Field of the invention

The present invention relates to a process to digest organic material.

Background of the invention

The production of biogas via the anaerobic digestion of organic material is a rapidly growing source of renewable energy. The process is complex; a combined action of several biotechnological processes determines the stability, efficiency and yield of the biogas produced. An optimal process design is still under active research done at laboratory and pilot plants. Substrates like grass, manure or sludge can be used as feed for the biogas production due to their high yield potential.

The digesting systems are often divided in one-stage and two-stage digesters. In one-stage digesters all the microbiological phases of the anaerobic digestion takes place in one tank or fermenter. In the two-stage digesters the hydrolysis and acidification will take place in the first reactor and acetogenesis and methanogenesis occurs in the second reactor. The two phase concept is often chosen for optimisation of the digestion process in order to produce more methane. Both one-stage and two-stage processes have in common that all the phases of the anaerobic digestion are a microbiological process, involving the presence of suitable consortia of microorganisms.

There are several reactor configurations used for the production of biogas from all kind of waste (water) treatment systems: CSTR (continuously stirred tank reactor), SBR (sequential batch reactor), involves periodic settling phases that allow microorganisms to remain longer, and in this way uncouples the growth rate to the hydraulic retention time. Another option is to apply an anaerobic membrane bioreactor (AnMBR). More sophisticated systems are UASB (upflow anaerobic sludge blanket), EGSB (expended granular sludge bed) or IC (Internal Circulation) reactor are often used for the production of biogas in all kind of waste water treatment systems, designs that are directed to the optimisation of higher OLR (organic loading rate), reduced HRT (hydraulic retention time) and higher methane (biogas) yields. A problem in biogas production is that they tend to be unstable. That is, the pH may drop due to acid production.

Summary of the invention

The present invention provides an improved process for the digestion of organic material in biogas. The process is a two-stage process whereby only the second stage is a microbiological digestion. In the first stage the organic material is pretreated and the pretreated organic material is enzymatically hydrolysed. The effluent of the first stage is separated in a liquid and in a, preferably washed, solid fraction. The liquid fraction is fed to the second stage to produce biogas. In the first stage a high ratio of oligomer-sugar to monosugar is obtained due to the enzymes added. The process of the invention makes lower overall retention times possible without loss of gas yield compared to prior art processes.

Therefore the present invention provides a process for the digestion of organic material into biogas which comprises:

- pretreating the organic material by thermal treatment, mechanical treatment, chemical treatment or any combination of these treatments;

- treating the pretreated organic material with at least two carbohydrases to obtain a weight ratio of soluble oligosugars to monosugars of between 1000: 1 and 1 :1 , preferably between 20: 1 and 1 :1 , and more preferably the weight ratio is higher than 2 : 1 ;

- separating the liquid fraction from the solid fraction of the enzyme-treated organic material; and

- digesting the liquid fraction to form biogas.

According to another embodiment of the invention a process for the digestion of organic material into biogas is provided which comprises:

- pretreating the organic material by thermal treatment, mechanical treatment, chemical treatment or any combination of these treatments;

- treating the pretreated organic material with a composition comprising at least an endo carbohydrase and an exo carbohydrase whereby the weight ratio of endo : exo carbohydrase is between 2 : 1 and 100 : 1 , preferably between 4 : 1 and 9 : 1 ;

- separating the liquid fraction from the solid fraction of the enzyme-treated organic material; and

- digesting the liquid fraction to form biogas.

According to another aspect of the invention a composition comprising at least an endo carbohydrase and an exo carbohydrase is provided whereby the weight ratio of endo : exo carbohydrase is between 2 : 1 and 100 : 1 , preferably between 4 : 1 and 9 : 1 .

Detailed description of the invention

By biogas is meant the product produced by the anaerobic digestion or fermentation of biodegradable materials. Biogas comprises primarily methane and carbon dioxide and may have small amounts of hydrogen sulphide, moisture and siloxanes. In special cases hydrogen is the targeted product.

By biodegradable, organic or biological material is meant matter that has come from a once-living or still-living organism; is capable of decay, or the product of decay. Preferably the organic material is microbial material such as sludge or biomass from purification, fermentation or digestion processes. Especially bacterial sludge from an aerobic purification process or bacterial biomass from an aerobic digestion can be advantageously treated according to the present invention.

By sludge or activated sludge is meant the solid waste or solid waste product or solid biomass of waste water or sewage treatment. This solid waste product consists mainly of bacteria. Preferably sludge of an aerobic purification step or system is used. Suitable organic waste streams that can be used in the present process are fermentation broths or fractions thereof from industrial fermentation industries. Another suitable organic waste stream is manure such as cow, pig, goat or horse manure.

By organic matter content of the organic material is meant the dry matter content of the organic material minus ash. COD (Chemical Oxygen Demand) test is commonly used to indirectly measure the amount of organic matter content of the organic material, see for example ISO 6060 (1989).

An oligosaccharide (sugar-oligomer, oligosugar or oligomer sugar) is a carbohydrate containing a relatively small number (typically two to ten) of component sugars, also known as simple sugars (monosaccharides or monosugars), joined through glycosidic linkages. By soluble oligomer sugar is meant oligomer sugar that is soluble in the liquid phase after a liquid/solid separation as well as finely dispersed oligomer or oligomer sugar aggregate which is present in the liquid phase after the liquid/solid separation. Nearly all oligosaccharides in the range of 2 to 10, or even higher number of sugar molecules, are water-soluble. The exception is beta-(1 ,4)-linked non-substituted oligosaccharides, such as those originating from cellulose, linear beta-mannan, and linear beta-xylans. Depending on the specific sugar moiety of these oligosaccharides the solubility of water is limited at degree of polymerization (= DP = number of sugar monomers in the molecule) of 6 for cellulose-originating oligomers, and can be at higher DP for the mannan and xylan originating oligomers.

The present invention provides a process for the digestion of organic material into biogas which comprises:

- pretreating the organic material;

- treating the pretreated organic material with one or more enzymes to obtain a high ratio of soluble oligomer-sugar to monosugar;

- separating the liquid fraction from the solid fraction of the enzyme-treated organic material; and

- digesting the liquid fraction to form biogas.

The enzyme compositions used in the process of the invention can extremely effectively hydrolyze lignocellulolytic material, for example corn stover or wheat straw, which can then be further converted into a useful product, such as ethanol, biogas, butanol, lactic acid, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock. Additionally, intermediate products from a process following the hydrolysis, for example lactic acid as intermediate in biogas production, can be used as building block for other materials. The present invention is exemplified with the production of biogas but this is done as exemplification only rather than as limitation, the other mentioned useful products can be produced equally well.

The process of the invention is capable to treat all kind of digestible organic material. The separation of the enzymatic step and the microbiological digestion allows an optimal control and the selection of conditions to treat the organic material. Examples of suitable substrates are energy crops like grass, farm waste like manure or agricultural waste, sludge from waste water treatment systems, the organic fraction of municipal waste, industrial organic waste streams including biomass from fermentation industries and bio refineries. Also mixes of several organic materials can be used in the process of the invention

The process of the invention is capable to treat all kind of digestible organic material such as sludge or other organic material, for example bacterial sludge or other bacterial organic waste. Especially bacterial sludge from an aerobic purification process or bacterial biomass from an aerobic digestion can be treated according to the present invention. The bacteria of these aerobic processes are found to be enzymatically digestible. The cell walls of these bacteria are found to be degradable by lytic enzymes optionally in combination with the pretreatment of the sludge or biomass as described herein. The separation of the enzymatic step and the optional microbiological digestion provides for an optimal control and selection of conditions to treat the organic material. Also fractions of sludge or mixes of several kinds of sludge or fractions thereof can be used in the process of the invention. Moreover sludge may be mixed or combined with other organic material or substrates like grass or manure. Apart from sludge also other microbial material such as biomass originating from for example yeast or fungal fermentation industries such as breweries or algae biomass from the cultivation of algae, can be used in the process of the present invention. The present process is found to be very useful for N-enriched substrates or digestible organic material.

