WO2010114481A1

WO2010114481A1 - Methods for improving biogas production in the presence of hard substrates

Info

Publication number: WO2010114481A1
Application number: PCT/SG2009/000121
Authority: WO
Inventors: Shinya Fumitaka; Jinchuan Wu
Original assignee: Agency For Science, Technology And Research
Priority date: 2009-04-02
Filing date: 2009-04-02
Publication date: 2010-10-07
Also published as: CN102459099A; SG175011A1

Abstract

The present invention provides systems and methods for increasing methane production from anaerobic digestion. By adding pentose or glycerol-degrading microbes into the anaerobic digesters, these hard substrates can be efficiently converted to biogas with greater methane content.

Description

METHODS FOR IMPROVING BIOGAS PRODUCTION IN THE PRESENCE OF HARD SUBSTRATES

FIELD OF THE INVENTION [0001] The present invention discloses methods for improving biogas production in the presence of pentose(s) and/or glycerol.

BACKGROUND OF THE INVENTION

[0002] There are some technologies to recover energy from organic wastes. Methane fermentation, one of the biological approaches for energy recovery, can readily convert conventional organic substances to methane. This biological decomposition process is regulated by metabolic interactions among at least three functional groups of microorganisms, hi general, organic materials are first hydrolyzed to simpler compounds that are then converted to volatile acids by acidogens. The volatile acids that contain more than 2 carbons are then converted to acetate and H₂ by obligate hydrogen-producing acetogens. Finally, acetate, H₂ and Cl compounds are converted to CH₄ by methanogens. {see, Speece, Anaerobic Biotechnology, 26 (1996)).

[0003] Vast quantities of C5 sugars occur as the structural polysaccharides of hemicellulose in lignocellulosic biomass. Lignocellulosic biomass is a much more abundant renewable material(s) than food crops and can be harvested without interference to the food supply chain and with less influence on environment. However, lignocellulosic biomass is unable to be directly utilized as a carbon source for microbial fermentation and needs to be treated to get fermentable sugars. Hydrolysis of lignocellulose gives a mixture of monomelic hexoses (glucose, mannose and galactose) and pentoses (D-xylose, L-arabinose). Among them, glucose is usually most abundant, followed by pentose (for hardwoods and agricultural residues) or mannose (for softwoods). Pentoses (D-xylose and L-arabinose) can represent up to 30% of the lignocellulosic biomass with xylose being the majority (about 80% of hemicellulose sugars). Although the glucose in the hydrolysate of lignocellulose can be easily fermented to ethanol and other useful chemicals as usual, it is usually hard for the conventional industrial strains to utilize pentose as the carbon source. Even though xylose has been successfully fermented to ethanol and other chemicals using some natural or genetically engineered strains in labs, efficient anaerobic digestion of xylose and other C5 sugars to biogas has not yet been achieved due to the difficulty in digesting pentose sugars by the naturally occurring microbes in sludge, (see, Lin, C. et al., Hydrogen Energy. 33:43-50 (2008)).

[0004] Glycerol is produced as a by-product in the biodiesel industry (production of 100 kg of biodiesel will generate about 10 kg of glycerol). The availability of crude glycerol is predicted to significantly increase in the coming years because of the rapidly expended biodiesel market. However, the utilization of cheap crude glycerol to make value-added chemicals is a challenge as the industrial chemistry of glycerol has not yet been well established. Anaerobic digestion of glycerol to biogas might provide an easier way to convert glycerol to fuels considering the simplicity of the anaerobic digestion process. However, it is also hard for the naturally occurring microbes in sludge to efficiently utilize glycerol as the substrate for biogas production.

[0005] Therefore, efficient conversion of pentose (D-xylose, L-arabinose) and glycerol to biogas will help reduce the total cost of bioethanol and biodiesel production processes where pentose sugars and glycerol are generated as "wastes," respectively.

[0006] As mentioned earlier, methanogens can utilize only very simple carbon sources such as acetate, CO₂, methanol, CO and H₂. Some microorganisms (bacteria such as Escherichia coli and yeasts such as Pichia stipitis) have stronger ability in utilizing pentose and/or glycerol to produce organic acids and other metabolites of smaller molecules. These smaller molecules might be better substrates for the methane-producing bacteria than pentose and glycerol. However, these microbes either do not exist in the virgin sludge or their contents in the virgin sludge are very low. A few researchers have reported the effect of E. coli naturally existing in the sludge on biogas production. Gonzalez, R. et al. Metabolic Engineering (2008)) found that E. coli can ferment glycerol in a 1,3 -propanediol ("1, 3-PDO")- independent manner. A few intermediates and the end-product 1, 3 -PDO generated in this way can be digested and converted to methane and CO₂ by the naturally existing microbes in digested sludge. Chiu et al. (2007) reported the utilization of xylose by H₂-producing bacteria. Prior to the hydrogen-producing process, xylose is first utilized by H₂-producing bacteria to generate some volatile fatty acid (VFAs) intermediates, which are subsequently converted to methane. The H₂-producing bacteria naturally co-exist with the methanogens in digested sludge. But H₂-producing bacteria can tolerate higher temperature (as high as 100 ⁰C) than methanogens due to their strong ability of forming spores. The ^-producing bacteria can be isolated from the methanogens by a simple heat treatment at higher temperatures. [0007] The yeast P. stipitis has been extensively used to produce ethanol from xylose in labs. Marques et al. (2007) reported the conversion of recycled paper sludge to ethanol using P. stipitis. Hahn-Hagerdal, B. et al. Enzyme Microb. Technol., 16 November (1994)) reported the physiology of xylose fermentation by this yeast. Pentose is metabolized via the pentose phosphate pathway, generating various VFAs (e.g. acetic acid) and other intermediates. As mentioned earlier, these might be better substrates for methanogens than xylose.

