprising at least one of a solubilized biomass, liquid effluent, and solid effluent.
The ASB environment provides an environment where microbes can thrive without compromising the environment for subsequent treatment in the AD environment by anaerobic methanogenetic microbes. Example microorganisms suitable for the ASB include bacteria or organisms capable of breaking down lignocellulose (e.g., cellulose, hemicellulose, lignin, etc.). Examples include thermophilic extremophiles found naturally in some hydrothermal pools. A study indicated that the bacteria anaerobic thermophile Caldicellulosiruptor bescii (“C. bescii”) solubilizes 85% of lignocellulose and cellular material. (See “Carbohydrate and lignin are simultaneously solubilized from untreated switchgrass by microbial action at high temperature,” Energy and Environmental Science, Issue 7, 2013.)
The principles described herein may be applied to carry out the reaction on an industrial scale, providing universal industrial conversion of biomass by extremely thermophilic microbes in a treatment that requires limited or no chemical additions. The ASB/AD digestion is suitable for any biomass containing lignocellulosic materials. Other hard to digest materials, such as bacterial cell material and algae may also be advantageously digested by the ASB/AS system. In an example, the ASB/AD digestion is applied to giant king grass, mixed green waste, paper, sewage, manure and/or several other feedstocks on a pilot plant scale. In another example, the ASB/AD digestion is scaled to commercial anaerobic digestion systems, such as electrical generation on a megawatt scale.
The environment suitable for organisms, such as C. bescii, to thrive and produce sufficiently large amounts of secretome, is thermophillic. For example, in a lab experiment, C. bescii solubilized up to 90% of lignocellulose and cellular material in an ASB environment to make the carbon accessible for anaerobic digestion. Accordingly, the biomass is heated, either in a previous tank, or in the ASB tank, to a high temperature that is suitable for the growth and flourishing of ASB organisms. The ASB environment is unsuitable for growth of anaerobic organisms usually found in conventional anaerobic digestion. Accordingly, the biomass is effectively pasteurized, with the exception of the ASB organisms.
The AD is operated in a similar manner as a conventional anaerobic digestion system. But, there are significant differences that derive from its use together with the ASB. Among others, the AD receives a non-conventional lignocellosic-depleted and optimized predigested feed that can there be more fully digested and converted to biogas.
Anaerobic digestion involves at least four processes-hydrolysis, acidogenesis, acetogenesis, and methanogenesis (biogas generation). A single anaerobic digestion cannot be optimized for all such processes. By separating digestion into two environments, processes such as hydrolysis, acidogenesis, acetogenesis in the ASB can be more quickly and more completely carried out, thus creating an ideal and readily available biomass for methanogenetic biogas generation in the AD.
The combined ASB and AD is an improved digestion reactor system which can achieve a much higher biogas conversion than a conventional anaerobic system. By digesting the biogas in two radically different anaerobic environments, rather than one, a more complete digestion of the biomass is achieved.
The ASB/AD digestion systems and methods have several advantages and differences over known anaerobic digestion systems, which will be described below and which include, but are not limited to:
The term “thermophilic” with respect to the ASB environment describes an environment where thermophiles or thermophilic microbes thrive, and NT microbes are killed or damaged. This can be a temperature above about 45 to 65° C., and includes temperatures at which extreme thermophiles thrive, such as 70° C.-75° C. and above. NT microbes, which include mesophilic microbes generally used in prior art biomass digestion processes at temperatures between 20 to 45° C. are killed or damaged in a thermophilic environment. Moreover, the thermophilic temperature is fatal to any microbes that are not adapted to high temperatures commonly found in natural thermal springs or pools which can have temperatures of, for example 70° C., 70-75° C., or 75° C. and greater.
The term “tank” refers to an actual tank or other reaction containment, space or environment in which suitable conditions are met. The term further includes a suitable continuous or semi-continuous flow reactor, plug-flow reactor, reaction tank, etc.
The term “reactor” refers to at least one tank or set of tanks used in relation to the treatment of lignocellulosic biomass.
The term “satellite reservoir” refers to a separate, independent reservoir that is configured to maintain and provide at least one microbe, nutrient solution, pH adjusting chemical, or other content to another environment.
The term “environment” is broadly used to include environments that include not only tanks and reservoirs, but other environments for applying principles discussed herein. Note that “tank”, “reactor”, “environment,” and “satellite reservoir” may be used interchangeably according to principles discussed herein. Each may further perform at least one of mixing and heating according to principles described herein. ASB and AD may be described in association with an environment or more generally as a process.
The term “biogas” refers to gas which may be combusted to generate electricity and heat, or further processed into renewable natural gas and transportation fuels. Biogas as described herein may include at least one of methane, pure methane, carbon dioxide, compressed natural gas (CNG), and other contents known or as described herein.
A 6% solids solution of dairy manure was ASB treated in the ASB tank for 48 hours. After ASB treatment, the material was anaerobically digested and the rate of biogas production and composition was measured. A control experiment was conducted by heating a 6% solids solution of dairy manure to 75° C. for 48 hours. Afterward, the solution was anaerobically digested and the rate of biogas production and composition was measured.
A 5% solids solution of WAS was ASB treated in the ASB for 48 hours. After ASB treatment, the material was anaerobically digested and the rate of biogas production and composition was measured. A control experiment was conducted by heating a 6% solids solution of WAS to 75° C. for 48 hours. Afterward, the solution was anaerobically digested and the rate of biogas production and composition was measured. FIG. A reproduced below shows ASB treated WAS (solid black line) produced 2.4× more biogas than the control (dashed black line). FIG. B reproduced below shows the rate of biogas production. The maximum rate of biogas production from ASB treated WAS (filled black circles) was 2.6× larger than the maximum rate of biogas production from the control (filled black triangles). The methane content in ASB treated, anaerobically digested WAS was 76% compared to 62% for the control. Clearly, it can be appreciated that the treated biomass increases biogas production.
In an example, an ASB tank receives biomass to carry out digestion. Turning to
The contents within the example ASB tank 208 are digested and may further be mixed with a mixer as represented by paddles 246 and powered by a mixing motor 232. Heat may also be applied in the ASB tank 208. ASB treated biomass 210 that is produced by the ASB tank 208 is supplied to the AD tank 212 for anaerobic digestion as indicated by a black arrow. The ASB tank 208 may further receive a supply of contents from the ASB satellite reservoir 236 that is functionally connected to the ASB tank 208 and powered by an ASB satellite motor 234. Satellite paddles 248 as shown may be used to stir its contents, such as at least one or more of bacteria, nutrients, and other matter described herein to facilitate the ASB treatment process within the ASB tank 208. Heat may also be applied to the ASB satellite reservoir 236.
In an example, contents such as CO2 and bicarbonate 211 that are produced in the AD tank 212 as powered by motor 238 are recycled back to the ASB tank 208 as indicated by a black arrow. Mixing may also occur in the AD tank 212, as provided by paddles 246. The AD treated biomass 240 is supplied to a biogas processor 254 which produces biogas 242.
The biomass effluent 209 provided to the ASB tank 208 may contain or be at levels approximating, for example, 10% of the influent solids content. Digestion within the ASB tank 208 is accomplished by a secretome of a class of high-temperature thermophilic microorganisms that cannot be present in sufficient numbers in conventional anaerobic digestion. A secretome is the set of proteins expressed by an organism and secreted into the extracellular space or onto the surface of the organism. Any secretome and digestion proteins produced by anaerobic microorganisms used in conventional anaerobic digestion only modestly react and break down biomass, thus achieving only the modest result that has been seen with current anaerobic digestion processes.
Within the ASB tank 208, the biomass effluent 209 receives exposure to at least one material comprising a thermophilic anaerobic microbe that digests and “solubilizes” at least a portion of the biomass effluent 209, including lignocellulose materials, essentially breaking down plant cell structure/walls within the material and making contents of the plant cells available for subsequent anaerobic digestion.
“Lignocellulose” or “lignocellulosic” is meant to describe materials that contain lignin, hemi-cellulose, and/or cellulose that are predominately not solubilized by NT microbes. These materials are not solubilized or are only partially solubilized, leaving a major portion as non-solubilized material that is not available to conversion to biogas. As discussed above, attempts to make these materials available to NT microbes often involve, for example, chemical and mechanical treatment, which is not necessary in the ASB tank 208 according to principles described herein.
Contents introduced into the ASB tank 208 having lignocellulosic biomass may include at least one of animal waste, human waste (e.g., biosolids, etc.), fats, oils, and grease (FOG); food waste/garbage, organic matter, plant matter (e.g., green waste, bio-energy crops, coconut husk, grass, etc.), waste activated sludge (WAS), and algae (e.g. algae grown in reactors, etc.), as well as other contents that may be digested and solubilized. Note that lignocellulosic biomass and other types of raw material or feedstock may be introduced into the ASB tank 208 being pre-mixed together (e.g. in the mixing tank 202, previously mixed before entering the mixing tank 202, mixed before entering the ASB tank 208, etc.) or added separately.
Besides lignocellulosic biomass, biomass effluent 209 contents may include non-lignocellulose biomass and waste paper that does not have lignin, such as slaughterhouse waste, bacteria cell walls that are sluffed off from a ruminant animal, waste activated sludge (WAS), algae, king's grass, waste paper, etc.
While anaerobic digestion by itself is effective, for example, on hemicellulose side chains, other chains like long cellulose chains are only slowly digested by anaerobic bacteria, and polyphenols like lignin are resistant or toxic to many microorganisms. The ASB tank 208 can access the chains and compounds inaccessible to prior art systems to solubilize the long cellulose chains and polyphenols. The use of thermophilic microbes solubilize biomass, up to 90% or more of lignocellulosic materials, making the carbon in the biomass accessible for anaerobic digestion.
The ASB tank 208 includes thermophiles that solubilize lignocellulose and other difficult to digest contents to produce products suitable for anaerobic digestion. The ASB tank 208 further includes at least a basic bicarbonate or other bicarbonate to promote production of biomass products suitable for the AD tank 212 and lower the concentration of lactic and acetic acids in the biomass products. The ASB tank 208 relationship with the AD tank 212 can be likened to a stomach to an intestine, both the ASB tank 208 and a stomach enhancing breakdown of contents for additional processing in the AD tank 212 and intestine. The thermophilic microbes in the ASB tank 208 can readily digest these materials under the thermophilic conditions.
The result is a biomass where a major portion of the lignocellulosic materials are converted (e.g., solubilized, metabolized, etc.) to at least one of lactic acid and acetic acid, which are then readily converted to biogas. In an example, the thermophilic anaerobic microbe is C. bescii or another bacteria. Possible other bacteria candidates for the ASB tank 208 include bacteria of at least one of the genus Caldicellulosiruptor, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum, and other bacteria with comparable or otherwise suitable properties for digesting at least one lignocellulosic material.
In another example, at least one of fungi, archaea, cellular organism, and an organism or mixture of organisms with comparable or otherwise suitable properties for digesting a lignocellulosic material is used.
At least one of the bacteria or other examples listed as candidates may be in a genetically modified form. In another example, at least one of the bacteria or other examples listed is found in at least one of a hot spring, a concentration of rotting wood, and a lignocellulose-degrading extremophile. The ASB tank 208 may find further use in medical industries. Viruses like those for flu and COVID-19 are inactivated through the ASB treatment. In an example, the virus is subject to two to three days at a temperature of 75° C.
The thermophilic anaerobic bacteria are adapted to derive energy from organic materials that happen to exist in the thermal pools, which are often the unsolubilized remains of wood. It has been found that the lignin/cellulosic materials in biomass can be digested by these same microbes in an industrial scale process that shows dramatically improved conversion of biomass to biogas, both in terms of short treatment times, and the high portion of biomass converted.
Unlike a system in which various compounds are metabolized in an oxygen-free environment, ASB conditions and microbes are chosen to target normally inaccessible ligneous and cellulosic portions of the biomass compounds. The contents in the ASB tank 208 are heated to become environmentally suitable for thermophile, i.e., thermophilic microbial action. Under these conditions, the contents introduced to the ASB tank 208 are pasteurized, eliminating a substantial portion of, or all of, the microbes except for thermophilic bacteria that survive and thrive under ASB heated conditions. This culture of thermophilic bacteria digests lignocellulosic materials, including materials previously inaccessible to other anaerobic digestion and creates an enhanced feed for the anaerobic digestion tank.
For the thermophile to thrive, consideration is taken for a desirable temperature. During the digestion process within the ASB tank 208, the ASB tank 208 is maintained at a desired temperature to provide a suitable environment for the C. bescii or other microbes to solubilize cellulose. An example temperature maintained may include 75° C. or approximately 75° C. In another example, a temperature range is maintained, for example, between 45-65° C., or 45-85° C. Narrower ranges include 45-50° C., 50-55° C., 55-60° C., 60-65° C., 65-70° C., 70-75° C., 75-80° C., 80-85° C., 60-70° C., 70-80° C., 70-85° C., or 60-85° C., or other ranges that are used for pasteurization of conventional anaerobic microbes and growth of ASB thermophilic anaerobic microbes. The higher temperature causes the reactions within the ASB tank 208 to go faster than non-thermophilic temperatures and can be done without killing ASB thermophilic anaerobic microbes, enzymes, and other microbes.
The ASB tank 208 is heated to maintain the temperature using any suitable means. Heat may be suitably recycled from other environments described herein as well as other environments and other processes, such as waste heat from engines and other mechanical devices, exhaust, combustion gas heat, solar, etc.
Another condition that is considered for the ASB tank 208 is the oxygen limit. The ASB tank 208 is configured to adhere to oxygen limits for C. bescii, based on C. bescii being a strict anaerobe, which is pO2=0.5%. The ASB may further be configured for higher oxygen levels. For example, C. bescii is capable to withstand oxygen levels as high as pO2=20% for 15-20 minutes. In an example, oxygen levels are thus maintained up to 20% for this duration of time.
Another condition for the ASB tank 208 is a suitable pH that should be maintained for the ASB bacterial growth, such as a range between 4.5 to 8.5. For optimal growth of C. bescii, the pH should be controlled between 6.5 and 8.5, or 6.8 and 7.2. Lower pH values do not immediately kill bacteria, but slowly starve it because it cannot acquire energy by metabolizing sugars from dissolved cellulose. Dissolution of the cellulose also stops because the products are not being metabolized.
In an example, the pH of the ASB tank 208 is controlled. In an example, a base is introduced for pH control and promotion of metabolism. In another example, a sufficient base is maintained to react with acids produced during metabolism. In another example, bicarbonate (e.g., HCO3—, etc.) is introduced or recycled from at least one of the AD tank 208 and other environments described herein to control pH of the ASB tank 208. Metabolism and growth of C. bescii are driven by production of CO2 gas or water. A base that does not produce sufficient Gibbs energy upon reaction with acids will not provide the energy required for optimum growth of the bacteria. Thus, an example includes that the base have a sufficient Gibbs energy to provide the energy needed for optimum growth.
