OA17181A - Methods and compositions for biomethane production. - Google Patents

Abstract

Methods of processing municipal solid wastes (MSW) are provided whereby concurrent enzymatic hydrolysis and microbial fermentation of wastes results in liquefaction of biodegradable components as well as accumulation of microbial metabolites. Liquefied biodegradable components are then separated from nondegradable solids to produce a bioliquid characterized in comprising a large percentage of dissolved solids of which a large fraction comprises some combination of acetate, ethanol, butyrate, lactate, formate or propionate. This bioliquid is, itself, a novel biomethane substrate composition, which permits very rapid conversion to biomethane. Methods of biomethane production are further provided using this bioliquid and using other biomethane substrate compositions produced by concurrent enzymatic hydrolysis and microbial fermentation of organic materials.

Description

Methods and compositions for blomethane production.

Municipal solid wastes (MSW), particularly including domestic household wastes, wastes from restaurants and food processing facilities, and wastes from office buildings comprise a very large 5 component of organic material that can be further processed to energy, fuels and other useful products. At présent only a small fraction of avallable MSW Is recyded, the great majority being dumped into landfills.

Considérable Interest has arisen In development of effident and environmentally friendly methods 10 of processing solid wastes, to maxlmize recovery of their Inhérent energy potential and, also, recovery of recydable materials. One sîgnificant challenge in waste to energy processing has been the heterogeneous nature of MSW. Solid wastes typically comprise a considérable component of organic, degradable material Intermingled with plastics, giass, me tais and other nondegradable materials. Unsorted wastes can be directly used In incinération, as is widely practiced 15 in countries such as Denmark and Sweden, which rely on district heating Systems. (Strehlik 2009).

However, indneration methods are associated with négative environmental conséquences and do not accomplish effective recyding of raw materials. Clean and effident use of the degradable component of MSW combined with recyding typically requires some method of sortlng to separate degradable from non-degradable material.

The degradable component of MSW can be used in waste to energy processing using both thermo-chemical and blological methods. MSW can be subject to pyrolysis or other modes of thermo-chemical gasificatlon. Organic wastes thermaliy decomposed at extreme high températures, produce volatile components such as tar and methane as well as a solid residue or 25 coke that can be bumed with less toxlc conséquences than those associated with direct

Incinération. Altematively, organic wastes can be thermaliy converted to syngas, comprising carbon monoxide, carbon dioxlde and hydrogen, which can be further converted to synthetîc fuels. See e.g. Malkow 2004 for review.

Blological methods for conversion of degradable components of MSW Include fermentation to produce spécifie useful end products, such as éthanol. See e.g. W02009/150455; W02009/095693; W02007/036795; Ballesteros et al. 2010; U et al 2007.

Altematlvely, biological conversion can be achieved by anaérobie digestion to produce biomethane or biogas. See e.g. Hartmann and Ahring 2006 for review. Pre-sorted organic component of MSW can be converted to biomethane directly, see e.g. US2004/0191755, or after a comparatively simple puiping process involving mindng in the presence of added water, see e.g. US2008/0020456.

However, pre-sorting of MSW to obtain the organic component is typlcally costly, Inefficient or impractical. Source-sorting requires large Infrastructure and operating expenses as well as the active participation and support from the community from which wastes are collected - an activity which has proved difficult to achieve in modem urban societies. Mechanicai sorting is typîcally capital Intensive and further associated with a large ioss of organic material, on the order of at least 30% and often mu ch hlgher. See e.g. Connsonni 2005.

Some of these problems with sorting Systems hâve been successfully avoided through use of liquéfaction of organic, degradable components In unsorted waste. Uquefied organic material can be readiiy separated from non-degradabie materials. Once liquefied Into a pumpable slurry, organic component can be readiiy used In thermo-chemlcai or biologica! conversion processes. Liquéfaction of degradable components has been widely reported using high pressure, high température autoclave processes, see e.g. US2013/0029394; US2012/006089; US20110008865; W02009/150455; W02009/108761 ; W02008/081028; US2005/0166812; US2004/0041301; US 5427650; US 5190226.

A rad ica üy different approach to liquéfaction of degradable organic components is that this may achieved using biologica) process, specifically through enzymatic hydroiysis, see Jensen et ai. 2010; Jensen et al. 2011; Toninl and Astrup 2012; WO2007/036795; WO2010/032557.

Enzymatic hydroiysis offers unique advantages over autodave methods for liquéfaction of degradable organic components. Using enzymatic liquéfaction, MSW processing can be conducted In a continuous manner, using comparatively cheap equipment and non-pressurized reactions run at comparatively low températures. In contrast, autodave processes must be conducted In batch mode and generaily Involve much hlgher capital costs.

A perceived need for steriiizatîon so as to reduce possible health risks posed by MSW-boume pathogenlc microogranlsms has been a prevalflng theme In support of the prédominance of autodave liquéfaction methods. See e.g. W02009/150455; W02000/072987; Ll et al. 2012;

Ballesteros et at. 2010; LJ et al. 2007. Similariy, It was previously believed that enzymatic liquéfaction requlred thermal pre-treatment to a comparatively high température of at least 90- 95o C. This high température was considered essentiel, in part to effect a sterilization of unsorted MSW and also so that degradable organic components could be softened and paper products 5 pulped. See Jensen et al. 2010; Jensen et al. 2011; Tonini and Astrup 2012.

We hâve discovered that safe enzymatic liquéfaction of unsorted MSW can be achieved without high température pre-treatment. Indeed, contrary to expectations, high température pre-treatment is not only unnecessary, but can be actively detrimental, since this kills ambient microorganisms 10 which are thriving in the waste. Promoting microbial fermentation concurrent^ with enzymatic hydrolysis at thermophillic conditions >45o C improves organic capture, either using ambient microorganisms or using selectlvely inoculated organisme. That is, concurrent thermophillic microbial fermentation safely Increases the organic yield of bioliquid, which is our term for the liquefied degradable components obtained by enzymatic hydrolysis. Under these conditions, 15 pathogenic microogranisms typically found in MSW do not thrive. See e.g. Hartmann and Ahring

2006; Déportés et al. 1998; Carrington et al. 1998; Bendixen et al. 1994; Kubler et al. 1994; Six and De Baerre et al. 1992. Under these conditions, typical MSW-boume pathogens are easily outcompeted by ubiqultous lactic acid bacteria and other safe organisme.

In addition to improving organic capture from enzymatic hydrolysis, concurrent microbial fermentation using any combination of lactic add bacteria, or acetate-, éthanol-, formate-, butyrate-, lactate-, pentanoate- or hexanoate- produdng microorganisms, pre-conditions the bioliquid so as to render It more effident as a substrate for biomethane production. Microbial fermentation produces bioliquid having a generally Increased percentage of dissolved compared with suspended solids, relative to bioliquid produced by enzymatic liquéfaction aione. Higher chain polysaccharides are generally more thoroughly degraded due to microbial pre-conditioning. Concurrent microbial fermentation and enzymatic hydrolysis dégradés biopolymers into readily usable substrates and, further, achleves metabolic conversion of primary substrates to short chain carboxylic adds and/or éthanol. The resultîng bioliquid comprising a high percentage of microbial métabolites provides a biomethane substrate which effectively avoids the rate llmiting hydrolysis step, see e.g. Delgenes et al. 2000; Angelidaki et al. 2006; Cysneiros et al. 2011, and which offers further advantages for methane production, particulariy using very rapld fixed filter anaérobie digestion Systems.

Summary.

Brief description ofthe figures.

Figure 1. Conversion of dry matter expressed as dry matter recovered In supematant as a percent of total dry matter in concurrent enzymatlc hydrolysis and microbial fermentation stimuiated by inoculation with EC12B bioliquid from exampie 5.

Figure 2. Bacterial metaboiites recovered in supematant following concurrent enzymatlc hydrolysis and fermentation induced by addition of bioliquid from example 5.

Figure 3. Graphical présentation of the REnescience test-reactor.

Figure 4. Schematic illustration of démonstration piant set-up.

Figure 5. Organic capture in bioliquid during different time period expressed as kg VS per kg MSW processed.

Figure 6. Bacterial metaboiites expressed as a percent of dissolved VS in bioliquid as well as aérobic bacterial counts at different time points during the experiment.

Figure 7. Distribution of bacterial spedes identified in bioliquid from example 3.

Figure 8 Distribution of the 13 prédominant bacteria in the EC12B sam pied from the test described in example 5.

Figure 9. Biomethane production ramp up and ramp down using bioliquid from example 5.

Figure 10 Biomethane production ramp up and ramp down characterization ofthe high lactate bioliquid from example 2.

Figure 11 Biomethane production ramp up and ramp down characterization of the low lactate bioliquid from example 2.

Figure 12 shows biomethane production ramp up characterization of the hydrolysed wheat straw bioliquid.

Detailed description of embodiments.

In some embodiments, the Invention provides a method of processing municipal solid waste (MSW) comprising the steps of (l). providing MSW at a non-water content of between 5 and 40% and at a température of between 45 and 75 degrees C, (il), enzymatically hydrolyslng the biodégradable parts of the MSW concurrently with microbial fermentation at a température between 45 and 75 degrees C resulting In liquéfaction of biodégradable parts of the waste and accumulation of microbial métabolites, followed by (iîi). sorting of the liquefied, biodegradble parts of the waste from non-biodegradable solids to produce a bioliquid characterized In comprising dissolved volatile solids of which at least 25% by welght comprise any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate, followed by (Iv). anaérobie digestion of the bioliquid to produce biomethane.

In some embodiments, the Invention provides an organic liquid blogas substrate produced by enzymatic hydrolysls and microbial fermentation of municipal solid waste (MSW) characterized In that

- at least 40% by welght of the non-water content exists as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate.

In some embodiments, the Invention provides a method of produdng blogas comprising the steps of (i). providing an organic liquld blogas substrate pre-conditioned by microbial fermentation such that at least 40% by weight of the non-water content exists as dissolved volatile soiids, which dissolved volatile soiids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, iactate and/or proplonate, (il), transferring the liquld substrate Into an anaérobie digestion System, followed by (lil). conducting anaérobie digestion of the liquld substrate to produce biomethane.

The metabolic dynamics of microbial communlties engaged in anaérobie digestion are complex. See Supaphoi et ai. 2010; Monta and Sasaki 2012; Chandra et al. 2012. in typlcal anaérobie digestion (AD) for production of methane blogas, bioiogical processes medlated by microorganisms achleve four primary steps - hydrolysis of bioiogical marcomolecuies into constituent monomers or other métabolites; acldogenesls, whereby short chaln hydrocarbon acids and aicohols are produced; acetogenesis, whereby availabie nutrients are catabolized to acetic acid, hydrogen and carbon dioxide; and methanogenesis, whereby acetic acid and hydrogen are catabolized by spedalized archaea to methane and carbon dioxide. The hydrolysis step Is typically rate-limitlng See e.g. Deigenes et al. 2000; Angeiidakl et al. 2006; Cysneiros et al. 2011.

Accordingly, It Is advantageous in preparing substrates for biomethane production that these be previously hydrolysed through some form of pretreatment. In some embodiments, methods of the Invention combine microbial fermentation with enzymatic hydrolysis of MSW as both a rapid bioiogical pretreatment for eventuai biomethane production as weli as a method of sortlng degradabie organic components from otherwise unsorted MSW.

Bioiogical pretreatments hâve been reported using solid biomethane substrates including sourcesorted organic component of MSW. See e.g. Fdez-Guelfo et ai. 2012; Fdez-Guelfo et ai. 2011 A; Fdez-Guelfo et ai. 2011 B; Ge et al. 2010; Lv et ai. 2010; Borghi et al. 1999. Improvements In eventuai methane yieids from anaérobie digestion were reported as a conséquence of increased dégradation of complex biopoiymers and Increased solubilisation of volatile soiids. However the levei of solubilisation of volatile soiids and the level of conversion to volatile fatty acids achleved by these previously reported methods do not even approach the levels achleved by methods of the invention. For example, Fdez-Guelfo et ai. 2011 A report a 10-50% relative Improvement in solubilisation of volatile soiids achleved through various bioiogical pretreatments of pre-sorted organic fraction from MSW - this corresponds to final absolute levels of solubilisation between about 7 to 10% of volatile solids. In contrast, methods of the Invention produce liquid blomethane substrates comprislng at least 40% dissolved volatile solids.

Two-stage anaérobie digestion Systems hâve also been reported In which the first stage process hydrolyses blomethane substrates Including source-sorted organic component of MSW and other spedalized blogenlc substrates. During the first anaérobie stage, which Is typically thermophillic, higher chaln polymère are degraded and volatile fatty acids produced. This is followed by a second stage anaérobie stage conducted in a physlcally separate reactor In which methanogenesls and acetogenesls domlnate. Reported two-stage anaérobie digestion Systems hâve typically utilized source-sorted, spedalized blogenlc substrates having less than 7% total solids. See e.g. Supaphol et al. 2011; Kim et al. 2011; Lv et al. 2010; Rlau et al. 2010; Kim et al. 2004; Schmlt and Ellis 2000; Lafitte-Trouque and Foreter 2000; Dugba and Zhang 1099; Kaiser et al. 1995; Harris and Dague 1993. More recently, some two stage AD Systems hâve been reported which utilize source-sorted, spedalized blogenlc substrates at levels as hlgh as 10% total solids. See e.g. Yu et al. 2012; Lee et al. 2010; Zhang et al.2007. Certalnly none ofthe reported two-stage anaérobie digestion Systems has ever contemplated use of unsorted MSW as a substrate, much less In order to produce a hlgh solids liquid blomethane substrate. Two stage anaérobie digestion seeks to convert solid substrates, continuously feeding additional solids to and continuously removing volatile fatty acids from the first stage reactor.