[0008] In view of the foregoing there is a need in the art for methods and systems to decompose "hard" substrates such as pentoses and glycerol to smaller molecular substances to promote their conversion to biogas and to increase methane yield. The present invention satisfies this and other needs.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides methods and systems that convert hard substrates such as pentoses and glycerol into biogas by anaerobic digestion, hi certain aspects, the methods include introducing exogenous microbes that can efficiently degrade and convert these "hard" substrates to smaller molecules. By introducing pentose-utilizing and glycerol- utilizing microbes exogenously into digested sludge, the inventive methods decompose these "hard" substrates (e.g., pentose and glycerol) to smaller molecular substances to promote conversion to biogas (e.g. , methane).

[0010] As such, in one embodiment, the present invention provides an anaerobic method for increasing the yield of conversion of a biomass to methane, wherein the biomass contains a hard substrate, comprising:

contacting the biomass containing the hard substrate with an exogenous microbe to generate a reaction mixture; and

fermenting the reaction mixture to increase the yield of methane.

[0011] In another embodiment, the present invention provides an anaerobic fermentation system for increasing the yield of conversion of a biomass to methane, the system comprising:

an anaerobic fermenter having a biomass with a hard substrate; and an exogenous microbe to convert the hard substrate, wherein the biomass is fermented to increase the yield of methane.

[0012] In certain instances, with the addition of exogenous microbes, the yield of biogas from pentoses or glycerol is about three times higher or more than without the addition of exogenous microbes. By modifying the operating conditions of anaerobic fermentation, it is possible to convert all of the hard substrates to biogas.

[0013] In certain preferred instances, any microbes that can degrade these hard substrates are applicable for the purpose of improving biogas production from these hard substrates.

[0014] In certain instances, the exogenous microbes are only added into the system once, for example, in the beginning preferably before hydrolysis. Afterwards, the microbes can grow for many generations. The medium for cultivating these microbes is very simple due to the ability of the microbe's ability of utilizing various carbon sources.

[0015] By adding pentose or glycerol-degrading microbes into the anaerobic digesters, these hard substrates can be efficiently converted to biogas. By selectively choosing the microbes that can degrade these substrates, and preferably do not generate CO₂ during the degradation, it is possible to generate increased yields of methane content in biogas. The efficient conversion of pentoses (e.g., D-xylose, L-arabinose) and glycerol to biogas can thereafter be used for bioethanol and biodiesel production processes.

[0016] Advantageously, the addition of exogenous microbes has been shown to significantly promote the digestion of organic substances that pre-exist in the digested sludge, but are unable to be efficiently digested by the methanogens in the digested sludge. The methods and systems herein lead to increased biogas production and methane yield.

[0017] In still yet another embodiment, the present invention provides a use of an anaerobic method for increasing the yield of conversion of a biomass to methane, wherein the biomass contains a hard substrate, the use comprising:

fermenting the reaction mixture to increase the yield of methane.

[0018] These and other objects, aspects and embodiments will become more apparent when read with the accompanying drawings and detailed description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 illustrates one embodiment of a flow chart and reaction process of the present invention.

[0020] FIG. 2 shows a graph of increased methane production (volume mL) from xylose by adding lactic acid bacteria.

[0021] FIG. 3 shows a graph of methane content (% v/v) in biogas produced from xylose with exogenous lactic acid bacteria.

[0022] FIG. 4 shows a graph of increased methane production (volume mL) from xylose by adding P. stipitis.

[0023] FIG. 5 shows a graph of methane content (% v/v) in biogas produced from xylose with exogenous P. stipitis.

[0024] FIG. 6 shows a graph of increased methane production (volume mL) from xylose by adding E. coli.

[0025] FIG. 7 shows a graph of methane content (% v/v) in biogas produced from xylose with exogenous E. coli.

[0026] FIG. 8 shows a graph of increased methane production (volume mL) from xylose by adding H₂-producing bacteria.

[0027] FIG. 9 shows a graph of methane content (% v/v) in biogas produced from xylose with exogenous H₂-producing bacteria.

[0028] FIG. 10 shows a graph of increased methane production (volume mL) from arabinose by adding lactic acid bacteria.

[0029] FIG. 11 shows a graph of methane content (% v/v) in biogas produced from arabinose with exogenous lactic acid bacteria.

[0030] FIG. 12 shows a graph of increased methane production (volume mL) from arabinose by adding P. stipitis.

[0031] FIG. 13 shows a graph of methane content (% v/v) in biogas produced from arabinose with exogenous P. stipitis.

[0032] FIG. 14 shows a graph of increased methane production (volume mL) from arabinose by adding E. coli. [0033] FIG. 15 shows a graph of methane content (% v/v) in biogas produced from arabinose with exogenous E. coli.

[0034] FIG. 16 shows a graph of increased methane production (volume mL) from glycerol by adding E. coli.

[0035] FIG. 17 shows a graph of methane content (% v/v) in biogas produced from glycerol with exogenous E. coli.

DETAILED DESCRIPTION OF THE INVENTION

I. Embodiments

[0036] The present invention provides methods and systems for the efficient conversion of hard substrates such as pentoses (e.g., xylose, arabinose) and/or glycerol to biogas. Advantageously, the methods and systems described herein reduce the total cost of bioethanol and biodiesel production processes where these hard substrates are generated. As used herein, the term "hard substrate" includes glycerol and pentoses such as D- and L- pentoses including xylose, arabinose, ribose, ribulose (preferably, L-) and lyxose. Preferred hard substrates also include glycerol, D-or L-xylose and D- or L-arabinose. The most preferred hard substrates are glycerol, D-xylose and L-arabinose. Suitable hard substrates also include cellulose, xylan, effluents of palm oil mills, mixtures of any of the above substrates, and the like.

