WO2024115642A1

WO2024115642A1 - Method for production of hydrogen gas from waste

Info

Publication number: WO2024115642A1
Application number: PCT/EP2023/083696
Authority: WO
Inventors: Bjarne Uller
Original assignee: Renescience AS
Current assignee: Renescience AS
Priority date: 2022-12-01
Filing date: 2023-11-30
Publication date: 2024-06-06
Anticipated expiration: 2025-06-01

Abstract

The present invention provides a method for producing hydrogen gas obtainable from one or more steps in an enzymatic and/or microbial waste treatment process. The hydrogen gas may be produced very fast with the present method and provides a beneficial set up for future hydrogen gas production at large scale.

Description

Method for production of hydrogen gas from waste

FIELD

The present invention relates to a method for production of hydrogen gas obtainable either from a process wherein waste, such as municipal solid waste (MSW) is subjected to enzymatic and/or microbial treatment in a reactor or from a downstream process wherein hydrogen gas is produced in an anaerobic digestion pre-process tank, wherein bioliquid product obtained from enzymatic and/or microbial treatment of waste, such as MSW, is collected for subsequent anaerobic digestion.

BACKGROUND

There is a great interest to employ methods in which the energy stored within waste comprising organic material is utilized to the fullest. Agricultural waste, household waste and municipal waste are examples of waste containing a high content of dry matter and a certain content of organic material, which is biodegradable. Considerable interest has arisen in development of efficient and environmentally friendly methods of processing such waste, to maximize recovery of their inherent energy potential (the bio-degradable material) and recovery of recyclable materials. One significant challenge in "waste to energy" processing has been the heterogeneous nature of waste, such as municipal solid waste (MSW).

An example of an environmentally friendly waste processing method is a biologically based method applied wherein waste comprising organic matter, such as ordinary unsorted and/or sorted/partially sorted household waste, is mixed with water, enzymes and/or microorganisms in order to liquefy and/or saccharify the organic fraction of the waste such as food waste, cardboard, paper, labels and similar. Such method is described in international patent application WO 2013/185778, which describes methods and compositions for biomethane production from MSW. MSW, which may be unsorted, is concurrently treated with enzyme and a bacterial culture to release the energy saved in the biodegradable material in MSW and turn it into a bioliquid that can be used for production of biogas via an anaerobic digestion process.

Waste comprises large variations of microbial populations depending on the type of waste being processed. In the enzymatic and/or microbial waste treating process, the enzymes added and the conditions wherein the waste is degraded will impact on the kind of microbial communities dominating in the reactor. Some fermenting microorganism communities, such as lactic acid producing bacteria, are preferred in order to provide bioliquid that is suitable as feed for biomethane production whereas other microbial communities are preferred for hydrogen gas production.

The main reactions in production of hydrogen being produced biologically (biohydrogen) involve fermentation of sugars. Important reactions start with glucose, which is converted to acetic acid: C₆HI₂O₆ + 2 H₂0 -> 2 CH₃CO₂H + 2 C0₂ + 4 H₂

A related reaction gives formate instead of carbon dioxide:

C₆HI₂O₆ + 2 H₂O -> 2 CH₃CO₂H + 2 HCO₂H + 2 H₂

These reactions are exergonic by 216 and 209 kcal/mol, respectively.

H₂ production is catalyzed by two hydrogenases. One is called [FeFe]-hydrogenase; the other is called [NiFe]-hydrogenase. Many organisms express these enzymes. Notable examples are members of the genera Clostridium, Desulfovibrio, Ralstonia, and the pathogen Helicobacter. Other studies have identified bacteria of the genera Enterobacter, Klebsiella, Citrobacter, and Bacillus.

Hydrogen has attracted worldwide attention as a feasible energy source that can be produced via biological processes and potentially replace fossil fuels. It is known that hydrogen can be produced from renewable raw materials such as organic wastes. Therefore, it would be beneficial to use hydrogen as a clean energy substitute for fossil fuels, particularly if this production can be combined with disposing wastes in an environmentally friendly manner.

For the last ten years, hydrogen gas has been produced from a wide range of sources, mostly from non-renewable feedstock such as oil, natural gas, and coal. Today, most hydrogen is obtained from natural gas via steam methane reforming using mostly fossil fuels. However, since the excessive use of fossil fuels contributes to the global warming, there is an interest in developing non-polluting and renewable energy sources. Hydrogen is a clean energy source, producing water as the only by-product when it burns. In addition, biohydrogen can be produced from diverse raw materials and requires low energy consumption.

Several scientific articles show a biological hydrogen production yield from various feedstocks, such as compost material (Khanal et al., 2004 International Journal of hydrogen Energy, 29, 1123-1131), wastes from the paper industry (Moreno-Davila et al., International Journal of Hydrogen Energy, 2019, 44, 12333-12338), and solid wastes such as MSW (Keskin et al., Biohydrogen, Chapter 12, 2019, https://doi.org/10.1016/B978-0-444-64203-5.00Q12-5) .

Although there are reports indicating that hydrogen can be generated from solid wastes, this technology needs further development in that hydrogen production from solid wastes has major challenges and there are as of yet no completed technology demonstrations.

One of the challenges are the various sources of feed for the hydrogen gas production. Agricultural residues are suitable for biohydrogen production and are very abundant and are rich in carbohydrates. However, their conversion processes to generate biofuel have been problematic due to the complexity of their structure, which is mainly composed of cellulose, hemicellulose and lignin which are crosslinked with the carbohydrates, thereby decreasing the accessibility of the polysaccharides for microbial attack. Thus, their cellulose and hemicellulose contents are a significant problem, requiring pre- treatment such as chemical, physical, or physiochemical methods in order to render this kind of waste suitable for hydrogen production.

Provision of a reactor suitable for providing continuous hydrogen production is also a challenge. Most studies have been conducted on small batch experiments although continuous stirred tank reactors, up flow anaerobic sludge blanket and membrane reactors have also been applied for biohydrogen production from solid waste.

Another challenge is the production cost for electricity and renewable transport fuels are much higher than for their fossil counterparts, normally two to three times higher. The costs for renewable energy production show a clear downward trend over the past decades due to technological improvements, but further improvements are needed if the production cost should match the production cost for the fossil counterparts.

Hydrogen also plays a role in the production of other renewable energy sources. For example, in methane production through anaerobic digestion of wastewater and residues (including sewage sludge, manure and the organic fraction of municipal waste) which is already broadly applied, hydrogen acts as an intermediary product in some microbial methane production processes and may be a limiting resource because it is rapidly taken up and converted into methane by methane- producing microorganisms.

Bio-hydrogen and bio-methane production are closely related processes through the interaction of microbial hydrogen and methane metabolism on both the physiological and process level where various species of microorganisms co-operate. Hydrogen is an intermediary product in the decomposition route of organic material to methane.

Hydrogen is also used to produce methane gas for combustion by combining hydrogen with carbon dioxide under high pressure and temperature.

Gaseous hydrogen storage requires high pressure reactors of up to 70 MPa while liquid storage needs cryogenic tanks maintained at -253°C. Compared to conventional fuels, hydrogen has a low volumetric energy density in both gas and liquid form. Hydrogen-enriched compounds which are liquid at mild conditions, such as ammonia, methane, and methanol, have recently gained attention as a distribution medium or for storage of hydrogen. In contrast to other forms of chemical storage, ammonia is the only carbon-free hydrogen carrier and can be synthesised from renewable sources. Ammonia is moreover a promising hydrogen carrier owing to its high hydrogen content (17.65 wt%), established distribution network and ability to be liquefied at 10 bar or -33°C. Although not without an energy penalty, hydrogen can be released on demand from ammonia through catalytic decomposition and consumed in a proton exchange membrane (PEM) fuel cell. Alternatively, ammonia can be combusted directly or used in an ammonia-fed fuel cell. In view of the desire to replace energy from fossil fuels with energy from renewable sources, the present obstacles for continued hydrogen production at large scale, it would be beneficial to obtain a continued production of hydrogen gas and methane gas, particularly if provided in a combined large- scale method.

We have identified how to provide a continuous process for production of hydrogen gas and optionally also methane gas at large scale and have developed a method for continuous hydrogen production at large scale (see Figure 1).

The present invention provides a method for producing hydrogen gas obtainable from one or more steps in an enzymatic and microbial waste treatment process. The hydrogen gas may be produced very fast with the present method and provides a beneficial set up for future hydrogen gas production at large scale. Further, hydrogen produced subject to enzymatic and microbial treatment of waste and/or the hydrogen produced in the anaerobic digestion pre-process tank can be added to the anaerobic digestion process thereby increasing the yield of methane production. Alternatively, the hydrogen can be collected and used directly or converted into ammonia for storage or for direct use.

SUMMARY

The present invention provides in a first aspect a method for producing hydrogen gas from waste, such as municipal solid waste, comprising the following steps: a) Adding waste, preferable municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferable municipal solid waste, to enzymatic and/or microbial treatment under anaerobic conditions while maintaining the pH of about 5.5 - 6.5, and c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor.

The present invention provides in a second aspect a method for producing hydrogen gas from waste, preferable municipal solid waste, in a reactor wherein bioliquid is produced subsequently in a separate reactor connected to the hydrogen producing reactor in a method comprising the following steps: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 -6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and/or microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained in step b) to continued enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions.

The present invention provides in a third aspect a method for producing hydrogen gas from waste, preferably municipal solid waste, in a reactor and subjecting the waste to enzymatic and/or microbial treatment at a pH below 5.5 at aerobic conditions (step d) followed by separation of the thus treated waste into solids and bioliquid. This step can optionally be followed by subjecting the bioliquid obtained from step e) to an anaerobic digestion process (step h) optionally including an additional hydrogen gas producing step prior to the anaerobic digestion (steps f and g): a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and/or microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained from step b) to enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions, e) subjecting the treated waste, preferably municipal solid waste, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank.