Any suitable solid waste may be used to practice methods ofthe Invention. As will be understood by one ski lied In the art, the term munldpal solid waste (MSW) refers to waste fradions which are typically available In a dty, but that need not corne from any munldpality per se. MSW can be any combination of celluloslc, plant, animal, plastic, métal, or glass waste Including but not limited to any one or more of the following: Garbage collected In normal munldpal collections Systems, optionally processed 1n some central sorting, shreddlng or pulplng device such Dewaster® or reCulture®; solid waste sorted from households, Including both organic fractions and paper rich fractions; waste fractions derived from Industry such as restaurant Industry, food processlng industry, general Industry; waste fractions from paper Industry, waste fractions from recycling fadlitles; waste fractions from food or feed industry; waste fraction from the medidnal Industry; waste fractions derived from agriculture or farmlng related sectors; waste fractions from processlng of sugar or starch rich produds; contamlnated or In other ways spolled agriculture produds such as grain, potatoes and beets not exploitable for food or feed purposes; garden refuse.

MSW Is by nature typicaliy heterogeneous. Statlstlcs conceming composition of waste materials are not wldely known that provide firm basis for compensons between countries. Standards and operating procedures for correct sampling and characterisation remain unstandardized. Indeed, only a few standardised sampling methods hâve been reported. See e.g. Riber et al., 2007. At least in the case of household waste, composition exhlblts seasonal and geographical variation. See e.g. Dahlen et al., 2007; Eurostat, 2008; Hansen et al., 2007b; Muhle et al., 2010; Riber et al., 2009; Slmmons et al., 2006; The Danlsh Environmental Protection agency, 2010. Geographical variation In household waste composition has also been reported, even over small distances of 200 — 300 km between munidpalities (Hansen et al., 2007b).

In some embodiments, MSW Is processed as 'unsorted* wastes. The term unsorted as used herein refers to a process In which MSW is not substantlaliy fractionated Into separate fractions such that blogenlc materiai Is not substantiaily separated from plastic and/or other inorganlc material. Wastes may be 'unsorted as used herein notwithstanding removal of some large objects or métal objects and notwithstanding some séparation of plastic and/or Inorganlc material. 'Unsorted waste as used herein refers to waste that has not been substantlaliy fractionated so as to provide a blogenlc fraction In which less than 15% of the dry weight Is non-biogenic material. Waste that comprises a mixture of blogenlc and non-biogenic material In which greater than 15% of the dry weight is non-blogenlc material Is unsorted as used herein. Typically unsorted MSW comprises blogenlc wastes, meaning wastes which can be degraded to blologically convertible substances, Including food and kltchen waste, paper- and/or cardboard-contalning materials, fbod wastes and the like; recydable materials, Including glass, bottles, caris, metals, and certain plastics; other bumable materials, which while not practically recyclable per se may give heat value In the form of refuse derived fuels; as well as Inert materials, Including ceramlcs, rocks, and various forms of débris.

In some embodiments, MSW can be processed as 'sorted waste. The term 'sorted' as used herein refers to a process In which MSW Is substantlaliy fractionated Into separate fractions such that blogenlc material Is substantiaily separated from plastic and/or other Inorganlc material. Waste that comprises a mixture of blogenic and non-blogenlc material In which less than 15% of the dry weight Is non-blogenlc material Is sorted as used herein.

In some embodiments, MSW can be source-separated organic waste comprislng predomlnantly fruit, vegetable and/or animal wastes. A variety of different sorting Systems can be used in some embodiments, Including source sorting, where househoids dispose of different waste materials separately. Source sorting Systems are currently In place In some munldpalities In Austria, Germany, Luxembourg, Sweden, Belglum, the Netheriands, Spain and Denmark. Altematively Industrial sorting Systems can be used. Means of mechanlcal sorting and séparation may Include any methods known In the art Including but not Iimited to the Systems described In US2012/0305688; W02004/101183; W02004/101098; WO2001/052993; W02000/0024531; WO1997/020643; WO1995/0003139; CA2563845; US5465847. In some embodiments, wastes may be lightly sorted yet still produce a waste fraction that is unsorted as used herein. In some embodiments, unsorted MSW Is used In which greater than 15% of the dry weight Is non-blogenlc 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 practldng methods of the invention, MSW should be provided at a non-water content of between 10 and 45%, or In some embodiments between 12 and 40%, or between 13 and 35%, or between 14 and 30%, or between 15 and 25%. MSW typlcaliy comprises considérable water content. Ail other solids comprising the MSW are termed non-water content as used herein. The level of water content used in practidng methods of the Invention relates to several interrelated variables. Methods of the invention produce a liquid blogenlc slurry. As will be readily understood byone skilled In the art, the capacity to render solid components into a liquid slurry Is Increased with Increased water content. Effective pulplng of paper and cardboard, which comprise a substantlal fraction of typlcal MSW, is typlcaliy Improved where water content Is Increased. Further, as Is well known in the art, enzyme activities can exhlblt dimlnlshed activity when hydrolysis is conducted under conditions with low water content. For example, cellulases typîcally exhibit dimlnished activity In hydrolysis mixtures that hâve non-water content hlgher than about 10%. In the case of cellulases, which dégradé paper and cardboard, an effectively iinear Inverse relationship has been reported between substrate concentration and yield from the enzymatic reaction per gram substrate. See Kristensen et al. 2009. Using commercially available Isolated enzyme préparations optimlzed for lignocelluloslc biomass conversion, we hâve observed in pilot scale studies that nonwater content can be as high as 15% without seelng cleariy detrimental effects.

In some embodiments, some water content should normally be added to the waste In order to achieve an appropriate non-water content For example, conslder a fraction of unsorted Danish household waste. Table 1, which describes characteristlc composition of unsorted MSW reported by Riber et al. (2009), Chemical composition of material fractions in Danish household waste, Waste Management 29:1251. Riber et al. characterized the component fractions of household wastes obtained from 2220 homes in Denmark on a single day in 2001. It will be readily understood by one skilled In the art that this reported composition Is simply a représentative example, useful In explalning methods of the Invention. In the example shown In Table 1, without any addition of water content priorto mlld heating, the organic, degradable fraction comprising vegetable, paper and animal waste would be expected to hâve approximately 47% non-water content on average, [(absolute % non-water)/(% wet weight)=(7.15 + 18.76 + 4.23)/(31.08 + 23.18 + 9.88) = 47% non5 water content ] Addition of a volume of water corresponding to one weight équivalent of the waste fraction being processed would reduce the non-water content of the waste Itself to 29.1% (58.2%/2) whlle reducing the non-water content of the degradable component to about 23.5% (47%/2). Addition of a volume of water corresponding to two weight équivalents of the waste fraction being processed would reduce the non-water content of the waste itself to 19.4% (58.2%/3) while reducing the non-water content of the degradable component to about 15.7% (47%/3).

Table 1 Summarised mass distribution of waste fractions from Denmark 2001 (a) Pure fraction.

(b) Sum of: newspaper, magazines, advertisements, books, office and clean/dirty paper, paper and carton containers, cardboard, carton with plastic, carton with Al foil, dirty cardboard and kltchen tissues.

(c) Sum of: Soft plastic, plastic bottles, other hard plastic and non-recyclable plastic.

(d) Sum of: Soit, Rocks etc., ash, ceramics, cat lifter and other non combustibles.

(e) Sum of: AI containers, al foil, metaf-like foil, métal containers and other métal.

(f) Sum of: Clear, green, brown and other glass.

(g) Sum of: The remalnlng 13 material fractions.

Waste fraction	Part of overall waste Part of overall waste quantlty expressed as absolute
%wet weight	contribution to total non water content of 58.2%
Vegetable waste (a)	31.08	7.15
Paper waste (b)	23.18	18.76
Animal waste(a)	9.88	4.23
Plastic waste (c)	9.17	8.43
Dlapers (a)	6.59	3.59

Non combustibles
(d)	4.05
Métal (e)	3.26
Glass (f)	2.91
Other (g)	9.88
TOTAL	100.00%

3.45

2.9

2.71

6.98

58.20%

One skilled in the art will readily be able to détermine an appropriate quantity of water content, if any, to add to wastes in practidng methods of the invention. Typlcally as a practical matter, notwithstandîng some varia billty in the composition of MSW belng processed, it Is convenant to add a relativeIy constant mass ratio of water, In some embodiments between 0.8 and 1.8 kg water per kg MSW, or between 0.5 and 2.5 kg water per kg MSW, or between 1.0 and 3.0 kg water per kg MSW. As a resuit, the actual non-water content of the MSW during processing may vary withln the appropriate range. Depending on the means being used to achleve enzymatic hydrolysis, the appropriate level of non-water content may vary.

Enzymatic hydrolysis can be achleved using a variety of different means. In some embodiments, enzymatic hydrolysis can be achleved using Isolated enzyme préparations. As used herein, the term Isoiated enzyme préparation refera to a préparation comprising enzyme adlvities that hâve been extracted, secreted or otherwise obtained from a blologlcal source and optionally partiaily or extensively purified.

A variety of different enzyme activlties may be advantageously used to practice methods of the invention. Considering, for example, the composition of MSW shown In Table 1, it will be readily apparent that paper-contalning wastes comprise the greatest single component, by dry weight, of the biogenic material. Accordlngly, as will be readily apparent to one skilled In the art, for typlcal household waste, cellulose-degrading activity will be partlculariy advantageous. In papercontalning wastes, cellulose has been previously processed and separated from its naturel occurrence as a component of lignoceiluloslc biomass, Intermingled with lignin and hemiceliulose. Accordingly, paper-contalning wastes can be advantageously degraded using a comparatively simple cellulase préparation.

Cellulase activity refera to enzymatic hydrolysis of 1,4-B-D-giycosldic linkages In cellulose. In Isolated cellulase enzyme préparations obtained from bacterial, fungal or other sources, cellulase activity typically comprises a mixture of different enzyme activities, Inciuding endoglucanases and exoglucanases (also termed celloblohydrolases), which respectively catalyse endo- and exohydrolysls of 1,4-B-D-glycosidic iinkages, along with B-glucosidases, which hydrolyse the oligosaccharide products of exoglucanase hydrolysis to monosaccharides. Complété hydrolysls of insoluble cellulose typically requires a synergistic action between the different activities.

As a practical matter, lt can be advantageous in some embodiments to slmply use a commerdally avallable Isolated cellulase préparation optimlzed for lignocelluloslc biomass conversion, slnce these are readily avallable at comparatively iow cost. These préparations are certalnly suitable for practicing methods of the invention. The term optimlzed for lignocellulosic biomass conversion refers to a product development process In which enzyme mixtures hâve been selected and modified for the spécifie purpose of Improvlng hydrolysls ylelds and/or reducing enzyme consumptlon In hydrolysls of pretreated lignocellulosic biomass to fermentable sugars.

However, commercial cellulase mixtures optimlzed for hydrolysis of lignocellulosic biomass typically contain hlgh ievels of additional and spedalized enzyme activities. For example, we determlned the enzyme activities présent in commerdally avaiiable ceiiuiase préparations optimlzed for lignocellulosic biomass conversion and provided by NOVOZYMES ™ under the trademarks CELLIC CTEC2 ™ and CELLIC CTEC3™ as well as similar préparations provided by GENENCOR ™ under the trademark ACCELLERASE 1500 ™ and found that each of these préparations contalned endoxylanase activity over 200 U/g, xylosidase activity at Ievels over 85 U/g, B-Larabinofuranosldase activity at Ievels over 9 U/g, amyloglucosldase activity at Ievels over 15 U/g, and a-amy!ase activity at Ievels over 2 U/g.

Slmpler isolated cellulase préparations may also be effedively used to practice methods of the invention. Suitable cellulase préparations may be obtained by methods well known In the art from a variety of mlcroorganisms, Induding aérobic and anaérobie bacteria, white rot fungi, soft rot fungl and anaérobie fungi. As described In ref. 13. R. Singhania et al., Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for mlcrobial cellulases, Enzyme and Mlcrobial Technoiogy (2010) 46:541-549, which Is hereby expressly incorporated by reference In entlrety, organisme that produce cellulases typically produce a mixture of different enzymes In appropriate proportions so as to be suitable for hydrolysls of lignocellulosic substrates. Preferred sources of cellulase préparations useful for conversion of lignocellulosic blomass Include fungi such as species of Trichoderma, Pénicillium, Fusarlum, Humlcola, Aspergillus and Phanerochaete.

In addition to ceiluiase activity, some additional enzyme activities which can prove advantageous In practicing methods of the invention Include enzymes which act upon food wastes, such as proteases, glucoamylases, endoamyiases, proteases, pectin esterases, pectin iyases, and lipases, and enzymes which act upon garden wastes, such as xylanases, and xylosidases. In some embodiments It can be advantageous to Include other enzyme activities such as lamlnarases, ketatinases or laccases.

In some embodiments, a selected microorganism that exhiblts extra-cellular ceiluiase activity may be directly Inoculated in performing concurrent enzymatic hydroiysis and microbial fermentation, Including but not limited to any one or more of the following thermophillic, cellulytic organisme can be Inoculated, aione or In combination with other organisme Paenibaciïlus barcinonensis, see Asha et al 2012, Clostridium thermocellum, see Blume et ai 2013 and Lv and Yu 2013, selected species of Streptomyces, Microbispora, and Paenibaciïlus, see Eida et ai 2012, Clostridium stramlnlsolvens, see Kato et al 2004, species of Firmlcutes, Actinobacteria, Proteobacteria and Bacteroidetes, see Maki et ai 2012, Clostridium clariflavum, see Sasaki et ai 2012, new species of Clostridiales phylogenetically and physioiogicaity related to Clostridium thermocellum and Clostridium straminlsolvens, see Shiratori et ai 2006, Clostridium clariflavum sp. nov, and Clostridium Caenicola, see Shiratori et ai 2009, Geobacilius Thermoleovorans, seeTai et ai 2004, Clostridium stercorarium, see Zveriov et ai 2010, or any one or more of the thermophillic fungi Sporotrichum thermophile, Scytalidium thermophiilum, Clostridium straminlsolvens and Thermonospora curvata, Kumar et al. 2008 for review. In some embodiments, organisme exhibiting other usefui extra cellular enzymatic activities may be inoculated to contribute to concurrent enzymatlc hydroiysis and microbial fermentation, for exampie, proteoiytic and keratinolytic fungi, see Kowaiska et ai. 2010, or lactlc acid bacteria exhibiting extra-cellular lipase activity, see Meyers et ai. 1996.