[0037] In certain instances, the present invention provides methods for converting hard substrates that methane-producing microbes can utilize by introducing exogenous microbes. Although the methods and systems herein are preferably applicable to pentoses and glycerol, other hard substrates or carbon sources can also be used. Moreover, it is possible to introduce exogenous microbes to all other types of fermentation processes, as the methods herein are not limited to biogas production.

[0038] Anaerobic methane fermentation of sewage and sludge is the consequence of a series of metabolic interactions among various groups of microorganisms or microbes. The first group of microorganisms secrete enzymes that hydrolyze polymeric materials to monomers such as glucose, pentoses and amino acids, which are subsequently converted to higher volatile fatty acids, H₂ and acetic acid. In the second group, hydrogen-producing acetogenic bacteria convert the higher volatile fatty acids e.g., propionic and butyric acids, produced, to H₂, CO₂, and acetic acid. Finally, methanogenic bacteria convert H₂, CO₂, and acetate, to CH₄ and CO₂.

[0039] More particularly, anaerobic methane fermentation comprises four stages:

i) the hydrolysis step which converts complex molecules into simpler molecules;

ii) the acidogenesis step, which transforms these simpler molecules into fatty acids, alcohols, carbon dioxide and hydrogen;

iii) the acetogenesis step which provides the conversion of the products of the acidogenesis into acetic acid; and

iv) the methanogenesis step, which converts acetic acid into methane.

[0040] FIG. 1 is an example flowchart illustrating a reaction process 100 in accordance with an embodiment of the present invention, hi operation, exogenous microbes 110 are added to an anaerobic digestor or reactor to process organic waste feedstock such as organic wastes 102 containing polysaccharides such as hemicelluloses, proteins and fats. In the process, hydrolysis fermentation 115 of the organic feedstock occurs which converts sugars, amino acids, fatty acids and hard substrates to acidic intermediates. The acid forming bacteria generate acids such as acetic acid by acetogenesis 125. Thereafter acidic intermediates 130 are converted by a step of methanogenesis 140 to useful gases, such as methane 150, by methane-producing organisms. Without the addition of the exogenous microbes, the conversion of hard substrates is not efficient.

[0041] The term "organic waste" as used herein includes organic sludge, all types of organic refuse, sewage sludge, animal waste, municipal waste, industrial waste, forestry waste, agricultural waste, and the like. In certain instances, the organic waste feedstock contains hemicellulose, which in turn is made up of pentoses such as D-xylose and D- or L- arabinose. Hemicellulose is the term used to denote non-cellulosic polysaccharides associated with cellulose in plant tissues. Hemicellulose frequently constitutes about 20-35% w/w of lignocellulosic materials, and the majority of hemicelluloses consists predominantly of polymers based on pentose (five-carbon) sugar units, such as D-xylose and D- or L- arabinose units, although more minor proportions of hexose (six-carbon) sugar units, such as D-glucose and D-mannose units, are generally also present. As a skilled artisan will appreciate, glycerol is present in the form of esters (glycerides) in all animal and vegetable fats and oils. It can be obtained commercially as a by-product when hydrolyzed to yield fatty acids or their metal salts (soaps). Glycerol is also synthesized on a commercial scale from propylene (obtained by cracking petroleum), as in certain instances, the supply of glycerol from natural sources is inadequate.

[0042] In certain other embodiments, the feedstock for the methods and systems of the present invention also includes biomass. Suitable biomass includes for example, plant material such as fresh harvested or stored plant material, and is typically untreated chemically or physically, except for size reduction. Both terrestrial and aquatic plants are suitable for use in the present invention.

[0043] hi certain embodiments, organic products, by-products or waste from human, animal or vegetable origin are optionally subjected to a mechanical, physical, chemical or microbiological preliminary treatment, for instance to a heat processing, a pounding, crushing, milling or a chopping through a chopper-projector, a quick depressurization or pressure drop, an anaerobic pre-fermentation to for instance promote the hydrolysis of the matter, the defiberization, shredding or delignification in particular in the case of cellulosic and ligno-cellulosic compounds.

[0044] As shown in FIG. 1 , the organic wastes 102 is subjected to a treatment resulting in at least partial hydrolysis 115 to obtain a slurry. The hydrolysis of the non-soluble organic compounds of the organic waste are depolymerized by the hydrolytic enzymes of the anaerobic bacteria of sewage. During hydrolysis, proteins are generally hydrolyzed to amino acids by proteases, and polysaccharides are hydrolyzed to monosaccharides by cellulases and amylases. The amino acids produced are thereafter degraded to fatty acids such as acetate, propionate, and butyrate. The hexoses and pentoses are generally converted to C2 and C3 intermediates. Most anaerobic bacteria undergo hexose metabolism via the Emden- Meyerhof-Parnas pathway (EMP) which produces pyruvate as an intermediate along with

NADH. The pyruvate and NADH are transformed into fermentation products such as lactate, propionate, acetate, and ethanol by other enzymatic activities which vary with microbial species.

[0045] As shown in FIG. 1, during hydrolysis and acidogenesis, sugars, ammo acids, and fatty acids produced by microbial degradation of biopolymers are successively metabolized by anaerobic bacteria and are fermented to acetate, propionate, butyrate, lactate, ethanol, carbon dioxide, and hydrogen. Although some acetate is formed in acidogenesis, the primary generation of acetate and hydrogen occur from the acetogenesis of fatty acids. In certain instances, acetogenic bacteria such as Syntrophobacter wolinii, and Sytrophomonos wolfei, are useful in this stage. After acetogenesis, methanogens convert acetate and H₂/CO₂ to methane.