Another condition for the ASB tank 208 is the alkalinity, or the amount of base to resist changes in pH that would make the contents more acidic. The base to be controlled within the ASB tank 208 may include carbonate and bicarbonate. In an example, bicarbonate is produced by the AD tank 212 and recycled to the ASB tank 208 to maintain a desired alkalinity or range of alkalinity.
C. bescii is believed to not form a biofilm on biomass particles. Instead, it produces a secretome with exozymes that catalyze dissolution of the lignocellulosic materials. In an example, the ASB tank 208 mixes the contents for C. bescii to increase contact between bacteria and biomass and support the production of exozymes that dissolve the lignocellulosic materials. An example mixing system includes a slow or low-level mechanical stirring or other suitable system for mixing. Means for mixing the contents may include one or more of a plurality of paddles and pumps 246.
ASB treatment examples include at least one of a batch process, semi-continuous process, and a continuous process. The reaction in the ASB tank 208 may depend on the time period depending on the feedstock and desired level of material destruction. In an example, ASB containment for reaction includes a period of time between 0 to 48 hours. In another example, the period of time is 0 to 7 days. The time for a reaction using C. bescii may be between 0.5 hour to 200 hours, but more typically 12-72 hours. C. bescii is suitable because it can rapidly depolymerize and solubilize lignocellulosic (plant material) and other cellular material. C. bescii further produces exozymes that catalyze hydrolysis of cellulose and lignin at a rapid rate. In an example, the products are sugars and phenolic compounds from lignin that are metabolized to acetic acid and lactic acid by C. bescii as a source of Gibbs energy for growth and activity.
In an example, at least a portion of the treated biomass and a portion of the biomass products are provided to the AD tank 212. To meet the conditions of the AD tank 212, the ASB effluent 210 may be cooled, for example, by a cooling reservoir or heat exchange system, prior to entering the AD tank 212 or other location.
ASB Treatment Reactions with C. bescii Include:
Lignocellulose→(acetate+lactate) ions+CO2 (g)+oxygenated aromatics from lignin+residual sugars. These reactions are catalyzed by exozymes produced by C. bescii. During treatment in the ASB tank 208, acetic and lactic acids in the presence of bicarbonate react to produce acetate ion, lactate ion, and CO2 gas. Acetic and lactic acids react with base in the pH buffer to produce acetate ion, lactate ion, and CO2 gas. The rate of ASB treatment can be obtained by monitoring the increase in CO2 gas pressure over the ASB treatment mixture in a sealed vessel or by one or more of measurement of the change in total suspended solids and volatile solids as ASB treatment progresses. Products are not toxic to anaerobic digestion microorganisms and are rapidly digested in the AD tank 208 to produce biogas.
Acetic acid+bicarbonate→acetate ion+CO2+H2O. The Acetate/Lactate ions and residual sugars are rapidly metabolized in the AD by anaerobic bacteria, producing CO2, and CH4, as follows;
Acetate/Lactate ions+sugars→methane+CO2+bicarbonate (which may be recycled back to the ASB tank 208).
At least one bacterial organism used in the ASB tank 208 can be introduced by any suitable method, and conditions should be maintained for optimum growth. For example, trace elements, nutrients, vitamins, and the like may be introduced to start or maintain the ASB treatment process.
For C. bescii, specific optimum conditions have been shown to include the following: 78° C., DSMZ media (0.33 g NH4Cl, 0.33 g KH2PO4, 0.33 g KCl, 0.33 g MgCl2*6H2O, 0.33 g CaCl2*2H2O, 0.50 g Yeast Extract, 0.50 mL Na-resazurin solution (0.1% w/v), 1.50 g NaHCO3, 0.50 g Na2S*9H2O, 1.00 ml of a trace element solution, 10.00 ml of a vitamin solution to 1000.00 ml distilled water.
The trace element solution (designated SL-10) was, in an example, composed of 10.00 mL HCl (25%; 7.7 M), 1.50 g FeCl2*4H2, 70.00 mg ZnCL2, 100.00 mg MnCl2*4 H2O, 6.00 mg H3BO3, 190.00 mg CoCl2*6 H2O, 2.00 mg CuCl2*2 H2O, 24.00 mg NiCl2*6 H2O, 36.00 mg Na2MoO4*2 H2O, and 990.00 ml Distilled water. The FeCl2 was dissolved in the HCl, which was then diluted in water. The remaining salts were added and the solution was diluted to 1000.0 mL. The vitamin solution was composed of 2.00 mg Biotin, 2.00 mg Folic acid, 10.00 mg Pyridoxine-HCl, 5.00 mg Thiamine-HCL*2H20, 5.00 mg Riboflavin, 5.00 mg Nicotinic acid, 5.00 mg D-Ca-pantothenate, 0.10 mg Vitamin B12, 5.00 mg Lipoic Acid, and 1000.00 mL Distilled water. The solutions should be stored under anaerobic conditions.
In prior art processes, a great expense may be involved in hauling away and safely disposing of waste material. The ASB tank 208 and other components described herein have great market potential because of the increase in quantity of commercially viable product that they yield over current methods. This reduces undigested waste material.
Another advantage in using the ASB tank 208 and other components is that they yield 1.5 to 10 times as much gas than if biomass is put directly into an AD tank 212.
After the ASB tank 208, at least a portion of the ASB treated biomass 210 enters the AD tank 212. Compared to conventional feed streams of an anaerobic digester, the ASB treated biomass 210 is more available for anaerobic metabolism and digestion than typical biomass feeds to anaerobic digestion. In particular, an advantage of extremely thermophilic ASB treatment is its pasteurization of the biomass 209 before being introduced into the AD tank 212, thus allowing better control of the AD microbes and processing.
For acetate only ASB effluent 210, CO2 may still be present in the biogas from digestion within the AD tank 212. It is speculated that such CO2 in the biogas may come from compounds other than acetate that were produced in the ASB tank 208.
Within the AD tank 212 are maintained suitable bacteria and archaea, such as acetogens and methanogens, that support production of biogas. Methanogens grow better with the ASB treated biomass 210 from the ASB tank 208 than with conventional untreated biomass. In an example, the AD tank 212 includes at least one of anaerobic bacteria and archaea to convert the treated biomass 210 into biogas under anaerobic digestive conditions. In another example, a portion of the AD treated biomass 211 is recycled back to the ASB tank 208 as indicated by the black arrow. The portion of AD treated biomass 211 may include, for example, basic bicarbonate or other bicarbonate produced within the AD tank 212 that may be recycled to control pH and promote metabolism with the ASB tank 208.
In other examples, at least one of the AD tank 212 and an AD satellite reservoir 236 provides the basic bicarbonate or other bicarbonate to the ASB tank 208. In an example, the bicarbonate is taken after AD treatment and can be a specified amount as needed or desired. In an example, the AD tank 212 is preferred for drawing bicarbonate with the AD satellite reservoir 236 being a back up resource.
Conditions in the AD tank 212 are maintained to allow anaerobic bacteria to thrive. An example temperature includes 40° C. Further examples include a temperature range between 15° C. to 85° C. The pH of the AD tank 212 may range from 6.5 to 8.5, with more narrow ranges including 6.5-7, 7, 7.5, 7.5-8, or 8-8.5. In an example, the pH of the AD tank 212 is maintained at 7 or slightly below 7 (e.g, 6.5-6.6, 6.6-6.7, 6.7-6.8, 6.8-6.9, 6.9-7, etc.). In another example, the temperature and pH are substantially different from the temperature and pH in the ASB tank 208. Maintaining disparate conditions where an organism of the ASB tank 208 and the bacteria in the AD tank 212 can thrive is at least one reason that separate tanks or digesters are provided for each part of the treatment.
The AD tank 212 may include at least one of a continuous stir, up flow anaerobic sludge blanket, induced bed reactor, dual anaerobic/aerobic digester, or any kind of anaerobic digester for the reaction. Treatment may be at least one of a batch process, semi-continuous process, and a continuous process.
In an example, the ASB tank 208 has a volume ratio with the AD tank 212 between 1:10 to 10:1 as defined by a relative rate of degradation of the biomass in the ASB tank 208 to the production of biogas in the AD tank 212.
In an example, the AD tank 212 has two main product streams including principally gas and liquid phase streams. The first main output stream (1), or biogas stream, contains at least one of methane (CH4) and carbon dioxide (CO2), but may also contain other reaction products and impurities (e.g., H2S, and H2O). The biogas stream is directed to suitable gas processing for its intended use. Bicarbonate is recycled back to the ASB tank 208. The second main output stream (2) is a slurry of undigested waste (dead bacteria, inorganic portions, dirt, etc) that has been pasteurized, and which therefore can be processed as a soil conditioner or compost. In an example, there is no biomass left. Because the biogas stream is considered to be pathogen free, pasteurization produces a higher quality, more valuable undigested waste than conventional AD treatment.
CH3COO−(aq)→CH4(g)+HCO3
2CH3CH(OH)COO−(aq)+H2O→3CH4(g)+2HCO3
2C1H2O→CH4(g)+CO2(g), (Sugars)
Production of acetate in the ASB tank 208 uses one bicarbonate and digestion of acetate produces one bicarbonate in the AD tank 212. Production of lactate uses one bicarbonate and digestion of lactate produces one bicarbonate ion. Thus, the reactions in the AD tank 212 produce the same amount of bicarbonate as the amount used in treatment in the ASB tank 208. Balancing bicarbonate (i.e. acid/base balance) would require recycling 100% of the biomass effluent from the AD tank 212. Efficiency of that nature is unlikely to be possible and not practical. Instead, bicarbonate may be introduced to the ASB tank 208 or the mixing tank 202 from other sources, or by adding another base to the ASB tank, such as ammonia, sodium carbonate, sodium bicarbonate, potassium hydroxide, and sodium hydroxide.
An application of ASB/AD digestion system will include optional systems designed to exploit the advantages of the ASB/AD system, and provide a suitable industrial operating environment. Below is a fuller description of exemplary systems.
Treating biomass with the ASB prior to anaerobic digestion is effective in promoting anaerobic digestion and does not introduce any harmful chemicals or otherwise harm the environment for the anaerobic bacteria in the AD tank 212 where biogas is produced. Note that C. bescii and other thermophiles are not considered to be pathogens because they cannot survive at low or mesophilic temperatures.
A mixing tank may be used to treat biomass before entering the ASB tank 212. The mixing tank 202 includes treatment that is to create a feedstock, or biomass effluent 209, for the ASB tank 212 that is suitable for and that promotes growth of the thermophilic microbes of the ASB tank 208. The mixing tank 202 may mix biomass with water, and possibly other reagents as well. In addition, the mixing tank 202 may heat the biomass 258. Mixing the biomass 258, or mixing and heating of the biomass 258 with water, may occur before treating the biomass 258 in the ASB tank 208 to mitigate pH changes and promote metabolism. Also, the mixing tank 202 may be used to remove oxygen from the biomass 258 and promote hydrolysis of biomass solids. In an example, the mixing and heating occurs prior to entry to the mixing tank 202.
Contents introduced to the mixing tank 202 having lignocellulosic biomass may include the same types of materials that enter the ASB tank 208, namely, at least one of animal waste (e.g. manure, etc.); human waste (e.g., biosolids, etc.); fats, oils, and grease (FOG); food waste/garbage; organic matter; plant matter (e.g., green waste, bio-energy crops, coconut husk, grass, etc.); waste activated sludge (WAS); and algae (e.g., algae grown in reactors, etc.), as well as other contents. Lignocellulosic biomass along with other types of raw material or feedstock may be introduced into the mixing tank being premixed together or added separately. Besides lignocellulosic biomass, biomass 258 contents may include non-lignocellulose biomass and waste paper that does not have lignin, such as slaughterhouse waste and waste paper.
Once the biomass 258 is treated with at least one of water, heat, and mixing, etc., the biomass effluent 209 from the mixing tank 202 is a treated biomass for the ASB tank 208 rather than its original form of biomass 258.
An example treatment includes the biomass 258 being ground to a 3 cm particle size and then being supplied to the mixing tank 202 before being introduced to the ASB tank 208. The contents of the mixing tank 202 are adjusted to a composition that is approximately 2% to 50% (e.g, 6% has been found suitable), and heated to approximately 60° C. to 100° C. for 1 to 6 hours. During this period of time, mixing can be accomplished, for example, with one or more of a plurality of paddles 244 and/or pumps. This process has the effect of pasteurizing the contents, expelling dissolved O2, and providing the contents with an optimal temperature for thermophilic microbial action.
The resultant biomass effluent 209 produced by the mixing tank 202 is sent to the ASB tank 208 in the form of a liquid suspension, slurry, or mash of solids. An example solids content of the effluent may represent approximately 10% of the effluent.
In an example, a solution or “tea” containing soluble materials in the biomass 258 or treated biomass 209 is separated from the solids content and sent directly to an AD tank 212. In another example, the biomass 258 is mixed with water to suspend at least a portion of the biomass 258 in the water and partially solubilize components of the biomass 258. In another example, the biomass effluent 209 includes at least a portion of one of suspended biomass and partially solubilized components of biomass being transferred to the ASB tank 208.
For some types of biomass, raw material, and feedstocks, the mixing tank 202 can also serve as a hydration tank in which hydration is provided. Also, two separate tanks are contemplated, one tank for hydration and one tank for mixing.
Satellite reservoirs may be provided that are associated with at least one of the tanks or environments, according to principles discussed herein. In an example, the ASB tank 208 benefits from a connection to a separate, independent ASB satellite reservoir 236. For example, there may be times when the ASB tank 208 becomes compromised due to a reinoculation failure. There may also be a bacterial, cultural, enoculum washout in which there is too short of a mean resonance time which pushes material out before bacteria grows. As a result, the whole community becomes washed out because the bacteria cannot grow fast enough. In another example, toxins and antiobiotics in the biomass are present. There may also be a chemical restrictions of the feedstock presented, or other problem. To counter occurrences of this nature and prevent failure of the ASB tank 208, an ASB satellite reservoir 236 as shown in
In addition, the ASB satellite reservoir 236 may also be used for at least one of the following—1) maintaining bacteria culture suited for the ASB tank 208, 2) alleviating the need for trace elements to be added to the ASB tank 208, depending on feedstock chemical characteristics, 3) adapting or conditioning C. bescii to utilize the feedstock present in the ASB tank 208, 4) speeding up the ASB treatment process in the ASB tank 208 by avoiding time that otherwise would be required for the C. bescii to grow in the ASB tank 208, and 5) adding a base, such as bicarbonate, to the ASB tank 208 to maintain pH and support metabolism.
In an example, the ASB satellite reservoir 236 facilitates the continuous inoculum of C. bescii. In addition, the satellite reservoir 236 contains nutrients necessary for C. bescii growth and a small amount of the ASB treatment feedstock (e.g., biosolids, green waste, energy crops, food waste, raw or organic materials, etc.). The C. bescii or other matter in the satellite reservoir is maintained at or near 1×106 cells per milliliter density.