The organic material is preferably plant material. A preferred organic material is brewer's spent grain (BSG). Brewer's spent grain (also called spent grain, brewer's grain or draff) is the residual grain which remains after the mashing in the beer brewing process. It mainly consists of carbohydrates and proteins, and is rich in phytic acid.

The organic waste material is pretreated by thermal, mechanical, chemical treatment or any combination thereof; this is to open up the organic waste material to make it accessible to enzymes, and it is preferred to reduce microbial growth in organic waste materials, such as manure, to prevent loss of compounds that can be converted to biogas later in the process. The organic material is preferably heat-treated or pasteurized at a temperature of 65 to 120°C, more preferably at 65 to 95°C for a suitable time. Pasteurization is a process of heating the organic material to a specific temperature for a definite length of time in a humid environment. For example pasteurization at 72°C for 30 seconds is sufficient. For example 1 hour at 120°C gives the same results as 4 hours at 90 °C with respect to the CFU count (see below). In general high temperatures may result in more protein denaturation as well as occurrence of toxic compounds. In general if the pasteurization time is longer, the pasteurization temperature can be lower. Water content at pasteurization should be sufficient to enable pasteurization effect. In general the water content is between 30 and 95 wt%, preferably between 50 and 90 wt%. This process slows microbial growth in the organic material. Pasteurization or heat-treatment is not intended to kill all micro-organisms in the organic material. Instead pasteurization or heat-treatment aims to reduce the number of viable microorganisms as they are unlikely to substantially produce biogas or other fermentation products like organic acids and alcohols, in the first stage (or first step or first phase or enzyme treatment) of the process. In general in the first stage less than 2%, preferably less than 1 %, of the total of biogas is formed. After the pasteurization or heat-treatment the CFU count is in general lower than 10 ⁶ , preferably less than 10 ⁵ , even more preferably less than 10 ⁴ and most preferably less than 10 ³ CFU/ml in the organic material present. In microbiology, colony-forming unit (CFU or cfu) is a measure of viable bacterial or fungal numbers of organisms. Unlike direct microscopic counts where all cells, dead and living, are counted, CFU measures viable cells. The pasteurization step also facilitates the use of enzymes or enzyme mixtures directly originating from harvested enzyme production fermentations.

Another way to characterize the efficacy of a pretreatment that reduces in the number of microorganisms is by calculating the logarithm of the number of CFUs of the starting material divided by the number of CFUs of the material after the treatment. The advantage of this method is that - since the killing of microorganisms is generally assumed to be a first-order reaction - the log reduction of a treatment is largely independent of the actual number of microorganisms present. A sterilization procedure may be required to deliver as much as log 10 reduction (which would kill off as many as 10 ⁸ microorganisms or more), but in the case of the present invention such high efficacy is not required, or not even desirable. An effective treatment procedure in the present invention would deliver at least a log 1 reduction in the number of CFUs, preferably log 2, even more preferably log 3.

In general, it is beneficial for the process to have the thermal treatment at low or high pH, for example a low pH treatment at pH < 4, more preferably at pH < 3, even more preferably at pH < 2, the low pH treatment is in general done at pH >-1 , or for example a high pH treatment at pH > 8, more preferably pH > 9, even more preferably pH > 10. The advantages of thermal treatment at high and low pH's, are for example solubilization and partial hydrolysis of polymers, such as proteins, carbohydrates, such as starch and hemicellulose, and lipids, but also the reduction of viable cells will be enhanced by extreme pH's, resulting in for example a need of lower temperature and/or less time for the thermal treatment. Additional advantages of high pH treatment are for example improving solid / liquid separation at the end of the thermal and enzyme treatments, improved solubilization of protein and fat, and ammonia stripping for feedstocks having high ammonia content. Chemicals to be used for adjustment of the pH can be for example hydrochloric acid, phosphoric acid, and sulphuric acid for lowering the pH, or for increasing the pH potassium hydroxide and sodium hydroxide.

An advantage of acidic pretreatment is that less enzyme may be used. For example, after acidic pretreatment the amount of hemicellulase may be reduced.

According to the present invention hardly any biogas is formed during the enzyme treatment of the organic material and the biogas production takes place in the biogas fermenter. Another advantage of the present process is that the enzymes used are hardly inactivated or consumed by microorganisms present. The low numbers of viable microorganisms present have hardly any effect on the enzymes added and their activity.

The heat-treatment needs the addition of energy to the organic material. It is noticed that the addition of this energy is compensated by an increased biogas production compared to the situation without this heat-treatment. In most cases even more energy is produced in the form of biogas than is needed for the heat-treatment.

Optionally before, during or after a pasteurization or heat-treatment (part of) the organic material can be pre-treated to make for example the material such as the cellulose present more accessible to the enzymes. The pretreatment can for example be a mechanical, chemical or thermal pretreatment or a combination thereof. A steam explosion treatment or a high temperature treatment of more than 120°C are examples of thermal treatment. Chemical oxidation or chemical hydrolysis (for example using strong an acid or alkaline compound) can be used as chemical pretreatment. Ultrasonic treatment or grinding (or blending or homogenizing) are examples of mechanical pretreatments. To the temperature treated organic material one or more enzymes are added. Said enzyme(s) make(s) the degradation of organic material possible in the pasteurized or heat-treated medium, in which microbial growth is limited. This will result in an improved biogas production compared to a process wherein no enzyme is used. By varying pH, temperature, and time of the pretreatment the skilled person can find suitable pre-treatment conditions which, combined with the enzyme treatment, results in suitable ratios of oligosugars and monosugars. For example, when acidic pretreatment is applied, it may be possible to reduce the level of hemicellulose. In case of alkaline pretreatment the hemicellulose level required to solubilize and produce oligosugars may be higher. However, when using alkaline pretreatment in combination with low level of hemicellulose the balance of oligomer versus monomer production may be easier to control, due to the mild and specific action of enzymes vs. chemical treatment.

After the pretreatment the material will be incubated with enzymes, which are able to solubilize the carbohydrates and proteins; optionally, pretreatment and enzyme incubation can be combined or partly combined in one step.

In general at least two enzymes are used, advantageously an exocarbohydrase and an endocarbohydrase are used. These enzymes preferably comprise cellulase, amylase and/or hemicellulase, and optionally a pectinase, a protease, a lipase, a phytase and/or a lysing enzyme is used. The enzymes decompose the long chains of the complex carbohydrates, proteins and lipids into shorter parts. For example, polysaccharides are converted into oligosaccharides and/or monosaccharides (monosugars). Proteases split protein into peptides and free amino acids. Also other enzyme compositions can be used which promote the degradation of the organic material. The enzymes can be mixed to form the selected combination or can be produced as a mixture by a selected strain during selected fermentation conditions. For example the enzyme mixture obtained from a fermentation broth of a fungus such as Trichoderma, Aspergillus or Talaromyces or a bacterium such as Bacillus can be used. The enzyme mixture can be designed in relation to the composition of the substrate or organic material added. For example in case high amounts of fatty material are present, lipase can be added to the process.

In the process of the present invention, the number of different enzymes for carbohydrate hydrolysis may be restricted. This restriction is chosen to result in a hydrolysis which results in a high ratio of soluble oligomer-sugars to monosugars in the first stage. In biogas processes known in the art, the aim is to produce as much monosugars as possible. Instead, in the process of the invention, carbohydrates are hydrolyzed such that a substantial amount of oligomers are formed. According to the present invention relative amounts of endo and exo carbohydrases are used in a weight ratio of endo : exo carbohydrase of between 2:1 and 100:1 , preferably between 4:1 and 9:1 .

Endo-acting enzymes are defined as enzymes capable of hydrolysing a polymeric substrate at random position in the polymeric chain. Exo-acting enzymes are defined as enzymes that can only hydrolyse a linkage in a polymeric substrate from a chain end, which can be the reducing chain end or the non-reducing chain end. Usually, these exo- acting enzymes are enzymes that release mono-, di-, tri-, up to hexamer from the chain ends.