[0046] Advantageously, the methods and systems of the present inventions provide exogenous microbes into the reaction to increase and improve biogas production. FIG. 2 clearly shows one embodiment of the present invention wherein addition of exogenous microbes (e.g., lactic acid bacteria) into an anaerobic reactor, which is capable of degrading xylose significantly improved biogas production.

[0047] The present invention can use any active producing psychrophilic, mesophilic or thermophilic microbial anaerobic digestion system. As used herein, the term "psychrophilic" includes relatively low temperatures. The term "mesophilic" includes microbes that grow or thrive best in an intermediate environment such as in one of moderate temperature, whereas the term "thermophilic" includes microbes that thrive or grow at a high temperature. Typically, the principal suitable non-methanogenic bacteria include species from genera including Aerobacter, Aeromonas, Alcaligenes, Bacillus, Bacteroides, Clostridium Escherichia, Klebsiella, Leptospria, Micrococcus, Neisseria, Paracolobactrum, Proteus, Pseudomonas, Rhodopseudomonas, Sarcina, Serratia, Streptococcus and Streptomyces.

[0048] Suitable methane-producing organisms include, but are not limited to, Methanobacterium, Methanococcus and Methanosarcina, specific members being

Methanobacterium formicicum, Methanosarcina barkerii, Methanobacterium omelianskii, Methanococcus vannielii, Methanobacterium sohngenii, Methanosarcina methanica, Methanococcus mazei, Methanobacterium suboxydans and Methanobacterium propionicum. In certain instances, mixed cultures can be used to obtain the most complete fermentation action. Nutritional balance and pH adjustments can be made to the digester system as necessary as is known to the art to optimize methane production from the culture used.

[0049] hi certain instance, the reaction conditions can be psychrophilic, mesophilic or thermophilic. Preferably, the reaction conditions are mesophilic, between an anaerobic temperature of about 15°C to about 55°C, preferably about 30°C to about 40°C. Preferably, the methods take place at atmospheric pressure, although higher pressures are suitable. In certain instances, the pH of the reaction is between pH 6 and pH 8 more preferably between pH 6.5 and pH 7.8, and most preferably between pH 6.8 and pH 7.5. [0050] In certain instances, the microorganism according to the present invention can be used as a biologically pure culture, or it can be used with other microorganisms in mixed culture. Biologically pure cultures are generally easier to optimize, but mixed cultures can utilize additional substrates. In certain preferred aspects, the exogenous microorganisms used include, but are not limited to, lactic acid bacteria, Pichia stipitis, Escherichia coli, hydrogen producing bacteria and a mixture thereof. The term "microorganisms" is used herein to include organisms such as bacteria, yeast, protozoa and fungi, which are able to metabolize hard substrates. The microorganisms can be naturally occurring or genetically modified. Naturally occurring microorganisms are typically maintained in a culture such as a starter culture. Genetically modified microorganisms can be mutated or manipulated by, for example, the introduction of genetic material such as a plasmid or vector.

[0051] In one preferred embodiment, the biomass containing a hard substrate is filled into the fermenter and biogas production is started using any of the known procedures and a reactor system a natural consortia of microorganisms is developed. The biogas producing fermenter is thereafter inoculated with a cultivated monoculture of the exogenous microorganisms such as lactic acid bacteria, Pichia stipitis, Escherichia coli, hydrogen producing bacteria and a mixture thereof. If necessary, the inoculation is repeated as the fermentation progresses. The methods and systems herein are applicable to biogas production modes in either liquid or solid state fermenters.

[0052] The use of microorganisms which have been genetically modified in order to create enhanced hard substrate metabolism is contemplated by the present invention. Such genetic modifications can include recombinant DNA technologies such as stable transfection of a bacterial cell line with an expression vector containing the mRNA for a protein(s) such as an enzyme which catalyzes the metabolism of a pentose, or the introduction into a host cell genome the gene or genes responsible for the conversion of the hard substrates to smaller molecules. For example, in one instance, the anaerobic metabolism of xylose via xylose isomerase and d-xylulokinase is optimized and is then transfected into a bacteria cell line.

[0053] In certain preferred aspects, the microbes that are used effectively metabolize pentoses and/or glycerol, without forming CO₂. In one instance, a preferred microbe degrades xylose without generating CO₂. In another aspect, the microbe degrades L- arabinose without generating CO₂. In still another aspect, the microbe degrade glycerol without generating CO₂. hi certain instances, additional microbes are introduced which effectively metabolize CO₂. In certain instance, microorganisms can metabolize carbon sources without generating large amounts OfCO₂. That is, the CO₂ emission is small or de minimus, by for example, the introduction of photosynthetic bacteria.

[0054] In certain aspects, the addition of exogenous bacteria can be done at various stages of the anaerobic fermentation process. For example, the addition of the microorganisms is preferably done at the beginning, before hydrolysis occurs. In certain other embodiments, the addition of the exogenous microbes can be done after hydrolysis, but before the acidogenesis step wherein pentoses, such as xylose, are converted to smaller molecular substances such as acetate, lactic acid, formic acid, ethanol and H₂, which are believed to be better substrates for the methanogens than xylose. The microbes can be individual cultures containing a single species or a populations which contain a combination of species. In certain instances, bacteria such as lactic acid bacteria, Escherichia coli, Bacilus subtilis and the like, including their combination are preferentially recommended considering their broad substrate range and their robust ability of growth as well as safety to the environment. In certain other instances, microorganisms such as yeast, fungi, archaea and the like and their combination are also suitable for use in the present invention. The exogenous microorganisms can be obtained from exogenous sources or obtained from the digested sludge itself by screening, purifying and cultivating.