The present invention provides in a fourth aspect a method for producing hydrogen gas from waste and an anaerobic digestion step wherein process water from the anaerobic digestion step is entered into the reactor in order to push the pH in the reactor in step a) to above about 5.5 (step i). Optionally, the hydrogen collected in step c) and/or step g) may be added to the anaerobic digestion tank in step h) in order to promote the anaerobic digestion process, thereby increasing the methane yield of the anaerobic digestion process: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained from step b) to enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions, e) subjecting the treated waste, preferably municipal solid waste, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH between 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank, i) optionally, adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combinations thereof to the reactor in step a) and/or b), j) optionally, adding the hydrogen obtained from step c) and/or step g) into the digestion tank in step h).

The present invention provides in a fifth aspect a method comprising an additional step k) wherein the hydrogen provided by the method in step c), in step g) or in both of steps c) and step g) is used for the production of ammonia or as an additive in an anaerobic digestion process for increasing the production of methane.

Accordingly, the present invention provides in the fifth aspect a method comprising: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained from step b) to enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions, e) subjecting the treated waste, preferably municipal solid waste, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH between 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank, i) optionally, adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combination thereof to the reactor in step a) and/or b), j) optionally, adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combinations thereof to the reactor in step a) and/or b), k) using the hydrogen provided by the method in step c) and/or step g) for the production of ammonia or as an additive in an anaerobic digestion process for increasing the production of methane.

The steps d) to h) are in some embodiments optional. Thus, in one embodiment, the method comprises steps a) to c) and the following additional steps: e) subjecting the treated waste from step b) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and h) subjecting said bioliquid fraction from step e) to anaerobic digestion in a digestion tank.

In another embodiment, the method comprises steps a) to c) and the following additional steps: e) subjecting the treated waste from step c) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank.

In another embodiment, the method comprises steps a) to c) and the following additional steps: e) subjecting the treated waste from step c) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and h) subjecting said bioliquid fraction step e) or from the anaerobic digestion pre-process tank in step f) to anaerobic digestion in a digestion tank.

In another embodiment, the method comprises steps a) to d) and the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre-process tank in step f) to anaerobic digestion in a digestion tank.

The steps e) to h) are in some embodiments optional. Thus, in one embodiment, the method comprises steps a) to d) and the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and h) subjecting said bioliquid fraction from step e) to anaerobic digestion in a digestion tank.

In another embodiment, the method comprises steps a) to d) and the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank.

In another embodiment, the method comprises steps a) to d) and the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre-process tank in step f) to anaerobic digestion in a digestion tank.

The present invention also provides for the use of hydrogen gas obtained from step c) and/or step g) of the methods of the invention for increasing the production of methane gas in an anaerobic digestion process or for the production of ammonia for storage or direct use.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a schematic overview of the enzymatic and microbial waste processing wherein hydrogen gas is produced.

Figure 2 shows CO2, H2 and pH profiles of fermentation in pure water.

Figure 3 shows vol % of gas evolution and pH change during fermentation in acidified reject water without further pH control over time

Figure 4 shows vol % gas evolution during the fermentation (at constant pH 6) in HsPCU-acidified reject water over time

Figure 5 shows the relative % of various bacterial classes over time

Figure 6 shows the relative % of various bacterial classes over time

Figure 7 shows MSW (tons), temperature and pH over time Figure 8 shows the relative % of various bacterial classes over time

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for hydrogen gas production in an enzymatic and microbial waste treatment process and use of hydrogen gas obtained by the method. The waste is preferably municipal solid waste (MSW). In presently applied large scale enzymatic/and or microbial waste treatment processes, one of the end products is a bioliquid suitable for further processing into for example bioethanol and methane. The present invention provides at method wherein hydrogen gas can be obtained independently from bioliquid production or in addition to the end products normally obtainable from enzymatic and/or microbial waste treatment processes. It is shown herein that the outcome of the enzymatic and/or microbial treatment of waste, preferably MSW, can be pushed towards hydrogen production primarily by controlling the pH of the enzymatic and microbial treatment of the waste. Moreover, it is shown that hydrogen gas can be produced in anaerobic digestion (AD) pre-process tanks for AD process wherein bioliquid obtained from an enzymatic and microbial treatment process is subjected to anaerobic conditions, temperatures of 35 - 55 °C and a pH of 5.5 - 6.5. That is, the pH in the pre-process tank must be higher than the pH of the bioliquid obtained from the enzymatic and/or microbial fermentation of waste in the reactor but lower than the methane producing AD process conditions of pH 6.8 - 8.2. The hydrogen obtained by this method can be used for the common purposes that hydrogen is applied for. Moreover, it is suggested herein that adding hydrogen to the AD methane gas producing process results in higher methane production by the extended use of the hydrogenotrophic methane producing process wherein microbes combine hydrogen and carbon dioxide under normal anaerobic process conditions to both form methane and consume carbon dioxide and thereby increase the methane content of the resulting biogas. Another specific use of the hydrogen produced by the method is conversion into ammonia for longterm storage or for direct use of ammonia.

The hydrogen gas can be collected at different steps in the method and the waste treatment plant can be set up accordingly.

The method comprises in a first aspect the following steps: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 -6.5, and c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor. The method according to the first aspect can be performed in an independent reactor or in a reactor that is connected to another reactor wherein other waste treatment steps may take place. The method according to the first aspect can for instance be performed in a reactor that is part of a larger waste treatment plant.

In a second aspect, the method comprises all of the above steps and in addition, the waste, preferably MSW, is subjected to further enzymatic and/or microbial treatment after hydrogen gas has been removed. In order to prevent hydrogen gas formation in the subsequent step, pH is lowered to 5.5 and maintained below 5.5 and oxygen is entered into the reactor. This change in pH and the change from anaerobic to aerobic conditions will provide for other microbial communities to thrive in the waste, such as fermenting microorganisms. Although not necessary in continuous processing of waste wherein the fermenting microorganism communities have already established, the waste can alternatively be inoculated with the desired bacterial populations in order to speed up the process. The second aspect accordingly provides a method for producing hydrogen gas from waste in a reactor, wherein bioliquid is produced subsequently in a separate reactor optionally connected to the hydrogen producing reactor in a method comprising the following steps: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, and d) subjecting the waste, preferably municipal solid waste, obtained in step b) to continued enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions.

In a third aspect, the method comprises all of the above steps and in addition, the pH of the method for producing hydrogen gas from waste, preferably MSW, in a reactor is kept at about 5.5 - 6.5 by adding process water or reject water obtained from an AD process by separation. The process water or reject water from a subsequent AD process can be obtained from an independent AD process, or from an AD process wherein the substrate of the AD process is bioliquid obtained from step d) and isolated by separation in step e). Accordingly, the method can comprise the following steps that may, however, not necessarily be carried out at the same plant: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste, preferably municipal solid waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 -6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained from step b) to enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions, e) subjecting the treated waste, preferably municipal solid waste, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained in step e) into an anaerobic digestion pre-process tank at a temperature of 35 - 55 °C and at a pH between 5.5 and 6.5, g) Collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) Subjecting said bioliquid fraction obtained from step e) or from the anaerobic digestion pre-process tank in step f) to anaerobic digestion in a digestion tank.

In a fourth aspect, the method comprises all of the above steps and in addition two optional steps i) and j) that can be added together or alone. In the optional step i) process water such as process water obtained from step h), or process water obtained from step h) further subjected to more solid-liquid separations to provide reject water, or water from external water sources, or any combination of process water and water from external water sources is added to the reactor in step a) and/or b). In optional step j) the hydrogen collected in step c) and/or in step g) is added to the AD reactor thereby improving the methane yield. Accordingly, in the fourth aspect, a method for producing hydrogen gas from an anaerobic digestion process of bioliquid comprising the following steps is provided: a) Adding waste, preferably municipal solid waste, and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting the waste, preferably municipal solid said waste, to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH at about 5.5 - 6.5, c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor, d) subjecting the waste, preferably municipal solid waste, obtained from step b) to enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions, e) subjecting the treated waste, preferably municipal solid waste, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH between 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank, i) optionally, adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combinations thereof to the reactor in step a) and/or b), j) Optionally, adding the hydrogen obtained from step c) and/or from step g) into the anaerobic digestion tank in step h).

The present invention further provides for the use of hydrogen gas obtained from step c) and/or step g) of the methods of the invention for increasing the production of methane gas in an anaerobic digestion process or for the production of ammonia for storage or direct use.

In another embodiment, the method comprises steps a) to c) and the following additional steps: e) subjecting the treated waste from step c) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre-process tank in step f) to anaerobic digestion in a digestion tank.

In other embodiments, step a) to d) are mandatory whereas steps e) to h) are optional.

Thus, on one embodiment the method comprises steps a) to d) and at least the following step e) and optionally one or more of the subsequent steps in consecutive order: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or f) to anaerobic digestion in a digestion tank.

According to this embodiment, hydrogen can be produced in step f)-g) in addition to steps a)- c). The hydrogen produced from the bioliquid obtained from step e) can be produced in an anaerobic pre- process tank at 35 - 55 °C and at a pH of 5.5 - 6.5 and then collected. Preferably, the pH in the pre- process tank is 5.5 - 6.0. This is because at a pH above 6 methane gas will also be produced in addition to the hydrogen gas. Thus, hydrogen gas can be collected without the presence of methane gas when the pH is maximum 6.

In one embodiment, the method comprises steps a) to d) and the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, and h) subjecting said bioliquid fraction from step e) to anaerobic digestion in a digestion tank.

The enzymatic and microbial waste treatment process wherein the hydrogen gas can be produced, may thus comprise the above steps a), b), and c) and optionally further steps. If the method comprises such further steps, the method comprises at least step e) and one or more of the subsequent steps f), g) and h) and optionally step i) and/or step j) when at least step h) is present and/or step k). For all steps, the preferred waste is MSW.