This general definition of endo and exo-acting enzymes is used herein, although some of the exo-enzymes are able to release the desired oligomers. Yet, as endo-acting enzymes are known to be unable to solubilize and hydrolyse polymeric carbohydrates completely, due to for example crystallinity in cellulose, and branching in hemicellulose, the present invention includes the presence of a relatively small amount of exo-acting enzymes. This small amount of exo-acting enzymes may include the enzymes that hydrolyse oligomers to monomers, but it may also include the enzymes releasing oligomers from the chain ends, such as β-amylase for starch hydrolysis and cellobiohydrolase for cellulose hydrolysis.

The presence of this relatively low amount of exo-acting enzymes in the present invention is required, at one hand because of the need of a limited amount of easily fermentable carbohydrates for the microbial consortia to survive and adapt in the feed of pretreated and hydrolysed organic waste material, at the other hand because it is known that polymeric cellulose cannot be solubilized by only endo-acting enzymes, and that endo- as well as exo-acting enzymes may be inhibited by the product they produce, which then results in insufficient solubilisation or hydrolysis of the polymer.

Amounts of endo and exo carbohydrases may be determined using APEX proteomics analysis as described in the "Methods and Materials" section. This is particularly suitable for complex (commercially available) enzyme mixes, such as Bakezyme ARA10.000 and Filtrase NL. It is also possible to make a suitable enzyme composition based on single (cloned) enzymes of known protein content and activity.

The relative contribution of oligosugars and monosugars of total solubilized sugars after pretreatment and enzyme hydrolysis according to the invention results in a weight ratio of soluble oligosugars to monosugars of between 1000:1 and 1 :1 , preferably between 500:1 and 1 :1 ; between 200:1 and 1 :1 , between 100:1 - 1 :1 ; between 20:1 and 1 :1 , and more preferably the weight ratio is higher than 2:1 , higher than 3:1 , or higher than 4:1.

In addition to the ratio of the endo and exo carbohydrases, the absolute amount of enzyme (the enzyme loading) may also be relevant to achieve suitable oligo and monosugar ratios. For example, at higher loadings, the ratio of endocarbohydrases to exocarbohyd rases may be higher.

Benefits of the process of the present invention are:

Less enzyme required, resulting in a more economic process to produce biogas By increasing the ratio of oligosugars to monosugars, a tool to manipulate or influence the bacterial consortium or population required for biogas production is at hand. The microbial consortium consists of fast-growers (fast growing microorganisms) which will acidify the liquid quickly, however the biogas will be produced by the slow-growers (methanogenic bacteria). In case, a lot of easily fermentable monosugars are present, such as glucose, xylose and arabinose, the fast-growers (acidogenic bacteria) will even grow faster, and out compete or outgrow the methanogenic bacteria. So a long process to achieve equilibrium conditions for growth of both types of micro-organisms is required. In case, low levels of monosugars are present, the fast-growers will grow and multiply more slowly, as they first need to produce enzymes that are able to hydrolyse those oligosugars to monomers, and hydrolyze the oligomers. The competition for carbon for both types of bacteria will then influence their relative abundance. This will result in a more stable biogas process.

Compared with processes known in the art, which aim for complete hydrolysis into monosugars, in the process of the present invention less enzyme is needed because complete hydrolysis is not aimed for, indeed not desired. Oligomer sugars may prevent the overload of easily fermentable sugars (monosugars) in the methane formation stage. Therefore the process of the present invention will result in:

More efficient biogas production

More stable biogas production

More consistent bacterial population in the methane producing reactor The enzyme step can be done as a one-step or a multi-step process. A multi-step process allows the further optimization of the process for, for example, the properties of the enzymes. A cellulase and hemicellulase treatment can be done separately, or a cellulase and hemicellulase treatment can be repeated. Preferably at least one of the enzymes used is thermostable. Preferably, the activities in the enzyme composition may be thermostable. Herein, this means that the activity has a temperature optimum of 60°C or higher, for example 70°C or higher, such as 75°C or higher, for example 80°C or higher such as 85°C or higher. All activities in the enzyme composition will typically not have the same temperature optima, but preferably will, nevertheless, be thermostable.

Amylases are enzymes that hydrolyse starch, which consists of amylose, which is a linear a-1 ,4-glucan, and amylopectin, which is an a-1 ,4 linked glucan in which branching occurs by an a-1 ,6 linked glucose. Products formed from starch hydrolysis are glucose, maltose, malto-oligosaccharides, also known as maltodextrins, and limit dextrins, which are oligosaccharides containing at least one branched a-1 ,6 glucose unit.

Examples of amylases are:

a. a-amylase (EC 3.2.1.1 ) is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4-a-D-glucosidic linkages in polysaccharides containing three or more (1→4)-a-linked D-glucose units, such as starch. This enzyme may also be referred to as glycogenase, endoamylase, Taka-amylase A, or 1 ,4-a-D-glucan glucanohydrolase

b. β-amylase (EC 3.2.1.2) is any polypeptide which is capable of catalysing the hydrolysis of (1→4)-a-D-glucosidic linkages in polysaccharides, such as starch, so as to remove successive maltose units from the non-reducing ends of the chains. This enzyme may also be referred to as saccharogen amylase, glycogenase, or 1 ,4-a-D-glucan maltohydrolase

c. amyloglucosidase (EC 3.2.1.3) is any polypeptide capable of hydrolysis of terminal (1→4)-linked a-D-glucose residues successively from non-reducing ends of the chains of polysaccharides, such as starch, with release of β-D-glucose. This enzyme also hydrolyses 1 ,6-a-D-glucose links, when in close approximation of 1 ,4- a-D-glucose links. This enzyme may also be referred to as glucoamylase, v- amylase, lysosomal a-glucosidase, acid maltase, exo-1 ,4-a-glucosidase, glucose amylase, Y-1 ,4-glucan glucohydrolase, 1 ,4-a-D-glucan glucohydrolase, or glucan

1 ,4-a-glucosidase

d. a-glucosidase (EC 3.2.1 .20) is any polypeptide capable of hydrolysis of terminal, non-reducing (1→4)-linked a-D-glucose residues with release of a-D-glucose from oligosaccharides and to a lesser extent polysaccharides, such as starch. This enzyme may also be referred to as maltase, glucoinvertase, glucosidosucrase, maltase-glucoamylase, a-glucopyranosidase, glucosidoinvertase, a-D-glucosidase; a-glucoside hydrolase, a-1 ,4-glucosidase, or a-D-glucoside glucohydrolase.

Pullulanase (EC 3.2.1 .41 ) is any polypeptide capable of hydrolysis of (1→6)-a-D- glucosidic linkages in pullulan, amylopectin and glycogen, and in the a- and β-limit dextrins of amylopectin and glycogen. This enzyme may also be referred to as amylopectin 6-glucanohydrolase, bacterial debranching enzyme, debranching enzyme, odextrin endo-1 ,6-oglucosidase, R-enzyme; pullulan a-1 ,6- glucanohydrolase, or pullulan 6-a-glucanohydrolase.

Glucan 1 ,4-a-maltotetraohydrolase (EC 3.2.1 .60) is any polypeptide capable of hydrolysis of (1→4)-a-D-glucosidic linkages in amylaceous polysaccharides, such as starch, to remove successive maltotetraose residues from the non-reducing chain ends. This enzyme may also be referred to as exo-maltotetraohydrolase, 1 ,4-oD-glucan maltotetraohydrolase, or 4-oD-glucan maltotetraohydrolase.

Isoamylase (EC 3.2.1 .68) is any polypeptide capable of hydrolysis of (1→6)-a-D- glucosidic branch linkages in glycogen, amylopectin and their β-limit dextrins. This enzyme may also be referred to as debranching enzyme or glycogen a-1 ,6- glucanohydrolase.