Enzymatic hydroiysis can be conducted by methods weli known in the art, using one or more Isolated enzyme préparations comprising any one or more of a variety of enzyme préparations including any of those mentioned previousiy or, altematively, by inocuiating the process MSW with one or more selected organisais capable of affecting the desired enzymatic hydroiysis. In some embodiments, enzymatic hydroiysis can be conducted using an effective amount of one or more isolated enzyme préparations comprising cellulase, B-glucosldase, amylase, and xylanase activities. An amount is an effective amount* where collectiveiy the enzyme préparation used achieves solubilisation of at least 40% of the dry weight of degradable biogenic material présent In MSW within a hydrolysis reaction time of 18 hours under the conditions used. In some embodiments, one or more Isolated enzyme préparations Is used In which collectiveiy the relative proportions of the various enzyme activities Is as follows: A mixture of enzyme activities Is used such that 1 FPU cellulase activity Is associated with at least 31 CMC U endoglucanase activity and such that 1 FPU cellulase activity Is associated with at least at least 7 pNPG U beta glucosidase activity. It will be readily understood by one skilled In the art that CMC U refers to carboxymethycellulose units. One CMC U of activity libérâtes 1 umol of reducing sugars (expressed as glucose équivalents) in one minute under spécifie assay conditions of 50° C and pH 4.8. It will be readily understood by one skilled in the art that pNPG U refers to pNPG units. One pNPG U of activity libérâtes 1 umol of nitrophenol per minute from para-nitrophenyl-B-Dglucopyranoside at 50° C and pH 4.8. It will be further readily understood by one skilled In the art that FPU of filter paper units provides a measure of cellulase activity. As used herein, FPU refers to filter paper units as determined by the method of Adney, B. and Baker, J., Laboratory Analytical Procedure #006, Measurement of cellulase activity, August 12,1996, the USA National Renewable Energy Laboratory (NREL), which Is expressly Incorporated by référencé herein In entirety.

In practidng embodiments of the invention, it can be advantageous to adjust the température of the MSW prior to initiation of enzymatic hydrolysis. As Is well known in the art, ceilulases and other enzymes typlcalfy exhibit an optimal température range. While examples of enzymes Isolated from extreme thermohillic organisais are certalnly known, having optimal températures on the order of 60 or even 70 degrees C, enzyme optimal température ranges typically fail within the range 35 to 55 degrees. In some embodiments, enzymatic hydrolysis are conducted within the température range 30 to 35 degrees C, or 35 to 40 degrees C, or 40 to 45 degrees C, or 45 to 50 degrees C, or 50 to 55 degrees C, or 55 to 60 degrees C, or 60 to 65 degrees C, or 65 to 70 degrees C, or 70 to 75 degrees C. In some embodiments it Is advantageous to conduct enzymatic hydrolysis and concurrent microbial fermentation at a température of at least 45 degrees C, because this is advantageous In discouraging growth of MSW-boume pathogens. See e.g. Hartmann and Ahring 2006; Déportés 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 will typica lly sacchartify celluloslc material. Accordingly, during enzymatic hydrolysis, solid wastes are both saccharified and llquefied, that Is, converted from a solid form Into a liquid slurry.

Previously, methods of processlng MSW using enzymatic hydrolysis to achleve liquéfaction of biogenlc components hâve envisioned a need for heating MSW to a température considerably higher than that requlred for enzymatic hydrolysis, specifically to achieve sterilization of the waste, followed by a necessary coollng step, to bring the heated waste back down to a température appropriate for enzymatic hydrolysis. In practicing methods of the Invention, It Is sufftdent that MSW be simply brought to a température appropriate for enzymatic hydrolysis. In some embodiments it can be advantageous to simply adjust MSW to an appropriate non-water content using heated water, adminîstered in such manner so as to bring the MSW to a température appropriate for enzymatic hydrolysis. In some embodiments, MSW Is heated, either by adding heated water content, or steam, or by other means of heating, within a reador vessel. In some embodiments, MSW is heated within a reactor vessel to a température greater than 30o C but less than 85o C, or to a température of 84oC or less, or to a température of 80oC or less, or to a température of 75o C or less, or to a température of 70o C or less, or to a température of 65o C or less, or to a température of 60o C or less, or to a température of 59o C or less, or to a température of 58o C or less, or to a température of 57o C or less, or to a température of 56o C or less, or to a température of 55o C or less, or to a température of 54o C or less, or to a température of 53o C or less, or to a température of 52o C or less, or to a température of 51 o C or less, or to a température of 50o C or less, or to a température of 49o C or less, or to a température of 48o C or less, or to a température of 47o C or less, or to a température of 46o C or less, or to a température of 45o C or less. In some embodiments, MSW is heated to a température not more than 10o C above the hîghest température at which enzymatic hydrolysis Is conduded.

As used herein MSW Is ‘heated to a température* where the average température of MSW Is Increased within a reador to the température. As used herein, the température to which MSW Is heated Is the hlghest average température of MSW achieved within the reador. In some embodiments, the highest average température may not be maintained for the entire period. In some embodiments, the heating reador may comprise different zones such that heating occurs in stages at different températures. In some embodiments, heating may be achieved using the same reador In which enzymatic hydrolysis Is conduded. The objed of heating is simply to render the majority of celluloslc wastes and a substantial fraction of the plant wastes in a condition optimal for enzymatic hydrolysis. To be in a condition optimal for enzymatic hydrolysis, wastes should ïdeally hâve a température and water content appropriate for the enzyme activities used for enzymatic hydrolysis.

In some embodiments, it can be advantageous to agltate during heating so as to achieve evenly heated waste. In some embodiments, agitation can comprise free-fall mixing, such as mixing In a reactor having a chamber that rotâtes along a substantially horizontal axis or In a mixer having a rotary axis lifting the MSW or in a mixer having horizontal shafts or paddles lifting the MSW. in some embodiments, agitation can comprise shaking, stirring or conveyance through a transport screw conveyor. In some embodiments, agitation continues after MSW has been heated to the desired température. In some embodiments, agitation is conducted for between 1 and 5 minutes, or between 5 and 10 minutes, or between 10 and 15 minutes, or between 15 and 20 minutes, or between 20 and 25 minutes, or between 25 and 30 minutes, or between 30 and 35 minutes, or between 35 and 40 minutes, or between 40 and 45 minutes, or between 45 and 50 minutes, or between 50 and 55 minutes, or between 55 and 60 minutes, or between 60 and 120 minutes.

Enzymatic hydrolysis Is Initiated at that point at which isolated enzyme préparations are added. Altematively, In the event that isolated enzyme préparations are not added, but instead microorganisms that exhibit desired extraceliuiar enzyme activities are used, enzymatic hydrolysis is Initiated at that point which the desired microorganism Is added.

In practidng methods of the invention, enzymatic hydrolysis Is conducted concurrently with microbial fermentation. Concurrent microbial fermentation can be achieved using a variety of different methods. In some embodiments, microorganisms naturally présent in the MSW are simply allowed to thrive in the reaction conditions, where the processed MSW has not previously been heated to a température that Is sufficient to effect a sterilization. Typicaliy, microorganisms présent in MSW will include organisme that are adapted to the local environment. The general bénéficiai effect of concurrent microbial fermentation is comparatively robust, meaning that a very wide variety of different organisais can, individually or collectively, contribute to organic capture through enzymatic hydrolysis of MSW. Without wishing to be bound by theory, we consider that cofermenting microbes Individually hâve some direct effect on dégradation of food wastes that are not necessarily hydrolysed by celiulase enzymes. At the same time, carbohydrate monomers and oligomers released by celiulase hydrolysis, in particular, are readily consumed by virtually any microbial spedes. Thls gives a benefidal synergy with celiulase enzymes, possibly through reiease of product inhibition of the enzyme activities, and aiso possibiy for other reasons that are not immediately apparent. The end products of microbial metabolism In any case are typicaliy appropriate for biomethane substrates. The enrichment of enzymatically hydroiysed MSW in microbial metaboiites is, thus, already, in and ofitself, an improvement In quality ofthe resulting biomethane substrate. Lactic acid bacteria In particular are ubiquitous in nature and lactic acid production Is typically observed where MSW Is enzymatically hydrolysed at non-water content between 10 and 45% within the température range 45-50. At higher températures, possibly other species of naturally occurring microorganisms may predominate and other microbial metaboiites than lactic acid may become more prévalent.

In some embodiments, microbial fermentation can be accompllshed by a direct inoculation using one or more microbial spedes. It will be readily understood by one skilled In the art that one or more bacterial spedes used for Inoculation so as to provide simultaneous enzymatic hydrolysis and fermentation of MSW can be advantageously selected where the bacterial spedes is able to thrive at a température at or nearthe optimum for the enzymatic activitîes used.

Inoculation of the hydrolysis mixture so as to induce microbial fermentation can be accompllshed by a variety of different means.

in some embodiments, it can be advantageous to Inoculate the MSW either before, after or concurrently with the addition of enzymatic activitîes or with the addition of microorganisms that exhlblt extra-cellular celluiase activity. In some embodiments, it can be advantageous to inoculate using one or more spedes of LAB induding but not limited to any one or more of the following, or genetlcally modified variants thereof: Lactobacillus plantarum, Streptococcus lactis, Lactobacillus casai, Lactobacillus lactis, Lactobacillus curvatus, Lactobacillus sake, Lactobacillus helveticus, Lactobacillus Jugurtl, Lactobacillus fermentum, Lactobacillus camls, Lactobacillus pisclcola, Lactobacillus corynlformls, Lactobacillus rhamnosus, Lactobacillus maltaromlcus, Lactobacillus psaudoplantarum, Lactobacillus agilis, Lactobacillus bavaricus, Lactobacillus alimentarius, Lactobacillus uamanashlensis, Lactobacillus amylophllus, Lactobacillus farclminls, Lactobacillus sharpaae, Lactobacillus divergans, Lactobacillus alactosus, Lactobacillus paracasal, Lactobacillus homohiochii, Lactobacillus sanfrandsco, Lactobacillus frudivorans, Lactobacillus bravis, Lactobacillus portti, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus vlridescans, Lactobacillus confusus, Lactobacillus mlnor, Lactobacillus kandlarl, Lactobacillus halotolarans, Lactobacillus hilgardl, Lactobacillus kafir, Lactobacillus collinoidas, Lactobacillus vacclnostarlcus, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus lelchmanni, Lactobacillus acldophilus, Lactobacillus salivarius, Lactobacillus saliclnus, Lactobacillus gassari, Lactobacillus suabicus, adobadllus oris, Lactobacillus bravis, Ladobadllus vaglnalis, Ladobacillus perttosus, Lactobacillus partis, Ladococcus cremoris, Ladococcus daxtranlcum, Ladococcus garviaaa,

Lectococcus hordniee, Lectococcus reffinolectis, Streptococcus diacetyiactis, Leuconostoc mesenteroides, Leuconostoc dextrenlcum, Leuconostoc cremoris, Leuconostoc oenos, Leuconostoc peremesenteroides, Leuconostoc pseudoesenteroides, Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc carnosum, Pediococcus damnosus, Pediococcus ecidiiactici, Pediococcus cervisiee, Pediococcus parvuius, Pediococcus haiophiius, Pediococcus pentosaceus, Pediococcus Intermedius, Bifidobectenum longum, Streptococcus thermophiius, Oenococcus oen! , Bifidobectenum brève, end Propionibacterium freudenreichli, or with some subsequently discovered spedes of LAB or with other species from the généra Enterococcus, Lectobeciiius, Lectococcus, Leuconostoc, Pediococcus, or Camobecterium that exhiblt useful capadty for metaboiic processes that produce lactic acid.

It will be readily understood by one skilled In the art that a bacterial préparation used for inoculation may comprise a community of different organisms. In some embodiments, naturally occurring b acte ri a which exist in any given géographie région and which are adapted to thrive In MSW from that région, can be used. As Is well known In the art, LAB are ubiqultous and will typlcally comprise a major component of any naturally occurring bacterial community withln MSW.

In some embodiments, MSW can be Inoculated with naturally occurring bacteria, by continued recyding of wash waters or process solutions used to recover resldual organic material from nondegradable solids. As the wash waters or process solutions are recyded, they gradualiy acquire higher microbe levels. In some embodiments, microbial fermentation has a pH lowering effect, espedally where métabolites comprise short chain carboxylic acids/ fatty adds such as formate, acetate, butyrate, proprionate, or lactate. Accordingly In some embodiments it can be advantageous to monltor and adjust pH of the concurrent enzymatic hydrolysls and microbial fermentation mixture. Where wash waters or process solutions are used to Increase water content of Incomlng MSW prior to enzymatic hydrolysls, inoculation Is advantageously made prior to addition of enzyme activitles, either as Isolated enzyme préparations or as mlcroorganlsms exhtblting extra-cellular cellulase activity. In some embodiments, naturally occurring bacteria adapted to thrive on MSW from a particular région can be cultured on MSW or on liquefied organic component obtained by enzymatic hydrolysis of MSW. In some embodiments, cultured naturally occurring bacteria can then be added as an Inoculum, either separately or supplémentai to inoculation using recyded wash waters or process solutions. In some embodiments, bacterial préparations can be added before or concurrently with addition of isolated enzyme préparations, or after some Initial period of pre-hydrolysis.

