1. A method of treating Municipal Solid Waste (MSW) or unsorted MSW, comprising the steps of:

(i) the MSW is provided with a list of MSWs,

(ii) enzymatic hydrolysis with cellulase activity while microbial fermentation of the biodegradable fraction of MSW at a temperature suitable for enzymatic hydrolysis, such that the biodegradable fraction of waste is liquefied and microbial metabolites accumulate, followed by

(iii) Sorting the liquefied biodegradable fraction of the waste with non-degradable solids to produce a biological liquid, wherein at least 25 or 40 wt% of the non-water content is present as dissolved volatile solids comprising at least 25 wt% of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate; optionally, then

(iv) Anaerobically digesting the biological fluid to produce biological methane,

wherein the simultaneous microbial fermentation is effected by bacteria naturally present in the MSW.

2. The process according to claim 1, wherein the simultaneous microbial fermentation is effected by one or more species selected from the group consisting of lactic acid bacteria, acetogenic bacteria, ethanogenic bacteria, propionogenic bacteria, or butanogenic bacteria.

3. The method of claim 1, wherein sorting the liquefied biodegradable fraction from the non-degradable solids in the waste is accomplished by employing at least two separation operations sufficient to provide a biological liquid having at least 0.10kg volatile solids/kg treated MSW.

4. A method for producing biomethane, comprising the steps of:

(i) providing an organic liquid biological methane substrate pretreated by microbial fermentation such that at least 40% by weight of the non-water content is present as dissolved volatile solids comprising at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate,

(ii) transferring the liquid substrate to an anaerobic digestion system, and then

(iii) Anaerobic digestion of the liquid substrate is performed to produce biomethane.

5. The method of claim 4, wherein the microbial fermentation is conducted at a pH of less than 5.5.

6. The method of claim 4, wherein the organic liquid biomethane substrate is produced by simultaneous enzymatic hydrolysis and microbial fermentation of unsorted MSW.

7. The method of claim 4, wherein the organic liquid biomethane substrate is produced by simultaneous enzymatic hydrolysis and microbial fermentation of a pretreated lignocellulosic biomass.

8. The method of claim 4, wherein the organic liquid biomethane substrate is produced by an autoclave process by simultaneous enzymatic hydrolysis and microbial fermentation of liquefied organic material obtained from unsorted MSW.

9. The method of claim 4, wherein the organic liquid biomethane substrate comprises at least 8% total solids.

10. The method of claim 4, wherein the anaerobic digestion system is a fixed filter anaerobic digester.

11. The process according to claim 6, wherein the enzymatic hydrolysis and microbial fermentation are carried out at a temperature in the range of 30-75 ℃ or 45-50 ℃.

12. The method of claim 4, wherein at least 40% by weight of dissolved volatile solids in the biomethane substrate comprise lactate and/or wherein the biomethane substrate comprises a dissolved methane content of less than 15mg/L at 25 ℃.

13. The method of claim 4, wherein the liquid biomethane substrate is produced by hydrothermal pretreatment of a lignocellulosic biomass and/or the lignocellulosic biomass is pretreated at a pH of 3.5 to 9.0 and a temperature of 120 ℃ or greater.

14. An organic liquid biomethane substrate produced by enzymatic hydrolysis and microbial fermentation of a pretreated lignocellulosic biomass, characterized by a non-aqueous content of at least 40% by weight present as dissolved volatile solids comprising at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate.

15. The liquid biomethane substrate of claim 14 comprising a total solids content of at least 8%.

16. The liquid biomethane substrate of claim 14 having a dissolved methane content of less than 15mg/L at 25 ℃.

17. The liquid biomethane substrate of claim 14 comprising a total solids content of at least 10%.

SUMMARY

Drawings

FIG. 1. Dry matter conversion, expressed as a percentage of the dry matter recovered in the supernatant of the total dry matter in the simultaneous enzymatic hydrolysis and microbial fermentation stimulated by inoculation of the EC12B biological fluid from example 5.

FIG. 2 bacterial metabolites recovered from the simultaneous enzymatic hydrolysis and fermentation supernatant subsequently induced by the addition of the biological liquid from example 5.

FIG. 3 is a schematic diagram of a RENeScience test reactor.

FIG. 4 shows a schematic diagram of a plant installation.

FIG. 5 organic capture of biological fluid during different time periods, expressed as kg VS/kg treated MSW.

FIG. 6 bacterial metabolites expressed as percent VS solubilized in biological fluid and aerobic bacterial counts at various time points during the experiment.

FIG. 7. distribution of bacterial species identified in biological fluids from example 3.

FIG. 8 distribution of 13 dominant bacteria in EC12B sampled from the test described in example 5.

Figure 9. increase and decrease in bio-methane production with bio-liquids from example 5.

FIG. 10 "increase" and "decrease" characterization of the biological methane production of the "high lactate" biological fluid from example 2.

FIG. 11 is a representation of the "increase" and "decrease" in the production of biomethane from the "low lactate" biological fluid of example 2.

Figure 12 shows a "increase" characterization of bio-methane production of hydrolyzed wheat straw bio-liquid.

Detailed description of the embodiments

In some embodiments, the present invention provides a method of treating Municipal Solid Waste (MSW), comprising the steps of:

(i) providing MSW with a non-water content of 5-40% at a temperature of 45-75 ℃,

(ii) enzymatic hydrolysis at a temperature of 45-75 ℃ while microbial fermentation of the biodegradable fraction of MSW results in liquefaction of the biodegradable fraction of the waste and aggregation of microbial metabolites, followed by

(iii) Sorting a biodegradable portion of the liquefied waste from non-biodegradable solids to produce a biological fluid characterized by comprising dissolved volatile solids, wherein at least 25% by weight of the volatile solids comprise acetate, butyrate, ethanol, formate, lactate, and/or propionate, in any combination, and then

(iv) Anaerobically digesting the biological fluid to produce biological methane.

In some embodiments, the present invention provides an organic liquid biogas substrate produced by enzymatic hydrolysis and microbial fermentation of Municipal Solid Waste (MSW), characterized in that

-a non-water content of at least 40 wt% is present as dissolved volatile solids comprising at least 25 wt% of acetate, butyrate, ethanol, formate, lactate and/or propionate, in any combination.

In some embodiments, the present invention provides a method of producing biogas comprising the steps of:

(i) providing an organic liquid biogas substrate pretreated by microbial fermentation wherein at least 40% by weight of the non-water content is present as dissolved volatile solids comprising at least 25% by weight of acetate, butyrate, ethanol, formate, lactate and/or propionate in any combination,

(ii) transferring the liquid substrate to an anaerobic digestion system, and then

(iii) Anaerobic digestion of the liquid substrate is performed to produce biomethane.

The metabolic dynamics of the microbial community involved in anaerobic digestion are very complex. See supapuol et al, 2010; morita and Sasaki 2012; chandra et al, 2012. In typical Anaerobic Digestion (AD) for methane biogas production, four major steps are accomplished by microorganism-mediated biological processes-hydrolysis of biological macromolecules into constituent monomers or other metabolites; acidification, wherein short chain hydrocarbon acids and alcohols are produced; acetoxylation, in which available nutrients are dissimilated into acetic acid, hydrogen and carbon dioxide; and methanation, in which acetic acid and hydrogen are dissimilated into methane and carbon dioxide by a specialized archaebacteria. The overall hydrolysis step is rate-limiting. See Delgenes et al, 2000; angelidaki et al, 2006; cysneiros et al 2011.

It is therefore advantageous to prepare the substrate for biomethane production by hydrolysis ahead of time by some form of pretreatment. In some embodiments, the inventive process combines microbial fermentation and enzymatic hydrolysis of MSW not only as a rapid biological pretreatment for final biomethane production, but also as a method of sorting degradable organic components from unsorted MSW.

Biological pretreatment with solid biomethane substrate containing organic components of the source classification in MSW has been reported. See Fdez-Guelfo et al, 2012; Fdez-Guelfo et al, 2011A; Fdez-Guelfo et al, 2011B; ge, etc., 2010; lv et al, 2010; borghi et al, 1999. The increase in final methane production in anaerobic digestion is reported as a result of increased degradation of complex biopolymers and increased dissolution of volatile solids. However, the level of dissolution of volatile solids and their conversion to volatile fatty acids achieved by these previously reported methods does not even approach the levels achieved by the present methods. For example, Fdez-Guelfo et al, 2011A report that a relative increase in solubility of the volatile solids of 10-15% was achieved by various biological pre-treatments of the pre-sorted organic fraction in MSW, which corresponds to a final absolute solubility level of 7-10% of the volatile solids. In contrast, the liquid biomethane substrate produced by the process of the present invention contains at least 40% dissolved volatile solids.

Two-stage anaerobic digestion systems have also been reported in which the first stage process hydrolyzes a biomethane substrate including source sorted organic components and other specific biological substrates in MSW. In the first anaerobic stage, which is usually thermophilic, the high chain polymers degrade to produce volatile fatty acids. This is followed by a second stage anaerobic phase in a physically separate reactor, in which methanation and acetoxylation predominate. The reported two-stage anaerobic digestion systems typically use source-sorted, specific, biologically derived substrates with less than 7% total solids. See supapuol et al, 2011; kim et al, 2011; lv et al, 2010; riau et al, 2010; kim et al, 2004; schmit and Ellis 2000; Lafit-Trouque and Forster 2000; dugba and Zhang 1999; kaiser et al, 1995; harris and Dague 1993. Recently, some two-stage AD systems have been reported that use source-sorted, specific, biologically derived substrates containing up to 10% total solids. See Yu et al, 2012; lee et al, 2010; zhang et al, 2007. Of course, none of the reported two-stage anaerobic digestion systems contemplate the use of unsorted MSW as a substrate, let alone for the production of high solids liquid biomethane substrate. Two-stage anaerobic digestion is to convert the solid substrate, continuously feed additional solids to the first stage reactor, and continuously remove volatile fatty acids from the first stage reactor.

Any suitable solid waste may be used to carry out the process of the invention. The skilled person will understand that the term "municipal solid waste" (MSW) refers to the waste fraction commonly found in cities, which itself does not need to come from a city. The MSW may be any combination of cellulosic, plant, animal, plastic, metal, or glass waste, including but not limited to one or more of the following: the refuse collected by normal urban collection systems, optionally in some central sorting, crushing or beating apparatus, e.g.OrPerforming intermediate treatment; solid waste sorted out at home, comprising an organic fraction and a paper-rich fraction; waste fractions from industries such as restaurant industries, food processing industries, general industries; a waste fraction from the paper industry; a waste fraction from a recycling facility; waste fractions from the food or feed industry; waste fractions from the pharmaceutical industry; waste fractions from agricultural or farm-related sectors; waste fractions from the processing of products rich in sugars or starches; contaminated or otherwise deteriorated agricultural products, such as grains, potatoes and sugar beets, which cannot be used to produce food or feed; garden garbage.

MSW is generally heterogeneous in nature. Statistical data that provide a solid foundation for comparisons between countries is not well known with respect to the composition of waste materials. The standards for proper sampling and characterization and the operational flow are not yet standardized. In fact, only few standardized sampling methods have been reported. See Riber et al, 2007. At least in the context of household waste, its composition exhibits seasonal and geographical variations. See Dahlen et al, 2007; eurostat, 2008; hansen et al, 2007 b; muhle et al, 2010; riber et al, 2009; simmons et al, 2006; the Danish Environmental Protection agency, 2010. Geographical variation in the composition of household waste is also reported, even between different cities at short distances of 200- & 300km (Hansen et al, 2007 b).

In some embodiments, the MSW is treated as "unsorted" waste. The term "unsorted" as used herein refers to a process in which MSW is not sufficiently separated into separate fractions, such that the material of biological origin is not sufficiently separated from plastic and/or other inorganic materials. By this definition, the waste may be "unsorted" as used herein, despite the removal of some large objects or metal objects, and despite some separation of the plastic and/or inorganic materials. As used herein, "unsorted" waste refers to waste that has not been separated sufficiently to provide a biologically derived fraction in which less than 15% by dry weight is non-biologically derived material. A waste product comprising a mixture of material of biological and non-biological origin, wherein more than 15% of the dry weight of the waste product is non-biological origin, is "unsorted" as used herein. Typically, unsorted MSW comprises waste of biological origin, i.e. waste that is degradable into biologically convertible material, including food and kitchen waste, paper and/or cardboard containing material, food waste, etc.; recyclable materials, including glasses, bottles, cans, metals, and certain plastics; other combustible materials, although by themselves somewhat non-recyclable, may provide thermal energy in the form of waste derived fuels; and inert materials including ceramics, rocks, and various forms of debris.

In some embodiments, MSW may be "sorted" waste disposal. The term "sorted" as used herein refers to a process in which MSW is sufficiently divided into but separate fractions so that the material of biological origin is sufficiently separated from the plastic and/or other inorganic material. A waste product comprising a mixture of material of biological and non-biological origin, wherein less than 15% of the dry weight of the waste product is non-biological origin, is "sorted" as used herein.