Glucan 1 ,4-a-maltohexaosidase (EC 3.2.1 .98) is any polypeptide capable of hydrolysis of (1→4)-a-D-glucosidic linkages in amylaceous polysaccharides, to remove successive maltohexaose residues from the non-reducing chain ends. This enzyme may also be referred to as exo-maltohexaohydrolase, 1 ,4-a-D-glucan maltohexaohydrolase, or 4-a-D-glucan maltohexaohydrolase.

Glucan 1 ,4-a-maltotriohydrolase (EC 3.2.1 .1 16) is any polypeptide capable of hydrolysis of (1→4)-a-D-glucosidic linkages in amylaceous polysaccharides, to remove successive maltotriose residues from the non-reducing chain ends. This enzyme may also be referred to as exo-maltotriohydrolase, maltotriohydrolase, 1 ,4-a-D-glucan maltotriohydrolase, or 4-a-D-glucan maltotriohydrolase

Glucan 1 ,4-a-maltohydrolase (EC 3.2.1 .133) is any polypeptide capable of hydrolysis of (1→4)-a-D-glucosidic linkages in polysaccharides so as to remove successive a-maltose residues from the non-reducing ends of the chains. This enzyme may also be referred to as maltogenic a-amylase, 1 , 4-a-D-glucan a- maltohydrolase, or 4-a-D-glucan a-maltohydrolase. Cellulases are enzymes that hydrolyze cellulose (β-1 ,4-glucan or β D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Also non-traditional glycosyl hydrolases such as the oxidative endo-acting GH 61 proteins formely named as cellulase-enhancing proteins are comprised by the term cellulase herein.

Examples of cellulases are:

a. endo-β-1 ,4-glucanase (EC 3.2.1 .4) is any polypeptide which is capable of catalysing the endohydrolysis of 1 ,4-3-D-glucosidic linkages in cellulose, lichenin or cereal β-D-glucans. Such a polypeptide may also be capable of hydrolyzing 1 ,4- linkages in β-D-glucans also containing 1 ,3-linkages. This enzyme may also be referred to as cellulase, avicelase, β-1 ,4-endoglucan hydrolase, β-1 ,4-glucanase, carboxymethyl cellulase, celludextrinase, endo-1 ,4^-D-glucanase, endo-1 ,4-β-ϋ- glucanohydrolase, endo-1 ,4^-glucanase or endoglucanase

b. β-(1 ,3)(1 (EC 3.2.1 .73) is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4^-D-glucosidic linkages in β-D-glucans containing 1 ,3- and 1 ,4-bonds. Such a polypeptide may act on lichenin and cereal β-D-glucans, but not on β-D-glucans containing only 1 ,3- or 1 ,4-bonds. This enzyme may also be referred to as licheninase, 1 ,3-1 ,4^-D-glucan 4- glucanohydrolase, β-glucanase, endo-β-1 ,3-1 ,4 glucanase, lichenase or mixed linkage β-glucanase. An alternative for this type of enzyme is EC 3.2.1 .6, which is described as endo-1 ,3(4 )-beta-glucanase. This type of enzyme hydrolyses 1 ,3- or 1 ,4-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolysed is itself substituted at C-3. Alternative names include endo-1 ,3-beta-glucanase, laminarinase, 1 ,3-(1 ,3;1 ,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates include laminarin, lichenin and cereal beta-D- glucans.

c. oxidative endo-acting glucan hydrolysing enzyme, classified as glycosyl hydrolase family 61 in the past, but not a true glycosyl hydrolase; also described as polysaccharide monooxygenase.

d. cellobiohydrolase (EC 3.2.1 .91 ) is any polypeptide which is capable of catalysing the hydrolysis of 1 ,4^-D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the ends of the chains. This enzyme may also be referred to as cellulase 1 ,4^-cellobiosidase, 1 ,4^-cellobiohydrolase, 1 ,4^-D-glucan cellobiohydrolase, avicelase, exo-1 ,4-3-D-glucanase, exocellobiohydrolase or exoglucanase. Cellobiohydrolases may be subdivided into cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II). CBH I is defined as cellobiohydrolases that hydrolyse cellulose predominantly from the reducing ends, splitting off cellobiose. CBH II is defined as cellobiohydrolases that hydrolyse cellulose from predominantly from the non-reducing ends, splitting of cellobiose.

e. β-glucosidase, abbreviated BG (EC 3.2.1 .21 ) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing β-D-glucose residues with release of β-D-glucose. Such a polypeptide may have a wide specificity for β-D- glucosides and may also hydrolyze one or more of the following: a β-D- galactoside, an a-L-arabinoside, a β-D-xyloside or a β-D-fucoside. This enzyme may also be referred to as amygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase. Hemicellulase is a collective term for a group of enzymes that break down hemicellulose.

Examples of hemicellulases are:

a. endoxylanase (EC 3.2.1 .8) is any polypeptide which is capable of catalyzing the endohydrolysis of 1 ,4^-D-xylosidic linkages in xylans. This enzyme may also be referred to as endo-1 ,4^-xylanase or 1 ,4^-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse 1 ,4 xylosidic linkages in glucuronoarabinoxylans.

b. β-xylosidase (EC 3.2.1 .37) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4^-D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1 ,4^-xylosidase, 1 ,4^-D-xylan xylohydrolase, exo-1 ,4^-xylosidase or xylobiase.

c. a-L-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on oL-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans. This enzyme may also be referred to as oN-arabinofuranosidase, arabinofuranosidase or arabinosidase α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which is capable of catalyzing a reaction of the following form: alpha-D-glucuronoside + H(2)0 = an alcohol + D-glucuronate. This enzyme may also be referred to as alpha- glucuronidase or alpha-glucosiduronase. These enzymes may also hydrolyse 4- O-methylated glucoronic acid, which can also be present as a substituent in xylans. Alternative is EC 3.2.1.131 : xylan alpha-1 ,2-glucuronosidase, which catalyses the hydrolysis of alpha-1 ,2-(4-0-methyl)glucuronosyl links

acetyl xylan esterase (EC 3.1 .1 .72) is any polypeptide which is capable of catalyzing the deacetylation of xylans and xylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically, not from triacetylglycerol. Such a polypeptide typically does not act on acetylated mannan or pectin

feruloyl esterase (EC 3.1 .1.73) is any polypeptide which is capable of catalyzing a reaction of the form: feruloyl-saccharide + H(2)0 = ferulate + saccharide. The saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in 'natural' substrates, p-nitrophenol acetate and methyl ferulate are typically poorer substrates. This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyi esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin,

coumaroyi esterase (EC 3.1.1 .73) is any polypeptide which is capable of catalyzing a reaction of the form: coumaroyl-saccharide + H(2)0 = coumarate + saccharide. The saccharide may be, for example, an oligosaccharide or a polysaccharide. This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme also falls within EC 3.1.1 .73 so may also be referred to as a feruloyl esterase.

a-galactosidase (EC 3.2.1 .22) is any polypeptide which is capable of catalyzing the hydrolysis of of terminal, non-reducing oD-galactose residues in a-D- galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing oD-fucosides. This enzyme may also be referred to as melibiase.

i. β-galactosidase (EC 3.2.1 .23) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing β-D-galactose residues in β-D- galactosides. Such a polypeptide may also be capable of hydrolyzing a-L- arabinosides. This enzyme may also be referred to as exo-(1 ->4)-3-D- galactanase or lactase.

j. β-mannanase (EC 3.2.1 .78) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-3-D-mannosidic linkages in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1 ,4-3-mannosidase or endo-1 ,4-mannanase.

k. β-mannosidase (EC 3.2.1 .25) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing β-D-mannose residues in β-D- mannosides. This enzyme may also be referred to as mannanase or mannase.