In some embodiments, spécifie stralns can be cultured for inoculation, including stralns that hâve been spedally modified or trained* to thrive under enzymatic hydrolysls réaction conditions and/or to emphaslze or de-emphasize particular metabolic processes. In some embodiments, it can be advantageous to inocuiate MSW using bacteriai stralns which hâve been Identified as capable of surviving on phthalates as sole carbon source. Such stralns include but are not limited to any one or more of the following, or genetically modified variants thereof: Chrysaomlcrobium Intachensa MW10T, LysInlbacclllusfusifonnisNBRC 157175, Tropiclbacter phthalicus, Gordonla JDC-2, Arthrbobactar JDC-32, Baclllus subtilis 3C3, Comamonas tastosteronli, Comamonas sp E6, Dalftla tsuruhatansis, Rhodoccoccusjostll, Burkholdaria cepacia, Mycobacterium vanbaalanii, Arthobactar kaysari, Baciïlus sb 007, Arthobactar sp. PNPX-4-2, Gordonla namiblansis, Rhodococcus phanolicus, Psaudomonas sp. PGB2, Psaudomonas sp. Q3, Psaudomonas sp. 1131, Psaudomonas sp. CAT1-8, Psaudomonas sp. Nitroreducens, Arthobactar sp AD38, Gordonla sp CNJ863, Gordonla rubrlparfmctus, Arthobactar oxydans, Aclnatobactar ganomosp, and Aclnatobactar calcoacatlcus. See e.g. Fukuhura et al 2012; Iwakl et al. 2012A; Iwakl et ai. 2012B; Latorre et ai. 2012; Uang et al. 2010; Llang et al. 2008; Navacharoen et al. 2011; Park et al. 2009; Wu et ai. 2010; Wu et al. 2011. Phthalates, which are used as plastidzers In many commercial poly vinyl chloride préparations, are leachable and, in our expérience, are often présent In liquefied organic component at levels that are undesirable. In some embodiments, stralns can be advantageously used which hâve been genetically modified by methods well known in the art, so as to emphaslze metabolic processes and/or de-emphasize other metabolic processes Including but not limited to processes that consume glucose, xylose or arablnose.

In some embodiments, it can be advantageous to Inocuiate MSW using bacteriai strains which hâve been Identified as capable of degradîng lignin. Such stralns include but are not limited to any one or more of the following, or genetically modified variants thereof: Comamonas sp B-9, Citrobacter fraundil, Cïtrobactar sp FJ581O23, Pandoraa norimbergensls, Amycolatopsis sp ATCC 39116, Straptomycas viridosporous, Rhodococcus jostil, and Sphlngoblum sp. SYK-6. See e.g. Bandounas et al. 2011; Bugg et al. 2011; Chandra et ai. 2011; Chen et ai. 2012; Davis et ai. 2012. In our expérience, MSW typically comprises considérable lignin content, which Is typically recovered as undigested resldual after AD.

In some embodiments, It can be advantageous to Inocuiate MSW using an acetate-produclng bacteriai strain, including but not iimlted to any one or more of the following, or genetically modified variants thereof: Acatltomaculum rumlnls, Anaarostipas caccaa, Acatoanaaroblum notaraa, Acetobactarlum carblnollcum, Acetobactarlum wiaringaa, Acetobactarlum woodil, Acatoganlum klvul, Acldamlnococcus farmentans, Anaarovibrio lipolytica, Bacteroldes coprosuls, Bacteroldes proplonidfaciens, Bacteroldes cellulosolvens, Bactaroldas xylanolyticus, Bifidobacterium catenulatum, Bifidobacterium blfidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium brave, Bifidobactarium gallicum, Bifidobacterium Înfantis, Bifidobactarium iongum, Bifidobactarium psaudolongum, ButyrMbrio fibrisolvens, Clostridium acatlcum, Clostridium acetobutylicum, Clostridium aclduricl. Clostridium bifermentans, Clostridium botulinum, Clostridium butyricium, Clostridium cellobioparum, Clostridium formlcaceticum, Clostridium histolyticum, Clostridium lochhaadil, Clostridium methylpentosum, Clostridium pasteurianum, Clostridium perfringens, Clostridium proplonicum, Clostridium putrefadens, Clostridium sporogenes, Clostridium tetanl, Clostridium tetanomorphum, Clostridium thermocellum, Desulfotomaculum orientis, Enterobacter eerogenes, Escherichia coli, Eubacterium limosum, Eubaderium rumlnantium, Fibrobacter succlnoganas, Lachnospira multiparus, Magasphaera aisdenii, Moorella thermoacetica, Pelobactar acetylenicus, Palobactar acldigallicl, Palobacter massiliensis, Pravotaiia rumlnocola, Proplonlbacterium freudenrelchiï, Rumlnococcus flavefaciens, Ruminobacter amylophilus, Rumlnococcus albus, Rumlnococcus bromii, Rumlnococcus champaneliensls, Selenomonas rumlnantium, Sporomusa paucivorans, Sucdnlmonas amylolytica, Sucdnivibrio daxtrinosolven, Syntrophomonas wolfei, Syntrophus additrophicus, Syntrophus gentianae, Treponema bryantii and Treponema primitia.

In some embodiments, it can be advantageous to Inoculate MSW using a butyrate-produdng bacterial strain, includïng but not limited to any one or more of the following, or genetically modified variants thereof: Acldaminococcus farmentans, Anaerostipas caccae, Bifidobacterium adolescentes, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Butyrivibrio hungatei. Clostridium acetobutylicum, Clostridium aurantibutyricum, Clostridium beijerinckii, Clostridium butyricium, Clostridium cellobioparum, Clostridium difficile, Clostridium Innocuum, Clostridium kluyveri. Clostridium pasteurianum. Clostridium perfringens, Clostridium protaoclasticum, Clostridium sporosphaeroidas, Clostridium symbiosum, Clostridium tertium, Clostridium tyrobutyricum, Coprococcus eutactus, Coprococcus cornes, Escherichia coli, Eubacterium barkeri, Eubacterium biforma, Eubacterium cellulosolvens, Eubacterium cylindroldes, Eubacterium doiichum, Eubacterium hadrum, Eubacterium haiii, Eubacterium limosum, Eubacterium moniliforme, Eubacterium oxldoraducens, Eubacterium ramulus, Eubacterium rectale, Eubacterium saburreum, Eubacterium tortuosum, Eubacterium ventriosum, Faecalibacterium prausnltzil, Fusobacterium prausnltzli, Peptostreptoccoccus vaglnalis, Peptostreptoccoccus tetradius, Pseudobutyrivibrio ruminis, Pseudobutyrivibrio xylanivorans, Roseburia cecicola, Roseburia intestlnalis, Roseburia hominis and Rumlnococcus bromii.

In some embodiments, it can be advantageous to inoculate MSW using a propionate-produdng bacterial strain, including but not limited to any one or more of the following, or genetically modified variants thereof: Anaerovibrio lipolytica, Baderddes coprosuis, Bacteroldes propionicifaciens, Blfidobacterium adolescentis, Clostridium acetobutylicum. Clostridium butyricium. Clostridium methylpentosum, Clostridium pasteurianum. Clostridium perfringens, Clostridium propionicum, Escherichia edi, Fusobacterium nudeatum, Megasphaera elsdenii, Prevotella rumlnocola, Proplonibacterium freudenreichii, Rumlnococcus bromil, Ruminococcus champanellensis, Selenomonas rumlnantium and Syntrophomonas wolfel.

In some embodiments, It can be advantageous to inoculate MSW using an ethanol-producing bacterial strain, Including but not limited to any one or more of the following, or genetically modified variants thereof: Acetobacterium carblnolicum, Acetobacterium wieringae, Acetobaderium woodil, Bacteroldes cellulosolvens, Bacteroldes xylandytlcus, Clostridium acetobutylicum. Clostridium beljerlnckli, Clostridium butyricium. Clostridium cellobioparum, Clostridium lochheadil, Clostridium pasteurianum, Clostridium perfringens, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermosacchardyticum, Enterobacter aerogenes, Escherichia edi, Klebsiella oxytoca, Klebsiella pneumonie, Lachnosplra multiparus, Lactobadllus brevis, Leuconostoc mesenteroldes, Paenibacillus macerans, Pelobacter acetylenlcus, Rumlnococcus albus, Thermoanaerobacter mathranil, Treponema bryantii and Zymomonas mobilis.

In some embodiments, a consortium of different microbes, optionally Including different spedes of bacteria and/or fungl, may be used to accomplish concurrent microbial fermentation. In some embodiments, suitable microorganisms may be selected so as to provide a desired metabolic outcome at the Intended reaction conditions, and then Inoculated at a high dose level so as to outcompete naturally occumng strains. For example, In some embodiments, It can be advantageous to Inoculate using a homofermentive lactate producer, since this provides a higher eventual methane potentiel In a resùlting biomethane substrate than can be provided by a heterofermentlve lactate producer.

In some embodiments, enzymatic hydrolysis and concurrent microbial fermentation are conducted using a hydrolysis reactor that provides agitation by free-fall mixing as described In W02006/056838, and In WO2011/032557.

Following some period of enzymatic hydrolysis and concurrent mlcroblal fermentation, MSW provided at a non-water content between 10 and 45% Is transformed such that blogenlc or •fermentable components become liquefied and mlcroblal métabolites accumulate In the aqueous phase, After some period of enzymatic hydrolysis and concurrent mlcroblal fermentation, the liquefied, fermentable parts of the waste are separated from non-fermentable solids. The liquefied material, once separated from non-fermentable solids, Is what we term a bloliquld.· In some embodiments, at least 40% of the non-water content of thls bloliquld 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 bloliquld comprise any combination of acetate, butyrate, 10 éthanol, formate, lactate, and/or proplonate. In some embodiments, at least 70% by weight of the dissolved volatile solids comprises lactate, or at least 60%, or at least 50%, or at least 40%, or at least 30%, or at least 25%.

In some embodiments, séparation of non-fermentable solids from liquefied, fermentable parts of the 15 MSW so as to produce a bloliquid characterized In comprising dissolved volatile solids of which at least 25% by weight comprise any combination of acetate, butyrate, éthanol, formate, lactate and/or proplonate Is conducted In less than 16 hours after the Initiation of enzymatic hydrolysis, or In less than 18 hours, or In less than 20 hours, or In less than 22 hours, or In less than 24 hours, or In less than 30 hours, or In less than 34 hours, or In less than 36 hours.

Séparation of liquefied, fermentable parts of the waste from non-fermentable solids can be achleved by a variety of means. In some embodiments, thls may be achleved using any combination of at least two different séparation operations, Including but not limited to screw press operations, balllstlc separator operations, vibrating sleve operations, or other séparation operations 25 known In the art. In some embodiments, the non-fermentable solids separated from fermentable parts of the waste comprise at least about 20% of the dry weight of the MSW, or at least 25%, or at least 30%. In some embodiments, the non-fermentable solids separated from fermentable parts of the waste comprise at least 20% by dry weight of recydable materials, or at least 25%, or at least 30%, or at least 35%. In some embodiments, séparation using at least two séparation operations 30 produces a bioliquid that comprises at least 0.15 kg volatile solids per kg MSW processed, or at least 010. It will be readily understood by one skilled in the art that the Inhérent blogenic composition of MSW Is variable. Nevertheless, the figure 0.15 kg volatile solids per kg MSW processed reflects a total capture of blogenlc material In typlcai unsorted MSW of at least 80%. The calculation of kg volatile solids captured in the bloliquld per kg MSW processed can be 35 estlmated over a time period In which total yields and total MSW processed are determined.

In some embodiments, after séparation of non-fermentable solids from liquefied, fermentable parts of the MSW to produce a bîotiquîd, the bioliquid may be subject to post-fermentation under different conditions, Including different température or pH.

The term dissolved volatile solids as used hère refers to a simple measurement calculated as follows: A sample of bioliquid 1s centrifuged at 6900 g for 10 minutes in a 50 ml Falcon tube to produce a pellet and a supematant. The supematant 1s decanted and the wet weight of the pellet expressed as a percentage fraction of the total Initial weight of the liquid sample. A sample of supematant Is dried at 60 degrees for 48 hours to détermine dry matter content. The volatile solids content of the supematant sample 1s determlned by subtracting from the dry matter measurement the ash remaining after fumace buming at 550 °C and expressed as a mass percentage as dissolved volatile solids In %. An Independent measure of dissolved volatile solids Is determlned by calculation based on the volatile solids content of the pellet. The wet weight fraction of the pellet is applied as a fractional estlmate of undlssolved solids volume proportion of total Intial volume. The dry matter content of the pellet Is determlned by drylng at 60 degrees C for 48 hours. The volatile solids content of the pellet Is determlned by subtracting from the dry matter measurement the ash remaining after fumace buming at 550 ’C. The volatile solids content of the pellet is corrected by the estlmated contribution from supematant liquid given by (1-wet fraction pe1let)x(measured supematant volatile solid %). From the total volatile solids % measured In the original liquid samples Is subtracted the (corrected volatile solids % of the pellet)x(fractional estimate of undissolved solids volume proportion of total Initial volume) to give an Independent estimate of dissolved volatile solids as %. The hlgher of the two estimâtes Is used In order not to overestimate the percentage of dissolved volatile solids represented by bacterial métabolites.

In some embodiments the invention provides compositions and methods for biomethane production. The preceding detaiied discussion conceming embodiments of methods of processing MSW may optionally be applied to embodiments providing methods and compositions for biomethane production. In some embodiments, the method of produdng biomethane comprises the steps of (i). providing an organic liquid biomethane substrate pre-conditioned by microbial fermentation such that at least 40% by weight of the non-water content exists as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate, (ii). transferring the liquid substrate Into an anaérobie digestion System, followed by (iîi). conducting anaérobie digestion of the liquid substrate to produce blomethane.

In some embodiments, the Invention provides an organic liquid blomethane substrate produced by enzymatlc hydrolysis and mlcroblal fermentation of municipal solid waste (MSW), orof pretreated lignocellusic biomass, altematively, comprislng enzymaticaily hydrolysed and mlcroblally fermented MSW, or comprislng enzymaticaily hydrolysed and mlcroblally fermented pretreated lignoceliulosic biomass characterized In that

- at least 40% by welght of the non-water content exists as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by welght of any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate.

As used herein the term anaérobie digestion System refers to a fermentation System comprising one or more reactors operated under controiled aération conditions In which methane gas Is produced In each of the reactors comprislng the System. Methane gas Is produced to the extent that the concentration of metabolically generated dissolved methane In the aqueous phase of the fermentation mixture wlthln the anaérobie digestion System Is saturating at the conditions used and methane gas Is emitted from the System,

In some embodiments, the anaérobie digestion System Is a fixed filter System. A fixed filter anaérobie digestion System refers to a System in which an anaérobie digestion consortium 1s Immobilized, optionally within a biofilm, on a physlcal support matrix.

In some embodiments, the liquid blomethane 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, oralleast 13%total solids. Total solidsas used hereinrefersto bothsolubleand Insoluble solids, and effectively means non-water content. Total solids are measured by drying at 60°C until constant weight Is achleved.