In some embodiments, the MSW may be a source-separated organic waste that primarily comprises fruit, vegetable, and/or animal waste. Various sorting systems may be used in some embodiments, including source sorting, in which a household separately processes different waste materials. Source sorting systems are currently in place in certain cities of austria, germany, ruthenburg, sweden, belgium, the netherlands, spain and denmark. Alternatively, an industrial sorting system may be used. Means for mechanical sorting and separation include any method known in the art, including but not limited to those described in US 2012/0305688; WO 2004/101183; WO 2004/101098; WO 2001/052993; WO 2000/0024531; WO 1997/020643; WO 1995/0003139; CA 2563845; the system described in US 5465847. In some embodiments, the waste may be sorted slightly, but still produce an "unsorted" waste fraction as used herein. In some embodiments, unsorted MSW is used wherein greater than 15% of the dry weight is non-biogenic material, or greater than 18%, or greater than 20%, or greater than 21%, or greater than 22%, or greater than 23%, or greater than 24%, or greater than 25%.

In the practice of the present invention, the MSW should be provided at a non-water content of 10-45%, or in certain embodiments 12-40%, or 13-35%, or 14-30%, or 15-25%. MSW typically contains a significant water content. All other solids in MSW are referred to as "non-water content" as used herein. The moisture level used in the practice of the present invention is related to several interrelated variables. The process of the invention produces a slurry of liquid biological origin. Those skilled in the art will appreciate that the ability to convert solid components into a liquid slurry increases with increasing water content. Efficient pulping of paper and board containing high amounts of typical MSW generally improves with increasing moisture content. Furthermore, it is well known that when hydrolysis is performed under conditions of low water content, the enzyme activity shows a decreased activity. For example, cellulases generally exhibit reduced activity when hydrolyzing mixtures with non-aqueous amounts above 10%. In the case of cellulases degrading paper and board, an efficient linear inverse relationship of substrate concentration and yield from enzymatic reactions per gram of substrate has been reported. See Kristensen et al, 2009. With commercially available isolated enzyme preparations optimized for lignocellulosic biomass conversion, we observed that non-aqueous content can be as high as 15% in pilot scale studies without observing clear detrimental effects.

In some embodiments, some amount of water should normally be added to the waste to achieve the appropriate non-water content. For example, consider a portion of unsorted danish household waste. Table 1 describes the unique composition of unsorted MSW reported in Riber et al (2009), "Chemical composition of material fractions in Danish household Waste," Waste Management 29: 1251. Riber et al characterized the component parts of household waste obtained from 2220 household in Denmark within one day 2001. It is readily understood by those skilled in the art that the composition reported is only one representative example for explaining the method of the present invention. In the example of table 1, without adding any water amount prior to mild heating, the organic degradable fraction containing vegetable, paper and animal waste is expected to have a non-water content of about 47% on average. [ (absolute nonaqueous%)/(% wet weight) — (7.15+18.76+4.23)/(31.08+23.18+9.88) — 47% nonaqueous content. Adding a volume of water corresponding to 1 weight percent of the waste fraction being treated reduces the non-water content of the waste itself to 29.1% (58.2%/2) while reducing the non-water content of the degradable component to about 23.5% (47%/2). The addition of a volume of water corresponding to 2% by weight of the waste fraction treated reduces the non-water content of the waste itself to 19.4% (58.2%/3) and simultaneously reduces the non-water content of the degradable component to about 15.7% (47%/3).

TABLE 1 Mass distribution of waste fractions summarized in Denmark 2001

(a) Pure fraction.

(b) Newspapers, magazines, advertisements, books, clean/dirty office paper, paper and carton containers, cardboard, plastic-bearing cartons, aluminum foil-bearing cartons, dirty cardboard, and kitchen paper towels.

(c) Soft plastics, plastic bottles, other hard plastics and non-recyclable plastics.

(d) Soil, rock, etc., ash, ceramics, cat litter, and other non-combustible materials.

(e) Aluminum containers, aluminum foil, metal containers, and other metals.

(f) Clear glass, green glass, brown glass and other glasses.

(g) The sum of the remaining 13 material fractions.

In the practice of the present invention, the appropriate amount of water to be added to the waste can be readily determined by one skilled in the art, if desired. Generally as a practical matter, despite some variability in the composition of the MSW being treated, it is convenient to add a relatively constant mass ratio of water, in some embodiments, from 0.8 to 1.8kg water/kg MSW, or from 0.5 to 2.5 water/kg MSW, or from 1.0 to 3.0 water/kg MSW. Thus, the actual non-water content in the MSW may vary within a suitable range during the treatment. Suitable levels of non-water content may vary depending on the manner in which enzymatic hydrolysis is effected.

Enzymatic hydrolysis can be achieved in a variety of different ways. In some embodiments, enzymatic hydrolysis may be achieved by an isolated enzyme preparation. As used herein, the term "isolated enzyme preparation" refers to a preparation comprising an enzyme activity, which preparation is extracted, secreted, or obtained by biological origin, optionally partially or extensively purified.

Various enzymatic activities can be advantageously used in practicing the methods of the invention. For example, with respect to the MSW composition shown in table 1, it is apparent that the paper-containing waste contains the largest dry weight of the biomaterial single component. Thus, it will be apparent to those skilled in the art that cellulose degrading activity will be particularly advantageous for typical household waste. In paper-containing waste, cellulose is pre-processed and separated from its natural components as lignocellulosic biomass components mixed with lignin and hemicellulose. Thus, paper-containing waste can be advantageously degraded with relatively "simple" cellulase preparations.

"cellulase activity" refers to the enzymatic hydrolysis of 1, 4-B-D-glucosidic bonds in cellulose. In isolated cellulase preparations obtained from bacteria, fungi or other sources, the cellulase activity typically comprises a mixture of different enzyme activities, including endoglucanases and exoglucanases (also called cellobiohydrolases) which catalyze the endo-and exo-hydrolysis of 1, 4-B-D-glycosidic linkages, respectively, and B-glucosidases which hydrolyze the exoglucanase hydrolyzed oligosaccharide products to monosaccharides. Complete hydrolysis of insoluble cellulose usually requires a synergy between the different enzymes.

As a practical matter, it is advantageous in some embodiments to simply use commercially available isolated cellulase preparations optimized for lignocellulosic biomass conversion, since these enzyme preparations are available at a fairly low cost. These formulations are of course suitable for use in carrying out the method of the invention. The term "optimized for lignocellulosic biomass conversion" refers to a product development process in which a mixture of enzymes is selected and modified for the specific purpose of increasing hydrolysis yield and/or reducing enzyme consumption for hydrolysis of pretreated lignocellulosic biomass to fermentable sugars.

However, commercially available cellulase enzyme mixtures optimized for hydrolysis of lignocellulosic biomass often contain high levels of other specialized enzyme activities. For example, we determined that commercially available NOVOZYMES^TMUnder the trademark CELLIC CTEC2^TMAnd CELLIC CTEC3^TMCellulase preparations optimized for lignocellulosic biomass conversion, and compositions and methods of use thereof^TMUnder the trademark ACCELLERASE 1500^TMThe enzyme activities present in similar preparations were provided and each of these preparations was found to compriseEndoxylanase activity at a level of more than 200U/g, xylosidase activity at a level of more than 85U/g, B-L-arabinofuranosidase activity at a level of more than 9U/g, amyloglucosidase activity at a level of more than 15U/g, and a-amylase activity at a level of more than 2U/g.

Simpler isolated cellulase preparations may also be effectively used in practicing the methods of the invention. Suitable cellulase preparations may be obtained from a variety of microorganisms by methods well known in the art, including aerobic and anaerobic bacteria, white rot fungi, soft rot fungi, and anaerobic fungi. Cellulolytic enzymes typically produce a mixture of different enzymes in suitable proportions for hydrolysis of lignocellulosic substrates as described in reference 13, R.Singhaia et al, "advanced and comparative profiles in the production technologies using solid-state and sub-polymerization for Microbial cells," Enzyme and Microbial Technology (2010)46:541-549, which is expressly incorporated herein by reference in its entirety. Preferred sources of cellulase preparations useful for lignocellulosic biomass conversion include fungi such as species of Trichoderma (Trichoderma), Penicillium (Penicillium), Fusarium (Fusarium), Humicola (Humicola), Aspergillus niger (Aspergillus), and Phanerochaete (Phanerochaete).

In addition to cellulase activity, some other enzyme activities that have proven advantageous in practicing the methods of the present invention include enzymes that act on food waste, such as proteases, glucoamylases, endo-amylases, proteases, pectinesterases, pectin lyases, and lipases, as well as enzymes that act on garden waste, such as xylanases and xylosidases. In some embodiments, other enzymatic activities such as laminarinase (laminarase), keratinase, or laccase are advantageously included.

In some embodiments, selected microorganisms exhibiting extracellular cellulase activity can be inoculated directly for simultaneous enzymatic hydrolysis and microbial fermentation, including but not limited to one or more thermophilic cellulolytic organisms, which can be inoculated alone or with other organisms: paenibacillus (Paenibacillus baccinonensis), see Asha et al, 2012; clostridium thermocellum (Clostridium thermocellum), see Blume et al, 2013 and Lv and Yu, 2013; selected species of Streptomyces (Streptomyces), microbacterium (Microbispora) and bacillus (Paenibacillus), see Eida et al, 2012; clostridium traminosolvens, see Kato et al, 2004; species of the genera Firmicutes (Firmicutes), actinomycetes (actinobacilla), Proteobacteria (Proteobacteria) and bacteroides (bacteroides), see Maki et al, 2012; clostridium clariflavum, see Sasaki et al, 2012; new species of Clostridium phylogenetically and physiologically related to Clostridium thermocellum (Clostridium thermocellum) and Clostridium straminisolvens, see Shiratori et al, 2006; clostridium clariflavum sp.nov. and Clostridium Caenicola, see Shiratori et al, 2009; geobacillus thermophilus (Geobacillus Thermoleovorans), see Tai et al, 2004; clostridium stercorarium, see Zverlov et al, 2010; or one or more of the thermophilic fungi Thermomyces thermophilus (Sporotrichum thermophile), Scytalidium thermophilum, Clostridium tramidium straminisolvens and Thermomonospora flexuosa (Thermomospora curvata), as reviewed in Kumar et al, 2008. In some embodiments, microorganisms exhibiting other useful extracellular enzyme activities may be inoculated for simultaneous enzymatic hydrolysis and microbial fermentation, for example, protein and keratin degrading fungi, see Kowalska et al, 2010, or lactic acid bacteria exhibiting extracellular lipase activity, see Meyers et al, 1996.

The enzymatic hydrolysis may be carried out by methods well known in the art, using one or more isolated enzyme preparations of any one or more of a variety of enzyme preparations, including any of those previously mentioned, or by treating the MSW with one or more selected biological inoculants capable of effecting the desired enzymatic hydrolysis. In some embodiments, the enzymatic hydrolysis is performed using an effective amount of one or more isolated enzyme preparations comprising cellulase, β -glucosidase, amylase, and xylanase activities. The amount is an "effective amount" in which the enzyme preparation used together achieves a dissolution of at least 40% of the dry weight of the degradable bio-sourced material present in the MSW within a hydrolysis reaction time of 18 hours under the conditions used. In some embodiments, one or more isolated enzyme preparations are used, wherein the relative proportions of the various enzyme activities are collectively as follows: the enzyme activity mixture is used such that the cellulase activity of 1FPU thereof is correlated with an endoglucanase activity of at least 31 CMC U and the cellulase activity of 1FPU is correlated with a beta-glucosidase activity of at least 7pNPG U. The person skilled in the art will readily understand that CMC U refers to carboxymethyl cellulose units. One CMC U activity released 1umol of reducing sugars (expressed as glucose equivalent) within 1 minute under specific assay conditions at 50 ℃ and pH 4.8. One skilled in the art will readily understand that pNPG U refers to a single bit of pNPG. One pNPG U activity released 1umol nitrophenol per minute from p-nitrophenyl-B-D-glucoside at 50 ℃ and pH 4.8. One skilled in the art will also readily appreciate that "filter paper units" FPU provide a measure of cellulase activity. As used herein, FPU refers to a unit of filter paper as determined using the method in Adney, B.and Baker, J., Laboratory Analytical Procedure #006, "Measurement of cellular activity", August 12,1996, the USA National Renewable Energy Laboratory (NREL), which is expressly incorporated herein by reference in its entirety.

In practicing embodiments of the invention, it may be advantageous to adjust the temperature of the MSW prior to starting the enzymatic hydrolysis. It is well known in the art that cellulases and other enzymes generally exhibit an optimal temperature range. Although examples of enzymes isolated from extreme thermophilic organisms are of course known, their optimum temperature is around 60 or even 70 ℃ and the optimum temperature range for enzymes generally falls within the range of 35-55 ℃. In some embodiments, the enzymatic hydrolysis is carried out at a temperature in the range of 30-35 ℃, or 35-40 ℃, or 40-45 ℃, or 45-50 ℃, or 50-55 ℃, or 55-60 ℃, or 60-65 ℃, or 65-70 ℃, or 70-75 ℃. In some embodiments, it is advantageous to perform the enzymatic hydrolysis and microbial fermentation simultaneously at a temperature of at least 45 ℃ as this is advantageous in hindering the growth of MSW-beren pathogenic bacteria. See, e.g., Hartmann and Ahring 2006; ports et al, 1998; carrington et al, 1998; bendixen et al, 1994; kubler et al, 1994; six and De Baerre et al, 1992.