Examples of pectinases are:

a. endo-polygalacturonase (EC 3.2.1 .15) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-oD-galactosiduronic linkages in pectate and other galacturonans. This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-a-1 ,4-galacturonide glycanohydrolase, endogalacturonase; endo-D-galacturonase or poly(1 ,4-a-D- galacturonide) glycanohydrolase.

b. pectin methyl esterase (EC 3.1 .1 .1 1 ) is any enzyme which is capable of catalyzing the reaction: pectin + n H ₂ 0 = n methanol + pectate. The enzyme may also been known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.

c. endo-galactanase (EC 3.2.1 .89) is any enzyme capable of catalyzing the endohydrolysis of 1 ,4^-D-galactosidic linkages in arabinogalactans. The enzyme may also be known as arabinogalactan endo-1 ,4^-galactosidase, endo-1 ,4^-galactanase, galactanase, arabinogalactanase or arabinogalactan 4^-D-galactanohydrolase. endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminative cleavage of (1→4)-a-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-0-methyl-a-D-galact-4-enuronosyl groups at their non-reducing ends. The enzyme may also be known as pectin lyase, pectin frans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1→4)-6-0-methyl-a-D-galacturonan lyase.

pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminative cleavage of (1→4)-a-D-galacturonan to give oligosaccharides with 4-deoxy-o D-galact-4-enuronosyl groups at their non-reducing ends. The enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, a-1 ,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo-a- 1 ,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin frans- eliminase, polygalacturonic acid frans-eliminase or (1→4)-a-D-galacturonan lyase.

alpha rhamnosidase (EC 3.2.1 .40) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing oL-rhamnose residues in o L-rhamnosides or alternatively in rhamnogalacturonan. This enzyme may also be known as a-L-rhamnosidase T, oL-rhamnosidase N or oL-rhamnoside rhamnohydrolase.

exo-poly-a-galacturonosidase (EC 3.2.1.82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate. The enzyme may also be known as exopolygalacturonosidase, poly (1 ,4-a-D- galactosiduronate) digalacturonohydrolase, or poly [1 ,4-a-D-galactosiduronate] digalactuonohydrolase.

Galacturonan 1 ,4-a galacturonidase (EC 3.2.1.67) is any polypeptide capable of catalyzing: (1 ,4-a-D-galacturonide),i + H ₂ 0 = (l ^-a-D-galacturonide)^ + D- galacturonate. The enzyme may also be known as exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase or poly(1 ,4-a-D-galacturonide) galacturonohydrolase. exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing eliminative cleavage of 4-(4-deoxy-a-D-galact-4-enuronosyl)-D- galacturonate from the reducing end of pectate, i.e. de-esterified pectin. This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exopectate lyase, exopolygalacturonic acid- frans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-a-D-galacturonan reducing-end-disaccharide-lyase.

rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic acid and rhamnopyranosyl in an endo-fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1 ,2-alpha-L-rhamnoyl-(1 ,4)-alpha-galactosyluronic acid], rhamnogalacturonan lyase is any polypeptide which is capable of cleaving a-L- hap-(1→4)-a-D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

rhamnogalacturonan acetyl esterase is any polypeptide which catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.

rhamnogalacturonan galacturonohydrolase is any polypeptide which is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion.

xylogalacturonase is any polypeptide which acts on xylogalacturonan by cleaving the β-xylose substituted galacturonic acid backbone in an endo- manner. This enzyme may also be known as xylogalacturonan hydrolase.

a-L-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on a-L-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans. This enzyme may also be referred to as oN-arabinofuranosidase, arabinofuranosidase or arabinosidase.

endo-arabinanase (EC 3.2.1 .99) is any polypeptide which is capable of catalyzing endohydrolysis of 1 ,5-oarabinofuranosidic linkages in 1 ,5-arabinans. The enzyme may also be known as endo-arabinase, arabinan endo-1 ,5-ol_- arabinosidase, endo-1 ,5-a-L-arabinanase, endo-a-1 ,5-arabanase; endo- arabanase or 1 ,5-oL-arabinan 1 ,5-oL-arabinanohydrolase.

According to the present invention an enzyme mixture of predominantly endo- type of enzymes can be used comprising preferably amylases types a, e and g, cellulases types a, b and c, hemicellulases types a and j, and/or pectinases types a, c, d, e, j, k, n, p. The content of the enzyme composition may be adapted to the feedstock or pretreatment used. Additionally minor or small amounts of exo-types of enzymes may be added

Proteases (protein degrading or modifying enzymes) are for instance endo-acting proteases (serine proteases, metalloproteases, aspartyl proteases, thiol proteases), exo- acting peptidases that cleave off one amino acid, or dipeptide, tripeptide etceteras from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of the polypeptide chain.

Lipases or fatty material splitting enzymes are for instance triacylglycerol lipases, phospholipases (such as A ₂ , B, C and D) and galactolipases.

A phytase (myo-inositol hexakisphosphate phosphohydrolase) is any type of phosphatase enzyme that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) which is an indigestible, organic form of phosphorus that is found in grains and oil seeds, and releases a usable form of inorganic phosphorus.

By lysing or lytic enzyme is meant an enzyme that is capable of lysis of the cell wall of a microorganism. Microorganisms include bacteria, fungi, archaea, and protists; algae; and animals such as plankton and the planarian. Preferably the microorganism is a bacterium, fungus, yeast or alga. A microorganism or microbe is an organism that is unicellular or lives in a colony of cellular organisms.

The lysing enzyme can for example be a protease. Examples of lysing enzymes are lysozyme or muramidase (from chicken egg), lysing enzyme from Trichoderma harzianum, lytic enzyme from Lysobacter such as Lysobacter enzymogenes, lyticase from Arthrobacter luteus and Mutanolysin from Streptomyces globisporus ATCC 21553, labiase from Streptomyces fulvissimus or lysostaphin from Staphylococcus. Examples of lysing enzymes can for example be purchased from Sigma-Aldrich®. By the lysis of sludge or other bacterial materials the cell contents of the cells are liberated which make enzymes present in the sludge cells or bacterial material available for use in the process of the invention on top of the added enzymes. The liberated enzymes may even contain lytic enzymes which may lyse other bacteria.

The organic material is preferably pasteurized or heat-treated at a temperature of 65 to 120°C, more preferably 65 to 95°C and therefore the enzyme has preferably an optimal activity at the pasteurization or heat-treatment temperature or at 0 to 10°C lower, so in general at a temperature of 55 to 90°C. The enzyme is preferably stable at the selected pasteurization or heat-treatment temperature for a sufficient time, so for example for at least 30 minutes, preferably at least for 1 hour. By stability is meant that after a certain period of incubation time activity under the reaction condition is at least half of the starting activity.

The pH during the enzyme treatment will be in general between 4 and 9, preferably between 4 and 8, more preferably between 5 and 8. The enzyme is preferably stable at the selected pH for a sufficient time, so for example for at least 30 minutes, preferably at least for 1 hour.

In order to arrive at an organic material with a weight ratio of soluble oligosugars to monosugars of between 1000: 1 and 1 :1 , the skilled person can, without undue burden, choose suitable enzymes and reaction conditions (temperature, time, pH). For example, he may use a small lot of a batch of organic material, apply one or more pre- treatment conditions, and subsequently vary and test several enzyme combinations and reaction conditions; often, a few grams of organic material is sufficient. Once such enzymes and reaction conditions have been defined, the remainder of the organic material may be treated in the same way. The enzyme / reaction conditions may change between different organic materials, for example between sources of different plant or crop species, but within one type these are expected to be essentially the same.