[0055] In certain embodiments, the exogenous microbes are derived from an anaerobic waste water sludge treatment facility as explained in the examples. In certain other instances, inocula can be obtained from any thermophilic anaerobic digester or can be prepared separately by using animal manure or waste water sludge and incubating it under anaerobic conditions at thermophilic temperature and incubating it until fermentation starts.

[0056] The methods and systems disclosed herein can be carried out in existing anaerobic digestion systems for organic substrate digestion. The methods and systems are suitable to any existing fermentation system by for example, supplementing cultivation and feeding equipment of exogenous microbes. The methods and systems cover all ranges of reactor size from laboratory bench top to wastewater treatment plants.

[0057] Both one phase systems and two phase systems are applicable to the inventive methods and systems disclosed herein. In the one phase systems, the organic substrate and the microorganisms are housed together. For example, the methods and systems herein can use an Upflow Anaerobic Sludge Blanket (UASB) process for bioconversion of feedstocks, which contain primarily soluble organic waste wherein small amounts of solids, ordinarily less than 1 percent of the feedstock, and the bacterial mass are allowed to settle in the reactor. In this system, waste water enters from the bottom of the reactor passes through a sludge bed and sludge blanket where organic materials are anaerobically decomposed. Gas produced is then separated by a gas-solid separator and the clarified liquid is discharged over a weir, while the granular sludge naturally settles to the bottom. UASB systems are primarily used in the treatment of waste water derived from the food processing industry.

[0058] Using the inventive methods in an UASB system, the exogenous microbes can be added to the sludge bed where the microorganisms are housed. In certain preferred aspects, the exogenous microbes are added to the sludge bed prior to hydrolysis and waste water addition to the UASB reactor.

[0059] In addition to UASB, upflow anaerobic filter process (UAFP) systems are also application for the inventive methods. Typically in these systems, the reactor contains a "medium", i.e., a microbial support. Granulated microorganisms exist not only in the spaces within the medium, but are also attached to its surface; hence, a high-density microbial population is retained within the reactor, creating a hybridization of microbial floe and adhesion. In certain instances, the microbial support can be added with microbes that digest hard substrates. Anaerobic filter-type reactors promote the retention of bacteria in the digester by attaching bacteria to fixed inert materials in the digester. These inert materials can be designed especially for hard substrate conversion.

[0060] In certain other aspects, anaerobic fluidized-bed reactors (AFBR) are used. In these systems, the medium to which the microbes adhere is fluidized within the reactor, resulting in conversion of organic materials to CH₄ and CO₂. Anaerobic microbes grow on the surface of the medium, expanding the apparent volume of the medium; hence this reactor is also designated an "expanded bed reactor." In the inventive methods, the exogenous microbes are added to the fluidized bed. In certain instances, continuous flow fluidized bed fermenters embodying a tower design or a supported film reactor can be used.

[0061] In addition to the one-phase systems, typical two phase anaerobic digester systems comprise an acid phase digester and a biogasification reactor. The acid phase digester is usually designed as a solid-bed batch reactor where solid waste is housed and leached soluble compounds are collected. In the acid first phase, the microbial population and operating conditions are selected to promote the conversion of organic carbonaceous materials to carbonaceous materials of lower molecular weight, primarily volatile fatty acids. In the two- phase systems, the exogenous microbes are preferably added to the acid phase digester. After the hard substrates are digested, the liquid and solid effluent from the acid phase is conveyed to a biogasification second phase. In this phase, the methanogenic organisms convert the volatile fatty acids to product gas that is composed primarily of methane and carbon dioxide. Product gas is removed from the biogasification reactor and processed, or scrubbed, to separate the methane component that is drawn off as pipeline gas.

II. EXAMPLES

Activation of digested sludge

[0062] The digested sludge was collected from a digester located in Water Reclamation Plant, Singapore. Activation of the digested sludge was conducted using the hydrolyzate of cooked rice (total solid: 8.4%) supplemented with (per liter) 1O g peptone, 5 g yeast extract and 1 mL nutrient solution. The nutrient solution is composed of (per liter) 4.5 g NH₄HCO₃, 0.25 g K₂HPO₄, 0.1 g MgCl₂ ^«6H₂O, 6.0 g NaHCO₃ and 10 mL of trace element solution. The trace element solution contains 0.4 g FeCl₂ ^«4H₂O, 0.12 g CoCl₂*6H₂0, 0.01 g Alk(SO₄)2-12H₂O, 0.01 g Na₂MoO₄«2H₂O, 0.01 g H₃BO₃, 0.01 g CuSO₄-5H₂O, 1.0 g NaCl, 0.02 g CaCl₂, 0.02 g NiCl₂ ^»6H₂O, O.lg MnCl₂-4H₂O, and 0.1 g ZnCl₂. The nutrient solution was sterilized by autoclaving at 121⁰C for 15 min to prevent bacterial contamination. The fermenter used for cultivation was controlled at 37°C, 150 rpm. After a steady state was established (constant gas production and methane content), the activated sludge was used for the batch experiments of biogas production.