Enzymatic hydrolysis using cellulase activity typically saccharifies cellulosic material. Thus, in the enzymatic hydrolysis process, the solid waste is saccharified and liquefied, i.e. converted from a solid form into a liquid slurry.

Previously, methods of processing MSW to achieve liquefaction of components of biological origin using enzymatic hydrolysis have anticipated the need to heat the MSW to temperatures much higher than those required for enzymatic hydrolysis, particularly in order to achieve "sterilization" of the waste, followed by a cooling step necessary to bring the heated waste back to a temperature suitable for enzymatic hydrolysis. In practicing the method of the invention, it is sufficient to simply subject the MSW to a temperature suitable for enzymatic hydrolysis. In some embodiments, it is advantageous to simply adjust the MSW to a suitable non-water content with hot water in such a way that the MSW is brought to a temperature suitable for enzymatic hydrolysis. In some embodiments, the MSW is heated by adding an amount of hot water, or steam, or by other heating means, to the reaction vessel. In some embodiments, the MSW is heated in the reaction vessel to a temperature of greater than 30 ℃ but less than 85 ℃, or to a temperature of 84 ℃ or less, or to a temperature of 80 ℃ or less, or to a temperature of 75 ℃ or less, or to a temperature of 70 ℃ or less, or to a temperature of 65 ℃ or less, or to a temperature of 60 ℃ or less, or to a temperature of 59 ℃ or less, or to a temperature of 58 ℃ or less, or to a temperature of 57 ℃ or less, or to a temperature of 56 ℃ or less, or to a temperature of 55 ℃ or less, or to a temperature of 54 ℃ or less, or to a temperature of 53 ℃ or less, or to a temperature of 52 ℃ or less, or to a temperature of 51 ℃ or less, or to a temperature of 50 ℃ or less, or to a temperature of 49 ℃ or less, or to a temperature of 48 ℃ or less, or to a temperature of 47 ℃ or less, or to a temperature of 46 ℃ or less, Or to a temperature of 45 c or less. In some embodiments, the MSW is heated to a temperature no higher than 10 ℃ above the maximum temperature at which the enzymatic hydrolysis is carried out.

As used herein, heating the MSW to "a temperature" refers to raising the average temperature of the MSW to that temperature in the reactor. As used herein, the temperature to which the MSW is heated is the highest average temperature of the MSW reached in the reactor. In some embodiments, the highest average temperature may not be maintained throughout the process. In some embodiments, the heating reactor may comprise different zones to allow heating to occur at stages of different temperatures. In some embodiments, heating may be accomplished in the same reactor in which the enzymatic hydrolysis is performed. The purpose of heating is to simply bring a large portion of the cellulosic waste and a large portion of the plant waste into an optimal environment for enzymatic hydrolysis. In order to be in optimal conditions for enzymatic hydrolysis, the waste should ideally have a temperature and water content suitable for the enzymatic activity employed in the enzymatic hydrolysis.

In some embodiments, it is advantageous to stir during heating to achieve uniformly heated waste. In some embodiments, agitation may include free-fall mixing, such as mixing in a reactor having a chamber that rotates substantially along a horizontal axis or in a mixer having a rotating shaft that raises and lowers the MSW or a mixer having a horizontal axis or paddle that raises and lowers the MSW. In some embodiments, agitation may include shaking, stirring, or conveying by a conveying screw conveyor. In some embodiments, stirring is performed after heating the MSW to the desired temperature. In some embodiments, the stirring is performed for 1 to 5 minutes, or 5 to 10 minutes, or 10 to 15 minutes, or 15 to 20 minutes, or 20 to 25 minutes, or 25 to 30 minutes, or 30 to 35 minutes, or 35 to 40 minutes, or 40 to 45 minutes, or 45 to 50 minutes, or 50 to 55 minutes, or 55 to 60 minutes, or 60 to 120 minutes.

The enzymatic hydrolysis is started when the isolated enzyme preparation is added. Alternatively, in the case where a microorganism exhibiting the desired extracellular enzyme activity is employed instead of adding a separate enzyme preparation, the enzymatic hydrolysis is initiated upon addition of the desired microorganism.

In practicing the methods of the invention, enzymatic hydrolysis is performed simultaneously with microbial fermentation. Concurrent microbial fermentation can be achieved by a variety of different methods. In some embodiments, the microorganisms naturally present in the MSW are simply grown under reaction conditions, wherein the treated MSW is not preheated to a temperature sufficient to produce a "sterilizing" effect. Typically, the microorganisms present in MSW include organisms that adapt to the local environment. The overall benefit of simultaneous microbial fermentation is greater, meaning that a very wide variety of different organisms can contribute to organic capture by enzymatically hydrolyzing MSW either individually or collectively. Without wishing to be bound by theory, we believe that the co-fermenting microorganisms alone have some direct effect on the degradation of food waste that is not hydrolysed by cellulase enzymes. At the same time, the carbohydrate monomers and oligomers released by the hydrolysis of cellulases are particularly susceptible to consumption by almost any microbial species. This provides a beneficial synergistic effect for the cellulase, possibly through the inhibited release of the enzyme activity product, and possibly for other reasons that do not immediately manifest. The end products of the microbial metabolism are in any case generally suitable as biological methane substrates. Thus, enrichment of microbial metabolites with enzymatically hydrolyzed MSW has itself led to an increase in the quality of the biomethane substrate. Lactic acid bacteria are ubiquitous in nature in particular, and lactic acid production is generally observed when MSW with a non-water content of 10-45% is enzymatically hydrolyzed in the 45-50 temperature range. At higher temperatures, other naturally occurring microbial species may dominate, and microbial metabolites other than lactic acid become more prevalent.

In some embodiments, microbial fermentation may be achieved by direct inoculation with one or more microbial species. One skilled in the art will readily appreciate that it may be advantageous to select one or more bacterial species for inoculation to provide simultaneous enzymatic hydrolysis and fermentation of MSW, wherein the bacterial species is capable of growing at or near the optimum temperature for the enzymatic activity employed.

Inoculation of the hydrolysis mixture to induce microbial fermentation can be accomplished by a variety of different methods.

In some embodiments, it is advantageous to inoculate the MSW before, after or simultaneously with the addition of the enzyme activity or the addition of a microorganism exhibiting extracellular cellulase activity. In some embodiments, it may be advantageous to employ one or more LAB species including, but not limited to, one or more of the following, or genetically modified variants thereof: lactobacillus plantarum (Lactobacillus plantarum), Streptococcus lactis (Streptococcus lactis), Lactobacillus casei (Lactobacillus casei), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus sake (Lactobacillus sake), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus delbrueckii (Lactobacillus juvenii), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus carnosus (Lactobacillus carnis), Lactobacillus piscicola (Lactobacillus piscicola), Lactobacillus corynebacterium clavatus (Lactobacillus piscicola), Lactobacillus corynebacterium (Lactobacillus corynebacterium parahaemolyticum), Lactobacillus rhamnosus (Lactobacillus rhamnophilus), Lactobacillus maltulinum (Lactobacillus malus), Lactobacillus pseudolactis (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus lactis), Lactobacillus plantarum (Lactobacillus), Lactobacillus casei (Lactobacillus lactis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus), Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus (Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus plantarum) and Lactobacillus (Lactobacillus), Lactobacillus plantarum) strain (Lactobacillus), Lactobacillus plantarum, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum, Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus plantarum, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus, lactobacillus casei (Lactobacillus lactis), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus homolyticus), Lactobacillus sanfrancisciensis (Lactobacillus sanfrancisco), Lactobacillus fructovorus (Lactobacillus fructivorans), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus ponti, Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus bulgaricus (Lactobacillus buchneri), Lactobacillus viridans (Lactobacillus virescens), Lactobacillus fuscus (Lactobacillus consortium), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus plantarum (Lactobacillus salivarius), Lactobacillus sanfrancisciensis), Lactobacillus kansuensis (Lactobacillus sanfranciscensis), Lactobacillus plantarum (Lactobacillus sancticus), Lactobacillus sancticus (Lactobacillus sanus), Lactobacillus sancus), Lactobacillus (Lactobacillus sancticus), Lactobacillus sanus, Lactobacillus sancticus (Lactobacillus sanus), Lactobacillus sancus), Lactobacillus sancticus (Lactobacillus sanus), Lactobacillus sanus, Lactobacillus sancticus), Lactobacillus (Lactobacillus sanus), Lactobacillus sancticus), Lactobacillus sanus (Lactobacillus sanus), Lactobacillus sanus, Lactobacillus sancticus), Lactobacillus sanus (Lactobacillus sanus), Lactobacillus sancticus (Lactobacillus sanus ), Lactobacillus sanus, Lactobacillus sancticus), Lactobacillus sanus, Lactobacillus sancticus (Lactobacillus sanus), Lactobacillus sancticus (Lactobacillus sanus, Lactobacillus sancticus), Lactobacillus sanus, Lactobacillus sancticus, Lactobacillus sanus, Lactobacillus sanoticus, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus sanctius, Lactobacillus sanus, Lactobacillus, Lactobacillus salivarius, Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus suenicus, Lactobacillus oralis (Lactobacillus oris), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus vaginalis (Lactobacillus vagiana), Lactobacillus pentosus (Lactobacillus pentosus), Lactobacillus bakeri (Lactobacillus panis), Lactococcus lactis (Lactobacillus cremoris), Lactococcus glucanus (Lactobacillus stranatus), Lactococcus gargaricus (Lactobacillus gargari), Lactococcus gargaricus (Lactobacillus garvieae), Lactococcus hollandii (Lactobacillus hominis), Lactococcus gossypii (Lactobacillus raffinosus), Streptococcus diacetylactis (Lactobacillus diacetatus), Leuconostoc mesenteroides (Leuconostoc), Leuconostoc lactis (Leuconostoc serosa), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis) and Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis) and Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis) and Leuconostoc lactis (Leuconostoc lactis) and Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis) and Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis) and Leuconostoc lactis (Leuconostoc lactis) and Leuconostoc lactis), Leuconostoc lactis (Leuconostoc lactis), Leuconostoc lactis, Pediococcus nociceps (Pediococcus damnosus), Pediococcus acidilactici (Pediococcus acidilactici), Pediococcus cerevisiae (Pediococcus cerivisiae), Pediococcus parvus (Pediococcus parvus), Pediococcus halophilus (Pediococcus halophilus), Pediococcus pentosaceus (Pediococcus pentosacus), Pediococcus intermedius (Pediococcus intecticus), Bifidobacterium longum (Bifidobacterium longum), Streptococcus thermophilus (Streptococcus thermophilus), Pediococcus oeni (Ocococcus oeni), Bifidobacterium breve (Bifidobacterium breve), and Bifidobacterium breve (Propionibacterium freudenreichii), or the subsequently found LAB species or inoculated with a strain from the genus Enterococcus (Enterobacter), Lactobacillus (Lactobacillus propionate), Lactobacillus paracasei (Lactobacillus), or a strain exhibiting a metabolic capability to produce other species, such as lactic acid bacteria (Streptococcus acidilactici).

One skilled in the art will readily appreciate that the bacterial preparation used for inoculation may comprise a collection of different organisms. In some embodiments, naturally occurring bacteria that are present in any given geographic region and are suitable for growth in MSW from that region may be employed. As is well known in the art, LAB are ubiquitous and typically comprise a major component of any naturally occurring bacterial population in MSW.

In some embodiments, the MSW may be inoculated with naturally occurring bacteria by continuously recovering wash water or process liquor used to recover residual organic material from non-degradable solids. With the recovery of the wash water or process liquid, it gradually acquires a higher level of microorganisms. In some embodiments, the microbial fermentation has a pH lowering effect, particularly when the metabolite comprises short chain carboxylic/fatty acids such as formate, acetate, butyrate, propionate, or lactate. Thus, in some embodiments, it is advantageous to monitor and adjust the pH of the simultaneous enzymatic hydrolysis and microbial fermentation mixture. When wash water or process liquor is used to increase the water content of the incoming waste prior to enzymatic hydrolysis, it is advantageous to perform the inoculation before adding the enzyme activity in the form of an isolated enzyme preparation or a microorganism exhibiting extracellular cellulase activity. In some embodiments, naturally occurring bacteria suitable for growth on MSW from a particular region may be cultured on MSW or on liquefied organic components obtained by enzymatic hydrolysis of MSW. In some embodiments, the cultured naturally occurring bacteria may then be added as an inoculum separately or in addition to inoculation with recovered wash water or process liquor. In some embodiments, the bacterial preparation may be added prior to or simultaneously with the addition of the isolated enzyme preparation, or after a period of time following the start of prehydrolysis.