Bacteria besides C. bescii that may be maintained in the ASB satellite reservoir 236 includes one or more of Caldicellulosiruptor bescii, Caldicellulosiruptor genus, Clostridium thermocellum, Thermoanaerobacterium saccharolyticum. Note that bacteria in the ASB satellite reservoir 236 is maintained at a pH range of 6.5-7, 7-. 7.5, 7.5-8, 8-8.5, 6.5-7.5, 7.5-8.5, or 6.5-8.5. Additionally, the bacteria may be cultured separately before being added to the ASB satellite reservoir 236.
In an example, the ASB satellite reservoir 236 maintains at least one of bacteria and archaea to be fed to the ASB tank 208. The ASB satellite reservoir 236 may maintain at least one of the following—a nutrient for bacterial growth, food waste, animal manure, biosolids, waste organic material, sewage, garbage, waste activated sludge, FOG, waste paper, lignocellulosic plant materials, and cellular material.
In addition to bacteria and nutrients, at least one of a trace nutrient and trace element may be maintained in the ASB satellite reservoir 236. In an example, at least one of a trace nutrient and trace element are maintained that is necessary for a bacteria in the ASB tank 208 to grow and divide. In another example, at least one of a trace nutrient and trace element are maintained to overcome a chemical restriction of the feedstock in the ASB tank 208. In operations that include C. bescii, at least one additional nutrient and trace metal may be provided to further facilitate C. bescii growth. Exemplary trace elements and proportionate amounts to obtain 1000 ml are shown in Table C below.
In addition, a sucrose or a carbon source may be maintained in the ASB satellite reactor 236 to promote optimal conditions for bacteria to grow and thrive and ultimately produce enzymes that will benefit the contents of the ASB tank 208. Also, a yeast, such as brewer's yeast, may be added to the ASB satellite reservoir 236 to promote growth of the bacteria by providing needed amino acids. An example sample of nutrients per liter maintained in the ASB satellite reservoir 236 include glucose (1 g), yeast extract (0.1 g) (e.g., brewer's yeast or another source of amino acids, etc.), NH4Cl (0.05 g), KH2PO4 (0.05 g), MgCl2 (0.05 g), CaCl2) (0.05 g), NaHCO3 (1.0 g), and Na2S (0.1 g to ensure anaerobic conditions). Note that the ASB satellite reservoir 236 may provide one specific type of content or a combination of at least one of a bacteria, nutrient, trace nutrient, trace element, sucrose, carbon matter, and other matter.
In an example, the contents in the ASB satellite reservoir 236 are grown on a substrate or other contents that are the same or similar to the biomass in the ASB tank 208. For example, if the biomass within the ASB tank 208 contains manure, the contents within the ASB satellite reservoir 236 are grown on manure or contents that include manure. If sludge is in the biomass, sludge is used as the substrate or as part of the substrate within the ASB satellite reservoir 236.
The similar contents enable similar bacterial propagation between the two environments. If the contents in the ASB satellite reservoir 236 are only given one type of biomass, the contents will have a difficult time being able to break down foreign types of biomass that are present in the ASB tank 208. The reason for this is that bacteria will not make enzymes that are not needed. For example, growing bacteria with only sucrose in the ASB satellite reservoir 236 will shut down genes that are not needed to metabolize sucrose. Introducing the sucrose fed bacteria into an ASB tank 208 that contains grass clippings will make the bacteria unable to break down the grass clippings because the bacteria were conditioned to metabolize sucrose. Maintaining similar conditions of bacteria and biomass between the ASB tank 208 and the ASB satellite reservoir 236 is therefore a support to the system as a whole.
In an example, the substrate comprises at least one of biomass effluent 209 from the mixing tank 202, ASB effluent 210 from the ASB tank 208, AD treated biomass 240 from the AD tank 212, nutrient for growth, food waste, human waste, animal manure (animal waste), biosolids, waste organic material, sewage, garbage, waste activated sludge, algae, FOG, organic matter, plant matter, waste paper, lignocellulosic plant materials, and cellular material. In addition to the substrate, the ASB satellite reservoir 236 may contain, for example, at least one acetoclastic methanogen.
Additional conditions of the ASB satellite reservoir 236 may by close or identical to that of the ASB tank. In an example, oxygen levels within the ASB satellite reservoir 236 are the same as, or similar to, the oxygen levels as the ASB tank 206. In another example, the ASB satellite reservoir 236 is configured to adhere to the oxygen limits for C. bescii, based on C. bescii being a strict anaerobe, which is pO2=0.5%. The satellite reservoir 236 may further be configured for higher oxygen levels, such as pO2=2% for 15-20 minutes. Maintaining a same oxygen level or maintaining certain oxygen levels for at least one or more specific constraint, such as a given set of contents and for certain time durations, may support the growth rates desired of the system.
Another condition of the ASB satellite reservoir 236 that may be close or identical to that of the ASB tank 206 is temperature. This may be a constraint that helps the ASB satellite reservoir 236 to stay inoculated with a particular bacteria that is suited for the ASB tank 206. Furthermore, maintaining a specific temperature or temperature range may enable the ASB satellite reservoir 236 to stay inoculated with a particular bacteria that is suited for the ASB tank 206. Temperature ranges that are maintained and that may be the same or similar to the temperature ranges of the ASB tank 206 may include, for example, 60-65° C., 65-70° C., 70-75° C., 75-80° C., 80-85° C., 60-70° C., 70-80° C., 70-85° C., or 60-85° C. degrees. Furthermore, temperature changes may be minimized during transport of contents from the ASB satellite reservoir 236 to the ASB tank 206 to protect the state of the bacteria or other matter.
Specific volume ratios or a range of volume ratios may also be observed between the ASB satellite reservoir 236 and the ASB tank 206. In an example, the satellite reservoir 236 has a volume ratio to the ASB tank 206 within a range of 1:10 to 10:1. In another example, the volume of the ASB satellite reservoir 236 is approximately 1/100 the volume of the ASB tank 206, or within a range of 1/200 to ½ of the volume of the ASB tank 206. In terms of mass per volume, the ASB satellite reservoir 236 may contain 0.5-1.0% of the ASB treated biomass. The volume ratios and percentages may be used to control and predict behavior of the contents within the ASB satellite reservoir 236 and the ASB tank 206.
In an example, the ASB satellite reservoir 236 provides at least one of a bacteria, nutrient, or other matter, primarily at two different times. The first time is at or near the beginning of the ASB treatment. The second time occurs when trace nutrients are depleted and not provided by the feedstock.
In another example, feeds to the ASB tank 206 occur at various times and in a manner so as to maintain an exponential growth culture. The mean doubling times for various feedstocks with C. bescii are shown in Table A1 below to provide information on the way to feed the ASB tank 206 so as to maintain their exponential growth culture. C. bescii growing on starch, for example, has a mean doubling time of 2.1 hours and therefore, quantities of the starch within the ASB tank 206 may be fed with contents in the ASB satellite reservoir 236 to double the amount of starch within that time frame.
C. bescii Mean
If a feedstock lacks the necessary requirements for C. bescii growth, contents from the ASB satellite reservoir 236 may provide specific chemical constituents to maintain such growth. For example, the C:N:P ratio of 500:10:1 dictates the addition of major nutrients, N and P, as NH4Cl, yeast extract, and/or KH2PO4.
An example ASB satellite reservoir 236 includes a monitoring system or device that monitors at least one of pH level, oxygen content, and type of bacteria present within the reactor. The monitoring system or a second monitoring system may further monitor at least one of the ASB tank 208 and AD tank 212. A delivery system manually, automatically, or semi-automatically delivers contents from the ASB satellite reservoir to the ASB tank 208 in quantities that are determined to be needed for the ASB tank 208. In an example, chemical analysis is performed so that trace metals that are lacking in the ASB tank 208 may be added including at least one of Fe, Zn, Mn, B, Co Cu, and Ni. In another example, product formation of acetate, lactate, oxygenated aromatic compounds, etc., is also monitored. The ASB satellite reservoir 236 may require that its entire volume be replaced every 72 hours or within a range of 50-80 hours.
Like the ASB tank 206, a separate, independent satellite reservoir may be connected to the AD tank 212. This is shown in
In
In an example, the ASB satellite reservoir 436 is used to supply one or more of a bacteria, nutrient, or other matter to the ASB tank 408. The contents within the ASB satellite reservoir 436 may be mixed by mixing paddles 448 and heated. The ASB satellite reservoir 436 may be powered by the ASB motor 434.
ASB treated biomass 410 that is produced by the ASB tank 408 is supplied to the AD tank 412, as indicated by a black arrow, for anaerobic digestion. The AD tank 412 includes mixing by mixing paddles 447 and heating capabilities to treat the contents therein, as powered by AD motor 438. In an example, at least a portion of the contents such as CO2 and bicarbonate 411 that are produced within the AD tank 412 are recycled back to the ASB tank 408, as indicated by a black arrow, or used for other purposes.
The AD satellite reservoir 437 is used to supply additional contents to the AD tank 412. The contents within the AD satellite reservoir 437 may be mixed by mixing paddles 449 and heated. The AD satellite reservoir 437 may be powered by an AD satellite motor 439. In an example, the AD satellite reservoir 437 contains at least one of the contents found in the AD tank 412. In another example, the AD satellite reservoir 437 contains substantially similar or identical content as found in the AD tank 412. In another example, at least one of archaea, acetoclastic consortium, and other matter is provided to the AD tank 412. In an example, the AD satellite reservoir 437 incubates bacteria that is used to augment desired bacteria in the AD tank 412 for processing of the ASB treated biomass in the AD tank 412. In another example, the AD satellite reservoir 437 maintains at least one of bacteria and archaea that are used to augment at least one bacteria and archaea in the AD tank 412. In a further example, the AD tank 412 is augmented from the AD satellite reservoir 437 with at least one of archaea and acetoclastic consortium isolated from WAS. In another example, the AD satellite reservoir 437 augments bacteria in the AD tank 412 that are specific to biogas production from the molecules being produced in the ASB tank 408. In another example, the AD satellite reservoir 437 supplies one or more of a nutrient solution and base to the AD tank 412.
The AD tank 412 may contain at least one of a synthetic content or biogenetically engineered content which are bio-augmented. The AD satellite reservoir 437 may also contain respective synthetic contents or biogenetically engineered contents.
Bioaugmentation with archaea has shown a significant reduction of acetate accumulation within seven days and the proportion of methane in biogas increased almost over a hundred-fold (Town and Dumonceaux, 2015). In an example, at least one of archaea and acetoclastic consortium is isolated during multiple successful digestions of WAS when methane production is relatively high to increase the likelihood that the archaea will thrive within similar AD conditions. The archaea or acetoclastic consortium may be cultured with DSMZ Medium 141 to capture local methanogens and an additional consortium is created that is spiked with two well-known Methanosarcina, Methanosarcina barkeri, DSMZ 800, and Methanosarcina acetivorans DSMZ 2834.
Consortia may be cultivated continuously at 55° C. for 6 months prior to bioaugmentation experiments in the AD tank and produced with consistent levels of methane in biogas. In an example, at least one of archaea and acetoclastic consortium may be added to the AD tank by an AD satellite prior to WAS/AD and periodically to reseed the AD tank when methane production drops.
There may be multiple satellite reservoirs for one tank, either duplicate, or with a different content, depending upon the specific content or purpose. For example, there may be two satellite tanks associated with the ASB, one for micronutrients, and another for C. bescii. In another example, there may be three types of satellite reservoirs, including at least one to deliver the bacteria, at least one to provide nutrients, and at least one to provide microbes, such as thermophilic microbes (C. bescii) for the ASB or NT microbes from the AD. In another example, nutrients provided by a satellite reactor include at least two main types of contents, such as two types of nutrients or two types of trace metals. In another example, the satellite reactor provides a combination of types of contents, such as at least one type of nutrient and at least one type of trace metal.
An example of a reactor system includes at least three satellite reservoirs that supply contents to the ASB and AD tanks, each satellite reservoir providing same or different contents at same or different times and rates. The first satellite reservoir is for the ASB tank and includes C. bescii, the second satellite reservoir is for the AD tank and includes at least one of archaea and acetoclastic bacteria, and the third satellite reservoir for the AD tank includes oxidative methanogenic bacteria. In another example, a specific type of bacteria, either the archaea, acetoclastic bacteria, or the oxidative bacteria, is delivered to the AD tank. In another example, predetermined quantities of bacteria from each tank are delivered to the AD tank. Further examples include that controls over at least one of an amount, time, or rate depends on determinations made by a monitoring process or other process.
In an example, the AD satellite reservoir contains bacteria grown on a substrate that is close or identical to effluent from the ASB tank. The contents may be grown on a substrate or other contents that are close or identical to the biomass effluent 409 in the ASB tank 408. In an example, the substrate comprises at least one of biomass effluent 409 received from the ASB tank 408, nutrient for growth, food waste, human waste, animal manure (animal waste), biosolids, waste organic material, sewage, garbage, WAS, algae, FOG, organic matter, plant matter, waste paper, lignocellulosic plant materials, and cellular material. In addition to the substrate, the AD satellite reservoir 437 may contain, for example, at least one of acetoclastic methanogen and archaea.
At least one condition of the AD satellite reservoir 437 may be close or identical to at least one of the AD tank 412 or other environment. Such conditions may include, for example, oxygen levels, temperature, volume ratios, and ranges thereof.
Maintaining a specific temperature or temperature range may enable the AD satellite reservoir 437 to stay inoculated with a particular bacteria that is suited for the AD tank 412. Temperature ranges that are maintained and that may be the same or similar to the temperature ranges of the AD tank 412 may include, for example, 60-65° C., 65-70° C., 70-75° C., 75-80° C., 80-85° C., 60-70° C., 70-80° C., 70-85° C., or 60-85° C. degrees. Furthermore, temperature changes may be minimized during transport of contents from the satellite reservoir to the AD tank 412 to protect the state of the contents.
The same or similar type of monitoring processes as the AD tank 412 may be implemented with the AD satellite reservoir 437, whether the monitoring be the same system or a separate, independent system.
In an example, the AD satellite reservoir 437 has a volume ratio to the AD tank 412 within a range of 1:10 to 10:1 as defined by a relative rate of degradation of the biomass in the ASB tank 408 to the production of biogas in the AD tank 412. In another example, the volume of the AD satellite reservoir 437 is approximately 1/100 or within a range of 1/200 to ½ of the volume of the AD tank 412. The volume ratios and percentages may be used to control and predict behavior of the contents within the AD satellite reservoir 437 and the AD tank 412.
Although reference is made specifically to the AD tank 412, the AD satellite reservoir 437 may supply contents to other environments and its principles may be applicable to other tanks and processes described herein and are not intended to be limited to the AD tank 412.