In some embodiments, microbial fermentation is conducted under conditions that discourage methane production by methanogens, for example, at pH less than 6.0, or at pH less than 5.8, or at pH less than 5.6, or at pH less than 5.5. In some embodiments, the liquid blomethane substrate comprises less than saturating concentatlons of dissolved methane. In some embodiments, the liquid biomethane substrate comprises less than 15 mg/L dissolved methane, or less than 10 mg/L, or less than 5 mg/L

In some embodiments, prior to anaérobie digestion to produce biomethane, one or more components of the dissolved volatile sollds may be removed from the liquid biomethane substrate by distillation, filtration, electrodialysis, spedfic blnding, précipitation or other means well known In the art In some embodiments, éthanol or lactate may be removed from the liquid biomethane substrate prior to anaérobie digestion to produce biomethane.

In some embodiments, a solid substrate such as MSW or fiber fraction from pretreated lignocelluloslc biomass, Is subject to enzymatic hydrolysis concurrents with microbial fermentation so as to produce a liquid biomethane substrate pre-condïtioned by microbial fermentation such that at least 40% by weight of the non-water content exlsts as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate. In some embodiments, a liquid biomethane substrate having the above mentioned properties Is produced by concurrent enzymatic hydrolysis and mlcroblal fermentation of liquefied organic material obtained from unsorted MSW by an autoclave process. In some embodiments, pretreated lignocelluloslc biomass can be mixed with enzymaticaliy hydrolysed and microbially fermented MSW, optionally in such manner that enzymatic activity from the MSW-derived biolioquid provides enzymatic activity for hydrolysis of the lignocelluloslc substrate to produce a composite liquid biomethane substrate derived from both MSW and pretreated lignocelluloslc biomass.

Soft lignocelluloslc biomass refers to plant biomass other than wood comprising cellulose, hemlcellulose and lignln. Any suitable soft lignocelluloslc biomass may be used, Induding biomasses such as at least wheat straw, corn stover, corn cobs, empty fruit bunches, rice straw, oat straw, bariey straw, canola straw, rye straw, sorghum, sweet sorghum, soybean stover, switch grass, Bermuda grass and other grasses, bagasse, beet pulp, corn fiber, or any combinations thereof. Lignocelluloslc biomass may comprise other lignocelluloslc materials such as paper, newsprint, cardboard, or other municipal or office wastes. Lignocelluloslc biomass may be used as a mixture of materials originating from different feedstocks, may be fresh, partialiy dried, fully dried or any combination thereof. In some embodiments, methods of the Invention are practiced using at least about 10 kg biomass feedstock, or at least 100 kg, or at least 500 kg.

Lignocellulosic biomass should generally be pretreated by methods known In the art priorto conducting enzymatic hydrolysis and microblal pre-conditioning. in some embodiments, biomass is pretreated by hydrothermal pretreatment. Hydrothermai pre-treatment refers to the use of water, either as hot liquid, vapor steam or pressurized steam comprising high température liquid orsteam or both, to cook biomass, at températures of 120o C or higher, either with or without addition of adds or other chemicals. in some embodiments, lignceilulosic biomass feedstocks are pretreated by autohydrolysis. Autohydrolysis refers to a pre-treatment process in which acetic add liberated by hemicellulose hydrolysis during pre-treatment further catalyzes hemicellulose hydrolysis, and applies to any hydrothermal pre-treatment of lignocellulosic biomass conducted at pH between 3.5 and 9.0.

In some embodiments, hydrothermally pretreated lignocellulosic biomass may be separated Into a liquid fraction and a solid fraction. Solid fraction and Uquld fraction’ refer to fractionation of pretreated biomass In solid/liquid séparation. The separated iiquid Is cdlectively referred to as liquid fraction. The residual fraction comprising considérable Insoluble solid content Is referred to as solid fraction. Either the solid fraction or the liquid fraction or both combined may be used to practice methods of the Invention or to produce compositions of the Invention, in some embodiments, the solid fraction may be washed.

Example 1. Concurrent mlcrobial fermentation Improves organic capture by enzymatic hydrolysis of unsorted MSW

Laboratory bench scale reactions were conducted with bioliquid sample from the test described In example 5.

The model MSW substrate for laboratory scale reactions was prepared using fresh produce to comprise the organic fraction (defined as the cellulosic, animal and vegetable fractions) of municipal solid waste (prepared as described In Jensen et al., 2010 based on Riber et al. 2009).

The model MSW was stored In aliquots at -20’C and thawed ovemight at 4C. The reactions were done In 50ml centrifuge tubes and the total reaction volume was 20g. Model MSW was added to 5% dry matter (DM) (measured as the dry matter content remaining after 2days at 60C).

The cellulase applied for hydrolysis was Cellic CTec3 (VDNI0003, Novozymes A/S, Bagsvaerd, Denmark) (CTec3). To adjust and maintaln the pH at pH5, a citrate buffer (0.05M) was applied to make up the total volume to 20g.

The reactions were incubated for 24hours on a Stuart Rotator SB3 (tuming at 4RPM) placed In a heating oven (Binder GmBH, Tuttlingen, Germany). Négative contrats were donc In parallel to assess backgraund release of dry matter from the substrate during Incubation. Following Incubation the tubes were centrifuged at 1350g for 1Ominutes at 4C. The supematant was then decanted off, 1ml was removed for HPLC analysis and the remaining supematant and pellet were dried for 2days at 60*C. The weight of dried material was recorded and used to calculate the distribution of dry matter. The conversion of DM In the model MSW was calculated based on these numbers.

The concentrations organic acids and éthanol were measured using an UltiMate 3000 HPLC (Thermo Sdentific Dionex) equipped with a refractive index detector (Shodex® RI-101) and a UV detector at 250nm. The séparation was performed on a Rezex RHM monosaccharide column (Phenomenex) at 80*C with 5mM H₂SO₄ as eluent at a flow rate of 0.6ml/min. The results were anaiyzed using the Chromeleon software program (Dionex).

To evaluate the effect of concurrent fermentation and hydrolysis, 2ml/20g of the bloliquld from the test described In example 5 (sampled on December 15^e1 and 1 β*¹) was added to the reactions with or without CTec3 (24mg/g DM).

Conversion of DM in MSW.

The conversion of soiids was measured as the content of soiids found In the supematant as a percent of total dry matter. Figure 1 shows conversion for MSW blank, Isolated enzyme préparation, microbial Inoculum alone, and the combination of microbial Inoculum and enzyme. The results shows that addition of EC12B from example 5 resulted in significantly hlgher conversion of dry matter compared to the backgraund release of dry matter In the reaction blank (MSW Blank) (Students t-Test p<0.0001). Concurrent microbial fermentation Induced by addition of the EC12B sample and enzymatic hydrolysis using CTec3 resulted In significantly hlgher conversion of dry matter compared to the reaction hydrolysed only with CTec3 and the reactions added EC12B alone (p<0.003).

HPLC anaiysis of glucose, lactate, acetate and EtOH.

The concentration of glucose and the microbial métabolites (lactate, acetate and éthanol) measured In the supematant are shown In Figure 2. As shown, there was a low background concentration of these In the model MSW blank and the lactic acid content presumably cornes from bacteria Indigenous to the model MSW since the material used to create the substrate was In no way stérile or heated to ktll bacteria. The effect of addition of CTec3 resulted In an Increase In glucose and lactic acid In the supematant. The highest concentrations of glucose and bacterial métabolites was found in the reactions where EC12B bioiiquid from example 5 was added concurrently with CTec3. Concurrent fermentation and hydrolysis thus Improve conversion of dry matter in mode! MSW and Increase the concentration of bacterial métabolites In the liquide.

Référencés: Jacob Wagner Jensen, Claus Felby, Henning Jergensen, Georg 0mskov Rensch, Nanna Dreyer Nerholm. Enzymatic processlng of municipal solid waste. Waste Management. 12/2010; 30(12):2497-503.

Rlber, C., Petersen, C., Christensen, T.H., 2009. Chemical composition of material fractions in Danish househoid waste. Waste Management 29,1251-1257.

Example 2. Concurrent microbial fermentation improves organic capture by enzymatic hydrolysis of unsorted MSW.

Tests were performed In a spedally deslgned batch reactor shown In Figure 3, using unsorted MSW with the aim to validate results obtained in lab scale experiments. The experiments tested the effect of adding an Inoculum of mlcroorganisms comprising bioiiquid obtained from example 3 bacteria in orderto achieve concurrent microbial fermentation and enzymatic hydrolysis. Tests were performed using unsorted MSW.

MSW used for smaii-scale trials were a focal point of the research and development at REnesdence. For the results of triais to be of value, waste was required to be représentative and reprodudble.

Waste was collected from Noml l/S Holstebro In March 2012. Waste was unsorted municipal solid waste (MSW) from the respective area. Waste was shredded to 30x30mm for use In small-scale trials and for collection of représentative samples for trials. Theory of sampllng was applied to shredded waste by sub-sampling of shredded waste In 22-litre buckets. Buckets were stored In a freezer container at -18*C until use. Real waste was composed of eight buckets of waste from the collection. The content of these buckets was remlxed and resampled In orderto ensure that variabllity between répétitions was as low as possible.

Ail samples were run under similar conditions regarding water, température, rotation and mechanlcal effect. Six chambers were used: three without Inoculation and three with Inoculation. Desïgnated non-water content during triai was set to 15 % non-water content by water addition. Dry matter In the Inoculating material was accounted for so the fresh water addition In the Inoculated chambers was smalier. 6 kg of MSW was added to each chamber, as was 84 g CTEC3, a commercial cellulase préparation. 2 liter of Inoculum was added to Inoculated chambers, with a corresponding réduction in added water.

pH was kept at 5.0 In the Inoculated chambers and at pH 4.2 in the non-inoculated chambers using respectively addition of 20% NaOH for increasing pH and 72% H₂SO< for decreaslng the pH. The lower pH in the non-inouclated chamber helped ensure that intrinslc bacteria would not flourish.

We hâve previously shown that, using the enzyme préparation used, CTEC3 Tm, In the context of MSW hydrolysls, no différence in activity can be discemed between pH 4.2 and pH 5.0 The reaction was continued at 50 degrees C for 3 days, with the pilot reactor provlding constant rotary agitation.

At the end ofthe reaction, the chamberswere emptied through a slave and bloliquld comprising Ilquefied material produced by concurrent enzymatic hydrolysls and mlcrobial fermentation of MSW.

Dry matter (TS) and volatile solids (VS) were determined Dry Matter (DM) method:

Samples were dried at 60 C for 48 hours. The weight of the sample before and after drylng was used to calculate the DM percentage.

Sample DM (%) (e)

X 100

Volatile solids method:

Volatile solids are calculated and presented as the DM percentage subtracted the ash content. The ash content of a sample was found by burning the pre-dried sample at 550 ‘C In a fumace for a minimum of 4 hours. Then the ash was calculated as:

Sample Ash percentage of dry matten

Samplf ash wight Çg)

Sample dry weight Cg) ^X

Volatile Solids percentage:

(1 - sample ash percentage) x

Sample DM percentage

Results were as shown below. As shown, a higher total solids content was obtalned In bioliquid obtalned In the inouculated chambers, Indlcatlng that concurrent microbial fermentation and enzymatic hydrolysis were superior to enzymatic hydrolysis atone.

	Bioliquid
	TS(kg)	VS (kg)
Std. iow lactate	1.098	0.853
Pode. High lactate	1.376	1.041
Added pode.TS +VS	TS	VS
Kg	0.228	0.17
Produced
	Bioliquid
	TS (kg)	stdev	VS (kg)	stdev
std. low lactate	1.098	0.1553	0.853	0.116
Pode. High lactate	1.148	0.0799	0.869	0.0799

	more %		more %
std. low lactate
Pode. High lactate	4.5579		1.8429

Sum metabolics (lactate acetate and éthanol) produced	% more
std avg.	92.20903	g/L
pode avg.	342.6085	g/L	271.5564
Sum metabolics (lactate acetate and éthanol) captured	% more
std avg. (low lac)	189.6075	g/L
pode avg. (high lac)	461.6697	g/L	143.4871

Example 3. Concurrent microbial fermentation Improves organic capture by enzymatic hydroiysis of unsorted MSW.

Experiments were conducted at the REnesdence démonstration plant placed at Amager ressource center (ARC), Copenhagen, Denmark. A schematic drawing showing prindple features of the plant Is shown in Figure 4. The concept of the ARC REnesdence Waste Refinery Is to sort MSW in to four products. A bio-iiquid for biogas production, inerts (glass and sand) for recyding and 2D and 10 3D fractions of inorganlc materials suitable for RDF production or recyding of metals, plastic and tree.

MSW from big dties is collected as is in plastic bags. The MSW Is transported to the REnesdence Waste Refinery where It is stored In a silo until processing. Depending on the character of the MSW 15 a sorting step can be installed In front of the REnesdence system to take out overslze partides (above 600 mm).

REnesdence technology as tested in this example comprises three steps.

The first step Is a mild heatlng (pretreatment, as shown in figure 4) of the MSW by hot water to températures In the range of 40-75° C for a period of 20-60 minutes. This heating and mixing period opens plastic bags and provides adéquate pulplng of degradabie components preparing a more homogenous organic phase before addition of enzymes. Température and pH are adjusted in the heatlng period to the optimum of isolated enzyme preparatons which are used for enzymatic hydrolysis. Hot water can be added as ciean tap water or as washlng water first used in the washing drums and then redrculated to the mild heating as Indicated in figure 4.

The second step is enzymatic hydrolysis and fermentation (liquéfaction, as shown in figure 4). in the second step of the REnescience process enzymes are added and optionally selected microorganlsms. The enzymatic liquéfaction and fermentation is performed continuously at a résidence time of app. 16 hours, at the optimal température and pH for enzyme performance. By this hydrolysis and fermentation the blogenic part of the MSW is liquefied in to a bio-liquid high in dry matter In between non-degradabie materials. pH is controiled by addition of CaCO₃.