In some embodiments, specific strains, including strains that have been specially modified or "trained" to grow under enzymatic hydrolysis reaction conditions and/or to enhance or de-enhance specific metabolic processes, may be cultured for inoculation. In some embodiments, it is advantageous to inoculate MSW with a bacterial strain identified as capable of surviving phthalate as the sole carbon source. Such strains include, but are not limited to, any one or more of the following, or genetically modified variants thereof: chrysomicrobium interchelnse MW10T, Lysinibacter fusformis NBRC 157175, Tropicobacter thermophilus, Gordonia JDC-2(Gordonia JDC-2), Arthrobacter JDC-32, Bacillus subtilis3C 3(Bacillus subtilis3C3), Comamonas testosteroni (Comamosonii), Comamonas comamonii E6(Comamonas E6), Delftia tsukutenensis (Delftia tsukutensis), Rhodococcus joris, Burkholderia cepacia (Burkholderia cepacia), Mycobacterium vaceae (Mycobacterium vanbaalenii), Arthrobacter arthricus, Bacillus subtilis 007, Pseudomonas inteiprocasiae 2P 23, Pseudomonas sp 23, Pseudomonas aeruginosa (Pseudomonas sp.g.), Pseudomonas aeruginosa P23, Pseudomonas sp.7, Pseudomonas sp.3, Pseudomonas sp. P368, Pseudomonas sp. 368, Pseudomonas sp 3, Pseudomonas sp. P3, Pseudomonas sp. 368, Pseudomonas sp. P3, Pseudomonas sp.3, Pseudomonas sp. P. 12, Pseudomonas sp.7, Pseudomonas sp.3, Pseudomonas sp.g. P. Pseudomonas sp.3, Pseudomonas sp.7, Pseudomonas sp.sp.sp.sp.sp.g. Pseudomonas sp.sp.sp.sp.sp.sp.3, Pseudomonas sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp, Pseudomonas sp, Pseudomonas sp.sp.sp.sp.sp, Pseudomonas sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp., Gordonia CNJ863(Gordonia sp CNJ863), Gordonia rubripecticus, Arthrobacter oxydans, Acinetobacter genoosp, and Acinetobacter calcoaceticus. See, e.g., Fukuhura et al, 2012; iwaki et al, 2012A; iwaki et al, 2012B; latore et al, 2012; liang et al, 2010; liang et al, 2008; navacharogen et al, 2011; park et al, 2009; wu et al, 2010; wu et al, 2011. Phthalates used as plasticizers in many commercial polyvinyl chloride products are leachable and, according to our experience, are often present in the liquefied organic component at undesirable levels. In some embodiments, strains that have been genetically modified by methods well known in the art may be advantageously employed to enhance metabolic processes and/or to de-enhance other metabolic processes, including but not limited to processes that consume glucose, xylose, or arabinose.

In some embodiments, it is advantageous to inoculate MSW with a bacterial strain identified as capable of degrading lignin. Such strains include, but are not limited to, any one or more of the following, or genetically modified variants thereof: comamonas sp B-9(Comamonas sp B-9), Citrobacter freundii (Citrobacter freundii), Citrobacter FJ581023(Citrobacter sp FJ581023), Pantoea henburgensis (Pandora norbergensis), Achromobacter amycolata ATCC39116(Amycolatopsis sp ATCC 39116), Streptomyces viridosporus, Rhodococcus jostii, and Sphingomonas SYK-6(Sphingobium sp. SYK-6). See, e.g., Bandounas et al, 2011; bugg et al, 2011; chandra et al, 2011; chen et al, 2012; davis et al, 2012. According to our experience, MSW typically contains considerable amounts of lignin that is typically recovered as undigested residue after AD.

In some embodiments, it is advantageous to inoculate MSW with a bacterial strain capable of producing acetate, including, but not limited to, any one or more of the following, or genetically modified variants thereof: rumen polyacetate bacteria (Acetobacter genus), Anaerobiosis caccae, Acetobacter xylinum (Acetoanaerobium nonaceae), Acetobacter methanolicus (Acetobacter carbinolicus), Acetobacter wegiae (Acetobacter vinelandii), Acetobacter woodiana (Acetobacter woonii), Acetobacter kanehensis (Acetobacter kii), Acetobacter fermentum (Acidococcus fermentum), Acetobacter lipolyticus (Anaerobiosystem), Bacillus coprinus, Bacillus propagulans, Bullobacterium cellulolyticum (Bifidobacterium cellulolyticum), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium longum bifidum (Bifidobacterium longum bifidum), Bifidobacterium longum (Bifidobacterium longum bifidum), Bifidobacterium longum bifidum (Bifidobacterium longum (Bifidobacterium longum) and Bifidobacterium longum (Bifidobacterium longum bifidum (Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum (Bifidobacterium longum) and Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum) including Bifidobacterium longum (Bifidobacterium longum including, Bifidobacterium longum including, Bifidobacterium longum including, Bifidobacterium longum including, clostridium acetate (Clostridium aceticum), Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium uricosulfans (Clostridium aceticum), Clostridium bifidum (Clostridium bifidum), Clostridium botulinum (Clostridium botium), Clostridium butyricum (Clostridium butyricum), Clostridium oligocellobiosus (Clostridium cellulolyticum), Clostridium formicoaceticum (Clostridium histolyticum), Clostridium Clostridium clostridia, Clostridium methylpenteneum (Clostridium methylpenticum), Clostridium bardii (Clostridium pasteurianum), Clostridium perfringens (Clostridium perfringens), Clostridium transporium, Clostridium natriensis (Clostridium propionicum), Clostridium putrefaciens (Clostridium propionicum), Clostridium sporogenes (Clostridium acidophylum), Clostridium butyricum (Clostridium acidum), Clostridium butyricum (Clostridium butyricum), Clostridium butyricum (Clostridium butyricum), Clostridium (Clostridium butyricum), Clostridium (Clostridium acidum) and Clostridium (Clostridium butyricum), Clostridium (Clostridium acidum) and Clostridium (Clostridium butyricum), Clostridium (Clostridium acidum) and Clostridium (Clostridium butyricum), Clostridium (Clostridium), Clostridium acidum) and Clostridium (Clostridium butyricum), Clostridium (Clostridium) and Clostridium), Clostridium (Clostridium acidum) are), Clostridium acidum) and Clostridium (Clostridium) are), Clostridium butyricum), Clostridium (Clostridium) are), Clostridium) and Clostridium), Clostridium acidum) are), Clostridium (Clostridium) are), Clostridium) and Clostridium (Clostridium) and Clostridium (Clostridium) are), Clostridium (Clostridium) are), Clostridium (Clostridium) and Clostridium (Clostridium) are), Clostridium (Clostridium) are), Clostridium) are used, Clostridium (Clostridium) are), Clostridium (Clostridium) and Clostridium) are), Clostridium) and Clostridium) are, Clostridium (Clostridium) and Clostridium (Clostridium) are), Clostridium (Clostridium) and Clostridium (Clostridium) are), Clostridium (Clostridium) are), Clostridium (Clostridium) are), Clostridium (Clostridium) and Clostridium (Clostridium) are, Clostridium) are), Clostridium) and Clostridium (Clostridium) are, Clostridium (Clostridium) are, filamentous bacteria producing succinic acid (Fibrobacter succinogenes), Paaspiria polysaccharea (Lachnospira multiparticulatus), Megasphaera elsdenii (Megasphaera elsdenii), Moorella thermoacetica (Moorella thermoacetica), Penicillium acetogenicus, Clavibacterium digallicum (Pelobacter acidalilliticum), Clavibacterium mosellatum (Pelobacter maliensis), Prevotella ruminocola, Corynebacterium freudenreichii (Propionibacterium freudenreichii), Ruminococcus xanthinum (Ruminococcus flaveacens), Ruminobacter amylovorans (Ruminobacter amylovorans), Ruminococcus albus (Ruminococcus alculatus), Ruminococcus bracteus (Ruminococcus bractensis), Streptococcus sphaericus (Streptococcus sphaeroides), Streptococcus sphaeroides, Streptococcus sphaeromonas campestris, Streptococcus sphaeroides, etc. strain, Streptococcus sphaeroides, etc. strain, etc. bacteria, etc. for producing strain, etc. bacteria, and strain, etc. bacteria, etc. which are included, etc. which are used for example, etc. which are used, etc. which are, etc.

In some embodiments, it is advantageous to inoculate the MSW with a bacterial strain capable of producing butyrate, including, but not limited to, any one or more of the following, or genetically modified variants thereof: fermented aminoacidococcus (Acidococcus fermentans), Anaerococcus caccae, Bifidobacterium adolescentis (Bifidobacterium adolescentis), Vibrio butyricum (Clostridium butyricum crocosum), Vibrio cellulolyticus (Clostridium cellulolyticus), Clostridium cellulolyticum (Clostridium cellulolyticum), Clostridium chrysogenum (Clostridium aurantiacum), Clostridium bailii (Clostridium bifidum), Clostridium butyricum (Clostridium butyricum), Clostridium cellulolyticum (Clostridium cellulolyticum), Clostridium difficile (Clostridium difficile), Clostridium innocuous bacterium (Clostridium butyricum), Clostridium butyricum (Clostridium butyricum), Clostridium difficile (Clostridium difficile), Clostridium difficile (Clostridium butyricum), Clostridium difficile (Clostridium difficile), Clostridium sporogenes (Clostridium butyricum), Clostridium difficile (Clostridium sporogenes (Clostridium butyricum), Clostridium (Clostridium difficile), Clostridium sporogenes (Clostridium butyricum), Clostridium sporogenes (Clostridium sporogenes), Clostridium (Clostridium sporogenes (Clostridium butyricum), Clostridium sporogenes (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium (Clostridium sporogenes), Clostridium sporogenes (Clostridium sporogenes), Clostridium (Clostridium sporogenes) and Clostridium sporogenes), Clostridium sporogenes) and Clostridium (Clostridium sporogenes) and Clostridium (Clostridium sporogenes) and Clostridium (Clostridium sporogenes) and Clostridium sporogenes) are included), Clostridium (Clostridium sporogenes) are), Clostridium (Clostridium sporogenes) are included), Clostridium (Clostridium sporogenes) are included), Clostridium (Clostridium sporogenes), Clostridium (Clostridium sporogenes) are), Clostridium sporogenes) are included), Clostridium (Clostridium sporogenes), Clostridium (Clostridium sporogenes), Clostridium (Clostridium sporogenes), Clostridium sporogenes) are), Clostridium (Clostridium sporogenes), Clostridium (Clostridium butyricum), Clostridium (Clostridium butyricum), Clostridium (Clostridium butyricum), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium) are), Clostridium butyricum) and Clostridium (Clostridium butyricum), Clostridium (Clostridium, Escherichia coli (Escherichia coli), Eubacterium pasteurianum (Eubacterium barospiri), Eubacterium diplodioides (Eubacterium biforme), Eubacterium cellulolyticum (Eubacterium cellulosolves), Eubacterium cylindricum (Eubacterium cylindroides), Eubacterium elongatum (Eubacterium dolichum), Eubacterium megaterium (Eubacterium hadrum), Eubacterium halii, Eubacterium limosum (Eubacterium limosum), Eubacterium candidum (Eubacterium moniliforme), Eubacterium oxydium redox (Eubacterium oxydorucans), Eubacterium ramosum (Eubacterium ramuscum), Eubacterium procumbens (Eubacterium rectalum), eubacterium arenarium (Eubacterium saburrum), Eubacterium polytrichum (Eubacterium torulosum), Eubacterium ventriosum (Eubacterium ventriosum), Clostridium molestanum (Fabalibacter prausnitzii), Clostridium prasuvianum (Fusobacterium prausnitzii), Peptostreptococcus vaculinis, Peptostreptococcus tetradifus, Vibrio ruminalis (Pseudobulbus pseudobutyric acid), Pseudobulbus pseudobutyric acid (Pseudobulbus cervi ruminans), Pseudobulbus maculans (Rosebularia ceicola), Rosebularia enterobacteriae (Rosebularia ceicola), Rosebularia enteralis (Rosebularia intestinalis), Rosebularia hominium and Ruminococcus bracteus (Ruminococcus bramii).

In some embodiments, it is advantageous to inoculate MSW with a bacterial strain capable of producing propionate, including but not limited to any one or more of the following, or genetically modified variants thereof: anaerobic vibrio lipolyticus (anaerobacterium lipolyticum), Bacteroides coprinus, Bacteroides prolifera, Bifidobacterium adolescentis (Bifidobacterium adolescentis), Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium butyricum (Clostridium butyricum), Clostridium methylpenteneum (Clostridium metpenticum), Clostridium bardii (Clostridium pasteurianum), Clostridium perfringens (Clostridium perfringens), Clostridium propionicum (Clostridium propionicum), Escherichia coli (Escherichia coli), Clostridium sp.