Cultivation of microbe

[0063] Four species of microbes were employed. Bac #4 (lactic acid bacterium, LAB) was screened from the local environment. Pichia stipitis (ATCC 58785) and Escherichia coli TOPlO were obtained commercially. H₂-ρroducing bacteria were isolated from the digested sludge by heat treatment at 80°C for 30 min. These microbes were cultivated respectively in their individual media. P. stipitis was grown in YDP medium (50 mL) consisting of 10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose. E. coli was grown in LB medium (50 mL) consisting of 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl. LAB was grown in modified MRS medium (50 mL) consisting of lOg/L peptone, 5g/L meat extract, 20 g/L xylose, 2 g/L dipotassium hydrogen phosphate, 5 g/L sodium acetate trihydrate, 2 g/L triamrnonium citrate, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganous sulfate tetrahydrate, 0.05g/L FeSO₄.7H₂O and 1 mL/L Tween 80. The H₂-producing bacteria obtained by heating digested sludge at 8O⁰C for 30 min were incubated in the nutrient solution (as described in 4.1) at 37°C for 2 days with shaking to activate the H₂ producing bacteria. All other microbes were incubated at 30⁰C for overnight with shaking. The cultures were washed with the same volume of water twice and then inoculated (5%, v/v) into the anaerobic digestion reactors.

Measurement of biogas production and methane content

[0064] The amount of biogas produced was measured using glass syringes, and the biogas composition was determined by a gas chromatograph (GC) packed with a Hayesep D (60/80) column and a thermal conductivity detector (TCD). The GC oven and inlet temperature were programmed to 35⁰C and 60⁰C, respectively. The temperature of TCD detector was set at 200⁰C with 50 mA current. Argon was used as the carrier gas at 25 mL/min.

Experimental procedures

[0065] The cultivated seed microbes were washed with water and inoculated (5 %, v/v) into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 15 g/L pentose (D-xylose or L-arabinose) or 20 mL/L glycerol. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen gas followed by sealing with butyl rubber stoppers prior to experiments. The reactors were placed in a water bath at 37⁰C with shaking at 150 rpm. Biogas production was measured using syringes and gas compositions were determined by GC.

Experimental results

Conversion of xylose to biogas

[0066] The theoretical methane production from xylose is 373 mL/g xylose under standard conditions (0⁰C, 760 mmHg).

XyIoSe (C₅H₁₀O₅) → 2.5CO₂ + 2.5CH₄

I g 373 mL 373 mL

Table 1

Summarized methane production, productivity, yield and recovery from xylose with exogenous microbes at day 7

* Methane recovery is defined as the ratio of actual methane produced to theoretical methane production X 100%

Table 2

Summarized methane content in biogas produced from xylose with exogenous microbes at day 7

Operation Digested sludge + xylose+

Digested mode Digested sludge + H₂ sludge LAB P. stipitis E. coli producing xylose bacteria

Methane

64.9 65.1 19.8 40.8 64.8 56.2 content (%)

Discussion

[0067] Figures 1-8 clearly show that the addition of exogenous microbes that are capable of degrading xylose significantly improved biogas production. The addition of xylose into the anaerobic reactors alone only slightly increased the biogas production compared to the control (digested sludge only), indicating the difficulty in converting xylose to biogas by the microbes naturally occurring in the digested sludge. The addition of xylose-degrading microbes alone (without addition of xylose) also slightly increased the biogas production, inferring that there existed some carbon sources that were unable to be efficiently degraded by the naturally occurring microbes in the digested sludge, but were able to be degraded by the exogenous microbes added. The highest methane production was achieved when both xylose and xylose-degradable microbes were added, indicating that the exogenous xylose was efficiently converted to biogas under the help of the exogenous microbes. This is ascribed to the conversion of xylose to smaller molecular substances such as acetate, lactic acid, formic acid, ethanol and H₂, which are believed to be better substrates for the methanogens than xylose. It is well known that the methane content in biogas is usually 60-70% (v/v) in conventional anaerobic digesters. However, in the cases of addition of some exogenous microbes (especially lactic acid bacteria and P. stipitis), the methane contents were significantly decreased. This might be ascribed to the production OfCO₂ accompanying the conversion of xylose to smaller molecular substances. A way to keep higher methane content in biogas is to utilize those microbes that can degrade xylose, but do not generate CO₂.

Examples of xylose conversion to biogas

Example 1

[0068] Lactic acid bacteria (LAB) were inoculated into 50 mL modified MRS medium at 30⁰C overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g xylose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were placed in a water bath at 37°C with shaking at 150 rpm. After 7 days, 61.4 mL methane was produced (methane recovery 13.7%, methane content in biogas 19.8%). m contrast, in the case with addition of xylose alone, the methane produced was only 21.2 mL (methane recovery 4.7%, methane content in biogas 65.1 %). hi the case with addition of LAB but without addition of xylose, the methane produced was 39.7 mL (methane content in biogas 70.7%).

Example 2

[0069] P. stipitis were inoculated into 50 mL YDP medium at 30⁰C overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four 4 mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g xylose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were then placed in water bath at 37⁰C with shaking at 150 rpm. After 7 days, 151 mL methane was produced (methane recovery 33.7%, methane content 40.8%). In contrast, in the case with addition of xylose but without addition of P. stipitis, the methane produced was only 21.2 mL (methane recovery 4.7%, methane content in biogas 65.1%). hi the case with addition of P. stipitis but without addition of xylose, the methane produced was 37.3 mL (methane content in biogas 80.5%) Example 3

[0070] E. coli TOP 10 were inoculated into 50 mL LB medium at 3O⁰C for overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four 4 mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g xylose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were then placed in a water bath at 37⁰C with shaking at 150 rpm. After 7 days, 121 mL methane was produced (methane recovery 27.0%, methane content in biogas 64.8%). In contrast, in the case with addition of xylose but without addition of E. coli, the methane produced was only 21.2 mL (methane recovery 4.7%, methane content in biogas 65.1%). In the case with addition of E. coli but without addition of xylose, the methane produced was 55.5 mL (methane content in biogas 56.2%)