In a preferred embodiment, the enzymatic and microbial waste treatment process wherein the hydrogen gas can be produced, comprises the above steps a), b), c), and d), and optionally further steps. If the method comprises such further steps, the method comprises at least step e) and one or more of the subsequent steps f), g) and h) and optionally step i) and/or step j) when at least step h) is present and/or step k). For all steps, the preferred waste is MSW.

Downstream processing steps can be added to the method when at least steps a) to c) are present, such as when steps a) to e) or such as when steps a) to h) are present. Downstream processing could be any process which takes place downstream of step c), such as the solid or the liquid fraction of the waste, preferably MSW, obtained from step e) which takes place downstream of the enzymatic and/or microbial treatment in the reactor in step d). Examples of other downstream processes are washing processes, evaporation processes, collection of bioliquid obtained in step e) and anaerobic digestion. Downstream processes also include processes wherein the liquid fraction of the waste, preferably MSW, obtained from step e) is converted into biogas, which can be combusted to generate electricity and/or heat, and processes wherein the solid and/or liquid fraction of the waste, preferably MSW, obtained from step e) or step f) is converted into, biomethane gas and/or transportation fuels. Preferably, the reactor hosting the process in step a) to c) is designed to ensure flow of the waste or comprises means for ensuring the flow of the waste, preferably MSW.

It is moreover preferred that the reactor hosting the process in step d) is designed to ensure forward flow of the waste or comprises means for ensuring the forward flow of the waste, preferably MSW.

Flow of the waste will help to maintain the temperature, pH, and microbial activity at the same level throughout the reactor.

Several common means for ensuring a forward flow in a reactor could be applied, such as rotation, Y- profile scoops, or screw conveyors.

Daily microbial monitoring was set up during developmental tests of the present method and has elucidated the differences in the microbial populations present in the reactor under conditions where hydrogen gas production prevails and where optimal conditions for providing a suitable bioliquid for AD prevails, respectively. The presence of specific classes of microbial communities have been found to provide a marker of the outcome of the enzymatic and microbial treatment of the waste. The detection of specific microbial communities was compared under the different temperature and pH conditions and retention times and gas production measurements of the available microbial feed sources, such as infeed waste, sugar sources and lactic acid present in the reactor that may stimulate growth of certain bacterial classes were registered.

It is important that the conditions in the reactor are stabilized to support growth of the desired microbial community providing either hydrogen gas or the fermented bioliquid. With the present invention, conditions for controlling growth of the microbial community providing hydrogen gas is provided.

In order to provide the preferred pH range for stimulating growth of the hydrogen gas producing bacteria, process water or reject water from a downstream AD process can be added to the reactor infeed tank or in the process water tanks thereby increasing pH in the reactor to 5.5 or above. The pH of process water obtained from an AD process (hereinafter referred to as reject water) is alkaline, normally within the range of pH 7-9.

Sampling the microbial flora under the conditions for hydrogen gas production showed that particularly species of Clostridium, bacillus and Megasphaera are possibly the main producers of the hydrogen gas. The output of hydrogen gas increases when the temperature is above approximately 30°C, preferably between 30°C - 65°C, more preferred between 35°C - 60°C, such as between 40°C and 55°C or between 45°C and 55°C, and most preferred around 50°C.

Sampling the microbial flora under the conditions for bioliquid production showed that fermenting microorganisms, particularly lactic acid producing species, were the main producers of the sugar compositions, short alcohol compositions, short acids, nitrogen compositions found in bioliquid. It was also found that when glucose in the reactor is depleted, such as when no waste is fed into the reactor regularly, the fermenting microbial populations producing valuable compounds for bioliquid production, such as lactic acid bacteria, will lose their food source and lactic acid will be the main substrate inside the reactor. The Megasphaera sp. bacteria will benefit from this and convert lactic acid into acetate (among others) and gas, particularly hydrogen gas. The hydrogen gas producing bacteria are strict anaerobes and do not tolerate O2 in any form, thus, in order for hydrogen gas production to dominate in the reactor, anaerobic conditions are required.

The method steps a) to d) according to the present invention could potentially provide for production of hydrogen gas and bioliquid simultaneously in the same reactor. However, due to the different requirements in relation to particularly the absence/presence of oxygen, it would at least for large scale production sites be easier to separate the hydrogen gas production from the fermenting process.

Initial pH of pure water prior to entering the reactor is normally within the range pH 6.5 - 7 while the initial pH of reject water applied here was pH 8 - 8.5.

The method according to the present invention may, in addition to step a), b), c), and d) additionally comprise steps e), f), g), and h): e) Subjecting the treated waste, preferably MSW, from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 -55 °C and at a pH between 5.5 - 6.5, g) Collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank.

The bioliquid obtained from step d) and as isolated in step e) provide an excellent feed for anaerobic digestion. Bioliquid obtained from step d) comprises sugar compositions, short alcohol compositions, short acids, and nitrogen compositions that has been provided by the enzymatic activity e.g., of the microbial fermenting population that are present under the conditions in step d). Hydrogen is an intermediary composition in some of the microbial processes for methane gas production and is used instantly by the methane producing bacteria during the AD process. Thus, in one embodiment of the invention bioliquid obtained from step d) is subjected to conditions for hydrogen production in pre- process tank, followed by anaerobe digestion of the bioliquid and methane production in a digestion tank.

Optionally, when the method of the present invention comprises step h), the method may additionally comprise one or two more steps: i) optionally, adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combinations thereof to the reactor in step a) and/or b), j) Optionally, adding the hydrogen obtained from step c) and/or step g) into the anaerobic digester in step h).

The optional steps i) and j) can be added to the process comprising steps a), b) and h) separately or both steps can be added and provide further optimization of the method. In step i) process water, such as reject water, from the anaerobic digestion step h) is entered into the reactor in order to push the pH in the reactor in step a) to above about 5.5 (step i). In step j) the hydrogen collected in step c) and/or step g) may be added to the anaerobic digestion tank in step h) in order to promote the anaerobic digestion process, thereby increasing the methane yield of the anaerobic digestion process.

In step i) process water or reject water, which can easily be obtained from the AD process subject to known and commonly applied means, is re-circulated into the reactor in step a) and/or b) in order to maintain pH within the range at about 5.5 - 6.5.

In optional step j), the hydrogen obtained in step c) and/or in step g) is added to the AD digester during the AD process in order to improve methane production.

In a preferred embodiment, the reject water is obtained from the anaerobic digestion process in step h). Process water from an AD process (e.g., reject water) has a basic pH above 7. Adding this to the reactor in step a) is a mean for increasing the pH of the water in step a) to between 5.5 - 6.5 as required. Similarly, the reject water can be added to the reactor in step b) in order to maintain the pH at 5.5 - 6.5.

However, pH of the water in step a) and/or b) can be adjusted to 5.5 - 6.5 by other means for increasing the pH, such as the addition of a base. In a preferred embodiment, pH in of the water in step a) and/or b) is between 5.5 - 6. In another preferred embodiment, the pH in the water in step a) and/or b) is kept at a pH value between 5.5 - 6 by the addition of process water or reject water.

In a preferred embodiment of the present invention, the temperature in step b) is between 30°C and 65°C. As shown in the examples section, hydrogen gas production is low when the temperature in the reactor is below 30°C. Accordingly, adjusting the temperature to above 30°C is a means for stimulating the growth of hydrogen gas producing bacteria when the other conditions for hydrogen gas production are met i.e., when pH is 5.5 or above and the conditions are anaerobic. Thus, temperature is an additional feature that can be used to control the conditions in the reactor for promoting hydrogen gas production.

The enzymatic and microbial treatment of waste, preferably MSW, in step b) will require addition of water if the waste to be treated has a dry matter content above approximately 20%. The water added in step a) and/or b) can be water from an external source such as tap-water and water from natural sources such as rivers and lakes, but it can also be process water, such as reject water as described above. In a preferred embodiment of the method of present invention, water added in in step a) and/or b) is selected from process water and water from external sources, and any combinations thereof.

In a preferred embodiment, the water entered into the reactor in step a) and/or b) comprises process water from an anaerobic digestion process and/or water from other sources such as water from external sources; and any combinations thereof. The process water comprises for example wash water or water obtained by evaporation of waste fractions or AD and bioliquid.

In another preferred embodiment, the water entered into the reactor in step a) and/or b) comprises reject water from an anaerobic digestion process and/or water from other sources such as process water other than reject water, water from external sources; and any combinations thereof. The process water other than reject water comprises for example wash water or water obtained by evaporation of waste fractions or AD and bioliquid.

In the examples disclosed herein, steps a) to d) may be performed in the same reactor either simultaneously in each separate end of the reactor or in the same reactor consecutively separated in time. However, the method need not be performed in the same reactor and for large scale production it is easier to control the conditions for step b) and c) when the steps are carried out in different reactors that are either connected and present in the same waste treatment plant or unconnected and present in the same or in a distant waste treatment plant. In a preferred embodiment, step a), b), and c) is performed in a reactor that is separated from the reactor in step d).

The hydrogen gas produced in step b) can be collected by any suitable means known in the art. Hydrogen will accumulate in the top of the headspace inside the reactors, and in one embodiment, the gas is extracted using an extraction pipe that is connected to and triggered by a gas detector. The gas is subsequently "cleaned" by bubbling it through a liquid phase, cryogenic distillation, pressure swing adsorption, or by use a gas membrane technology.

Hydrogen gas cannot only be produced in step b) but can also be produced in step f) in an anaerobic digestion pre-process tank prior to the anaerobic digestion process in step h). In order to provide hydrogen gas production in the anaerobic digestion pre-process tank, the tank must be anaerobic, the temperature should be between 35 - 55 °C, preferably between 40 - 50 °C and the pH should be between 5.5 - 6.5 or between 6.0 -6.5, preferably about 6. The hydrogen gas produced in step f) can be collected by any suitable means known in the art. In one embodiment, the gas produced in step f) is distributed into the anaerobic digester liquid by means of small bubble aeration pans on the AD tank, for instance at the bottom of the tank. In another embodiment, the hydrogen rich gas is added into a gas scrubber with biogas passing over biofilm covered fill-bodies under anaerobic conditions. The hydrogen gas obtained by the present method could be applied for the same purposes that hydrogen gas is normally used for. In a preferred embodiment, the H2 obtained from step c) and/or step g) according to the method of the present invention is used for production of ammonia according to methods applied in the art for this conversion.