In some embodiments, it is advantageous to inoculate MSW with a bacterial strain capable of producing ethanol, including, but not limited to, any one or more of the following, or genetically modified variants thereof: acetobacter methanolicus (Acetobacter carinatus), Acetobacter westermanii (Acetobacter wieriensis), Acetobacter woodwardii (Acetobacter woododii), Bacteroides cellulolyticus (Bacteroides cellulosolveus), Bacteroides xylanolyticus (Bacteroides xylolyticus), Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii (Clostridium bifidus), Clostridium butyricum (Clostridium butyricum), Clostridium cellulolyticum (Clostridium cellulolyticum), Clostridium sporogenes (Clostridium butyricum), Clostridium sporogenes (Clostridium pasteurianum), Clostridium pasteurianum (Clostridium sporogenes), Clostridium thermocellum (Clostridium thermocellum), Clostridium thermocellum sulfide (Clostridium thermocellum), Clostridium thermocellum (Clostridium thermocellum), Clostridium sporogenes (Clostridium sporogenes), Clostridium thermocellum), Clostridium sporogenes (Clostridium sporogenes), Clostridium sporogenes (Clostridium thermocellum), Clostridium thermocellum (Clostridium perfringens), Clostridium (Clostridium thermocellum), Clostridium (Clostridium thermocellum) and Escherichia coli (Lactobacillus) bacteria (Lactobacillus) and Escherichia coli (Lactobacillus acidophylum), Clostridium perfringens, Clostridium (Clostridium thermocellum) bacteria (Lactobacillus) are used in the strains, bacteria, Pelobacter acetylenicus, Peulococcus albus (Ruminococcus albus), Thermoanaerobacter mathranii, Treponema bryantii, and Zymomonas mobilis (Zymomonas mobilis).

In some embodiments, a combination of different microorganisms, optionally including different species of bacteria and/or fungi, may be used to achieve simultaneous microbial fermentation. In some embodiments, a suitable microorganism is selected to provide the desired metabolic result under the expected reaction conditions, and then inoculated with high doses horizontally to outweigh the naturally occurring strains. For example, in some embodiments, it may be advantageous to inoculate with homofermentative lactic acid producing bacteria because it can provide a higher final methanogenic capacity in the produced biomethane substrate than that provided by heterofermentative lactic acid producing bacteria.

In some embodiments, the simultaneous enzymatic hydrolysis and microbial fermentation is carried out in a hydrolysis reactor capable of being stirred by free fall mixing, as described in WO2006/056838 and WO 2011/032557.

After a period of time in which the simultaneous enzymatic hydrolysis and microbial fermentation takes place, the MSW, which is provided in a non-aqueous amount of 10-45%, is converted, the biologically derived or "fermentable" component becomes liquefied, and the microbial metabolites accumulate in the aqueous phase. After a period of time in which the simultaneous enzymatic hydrolysis and microbial fermentation takes place, the liquefied fermentable part of the waste is separated from the non-fermentable solids. Liquefied material, once separated from non-fermentable solids, is what we call a "biological fluid". In some embodiments, at least 40% of the non-water content of such biological fluids comprises dissolved volatile solids, or at least 35%, or at least 30%, or at least 25%. In some embodiments, at least 25% by weight of the dissolved volatile solids in the biological fluid comprise any combination of acetate, butyrate, ethanol, formate, lactate, and/or propionate. In some embodiments, at least 70% by weight of the dissolved volatile solids comprise lactate, or at least 60%, or at least 50%, or at least 40%, or at least 30%, or at least 25%.

In some embodiments, the separation of non-fermentable solids and liquefied fermentable portion of MSW to produce a biological fluid characterized by comprising dissolved volatile solids at least 25 wt.% of which contain any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate is performed less than 16 hours, or less than 18 hours, or less than 20 hours, or less than 22 hours, or less than 24 hours, or less than 30 hours, or less than 34 hours, or less than 36 hours after the start of the enzymatic hydrolysis.

Separation of the liquefied fermentable portion from the non-fermentable solids in the waste can be accomplished by various means. In some embodiments, this may be accomplished using any combination of at least two different separation operations, including but not limited to screw press operations, ballistic separator operations, shaker screen operations, or other separation operations known in the art. In some embodiments, the non-fermentable solids separated from the fermentable portion of the waste comprise at least about 20% dry weight of MSW, or at least 25%, or at least 30%. In some embodiments, the non-fermentable solids separated from the fermentable portion of the waste comprise at least 20% by dry weight of recoverable material, or at least 25%, or at least 30%, or at least 35%. In some embodiments, the biological liquid produced by the separation of at least two separation operations contains at least 0.15kg volatile solids per kg treated MSW or at least 010. One skilled in the art will readily appreciate that the composition of biological origin inherent in MSW is variable. Despite this, the data for 0.15kg volatile solids/kg treated MSW reflects at least 80% total capture of biogenic material in typical unsorted MSW. Calculation of kg of volatile solids captured per kg of MSW biological fluid processed can be estimated over a period of time to determine total yield and total MSW processed.

In some embodiments, after separation of the non-fermentable solids and the liquefied fermentable portion of the MSW to produce a biological liquid, the biological liquid may be post-fermented under different conditions including different temperatures or pH.

The term "dissolved volatile solids" as used herein refers to a simple measurement calculated as follows: a sample of biological fluid in a 50ml centrifuge tube was centrifuged at 6900g for 10 minutes to produce a pellet and supernatant. The supernatant was decanted and the wet weight of the pellet was expressed as a percentage of the total weight of the initial liquid sample. The supernatant samples were dried at 60 ℃ for 48 hours to determine the dry matter content. The ash remaining after the 550 ℃ furnace combustion was subtracted from the dry matter measurement to determine the volatile solids content of the supernatant sample and expressed as a mass percent of dissolved volatile solids. Independent measurements of dissolved volatile solids were determined by calculation based on the volatile solids content in the precipitate. The precipitated wet weight fraction was used as a fraction of the volume of undissolved solids in the total initial volume. Dried at 60 ℃ for 48 hours to determine the dry matter content of the precipitate. The ash remaining after the 550 ℃ furnace firing was subtracted from the dry matter measurement to determine the volatile solids content of the precipitate. The volatile solids content in the precipitate was corrected by the estimated contribution from the supernatant as (1-moisture of the precipitate) X (measured% supernatant volatile solids). Subtraction (corrected volatile solids content in the precipitate) of X (fractional estimate of the volume of undissolved solids as a proportion of the total initial volume) from the total volatile solids detected in the original liquid sample gives an independent estimate of the% dissolved volatile solids. The higher of the two estimates was used in order not to overestimate the percentage of dissolved volatile solids represented by the bacterial metabolites.

In some embodiments, the present invention provides compositions and methods for biomethane production. The foregoing detailed discussion regarding embodiments of methods of treating MSW can optionally be applied to embodiments that provide methods and compositions for biomethane production. In some embodiments, a method of producing biomethane comprises the steps of:

(i) providing an organic liquid biomethane substrate pretreated by microbial fermentation such that at least 40% by weight of the non-water content is present as dissolved volatile solids comprising at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate salts,

(ii) transferring the liquid substrate to an anaerobic digestion system, and then

(iii) Anaerobic digestion of the liquid substrate is performed to produce biomethane.

In some embodiments, the present invention provides an organic liquid biomethane substrate produced by enzymatic hydrolysis and biological fermentation of Municipal Solid Waste (MSW) or pretreated lignocellulosic biomass, said biomethane substrate comprising enzymatically hydrolyzed and microbially fermented MSW, or comprising enzymatically hydrolyzed and microbially fermented pretreated lignocellulosic biomass, characterized in that-at least 40% by weight of the non-aqueous content is present as dissolved volatile solids comprising at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate.

As used herein, the term "anaerobic digestion system" refers to a fermentation system comprising one or more reactors operated under controlled aeration conditions, wherein methane gas is produced in each reactor comprising the system. The extent of methane gas production is such that the concentration of dissolved methane produced metabolically in the aqueous phase of the fermentation mixture in the "anaerobic digestion system" is saturated at the conditions used and methane gas is released from the system.

In some embodiments, an "anaerobic digestion system" is a fixed filtration system. By "immobilized filtered anaerobic digestion system" is meant a system in which an anaerobic digestion population is immobilized (optionally within a biofilm) on a physical support matrix.

In some embodiments, the liquid biomethane substrate comprises at least 8% total solids, or at least 9% total solids, or at least 10% total solids, or at least 11% total solids, or at least 12% total solids, or at least 13% total solids. As used herein, "total solids" refers to both soluble and insoluble solids, and in fact refers to "non-water content. The total solids were measured by drying at 60 ℃ until a constant weight was obtained.

In some embodiments, the microbial fermentation is conducted under conditions that inhibit the production of methane by methanogens, e.g., at a pH of less than 6.0, or at a pH of less than 5.8, or at a pH of less than 5.6, or at a pH of less than 5.5. In some embodiments, the liquid biomethane substrate comprises dissolved methane at a concentration less than the saturation concentration. In some embodiments, the liquid biomethane substrate comprises less than 15mg/L dissolved methane, or less than 10mg/L, or less than 5 mg/L.

In some embodiments, one or more components of dissolved volatile solids are removed from the liquid biomethane substrate by distillation, filtration, electrodialysis, specific binding, precipitation, or other methods known in the art prior to anaerobic digestion to produce biomethane. In some embodiments, ethanol or lactate is removed from the liquid biomethane substrate prior to anaerobic digestion to produce biomethane.

In some embodiments, a solid substrate, such as MSW or a fibrous portion from pretreated lignocellulosic biomass, is simultaneously subjected to enzymatic hydrolysis and microbial fermentation to produce a liquid biomethane substrate pretreated by microbial fermentation such that at least 40% by weight of the nonaqueous content is present as dissolved volatile solids comprising at least 25% by weight of any combination of acetate, butyrate, ethanol, formate, lactate and/or propionate. In some embodiments, a liquid biomethane substrate having the above-described properties is produced from liquefied organic material obtained from unsorted MSW by a thermo-compressor process with simultaneous enzymatic hydrolysis and microbial fermentation. In some embodiments, the pretreated lignocellulosic biomass is mixed with enzymatically hydrolyzed and biofermented MSW, optionally in such a way that the enzymatic activity of the MSW-derived biological liquid provides enzymatic activity for hydrolysis of the lignocellulosic substrate to produce a composite liquid biomethane substrate derived from both the MSW and the pretreated lignocellulosic biomass.

"Soft lignocellulosic biomass" refers to plant biomass other than wood containing cellulose, hemicellulose, and lignin. Any suitable soft lignocellulosic biomass may be employed, including, for example, biomass of at least wheat straw, corn stover, corn cobs, empty fruit clusters, rice straw, oat straw, barley straw, canola straw, rye straw, sorghum, sweet sorghum, soybean stover, switchgrass, bermuda grass and other grasses, sugar cane bagasse, sugar beet pulp, corn fiber, or any combination thereof. Lignocellulosic biomass includes other lignocellulosic materials such as paper, newsprint, cardboard, or other municipal or office waste. Lignocellulosic biomass may be used as a mixture of materials derived from different feedstocks, and may be fresh, partially dried, completely dried, or any combination thereof. In some embodiments, the methods of the present invention are practiced using at least about 10kg of biomass feedstock, or at least 100kg, or at least 500 kg.

Lignocellulosic biomass is generally pretreated using methods known in the art prior to enzymatic hydrolysis and microbial pretreatment. In some embodiments, the biomass is pretreated by hydrothermal pretreatment. "hydrothermal pretreatment" refers to "cooking" of biomass with water, in the form of hot liquid, steam or high pressure steam containing high temperature liquid or steam or both, at temperatures of 120 ℃ or higher, with or without the addition of acids or other chemicals. In some embodiments, the lignocellulosic biomass feedstock is pretreated by spontaneous hydrolysis. "spontaneous hydrolysis" refers to a pretreatment process during which the hydrolysis of hemicellulose is further catalyzed by acetic acid released by the hydrolysis of hemicellulose during pretreatment, and is applied to any hydrothermal pretreatment of lignocellulosic biomass carried out at ph 3.5-9.0.

In some embodiments, the hydrothermally pretreated lignocellulosic biomass is separated into a liquid fraction and a solid fraction. "solid fraction" and "liquid fraction" refer to the fractionation of pretreated biomass in a solid/liquid separation. The separated liquids are collectively referred to as the "liquid fraction". The residual fraction containing a substantial amount of insoluble solids content is referred to as the "solids fraction". The solid portion or the liquid portion or a combination of both may be used to carry out the method of the invention or to produce the composition of the invention. In some embodiments, the solid portion may be washed.

Example 1 Simultaneous microbial fermentation improves organic Capture of enzymatic hydrolysis of unsorted MSW

Laboratory scale reactions were performed with samples of biological fluids from the tests described in example 5.

Model MSW substrates for laboratory scale reactions were prepared using organic fractions (defined as cellulose, animal and plant fractions) freshly generated to contain municipal solid waste (as described in Jensen et al, 2010, based on Riber et al, 2009).

Model MSW was stored in aliquots at-20 ℃ and thawed at 4 ℃ overnight. The reaction was carried out in 50ml centrifuge tubes and the total reaction volume was 20 g. Model MSW was added to 5% Dry Matter (DM) (measured as dry matter content remaining after 2 days at 60 ℃).

The cellulase used for hydrolysis was Cellic CTec3(VDNI0003, Novozymes A/S, Bagsvaerd, Denmark) (CTec 3). To adjust and maintain the pH at 5, citrate buffer (0.05M) was added to make the total volume 20 g.