The third step of REnescience technology as practiced in this example is a séparation step where the bio-liquid is separated from the non-degradabie fractions. The séparation fs performed in a ballistic separator, washing drums and hydrauiic presses. The ballistic sépara tor séparâtes the enzymatic treated MSW into the bio-liquid, a fraction of 2D non-degradabie materials and a fraction of 3D non-degradabie materials. The 3D fraction (physical 3 dimensionai objects as cans and plastic bottles) does not bind large amounts of bio-liquid, so a single washing step is sufficient to ciean the 3D fraction. The 2D fraction (textiles and foüs as examples) binds a slgnificant amount of bio-liquid. Therefore the 2D fraction is pressed using a screw press, washed and pressed again to optimize the recovery of bio-liquid and to obtain a ciean and dry 2D fraction. Inert material which is sand and glass Is sleved from the bio-liquid. The water used In ali the washing drums can be redrculated, heated and then used as hot water in the first step for heating.

The trial documented in this example was split up in three sections as shown In table 1

Table 1.

Time (hours)

Rodaion

Tap water/Washing water to mild heatlng

27 - 68	+	tap water
86 -124	-	tap water

142-J 87

-

washing water

In a 7-day trial, unsorted MSW obtained from Copenhagen, Denmark was loaded continuously by 335 kg/h In to the REnesdence demo plant. In the mlld heating was added 536 kg/h water (tap water or washing water) heated to app. 75°C before ente ring the mild heating reactor. Température Is hereby adjusted to app. 50*C in the MSW and pH Is adjusted to app. ^.5 by addition of CàCOy

In the first section the surface-active anti-bacteriai agent Rodalon ™ (benzyl alkyl ammonium chloride) was Induded in the added water at 3 g active ingrédient per kg MSW.

In the liquéfaction reactor is added app. 14 kg of Cellic Ctec3 ( commercially availabnle ceiluiase préparation from Novozymes) per wet ton of MSW. The température was kept In the range from 4550*C and the pH was adjusted In the range from £.2^475 by adding ’CaCOl Enzyme reactor rétention time is app. 16 hours.

In the séparation System of ballistic separator, presses and washing drums the bio-liquid (iiquefied degradable material) is separated from non-degradable materials.

Wash waters were selectively either poured out, recording organic content, or reclrculated and reused to wet incomlng MSW in the mild heating. Recirculation of wash water has the effect of accomplishing bacteriai Inoculation using organisais thriving at 50’C reaction conditions to levels higher than those initially présent. In the process schéma used, redrcuiated wash water were first heated to approximatety 70*C, In order to bring Incomlng MSW to a température appropriate for enzymatic hydroiysis, In this case, about 50’C. Particulariy in the case of lactic add bacteria, heating to 70C has prevtously been shown to provide a sélection and înducement of thermal tolérance expression.

Samptes were obtained at selected time points at the following places:

- The bio-liquid leaving the smali sieve, which is termed EC12B*

- The bio-liquid in the storage tank

- Washing water after the whey sieves

- 2D fraction

- 3D fraction

- Inert bottom fraction from both washing units

The production of bioliquid was measured with load cells on the storage tank. The input flow of fresh waters was measured with flowmeters, the recyded or drained washing waste was measured with load cells.

Bacterial counts were examined as follows: Selected samples of bioliquid were diluted 10-fold In the SPO (peptone sait solution) and 1 ml of the dilutions are plated at sowing depth on beaf Extract Agar (3.0 g / L of Beef extract (Fluka, Cas.: B4888), 10.0 g / L Tryptone (Sigma, cas.no.: T9410), 5.0 g / L NaCI (Merck, cas.no. 7647-14-5), 15.0 g / L agar (Sigma, cas. no. 9002-18-0)). The plates were Incubated at 50 degrees, respectively. aérobic and anaérobie atmosphère. Anaérobie cultivation took place in appropriate containers were kept anaérobie by gassing with Anoxymat and adding lltfjemende letters (AnaeroGen from Oxoid, cat.no AN0025A). The aérobic colonies were counted after 16 hours and again after 24 hours. The anaérobie growing bacteria were quantified after 64-72 hours.

Figure 5 shows total volatile solids content In bioliquid samples at EC12B as kg per kg MSW processed. Point estimâtes were obtained at different tlme points during the experiment by considering each of the three separate experimental periods as a separate time period. Thus, a point estimate during period 1 (Rodalon) Is expressed relative to the mass balances and matériel flows during period 1. A shown In Figure 5, during period 1, which was initiated after a prolonged stop due to complications in the plant, total solids captured In bioliquid are seen to drop steadily, consistent with a sllght antl-bacterial effect of Rodalon During period 2, total captured solids retums to slightly higher levels. During period 3, where recirculation provides an effective inoculation of Incoming MSW, bioliquid kg VS/kg affald rises to conslderably higher levels around 12%.

For each of the 10 tlme points shown In Figure 5, bioliquid (EC12B) samples were taken and total solids, volatile solids, dissolved voilatile solids, and concentrations of the presumed bacterial métabolites acetate, butyrate, éthanol, formate, and proplonate were determlned by HPLC. These results Including glycero! concentrations are shown In Table 1 below.

Table 1. Analysis of bloliquld samples.

Time	Total solide	VS	Dlssolved VS	Lactat e	Forml c acid	Acetat e	Proplonat e	Ethano I	Glycero I
hours	%	%	%	%	%	%	%	%	%
45	10,30	8,6 9	7,00	3,22	0,00	0,35	0,00	0,12	0,4165
53	9.77	8,2 2	6,62	3,00	0,00	0,42	0,00	0,17	0
63	9,31	7,7 4	6,07	2,74	0,09	0,41	0,03	0,17	0,415
67	8,66	7.1 5	5,54	2,82	0,00	0,39	0,03	0,20	0,475
88	9,57	7,9 7	6,02	3,24	0,00	0,31	0,04	0,13	0,554
116	10,57	8,9 0	6,77	3,27	0,01	0,25	0,00	0,11	0,5635
130	9,93	8,3 3	6,43	3,39	0,00	0,25	0,00	0,11	0
141	12,07	9,0 8	6,76	4,16	0,00	0,28	0,00	0,14	0,6205
159	11,30	8.6 8	6,33	4,63	0,00	0,31	0,00	0,11	0
166	11,04	8,1 7	5,72	4,50	0,00	0,32	0,03	0,12	0,646
181	11,76	8,7 5	6,11	5,48	0,12	0,37	0,00	0,11	1,38

188

11,20

8,0 5

6,20

5,40

0,00

0,40

0,00

0,11

0

For bloliquld samples taken at each of the ten time points, Figure 6 shows both lîve bacterial counts determined under aérobic confitions and also the weight percent bacterial métabolites (meanlng the sum of acetate, butyrate, éthanol, formate, and proprionate) expressed as a percentage of dissolved volatile solids. As shown, the weight percent bacterial métabolites deariy increases with Increased bacterial activity, and Is associated with increased capture of solids In the bioliquid.

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

Samples of bloliquld obtained from example 3 were analysed for mlcroblal composition.

The mlcroblal species présent in the sample were identified by comparing their 16S rRNA gene sequences with 16S rRNA gene sequences of well-characterized species (référencé species). The normal cut-off value for species Identification is 97% 16S rRNA gene sequence similarity with a référencé species. if the similarity Is below 97%, It is most iikeiy a different species.

The resulting sequences were queried In a BlastN against the NCBi databasese. The database contalns good quality sequences with at least 1200bp in iength and a NCBI taxonomie association. Only BLAST hits 295% identity were induded.

The sampled bloliquld was directly transferred to analysis without freezing before DNA extraction.

A total of 7 bacterial spedes were Identified (Figure 7) and 7 species of Archea were Identified. in some cases the baderial spedes the subspedes could not be asslgned (L acldophilus, L amyfovorus, L sobrius, L reuterl, L frumenti, L fermantum, L fabifermentans, L plantarum, L pantosus)

Exemple 5. Detalled analysis of organic capture using concurrent mlcroblal fermentation and enzymatic hydrolysis of unsorted MSW.

The REnesclence démonstration plant described in example 1 was used to make a detailed study of total organic capture using concurrent bacterial fermentation and enzymatic hydrolysis of unsorted MSW.

Trash from Copenhagen was characterized by Econet to détermine its content.

Waste analysis hâve been analysed to détermine the content and variation. A large sample of MSW was delivered to Econet A/S, which performed the waste analyses. The primary sample was reduced to a sub sample around 50 - 200 kg. This subsample was the sorted by tralned personnel 10 Into 15 different waste fractions. The weight of each fraction was recorded and a distribution calculated.

Table x Waste composition as (%) of total, analysed by Econet during the 300 hours test

Sample:	1.	2.	3.	4.	5.	6.	7.	8.	9.	averag e	Standard devlatio n
	%	%	%	%	%	%	%	%	%	%
Plastic packaging	5.1	6.7	8.0	4.9	6.2	2.5	6.2	7.5	6.4	5.9	1.64
Plastic foll	10.8	8.6	10.7	7.9	10.1	7.8	8.8	8.5	9.5	9.2	1.13
Other plastic	0.7	0.8	0.5	0.7	1.0	0.7	1.6	0.4	0.9	0.8	0.33
Métal	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
Yard waste	0.7	3.5	1.9	1.8	0.9	2.7	0.6	4.5	2.8	2.1	1.33
WEEE (batteries etc.)	0.7	0.1	0.6	0.4	0.7	0.8	1.1	0.1	0.5	0.6	0.33
Paper	14.8	8.3	13.3	8.8	10.5	5.6	10.2	12.6	12.4	10.7	2.86
Plastic and cardboard packaging	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

Diapers	8.0	10.3	6.9	18.8	8.1	25.1	15.2	10.1	14.0	12.9	6.00
Dlrty paper	8.5	6.7	7.3	7.4	8.5	8.6	7.9	5.7	6.3	7.4	1.03
Fines	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 non* combustibles	6.2	11.1	5.0	7.3	7.2	7.6	5.6	3.7	6.2	6.7	2.07
sum	100	100	100	100	100	100	100	100	100	100.0

The composition of waste varies from time to time, presented in table 2 Is waste analysis resuit from different samples collected over 300 hours. the larges variation Is seen en the fractions diapers plastic and cardboard packing and food waste which is ail fractions that affect the content 5 of organic material that can be captured.

Over the entire course of the 300 Hours Test, the average captured biodégradable material expressed as kg VS per kg MSW processed was 0.156 kg VS/kg MSW Input.

Représentative samples of bioliquid were taken at various time points during the course of the experiment, when the plant was in a period of stable operation. Samples were analysed by HPLC and to détermina volatile solids, total solids, and dissolved solids as described In example 3. Results are shown In Table 2 below.

Table 2. Analysis of bioliquid samples.

Time	Total solids	VS	Dissolved VS	Forml cacld	Lactat e	Acetat e	Propion at e	Ethano I	Glycero I
hours	%	%	%	%	%	%	%	%	%
212	10,45	8,3 6	5,95	0,00	5,36	0,46	0,03	0,46	0,82
239	10,91	8,6	5,85	0,00	6,08	0,33	0,00	0,33	0,77

		4
264,5	11,35	8.8 2	6,25	0,00	4,97	0,49	0,00	0,49	1,06
294	10,66	8,4 8	5,60	0,08	3,37	0,39	0,00	0,39	0,55

Exampie 6. Identification of microorganisms contributing to concurrent fermentation în example 5,

A sample ofthe bioliquld ‘EC12B’was withdrawn during the test described In example 5 on December the 15^m and 16⁹¹2012 and stored at -20’C for the purpose of performing 16S rDNA analysis to Identify the microorganisms In the sample. The 16S rDNA analysis Is widely used to Identification and phylogénie analysis of prokaryotes based on the 16S component of the small ribosomal subunit. The frozen samples were shipped on dry Ice to GATC Biotech AB, Solna, SE where the 16S rDNA analysis was performed (GATC_Biotech). The analysis comprised: extraction of genomic DNA, amplicon library préparation using the universal primera primer pair spanning the hypervariable régions V1 to V3 27F: AGAGTTTGATCCTGGCTCAG / 534R: ATTACCGCGGCTGCTGG; 507 bp length), PCR tagging with GS FLX adaptera, sequenclng on a Genome Sequencer FLX Instrument to obtain 104.000-160.000 number of reads pr. sample. The resulting sequences were then queried In a BlastN against the rDNA database from Ribosomal Database Project (Cole et al., 2009). The database contains good quality sequences with at least 1200bp In length and a NCBI taxonomie association. The current release (RDP Release 10, Updated on September 19, 2012) contains 9,162 bacteria and 375 archaeal sequences. The BLAST results were filtered to remove short and low quality hits (sequence Identity £ 90%, alignment coverage £ 90%).

A total of 226 different bacteria were identified.

The prédominant bacteria In the EC12B sample was PaludibacterpropionlcigenesWB4. a propionate producing bacteria (Ueki et al. 2006), which comprised 13% ofthe total bacteria identified. The distribution of the 13 prédominant bacteria identified (Paludibacterpropionidgenes WB4, Protelniphilum acatatlganas, Actlnomycas europaeus, Levilinea saccharolytica, Cryptanaarobactar phanolicus, Sadlmantibacter hydroxybenzoicus, Clostridium phytofermentans

ISDg, Petrimonas sulfuriphila, Clostridium lactatiïermentans, Clostridium caenicola, Garciella nitratireducens, Dehalobacter restrictus DSM 9455, Marinobacter lutaoensis) Is shown in Figure 8.

Comparing the bacteria identified at genus ievei showed that Clostridium, Paludibacter, Protelnlphllum, Actinomyces and Levilinea (al! anaerobes) represented approximately half of the généra Identified. The genus Lactobacillus comprised 2% of the bacteria Identified. The prédominant bacteria! specle P. propionlclgenes\NB4 belong to the second most predominating généra (Paludibacter) In the EC12B sample.

The prédominant pathogenic bacteria In the EC12B sample was Streptococcus spp., which comprised 0.028% of the total bacteria Identified. There was not found any spore formlng pathogenic bacteria In the blo-liquid.