In another preferred embodiment, the H2 obtained from step b) and/or step f) according to the method of the present invention is used as an additive in an anaerobic digestion process for increasing the production of methane. In one embodiment, the hydrogen produced in the anaerobic digestion pre- process tank is injected into the AD digesters in very small and heavily dispersed bubbles. The added H2 will contribute to the methane concentration in the produced gas by means of hydrogenotrophic methane production from CO2 and H2.

DEFIN ITIONS

Aerobic means, in the context of the present invention, presence of free oxygen. The aerobic fermenting microbial populations are accordingly able to live, being active and occur under conditions where free oxygen is present. Aerobic microorganisms have different levels of sensitivity to absence of oxygen. In the context of the present invention, aerobic microbial populations refer to microbial populations that are not capable of growth and of fermenting the organic compounds of the waste subject to conditions absent to free oxygen.

Anaerobic means, in the context of the present invention, absence of free oxygen. The anaerobic microbial populations providing the hydrogen gas is accordingly able to live, being active and occur under conditions where free oxygen is absent. Anaerobic microorganisms have different levels of sensitivity to oxygen. In the context of the present invention, anaerobic microbial populations refer to microbial populations that are not capable of growth and of producing hydrogen gas subject to conditions where free oxygen is present.

"Anaerobic Digestion (AD)" refers to a fermentation process operated in a system comprising one or more digestion tanks or digestors operated under anaerobic conditions in which methane gas is produced in each of the digestion tanks. Methane gas is produced to the extent that the concentration of dissolved methane in the aqueous phase of the fermentation mixture within the AD system is saturated at the conditions used and methane gas and other over saturated gaseous compounds such as CO2, H2S and NH3 are emitted from the system.

"Anaerobic digestion pre-process tank" is the tank holding the AD feed e.g., bioliquid, prior to the feed being fed into the anaerobic digestion tank for methane gas production. The feed in the anaerobic digestion pre-process tank is bioliquid obtained from the enzymatic and/or microbial treatment of waste, preferably MSW. The anaerobic digestion pre-process tank may serve as a pre-conditioning tank where properties like pH, temperature and retention time can be manipulated before allowing the AD process to occur. The AD process will establish a very solid pH buffer and be quite expensive to pH adjust due to conversion of organically bound elements to ammonia and carbon dioxide when allowed to run its course. In the pre-process tank, the acids are still unconverted, and the pH is thus much easier and/or cheaper to adjust. Moreover, according to the present invention, the conditions in the anaerobic digestion pre-process tank can be made to promote hydrogen gas production.

"Bioliquid" is the liquefied and/or saccharified degradable components obtained by enzymatic treatment of waste, such as MSW, comprising organic matter. Bioliquid also refers to the liquid fraction obtained by enzymatic treatment of waste comprising organic matter once separated from non- fermentable solids. Bioliquid comprises water and organic substrates such as protein, fat, galactose, mannose, glucose, xylose, arabinose, lactate, acetate, ethanol and/or other components, depending on the composition of the waste (the components such as protein and fat can be in a soluble and/or insoluble form). Bioliquid comprises also fibers, ashes, and inert impurities. The resulting bioliquid comprising a high percentage of soluble microbial metabolites provides a substrate for gas production, a substrate suitable for anaerobic digestion e.g., for the production of biogas.

"Reactor" is simply denoting a vessel suitable for housing biological processes, such as degradation, fermentation etc.

Cellulolytic background composition (CBC) or Cellulolytic Enzyme Blend" means an enzyme composition comprising a mixture of two or more cellulolytic enzymes.

The CBC may comprise two or more cellulolytic enzymes selected from: i. an Aspergillus fumigatus cellobiohydrolase I; ii. an Aspergillus fumigatus cellobiohydrolase II; ill. an Aspergillus fumigatus beta-glucosidase or variant thereof; and iv. a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity; or homologs thereof.

The CBC may further comprise one or more enzymes selected from: a) an Aspergillus fumigatus xylanase or homolog thereof, b) an Aspergillus fumigatus beta-xylosidase or homolog thereof; or c) a combination of (a) and (b) (as described in further detail in WO 2013/028928).

The major activities of the CBC may comprise endo-l,4-beta-glucanases (E.C. 3.2.1.4); endo-l,4-beta- xylanases (E.C. 3.2.1.8); endo-l,4-beta-mannanase (E.C. 3.2.1.78), beta-mannosidase (E.C 3.2.1.25), whereas other enzymatic activities may also be present in the CBC such as activity from glucanases, glucosidases, cellobiohydrolase I cellobiohydrolase II; beta-glucosidase; beta-xylosidase; beta-L- arabinofuranosidase; amyloglucosidase; alpha-amylase; acetyl xylan esterase. The CBC may be any CBC described in WO2013/028928 (the content of which is hereby incorporated by reference). The CBC may be from T. reesei. The CBC may be from Myceliophtora thermophilae. The CBC may be Cellic® CTec3 obtainable from Novozymes A/S (Bagsvaerd, Denmark).

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in pre-treated corn stover (PCS) (or other pre-treated cellulosic material) for 3-7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, preferably in the range 5.5-6.5 or even more preferably in the range 6.0-6.5 compared to a control treatment without addition of cellulolytic enzyme protein.

"Digestion tank" or "Digester" is a reactor and/or container suitable for anaerobic digestion.

"Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. The fermenting microorganism can e.g., produce glucosebased chemicals, such as lactic acid, 3- hydroxypropionic acid (3-HPA), 1,4-butanediol (BDO), butanedioic acid (succinic acid), ethane-l,2-diol (ethylene glycol), butanol and/or 1,2-propanediol (propylene glycol).

"Hydrogen gas" is at standard temperature and pressure, a colorless, odorless, tasteless, non-toxic, nonmetallic, highly combustible diatomic gas with the molecular formula Hz. Industrial production of hydrogen gas is mainly obtained from steam reforming natural gas, and less often from more energy- intensive methods such as the electrolysis of water. Most hydrogen gas is used near the site of its production, mainly for fossil fuel processing (e.g., hydrocracking) and ammonia production, mostly for the fertilizer market.

"Municipal solid waste" (MSW) refers to waste fractions which are typically available in a city, but that need not come from any municipality per se, i.e., MSW refers to every solid waste from any municipality but not necessarily being the typical household waste - could be waste from airports, universities, campus, canteens, general food waste, among others. MSW may be any combination of one or more of cellulosic, plant, animal, plastic, metal, or glass waste including, but not limited to, any one or more of the following: Garbage collected in normal municipal collections systems, optionally processed in a central sorting, shredding or pulping device, such as e.g., a Dewaster® or a reCulture®; solid waste sorted from households, including both organic fractions and paper rich fractions; Generally, municipal solid waste in the Western part of the world normally comprise one or more of: animal food waste, vegetable food waste, newsprints, magazines, advertisements, books, office paper, other clean paper, paper and carton containers, other cardboard, milk cartons and alike, juice cartons and other carton with alu-foil, kitchen tissues, other dirty paper, other dirty cardboard, soft plastic, plastic bottles, other hard plastic, non-recyclable plastic, yard waste, flowers etc., animals and excrements, diapers and tampons, cotton sticks etc., other cotton etc., wood, textiles, shoes, leather, rubber etc., office articles, empty chemical bottles, plastic products, cigarette buts, other combustibles, vacuum cleaner bags, clear glass, green glass, brown glass, other glass, aluminum containers, alu-trays, alu-foil (including tealight candle foil), metal containers (-AI), metal foil (-AI), other sorts of metal, soil, rocks, stones and gravel, ceramics, cat litter, batteries (button cells, alkali, thermometers etc.), other non-combustibles and fines. Normally, waste such as MSW inherently comprise microbial activity before entry and the waste is therefore already partly degraded by natural degradation processes when it is subjected to the first step in the present method. Moreover, the enzymatic and microbial treatment in step b) of the present method and the enzymatic and/or microbial treatment in step d) of the present method further contributes to the degradation of the waste. Accordingly, in the context of the present method, the terms "MSW" and "waste" comprise waste that is degraded to different extend by microbial and/or enzymatic activity.

"Process water" may comprise water that is recycled from an industrial process, wherein e.g., waste undergo an enzymatic and/or microbial treatment, such as a process according to some of the embodiments of the present invention including wash water, reject water and bioliquid. Process water is of lower quality than drinking water such as in terms of e.g., any one of organic and/or inorganic salt(s), microbial organisms / plate counts, suspended solids, DM, and/or pH, including any combination thereof. Process water may be adjusted in terms of mineral/salt content, pH and the like. Process water includes bioliquid, reject water and wash water as described above.

"Reject water" is defined as the liquid fraction obtained after one or more solid-liquid separations of the AD digestate and is accordingly the term applied to denote process water obtained from an AD process. The one or more solid liquid separations can comprise one or more of decantation, centrifugation, filtering, flocculation, pressing and sedimentation. Like the AD digestate, reject water has an alkaline pH, and comprises dissolved matter, such as salts which may include both inorganic salts and organic salts. Reject water may also comprise some suspended matter and live microorganisms from the AD process. Such water may be subject to hygienization and/or other purification steps in accordance with national requirements prior to being released from the AD plant.

"Water from external sources" includes water obtained from any source wherein said water has not previously been subjected to any steps in an enzymatic and/or microbial waste treatment process. Thus, water from external sources comprise tap water, wastewater that has not been subjected to an enzymatic and/or microbial waste treatment process, and water from natural sources. "Water from natural sources" is water obtained from natural sources such as rivers, lakes and ponds.