The reaction was incubated on a Stuart Rotator SB3 (4 RPM) in an oven (Binder GmBH, Tuttlingen, Germany) for 24 hours. Negative controls were set up in parallel to assess the background of dry matter released from the substrate during incubation. After incubation, the tubes were centrifuged at 1350g for 10 min at 4 ℃. The supernatant was then decanted, 1ml was taken for HPLC analysis, and the remaining supernatant and pellet were dried at 60 ℃ for 2 days. The dry matter weight was recorded and used to calculate the dry matter distribution. The conversion of DM in the model MSW is calculated based on these numbers. By means of a detector equipped with refractive index detector (RI-101) and Ultimate 3000 HPLC (thermo Scientific Dionex) with a 250nm UV detector. On a Rezex RHM monosaccharide column (Phenomenex) at 80 ℃ with 5mM H₂SO₄The eluate was separated at a flow rate of 0.6 ml/min. The results were analyzed using Chromeleon software program (Dionex).

To evaluate the effect of simultaneous fermentation and hydrolysis, 2ml/20g of the biological fluid from the test described in example 5 (sampled on days 15 and 16 of december) was added to the reaction, with or without addition of CTec3(24mg/g DM).

DM conversion in MSW

The conversion of solids was determined as the percentage of the total dry matter found in the supernatant. Figure 1 shows the conversion in MSW blanks, isolated enzyme preparations, inoculated microorganisms alone, and combinations of microorganism inoculation and enzymes. The results show that the addition of EC12B from example 5 resulted in significantly higher dry matter conversion compared to the background of dry matter released in the blank reaction (MSW blank) (Students t test p < 0.0001). The simultaneous microbial fermentation induced by the addition of the EC12B sample and the enzymatic hydrolysis with CTec3 resulted in significantly higher dry matter conversion rates compared to the reaction with hydrolysis with CTec3 alone and the reaction with EC12B alone.

HPLC analysis of glucose, lactate, acetate and EtOH

The concentrations of glucose and microbial metabolites (lactate, acetate and ethanol) measured in the supernatant are shown in figure 2. As shown, the background concentration of these in the model MSW blank is low and the lactic acid content may be from bacteria inherent in the model MSW, as the material used to produce the substrate may not be sterilized or heat sterilized. The effect of adding CTec3 resulted in an increase in glucose and lactate in the supernatant. The highest concentrations of glucose and bacterial metabolites were found in the reaction with the simultaneous addition of EC12B bioliquid from example 5 and CTec 3. The simultaneous fermentation and hydrolysis thus improves the conversion of dry matter in the model MSW and increases the concentration of bacterial metabolites in the liquid.

Reference documents: jacob Wagner Jensen, Claus Felby, Henning GeorgNanna DreyerEnzymatic processing of municipal solid waste.Waste Management.12/2010； 30(12):2497-503。

Riber,C.,Petersen,C.,Christensen,T.H.,2009.Chemical composition of material fractions in Danish household waste.Waste Management 29,1251–1257。

Example 2 Simultaneous microbial fermentation improves organic Capture of unsorted MSW

Tests were performed with unsorted MSW in a specially designed batch reactor as shown in fig. 3 to confirm the results obtained in the laboratory scale experiments. This experiment tested the effect of adding an inoculated microorganism comprising a biological fluid obtained from the bacteria of example 3 in order to achieve simultaneous microbial fermentation and enzymatic hydrolysis. The test was performed using an unsorted MSW.

MSW for small scale experiments was the focus of renesccience research and development. In order for the test results to be valuable, it is required that the waste is representative and renewable.

Waste was collected from Nomi I/S Holstebro in March 2012. The waste is unsorted Municipal Solid Waste (MSW) from various regions. The waste was cut into 30x30mm pieces for small scale testing and collection of representative samples. Sampling theory was applied to the shredded waste by sub-sampling the shredded waste into a 22 liter bucket. The barrels were stored in a freezer at-18 ℃ until use. The "real waste" consists of eight barrels of waste collected. The contents of these barrels were remixed and re-sampled to ensure as low as possible variability between replicates.

All samples were processed under similar conditions of water, temperature, rotation speed and mechanical effect. Six boxes were used: three were not inoculated and three were inoculated. The non-water content during the test was designated as 15% non-water content by adding water. The dry matter in the inoculation material was calculated and less water was newly added in the inoculation tank. 6kg of MSW, and 84g of the commercially available cellulase preparation CTEC3 were added to each box. 2 liters of inoculum was added to the inoculation chamber and the water added was reduced accordingly.

Separately, 20% NaOH was added to raise the pH and 72% H₂SO₄To lower the pH to maintain the pH of the inoculation chamber at 5.0 and the pH of the non-inoculation chamber at 4.2. The lower pH of the non-inoculation chamber helps to ensure that indigenous bacteria do not multiply. We previously showed that the use of the enzyme preparation CTEC3 Tm in MSW hydrolysis did not distinguish between differences in activity at ph4.2-ph 5.0. The reaction was continued for 3 days at 50 ℃ in an experimental reactor capable of providing constant rotating stirring.

At the end of the reaction, the tank was emptied through the sieve and the biological liquid containing the liquefied material produced by simultaneous enzymatic hydrolysis and microbial fermentation of MSW.

Dry matter (TS) and volatility fixing (VS) were determined.

Dry Matter (DM) method:

the samples were dried at 60 ℃ for 48 hours. The sample weight before and after drying was used to calculate the DM percentage.

Sample DM (%): sample dry weight/wet weight (g). times.100

Volatile solids method:

volatile solids were calculated and expressed as percent DM minus ash content. The ash content of the samples was determined by burning the pre-dried samples in a furnace at 550 c for at least 4 hours. Then, the ash is calculated as follows:

ash percentage in dry matter of sample: sample ash weight (g)/sample dry weight (g). times.100

Percent volatile solids: (1 sample Grey percent) × (sample DM percent)

The results are shown below. As shown, the higher total solids content was obtained for the biological fluid obtained in the inoculum tank, indicating that simultaneous microbial fermentation and enzymatic hydrolysis were superior to enzymatic hydrolysis alone.

Example 3 Simultaneous microbial fermentation improves organic Capture of enzymatic hydrolysis of unsorted MSW

Experiments were performed at the renesccience exemplar factory at copenhagen Amalgarian Resource Center (ARC) in denmark. Figure 4 shows a schematic diagram of the main features of the plant. The concept of ARC renescence waste smelter is to sort MSW into four products. Biological fluids for biogas production, inerts for recycling (glass and sand), and 2D and 3D parts of inorganic materials suitable for RDF production or recycling of metals, plastics and trees.

MSW from large cities was collected in plastic bags. MSW is shipped to renescence waste smelters and stored in silos until processed. According to the characteristics of MSW, a sorting step may be installed before renescence system to remove oversize particles (larger than 600 mm).

The renesccience technique as tested in this example includes three steps. The first step is to gently heat (pre-treat, as shown in fig. 4) the MSW with hot water for a temperature range of 20-60 minutes to 40-75 ℃. This heating and mixing stage opens the plastic bag and provides a sufficient slurry of the degradable components to produce a more homogeneous organic phase prior to addition of the enzyme. The temperature and pH are adjusted during heating to make them optimal for the isolated enzyme preparation used for the enzymatic hydrolysis. As shown in fig. 4, the added hot water may be clean tap water or wash water that was first used in the tub and recycled in mild heating.

The second step is enzymatic hydrolysis and fermentation (liquefaction, as shown in figure 4). Enzymes and optionally selected microorganisms are added in the second step of renesccience treatment. The enzymatic liquefaction and fermentation are carried out continuously at a temperature and pH optimal for the enzymatic performance, with a residence time of about 16 hours. By this hydrolysis and fermentation, the biogenic part of the MSW is fractionated into a biological liquid with a high dry matter between the non-degradable materials. pH by addition of CaCO₃And (5) controlling.

The third step of the renesccience technique as implemented in this example is a separation step that separates the biological fluid from the non-degradable fraction. The separation is carried out in a ballistic separator, a wash tank and a hydraulic press. The ballistic separator separates the enzyme-treated MSW into a biological fluid, a 2D non-degradable material fraction and a 3D non-degradable material fraction. The 3D parts (physically three-dimensional objects such as cans and plastic bottles) do not bind large amounts of biological fluid, so a single washing step is sufficient to clean the 3D parts. The 2D parts (e.g. textiles and foils) are bound to a large amount of biological fluid. Thus, the 2D fraction is pressurized, washed and repressurized using a screw press to optimize the recovery of the biological liquid and obtain a "clean" and dry 2D fraction. The biological fluid is screened for inert materials such as sand and glass. All the water used in the wash tank can be recycled, heated and used as hot water for heating in the first step.

The tests reported in this example are divided into three sections, as shown in table 1.

TABLE 1

Time (hours)	Rodalon	Mildly heated tap/wash water
			27–68	+	Tap water
86–124	-	Tap water
			142-187	-	Washing water

In a 7 day trial, unsorted MSWs from Copenhagen, Denmark were continuously loaded at 335kg/h into a RENeScience demonstration plant. In the mild heating, 536kg/h of water (tap or wash) are added before entering the mild heating reactor and heated to about 75 ℃. Adjusting the MSW temperature to about 50 ℃ by adding CaCO₃The pH was adjusted to about 4.5.

In the first part, a surface active antibacterial agent Rodalon comprising 3g active ingredient/kg MSW in the added water^TM(benzylalkylammonium chloride).

Approximately 14kg Cellic Ctec3 (a cellulase preparation commercially available from Novozymes) per wet ton of MSW was added to the liquefaction reactor. Maintaining the temperature in the range of 45-50 deg.C by adding CaCO₃Adjusting the pH to a range of 4.2-4.5. The residence time of the enzyme reactor was about 16 hours.

In the separation system of ballistic separators, hydraulics and wash tanks, biological fluids (liquefied degradable materials) are separated from non-degradable materials.

The wash water is optionally decanted, the organic content recorded, or recycled and reused in the wet feed MSW in mild heating. The recycling of the wash water has the effect of achieving a higher level of bacterial inoculation with organisms growing under 50 ℃ reaction conditions than with the organisms initially present. In the process scheme used, the recycled wash water is first heated to about 70 ℃, in this case to about 50 ℃ in order to bring the feed MSW to a temperature suitable for enzymatic hydrolysis. In particular for lactic acid bacteria, heating to 70 ℃ has been shown to provide a selective and "inducible" thermotolerant expression.

Samples were obtained at selected time points from the following locations:

-biological fluid passing through a small sieve, referred to as "EC 12B";

-a biological liquid in a tank;

-wash water after the whey sieve;

-a 2D moiety;

-a 3D part;

-bottom inert parts from both scrubbing units.

Production of biological fluids is measured using the loaded cells on the reservoir. The input flow is measured using a flow meter and the recovered or drained wash waste is measured using the loaded cells.

The number of bacteria was checked as follows: the selected biological fluid sample was diluted 10-fold with SPO (peptone salt solution), and 1ml of the dilution was spread at a seeding depth on beef extract agar (3.0g/L beef extract (Fluka, Cas.: B4888), 10.0g/L tryptone (Sigma, cas. No.: T9410), 5.0g/L NaCl (Merck, cas. No.7647-14-5), 15.0g/L agar (Sigma, cas. No. 9002-18-0)). Plates were incubated at 50 ℃ in aerobic and anaerobic environments, respectively. Anaerobic cultivation in a suitable vessel was maintained anaerobically by aeration of anaerobat and addition of iltfjernender cutter (anaerobgen from Oxoid, cat. No. AN 0025A). Aerobic colonies were counted after 16 hours and again after 24 hours. Anaerobically growing bacteria were quantified after 64-72 hours.

FIG. 5 shows the total volatile solids content of the EC12B biological liquid sample, expressed in kg/kg of treated MSW. Point estimates are obtained at different time points during the experiment by considering each of the three independent experimental phases as independent time periods. Thus, the point estimates during phase 1(Rodalon) are represented in terms of mass balance and material flow relative to those during phase 1. As shown in FIG. 5, during phase 1 of start-up after a long period of stop due to plant difficulties, a steady decrease in total solids captured in the biological fluid was observed, with Rodalon^TMThe slight antibacterial effect of (1) was consistent. During stage 2, the total captured solids returned to a slightly higher level. During stage 3, the recycle provided an effective "inoculation" for the feed MSW, with the biological fluid kg VS/kg rising to a fairly high level of about 12%.

For each of the 10 time points shown in fig. 5, a sample of biological fluid (EC12B) was taken and the concentrations of total solids, volatile solids, dissolved volatile solids, and putative bacterial metabolites acetate, butyrate, ethanol, formate, and propionate were determined by HPLC. These results, including the glycerol concentration, are shown in table 1 below.

TABLE 1 analysis of biological fluid samples

For the biological fluid samples sampled at each of the 10 time points, fig. 6 shows the viable count under aerobic conditions and the weight percentage of "bacterial metabolites" (referring to the sum of acetate, butyrate, ethanol, formate, and propionate) expressed as dissolved volatile solids. As shown, the weight percentage of bacterial metabolites increased significantly with increasing bacterial activity and was associated with increased solid capture in the biological fluid.