Example 4

[0071] H₂ producing bacteria were obtained by heating digested sludge at 80⁰C for 30 min. The heat-treated sludge (40 mL) was added into 10 mL nutrient solution (described in 4.1) followed by incubation at 37°C for 2 days with shaking to activate the H₂ producing bacteria. Four 4 mL of the activated H₂ producing bacteria was added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g xylose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were then placed in water bath at 37°C with shaking at 150 rpm. After 7 days, 123 mL methane was produced (methane recovery 27.5%, methane content in biogas 56.2%). In contrast, in the case with addition of xylose but without addition of E. coli, the methane produced was only 21.2 mL (methane recovery 4.7%, methane content in biogas 65.1 %). In the case with addition of E. coli but without addition of xylose, the methane produced was 86.3 mL (methane content in biogas 80.0%)

Conversion of arabinose to biogas

[0072] The theoretical methane production from arabinose is also 373mL/g-arabinose under standard conditions (0⁰C, 760 mmHg). Arabinose (C₅H₁₀O₅) → 2.5CO₂ + 2.5CH₄

Ig 373 mL 373 mL

Table 3

Summarized methane production, productivity, yield and recovery from L-arabinose with exogenous microbes

Table 4 Summarized methane content in biogas produced from L-arabinose with exogenous microbes

Discussion

[0073] Figures 9-14 clearly indicate that the addition of exogenous microbes that are capable of degrading arabinose into the anaerobic reactors significantly improved the biogas production. The addition of arabinose into the anaerobic reactors alone only slightly increased the biogas production compared to the control (digested sludge only), indicating the difficulty in converting arabinose to biogas by the microbes naturally occurring in the digested sludge. The addition of arabinose-degrading microbes alone (without addition of arabinose) also slightly increased the biogas production, inferring that there existed some carbon sources that were unable to be degraded by the naturally occurring microbes in the digested sludge but were able to be degraded by the exogenous microbes added. The highest methane production was achieved when both arabinose and arabinose-degradable microbes were added, indicating that the exogenous arabinose was efficiently converted to biogas under the help of the exogenous microbes. This is ascribed to the conversion of arabinose to smaller molecular substances such as acetate, lactic acid, formic acid, ethanol and H₂, which are believed to be better substrates for the methanogens than arabinose. In the cases of addition of the exogenous microbes, the methane contents in biogas products were also significantly decreased. Similarly, a way to get higher methane content to add those microbes that can degrade arabinose without generating CO₂.

Examples of arabinose conversion to biogas

Example 5

[0074] Lactic acid bacteria (LAB) were inoculated into 50 mL modified MRS medium at 30⁰C overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g L-arabinose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were placed in a water bath at 37°C with shaking at 150 rpm. After 7 days, 78.3 mL methane was produced (methane recovery 17.5%, methane content in biogas 21.6%). In contrast, in the case with addition of L-arabinose but without addition of LAB, the methane produced was only 20.7 mL (methane recovery 4.6%, methane content in biogas 65.1 %). hi the case with addition of LAB but without addition of L-arabinose, the methane produced was 39.7 mL (methane content in biogas 70.7%).

Example 6

[0075] P. stipitis were inoculated into 50 mL YDP medium at 30⁰C overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g L-arabinose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were placed in a water bath at 37°C with shaking at 150 rpm. After 6 days, 58.0 mL methane was produced (methane recovery 12.9%, methane content in biogas 18.7%). In contrast, in the case with addition of L-arabinose but without addition of P. stipitis, the methane produced was only 18.7 mL (methane recovery 4.2%, methane content in biogas 65.1%). In the case with addition of P. stipitis but without addition of L-arabinose, the methane produced was 33.5 mL (methane content in biogas 80.5%).

Example 7

[0076] E. coli TOP 10 were inoculated into 50 mL LB medium at 30⁰C for overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Four 4 mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.2 g L-arabinose. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were then placed in a water bath at 37°C with shaking at 150 rpm. After 6 days, 82.3 mL methane was produced (methane recovery 18.4 %, methane content in biogas 26.6%). In contrast, in the case with addition of L-arabinose but without addition of E. coli, the methane produced was only 18.7 mL (methane recovery 4.2%, methane content in biogas 65.1%). In the case with addition of E. coli but without addition of L-arabinose, the methane produced was 47.5 mL (methane content in biogas 56.2%).

Conversion of glycerol to biogas

[0077] The theoretical methane production from glycerol is 426 mL/g glycerol under standard conditions (0⁰C, 760 mmHg).

Glycerol (4C₃H₈O₃) → 5CO₂ + 7CH₄ + 2H₂O

1 g 304 mL 426 mL

Discussion

Table 5

Summarized methane production, productivity, yield and recovery from glycerol with exogenous E. coli.