DESCRIPTION OF STEPS a) TO k) IN TH E ENZYMATIC AN D/OR M ICROBIAL TREATM ENT OF WASTE

Step a)

In step a) waste, preferably MSW, and water at a pH of 5.5 -6.5 are added to a reactor. The waste to be processed, such as e.g., MSW, may preferably have a non-water content of above 10% and below 80%, in some embodiments between 40% and 70%. Waste such as MSW may often comprise a considerable amount of water. However, the water content may be adjusted in order to achieve appropriate water activity.

Waste suitable for the present enzymatic and microbial treatment comprises biodegradable material, which is organic material that can be degraded by enzymes and/or microorganisms. The organic material may comprise carbohydrates, proteins, fat and mixtures thereof, which are organic matter that are typical present in household waste. The waste further comprises material that is not biodegradable, such as plastic or metal. Suitable waste could be municipal solid waste (MSW), agriculture waste, hospital waste, industrial waste, e.g., waste fractions derived from industry such as restaurant industry, food processing industry, general industry; waste fractions from paper industry; waste fractions from recycling facilities; waste fractions from food or feed industry; waste fraction from the medicinal or pharmaceutical industry; waste fractions from hospitals and clinics, waste fractions derived from agriculture or farming related sectors; waste fractions from processing of sugar or starch rich products; contaminated or in other ways spoiled agriculture products such as grain, potatoes and beet roots not exploitable for food or feed purposes; or garden refuse

The waste can be sorted or unsorted.

In a preferred embodiment, the waste subjected to the reactor in step a) is municipal solid waste (MSW).

The waste, e.g., MSW, may have a Dry Matter (DM) content in the range 10%-90%; 20%-85%; 30%- 80%; 40%-75%; 50%-70%; or 55%-65 % (w/w); and/or around 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; or 90% (w/w). Preferably, the dry matter content in MSW for the present method is at least 20%. The amount of water added in step a) depends on the amount of dry matter of the waste when the dry matter content is low the need of adding water to the process of step a) is also low.

The dry matter may be measured as follows: prepare crucibles by adding 1.5 g of mineral based (heat resistant) litter e.g., cat litter. Heat in furnace for 1 hour at 550°C then allow to cool to 200°C before transferring the crucibles into a desiccator filled with silica gel using metal tongues. Allow to cool to room temperature. Weigh the crucible W_crucibie and add 25 g of sample and reweigh and note the weight Wsampie- Place crucible on a suitable tray and place in the preheated oven and heat at 105°C for 24 hours. Take crucibles from the oven and return them into the desiccator. When cooled to room temperature weigh crucible plus contents and note the weight Wdry. Dry matter (DM) is calculated as DM — ((Wdry " Wcrucibie/ Wsampie " Wcrucibie) * 100).

Thus, the DM content of the waste may be measured or assessed at one or more of the following points in time: (i) before entry into the reactor in step a); ii) at the onset of said enzymatic and microbial treatment of the waste entering the reactor in step a); (iii) before provision of the bioliquid obtained in step d) through one or more solid/liquid separation step(s).

The water added in step a) and/or step b) must have a pH of at least 5.5 and is preferably above 5.5, such as pH 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4 or 6.5. When pH is above 6, conditions for methane producing microorganisms were found to be improved. Thus, in order to avoid methane gas production, pH is preferably not higher than 6.5 in step a) and/or b). Accordingly, the preferred pH in step a) and/or b), is 5.5 - 6.5 since this pH will promote hydrogen production of microbial communities over the fermenting microbial communities and over the methane gas producing microbial communities.

The pH of the water in step a) may be adjusted prior to being entered into the reactor or after entry into the reactor, but it should have a pH of 5.5 or above before addition of the enzymes in step b) to optimize hydrogen production.

The pH of the water (process, reject or water from an external source) can optionally be adjusted by any known means, such as by the addition of acid or base if the pH of the water is not within the optimum pH range of the hydrogen producing process in step b). In one embodiment, the pH of the water is adjusted by adding base until pH of the water is between pH 5.5 - 6.5 such as pH 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, or 6.4. Any base could be used to adjust pH. Preferably, the base is an organic base since such bases are less likely to accumulate water soluble salts in the recirculation loop when recirculation of process water is applied.

Step b)

In step b), the waste, preferably MSW, is subjected to enzymatic and microbial treatment while maintaining pH at about 5.5 - 6.5 under anaerobic conditions. The enzymatic and microbial treatment of the waste in step b) may be performed in a reactor. The treatment is performed by adding one or more enzymes and by the bacteria present in the waste. Optional ly, standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of converting the organic matter present in the waste into hydrogen gas may be added to the reactor. The enzymes are supplied in either native form or in form of microbial organisms expressing the enzymes. The enzymatic and microbial treatment in step b) may be performed by adding one or more enzymes, supplied in either native form and/or in form of microbial organisms giving rise to the expression of such enzymes; and/or by the bacteria present in the waste and/or optionally by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of converting the organic matter present in the waste into hydrogen gas, such as species of Megasphaera, Clostridium and Bacillus.

In one embodiment, the method of the invention is a method wherein said enzymatic and microbial treatment in step b) is performed by adding enzymes, supplied in either native form or in form of microbial organisms giving rise to the expression of such enzymes, and/or by the bacteria present in the waste and optionally by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing hydrogen.

Microorganisms that may be added to the reactor in step b) (and in step d)) include yeasts, and/or fungi and/or bacteria.

In step b) the waste is treated with an enzyme composition. Suitable enzyme compositions for enzymatic and microbial treatment of waste are well known in the art and are commercially available e.g., such as cellulolytic background composition. Such compositions assist the micro bacterial turnover of the waste by degrading complex compounds that can more easily be assessed by the bacteria to provide smaller entities such as monomers of sugars, amino acids and lipids. However, most of these enzyme compositions have a pH optimum lower than pH 5.5, normally around pH 4.8. Nevertheless, enzyme compositions normally applied for producing bioliquid was applied when developing the present method and are thus suitable for use in step b), see description of step d). However, in order to optimize the hydrogen gas yield, and to reduce the required amount of enzyme, enzyme compositions with similar targets but with higher pH optimum is preferred. Such a composition could for instance be obtained by adding additional enzymes with a higher pH optimum to the cellulolytic background composition.

Step b) must be performed under anaerobic conditions, that is in the absence of oxygen. When waste, water and enzymes are added into an airtight reactor, the oxygen present is easily accessed and consumed by the aerobic microorganisms naturally present in the waste. In order to ensure and/or promote anaerobic conditions, common measures for avoiding entry of air at the inlet and outlet of the tank could be applied. If applicable, removal of oxygen by membrane filtration of the liquids in the reactor could be applied. Avoidance of ventilation is preferred. pH should be maintained at pH 5.5 - 6.5 during step b). The pH can be maintained by any common means. In a preferred embodiment, the pH is maintained at 5.5 - 6.5 during step b) by the addition of process water or reject water obtained from an anaerobic digestion process, such as the AD in step h). Water from an AD process (reject water) has an alkaline pH above 7. Adding this to the reactor in step a) is a mean for maintaining the pH of the water in step a) to between 5.5 - 6.5 as required.

When the conditions for step b) is provided in the reactor at the upstart of a first batch or at the upstart of a continuous process, it will take some time for the hydrogen producing microbial populations to become the dominating population and thus, for the hydrogen to develop. When the hydrogen producing microbial populations have been established, hydrogen production is fast. For example, when waste is fed continuously into the reactor with a retention time of 1- 72 hours, such as 1 to 15 hours, such as 1 to 20 hours, such as 5 to 50 hours, such as 5 to 20, or such as 3 to 20 hours, or such as 3 to 15 hours or such as 3-6 hours inside the reactor, hydrogen gas may be produced continuously.

Step c)

In step c) the hydrogen produced in step b) may be removed from the headspace of the reactor by extraction or separation of the gas phase. Any applicable method known in the art for removing hydrogen gas could be applied.

Step d)

Steps d), e), and h) according to the present invention are steps currently applied for processes wherein waste comprising organic matter has been subjected to enzymatic degradation and/or microbial fermentation producing a bio-liquid and various solid fractions. Examples of such waste treatment processes are disclosed in W02006056838, W02007036795, WO2011032557, WO2013185778, WO2014198274, W02016030480, W02016030472, W02016050893 and WO2017/174093.

In step d), the waste, preferably MSW, obtained from step b) is optionally subjected to continued enzymatic and/or microbial treatment in a reactor at a pH below 5.5 at aerobic conditions.

In order for the enzymatic and/or microbial liquefaction of the waste in the reactor in step d) to provide the conditions for the microbial communities producing a bioliquid comprising an optimum amount of short chain carboxylic acids and sugars such as glucose, xylose, arabinose, lactic acid/lactate, acetic acid/acetate and/or ethanol to thrive instead of the hydrogen gas producing microbial communities, the pH in the reactor should be below 5.5.

In some embodiments, it is necessary to adjust the pH to below 5.5. In other embodiments, the pH of the waste/water/enzyme mixture will drop to below 5.5 due to the acidification of the waste during the hydrogen gas production, such as the formation of lactate or due to the aerobic conditions required for the process in step d). This will promote the growth of the fermenting microorganisms providing the valuable degradation compounds of the bioliquid. When these bacteria become active and take over the process, the pH will drop further due to compounds such as lactic acid being one of the primary compounds produced when the conditions in step d) is applied.

The pH of the waste/water/enzyme mixture can be adjusted, if necessary, by any known means such as by the addition of an acid.

In one embodiment, the pH of the waste/water/enzyme mixture is adjusted by adding acid until pH of the process water is between pH 3 - 5.5 such as pH 3, pH 3.5, pH 4, pH 4.5 and pH 5 or any pH value in between these pH values. Any acid could be used to adjust pH. Preferably, the acid is an organic acid since such acids are less likely to accumulate water soluble salts if bioliquid is used in a recirculation loop.

The enzymatic and/or microbial treatment of the waste in step d) may be performed in a reactor. The treatment is performed by adding one or more enzymes and by the bacteria present in the waste.

Optionally, standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of converting the organic matter present in the waste into compositions suitable for subsequent biogas production in an anaerobic digestion process may be added to the reactor.