Streptococcus spp. was the only pathogenic bacteria présent in the blo-liquid In example 5. Streptococcus spp. Is the bacteria with the hlghest température tolérance (of the non-spore formlng) and D-value, which indicates that the amount of time needed ata given température to reduce the amount of living Streptococcus spp. cells tenfold, is hlgher than any of the other pathogenic bacteria reported by Déportes et al. (1998) In MSW. These resuits show that the conditions applied in example 5 are able to sanitize MSW during sorting In the REnesclence process to a ievei where only Streptococcus spp. was présent.

The compétition between organism for nutrients, and the increased In température during the process will decrease the number of pathogenic organisms significantly and as shown above eliminate presence of pathogens in MSW sorted in the REnesclence process. Other factors like pH, a_w, oxygen tolérance, CO₂. NaCI, and NaNO₂ also influence growth of pathogenic bacteria in bioliquid. The interaction between the above mentioned factors, might lower the time and température needed to reduce the amount of living cells during the process.

Example 7. Detailed analysis of organic capture using concurrent mlcroblal fermentation and enzymatic hydrolysis of unsorted MSW obtained from a distant géographie location.

The REnesclence démonstration plant described In example 3 was used to process MSW imported from the Netheriands. The MSW wsas found to hâve the following composition:

Table Y waste composition (5) of total, analysed by Econet during the van Gansewlnkel test

	%
Plastic packaging	5
Plastic foll	7
Other plastic	2
Métal	4
Glass	4
Yard waste	4
WEEE (batteries etc)	1
Paper	12
Cardboard	12
Diapers	4
Dirty paper	2
Other combustibles	15
Other non-combustibles	5
Food waste	13
Fines	9
Total	100

The material was subject to concurrent enzymatic hydrolysis and microbial fermentation as described In example 3 and 5 and tested for a plant run of 3 days. Samples of bloloiquld obtained at various tlme points were obtained and characterized. Results are shown In Table 3.

Table 3. Analysis of bioiiquid.

	Total		Dissolved	Lactat	Forml	Acetat	Proplonat	Ethano	Glycero
Tlme	sollds	VS	VS	e	c acid	e	e	I	I

hours	%	%	%	%	%	%	%	%	%
76	7.96	6,0 8	3,07	4,132	0,08	0,189	0	0,298	0,4205
95	9,19	6,9 9	6,66	6,943	0	0,352	0,034	0,069	0,6465
The dissolved VS has been corrected w	th 9% according to loss of	actate during drying.

Example 8. Blomethane production using bioiiquid obtained from concurrent microbial fermentation and enzymatic hydrolysis of unsorted MSW.

Bioiiquid obtained in the experiment described in exampie 5 was frozen in 20 liter buckets and stored at -18o C for later use. This material was tested for biomethane production using two identical well prepared fixed filter anaerboic digestion Systems comprising an anaérobie digestion consortium within a biofilm Immobilized on the filter support

Initial samples were collected for both the feed and the liquid inside the reactor. VFA, tCOD, sCOD, and ammonia concentrations are determined using HACH LANGE cuvette tests with a DR 2800 Spectrophotometer and detailed VFAs were determined daily by HPLC. TSVS measurements are also determined by the Gravimétrie Method.

Gas samples for GC analysis are taken daily. Vérification of the feed rate Is performed by measuring headspace volume in the feed tank and aiso the amount of effluent coming out of the reactor. Sampling during the process was performed by collecting with a syringe of liquid or effluent.

Stable blogas production was observed using both digester Systems for a period of 10 weeks, corresponding to between 0.27 and 0.32 L/g COD, or between R and Z L/g VS.

Feed of bioiiquid was then discontinued on one of the two system and the retum to baseline monitored, as shown In Figure 9. Stable gas production level Is shown by the horizontal line Indicated as 2. The time point at which feed was discontinued is shown at the vertical iines

Indicated as 3. As shown, after months of steady operation, there remained a resldual résilient matériel which was converted during the period indicated between the vertical lines Indicated as 3 and 4. The retum to baseline or ramp down Is shown In the period following the vertical line Indicated as 4. Following a baseline period, feed was again Initiated at the point Indicated by the 5 vertical line Indicated as 1. The rise to steady state gas production or ramp up Is shown In the period following the vertical line Indicated as 1.

Pa ram et ers of gas production from the bioliquld, Including ramp up and ramp down” measured as described are shown below.

Parameter Unit Sample name

300 hour Amager waste

Feed rate	LVday	1.85
Total feed	Uter	3.7
Ramp-up time *	Hours	15
Ramp-down time	Hours	4
Bum-down time ***	Days	4
Gas production In stable phase ****	Uday	122
Total gas produced	L	244
CH4 %	%	60
Total yield	Lgas/Lfeed	66
Gas from the easy convertible organics	%	53
Feed COD	fl/L	124
Total COD feed-ln	g	459
COD yield	Lgas/gCOD	0.53
Spedfic COD yield	L CHVgCOD	0.32

COD accounted for by mass balance	% of feed COD	96
COD to gas	g	418
COD to gas	%	91

*Ramp-up time ls the time from first feed till gas production seize to increase and stabilises. The ramp-up time indicates the level of easy convertible organics in the feed.

**Ramp-down time is the time from last feed till gas production seizes to fali steeply. The ramp· down time shows the gas production from easily convertible organics.

***Bum-down is the time after the Ramp-down time until the gas production seizes totally at base level. The bum-down time shows the gas production from slowly convertible organics.

****Corrected for background gas production of 2 L/day.

Example 9. Comparative biomethane production using bioliquid obtained from enzymatic hydrolysis of unsorted MSW with and without concurrent microbial fermentation.

High lactate’ and low lactate bioliquid obtained In example 2 were compared for biomethane production using the fixed filter anaérobie digestion System described In example 8. Measurements were obtained and ramp up and ramp down times were determined as described in example 8.

Figure 10 shows ramp up and ramp down characterization of the hlgh lactate bioliquid. Stable gas production level is shown by the horizontal line Indicated as 2. The time point at which feed was Initiated ls shown at the vertical Unes indicated as 1. The rise to steady state gas production or ramp up is shown in the period following the vertical line Indicated as 1. The time point at which feed was discontinued ls shown at the vertical line Indicated as 3. The retum to baseline or ramp down ls shown In the period following the vertical line Indicated as 3 to the period at the vertical line Indicated by 4.

Figure 11 shows the same characterization of the low lactate bioliquid, with the relevant points Indicated as described for Figure 11.

Comparative para met ers of gas production from the hlgh lactate and low lactate bioliquid, including ramp up and ramp down measured as described are shown below.

The différence In ramp upTramp down tlmes show différences in ease of blodegradability. The fastest bioconvertible biomasses will ultimately hâve the highest total organic conversion rate In a blogas production application. Moreover, the faster biomethane substrates are more ideally suited for conversion by very fast anaérobie digestion Systems, such as fixed filter digesters.

As shown, the hlgh lactate bioliquid exhibits a much faster ramp up and ramp down time In 10 biomethane production.

Parameter Unit Sample name

		High lactate Holstebro waste	Low lactate control Holstebro
Feed rate	L/day	1.0	1.0
Total feed	Liter	2.83	3.95
Ramp-up time *	Hours	16	48
Ramp-down time	Hours	6	14
Bum-down time ***	Days	2	2
Gas production In stable phase ***	L/day	59	40
Total gas produced	L	115	140
CH< %	%	60	60
Total yield	Lgas/Lfeed	41	35
Gas from the easy convertible organics	%	86	82
Feed COD	g/L	106	90
Total COD feed-ln	g	300	356
COD yield	Lgas/gCOD	0.38	0.39

Spécifie COD yield	L CHVgCOD	0.23	0.24
COD accounted for by mass balance	% of feed COD	91	95
COD to gas	g	197	240
COD to gas	%	66	68

*Ramp-up time Is the time from first feed tili gas production seize to Increase and stabilises. The ramp-up time Indicates the ievel of easy convertible organics in the feed.

**Ramp-down time Is the time from last feed till gas production selzes to fall steeply. The rampdown time shows the gas production from easlly convertible organics.

***Bum-down is the time after the Ramp-down time until the gas production selzes totally at base levei. The bum-down time shows the gas production from slowly convertible organics. ****Corrected for background gas production of 2 L/day.

Example 11. Biomethane production using bioliquid obtained from concurrent microbial fermentation and enzymatic hydrolysls of hydrothermally pretreated wheat straw.

Wheat straw was pretreated, separated into a fiber fraction and a iiquld fraction, and then the fi ber fraction was separateiy washed. 5 kg of washed fiber were then Incubated In a horizontal rotary drum reactor with dose of Cellic CTEC3 with an Inoculum of fermenting mlcroorganlsms consisting of blovæske obtained from exemple 3. The wheat straw was subject to slmuitaneous hydrolysls and microbial fermentation for 3 days at 50 degrees.

This bioliquid was then tested for biomethane production using the fixed filter anaérobie digestion system described in example 8. Measurements were obtained for ramp up time as described In example 8.

Figure 12 shows ramp up characterization of the hydrolysed wheat straw bioliquid. Stable gas production ievel Is shown by the horizontal line Indicated as 2. The time point at which feed was initiated Is shown at the vertical Unes indicated as 1. The rise to steady state gas production or ramp up is shown in the period following the vertical line indicated as 1.

Parameters of gas production from wheat straw hydrolysate bioliquid are shown below.

As shown, pretreated lignocellulosic biomass can also readily be used to practice methods of blogas production and to produce novei biomethane substrates of the invention.

Parameter Unit Sample name

Wheat hydrolysate + Bioliquid

Feed rate	L/day	1
Total feed	Liter	1.2
Ramp-up time *	Hours	29
Ramp-down time **	Hours	N/A
Bum-down time ***	Days	N/A
Gas production In stable phase ***·	L/day	56
Total gas produced	L	N/A
CH4 %	%	60
Total yield	Lgas/Lfeed	N/A
Gas from the easy convertible organics	%	N/A
Feed COD	g/L	144
Total COD feed-in	9	173
COD yield	Lgas/gCOD	N/A
Spécifie COD yield	L CHVgCOD	N/A
COD accounted for by mass balance	% offeed COD	N/A
COD to gas	g	N/A
COD to gas	%	N/A

•Ramp-up time Is the time from first feed tili gas production seize to Increase and stabilises. The 5 ramp-up time Indicates the levei of easy convertible organics In the feed.

**Ramp-down time Is the time from last feed till gas production seizes to fall steeply. The rampdown time shows the gas production from easily convertible organics.

***Bum-down is the time after the Ramp-down time until the gas production seizes totally at base ievel. The bum-down time shows the gas production from slowly convertible organics.

****Corrected for background gas production of 2 L/day.

Example 12. Concurrent mlcrobial fermentation and enzymatic hydrolysis of MSW using selected organisms.

The concurrent microbial and enzymatic hydrolysis reactions using spedfic, monoculture bacteria were carried out in laboratory scale using model MSW (described in example 1) and the procedure described In following the procedure In example 1. The reaction conditions and enzyme dosage are spedfied In Table 4.

Uve bacteria! stralns of Lactobaccillus amylophiles (DSMZ No. 20533) and proplonlbacterium 15 eddiproplonld (DSMZ No. 20272) (DSMZ, Braunswelg, Germany) (stored at 4^eC for 16hours until use) were used as Inoculum to détermine the effect of these on the conversion of dry matter In mode! MSW with or without addition of CTec3. The major métabolites produced by these are lactic add and propionic add, respectively. The concentration of these métabolites were detected using the HPLC procedure (described in example 1).

Slnce propionibacterium addiproplonld Is an anaerobe, the buffer applied In the reactions were this strain was applied, was purged using gaseous nitrogen and the live culture was Inoculated to the reaction tubes inslde a mobile anaérobie chamber (Atmos Bag, Sigma Chemlca! CO, St. Louis, MO, US) also purged with gaseous nitrogen. The reaction tubes with P. propionid were dosed before transferred to the incubator. The reactions were Inoculated with 1 m! of either P. propionid or L amylophilus.

The results displayed in table 4 deariy show that the expected métabolites were produced; propionic add was detected in the reactions inoculated with p. eddipropionicwhïïe propionic add 30 was not detected In the control containîng model MSW with or without CTec3. The concentration of lactic add In the control reaction added only model MSW was almost the same as in the reactions added only L amylophilus. The production of lactic add In this control reaction is attributed to bacteria indigenous to the model MSW. Somce background bacteria were expected since the indlvidua! components ofthe mode! wastewere fresh produce, frozen, butnotfurthersterilised In any way before préparation of the model MSW. When L amylophllus was added concurrent^ with CTec3, the concentration of iactic acid was almost doubled (Table4).

The positive effect on release of DM to the supematant following hydrolysis was demonstrated as a hlgher DM conversion In the réactions added either L amylophllus or P. proplonlcl In conjunction with CTec3 (30-33% Increase compared to the réactions added only CTec3).

Table 4. Bacterial cultures tested in lab scale atone or concurrently with enzymatic hydrolysis. The température, pH and CTec3 dosage 96mg/g is shown. Control reactions with MSW in buffer with or 10 without CTec3 were done in parallel to evaluate the background of bacterial métabolites in reaction.

(Average and standard déviation of 4 reactions are shown except for the MSW control which were done as singles).

Nd. Not detected, below détection iimit.

Température	PH	Organism	CTec3	Conversion of DM	Proplonlc acid (g/L)	Lactic acid (g/L)
				17.011,0	6.211.8
		Proplonibacterium acidiproplonici	96mg/g DM	40.812.2	3.710.09
	7
				21	Nd.
30*C		MSW control	96mg/g DM	30.6	Nd.
	Lactobacillus		19.712.2		8.410.8
		amylophllus	96mg/g DM	41.716.5		21.210.7
	6.2			21		10.3
		MSW control	96m g/g DM	32		16.9

Exampie 13. Identification of microorganisms contributing to concurrent fermentation in example 7.