Applying these findings, a synthetic microbial community may be incorporated in at least one of the ASB tank 408 and in the AD tank 412. A synthetic microbial community is a systems approach to reducing the complexity, while increasing the controllability, by selecting genetically-engineered or wild type microorganisms that cooperate metabolically. The combination of different microbial species may decrease the potential competition between species, often leading to the co-metabolism of a substrate in an independent manner. In the ASB tank 408, for example, depending on feedstock, an additional species, such as Clostridium thermocellum, may be provided by the ASB satellite reservoir 436 or AD satellite reservoir 437 to help decompose difficult to digest biomass (e.g. lignocellulolytic materials). Also, a synthetic community of substrate dependent microbes configured specifically to digest acetate and lactate and the products of ASB treatment may be provided by the AD satellite reservoir 437.
In an example, the ASB tank 408 favors production of acetate ion over lactate ion to produce a reduced CO2 content in the biogas from the AD tank 412 and to produce increased bicarbonate which may be used in at least one of the ASB tank 408 and AD tank 412. Please see Section A entitled EXAMPLE KINETIC MODEL to see a model that shows that ASB treatment results in ASB effluent with acetate as the major ASB treatment product.
If desired, at least a portion of AD biogas produced in an AD tank may be used by itself or be subjected to a at least one of a biogas processor or conditioner to process or condition biogas suitable for its intended use. The biogas processor may remove at least one of siloxanes, carbon dioxide, hydrogen sulphide, moisture (e.g. water, etc.), and contaminants from the AD biogas to make it suitable for at least one intended use. Further processing is also anticipated with the biogas conditioner.
Turning to
In another example, at least a portion of the contents within the biogas processor 42 are recycled back or directed back to at least one of the mixing tank 19, ASB tank 18, or any other process tank or environment. Recycling lines are showed as dashed lines. The contents may include at least a portion of the more purified gas form or at least one specific content that is separated from the more purified gas form. As shown, contents 53 and 54 are directed to respective mixing tank 19 and the ASB tank 18. Specific contents separated from the more purified gas form may include, for example, at least one of CH4 and CO2 or other content that is used to aid its respective tank in processing its biomass content, as shown, biomass 58 treated within the mixing tank 19 and biomass effluent 6 treated within the ASB tank 18.
In another example, CO2 is recycled to one or more of the mixing tank 19 or ASB tank 18. Gas processing requirements may be significantly reduced or eliminated by choice of the ASB bacteria and the processing conditions. As noted elsewhere, lactate in the AD tank 12 is metabolized to methane (CH4), bicarbonate (HCO3−), and carbon dioxide (CO2). Acetate is metabolized to methane and bicarbonate. Sugars are metabolized to methane and carbon dioxide. If carbon dioxide is reduced or eliminated as an AD tank product, gas processing to remove carbon dioxide can be correspondingly reduced or eliminated.
An experiment was conducted to test the modification of the bacteria and/or conditions in the ASB tank for production of only acetate instead of a mixture of acetate, lactate, and sugars. In an example, acetate is the feed, and may be the only feed, to the AD tank 12, such that modification of the AD bacteria and/or conditions enable the production of methane with little or no CO2 in the AD biogas. This eliminates gas processing to remove CO2, which is a costly process. In an example, at least one of a majority of acetate or only acetate is produced by ASB treatment with C. bescii.
In an example, the biogas is used to produce power generation. This involves combustion of the CH4. The combustion gas, which contains mostly nitrogen, but also CO2, may also advantageously be recycled to the mixing tank 19 or the ASB tank 18 to displace oxygen or air.
In another example, the biogas is processed and compressed for compressed natural gas (CNG). It may also be used as a feed stock for chemical processing, such as a Fischer-Tropsch process for production of biodiesel or other fuel.
To increase process efficiency, reduce material costs, and reduce parasitic losses of material and energy, at least one of heat recovery, recycling, and purification can be employed. In an example, recycling streams may be implemented such that at least one environment provides at least a portion of its contents to at least one other environment before, during, or after a digestion related process occurs in that environment. Turning to
An example includes that heat be recycled to heat at least one of the tanks. Also, a heat exchanger may be used to heat the contents being recycled from at least one environment to at least one other environment. For example, a heat exchanger may heat the contents from the AD tank 12 to the ASB tank 18. Solar energy may also be used to heat contents from one environment to the other environment. For example, solar energy may be used to heat the contents from the AD tank 12 to the ASB tank 18.
Heat recovery may be significant. Recycling heat back to the ASB tank 18 or mixing tank 19 may help maintain respective temperatures. With an ASB tank 18 that is thermophilic, for example, at a temperature of about 75° C. and an AD tank 12 that is non-thermophilic, for example, at a temperature of about 40° C., the heat directed to the ASB tank 18 may be significant. In addition to recycling heat to an environment, heat recovery systems may be implemented to power another environment.
In addition to recycling and heating, example reactors may include purification treatments. For example, biomass 58 or biomass effluent 6 may first go through a purification processing treatment before being treated within the ASB tank 18. Separation and purification of the treated biomass 58 from the ASB tank 18 can occur, for example, by one or more of semi-permeable membranes, centrifuge purification, distillation, filtration, industrial chromatography with zeolites, sorption, and other known mechanical and chemical means.
Also, various contents from environments may be purified for a separate process. Turning to
In an example, at least a portion of the ASB treated biomass serves as a feedstock or reagent for crude oil or fuel production or as at least one precursor for at least one synthetic process or other process. In another example, biogas methane is the portion of AD biogas that is separated and used as one or more precursors for at least one synthetic process or other process. Examples of synthetic processes include oxygenated aromatic compounds for synthesis of medicines and new materials.
Purification may occur before separation as well. In an example, at least a portion of the AD biogas from the AD tank 12 is purified in a respective processing treatment (not shown) before being separated into different contents 22a and 22b or before being used to produce a pure biogas.
In another example, contents recycled as shown in
In an example, the AD tank 12 may provide at least one nutrient to the ASB tank 18 before the ASB tank 18 treats the biomass. In another example, the AD satellite tank 14 provides at least one nutrient to the ASB tank 18 before the ASB tank 18 treats the biomass 6. In another example, the AD tank 12 mixes and heats biomass with water and provides CO2 and nutrients from the AD tank 12 to the ASB tank 18 before the ASB tank 18 treats the biomass. The contents being provided may or may not have been recycled by the environment providing them.
In one example, bicarbonate or another base from the AD tank 12 is recycled to the ASB tank 18 to maintain growth conditions. In another example, at least a portion of CO2 and nutrients are removed from the AD tank 12 and recycled to at least one of the mixing tank 19 and the ASB tank 18 to displace oxygen or air.
In another example, oxygen is removed from at least one of the AD tank 12 and ASB tank 18 and any input streams with or without recycling. In an example, CO2 and bicarbonate produced in the AD environment are recycled to the ASB environment to mitigate pH changes and reduce oxygen concentration in the ASB environment. Oxygen removal can be accomplished by flushing the AD 12 and ASB tanks 18 with CO2 from combustion or gas processing or by other removal means. Note that contents being recycled or released may be in a gas phase, liquid phase, dissolved in a solution, or in another form.
During ASB treatment in the ASB tank 18, the pH level naturally drops and becomes acidic. Bicarbonate ions that are formed in the AD tank 12 may be removed from the AD tank 12 and put back into the ASB tank 18, which neutralizes the pH level of the ASB tank 12. In this manner, matter in the AD tank 18 is recycled in the ASB tank 12. This act may make it unnecessary to buy a base to neutralize the pH level in the ASB tank 12, and is thus a cost-saving step. In another example, an ammonia scrubber (not shown) is included in the system to scrub contents that are recycled from the AD tank 12. The scrubber strips out ammonia and keeps the concentration below a toxic level.
The description and examples below are provided, followed by reference to
An example reactor for conversion of biomass into biogas comprises an ASB treatment tank containing anaerobic organisms and an AD tank that receives ASB tank effluent. The ASB tank receives biomass effluent that includes non-solubilized lignocellulosic components and treats the biomass effluent under conditions such that the anaerobic organisms reproduce and solubilize the lignocellosic components. Upon receiving the ASB tank effluent, the AD tank contains anaerobic bacteria that convert organism metabolic products of the lignocellulosic components into biogas under anaerobic digestion conditions. Outputs from the AD tank including the biogas and a slurry of undigested biomass.
An example reactor may further comprise a mixing tank where the biomass is mixed with water, heated, and components in the biomass are solubilized. The effluent of biomass suspended in water from the mixing tank is then transferred to the ASB tank.
An example reactor may further comprise at least one satellite reservoir that provides at least one of a bacteria, nutrient, and other content discussed herein, to at least one of the ASB tank and AD tank according to principles discussed herein.
An example reactor further comprises an AD tank that contains anaerobic bacteria and that receives contents comprising solubilized biomass. The anaerobic bacteria is to convert organism metabolic products of lignocellulosic components of the solubilized biomass into biogas under anaerobic digestion conditions. Outputs from the AD tank include the biogas and a slurry of undigested biomass. An AD satellite reservoir is used to supply one or more microbial species to the AD tank to support conversion of the organism metabolic products.
An example method of converting biomass into biogas includes 1) treating biomass in an ASB environment with anaerobic organisms that solubilize and metabolize lignocellulosic components of the biomass and then 2) treating the treated biomass in an AD environment with archaea and anaerobic bacteria that convert products of the lignocellulosic components in the treated biomass into biogas under anaerobic digestive conditions.
Another example method of converting biomass into biogas includes that biomass received by the ASB environment is 1) at least partially solubilized by at least one of chemical or mechanical treatment prior to the biomass being introduced into the ASB environment. Further examples include that the biomass is solubilized by at least one of chemical or mechanical treatment in the mixing tank prior to the biomass being introduced into the ASB tank.
An example method may further comprise providing conditions to produce bicarbonate in the AD environment. The bicarbonate may be recycled to the ASB environment.
An example method further comprises mixing and heating the biomass with water before treating the biomass in the ASB environment.
An example method further comprises recovering heat from one of more of the AD environment and the ASB environment.
An example method further comprises buffering the ASB environment to produce acetate and lactate as part of the solubilized components. The ASB environment may further be operated to produce predominantly acetate, with little or no carbon dioxide.
An example method further comprises favoring ASB acetate ion production over lactate ion production within the ASB environment to produce a reduced CO2 stream in the AD environment.
Large scale production of converting biomass into biogas is anticipated based on examples and principles discussed herein.
Alternatives include no power generation, such that the motors are replaced by, or used in conjunction with, electrical generators.
In an example, CO2 and bicarbonate that are produced in the AD tank are recycled back to the ASB tank.
At least a portion of the biogas may be biogas methane that is burned by an AD electrical generator. In this manner, the AD tank provides its own power to the system.
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Other reactors are anticipated that incorporate principles discussed herein.
A possible model describes ASB treatment with acetate as the major ASB treatment product.
The ASB Tank in this Model Includes the Following Example Reactions:
Cellulose(s)+H2O(I)→glucose(aq) and glucose(aq)+3OH−(aq)→3CH3COO−(aq)+3H2O(I)
d[CH3COO−]/dt=−ΔGc=−(ΔG°+RTln([CH3COO−]3/[OH—]3[glucose])[CB]
d[glucose]/dt=k4[active enzyme][substrate sites]−(⅓)d[CH3COO−]/dt
d[CB]/dt=k5[CB]→d[CB]/[CB]=k5t→ln([CB]/[CB]0)=k5(t−t0)→[CB]t=[CB]0ek5(t−t0)
d[substrate sites]/dt=a[(TSS)0−(d(TSS)t/dt)]
d[active enzyme]/dt=d[CB]/dt=(nCB0ek7t)/VASB
An example kinetic model for an ASB treated substrate may be used for the ASB effluent that enters the AD tank.
The AD Tank in this Model Includes the Following Example Reactions:
CH3COO−(aq)→CH4(g)+HCO3−(aq)
and VS→CH4(g)+CO2(g)
d[CH3COO−]Vl/dt=−d(PCH4Vg/RT)/dt=Δ8Gc=(Δ8G°+RTln(PCH4[HCO3−]/[CH3COO−]))[acetoclastic methanogens]
d[CH3COO−]/dt=−k8[CH3COO−][acetoclastic methanogens]
d[VS]=−k9[VS][oxidative methanogens]=d(2bPCO2Vg/VlRT)/dt
Some possible results are anticipated as follows—
The ASB treatment tanks in the model may be filled according to
Table B shows exemplary k value corresponding to doubling time.
Implementing the model above should maintain an exponentially growing culture and maximize output of exozymes.
To further design the treatment, the following formulas may be helpful-Gas composition from stoichiometry of carbon going into AD tank.
Cin=2xCH3COO−+3yCH3CH(OH)COO−+6zC6H12O6 (products of ASB treatment)
Cout=aCH4+bCO2+cHCO3− (products of methanogenesis)
xCH3COO−+xH2O=xCH4+xHCO3−yCH3CH(OH)COO−+yH2O=yHCO3−+1.5yCH4+0.5yCO2
zC6H12O6=3zCH4+3zCO2
If x, y and z (acetate, lactate, and glucose) are measured and total C in the filtered solution come from ASB treatment, gas composition and bicarbonate production can be predicted.
This is a discussion of chemistry and thermodynamics of anaerobic and thermophilic pre-digestion with C. bescii followed by anaerobic digestion to produce biogas from organic wastes as a renewable energy source.
ASB Digestion with C. bescii.
An example of the present process according to principles discussed herein takes place in three consecutive tanks:
Lignin+H2O→oligomers of substituted phenolics
Cellulose+H2O→glucose, C(H2O)
Polyhydroxyalkanoates+H2O→hydroxyalkanoates
These hydrolysis reactions are all exergonic, i.e. the Gibbs energy change is negative and relatively rapid at 75° C. The rate of reaction is proportionate to the concentration of enzyme and number of sites for attack on the polymeric substrate.
d[products]/dt=kcat[exozymes][polymer surface area]
For the reaction to proceed, conditions must be such that ΔG is negative. ΔH0≈0 and ΔS0 is also small, so ΔG0 is also small, particularly at low temperature. Therefore, to obtain a significantly negative ΔG requires removal of products to keep [products] small and an elevated temperature to obtain a significant rate. Use of a hyperthermophile is required to obtain commercially viable rates. (Fungi that do this at low temperature are all extremely slow growing.) Products are removed by metabolism by C. bescii and by dilution by incoming material from the mixing-hydrolysis tank.