Example 4 identification of microorganisms contributing to the Simultaneous fermentation in example 3

The biological fluid samples from example 3 were analyzed for microbial composition.

The microbial species present in the sample are identified by aligning the 16S rRNA gene sequence of the microbial species present in the sample with the 16S rRNA gene sequence of a well-characterized species (reference species). The species identified a general cut-off with 97% similarity to the reference species 16S rRNA gene sequence. If the similarity is less than 97%, it is likely to be a different species.

The resulting sequences were queried in the NCBI database using BlastN. The database contains high quality sequences of at least 1200bp in length and has a classification relationship with NCBI. Only sequences with BLAST clicks > 95% identity are included.

The sampled biological fluid was transferred directly for analysis without freezing prior to DNA extraction.

In total 7 bacterial species were identified (fig. 7) and 7 archaebacteria species were identified. In some cases of bacterial species, subspecies cannot be determined (lactobacillus acidophilus (l.acidophilus), lactobacillus amylovorus (l.amylovorus), lactobacillus suis (l.sobrius), lactobacillus reuteri (l.reuteri), l.frumenti, lactobacillus fermentum (l.fermentum), l.fabiferentins, lactobacillus plantarum (l.planterum), lactobacillus pentosus (l.pentosus)).

Example 5 detailed analysis of organic harvesting Using Simultaneous microbial fermentation and enzymatic hydrolysis of unsorted MSW

The renescence demonstration plant described in example 1 was used to investigate in detail the total organic capture carried out with simultaneous microbial fermentation and enzymatic hydrolysis of unsorted MSW.

Waste from copenhagen was characterized by Econet to determine its content.

The waste analysis was analyzed to determine content and variation. Large samples of MSW were transported to Econet A/S for waste analysis. The primary sample is reduced to about 50-200kg of subsamples. The subsamples were sorted by trained personnel into 15 different waste fractions. The weight of each part was recorded and the distribution calculated.

Table x waste constituents (%) total amount analyzed by eco net during the 300 hour test

Sample (I)	1.	2.	3.	4.	5.	6.	7.	8.	9.	Average	Standard deviation of the mean
													％	％	％	％	％	％	％	％	％	％
Plastic package	5.1	6.7	8.0	4.9	6.2	2.5	6.2	7.5	6.4	5.9	1.64
												Plastic foil	10.8	8.6	10.7	7.9	10.1	7.8	8.8	8.5	9.5	9.2	1.13
Other plastics	0.7	0.8	0.5	0.7	1.0	0.7	1.6	0.4	0.9	0.8	0.33
												Metal	2.5	3.6	2.7	2.0	2.5	2.1	3.6	2.1	3.6	2.7	0.68
Glass	0.2	0.0	0.5	0.6	0.6	0.0	0.6	0.4	0.0	0.3	0.27
												Courtyard waste	0.7	3.5	1.9	1.8	0.9	2.7	0.6	4.5	2.8	2.1	1.33
WEEE (Battery, etc.)	0.7	0.1	0.6	0.4	0.7	0.8	1.1	0.1	0.5	0.6	0.33
												Paper sheet	14.8	8.3	13.3	8.8	10.5	5.6	10.2	12.6	12.4	10.7	2.86
Plastic and paperboard package	10.4	21.4	11.9	8.6	11.0	6.7	10.7	11.8	13.9	11.8	4.13
												Food waste	19.8	15.6	25.9	27.6	26.3	24.5	24.5	23.3	18.0	22.8	4.09
Diaper	8.0	10.3	6.9	18.8	8.1	25.1	15.2	10.1	14.0	12.9	6.00
												Dirty paper	8.5	6.7	7.3	7.4	8.5	8.6	7.9	5.7	6.3	7.4	1.03
Fine dust	9.7	2.5	4.2	2.1	4.5	4.7	2.7	7.0	4.9	4.7	2.40
												Other combustibles	2.0	0.9	0.8	1.2	1.8	0.7	0.7	2.2	0.8	1.2	0.61
Other incombustibles	6.2	11.1	5.0	7.3	7.2	7.6	5.6	3.7	6.2	6.7	2.07
												Sum of	100	100	100	100	100	100	100	100	100	100.0

The composition of the waste was varied frequently and table 2 shows the results of waste analysis of different samples collected over 300 hours. The greatest changes were observed in diapers, plastic and cardboard packaging and food waste as all parts affecting the amount of captive organic material.

Throughout the "300 hour test", the average "capture" biodegradable material expressed as kg VS/kg treated MSW was 0.156kg VS/kg MSW delivered.

Representative samples of biological fluids were taken at various time points during the experiment while the plant was in steady operation. Samples were analyzed by HPLC to determine volatile solids, total solids and dissolved solids as described in example 3. The results are shown in table 2 below.

TABLE 2 analysis of biological fluid samples

Example 6 identification of microorganisms contributing to the fermentation carried out simultaneously in example 5

Samples of biological fluid "EC 12B" were removed from the test period described in example 5 at 12 months, 15 days and 16 days 2012 and stored at-20 ℃ to facilitate 16S rDNA analysis for the identification of microorganisms in the samples. The 16S rDNA analysis is widely applied to identification of prokaryotes and phylogenetic analysis based on the 16S component of the ribosomal small subunit. Frozen samples on dry ice were transported to GATC Biotech AB, Solna, SE for 16S rDNA analysis (GATC _ Biotech). The analysis includes: genomic DNA was extracted, an amplicon library was prepared with a universal primer pair (27F: AGAGAGTTTGATCCTGGCTCCAG/534R: ATTACCGCGGCTGCTGG; 507bp long) spanning the hypervariable regions V1 to V3, PCR-labeled with the GS FLX adapter, and sequenced on the genome sequencer FLX instrument to obtain reads of 104.000-160.000 for each sample. The resulting sequences were then queried in the rDNA database from the ribosomal database entry (Cole et al, 2009) with BlastN. The database contains high quality sequences of at least 1200bp in length and has a taxonomic relationship to NCBI. The current Release (RDP Release10, updated at 9/19/2012) contains 9162 bacterial and 375 archaeal sequences. BLAST results were filtered to remove short and low quality hits (sequence identity ≧ 90%, comparison coverage ≧ 90%).

A total of 226 different bacteria were identified.

The dominant bacterium in the EC12B sample was a propionate-producing bacterium, Paludibacter propiocigens WB4(Ueki et al, 2006), which accounted for 13% of the total identified bacteria. FIG. 8 shows the distribution of the identified 13 dominant bacteria (Paludibacterium propionicigenes WB4, Proteinophilum aceticates, Actinomyces europaeus, Levilinea saccharolytica, Cryptobacterium phenolyticus, Sediberibacter hygrophicus, Clostridium phytofermentans ISDg strain (Clostridium phytofermentans ISDg), Petrias monosulfuriphilia, Clostridium lactofermentum (Clostridium lacticitigenes), Clostridium caenicola, Garcinia orientalis, Corynebacterium halodurans 9455 (Corynebacterium Restrictus 9455), Marinibacter 9455).

Comparison of the identified bacteria at the genus level indicates that clostridia (Clostridium), Paludibacter, Proteiniphilum, actinomycetes (Actinomyces) and leviline (all anaerobic bacteria) represent about half of the identified genera. Lactobacillus (Lactobacillus) genus accounts for 2% of the identified bacteria. The dominant species p. propiocigens WB4 belongs to the second major genus (Paludibacter) in the EC12B sample.

The predominant pathogen in the EC12B sample was Streptococcus (Streptococcus spp.) which accounted for 0.028% of the total identified bacteria. No sporulated pathogenic bacteria were found in the biological fluid.

Streptococcus is the only pathogenic bacterium present in the biological fluid of example 5. Streptococci have the highest temperature tolerance and D-value (non-sporulation), indicating that the amount of time required to reduce the number of viable streptococcal cells by ten-fold at a given temperature is higher than any other pathogenic bacterium in the MSW reported by D portes et al (1998). These results show that the conditions used in example 5 are capable of disinfecting MSW during the renescence process sorting to a level where only streptococci are present.

Competition for nutrients among organisms, and increased temperature during the process, will significantly reduce the number of pathogenic organisms and eliminate the presence of pathogens in the sorted MSW during renescence process as described above. Other factors such as pH, a_wOxygen and CO resistance₂NaCl, and NaNO₂Also affects the growth of pathogenic bacteria in the biological fluid. The interaction between the above-mentioned factors may reduce the time and temperature required to reduce the number of living cells in the process.

Example 7 detailed analysis of organic Capture by Simultaneous microbial fermentation and enzymatic hydrolysis of unsorted MSW obtained from remote geographical locations

MSW imported from the netherlands was processed using the renesccience demonstration plant described in example 3. The MSW was found to contain the following composition:

table Y: waste constituents account for the total (%), and ECOnet analysis was used in the van Gansewinkel test.

This material was subjected to simultaneous enzymatic hydrolysis and microbial fermentation as described in examples 3 and 5 and tested in the plant run for 3 days. Samples of biological fluid were obtained at various time points and characterized. The results are shown in Table 3.

TABLE 3 analysis of biological fluids

The dissolved VS was corrected with 9% based on lactate lost during drying.

Example 8 biomethane production Using a biological liquid obtained from Simultaneous microbial fermentation and enzymatic hydrolysis of unsorted MSW

The biological fluid obtained in the experiment described in example 5 was frozen in 20 liter buckets and stored at-18 ℃ for later use. The material was tested for biomethane production using two identical well-prepared fixed filter anaerobic digestion systems comprising anaerobic digestion populations immobilized within a biofilm on a filter support.

Initial samples were taken of the feed and liquid in the reactor. VFA, tCOD, SCOD and ammonia concentrations were determined by HACH LANGE test tube testing on a DR2800 spectrophotometer, and specific VFA were determined by HPLC daily. TSVS measurements were also determined gravimetrically. Gas samples for GC analysis were collected daily. Confirmation of the feed rate was performed by measuring the headspace volume of the feed tank and the effluent discharge from the reactor. Sampling during the process is performed by collecting the liquid or effluent with a syringe.

Stable biogas production was observed for 10 weeks with both digestion systems, corresponding to 0.27-0.32L/g COD, or R-Z L/g VS.

The biological fluid feed was interrupted in one of the two systems and monitored back to baseline as shown in fig. 9. Horizontal line 2 indicates a steady gas production level. Vertical line 3 represents the point in time when the feed is interrupted. As shown, after several months of steady operation, there is still residual converted elastomeric material remaining during the time periods shown by vertical lines 3 and 4. The time period after vertical line 4 represents a return to baseline or "dip". After the baseline period, the feed was started again at the point indicated by vertical line 1. The time period after vertical line 1 represents an increase in gas production or "ramp up" to steady state.

The parameters for the production of gas from a biological liquid (including "up" and "down" as measured) are as follows.

Rise time is the time from the first feed until gas production increases and stabilizes. The rise time indicates the level of convertible organics in the feed.

Drop time is the time from the last feed until the steady drop in gas production. The fall time indicates gas production from the convertible organic.

Burnout time is the time after the drop time until gas production is completely at baseline level. The burn-off time indicates gas production from slowly converted organics.

Corrected for 2L/day of background gas production.

Example 9 comparative biomethane production using bioliquids obtained from enzymatic hydrolysis of unsorted MSW with and without concurrent microbial fermentation

The biological methane production of the "high lactate" and "low lactate" biological fluids of example 2 was compared using the fixed filter anaerobic digestion system described in example 8. Measurements were obtained and "rise" and "fall" times were determined as described in example 8.

FIG. 10 shows the "rise" and "fall" of "high lactate" biological fluid. Horizontal line 2 indicates a steady gas production level. Vertical line 1 represents the point in time at which the feed begins. The time period after vertical line 1 represents an increase in gas production or "ramp up" to steady state. Vertical line 3 represents the point in time when the feed is interrupted. The time period shown by vertical lines 3 to 4 represents a return to baseline or "dip".

Fig. 11 shows the same characteristics of "low lactate" biological fluids, with the relevant points as described in fig. 11.

Comparative parameters for gas production from "high lactate" and "low lactate" biological fluids (including "rise" and "fall" as measured as described) are shown below.

The difference in "rise"/"fall" times indicates a difference in the ease of biodegradability. The fastest bioconvertable biomass will ultimately have the highest overall organic conversion in biogas production applications. Also, a "faster" biomethane substrate is more suitable for conversion in very fast anaerobic digestion systems such as fixed filter digesters.

As shown, the "high lactate" biological fluid shows much faster "rise" and "fall" times in biological methane production.

Rise time is the time from the first feed until gas production increases and stabilizes. The rise time indicates the level of convertible organics in the feed.

Drop time is the time from the last feed until the steady drop in gas production. The fall time indicates gas production from the convertible organic.

Burnout time is the time after the drop time until gas production is completely at baseline level. The burn-off time indicates gas production from slowly converted organics.

Corrected for 2L/day of background gas production.