The enzymes are supplied in either native form or in form of microbial organisms expressing the enzymes.

The enzymatic and/or microbial treatment in step d) may be performed by adding one or more enzymes, supplied in either native form and/or in form of microbial organisms giving rise to the expression of such enzymes; and/or by the bacteria present in the waste and/or optionally by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of converting the organic matter present in the waste into organic acids or other compositions, such as lactic acid, 3-hydroxypropionic acid (3-HPA), 1,4-butanediol (BDO), butanedioic acid (succinic acid), ethane-1,2- diol (ethylene glycol), butanol or 1,2-propanediol (propylene glycol), suitable for subsequent biogas production in an anaerobic digestion process.

In one embodiment, the method of the invention is a method wherein said enzymatic and/or microbial treatment in step d) is performed by adding enzymes, supplied in either native form or in form of microbial organisms giving rise to the expression of such enzymes, and/or by the bacteria present in the waste and optionally by adding standard, cultivated, or manipulated yeast, bacteria, or any other microorganism capable of producing biochemicals, ethanol, or biogas.

Microorganisms that may be added to the reactor in step d) include yeasts, and/or fungi and/or bacteria.

Other microorganisms that may be added to the reactor in step d) include bacteria that can efficiently ferment hexose and pentose including but not limited to cellobiose, glucose, xylose and arabinose to short chain organic acids including but not limited to citric acid, lactic, formic acid, acetic acid, butyric acid, valeric acid, isovaleric acid and propionic acid as well as alcohols including but not limited to ethanol.

In order to find out how much enzyme of a given enzymatic composition may be added, a solubilization test of the enzyme composition on model waste may be applied to provide an optimum enzymatic solubilization process.

When added to the process the cellulolytic background composition (CBC) may comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the method according to the present invention include but is not limited to, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC®CTec3 (Novozymes A/S), CELLUCLAST® (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP (Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic International, Inc.).

When the enzyme composition comprises further enzymatic activity apart from the activities present in the CBC, such enzyme activity may be added from individual sources or together as part of enzyme blends. Suitable blends include but are not limited to the commercially available enzyme compositions Cellulase PLUS, Xylanase PLUS, BrewZyme LP, FibreZyme G200 and NCE BG PLUS from Dyadic International (Jupiter, FL, USA) or Optimash BG from Genencor (Rochester, NY, USA).

In another preferred embodiment, the activity of the CBC is in accordance with the activity of ACCELLERASE® TRIO™ (Genencor Int.), Cellic CTec2 (Novozymes A/S) or Cellic CTec3 (Novozymes A/S) or Cellic CTec3 (Novozymes A/S).

Step e)

In step e) the treated waste, preferably MSW, from step d) is subjected to one or more separation step(s), whereby a bioliquid and a solid fraction is provided.

In this separation step, the bioliquid is separated from the non-degradable fractions. Clean and efficient use of the degradable component of waste, such as MSW, combined with recycling typically requires some method of sorting or separation to separate degradable from non-degradable material. The separation in step d) may be performed by any means known in art, such as in a ballistic separator, washing drums and/or hydraulic presses. In one embodiment the separation is performed before the enzymatic treatment. Separation of liquid and solids can e.g., be done in different presses (such as screw and/or piston presses) or e.g., using a simpler sieve function. A ballistic separator is typically used to separate the solids into 2D and 3D fractions and only secondarily a liquid separation.

The 3D fraction (such as cans and plastic bottles) does not bind large amounts of bioliquid, so a single washing step is often sufficient to clean the 3D fraction. The 2D fraction (textiles and foils as examples) typically binds a significant amount of bioliquid. Therefore, the 2D fraction is typically pressed using e.g., a screw press, washed and pressed again to optimize the recovery of bioliquid and to obtain a cleaner and drier 2D fraction. Inert material which is, sand and glass is typically removed e.g., sieved from the bioliquid. Metals are typically removed from all mentioned fractions. The 2D fraction can be further separated into recyclables and/or residuals such as SRF (Solid Recovered Fuel), RDF (Refused Derived Fuel) and/or inerts. The 3D fraction can also be further separated into recyclables and/or residuals such as metals, 3D plastic and/or RDF.

Step e) can be conducted in one separation operation or in a combination of at least two different separation operations.

Step )

In step f) the bioliquid fraction obtained provided in step d) and isolated in step e) is entered into an anaerobic digestion pre-process tank for subsequent use as a feed in anaerobic digestion (step h)). We have identified the conditions under which it is possible to produce hydrogen gas in an amount suitable for extraction. When the bioliquid from a fermentation process is fed into the anaerobic digestion pre- process tank, the temperature is 45 - 55 °C, the pH is within the range of 4.3 - 5.3, and the microbial content is dominated by fermenting microorganisms. It is shown herein that hydrogen may be produced in significant amounts when the temperature in the anaerobic digestion pre-process tank is 35 - 55 °C and the pH is increased to be within the range of pH 5.5 - 6.5.

Step g)

In step g) hydrogen produced in the anaerobic digestion pre-process tank is collected from the headspace of the anaerobic digestion pre-process tank by extraction or separation of the gas phase. Any applicable method known in the art for removing hydrogen gas could be applied.

Step h)

In step h) the bioliquid obtained from step e) or f)) is subjected to downstream anaerobic digestion in an anaerobic digester. In step f), when applied, hydrogen from the bioliquid is optionally collected before the bioliquid is subjected to step h).

In Step h), the bioliquid obtained in step e) or f) is processed further for the subsequent use in methods for providing energy or biochemicals. Such methods provide the thermo-chemical conversion of the solubilized waste to electricity, heat, methanol, hydrogen, dimethyl ether, petrol, biodiesel, and/or bio-chemical conversion of the solubilized waste to biogas, hydrogen, bioethanol, biodiesel and the like.

A bioliquid comprising a higher amount of acids will contribute to a faster anaerobic degradation process. Anaerobic digestion (AD) is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen. One of the end products is biogas, which can be combusted to generate electricity and/or heat or can be processed into biomethane and/or transportation fuels. A range of anaerobic digestion technologies exists in the state of the art for converting waste, such as municipal solid waste, municipal wastewater solids, food waste, high strength industrial wastewater and residuals, fats, oils and grease (FOG), and various other organic waste streams into biogas. Many different anaerobic digester systems are commercially available, and the skilled person will be familiar with how to apply and optimize the anaerobic digestions process. The metabolic dynamics of microbial communities engaged in anaerobic digestion are complex.

In typical anaerobic digestion (AD) for production of methane biogas, biological processes mediated by microorganisms achieve four primary steps - hydrolysis of biological macromolecules into constituent monomers or other metabolites; acidogenesis, whereby short chain hydrocarbon acids and alcohols are produced; acetogenesis, whereby available nutrients are catabolized to acetic acid, hydrogen and carbon dioxide; and methanogenesis, whereby acetic acid and hydrogen are catabolized by specialized archaea to methane and carbon dioxide. The hydrolysis step is typically rate-limiting and dependent on the biomass type. When the feed is bioliquid it is the methanogens that limit the processing rate. End products of the AD is furthermore digestate, comprising a solid fraction and a liquid fraction (reject water), in particular comprising a water-like liquid with separable suspended particles. The reject water can be isolated by common separation means and optionally recirculated into step a) and/or b) or f).

In order for a common AD process to work efficiently, the pH should generally remain between 6.0 and 9.0, preferably between 6.5 and 8.3. This can be largely affected by the carbon dioxide produced within the biogas. The process itself produces the pH buffer (alkalinity concentration) by the production/release of HCOs" and NH₄ ⁺. Stability may be increased by maintaining high alkalinity concentrations. Decreases in pH may be due to accumulation of organic acid intermediates, often due to substrate overfeeding or the presence of wastes that reduce the ability of methanogens to turn those wastes into biogas, causing inhibition of the methanogenic conversion of previous process products into biogas. Ammonia is passively released as proteins are broken down. Bicarbonates are the primary buffer for balancing alkalinity with pH. Bicarbonate is produced in the same process as methane. Ammonia ions can be released into the liquid from protein breakdown. Ammonia is always present as an equilibrium of ammonia to ammonium-ion in a liquid. When temperature increases, more is available as free ammonia which can act as a methanogen inhibitor at certain concentrations. Acetate/ acetic acid is the direct precursor of methane through the aceticlastic process. During the process, acetate is removed and turned into methane. Only if the process is unbalanced and the methanogenic microbes cannot remove the produced acetate, the pH goes down. The sugar content of the substrate affects the methane percentage of the resulting biogas as the breakdown of glucose yields less hydrogen for the hydrogenotrophic formation of methane from H2 and CO2 than does more complex organics.

To ensure proper pH maintenance, alkalinity can be added at the beginning of the digestion batch. Common alkaline additives include sodium bicarbonate, potassium bicarbonate, potassium carbonate, sodium nitrate, and anhydrous ammonia. The AD digestate released from the AD process is accordingly alkaline, typically with a pH around 8.

Step i)

In step i) water is added to the reactor in step a) and/or b). The water is selected from different sources that may be combined. In a preferred embodiment, the water is process water i.e. water that is recycled from an industrial process, wherein e.g., waste undergo an enzymatic and/or microbial treatment, such as wash water, reject water and bioliquid. Process water is of lower quality than drinking water in terms of for example organic and/or inorganic salt(s), microbial organisms / plate counts, suspended solids, DM, and/or pH. In another preferred embodiment, the process water is obtained from step h) in the present method. In another preferred embodiment, the water is reject water obtained from step h) from solid-liquid separations of the AD digestate. In another embodiment, the water is from an external water source. In another embodiment, the water is a combination of process water, such as process water or reject water obtained from step h) and water from an external water source.

The process water or reject water obtained from the downstream anaerobic digestion in step h) is collected by any known means for separating the process or reject water from the rest of e.g., the AD digestate. The reject water has a high pH of 6 - 9 and will accordingly increase the pH of the incoming waste e.g., MSW in step a) when re-circulated into the reactor in step a) or when added to maintain the pH at 5.5 - 6.5 in step b).