Some of the products of hydrolysis are then metabolized by C. bescii as follows—
C(H2O)→acetic acid, CH3COOH and lactic acid, CH3CH(OH)COOH
CH3COOH+HCO3−→CH3COO—+H2O+CO2(g)
CH3CH(OH)COOH+HCO3—→CH3CH(OH)COO—+H2O+CO2(g)
Other saccharides are metabolized similarly to glucose. Hydroxyalkanoates are also metabolized by C. bescii, but the products are unknown. All of these reactions are intracellular. The first reaction is a disproportionation reaction and has near zero Gibbs energy change. The second and third reactions are acid-base reactions that produce gas and have a significant negative Gibbs energy change that powers the growth and activities of C. bescii. ΔG0=−9 KJ/mole, ΔH0 is −9 KJ/mole, and ΔS0 is ≈0 for the reaction to produce CO2(aq). The entropy of CO2(g) is 158 J/K mole, so ΔG0≈−56 KJ/mole at 25° C. for the reactions as written. ΔG0 at 80° C. is ≈−65 KJ/mole.
ΔG0 values (KJ/mole) for reaction of various bases with acetic and lactic acid are given in Table D.
Because these reactions in the sequence are the only reactions with relatively large negative ΔG values, production of CO2 gas, water, and other products from reaction of the acids produced with a base is essential for growth of C. bescii. Note that −ΔG gets numerically smaller as the concentration of acid anion increases, so the reaction may slow as these concentrations increase and there may be a practical limit for bases other than bicarbonate. ΔG is sufficiently large and negative for bicarbonate ion that this limit will not be reached in realistic systems buffered with this base.
These processes are related to the growth rate of C. bescii which is also relatively fast with doubling times of 2 to 5 days depending on the substrate (See Table A1 above).
3. The products of the reactions in the ASB tank are transferred to the anaerobic digestion (AD) tank where the reactions may be as follows—
CH3COO−+H2O→CH4(g)+HCO3−(100% CH4)
2CH3CH(OH)COO−+2H2O→3CH4(g)+2HCO3—+CO2(g)(75% CH4)
2C(H2O)→CH4(g)+CO2(g)(50% CH4)
The methane may thus be increased. The methane content of the biogas produced from anaerobic digestion of each of these substrates is given in parentheses. When catalyzed by acetoclastic methanogens, these reactions are relatively fast with half times of 2 to 5 days depending on the concentrations of substrates. The oligomers of substituted phenolics are probably not metabolized by acetoclastic methanogens, but are partially metabolized by oxidative methanogenesis to produce biogas with about 60% methane. The chart in
The bicarbonate produced in these reactions maintains the pH in the AD tank at slightly basic levels, so no further pH control is necessary. Note that bicarbonate ion is not volatile and will not contribute a significant amount of CO2 to the biogas as long as the AD pH is above neutral. The amount of bicarbonate produced by AD is the same as the amount of bicarbonate consumed by the reactions in the ASB tank. Therefore, bicarbonate from the AD can be recycled to supply most or all of the bicarbonate needed in the ASB reactions.
Note that there are at least two processes in the ASB Tank, namely, hydrolysis of the feedstock and metabolism of the products of hydrolysis. The rates of these two processes have differing dependencies on variables 1 through 5. The composition of the effluent that is fed to the AD tank thus has a multivariate dependence on all of the above variables. The composition of the effluent from ASB tank determines the composition of the biogas produced by anaerobic digestion and the optimum conditions for anaerobic digestion operation.
Governing equations for ASB
Measurement as f(time)
d[n]/dt=kn[n]
Rate of metabolism d[Pm]/dt=−km[n][Sm]
Rate of hydrolysis d[Ph]/dt=−kh[E][Sh]
The ks are all rate constants with units determined by variables in the equation.
Solution to these differential equations will depend on feedstock, but in general there are two maxima, one that maximizes [Pm] and one that maximizes [Ph]. Short ASB retention times (around 12-20 hours) maximizes [Pm] and longer ASB retention times (around 48+ hours) maximizes [Ph]. The output from AD depends on which you maximize. Maximizing [Pm] maximizes the methane content in biogas and maximizing [Ph] maximizes total biogas and waste destruction.
These equations are based on an assumption that all of the volatile solids can be hydrolyzed and solubilized. That can easily be fixed if necessary by adding a constant multiplier on Sh.
The output from the ASB is the input to AD, which has two components:
Products of C. bescii metabolism, i.e. acetate and lactate, at concentration [Pm].
Products of hydrolysis from enzymatic action, i.e. saccharides, polyphenols, hydroxyalkanoates, etc. with a total concentration of [Ph].
These equations are based on an assumption that all volatile solids are digested.
Recycle to recover bicarbonate has not been included, but could be by adding more terms to the equations.
Relative tank sizes determine retention time in a continuous flow system
is the time required to obtain a set percentage of potential biogas production.
The invention has been described with reference to various specific and preferred embodiments and techniques. Nevertheless, it is understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
This additional disclosure, in conjunction with the foregoing paragraphs of this application, relates to an advanced thermophilic synthetic microbial community specifically optimized for the simultaneous and efficient degradation of: (1) lignocellulosic materials, (2) fats, oils, and grease (FOG), and (3) waste activated sludge (WAS). This community functions within an Anaerobic Secretome Bioreactor (ASB) environment, encompassing ASB configurations disclosed in this application. Operating under high-temperature anaerobic conditions (e.g., 65° C. to 85° C.) and at moderately alkaline pH levels between 6.5 to 8.0, this microbial community provides significant improvements over conventional anaerobic processing methods, particularly in the efficient breakdown of complex and diverse organic substrates with enhanced efficiency.
The following terms are defined as they apply specifically to this additional disclosure and continuing application. These definitions may differ from their conventional meanings.
“Endozymes” (also known as endoenzymes or intracellular enzymes) typically refer to the enzymes that function within the cell of microorganisms, such as bacteria and fungi. These enzymes catalyze internal metabolic reactions, converting substrates into energy, and building cellular components for the organism's growth, maintenance, and overall function. This contrasts with exozymes, which are either secreted into the surrounding environment or bound to the external cell membrane, where they operate outside the microbial cell to break down complex organic materials. Endozymes also catalyze the production of metabolic byproducts, which can serve as substrates for other microbial communities.
“Exozymes” (also known as exoenzymes or extracellular enzymes) typically refers to enzymes that are either secreted by microorganisms, such as bacteria and fungi, into their surrounding environment or bound to the outer cell membrane, where they catalyze the breakdown of complex organic materials outside the microbial cell. This contrasts with endozymes, which function inside the cell to catalyze metabolic reactions within the organism itself.
“Extracellular enzyme system” refers to the collection of enzymes that are synthesized by microorganisms, such as bacteria and fungi, and either secreted outside of the cell or anchored to the outer cell membrane. These enzymes function externally to catalyze the breakdown of complex organic compounds into simpler, more accessible substrates, producing intermediate products. In the context of anaerobic digestion, these breakdown products are essential substrates for other microbial communities, facilitating further conversion into biogas and enhancing overall system efficiency.
“Feedstock” typically refers to the raw organic materials introduced into a digestion system to be broken down by microorganisms during the digestion process. Feedstocks provide the source of carbon and nutrients necessary for microbial activity, ultimately leading to the production of biogas and digestate.
“FOG” typically refers to Fats, Oils, and Grease, which are organic compounds commonly present in wastewater, food waste, and industrial byproducts. FOG is primarily composed of glycerides, free fatty acids, and glycerol. In anaerobic systems, FOG can create operational challenges, such as scum formation, clogging of equipment, and inhibition of microbial processes, particularly due to the accumulation of long-chain fatty acids (LCFAs). Despite these challenges, FOG is an energy-dense material and, when properly managed, can significantly enhance biogas production.
“Intracellular enzyme system” refers to the collection of enzymes that are synthesized and remain within the cell of microorganisms, such as bacteria and fungi. These enzymes function internally to catalyze metabolic processes that convert substrates into Gibbs energy and cellular components necessary for growth and maintenance. As a result of these metabolic processes, waste products such as carbon dioxide, ammonia, organic acids, and hydrogen gas are produced. In the context of anaerobic digestion, these waste products are valuable intermediates, as they serve as substrates for other microbial communities, enhancing biogas production and overall digestion efficiency.
“Lignocellulosic materials” and “LM” typically refer to plant-based biomass composed primarily of three structural polymers: cellulose, hemicellulose, and lignin. These materials are commonly found in agricultural residues (e.g., straw, corn stover), forestry byproducts (e.g., wood chips, sawdust), and energy crops (e.g., switchgrass), and represent a significant portion of organic waste streams.
“Mesophilic” usually refers to processes or microorganisms that operate optimally at moderate temperatures, typically between 30° C. to 40° C. (86° F. to 104° F.). However, in the context of this invention, “mesophilic” refers to temperatures ranging from about 20° C. and 55° C. (68° F. to 131° F.), essentially spanning the transition zone between conventional psychrotrophic and thermophilic conditions.
“Methanogenic digestion” typically refers to the final step in the process of anaerobic digestion, where methane-producing microorganisms (methanogens) convert intermediate byproducts into methane (CH4) and carbon dioxide (CO2), resulting in biogas production. This stage relies on the preceding stages of anaerobic digestion: hydrolysis, acidogenesis, and acetogenesis. Methanogenic digestion completes the anaerobic process, transforming the intermediates from earlier stages into methane-rich biogas.
“Scum” typically refers to a layer of solid or semi-solid material that floats on the surface of a digester due to the accumulation of substances like fats, oils, grease (FOG), fibrous materials, or gas bubbles trapped in organic matter.
“Secretome of exozymes” typically refers to the collection of enzymes synthesized and either secreted into the extracellular environment or bound to the outer cell membranes of microbes, such as bacteria and fungi, within a microbial community. Such secretomes typically include various exozymes that catalyze the breakdown of complex organic substrates. A secretome of exozymes facilitates the extracellular deconstruction of such substrates into intermediate products that can be readily absorbed by individual microbes or further metabolized within the microbial community.
“Sludge” typically refers to the semi-solid byproduct that results from the treatment of wastewater, industrial effluents, or other organic materials. Sludge is primarily composed of water, organic matter, microorganisms, and various inorganic particles.
“Substrate” typically refers to organic material that serves as a reactant in metabolic processes carried out by individual microorganisms or within a microbial community at any stage of a digestion process, anaerobic or otherwise. Substrates may include feedstocks, partially digested materials, and effluents from other processing stages. These substrates provide the nutrients and energy required for microorganisms to break down complex organic matter into simpler compounds, supporting both extracellular reactions in a digester environment and enzymatic reactions within cells.
“Synthetic” in the context of microbial communities typically refers to a deliberately designed and engineered community of microorganisms that collaborate to optimize the breakdown of complex organic materials, particularly under anaerobic conditions. Unlike natural microbial communities, which evolve without human intervention, synthetic microbial communities are custom-tailored to achieve targeted outcomes, such as enhancing the efficiency of organic waste degradation, maximizing biogas production, and/or mitigating process inhibitors.
“Thermophilic” usually refers to processes or microorganisms that operate optimally at high temperatures, typically between 50° C. and 70° C. (122° F. to 158° F.). However, in the context of this invention, “thermophilic” refers to higher temperatures, specifically ranging from about 65° C. to 85° C. (149° F. to 185° F.), essentially spanning the transition zone between conventional thermophilic and hyperthermophilic conditions.
“WAS” typically refers to Waste Activated Sludge, which is the biomass consisting of microbial cells and organic matter removed from the aeration stage of wastewater treatment plants. WAS is a byproduct of the activated sludge process, where microorganisms metabolize organic pollutants in wastewater. Once the biological treatment is complete, the excess microbial biomass, or WAS, is separated from the treated water. For the purposes of this invention, the term “WAS” encompasses not only conventional Waste Activated Sludge but also primary sludge, mixtures of primary sludge and WAS, and sludge resulting from the anaerobic digestion of primary sludge.
Enhanced Degradation of Diverse Feedstocks. Unlike traditional anaerobic digestion, which often struggles with feedstock heterogeneity, the disclosed microbial community is engineered to degrade a wide range of organic materials—including lignocellulosic biomass (e.g., agricultural residues, wood), lipid-rich substrates like FOG, and nutrient-dense WAS. The community's multi-functional enzymatic capacity facilitates efficient breakdown of these diverse substrates within a single system, potentially reducing the need for separate treatment streams. Thermophilic operation further enhances the breakdown of polymeric materials which enhances methane yields in subsequent methanogenic digestion.
Thermophilic Efficiency. Operating at thermophilic temperatures offers several advantages. First, elevated temperatures accelerate enzymatic hydrolysis, enabling more rapid breakdown of recalcitrant substrates such as lignin and cellulose. Additionally, thermophilic conditions facilitate pathogen elimination in WAS and other feedstocks, improving the safety of the resulting digestate. This faster processing can support smaller facility designs, thereby reducing capital costs.
Simultaneous Degradation of Complex Substrates. The microbial community functions synergistically to process diverse substrates simultaneously. Lipase-producing microbes break down FOG into glycerol and various fatty acids, mitigating scum formation and reactor clogging. Cellulase, hemicellulase, and ligninase-producing microbes degrade lignocellulosic materials into metabolizable sugars, leaving behind resistant lignin residues. Additionally, proteolytic microorganisms target proteins and carbohydrates in WAS, supporting efficient processing of mixed organic waste streams in a single-stage system, which creates optimal conditions for subsequent anaerobic digestion stages.
Reduction of Process Inhibition. This technology mitigates common inhibitors, such as long-chain fatty acids (LCFAs) and ammonia, to enhance anaerobic digestion efficiency. Lipase-producing microbes rapidly hydrolyze mono-, di-, and triglycerides, reducing lipid accumulation that can cause scum formation. This hydrolysis facilitates the microbial breakdown of LCFAs, which become more soluble in their ionized form (LCFAAs) under alkaline conditions. Furthermore, the co-digestion of lignocellulosic materials with WAS improves the carbon-to-nitrogen (C/N) ratio, mitigating ammonia toxicity and fostering stable methanogenesis.
Improved Process Stability and Flexibility. The microbial community is robust and adaptable to fluctuations in feedstock composition, supporting stable performance in industrial-scale operations. The thermophilic ASB environment promotes rapid microbial growth, enabling quick adaptation to varying levels of lignocellulosic materials, FOG, and WAS. This flexibility allows the system to process a broad range of organic inputs with minimal adjustments.
Single-Step, High-Efficiency Processing. The thermophilic ASB allows for single-step processing, eliminating the need for energy-intensive pre-treatments (e.g., chemical hydrolysis or mechanical disruption) commonly required in conventional anaerobic digestion systems. This streamlined approach reduces system energy consumption, making the process more sustainable and cost-effective.
Pasteurization for Pathogen Reduction and Enhanced Next-Stage Digestion. Operating at thermophilic temperatures provides a pasteurization effect, eliminating harmful bacteria, viruses, parasites, and other pathogens in WAS and other organic materials. This enhances the safety of the digestate for agricultural and other uses. Additionally, pre-digestion pasteurization partially degrades complex organic structures, making the remaining content more readily available for microbial breakdown in the next anaerobic digestion stage. This leads to faster access to nutrients for microbes, improving digestion efficiency and higher biogas yields, as more easily digestible substrates become available for methane conversion.