The use of enzymes for hydrolysis of organic material containing polymer substrates was found to produce a high extraction yield of the organic matter in the liquid phase, in general higher than 75%. For (pig) manure or sludge this will be higher than 40%. The enzymes hydrolyze water-binding structures (proteins, polysaccharides), without producing polymer structures by themselves, like microorganism do. The enzymes produce the reduction of viscosity of the phases, facilitating solid/liquid separation. Another way in which the enzymes can facilitate solid/liquid separation is by lowering the emulsification properties. For example proteases and lipases are known to be helpful in this respect. In this way the volume of solid phase is reduced. Processing and disposal of solid residues, which are important cost factors in waste water plants, are facilitated and cheaper. It was found that these properties of the enzymatic process simplify largely the solid/liquid separation and are surprisingly very advantageous in comparison to processes based on microbial hydrolysis. Moreover, the ability in the present invention of extracting with high yield the organic matter in soluble form is advantageous for the processing of this organic matter to biogas. Using such soluble substrate, it is possible to apply the technology of high load anaerobic reactors based on biomass retention (for example UASB and EGSB). This technology is not applicable in conventional processes using partially and slowly degradable insoluble substrates.

The pasteurization or heat-treatment step can be done before or (partly) during the enzyme treatment. If pasteurization or heat-treatment (partly) coincides with the enzyme treatment, the pasteurization or heat-treatment time may be as long as the enzyme treatment time. The enzyme treatment time will depend on for example the temperature used, the substrate, the enzymes(s) used, and the concentration of the enzymes. In general the enzyme treatment will take 2 to 50 hours, preferably 3 to 30 hours. The enzyme treatment can be batch wise or continuously, for example a CSTR reactor can be used.

Optionally at the end of the enzyme treatment the treated organic material is treated to deactivate at least part of the enzyme(s) present. For example a heat shock and/or a pH change can be applied. Also the enzymes used may be selected to become de-activated during the enzyme treatment process after the enzymes have fulfilled their job.

In general the enzymes used are chosen not to have a substantial negative effect on the biogas production later on in the process or even to contribute in a positive way in the biogas production phase.

After a solid/liquid separation step the liquid fraction is separated from the solid fraction of the treated organic material. Preferably optimal conditions are chosen during the solid/liquid separation such as pH, temperature, addition of flocculants or filter aids etc. All kinds of suitable separation techniques can be used such as decantation, filtration, centrifugation or combinations thereof. Optionally flocculant or filter aid is added before the separation takes place in order to improve the separation. Especially flocculants and filter aids which are biologically degradable such as cellulose are advantageously applied. To prevent loss of soluble digestible material the obtained filter cake or centrifuge sludge may be washed. The wash liquor is combined with the primary obtained filtrate or supernatant. To perform these process steps at the enzyme incubation temperatures will facilitate the separation process.

The solid fraction from the solid/liquid separation can be processed or used for example by incineration (combustion), composting or spreading on cultivated areas, or forests. The present process having a temperature treatment step, allows composting or spreading of the solid fraction without a further thermal treatment of the solid fraction which is often required in case of spreading of sludge or other biomass. During the enzyme treatment and/or separation step anaerobic or aerobic conditions can be maintained. In general no special measures have to be taken to keep anaerobic conditions.

The liquid fraction from the solid/liquid separator is introduced in a biogas reactor. Upflow anaerobic filters, UASB, anaerobic packed bed and EGSB reactors are examples of high-rate digesters on industrial scale. Especially UASB and EGSB reactors offer benefits of high-rate digesters when applied at high organic loading rates. The use of liquid and solubilized substrate in the biogas reactor enables a very high loading of the reactor. In general 2 to 70 kg COD/m ³ /day, preferably at least 10 COD/m ³ /day and/or less than 50 kg COD/m ³ /day can be introduced in the biogas reactor. More preferably at least 20 kg COD/m ³ /day can be introduced in the biogas reactor. Preferably the HRT in the EGSB digester is between 3 to 100 hours, more preferably between 3 and 75 hours, even more preferably between 3 and 60 hours and most preferably between 4 and 25 hours. Preferably the HRT in an IC reactor is between 3 to 100 hours, more preferably between 10 and 80 hours and most preferably between 15 and 60 hours. Preferably the HRT in the UASB digester is between 10 to 100 hours, more preferably between 20 and 80 hours and most preferably between 20 and 50 hours. Preferably the HRT in the CSTR digester is between 1 to 20 days, more preferably between 2 to 15 days and most preferably between 2 to 10 days. In general no recycling of liquid to the first stage (enzyme treatment) will take place. In a CSTR system measures can be taken to keep the biomass in the reactor. Preferably the HRT in the anaerobic membrane bioreactor is between 3 to 12 days, more preferably between 4 and 10 days.

In general the biogas reactor wherein the digestion of the liquid fraction takes place has a volume of more than 10 m ³ preferably more than 25 m ³ .

In recycling liquid, microorganisms will be present that will start producing biogas in the first phase in case of liquid recycling. If recycling of liquid is desired, measures have to be taken that no biogas production will occur in the first phase due to introducing anaerobic microorganisms, for example the recycling liquid can be pasteurized or sterilized.

The pH of the biogas reactor will in general be between pH of 3 and 8, preferably between pH of 6 and 8. Generally no measures have to be taken to control the pH as the system is capable to maintain this pH itself. If the pH of the substrate of the biogas reactor is outside this pH range (for example pH of 5 or lower or pH of 9 or higher) the pH of this substrate is preferably neutralized to for example between 6 and 8. The process of the invention is directed to an optimal use of the energy that is applied for the thermal treatment of the organic material. Directly applying the next steps of the process of the invention may reduce energy losses in the form of heat that is lost in the enzymatic treatment, liquid/solid separation and biogas production. The enzymatic treatment, liquid/solid separation and biogas production may take place at temperatures almost the same as the thermal treatment temperature without the addition of extra heating or other forms of energy supply. Therefore the solid/liquid separation preferably takes place at 70 to 50 °C. The biogas production preferably takes place at 65 to 30 °C and most preferably takes place at 65 to 40 °C.

The process of the invention can be performed in many ways including batch, fed batch or continuously loaded reactors or fermenters or a combination thereof. For the enzymatic treatment batch reactors are preferred. In the biogas production phase continuous reactors like UASB or EGSB are preferred. EXAMPLES

Methods and Materials

Enzymes

Enzymes used for the incubations of the various feedstocks are commercially available enzyme samples of the classes of hemicellulases, and cellulases. The hemicellulase product used was Bakezyme ^® ARA10.000, which is a mix of primarily exo-type of hemicellulases, and includes some endo-type of enzymes. The cellulase product was Filtrase ^® NL, which includes both endo and exo-type of enzymes. In addition, a protease, Delvolase ^® , was applied. These commercially available enzyme products can be obtained from DSM Food Specialties (The Netherlands). Additionally, a series of other enzymes were applied:

• a cellobiohydrolase I (CBHI) from Talaromyces emersonii expressed in Aspergillus niger and described in WO201 109858

· a cellobiohydrolase II (CBHII) from Talaromyces emersonii expressed in

Aspergillus niger and described in WO2010122141

• a beta-glucosidase (BG) from Talaromyces emersonii expressed in Aspergillus niger and described in WO201 1054899 • an endo-3-1 ,4-glucanase (EG) from Talaromyces emersonii expressed in Aspergillus niger and described in EP1621628

• an oxidative endo-acting glucan hydrolysing enzyme (OEG) from Talaromyces emersonii expressed in Aspergillus niger and described in WO2012000892 · an endoxylanase (EX), originating from Talaromyces emersonii and overexpressed in Aspergillus niger, as described in EP1319079

• A complex cellulose mix derived from Talaromyces emersonii (CCM), which differs from the naturally produced mix as a beta-glucosidase gene was deleted. Method for determination of monosugars and oligosugars

Monosugars are determined using an HPLC-system (Agilent 1 100) equipped with a refractive index detector. Separation was achieved using a Biorad Aminex HPX-87P column (300 X 7.8 mm). The mobile phase was Milli-Q water at a flow of 0.6 ml/min and a column temperature of 85°C. The injection volume was 10 μΙ. Monosugars were identified and quantified according to their retention times, which were compared to external standards (arabinose, xylose, glucose). Oligosugars were quantified after acid hydrolysis of the solubilized supernatant, using 1 M sulphuric acid, at 100°C for 1 h. The acid hydrolysate was analysed using HPLC system as described above. The difference between monosugars before acid hydrolysis and the total solubilized sugar content as determined after acid hydrolysis is a measure for the oligosugars content of the supernatant. Apart from analysis of the monosugars according to the HPLC method described, they can also be determined using flow-NMR. The ¹ H NMR spectra were recorded on a Bruker AVANCE II BEST NMR system operating at proton frequency 500MHz and probe temperature 27°C.