The end product of the AD process, the AD digestate, comprises both solids and liquids and these fractions may be used for various purposes.

Solid-liquid separations can for instance be done by decantation, centrifugation and/or sedimentation. In a preferred embodiment the liquid digestate, which may be hygienized, is dewatered e.g., through a decanter centrifuge, which produce reject water, which is re-circulated into the process according to step i). Usually, the liquid digestate has alkaline pH, and comprises mainly water, but also dry suspended solids and dissolved matter such as salts. "Process water" being defined as the liquid fraction obtained after one or more solid-liquid separations in a process downstream of the enzymatic and/or microbial treatment of waste, such as a water resulting from dewatering of digestate from an AD process, which is also defined as "reject water". Thus, process water comprises reject water. Process water may comprise various salts and dissolved matter and live microorganisms.

Recirculation of process water in waste treatment processes as such have been applied in various treatment process but not in a process where the process water or reject water is added to the reactor actively promoting hydrogen gas production.

The reject water obtained from dewatering digestate comprises microorganisms, mainly archaea when it is entered into the reactor without prior hygienization. It has in relation to the present invention been found that when reject water is entered into the reactor, the hydrogen gas producing population increases whereas the archaea population declines steadily upon entry into the reactor (not shown). Thus, the use of non-hygienized process water such as reject water, comprising e.g., archaea, does not inhibit the hydrogen gas producing process. Without being bound to this theory, we believe that the main producers of hydrogen are the acetogenic bacteria that break down carboxylic acids and alcohols to acetate, H2 and CO2. Some archaea can convert between acetate and H2/CO2. The balance believed to be relevant to ensure hydrogen production over methane production in step b) is to not allow the acetate to be consumed by aceticlastic archeae before the maximum amount of hydrogen has been released to the surroundings. At pH below 6 the methane producing process is slow and inefficient, pushing the Methanosarcina microbes to consume acetate and form H2 and CO2.

In some embodiments of the present invention, the process water or reject water and optionally water from one or more external sources or process water is continuously entered into a reactor with ongoing enzymatic and microbial treatment of waste in step a) and/or step b).

In other embodiments of the present invention, the process water or reject water and optionally water from one or more external sources or process water is discontinuously entered into a reactor with ongoing combined enzymatic and microbial treatment of waste in step a) and/or step b).

That is, the process water or reject water and optionally water from an external source or process water is entered into the reactor when required optionally subject to the monitored pH in the bioliquid present in the reactor. In one embodiment of the invention the process water is added to step a) and/or step b) in batches, such that pH in the reactor is at least 5.5 and not more than 6.5.

Stepj) In step j) the hydrogen obtained from step c) and/or step g) is optionally added into the anaerobic digester in step h). In the presently applied method for anaerobic digestion where bioliquid from enzymatic and/or microbial treatment of waste, e.g., MSW is used as feed for the AD process, the hydrogen is consumed very fast once the AD process begins. Hydrogen formation and consumption is not a requirement for biomethane production, but it is a side-stream that can be utilized by hydrogenotrophic archaea bacteria together with carbon dioxide to produce more methane than can be produced solely by the consumption of acetate. The presence of hydrogen increases the methane percentage in the resulting biogas by converting COjto methane. The speed of the methane formation is not subjective to the hydrogenotrophic methane formation but the methane percentage and thus the energy content of the biogas is improved by hydrogenotrophic methane formation. Hydrogen thus improves the production of biomethane, and we have found that by adding hydrogen to the AD process biomethane production is increased.

Step k)

In step k), the hydrogen collected in step c), in step g) or in both of steps c) and step g) is used. Either the hydrogen is used for the production of ammonia; or the hydrogen is used as an additive in an anaerobic digestion process for increasing the production of methane.

EXAMPLES

Model substrate and tests

Preparation of municipal solid waste ("model MSW") model substrate

In the below examples wherein MSW model substrate was used, 50 kg "model MSW" was prepared in order to mimic the composition of real municipal solid waste. The model substrate was prepared essentially as disclosed in e.g., W02016/030480.

The model waste consisted of 3 fractions based on fresh products supplemented with water:

• 41% vegetable fraction (Mix of fresh vegetables (onions, cabbage, carrots, cucumber etc.), cereals (oatmeal, corn flakes etc.), bread, cake, flowers, boiled rice, boiled pasta fruit, ketchup etc.,

• 13% protein/fat fraction (animal origin) Mix of pate, sausage, hot wings, spareribs, crude meats from chicken, pork and beef etc.

• 46% cellulosic fraction Mix of newsprints, office paper, magazines, cardboard, juice cartons, kitchen tissue, cotton, wood, textiles etc.

Enzymes Cellic® CTec3TM was purchased from Novozymes A/S. Cellic® CTec3TM is a state-of-the-art cellulase and hemicellulase complex comprising GH61 compounds and beta-glucosidases. In some experiments, an enzyme composition with enzymatic activities similar to CTec3 was used.

The amount of enzymatic composition to be added to the waste for sufficient enzymatic treatment was determined by a solubilization test.

The model substrate described above consisting of 41% mixed food waste of vegetable origin, 13% mixed food waste of animal origin and 46% mixed cellulosic waste is shredded, mixed and milled several times until homogeneous, passed through a 3 mm screen, divided into smaller portions and stored frozen at < -18 °C.

A set of pre-tared 50 mL centrifuge tubes, each containing 1.500 ± 0.010 g TS (Total Solids at 60 °C) of the above-mentioned model substrate in a 50 mM Sodium acetate buffer and pH 4.50 ± 0.05, are added various amounts of the enzyme to test (typically 5 - 60 mg EP (Enzyme Protein)/g TS of model substrate) for a final total weight of 20.000 ± 0.025 g in each tube.

The tubes are closed with tight fitting lids and the reaction mixtures are incubated at 50 ± 1 °C for 24 hours ± 10 minutes with agitation by inverting the test tubes (end-over-end) at 10.0 ± 0.5 revolutions per minute.

Immediately after finished incubation the tubes are centrifuged at 2100 ± 10 G for 10 minutes, and immediately after centrifugation (and within less than 5 minutes) the supernatant is decanted into another set of pre-tared tubes. The first set of tubes (including lids), with the residual undissolved model substrate, and the second set of tubes, with the decanted supernatant containing the solubilized model substrate, are weighed on a 4 decimal analytical balance, and then left to dry at 60 ± 1 °C for 6 days in a well-ventilated drying cabinet.

After drying the tubes (including lids) are weighed again, the TS amounts in pellet and supernatant are determined and the mass balance is calculated as:

Mass balance% = ((TS pellet + TS supernatant - TS Enzyme) / TS model substrate) * 100%

The mass balance based on TS model substrate (1.500 ± 0.010 g), to assure for no loss of material and proper drying, will typically be in the interval of 95-105%.

Based on the Total amount and TS amount of the decanted supernatant, TS% in the decanted supernatant is calculated as:

TS% = (TS decanted supernatant - TS Hydrochloric acid/ Total decanted supernatant) * 100%

Finally, the solubilization is calculated as:

Sol u bi I ization% = (((TS% * Residual water / (1 - TS%)) - TS Enzyme) / TS model substrate) * 100% By calculating solubilization based on TS% of the decanted supernatant and the Residual water amount (weight of decanted supernatant and residual wet pellet subtracted weight of the empty tubes and TS of model substrate), the liquid phase that is trapped in the centrifugation pellet will also be accounted for.

A graph of solubilization versus enzyme dose will show the characteristics of enzyme efficacy (maximum solubilization at high enzyme dosages) and enzyme potency (dose required for obtaining a certain level of solubilization).

Enzyme efficacy was measured to be around 70% solubilization and the dose used corresponded to approximately 90% efficacy.

All of the experiments in Examples 1 - 3 were set up in a 1-L fermenters with 166 g model waste and 7.2 g enzymes at 50 °C. Each fermentation was inoculated with 8 mL of bioliquid sampled at a waste treatment facility in Northwich, England. In the fermentations with pure water 1 L of milliQ. water was used. In the experiments with acidified reject water a bit more that 1 L of reject water from the AD process was acidified with acetic acid (80%) or phosphoric acid (85%) until pH 5,7. To control pH during the experiments sodium hydroxide or hydrochloric acid were dosed into the fermenters as controlled by the fermenter software.

To analyze the gases produced during the experiments, nitrogen was continuously blown through the headspace (flow rate 150 cm3/min) and the exhaust was connected to a Gas Chromatograph (GC). To record IR spectra, a Fourier-transform infrared spectroscopy (FTIR) probe was immersed in the fermenters and spectra were recorded every minute. The spectra were processed in PLS_Toolbox by applying a PLS model (PLS= Partial Least Squares), which was built from the spectra recorded in situ and the corresponding samples taken from the fermenters. Chemical Oxygen Demand (COD) and High Pressure Liquid Chromatography (HPLC) were measured for each sample.

Gas detectors are cross sensitive to several gasses. For example, the CO electrochemical cell inside a gas detector is cross sensitive to Hz. Manufactures of gas detectors report that CO electrochemical cells are cross sensitive to H? in a 1:4 scale, i.e., lOOppm CO equals 25ppm H?. H? production detected in the present method far exceeds the levels of CO that microorganisms can produce. Analysis of the gas by use of a high spectrometry gas chromatography system, confirmed that the elevated gasses produced in the reactors was H?.

Example 1 - H2 production in pure water without pH control (Lower pH limit)

Fermentation in pure water without any pH adjustment was made. Inoculation with fresh bioliquid from a waste treatment facility in Northwich, England as described above. The microbial activity leading to the conversion of glucose to lactate started approximately 8 h after the beginning of the fermentation (Figure 2). At the same time CO? and H? started to be formed (Figure 2).

The H2 formation peaked above pH 5.5- below which it slowly declined. Figure 2 shows the CO2, H2 and pH profiles of fermentation in pure water measured in this experiment. The rate of H2 formation is noticeably lower below pH 5.5 as opposed to higher pH values.