Post-digestion Treatment with Recycle for Enhanced Digestion. Effluent from an earlier stage of anaerobic digestion may be processed in a thermophilic ASB, where microbial activity promotes the further degradation of residual organic material. A portion of the ASB-treated effluent may be recycled back to the earlier digestion stage, reducing energy and water consumption, diluting concentrated feedstock streams, and enhancing overall process stability and efficiency.
The disclosed synthetic microbial community presents a uniquely integrated solution for the simultaneous degradation of complex organic substrates that are traditionally difficult to process in conventional systems. This innovative approach offers several key features that set it apart from standard methods.
First, synthetic microbial selection, as described earlier in paragraph [00149], allows for the inclusion of thermophilic microbes that produce a broad range of exozymes—such as lipases, cellulases, hemicellulases, ligninases, and proteases—which are specifically targeted to break down LM, FOG, and WAS. This diversity of exozymes ensures efficient breakdown of various complex substrates, improving overall system performance.
Next, thermophilic conditions foster the rapid and thorough conversion of diverse organic materials into readily digestible precursors for biogas production in a subsequent methanogenesis stage. This rapid adaptability enables the microbial community to efficiently process a wide range of feedstocks, including those typically resistant to degradation, thereby increasing operational efficiency and simplifying the process.
Finally, simultaneous degradation is achieved by processing multiple substrate types in parallel within a single anaerobic secretome bioreactor (ASB). This eliminates the need for multi-stage systems or complex pre-treatment methods, which are commonly required in conventional anaerobic digestion. The result is a more streamlined and efficient process, capable of handling varied feedstocks in a single-step system.
In one example of a thermophilic ASB environment, thermophilic microorganisms are selected for their ability to thrive under high-temperature anaerobic conditions, specifically at temperatures between approximately 65° C. and 85° C. These microorganisms play a crucial role in the breakdown and solubilization of lignocellulosic materials (including cellulose, hemicellulose, and lignin) into simpler compounds that are accessible for further anaerobic digestion.
The ASB environment employs a synthetic microbial community comprising thermophilic microorganisms that primarily function as acidogens and acetogens, thriving under non-methanogenic conditions. These microorganisms are selected for their complementary roles in hydrolyzing complex polymers, fermenting sugars, and producing intermediate metabolites such as volatile fatty acids (VFAs), acetate, and hydrogen. The community is designed to support synergistic interactions, ensuring the efficient breakdown of lignocellulosic materials (LM) while maintaining long-term system stability. Each microorganism contributes unique extracellular enzymes (exozymes) or metabolic pathways essential for the degradation of cellulose, hemicellulose, lignin, and any lipid components present in mixed feedstocks. The following thermophilic organisms have been identified as suitable for LM degradation in the ASB based on their lignocellulosic-degrading capabilities and cooperative potential:
1. Caldicellulosiruptor spp.: Known for producing ligninases, cellulases and hemicellulases that degrade cellulose and hemicellulose efficiently at high temperatures, Caldicellulosiruptor bescii is particularly effective at solubilizing up to about 90% of lignocellulose under thermophilic anaerobic conditions.
2. Clostridium thermocellum: A highly efficient cellulose-degrading bacterium that produces a range of cellulolytic exozymes, making it a strong candidate for inclusion in the microbial community for the breakdown of lignocellulosic biomass.
3. Thermoanaerobacterium saccharolyticum: This bacterium is adept at producing cellulases and hemicellulases, facilitating the breakdown of both cellulose and hemicellulose components of lignocellulosic material in high-temperature environments.
4. Thermoclostridium stercorarium: A thermophilic anaerobe capable of degrading both cellulose and hemicellulose, producing fermentable sugars and volatile fatty acids. This bacterium complements other cellulolytic microbes, enhancing the overall efficiency of lignocellulose hydrolysis.
5. Anaerobacillus thermoterrificus: A thermophilic bacterium that produces extracellular lipases, enabling the hydrolysis of glycerides into glycerol and long-chain fatty acids. It plays a critical role in managing lipid components of mixed feedstocks under anaerobic conditions.
6. Syntrophomonas wolfei: A strict anaerobe specializing in the breakdown of LCFAs into acetate and hydrogen. This bacterium forms syntrophic associations with hydrogen-consuming microbes, preventing LCFA accumulation and supporting system stability.
7. Thermoanaerobacter ethanolicus: Known for its ability to ferment sugars into ethanol, acetate, and hydrogen under thermophilic anaerobic conditions. This bacterium contributes to the production of intermediate metabolites essential for downstream methanogenesis.
8. Caloramator fervidus: A thermophilic anaerobe that ferments carbohydrates into lactate and hydrogen. Its acidogenic activity supports the fermentation of lignocellulosic hydrolysis products, enhancing intermediate metabolite production.
10. Thermotoga maritima: A thermophilic anaerobe that thrives at temperatures between 80° C. and 90° C. This bacterium ferments sugars derived from lignocellulose hydrolysis into acetate, hydrogen, and carbon dioxide, producing methane-ready intermediates. Its robust metabolic pathways and thermostable enzymes make it an excellent complement to cellulolytic bacteria in the ASB.
In one example, the selected LM-targeting microorganisms secrete a secretome rich in cellulases, hemicellulases, ligninases, esterases, and pectinases. These exozymes work synergistically to hydrolyze plant cell wall components, breaking down cellulose, hemicellulose, lignin, and cross-linked structures into simpler, more accessible substrates, including soluble sugars, aromatic compounds, and organic acids in their anionic forms. Esterases and pectinases specifically target ester-linked and pectin components, increasing access to cellulose and hemicellulose and facilitating a more complete solubilization of the LM before it enters the methanogenic digester (MD) environment.
Additionally, a portion of the synthetic microbial community metabolizes the soluble sugars and other hydrolysis products, further enriching the liquid effluent with soluble anions of organic acids.
In another example, the ASB environment employs a synthetic microbial community optimized for the breakdown of complex lipid structures found in fats, oils, and grease (FOG). These thermophilic microorganisms, thriving at high temperatures between 65° C. and 85° C. under strict anaerobic conditions, secrete lipolytic exozymes that hydrolyze glycerides and other lipid compounds into glycerol and LCFAs. VFA-producing bacteria ferment glycerol into VFAs, which remain in their anionic form due to the ASB's moderately alkaline environment. Together, these processes enable efficient conversion of hydrolysis products into methane-ready precursors such as VFAs, acetate, and hydrogen. The following thermophilic organisms have been identified as suitable for FOG degradation in the ASB based on their robust enzymatic activity and cooperative potential:
1. Anaerobacillus thermoterrificus: This thermophilic bacterium produces extracellular lipases that effectively break down lipids at temperatures of about 65° C. and 85° C., converting them into fatty acids and glycerol, which can then be efficiently metabolized by anaerobic microorganisms in subsequent digestion stages.
2. Clostridium spp.: A thermophilic bacterium that converts glycerol and other hydrolysis products into VFAs (such as acetic acid and butyric acid), which are crucial intermediates for biogas production.
3. Syntrophomonas wolfei: A strict anaerobic bacterium specializing in the breakdown of long-chain fatty acids (LCFAs) into acetate and hydrogen under thermophilic conditions. This organism forms syntrophic associations with hydrogen-consuming microbes, preventing LCFA accumulation and ensuring process stability in lipid-rich environments.
4. Thermoanaerobacter ethanolicus: A thermophilic anaerobe that ferments glycerol and sugars into acetate, ethanol, and hydrogen. This microorganism is highly efficient at producing methane-ready intermediates, making it a useful component for FOG degradation in anaerobic systems.
6. Thermotoga maritima: A thermophilic anaerobe thriving at temperatures of about 80° C. to 90° C. This microorganism ferments glycerol and sugars into acetate, hydrogen, and carbon dioxide, complementing other fermenters and enhancing the production of methane-ready intermediates in lipid degradation systems.
In one example, the selected thermophilic microorganisms targeting FOG secrete a secretome rich in lipases, phospholipases, esterases, and amylases. These exozymes work synergistically to hydrolyze a substantial portion of FOG, including glycerides, phospholipids, and ester-linked compounds, enriching the liquid effluent with free fatty acid anions (FFAAs), including long-chain fatty acid anions (LCFAAs), and glycerol—key intermediates for methanogenic digestion. Phospholipases specifically target phospholipids within FOG, further breaking down complex lipid structures, while esterases hydrolyze ester bonds across various lipid compounds, enhancing lipid accessibility for further metabolism. Amylases contribute by breaking down carbohydrate contaminants in FOG, ensuring thorough degradation of mixed waste components.
The LCFAAs are subsequently degraded in a downstream methanogenic digestion stage for biogas production or directed into other metabolic pathways. Concurrently, glycerol metabolization by the microbial community in the ASB generates SCFAAs, VFAAs, and other anionic fatty acid intermediates. These processes collectively optimize the substrate profile for subsequent methanogenic digestion, enhancing biogas yield and process efficiency.
In another example, the ASB environment employs a synthetic microbial community optimized for the efficient degradation of waste activated sludge (WAS). These thermophilic microorganisms, thriving at high temperatures between about 65° C. and 85° C. under strict anaerobic conditions, are selected for their ability to secrete proteolytic, lipolytic, and polysaccharide-degrading enzymes, enabling the breakdown of complex organic materials such as proteins, lipids, and polysaccharides from microbial cell walls and other organic matter. The thermophilic conditions of the ASB not only enhance enzymatic activity but also eliminate pathogens and mesophilic bacteria, ensuring efficient degradation and preparing the remaining substrates for downstream processing. The following thermophilic microorganisms have been identified as suitable for efficient WAS degradation in the ASB:
1. Thermoanaerobacterium thermosaccharolyticum: Known for its ability to produce exozymes that break down complex carbohydrates and proteins, this bacterium thrives at thermophilic anaerobic conditions and contributes to the degradation of organic material present in WAS.
2. Anaerobacillus thermoterrificus: This thermophilic bacterium produces exozymes, including proteases and lipases, which are essential for breaking down proteins and lipids in WAS in thermophilic anaerobic conditions.
3. Caldicellulosiruptor spp.: These thermophilic microorganisms are adept at degrading cellulose, hemicellulose, and other polysaccharides present in WAS. By producing a wide array of polysaccharide-degrading exozymes, Caldicellulosiruptor spp. complement other hydrolyzers in breaking down complex carbohydrates under thermophilic anaerobic conditions.
4. Thermoanaerobacter spp.: These bacteria exhibit robust metabolic activity in thermophilic anaerobic conditions, efficiently fermenting sugars, amino acids, and fatty acids into VFAs, acetate, and hydrogen. Their versatility enhances the production of methane-ready intermediates from WAS.
5. Clostridium spp.: A thermophilic bacterium that ferments glycerol, amino acids, and sugars into VFAs such as acetic acid and butyric acid, which are critical intermediates for downstream methane production.
6. Thermoanaerobacter ethanolicus: Known for its ability to ferment glycerol and sugars into ethanol, acetate, and hydrogen, this thermophilic anaerobe enhances the diversity of VFAs and hydrogen in the ASB, supporting efficient downstream methanogenesis.
7. Syntrophomonas wolfei: A strict anaerobe specializing in the degradation of LCFAs into acetate and hydrogen through syntrophic relationships. This bacterium prevents LCFA accumulation, which can inhibit anaerobic processes, and ensures system stability.
9. Thermotoga maritima: A highly thermophilic anaerobe that ferments sugars into acetate, hydrogen, and carbon dioxide, complementing other fermenters in producing methane-ready intermediates, enhancing the efficiency of WAS degradation.
In one example, the selected WAS-targeting microorganisms secrete a diverse secretome of hydrolytic exozymes, including proteases, lipases, chitinases, nucleases, and polysaccharide-degrading enzymes, which work synergistically to break down the complex organic matter in waste activated sludge (WAS). Proteases degrade proteins into soluble peptides and amino acids, which can then be metabolized by the synthetic microbial community. Lipases hydrolyze fats and oils into glycerol and FFAAs, including LCFAAs. Polysaccharide-degrading enzymes target carbohydrates in WAS, hydrolyzing and solubilizing them to yield an effluent enriched with soluble sugars and anions of organic acids. Additionally, chitinases break down chitin in fungal cell walls, while nucleases degrade nucleic acids, contributing nucleotides and nitrogenous bases to the effluent, which further supports microbial metabolism.
Complex carbohydrates are partially hydrolyzed into oligosaccharides and anions of uronic acids, which the synthetic microbial community further processes into soluble sugars and organic acids. A significant portion of the ammonia released during degradation is assimilated by the microbial community, enriching the effluent with soluble nitrogen compounds essential for microbial growth. Glycerol metabolization by the microbial community generates additional VFAAs, further optimizing the effluent for downstream methane production.
In this configuration, the anaerobic secretome bioreactor (ASB) effectively solubilizes WAS, producing an effluent rich in digestible intermediates. This effluent is primed for enhanced biogas production in a subsequent methanogenic digestion stage, optimizing the conversion of WAS into valuable energy sources.
To ensure optimal microbial activity for the simultaneous degradation of lignocellulosic materials (LM), fats, oils, and grease (FOG), and waste activated sludge (WAS), the ASB environment must be carefully controlled. Key environmental factors that support the efficiency and stability of the synthetic microbial community include temperature, pH, and anaerobic conditions.
The ASB is maintained at thermophilic temperatures, typically between 65° C. and 85° C., to promote the metabolic activity of thermophilic microorganisms selected for degrading lignocellulosic materials (LM), fats, oils, and grease (FOG), and/or waste activated sludge (WAS). These elevated temperatures accelerate the enzymatic hydrolysis of complex substrates and also serve as a pasteurization step, eliminating non-thermophilic microorganisms and pathogens to ensure a safer digestate for downstream processes.
pH is another critical factor, and the ASB environment is typically regulated between 6.5 and 8, providing an optimal range for enzyme stability and microbial metabolism. Maintaining this pH range ensures that hydrolytic exozymes—including cellulases, hemicellulases, ligninases, lipases, proteases, and other polysaccharide-degrading enzymes—function effectively, promoting efficient degradation of diverse substrates. In one example, the pH is regulated by an ammonium bicarbonate buffer.
The specified pH range of 6.5 to 8 is particularly advantageous for managing the organic acids produced during substrate breakdown, including VFAs, FFAs, SCFAs, and LCFAs. Within this range, these acids remain in their anionic forms. VFAs, in particular, are ideal substrates for methanogenesis in downstream methanogenic digestion. Altogether, maintaining this pH promotes efficient substrate breakdown and ensures compatibility with downstream biogas production processes.
For clarity, the various anions produced under these pH conditions will be referred to by the following acronyms: VFAAs for volatile fatty acid anions, FFAAs for free fatty acid anions, SCFAAs for short-chain fatty acid anions, and LCFAAs for long-chain fatty acid anions. Specific organic acid anions, such as acetate and lactate ions, will be referred to by their respective names, e.g., acetate and lactate.