Samples of the bioliquid EC12B’ and of the redrculated water ΈΑ02* were taken during the test described In example 7 (samplîng was done on March 21^rt and 22™¹). The liquld samples were frozen In 10% glycerol and stored at -20’C for the purpose of performlng 16S rDNA analysis to Identlfy the mlcroorganisms in the which Is wldely used to Identification and phylogénie analysis of prokaryotes based on the 16S component of the small ribosomal subunit. The frozen samples were shlpped on dry Ice to GATC Biotech AB, Solna, SE where the 16S rDNA analysis was performed (GATC_Biotech). The analysis comprised:

extraction of genomic DNA, amplicon library préparation using the universal primers primer pair spannlng the hypervariable réglons V1 to V3 27F: AGAG111GATCCTGGCTCAG / 534R: ATTACCGCGGCTGCTGG; 507 bp length), PCR tagglng with GS FLX adaptera, sequencing on a Genome Sequencer FLX Instrument to obtain 104.000-160.000 number of reads pr. sample. The resulting sequences were then querîed In a BlastN against the rDNA database from Ribosomal Database Project (Cole et al., 2009). The database contains good quality sequences with at least 1200bp In length and a NCBI taxonomie association. The current release (RDP Release 10, Updated on September 19, 2012) contains 9,162 bacteria and 375 archaeal sequences The BLAST results were filtered to remove short and iow quality hits (sequence Identity £ 90%, alignaient coverage S 90%).

In the samples EC12B-21/3, EC12B-22/3 and EA02B 21/3, EA02-22/3 a total of 452, 310,785, 594 different bacteria were Identified.

The analysis dearly showed, at a species level, that Lectobecillus amylolyticus was by far the most dominating bacterîum accounting for 26% to 48% of the entire microbiota detected. The mlcroblota in the EC12B samples was simiiar; the distribution of the 13 prédominant bacteria (Ladobacillus amylolyticus DSM 11664, Ladobadllus delbrueckii subsp. delbrueckil, Ladobadllus amylovorus, Lactobacillus delbrueckii subsp Indicus, Lactobadllus similis JCM 2765, Lactobadllus delbrueckii subsp. Lactis DSM 20072, Bacillus coagula ns, Lactobadllus hamster!, Ladobacillus parabuchnerl, Lactobadllus plantarum, Ladobacillus brevis, Lactobadllus pontis, Ladobacillus buchneri) was practïcaliy the same comparing the two different sampiing dates.

The EA02 samples were simiiar to the EC12B although L amylolyticus was less dominant. The distribution of the 13 prédominant bacteria (Ladobacillus amylolyticus DSM 11664, Ladobacillus delbrueckii subsp delbrueckii, Ladobadllus amylovorus, Ladobacillus delbrueckii subsp. Ladis DSM 20072, Ladobacillus similis JCM 2765, Ladobacillus delbrueckil subsp. Indicus, Ladobacillus paraplantarum, Weissella ghanends, Ladobacillus oligofermentans LMG 22743, Welssella benlnensis, Leuconostoc gasicomitatum LMG 18811, Welssella soli, Lactobacillus paraplantarum) was also similar with the exception of the presence of with the exception of the occurrence of Pseudomonas extremaustralis 14-3 In the 13 prédominant bacterial species. This Pseudomonas found In EA02 (21/3) has previously been Isolated from a temporary pond In Antarctica and should be able to produce polyhydroxyalkanoate (PHA) from both octanoate and glucose (Lopez et al. 2009; Tribelli étal., 2012).

Comparing the results at a genus level showed that lactobacillus comprised 56-94 % of the bacteria Identifi ed In the samples Again the distribution across généra is extremely similar between the two sampling dates of EC12B and EA02. Interestingly, In the EA02 samples the généra Welsella, Leuconostoc and Pseudomonas are présent to large extent (1.7- 22%) while these are only found as minor constituées of the EC12B sample (>0.1%). Welsella and Leuconostoc both belong to the order lactobadllales, the same as the lactobacillus.

The prédominant pathogenlc bacteria In the EC12B and EA02 sampled during the test described In example 7 comprised 0.281-0.539% and 0.522-0.592%, respectively of the total bacteria Identified. The prédominant pathogenlc bacteria in the EC12B samples were Aeromonas spp., Bacillus cereus, Brucella sp., Citrobacter spp., Clostridium perfrigens, Klebsiells sp., Proteus sp., Providencia sp., Salmonella spp., Serratia sp., Shlgellae spp. and Staphylococcus aureus. No spore forming pathogenlc bacteria were identified in the EC12B and EA02 described In example 7. The total amount of pathogen bacteria Identified In both EC12B and EA02 was reduced during time, almost dismlsslng the amount of total bacteria In EC12B In one day.

In Déportes et al. (1998) an overvlew of the pathogens know to be présent In MSW was made. The pathogens présent in the MSW described In exemples 3, 5 and 7 are shown In Table 5 (Déportes et al. (1998) and 16S rDNA analysis). In addition to the pathogens described by Déportes et al. (1998), Proteua sp. and Providencia sp. were both found In EC12B and EA02 sampled during the test described in example 7. Whereas the Streptococcus spp. the only pathogenlc bacteria présent In the blo-liquld In example 5, was not présent hère. This indicate that another bacterial community is présent in EC12B and EA02 In exampie 7, which might be due to compétition between organism for nutrients, and a slight decrease in température during the process which will favor the growth of another bacteria community.

Table 5. Overvlew of pathogens présent In examples 3,5 and 7

ΟηρηΙκη Bacteria	Opfcnri	Mu (growtO	Tamparatte» Bactarioaldri nneraq. D-wtue Imlnl frnlnl	pH range Mn kta	9* Mn	BtoaaWytovri	Sources Found h MSW	Ref on grwtng eondfcne
Aaromonaa tu	37	55	55		0,25		0,84	1-2	(OéportM ei ai 1998)	Rote and Rignay 1871, Sorti et ri 2008, Sateoe et ri 1994
Baclueceraut	37	50	»5	10		4,8 8.3	0,951	2	(Déportés H al 1998)	Lanctott et ri 2001
Brucateea			3	(Déportés,*! al 1998)
C*oteeWtA			52,5	7		4-5	0.84	1-2	(Déportes, et ai 19%)	Wrrfca and Kwapa 1977, SmW> and Bhagwat 2013, Cotante et ri 2003
ClottUun ptrUngint	37	50	SI	23		5 8,5	O.»	2	(Déportes, el al 19%)	Jey. JM 1991
KMel^tttfi			55		8,5	<3		1-2	(Déportés, et ai 1998)
SrimonaUtA	37	45	55	2.5		3.7'0,5	0,84	2-3	(Déportés, et al 1998)	Jay, J M 1991, Splnks al ri 2008
S«rtMn>.			55	1.5		2	(Déportes, étal 1998)	Solnks et ri 2006
SNoaitatm Stoph0ococcut«raua	37	48 47.8	____80_____			5 5 4 0	0.88	2-3 2	(Déportés, et al 1998) Spk** et ri 2006 (Déportés, étal 1998) Jay, JM 1991
Staptococcu» tPP			65	20				2	(Déportes, ef al 1998)	Francia, A E 1959

Strain identification and DSMZ deposlts

Samples of EA02 from March 21** and 22⁰ retrieved from the test described In example 7, were sent for platîng at the Novo Nordic Centre for Biosustalnability (NN Center)(Hoershoim, Denmark) with the purpose of Identifying and obtaining monocultures of Isolated bacteria. Upon arrivai at the NN center, the samples were Incubated ovemight at 50*C, then plated on different plates (GM17, tryptic soy broth, and beef extract (GM17 agan 48.25g/L m17 agar, after 20 min. autoclaving added Glucose to final concentration at 0.5%, Tryptic soy agan 30g/L Tryptic soy broth, 15g/L agar, Beef broth (Statens Sérum Institute, Copenhagen, Denmark ) added 15 g/l agarose) and grown aeroblcally at 50’C.

After one day, the plates were vlsually Inspected and seiected colonies were re-streaked on the corresponding plates and send to DSMZ for Identification.

The following stralns Isolated from the redrculated water from EA02 hâve been put In patent deposlt at DMSZ, DSMZ, Braunswelg.Germany:

Identlfled samples

Sample ID: 13-349 (Bacillus safensls) originating from (EA02-21/3), DSM 27312 Sample ID: 13-352 (Brevibacillus bravis) originating from (EA02-22/3), DSM 27314 Sample ID: 13-353 (Bacillus subtilis sp. subtilis) originating from (EA02-22/3), DSM 27315 Sample ID: 13-355 (Bacillus licheniformls) originating from (EA02-21/3), DSM 27316 Sample ID: 13-357 (Actinomyces bovis) originating from (EA02-22/3), DSM 27317

Not Identlfled samples

Sample ID: 13-351 originating from (EA02-22/3), DSM 27313

Sample ID: 13-362A originating from (EA02-22/3), DSM 27318

Sample ID: 13-365 origlnating from (EA02-22/3), DSM 27319

Sample ID: 13-367 origlnating from (EA02-22/3), DSM 27320

Référencés:

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

GATC_Biotech supporting material. Defining the Microbial Composition of Envlronmental Samples Using Next Génération Sequencing. Version 1.

Tribelll, P. M., lustman, L J. R., Catone, Μ. V., Di Martino, C., Reyale, S., Méndez, B. S., Lôpez, N.

I. (2012). Genome Sequence of the Polyhydroxybutyrate Producer Pseudomonas extremaustralls, a Highly Stress-Résistant Antarctic Bacterium. J. Bacterioi. 194(9):2381.

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

The embodiments and examples are représentative only and not intended to limit the scope of the daims.

Claims (15)

1. A method of processing municipal solid waste (MSW) or unsorted MSW comprising the steps of (i) providing MSW, (li) enzymatically hydrolyslng the biodégradable parts of the MSW using celiulase activity concurrently with microbial fermentation at a température appropriate for enzymatic hydrolysis resulting In liquéfaction of biodégradable parts of the waste and accumulation of microbial métabolites, followed by (lii) sortlng of the liquefied. biodégradable parts of the waste from non-blodegradable solids to produce a bioliquid In which at least 25 or 40% by welght of the non-water content exists as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or propionate; optionally, followed by (iv) anaérobie digestion of the bioliquid to produce biomethane, wherein concurrent microbial fermentation is accomplished by Inoculation of the MSW using one or more species from the group comprising lactic acid bacteria, acetate-produdng bacteria, éthanolproducing bacteria, propionate-produclng bacteria, butyrate-produdng bacteria, or bacteria naturaliy présent in the MSW.

2. The method of claim 1, wherein the sorting of liquefied, biodégradable parts of the waste from non-blodegradable solids is achieved using at least two séparation operations suffident to provide a bioliquid having at least 0.10 kg volatile solids per kg MSW processed.

3. The method of daim 1 or 2, wherein Inoculation Is provided by recyding wash waters or process solutions used to recover residual organic matériel from non-degradabie solids.

4. A method of produdng biomethane comprising the steps of (i) a process of providing a liquld biomethane substrate produced by concurrent enzymatic hydrolysis using celiulase activity and microbial fermentation of munidpal solid waste (MSW), unsorted MSW and/or pretreated lignoceliulosic biomass at a température appropriate for enzymatic hydrolysis. wherein concurrent microbial fermentation Is accomplished by Inoculation using one or more spedes from the group comprising lactic add bacteria, acetate-produdng bacteria, ethanol-produdng bacteria, proplonate-produdng bacteria, and butyrate-produdng bacteria, In which biomethane substrate at least 25 or 40% by weight of the nonAvater content exists as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or proplonate, (il) transferring the liquid substrate Into an anaérobie digestion System, followed by (iil) conducting anaérobie digestion of the liquid substrate to produce biomethane.

5. The method of any of the preceding claims, wherein the non-water content of the municipal solid waste is between 10 and 45%.

6. The method of any of the preceding claims, wherein Inoculation Is made before or concurrently with the addition of enzymatic activities or with the addition of microorganlsms that exhiblt extracellular ceiluiase activity.

7. The method of any of the preceding daims, wherein ceiluiase activity Is added (I) by Inoculation with a selected mlcroorganism that exhlblts extra-cellular ceiluiase activity and/or (ii) as an Isolated ceiluiase préparation.

8. The method of any of the preceding daims, wherein microbial fermentation Is accomplished by

Inoculation using one or more spedes of lactic acid baderia. -

9. The method of any of the preceding daims, wherein enzymatlc hydroiysis and microbial fermentation are conducted wïthin the température range of 30-75 or 45-50 degrees C.

10. The method of any of the preceding daims, wherein concurrent enzymatic hydroiysis and microbial fermentation are conducted at a pH of less than 6.0.

11. The method of any of the preceding daims, wherein at least 40% by weight of the dissdved volatile solids of the biomethane substrate or of the bloliquid comprises lactate, and/or wherein the liquid biomethane substrate or bloliquid comprises a dissolved methane content at 25 degrees C of less than 15mg/L

12. The method of claims 4 to 11, wherein the liquid blomethane substrate Is produced using hydrothermally pretreated ügnocelluloslc biomass and/or wherein the iignocelluloslc biomass has been pretreated at pH between 3.5 and 9.0 and at températures of 120 degrees C or higher.

13. A bloliquld obtalnable by the method according to claims 1 to 3 or 5 to 12 or the liquid blomethane substrate obtalnable by the method according to daims 4 to 12.

14. A liquid biomethane substrate obtained by a process of concurrent enzymatic hydrolysis using cellulase activity and mlcroblal fermentation of munidpal solid waste (MSW), unsorted MSW and/or pretreated iignocelluloslc biomass at a température appropriate for enzymatic hydrolysis, In which biomethane substrate at least 25 or 40% by weight of the non-water content exlsts as dissolved volatile solids, which dissolved volatile solids comprise at least 25% by weight of any combination of acetate, butyrate, éthanol, formate, lactate and/or proplonate, wherein concurrent mlcroblal fermentation Is accomplîshed by inoculation using one or more spedes from the group comprising lactic add bacteria, acetate-produdng bacteria, ethanolprodudng bacteria, propionate-produclng bacteria, and butyrate-produdng bacteria.

15. The blomethane substrate of daim 14, wherein the non-water content of the munidpal solid waste Is between 10 and 45% and/or wherein concurrent enzymatic hydrolysis and mlcroblal fermentation are conducted at a pH of iess than 6.0 and/or wherein concurrent enzymatic hydrolysis and mlcroblai fermentation are conducted within the température range of 30 to 75 degrees C and/or wherein at least 40% by weight of the dissolved volatile solids comprises lactate.