Table 6 Summarized methane content in biogas produced from glycerol with exogenous

E. coli

Operation _{^ j 1} , Digested sludge+ Digested sludge + mode D'gestt** ^dge ^^ ^^ _{+ R ∞u}

co "nftenft (Z%), 64.9 24.0 19.7

[0078] From Figures 15 and 16 it is shown that the addition of exogenous E. coli that is able to utilize glycerol as the carbon and energy sources into the anaerobic reactors significantly improved the biogas production from glycerol. The addition of glycerol into the anaerobic reactor alone only slightly increased the biogas production compared to that of the control (digested sludge only), indicating the difficulty in converting glycerol to biogas by the microbes naturally occurring in the digested sludge. The addition of E. coli alone (without addition of glycerol) also slightly increased the biogas production, inferring that there existed some carbon sources that were unable to be degraded by the naturally occurring microbes in the digested sludge but were able to be degraded by E. coli. The highest methane production was achieved when both glycerol and E. coli were added, indicating that the exogenous glycerol was efficiently converted to biogas under the help of E. coli. This is ascribed to the conversion of glycerol to other smaller molecules that are easier substrates than glycerol for the anaerobic digestion by methanogens. Similarly, in the case of the addition of exogenous E. coli, the methane content was also significantly decreased due to the formation of CO₂ accompanying the glycerol degradation by E. coli.

Examples of glycerol conversion to biogas

Example 8

[0079] E. coli TOPlO was inoculated into 50 mL LB medium at 30⁰C overnight followed by washing with water (50 mL) twice. Then 50 mL water was added to re-suspend the cells. Then 4 mL of the re-suspended cells were added into 64 mL of digested sludge in 135 mL serum bottles followed by addition of 1.6 mL (2.02g) glycerol. The total volume of each reactor was adjusted to 80 mL using water. The headspace air of the bottles was replaced with nitrogen followed by sealing with butyl rubber stoppers. The reactors were then placed in water bath at 37⁰C with shaking at 150 rpm. After 7 days, 77.1 mL methane was produced (methane recovery 9.0%, methane content in biogas 19.7%). In contrast, in the case with addition of glycerol but without addition of E. coli, the methane produced was only 23.1 mL (methane recovery 2.7%, methane content in biogas 24.0%). hi the case with addition of E. coli but without addition of glycerol, the methane produced was 55.5 mL (methane content in biogas 56.2%)

[0080] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An anaerobic method for increasing the yield of conversion of a biomass to methane, wherein said biomass contains a hard substrate, said method comprising: contacting said biomass containing said hard substrate with an exogenous microbe to generate a reaction mixture; and fermenting said reaction mixture to increase the yield of methane.

2. The method of claim 1, wherein said hard substrate is a member selected from the group consisting of a pentose, glycerol and a mixture thereof.

3. The method of claim 2, wherein said pentose is a member selected from the group consisting of xylose, arabinose, lyxose, ribose and a mixture thereof.

4. The method of claim 2, wherein said pentose is a member selected from the group consisting of D-xylose and L-arabinose.

5. The method of claim 2, wherein said exogenous microbe is a member selected from group consisting of lactic acid bacteria, Pichia stipitis, Escherichia coli, hydrogen producing bacteria and a mixture thereof.

6. The method of claim 2, wherein said hard substrate is glycerol.

7. The method of claim 1, wherein said reaction mixture is fermented at a member selected from the group consisting of a psychrophilic temperature, a mesophilic temperature and a thermophilic temperature.

8. The method of claim 1, wherein said reaction mixture comprises a 5-20 volume % inoculum of said exogenous microbe.

9. The method of claim 1, wherein said method comprises: i) a hydrolysis step which converts complex molecules into simpler molecules; ii) an acidogenesis step, which transforms said simpler molecules into a product comprising fatty acids, alcohols, carbon dioxide and hydrogen; iii) an acetogenesis step, which provides the conversion of said products of the acidogenesis into acetic acid; and iv) a methanogenesis step, which converts acetic acid into methane.

10. The method of claim 1, wherein said method takes place in a one phase system.

11. The method of claim 10, wherein said one phase system is a member selected from the group consisting of UASB, UAFP and AFBR.

12. The method of claim 1, wherein said method takes place in a two phase system.

13. An anaerobic fermentation system for increasing the yield of conversion of a biomass to methane, said system comprising: an anaerobic fermenter having a biomass with a hard substrate; and an exogenous microbe to convert said hard substrate, wherein said biomass is fermented to increase the yield of methane.

14. The fermentation system of claim 13, wherein said hard substrate is a member selected from the group consisting of a pentose and glycerol.

15. The fermentation system of claim 14, wherein said pentose is a member selected from the group consisting of xylose, arabinose, lyxose and ribose.

16. The fermentation system of claim 14, wherein said pentose is a member selected form the group consisting of D-xylose and L-arabinose.

17. The fermentation system of claim 14, wherein said exogenous microbe is a member selected from group consisting of lactic acid bacteria, Pichia stipitis, Escherichia coli, hydrogen producing bacteria and a mixture thereof.

18. The fermentation system of claim 13, wherein said hard substrate is glycerol.

19. The fermentation system of claim 18, wherein said exogenous microbe is Escherichia coli.

20. The fermentation system of claim 13, wherein said reaction mixture is fermented at a member selected from the group consisting of a psychrophilic temperature, a mesophilic temperature and a thermophilic temperature.

21. The fermentation system of claim 13, wherein said reaction mixture comprises a 5-20 volume % inoculum of said exogenous microbe.

22. Use of an anaerobic method for increasing the yield of conversion of a biomass to methane, wherein said biomass contains a hard substrate, said use comprising: contacting said biomass containing said hard substrate with an exogenous microbe to generate a reaction mixture; and fermenting said reaction mixture to increase the yield of methane.

23. Use of an anaerobic method for increasing the yield of conversion of a biomass to methane of any claim 2-12, wherein said biomass contains a hard substrate and an exogenous microbe to create a reaction mixture and fermenting said reaction mixture to increase the yield of methane.

24. Use of an anaerobic method for increasing the yield of conversion of a biomass to methane of any claim 14-21, wherein said biomass contains a hard substrate and an exogenous microbe to create a reaction mixture and fermenting said reaction mixture to increase the yield of methane.