By carefully controlling these environmental factors—such as maintaining anaerobic conditions, thermophilic temperatures, and a stable pH—the ASB creates an optimal environment for the synthetic microbial community to efficiently degrade LM, FOG, and WAS. These optimized conditions promote high substrate breakdown rates, enhance intermediate metabolite production, and improve overall system efficiency, ultimately maximizing biogas yield
Anaerobic conditions within the ASB tend to be self-sustaining, primarily due to the thermophilic conditions which limit oxygen solubility, the metabolic characteristics of the microbial community and the high biochemical oxygen demand (BOD) of introduced feedstocks. The microbial community—particularly facultative anaerobes—along with readily oxidizable organic compounds, rapidly consumes residual oxygen upon feedstock introduction, quickly establishing anaerobic conditions. This rapid oxygen depletion favors anaerobic organisms and suppresses aerobic competitors. The high BOD of the organic-rich feedstock further accelerates oxygen depletion, supporting both the rapid establishment and long-term maintenance of anaerobic conditions. These biological and chemical interactions naturally sustain anaerobic conditions, reducing the need for external controls and allowing the microbial community to thrive in a stable, optimized environment that prepares substrates for biogas production in a subsequent methanogenic stage. Additionally, the presence of ammonium and bicarbonate ions in most feedstocks (e.g., WAS) provides a pH buffer, helping to maintain pH at moderately alkaline levels.
For LM substrates, maintaining a pH range of 6.5 to 8.0 in the ASB environment offers several critical advantages for efficient degradation. This range optimizes the activity of key cellulolytic and hemicellulolytic enzymes, including cellulases, hemicellulases, and ligninases, which break down the complex polymer structures of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are hydrolyzed and metabolized into simpler sugars and organic acids that are readily converted into biogas in subsequent methanogenic digestion. By sustaining this controlled pH range, the ASB ensures effective hydrolysis of LM components, accelerating their conversion into substrates that the microbial community can utilize efficiently. Furthermore, this pH range promotes the stability of organic acids, particularly VFAs, in their anionic forms, minimizing acidification within the reactor. This stabilization not only prevents pH fluctuations but also supports continuous enzymatic activity and overall system stability.
The pH range promotes a stable environment that maintains the breakdown products from LM, especially VFAs generated through microbial sugar metabolism, in their anionic forms. This configuration creates optimal conditions for methanogenesis in downstream processes. Methanogenic microbes can efficiently convert these anionic VFAs into methane, CO2, and bicarbonate, maximizing energy yield from lignocellulosic materials and enhancing the methane content of the resulting biogas.
Overall, by maintaining the ASB pH between 6.5 and 8.0, the system achieves effective LM degradation through enhanced enzymatic activity and improved compatibility with biogas production pathways. This carefully regulated pH range enables stable and efficient processing of lignocellulosic materials, contributing to the overall productivity and energy efficiency of the ASB system.
Regarding FOG substrates, the pH range of 6.5 to 8.0 offers several distinct advantages. First, it optimizes the activity of lipolytic enzymes, such as lipases and esterases, which break down the glycerides in FOG—triglycerides, diglycerides, and monoglycerides—into glycerol and LCFAAs, making them ideal substrates for methanogenesis in downstream digestion. Methanogenic microbes can efficiently convert these LCFAAs into biogas, enhancing methane production and optimizing energy yield.
Altogether, maintaining the ASB PH between 6.5 and 8.0 enables efficient FOG degradation, minimizes potential inhibitory effects by converting LCFAs into LCFAAs, and maximizes the biogas production potential of the ASB system.
Regarding WAS as a substrate, the pH range of 6.5 to 8.0 offers several important benefits. First, it supports the optimal activity of proteolytic and polysaccharide-degrading enzymes, such as proteases and chitinases, which break down complex proteins, polysaccharides, and other organic components in WAS into simpler, soluble forms. Maintaining this pH range enhances the efficiency of hydrolytic reactions within the ASB, facilitating the conversion of WAS components into accessible substrates for further microbial metabolism.
Additionally, the pH range of 6.5 to 8.0 helps ensure that the breakdown products from WAS, such as amino acids, peptides, and short-chain fatty acids (SCFAs), are maintained in their anionic forms. These products become suitable intermediates for downstream methanogenic processes-methanogenic microbes can efficiently convert these intermediates into biogas, increasing methane yield and optimizing the energy production potential of the system.
Maintaining the ASB pH between 6.5 and 8.0 facilitates efficient WAS degradation by optimizing enzymatic activity and ensuring compatibility with downstream biogas production processes. This controlled pH range enhances process stability, supports continuous microbial activity, and maximizes the ASB's efficiency in producing intermediates critical for downstream methanogenesis, ultimately improving biogas yield.
The pH range of 6.5 to 8.0 in the ASB is particularly beneficial for setting up substrate conditions that promote acetoclastic methanogenesis in a downstream methanogenic digestion (AD) stage. In this downstream stage, acetate produced in the ASB is converted directly into methane and bicarbonate by acetoclastic methanogens. The ASB's controlled pH range ensures that organic acids like acetate remain in their anionic forms, making them ideal substrates for methanogens in the MD stage. By maintaining these acids in their anionic forms, the ASB fosters stability, ensuring substrates are well-suited for efficient conversion in the subsequent MD stage without acidifying the environment or disrupting microbial activity in either system.
Additionally, the bicarbonate generated in the downstream MD stage can be recycled back into the ASB as a natural buffer, helping to maintain the pH within the ideal range of 6.5 to 8.0. This bicarbonate recycling supports enzyme activity in the ASB, enabling the efficient breakdown of LM, FOG, WAS, and other substrates. By sustaining a stable microbial environment within the ASB, the bicarbonate buffer reduces the need for external pH adjustments, supporting consistent microbial activity and stabilizing the digestion process. Bicarbonate recycling supports a balanced environment, minimizing operational costs and reducing dependence on added chemical buffers.
Moreover, because bicarbonate ions are non-volatile, the formation of bicarbonate instead of CO2 during acetoclastic methanogenesis at a pH of 6.5 to 8.0 reduces CO2 concentration and increases methane concentration in the biogas. This shift improves the efficiency of CO2 removal from the biogas.
Altogether, maintaining the ASB pH within the range of 6.5 to 8.0 optimizes substrate conditions for acetoclastic methanogenesis in the downstream MD stage, enables efficient bicarbonate buffering, and promotes sustainable carbon management within the ASB system.
The selected pH range of 6.5 to 8.0 in the ASB provides significant advantages for managing and reducing ammonia levels. Within this range, ammonia (NH3) predominantly exists as ammonium ions (NH4+), reducing the potential for ammonia toxicity. Maintaining a pH closer to the lower end of this range, around 6.5, further limits the presence of free ammonia, creating a more stable and supportive environment for substrate breakdown. This pH-controlled approach to reducing free ammonia allows the ASB to more effectively process high-protein substrates, such as waste-activated sludge (WAS) and manure, optimizing conditions for enzyme activity while minimizing ammonia-related disruptions. Altogether, these pH benefits enhance the ASB's overall efficiency and operational stability.
In the ASB, selecting microorganisms that predominantly convert complex organic substrates, such as cellulose, into acetate with minimal residual sugars significantly influences the composition of the resulting biogas. When acetate is the primary organic acid in the liquid effluent, it supports methanogenic pathways that efficiently generate methane (CH4), with nonvolatile bicarbonate ions contributing to pH stability. Acetoclastic methanogens, such as Methanosaeta spp. and Methanosarcina spp., are particularly effective at converting acetate into methane with limited carbon dioxide (CO2) production, unlike hydrogenotrophic methanogens, such as Methanobacterium spp. and Methanobrevibacter spp., which typically yield higher CO2 levels in biogas due to their reliance on hydrogen and CO2 produced from VFAAs for methane production.
The reduction of residual sugars in the liquid effluent from the ASB further supports this efficiency. Residual sugars can be metabolized into a variety of VFAs, which, in downstream processes, result in both methane and CO2. By selecting microorganisms that convert sugars almost entirely to acetate, the anaerobic digestion system limits CO2 levels in the biogas. The presence of acetate in its anionic form, maintained by the pH range of 6.5 to 8.0, enhances its stability and suitability as a substrate for methanogenic microbes, supporting methane-rich biogas production with minimal CO2.
Furthermore, the combined processes of hydrolyzing, solubilizing, and metabolizing LM ensure that complex polymers are thoroughly broken down into simpler compounds, yielding an acetate-rich effluent. This comprehensive substrate breakdown minimizes partially metabolized intermediates that might otherwise contribute to CO2 formation. By tailoring the microbial community to maximize acetate production and limit residual sugars, the process enables downstream production of biogas with a high methane concentration, ideally suited for energy production.
To produce biogas that is rich in methane and substantially low in carbon dioxide, the method promotes a microbial community configured to primarily generate acetate, with minimal residual sugars and other non-acetate VFAs. By structuring the microbial community and enzymatic pathways to yield a liquid effluent where acetate comprises at least 80-95% of the total soluble organic acids, the process maximizes acetate-to-methane conversion efficiency through acetoclastic methanogenesis. This pathway, which converts acetate directly into methane and bicarbonate ion, inherently limits CO2 production by avoiding metabolic byproducts that typically increase CO2, thereby enhancing the methane concentration of the resulting biogas.
Additionally, by maintaining residual sugar levels below 5% of the total dissolved organic carbon (DOC) in the ASB effluent, the method minimizes alternate pathways that could contribute to carbon dioxide formation in the MD. The resulting high-methane, low-CO2 biogas is thus optimized for energy density and requires minimal post-treatment, making it suitable as a high-quality renewable fuel.
Carbon Dioxide Content: The phrase “substantially low in carbon dioxide” is defined here as biogas containing less than 40% carbon dioxide by volume. The described metabolic processes, particularly the acetoclastic methanogenic pathway, are designed to minimize CO2 output, ideally achieving CO2 concentrations as low as 5%. By reducing alternate metabolic pathways that produce CO2, the process yields low-CO2 biogas suitable for direct use or requiring minimal purification.
In the ASB stage, maintaining a pH range of 6.5 to 8.0 is crucial for minimizing odor formation. This controlled pH stabilizes organic acids, such as volatile fatty acids (VFAs) produced during substrate breakdown, in their anionic forms, while ammonia and amines are stabilized in their cationic forms. These non-volatile states significantly reduce the release of these compounds as gaseous odors, as acids in anionic form and ammonia and amines in cationic form do not evaporate as readily as their neutral counterparts, which are responsible for strong smells. Additionally, the stable pH suppresses sulfate-reducing bacteria and other odor-producing microbes that thrive in more acidic conditions and generate hydrogen sulfide (H2S), a particularly pungent gas. By providing a controlled environment, the ASB effectively supports a clean, low-odor processing stage, even when handling complex substrates like FOG and WAS.
Moreover, the ASB's near neutral pH range creates a stable environment for efficient substrate breakdown, preventing the buildup of partially degraded, odorous intermediates. By minimizing the accumulation of volatile organic compounds (VOCs) and other intermediate byproducts that could contribute to odors, the ASB ensures a clean and low-odor process. This controlled environment is particularly effective for processing LM, FOG, and WAS, as the stabilized pH maintains an optimal balance of anions and cations, reducing the release of unpleasant smells throughout the ASB stage.
The odor control benefits established in the ASB stage carry over into the downstream MD stage by delivering a pre-conditioned substrate. Since the ASB produces an acetate-rich effluent with minimal residual sugars and other VFAs, the substrate entering the MD stage is already low in odor-producing compounds. Acetate, the primary product stabilized in the ASB, exists in its anionic form and is efficiently converted to methane and bicarbonate ion by acetoclastic methanogens in the MD stage. This direct conversion process is largely odor-free and produces high-methane biogas without generating additional volatile compounds that could contribute to unpleasant smells.
Additionally, the stable, anion-rich effluent from the ASB minimizes the potential for production of volatile compounds that might otherwise elevate odor levels in the MD stage. By limiting residual sugars and intermediate compounds that could contribute to odor formation, the MD stage can continue processing the effluent with reduced odor emissions. As a result, the biogas produced in the MD stage benefits from the pre-conditioning achieved in the ASB, with a high methane concentration and a minimized CO2 and odor content. This setup not only yields biogas suitable for energy applications but also creates an efficient, low-odor process across both stages, enhancing the overall operational environment.
Benefits for pH-Aligned Substrates
The selected pH range of 6.5 to 8.0 in the Anaerobic Secretome Bioreactor (ASB) closely aligns with the natural PH levels of key substrates-FOG, WAS, and manure. These materials generally have pH values that either fall within or near this target range, reducing the need for significant pH adjustments before they enter the ASB. This natural compatibility is particularly advantageous for efficient processing and cost-effective operation, as it minimizes the need for buffering agents or other chemical treatments that would otherwise be necessary to bring these substrates into the optimal range.
For FOG, which often has a slightly acidic pH, only minor adjustments are needed to bring it into the ASB's target range. In some examples, such minor adjustments can be achieved by recycling bicarbonate. This slight tuning allows the ASB to efficiently hydrolyze the complex lipids in FOG without producing odorous VFAs that would volatilize in a more acidic environment. By maintaining FOG in a stable, near-neutral state, the ASB reduces the volatilization of odorous compounds and supports optimal enzyme activity for lipid breakdown, creating favorable conditions for further processing in downstream methanogenic digestion.
WAS and manure generally already fall within or near the 6.5 to 8.0 pH range, making them particularly well-suited for the ASB's operating conditions. Treatment of WAS benefits from the ASB's controlled pH by maintaining stable microbial communities, which are essential for breaking down complex organic materials. Manure, with its naturally variable but generally near-neutral pH, also integrates well with minimal adjustment, allowing the ASB to capitalize on its rich organic content for efficient degradation. This compatibility promotes balanced microbial activity within the ASB, supporting an odor-controlled breakdown process across multiple substrates.
Overall, the alignment of the ASB's pH range with the natural pH of FOG, WAS, and manure reduces the need for external buffering, streamlines substrate integration, and enhances system efficiency. By operating within this compatible range, the ASB fosters a stable environment that encourages efficient substrate breakdown, minimizes odors, and reduces operational costs associated with pH adjustment. This setup not only simplifies the ASB's preparation process but also establishes optimal conditions for subsequent biogas production in downstream digestion, where a well-balanced, pre-conditioned effluent supports higher methane yields and improved process stability.
Lignocellulosic material (LM) generally has a slightly acidic to neutral pH, typically around 5 to 7. While this natural pH places LM close to the ASB's target range, minor adjustments—such as using bicarbonate recycled from downstream effluent—can bring it fully into the ideal 6.5 to 8.0 window. This bicarbonate feedback not only buffers LM's pH but also optimizes conditions for the hydrolysis and breakdown of its complex fibrous structures. Maintaining this adjusted pH enhances the activity of enzymes responsible for degrading cellulose, hemicellulose, and lignin, which are otherwise challenging to process. This adjustment supports efficient conversion of LM into simpler, metabolizable compounds while minimizing the formation of intermediate organic acids that could contribute to odors. By using bicarbonate effluent feedback to bring LM into the ASB's compatible pH range, the process allows for smoother integration with FOG, WAS, and manure, creating a stable, balanced environment that promotes efficient, odor-controlled substrate degradation.