Method for determination of total protein content of enzyme solutions

The method was a combination of precipitation of protein using trichloroacetic acid (TCA) to remove disturbing substances and allow determination of the protein concentration with the colorimetric Biuret reaction. In the Biuret reaction, a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogen atoms and carbon atoms of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color, and hence the absorption at 546 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Standardisation was performed using BSA (Bovine Serum Albumine) and the protein content was expressed in g protein as BSA equivalent/L or mg protein as BSA equivalent /ml. The protein content was calculated using standard calculation protocols known in the art, by plotting the OD ₅₄₆ versus the concentration of samples with known concentration, followed by the calculation of the concentration of the unknown samples using the equation generated from the calibration line.

Total solids or dry matter (DM) volatile solids (VS) (=organic dry matter)

These parameters were determined according to standard procedures known in the art and as described by Standard Methods of American Public Health Association (APHA, 1995). Biogas composition (CH4 and C02) was measured using gas chromotography, equipped with a thermal conductivity detector (TCD). Volatile fatty acids (VFA) were measured using a gas chromatograph (Shimadzu GC-2010 AF, Kyoto, Japan), equipped with a flame ionization detector (FID) (Angelidaki et al., 2009). Method for determination of carbohydrates

Carbohydrates and lignin content were determined according to "Determination of structural carbohydrates and lignin in biomass", A. Sluiter et al. Technical report NREL/TP-510-42618. Individual proteins using APEX proteomics analysis

These analyses were performed as described in WO201 1/054899.

Inulin was obtained from Beneo Orafti as Raftiline® ST ("OS1 "; also known as Orafti® GR) and as Raftilose® P95 ("OS2"; also known as Orafti® P95, from Beneo Orafti) These oligosaccharide mixtures differ in the degree of polymerization: for Raftiline it ranges from 2-30, for Raftilose it ranges from 2-8. Furthermore, the ratio of oligosugar to monosugar differs for these two products. The ratio ranges from 1000:1 to 24:1 for Raftiline, and from 32:1 to 13:1 for Raftilose, according to the specification of the supplier.

Example 1A

Effect of different enzyme combinations on ratio of oligo- and monosugars

Brewers spent grain (BSG), as milled and dried material, is obtained from a commercial brewery. The material is suspended in distilled water to a dry matter content of 10%, in a double-walled closed glass reaction chamber, which is connected to a circulating waterbath, in which the water temperature is set to the desired temperature, i.e. 90°C. The pH of the suspension as such is pH 6.6, and is adjusted to pH 1 1.5, using 4N NaOH. Subsequently, the slurry is incubated for 4 h, while stirred. After this pretreatment, the slurry is cooled down, and adjusted to pH 4.5 using HCI, and incubated for 24 h at 60°C, with the addition of 7.5 mg protein derived from Bakezyme ^® ARA10.000 per g BSG dry matter and 9 mg protein derived from Filtrase ^® NL per g BSG dry matter. A similar treatment of the BSG is performed in which the enzyme incubation is done with the addition of 3.5 mg endoxylanase protein of Talaromyces emersonii expressed in Aspergillus niger per g BSG dry matter and 9 mg protein derived from Filtrase ^® NL per g BSG dry matter. The actual ratio of endo to exo carbohydrases applied in the mix containing Filtrase ^® NL and Bakezyme ^® ARA10.000 is 0.9:1. For the mix containing Filtrase ^® NL and the endoxylanase ex Talaromyces emersonii this ratio is 6:1 . For each of the two samples the monosugar and oligosugar content of the supernatant is determined. In the case of the Filtrase ^® NL and Bakezyme ^® ARA10.000 incubation almost all the sugars are present as monosugars, whereas over half of the total sugars originates from oligomers in the Filtrase ^® NL and endoxylanase combination.

Example 1 B

Effect of different mixtures of endo- and exo-carbohyd rases on ratio of oligo- and monosugars

Brewers spent grain (BSG) was pretreated as described in Example 1A. After the 4 h incubation time at 90°C, and cooling down the pH of the BSG slurry was adjusted to pH 8.0 using 4N HCI and incubated with 3.85 mg protein of Delvolase ^® per g of BSG dry matter for 20 hours at 65°C. Next, the slurry was adjusted to pH 4.5 using 4N HCI and incubated for 22 hours at 65°C with one of the following enzyme mixtures of endo- and exocarbohydrases added per g of BSG dry matter:

- a mixture with an endo:exocarbohydrase ratio of 0.9:1 , comprising 1.75 or 3.5 mg

Filtrase ^® NL protein from and with an endo:exocarbohydrase ratio of 0.7:1 , plus 1 .75 or 3.5 mg Bakezyme ^® ARA10.000 protein from Aspergillus niger with an endo:exocarbohydrase ratio of 1.1 :1 - 3.5 or 7.0 mg of mixture A with an endo:exocarbohydrase ratio of 5:1 , comprising 16.7% EG, 58.3% OEG, 8.3% EX, 8.3% CBHI and 8.3% CBHII

- 3.5 or 7.0 mg of a mixture B with an endo:exocarbohydrase ratio of 25:1 , comprising 38.5% EG, 38.5% OEG, 19.2% EX, 1.9% CBHI and 1 .9% CBHII - 3.5 or 7.0 mg of a mixture C with an endo:exocarbohydrase ratio of 150:1 , comprising 33.1 % EG, 39.7% OEG, 26.5% EX, 0.1 % BG, 0.3% CBHI and 0.3% CBHII

- 3.5 or 7.0 mg of cellulase mix CCM protein with an endo-exocarbohydrase ratio of 0.7. This cellulase enzyme mixture deviated from Filtrase NL as one gene encoding an exo-carbohydrase was knocked out.

Results in Table 1 .

Table 1. Oligo-monosugar ratios obtained after thermochemical pretreatment of BSG and subsequent enzyme treatment with different endo : exocarbohydrase ratios

Enzyme mixture Endo:exocarbohydrase Oligo:monosugar ratio ratio

3.5 Filtrase NL / g DM 0.7:1 1 .5

7.0 Filtrase NL / g DM 0.7:1 1 .2

1 .75 Filtrase NL +

0.9:1 0.7

1 .75 Bakezyme ARA10.000 / g DM

3.5 Filtrase NL +

0.9:1 0.6

3.5 Bakezyme ARA10.000 / g DM

3.5 mix A / g DM 5:1 4.3

7.0 mix A / g DM 5:1 3.0

3.5 mix B / g DM 25:1 5.1

7.0 mix B / g DM 25:1 3.4

3.5 mix C / g DM 150:1 2.9

7.0 mix C / g DM 150:1 2.3

3.5 mix CCM / g DM 0.7:1 5.0

7.0 mix CCM / g DM 0.7:1 4.4 Example 2

Effect of different ratio of oligo- and monosugars on the acidification rate in biogas production The two different batches of pretreated and hydrolysed BSG, described in Example 1 , are used for biogas production in batch process. From this experiment it is observed that the rate of acidification in the high monosugar feed is much higher than that in the mixed oligo- and monosugar feed, and will allow processing higher organic loads by the same population without acidification, and thereby inhibition. Anaerobic batch tests will be performed with increasing amounts of hydrolysate and the two different fractions of oligomers and monomers (as supplied by example 1 ). Biogas evolution will be monitored and quantified, and pH at the end of each test will be measured. Example 3

Effect of mono- and oligosugars on methane and acids production

In a small scale batch anaerobic digestion set-up were tested:

- a synthetic mixture of monosaccharides, consisting of glucose, arabinose and xylose at a ratio of 2:1 :2 (w/w) ("Glc+Ara+Xyl"); and

- inulin OS1 and OS2.