Example 11 biological methane production Using a biological liquid obtained by simultaneous microbial fermentation and enzymatic hydrolysis of hydrothermally pretreated wheat straw

Wheat straw is pretreated, separated into a fiber fraction and a liquid fraction, and then the fiber fraction is washed separately. 5kg of the washed fiber was then cultured in a horizontal rotary drum reactor containing a dose of Cellic CTEC3 and the fermenting microorganism consisting of the bacteria obtained from example 3. The wheat straw is subjected to simultaneous hydrolysis and microbial fermentation at 50 ℃ for 3 days.

The biological fluid was then tested for biological methane production using the fixed filter anaerobic digestion system described in example 8. The "rise" time of the measurement was obtained as described in example 8.

Fig. 12 shows the "rise" characteristic of hydrolyzed wheat straw biofluid. Horizontal line 2 represents a steady gas production level. Vertical line 1 represents the point in time at which the feed begins. The time period after vertical line 1 represents an increase in gas production or "ramp up" to steady state.

The gas production parameters of the biological liquid from wheat straw hydrolysis are shown below.

As shown, pretreated lignocellulosic biomass can also be readily used to practice the biogas production process of the present invention and to produce novel biological methane substrates.

Rise time is the time from the first feed until gas production increases and stabilizes. The rise time indicates the level of convertible organics in the feed.

Drop time is the time from the last feed until the steady drop in gas production. The fall time indicates gas production from the convertible organic.

Burnout time is the time after the drop time until gas production is completely at baseline level. The burn-off time indicates gas production from slowly converted organics.

Corrected for 2L/day of background gas production.

Example 12 Simultaneous microbial fermentation and enzymatic hydrolysis of MSW by selected organisms

The microbial fermentation and enzymatic hydrolysis reactions were carried out simultaneously on a laboratory scale on model MSW (as described in example 1) using specific monocultured bacteria, the procedure being as described in example 1. The reaction conditions and enzyme dosages are shown in Table 4.

The effect on dry matter conversion of model MSW with or without CTec3 was determined using the viable strains lactobacillus amyloliquefaciens (DSMZ No.20533) and propionibacterium acidipropionici (DSMZ No.20272) (DSMZ, No. rel, germany) (stored at 4 ℃ for 16 hours until use) as inocula. The major metabolites produced are lactic acid and propionic acid, respectively. The concentration of these metabolites was measured using an HPLC procedure (as described in example 1).

Since propionibacterium is an anaerobe, the buffers used for the strain reaction were purged with gaseous nitrogen, and the live culture was inoculated into a reaction tube inside a mobile anaerobic chamber (Atmos Bag, Sigma Chemical CO, st. The reaction tube containing p. propionici was closed before transfer to the incubator. The reaction was inoculated with 1ml of P.propioci or L.amylophilus.

The results shown in table 4 clearly indicate the production of the expected metabolites; propionic acid was detected in the reaction inoculated with p. acidipropionic, whereas no propionic acid was detected in the control containing model MSW with or without CTec 3. The lactic acid concentration in the control reaction with model MSW alone was almost the same as the reaction with l. Lactic acid production in the control reaction was attributed to the bacteria inherent in the model MSW. Some background bacteria were expected since the individual components of the model waste were fresh products that were frozen but not subjected to any further sterilization prior to preparation of the model MSW. When l.amylophilus and CTec3 were added simultaneously, the lactic acid concentration almost doubled (table 4).

The positive effect of DM release to the supernatant after hydrolysis was demonstrated by higher DM conversion in the reaction with the simultaneous addition of l.amylophilus or p.propionici with CTec3 (30-33% increase compared to the reaction with addition of CTec3 only).

TABLE 4 bacterial cultures tested on a laboratory scale with enzymatic hydrolysis performed separately or simultaneously. The temperature, pH, and dose of CTec3, 96mg/g, are shown. MSW control reactions were performed in parallel in buffer with or without CTec3 to assess the background of bacterial metabolites in the reactions (mean and standard deviation of 4 reactions are shown except for the MSW control which was performed once).

Nd. not detected, below the detection limit.

Example 13 identification of microorganisms contributing to the Simultaneous fermentation of example 7

Samples of biological fluid "EC 12B" and recycled water "EA 02" were taken during the testing described in example 7 (samples taken on days 21 and 22 at 3 months). The liquid sample was frozen in 10% glycerol and stored at-20 ℃ to facilitate 16S rDNA analysis for the identification of microorganisms in the sample, which 16S rDNA analysis is widely used for the identification of prokaryotes and phylogenetic analysis based on its 16S component of the ribosomal small subunit. Frozen samples on dry ice were transported to GATC Biotech AB, Solna, SE for 16S rDNA analysis (GATC _ Biotech). The analysis includes:

genomic DNA was extracted, an amplification library was prepared with a universal primer pair (27F: AGAGAGTTTGATCCTGGCTCCAG/534R: ATTACCGCGGCTGCTGG; 507bp long) spanning the hypervariable regions V1 to V3, PCR-labeled with the GS FLX adapter, and sequenced on the genome sequencer FLX instrument to obtain reads of 104.000-160.000 for each sample. The resulting sequences were then queried in the rDNA database from the ribosomal database entry (Cole et al, 2009) with BlastN. The database contains high quality sequences of at least 1200bp in length and has a taxonomic relationship to NCBI. The current Release (RDP Release10, updated at 9/19/2012) contains sequences of 9162 bacteria and 375 archaea. BLAST results were filtered to remove short and low quality hits (sequence identity ≧ 90%, alignment coverage ≧ 90%).

A total of 452, 310, 785, 594 different bacteria were identified in samples EC12B-21/3, EC12B-22/3 and EA02B 21/3, EA 02-22/3.

The analysis clearly shows that, at the species level, lactobacillus amyloliquefaciens is by far the most dominant bacterium, accounting for 26% -48% of all microorganisms tested. The microorganisms in the EC12B samples were similar, with 13 dominant bacteria (Lactobacillus amylovorus DSM11664, Lactobacillus delbrueckii subsp. delbrueckii), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus simus, Lactobacillus delbrueckii subsp. lactis DSM20072(Lactobacillus delbrueckii subsp. lactis DSM 20072), Bacillus coagulans (Bacillus coagulons), Lactobacillus bulgaricus (Lactobacillus halsser), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus buchneri (Lactobacillus paracasei)), and the two types of samples were of the same distribution.

EA02 sample was similar to EC12B, although the l. The distribution of the 13 dominant bacteria (Lactobacillus amylovorus DSM11664, Lactobacillus delbrueckii subspecies, Lactobacillus amylovorus, Lactobacillus delbrueckii subspecies lactis DSM20072, Lactobacillus simlis JCM 2765, Lactobacillus delbrueckii subspecies pterocarpus, Lactobacillus plantarum (Lactobacillus parapatarum), Weissella ganesei (Weissella ghansensis), Lactobacillus fermentum oligosaccharide LMG22743(Lactobacillus oligoformans LMG 22743), Weissella beiningensis (Weissella benensis), Leuconosoc gasicomatatum LMG 18811, Weisella soli, Lactobacillus delbrueckii) was also very similar, except for the presence of Pseudomonas psychrophila 14-3(Pseudomonas existralis 14-3) among the 13 dominant bacterial species. This pseudomonas, found in EA02(21/3), was previously isolated from temporary ponds in antarctic states and should be capable of producing Polyhydroxyalkanoates (PHA) from caprylate and glucose (Lopez et al, 2009; Tribelli et al, 2012).

Comparison of the results at the genus level showed that Lactobacillus (Lactobacillus) accounted for 56-94% of the identified bacteria in the samples. Again, the distribution of genera between the two sampling days of EC12B and EA02 is very similar. Interestingly, in the EA02 sample, the genera weissella (Weisella), Leuconostoc (Leuconostoc) and Pseudomonas (Pseudomonas) were present in large amounts (1.7-22%), but in the EC12B sample only a minor constituent (> 0.1%). Weissella (Weissella) and Leuconostoc (Leuconostoc) belong to the order Lactobacillales, as do lactobacilli.

The dominant pathogens in EC12B and EA02 sampled during the test described in example 7 accounted for 0.281-0.539% and 0.522-0.592% of the total bacteria identified, respectively. The predominant pathogenic bacteria in the EC12B sample were Aeromonas (Aeromonas spp.), Bacillus cereus (Bacillus cereus), Brucella (Brucella sp.), Citrobacter (Citrobacter spp.), Clostridium perfringens (Clostridium perfringens), klebsiella sp., Proteus (Proteus sp.), Providencia sp., Salmonella (Salmonella spp.), Serratia (Serratia sp.), shigella sp., and Staphylococcus aureus (Staphylococcus aureus). Sporulated pathogens were not detected in EC12B and EA02 as described in example 7. The total amount of pathogenic bacteria detected in EC12B and EA02 decreased over time and the total number of bacteria in EC12B almost disappeared within one day.

D portes et al (1998) outline pathogenic bacteria known to be present in MSW. Table 5 shows the presence of pathogenic bacteria in the MSW described in examples 3, 5 and 7 (D portes et al (1998) and 16S rDNA analysis). Proteua sp and Providencia sp (Providencia sp.) were also found in EC12B and EA02 sampled during the tests described in example 7, in addition to the pathogens described by Disportes et al (1998). However, the only pathogenic bacteria present in the biological fluid of example 5, streptococci, were not present here. This indicates the presence of other bacterial populations in EC12B and EA02 of example 7, which may be due to inter-organism competition for nutrients and a slight drop in temperature during the process that will promote the growth of the other bacterial populations.

TABLE 5 review of pathogens present in examples 3, 5 and 7

Strain identification and DSMZ Collection

Samples of EA02 taken from the test period described in example 7 on days 3 and 22 were plated to bio-persistent nordic center (NN center) (helschlemm, denmark) to identify and obtain a single culture of isolated bacteria. After reaching the NN center, the samples were incubated at 50 ℃ overnight, then plated on different plates (GM17, tryptic Soy Medium and beef extract (GM17 agar: 48.25g/L m17 agar sterilized at high temperature for 20 minutes, glucose was added to a final concentration of 0.5%, tryptic Soy agar: 30g/L tryptic Soy Medium, 15g/L agar, beef Medium supplemented with 15g/L agar (national serum institute, Copenhagen, Denmark)) and grown aerobically at 50 ℃ after one day, the plates were visually inspected, and the selected clones were reapplied to the corresponding plates and sent to the DSMZ for identification.

The following strains isolated from EA02 recycled water have been patented in DMSZ of beronsweck DSMZ, germany:

identified sample

Sample ID: bacillus safensis (Bacillus safensis) 13-349 was derived from (EA02-21/3), DSM 27312, and deposited at DSMZ on 11/06/2013.

Sample ID: 13-352 Brevibacillus brevis (Brevibacillus brevis) was derived from (EA02-22/3), DSM 27314, and deposited at DSMZ on 11/06/2013.

Sample ID: 13-353 Bacillus subtilis sp. subtilis, derived from (EA02-22/3), DSM 27315, deposited at DSMZ at 11.06.2013.

Sample ID: 13-355 Bacillus licheniformis (Bacillus licheniformis) originated from (EA02-21/3), DSM 27316, deposited at DSMZ on 11/06/2013.

Sample ID: 13-357 Actinomyces bovis (Actinomyces bovis) originated from (EA02-22/3), DSM 27317, deposited at DSMZ on 11/06/2013.

Unidentified sample

Sample ID: 13-351 were derived from (EA02-22/3), DSM 27313, deposited at DSMZ on month 06 and 11 of 2013.

Sample ID: 13-362A was derived from (EA02-22/3), DSM 27318, and was deposited at DSMZ on 11/06/2013.

Sample ID: 13-365 were derived from (EA02-22/3), DSM 27319, deposited at DSMZ on month 06 and 11 of 2013.

Sample ID: 13-367 was derived from (EA02-22/3), DSM 27320, deposited at DSMZ on month 11 of 2013 on 06.

Reference to the literature

Cole,J.R.,Wang,Q.,Cardenas,E.,Fish,J.,Chai,B.,Farris,R.J.,&Tiedje, J.M.(2009).The Ribosomal Database Project:improved alignments and new tools for rRNA analysis.Nucleic acids research,37(suppl 1), (D141-D145).

GATC_Biotech supporting material.Defining the Microbial Composition of Environmental Samples Using Next Generation Sequencing.Version 1. Tribelli,P.M.,Iustman,L.J.R.,Catone,M.V.,Di Martino,C.,Reyale,S., Méndez,B.S.,López,N.I.(2012).Genome Sequence of the Polyhydroxybutyrate Producer Pseudomonas extremaustralis,a Highly Stress-Resistant Antarctic Bacterium.J.Bacteriol.194(9):2381.

Nancy I.López,N.I.,Pettinari,J.M.,Stackebrandt,E.,Paula M.Tribelli,P. M.,M.,Steinbüchel,A.,Méndez,B.S.(2009).Pseudomonas extremaustralis sp.nov.,a Poly(3-hydroxybutyrate)Producer Isolated from an Antarctic Environment.Cur.Microbiol.59(5):514-519.