Due to the high thermophilic temperatures of the Anaerobic Secretome Bioreactor (ASB), typically ranging from 65° C. to 85° C., the effluent entering a downstream methanogenic digestion (MD) stage is likely void of active methanogens. Most methanogenic microbes, especially those critical for methane production, thrive in mesophilic conditions (around 30° C. to 40° C.) or moderate thermophilic conditions up to 60° C. The elevated temperatures in the ASB may inhibit or eliminate these methanogens, making it necessary to inoculate the MD stage with a robust community of suitable methanogens, both syntrophic and acetoclastic, to optimize methane production.
Inoculating the MD with acetoclastic methanogens, such as Methanosaeta spp. and Methanosarcina spp., ensures efficient conversion of the acetate-rich effluent into methane. These acetoclastic methanogens directly metabolize acetate to produce methane, aligning with the ASB's output, which is high in organic acids stabilized in their anionic forms. Additionally, introducing syntrophic bacteria fosters a balanced microbial community by facilitating the breakdown of any remaining VFAs into hydrogen, CO2, and bicarbonate ions. Hydrogenotrophic methanogens then convert hydrogen and CO2 into methane. This initial inoculation establishes a stable mesophilic environment in the MD stage, tailored to maximize methane yield while efficiently processing the effluent from the ASB, producing high-quality biogas with minimal CO2 content.
The thermophilic synthetic microbial community disclosed herein offers a transformative approach to anaerobic digestion, enabling the efficient and simultaneous degradation of lignocellulosic materials (LM), fats, oils, and grease (FOG), and waste activated sludge (WAS) in a single, streamlined process. Operating within the high-temperature range of about 65° C. to 85° C., this innovative microbial community in the ASB leverages the natural enzymatic abilities of thermophilic microorganisms, each selected for their specific capabilities in breaking down complex organic substrates.
The microorganisms within this community are not only capable of thriving under thermophilic conditions but also coexist and interact synergistically, complementing each other's metabolic activities. By producing a wide range of extracellular enzymes—a secretome of exozymes, such as cellulases, lipases, proteases, and hemicellulases—these microbes work together to efficiently degrade diverse substrates. Their coordinated activity ensures that complex polymers from LM, lipids from FOG, and proteins from WAS are broken down simultaneously without the risk of inhibiting each other's metabolic functions. This functional compatibility and cooperative metabolism within the microbial community significantly enhance the rate of substrate breakdown, allowing for higher overall biogas yields and more stable anaerobic digestion processes.
The core advantages of this system stem from its ability to accelerate reaction rates, achieve simultaneous degradation, prevent process inhibition, optimize energy use, and enhance safety through pasteurization. Thermophilic conditions allow for faster degradation of recalcitrant materials like lignin and cellulose while promoting efficient lipid hydrolysis and protein breakdown. This results in shorter retention times and higher biogas yields. Additionally, the system's ability to break down multiple substrate types simultaneously—including the fibrous components of lignocellulosic materials, the lipid-rich content of FOG, and the protein and carbohydrate components of WAS—represents a significant improvement over conventional anaerobic digestion systems, which often require multi-stage or pre-treatment processes to handle such diverse feedstocks.
Preventing process inhibition is another critical advantage. The tailored microbial species are capable of degrading LCFAs from FOG through anaerobic beta-oxidation, producing methane-ready intermediates such as acetate and hydrogen, while also balancing the carbon-to-nitrogen (C/N) ratio from WAS. The microbial community works synergistically to ensure continuous substrate degradation and intermediate production, maintaining optimal conditions for stable methanogenesis. This mitigates common issues like LCFA buildup and ammonia toxicity, promoting process stability. Furthermore, the thermophilic ASB system optimizes energy use through single-step, high-efficiency processing, eliminating the need for energy-intensive pre-treatments such as chemical hydrolysis or prolonged retention times. This leads to a lower energy footprint, making the process more sustainable and cost-effective.
The high-temperature operation also serves as a natural pasteurization step, ensuring the destruction of non-thermophilic microorganisms and pathogens in the WAS and other feedstocks. This results in a safer, pathogen-free downstream methanogenic digestion and digestate that can be repurposed for agricultural or environmental use. Additionally, the microbial community's adaptability to feedstock variability ensures that the system can handle a wide range of organic inputs, making it ideal for large-scale, industrial applications where waste streams vary significantly in composition.
By integrating a synergistic microbial community capable of producing a wide range of exozymes—cellulases, hemicellulases, ligninases, lipases, and proteases—the ASB environment sets a new standard for the complete and efficient degradation of organic waste. This system not only leads to increased biogas production but also improves the overall sustainability and effectiveness of downstream methanogenic digestion as a key waste-to-energy technology.
The following are examples of various aspects of the disclosed invention.
In a first example, a method of processing feedstock for biogas production, the feedstock comprising lignocellulosic materials, the method comprising: maintaining, in an anaerobic secretome bioreactor environment of a system, the feedstock and a synthetic microbial community at a temperature substantially within a thermophilic temperature range and at a pH substantially within a pH range of 6.5 and 8.0, wherein the synthetic microbial community comprises at least one type of microorganism selected from thermophilic anaerobic microorganisms capable of acting as acidogens and acetogens and of producing a secretome of exozymes capable of hydrolyzing and solubilizing a substantial portion of the lignocellulosic materials; hydrolyzing and solubilizing, in the anaerobic secretome bioreactor environment by the secretome of exozymes, the substantial portion of the lignocellulosic materials, resulting in liquid effluent comprising soluble sugars and organic acid anions; and metabolizing, in the anaerobic secretome bioreactor environment by the synthetic microbial community, a portion of the soluble sugars and organic acid anions, resulting in the liquid effluent further comprising additional organic acid anions.
In the first example, further comprising pasteurizing, by virtue of the temperature, the liquid effluent comprising the soluble sugars, organic acid anions, and additional organic acid anions, such that the liquid effluent is rendered essentially free of pathogens and is suitable for biogas production via methanogenic digestion; wherein the method further comprises communicating the pasteurized liquid effluent from the anaerobic secretome bioreactor environment into a separate methanogenic digestion bioreactor environment of the system; and maintaining, in the methanogenic digestion bioreactor environment, the communicated liquid effluent and a community of methanogens within a mesophilic temperature range suitable for digestion of the communicated liquid effluent by the community of methanogens resulting in the production of biogas; wherein the thermophilic temperature range is 65-85° C.; wherein the at least one type of microorganism selected from the thermophilic anaerobic microorganisms includes Caldicellulosiruptor spp., Clostridium thermocellum, Thermoanaerobacterium saccharolyticum, Thermoclostridium stercorarium, Anaerobacillus thermoterrificus, Syntrophomonas wolfei, Thermoanaerobacter ethanolicus, Caloramator fervidus, and/or Thermotoga maritima; wherein the feedstock further comprises fats, oils, and grease (FOG); wherein the feedstock further comprises waste activated sludge (WAS); and wherein the method further comprises hydrolyzing and solubilizing, in the anaerobic secretome bioreactor environment by the secretome of exozymes, the substantial portion of the FOG and/or WAS, wherein the secretome of exozymes is further capable of hydrolyzing and solubilizing a substantial portion of the FOG and/or WAS.
In a second example, a system for processing feedstock for biogas production, the feedstock comprising lignocellulosic materials, the system comprising: an anaerobic secretome bioreactor environment configured to maintain the feedstock and a synthetic microbial community at a temperature substantially within a thermophilic temperature range and at a pH substantially within a pH range of 6.5 and 8.0, wherein the synthetic microbial community comprises at least one type of microorganism selected from thermophilic anaerobic microorganisms capable of acting as acidogens and acetogens and of producing a secretome of exozymes capable of hydrolyzing and solubilizing a substantial portion of the lignocellulosic materials; the anaerobic secretome bioreactor environment further configured to support hydrolyzing and solubilizing, by the secretome of exozymes, the substantial portion of the lignocellulosic materials, resulting in liquid effluent comprising soluble sugars and organic acid anions; and the anaerobic secretome bioreactor environment further configured to support metabolizing, by the synthetic microbial community, a portion of the soluble sugars and organic acid anions, resulting in the liquid effluent further comprising additional organic acid anions.
In the second example, the anaerobic secretome bioreactor environment further configured to support pasteurizing, by virtue of the temperature, the liquid effluent comprising the soluble sugars, organic acid anions, and additional organic acid anions, such that it is rendered essentially free of pathogens and suitable for biogas production via methanogenic digestion; wherein the anaerobic secretome bioreactor environment is further configured to support: communicating the pasteurized liquid effluent from the anaerobic secretome bioreactor environment into a separate methanogenic digestion bioreactor environment of the system; and maintaining, in the methanogenic digestion bioreactor environment, the communicated liquid effluent and a community of methanogens within a mesophilic temperature range suitable for digestion of the communicated liquid effluent by the community of methanogens resulting in the production of biogas; wherein the thermophilic temperature range is 65-85° C.; and wherein the at least one type of microorganism selected from the extremophile thermophilic anaerobic microorganisms includes Caldicellulosiruptor spp., Clostridium thermocellum, Thermoanaerobacterium saccharolyticum, Thermoclostridium stercorarium, Anaerobacillus thermoterrificus, Syntrophomonas wolfei, Thermoanaerobacter ethanolicus, Caloramator fervidus, and/or Thermotoga maritima; wherein the feedstock further comprises FOG; wherein the feedstock further comprises WAS; and wherein the anaerobic secretome bioreactor environment is further configured to support hydrolyzing and solubilizing, by the secretome of exozymes, the substantial portion of the FOG and/or WAS, wherein the secretome of exozymes is further capable of hydrolyzing and solubilizing a substantial portion of the FOG and/or WAS.
In a third example, a method of processing feedstock comprising (1) lignocellulosic materials (LM), (2) fats, oils, and grease (FOG), and (3) waste activated sludge (WAS) for biogas production, the method comprising: maintaining, in an anaerobic secretome bioreactor environment of a system, the feedstock and a synthetic microbial community at a temperature substantially within a thermophilic temperature range and at a pH substantially within a pH range of 6.5-8.0, wherein the synthetic microbial community produces a secretome of exozymes comprising at least: cellulases, hemicellulases, ligninases, esterases, and pectinases from at least one selected first type of thermophilic anaerobic microorganism capable of hydrolyzing and solubilizing a substantial portion of the lignocellulosic materials; lipases, phospholipases, esterases, and amylases from at least one selected second type of thermophilic microorganism capable of hydrolyzing and solubilizing a substantial portion of the FOG; and proteases, lipases, chitinases, nucleases, and polysaccharide-degrading enzymes from at least one selected third type of thermophilic anaerobic microorganism capable of hydrolyzing and solubilizing a substantial portion of the WAS; hydrolyzing and solubilizing, in the anaerobic secretome bioreactor environment and by at least the cellulases, hemicellulases, ligninases, esterases, and pectinases in the secretome of exozymes, the substantial portion of the lignocellulosic materials, resulting in liquid effluent comprising LM soluble intermediaries; metabolizing, in the anaerobic secretome bioreactor environment by at least a portion of the synthetic microbial community, a portion of the LM soluble intermediaries, resulting in the liquid effluent further comprising LM additional soluble intermediaries; hydrolyzing, in the anaerobic secretome bioreactor environment by at least the lipases, phospholipases, esterases, and amylases in the secretome of exozymes, the substantial portion of the FOG, resulting in the liquid effluent further comprising FOG soluble intermediaries; hydrolyzing, in the anaerobic secretome bioreactor environment by at least the proteases, lipases, chitinases, nucleases, and polysaccharide-degrading enzymes in the secretome of exozymes, the substantial portion of the WAS, resulting in the liquid effluent further comprising WAS soluble intermediaries; pasteurizing, by virtue of the temperature, the liquid effluent comprising the LM, FOG, and WAS soluble intermediaries, such that it is rendered essentially free of pathogens and suitable for biogas production via methanogenic digestion; communicating the pasteurized liquid effluent from the anaerobic secretome bioreactor environment into a separate methanogenic digestion bioreactor environment of the system; and maintaining, in the methanogenic digestion bioreactor environment, the communicated liquid effluent and a community of methanogens within a mesophilic temperature range suitable for digestion of the communicated liquid effluent by the community of methanogens resulting in the production of biogas.
In the third example, wherein the thermophilic temperature range is 65-85° C.; wherein the mesophilic temperature range is 20-55° C.; wherein the at least one selected first type of thermophilic anaerobic microorganism includes Caldicellulosiruptor spp., Clostridium thermocellum, Thermoanaerobacterium saccharolyticum, Thermoclostridium stercorarium, Anaerobacillus thermoterrificus, Syntrophomonas wolfei, Thermoanaerobacter ethanolicus, Caloramator fervidus, and/or Thermotoga maritima; wherein the at least one selected second type of thermophilic microorganism includes Anaerobacillus thermoterrificus, Clostridium spp., Syntrophomonas wolfei, Thermoanaerobacter ethanolicus, and/or Thermotoga maritima; and wherein the at least one selected third type of thermophilic anaerobic microorganism includes Thermoanaerobacterium thermosaccharolyticum, Anaerobacillus thermoterrificus, Caldicellulosiruptor spp., Thermoanaerobacter spp., Clostridium spp., Thermoanaerobacter ethanolicus, Syntrophomonas wolfei, and/or Thermotoga maritima.
This application is a continuation-in-part of U.S. application Ser. No. 17/706,569, filed on Mar. 28, 2022, which is a divisional of U.S. application Ser. No. 16/875,977, filed on May 15, 2020, which is a continuation-in-part of U.S. application Ser. No. 16/194,271, filed on Nov. 16, 2018, and of PCT Application No. PCT/US2018/061695, filed on Nov. 16, 2018. Each of U.S. application Ser. No. 16/194,271 and PCT Application No. PCT/US2018/061695 claims priority to U.S. Provisional Application No. 62/750,221, filed on Oct. 24, 2018, and U.S. Provisional Application No. 62/587,417, filed on Nov. 16, 2017. Each of the aforementioned applications is incorporated herein by reference in its entirety.
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| 62750221 | Oct 2018 | US | |
| 62587417 | Nov 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16875977 | May 2020 | US |
| Child | 17706569 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17706569 | Mar 2022 | US |
| Child | 18957723 | US | |
| Parent | 16194271 | Nov 2018 | US |
| Child | 16875977 | US | |
| Parent | PCT/US2018/061695 | Nov 2018 | WO |
| Child | 16875977 | US |