The digestion set up contained 18 ml fermentation media, and used an inoculum of a culture obtained from maize silage, at a load 19 g oDM/L. In the Glc+Ara+Xyl mixture no oligosugars are present. The mixtures were tested at concentrations of 6 to 33 g/L. A blank, containing only the inoculum, was also included. Anaerobic digestion was performed at 39°C for 15 days. Each condition was tested in triplicate. During the digestion pressure build up was monitored as indication for biogas production, and at end of digestion gas composition, pH and acids in the medium were analysed. In Table 2, the cumulative normed gas production, ratio of CH ₄ over CH ₄ +C0 ₂ (v/v), pH at end of digestion, sum of acids and are given. Table 2. Data from anaerobic digestion of monosaccharide mixture gl arabinose, xylose at ratio 2:1 :2, and inulin mixtures OS1 and OS2.

The data show that OS1 and OS2 give higher biogas production than Glc+Ara+Xyl at similar sugar concentration. Since the oligosugar to monosugar ratio in both inulin mixtures is in the range of 1000:1 to approximately 10:1 , this clearly indicates that oligosugars positively influence the gas production yield. Even at very high sugar loading, biogas production with OS1 and OS2 is at the expected 50% methane content, and no acidification occurs. Thus, the biogas process with OS1 and S02 is very stable. Instead, with Glc+Ara+Xyl, which is only containing monosugars, the biogas production is poor and acidification occurred, which is an indication that the process is not stable. From the evolution of gas production in time (data not shown), it was clear that acid production and breakdown of the culture occurred after 5 days of digestion for the monosugars mixture, at 33 g/L sugar load, whereas no breakdown of the culture could be noticed for the oligosaccharide mixtures at all loads tested, again pointing to stabilizing effect of the oligomer sugars.

Example 4

Effect of mono-, oligo- and polysugars on biogas production rate and yield

A comparison of the gas production rate and yield on sugars available as mono-, oligo- or polysugars was tested in a similar set-up as given in Example 3: As monosugars were used glucose, arabinose and xylose, at a ratio of 2:1 :2, in Table 3 indicated as 'mono'.

Arabinoxylo- oligosugars were produced from a mix of wheat arabinoxylan and oat spelt xylan (purchased from Megazyme and Sigma, respectively); at a ratio of 1 ;1 to arrive at an Ara:Xyl ratio of approximately 1 :2, as was present in the monosugar mix. The mixture of these polymeric xylans in sterile MilliQ water was adjusted to pH 4.5 using 4N HCI and incubated with 1.5 mg endoxylanase EX per g xylan.for 4 hours at a temperature of 65°C. After incubation, the enzyme was inactivated by heating the mixture for 20 min at 95°C and the pellet was separated from the dissolved arabinoxylose-oligomers in the supernatant by centrifugation for 30 min at 4000 rpm.

Gluco-oligomers were produced from Avicel cellulose (Sigma-Aldrich) similarly as described for the arabinoxylo-oligomers, however instead of endoxylanase, cellulase enzyme mixture CCM was applied at 20 mg of protein per g of Avicel, and incubation lasted for 4 hours at 65°C and pH 4.5.

The arabinoxylo- and gluco-oligomers from the two separately prepared supernatants were mixed at ratio corresponding to the ratio mentioned for the monosugars. This mixture of arabinoxylo- and gluco-oligomers is indicated in Table 3 as 'oligosugars'. Furthermore, the original non-enzyme-incubated polysaccharides, as were used for the preparation of the oligomer mixture, were also used for anaerobic digestion studies. These original polysaccharides were mixed in a ratio as mentioned for the monosugars, and are indicated in Table 3 as 'Poly'. A total volume of 19 mL of medium was fermented during 13 days at 39° C, using 32 g oDM/L load of the inoculum of the same culture as described in Example 3, while recording the gas production in time. In Table 3 the cumulative normed gas production after 2 and 3 days of fermentation are shown, when applying 5 and 13 g/L of sugars load. Table 3. Cumulative normed gas production after 2 and 13 days of anaerobic digestion, using 5 and 13 g/L of sugar load, originating from mono-, oligo- or polysugars.

Table 3 clearly shows that gas production rate and gas production yield is higher when using oligosugars, than with monosugars or polysaccharides. This indicates that using a mixture of oligosugars allows applying higher substrate loads and increases productivities and performance of the process.

Example 5

Effect of mono- and oligosugars on biogas production rate and yield, in presence of solubilized protein

Similar experiments as described in Example 3 and 4 on anaerobic digestion of mono- and oligosugars were performed, while comparing the sugar mixtures in presence of solubilized protein. Two mixtures of sugar and protein were produced by thermochemical and enzymatic treatment of Brewers Spent Grains, as described in Example 1 B, applying enzyme incubations for 22 h at 65°C with 3.5 mg Filtrase® NL protein per g DM + 3.5 mg Bakezyme ARA10.000 protein per g DM (BSG1 ), or 7 mg CCM protein per g DM (BSG2). In one mixture (BSG1 ) the solubilized sugars were nearly completely hydrolysed to monosugars with an oligo- to monosugar ratio of approximately 1 , whereas in the other mixture (BSG2) the sugars were present at oligo- to monosugars at a ratio of approximately 4. The anaerobic digestions were carried out in a total volume of 9.4 ml_, with a load of 15 g oDM/L of the inoculum of the culture described in Example 3. The different sugar mixes BSG1 and BSG2 were applied at concentrations of 16, 19 and 22 g/L. In Table 4 the pH and acid production after 2 and 10 days of digestion are given. Table 4. Sum of acids and pH after 2 and 10 days of anaerobic digestion, using 16, 19 and 22 g/L of sugar load, originating from BSG1 and BSG2.

The data in Table 4 show that in case of a relatively high level of monosugars compared to oligosugars, as is present in BSG1 , the initial pH drop due to acid production in the early stage of digestion is larger than for the BSG2 mixture, which contains relatively more oligosugars, when comparing both sugar mixes at equal concentrations. The culture shows to be able to recover from this initial pH drop, since pH is increased after 10 days of digestion. The pH drop is less and the pH recovery is clearly more pronounced for the BSG2 mix as compared to the BSG1 mix at the substrate loads of 16 and 19 g/L. A substrate load of 22 g/L is clearly too high for both sugar mixes. The cumulative normed gas production on BSG2 at 19 g/L load is 82 Nml, and for BSG1 at similar load it is 64 Nml, which shows gas production on oligosugars is higher. Furthermore, it was noticed that during the start-up of the digestion the gas production velocity for BSG2 as substrate was 3 Nml/h, whereas it was 4 Nml/h for BSG1 . The slower initial velocity in combination with the higher gas yield are clear indicators for a more stable and robust anaerobic digestion, when using a oligomer rich substrate. In Table 5 more detailed data on the four main acids produced in the anaerobic digestions using BSG1 and BSG2 is given. Table 5. Contents of the 4 main acids present during anaerobic digestion at day 2 and day 10, using BSG1 and BSG2 as substrates.

These data show that when BSG2 is digested the major acid formed is acetic acid, whereas in presence of BSG1 , having relatively more monosugars also high levels of butyric acid are formed. The presence of acetic acid is not a problem for the methanogenesis at concentrations in the range mentioned for the 16 and 19 g/L substrate loads, as it is a direct substrate. The presence of butyric acid is less favourable, because it needs to be converted first to acetic acid and hydrogen, before further conversion to methane. The high hydrogen partial pressure that is present at high substrate load slows down this conversion. Additionally, butyric acid is a known inhibitor of methanogenesis. So, methane production from oligomer-rich substrate, such as BSG2 is more advantageously, since the relative low levels of butyric acid production make the process more robust, and less prone to breakdown.