Example 2 - H2 production in reject water at pH 5.7

Fermentation in reject water acidified with acetic acid to pH 5.7 was set up. As in Example 1 the mixture was inoculated with fresh bioliquid. Despite the initial pH adjustment, the pH of the reaction media quickly increased to pH 6.7 which created favorable conditions for the H2 production. Similarly, to the fermentation in pure water, H2 and CO2 started being produced when the production of acids started (see the pH decline, Figure 3). Another similarity between these two experiments is the slow-down of the H2 and CO2 formation after several hours. However, unlike in pure water, in the acidified reject water the gas production has stabilized after a while and a constant 1% of H2 was detected until the end of the experiment. The maximal concentration of H2 in the current experiment was twice as high as in the fermentation in pure water.

Figure 3 shows the CO2, H2 and pH profiles of fermentation in reject water acidified with acetic acid to pH 5.7 measured in this experiment. pH during the experiment did not drop below 5.6 which was accompanied by the stable production of hydrogen gas. As in the previous experiment (Figure 2), the start of the H2 and CO2 formation coincided with the beginning of microbial activity (leading to the pH decrease).

Even though these two fermentations had some similarity, the difference in H2 production and in pH during the reaction suggest that the different microbial communities were present during the fermentations. Also, the H2 profile under the two conditions looked different: a total of 50 mmol of H2 was produced in pure water and the H2 formation stopped quickly (9 h after H2 started being produced) while more than 140 mmol of H2 was produced in acidified reject water and its production was still active when the fermentation was stopped.

In conclusion, keeping the reaction mixture at pH 6.7 for as short as 7 h was enough to form a good hydrogen-producing microbial community despite the mixture being inoculated with lactic-acid- bacteria-containing bioliquid.

Example 3 - H2 production under continuous pH 6 in reject water

The previous Examples show a decline in hydrogen production as pH decreases. The present Example was performed to determine the pH level at which no hydrogen can be produced as exactly as possible. To do so a series of fermentations at fixed pH values in acidified reject water and pure water was set up.

A fermentation at pH 6 in HsPCU-acidified reject water confirmed a stable H2 production at high pH (Figure 4). Figure 4 shows H2, CO2 and H2S profiles during fermentation at pH 6 in HsPCU-acidified reject water. Both CO2 and H2 are steadily produced starting at 10 h. Example 4 - detection of microflora at various pH in full scale reactor

In a full-scale reactor for enzymatic and microbial treatment of around 10 tons waste, primarily MSW, the microflora at various pH was examined while hydrogen gas production and lactic acid production was detected during a period of 29 consecutive days. The frequency of the microbial feed (MSW) was varied in order to observe any changes between the prevailing microorganism communities, particularly any competition between fermenting and hydrogen gas producing communities and to identify the optimal conditions for these two groups of microorganisms.

The reactor was subjected to continuous forward flow by rotation.

On day one, 10 tons of unsorted MSW was fed into the reactor and the same enzyme composition as was used in examples 1-3 at a dose providing 85-95% efficacy as found by the solubilization test described in the introductory section to the examples.

The retention time of the waste in the reactor was 72 hours. No waste was added to the reactor in the following days 2 - 5.

On day 6, in feeding of waste was resumed with the addition of reject water of pH 7-9 obtained from an AD process.

From day 13 to day 22 no waste was fed into the reactor. On day 23 in feeding of waste was resumed with the addition of water from a natural source of pH 6 - 7.

Sampling of the liquid fraction of the waste was made every day for determining the identity of the microflora, at the outlet of the reactor.

The development of the relative percentage of the various microbial flora tested for during the 29 days is shown in Figure 5.

As shown in Figure 5, the microbiology inside the reactor shifted from fermenting microorganisms to hydrogen producing bacteria when daily in feeding was not carried out. Furthermore, reject water increased pH significantly, but did not shift the bacterial population away from fermenting microorganisms when pH was kept below pH 5.5.

Example 5 - Simultaneous production of hydrogen gas and bioliquid in the reactor

Simultaneous production of hydrogen gas and bioliquid in the same reactor was tested during this 30- day period in a reactor similar to the reactor applied in Example 4. Prior to this 30-day test period, the entire reactor had been optimized for growth of fermenting microorganisms and therefore, fermenting microorganisms was expected to dominate the bacterial population in the reactor as a point of departure. In order to test whether conditions for favoring hydrogen gas producing microbial communities could be instituted at the inlet end of the reactor, the reactor was set for forward flow by rotation avoiding mixing of contents between the inlet and outlet ends of the reactor. The previous experiments had indicated that pH should be above 5.5 in order to obtain hydrogen gas production. Thus, pH was kept at 5.5 at the inlet of the reactor by addition of reject water of pH 7-9 from a downstream AD process reactor.

As expected, lactic acid bacteria in the form of Lactobacillus ultunensis dominated the reactor (Figure 6) for the first 21 days. This domination of lactic acid bacteria and the production of lactic acid resulted in a stable low pH (Figure 7). Figure 7 shows that pH is stable from day 1 to day 21, with MSW present in the reactor. Sampling was made at the outlet of the reactor, which accordingly reflects a delay of the situation at the inlet of the reactor. Accordingly, Figure 6 is a description of the reactor microbiological population near the outfeed end of the reactor not the infeed end.

At the end of the test period, hydrogen gas was detected at the inlet end of the reactor. Accordingly, this test confirmed that hydrogen gas producing microbial communities present in the incoming waste can be promoted by keeping pH above 5.5 at anaerobic conditions.

Figure 6 shows the development in the bacterial populations during this 30-day testing period. LAB denotes relative % of lactic acid bacteria. Clostridia denote the relative % of the class of Clostridia with a wide range Clostridium present (in this class). Megasphaera denote the relative % of Megasphaera sp. Enterobacteriaceae denote the relative % of Enterobacteriaceae (pathogenic bacteria). Bacillales denote the relative % of all bacillus species. Remaining denote the % all other bacteria present.

From literature, Megasphaera is known as being able to convert lactic acid into gasses and butyrate. The lower pH limit of 5.5 found in these tests might reflect the lower limit for the conditions required for hydrogen gas production of hydrogen gas producing microorganisms such as Megasphaera microorganisms. The upper limit of pH if bioliquid is to be produced simultaneous might relate to the fermenting microorganisms, such as lactic acid producing microorganisms, having an upper pH limit for converting glucose into lactate.

Example 6 - Determination of the most frequent bacterial classes found during hydrogen gas production

Zooming in on the top bacterial species identified in the reactor in Example 5 provides a clearer picture of the microbiological population producing the elevated amounts of Hz and H?S gasses detected in Example 5.

Of the 5 top contributors to the bacterial population (figure 8), 4 species are part of the class named Clostridia (Clostridia, Clostridiaceae, Clostridium and Tepidimicrobium ferriphilum) and one a member of the bacillus family (Anoxybacillus).

Anoxybacillus denote relative % of all Anoxybacillus species not identified to species level by the sequencing. Clostridia denote the relative % of the class of Clostridia (not identified to family, genus and species level by the sequencing) with a wide range Clostridium present (in this class). Clostridiaceae denote the relative % of clostridiaceae (not identified to genus and species level by the sequencing) with a wide range Clostridium present (in this family). Clostridium denote the relative % of all Clostridium species not identified to species level by the sequencing. Tepidimicrobium ferriphilum denote the relative % of Tepidimicrobium ferriphilum a species belonging to the order Clostridiales.

Anoxybacillus sp. has only indirectly been associated with production of hydrogen in literature i.e., being member of a consortia of bacterial species (the others were Clostridium sp. and Bacillus sp.) from which H2 was produced (A. Hniman et al., 2011, Community analysis of thermophilic hydrogenproducing consortia enriched from Thailand hot spring with mixed xylose and glucose). Thus, either it is a hydrogen producing bacteria or it plays a secondary support role of releasing nutrients for the real gas producers in a symbiotic relationship. This has been shown for another bacillus species e.g., for Bacillus thermoamylovorans (CH. Chou et al., 2011. Co-culture of Clostridium beijerinckii L9, Clostridium butyricum Ml and Bacillus thermoamylovorans B5 for converting yeast waste into hydrogen.

Clostridia (Class), hereunder Clostridiaceae (family) and Clostridium (Genus) have all been heavily associated with both H2 production in numerous studies.

Not all bacteria were identified to species level in the present experiment. However, sequencing of the bacterial population identified that several species associated with H2 production were present in the reactor during H2 production. Moreover, live bacterial counts sampled from the reactor output revealed several species of Clostridium.

Claims

1. Method for producing hydrogen gas from municipal solid waste comprising the following steps: a) Adding waste and water at a pH of about 5.5 - 6.5 into a reactor, b) subjecting said waste to enzymatic and microbial treatment under anaerobic conditions while maintaining the pH of about 5.5 - 6.5, and c) collecting the hydrogen gas produced in step b) by separation or extraction of the gas phase from the reactor.

2. Method according to claim 1, comprising the following additional step: d) subjecting the waste obtained in step b) to continued enzymatic and/or microbial treatment in a separate reactor at a pH below 5.5 at aerobic conditions.

3. Method according to claim 1 or 2, comprising at least the following step e) and optionally one or more of the subsequent steps in consecutive order: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank.

4. Method according to claim 2, comprising the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, h) subjecting said bioliquid fraction from step e) to anaerobic digestion in a digestion tank.

5. Method according to claim 2, comprising the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank.

6. Method according to claim 2, comprising the following additional steps: e) subjecting the treated waste from step d) to one or more separation step(s), whereby a bioliquid and a solid fraction is provided, f) entering the bioliquid obtained from step e) into an anaerobic digestion pre-process tank at 35 - 55 °C and at a pH of 5.5 - 6.5, g) collecting the hydrogen gas produced in step f) by separation or extraction of the gas phase from the anaerobic digestion pre-process tank, and h) subjecting said bioliquid fraction from step e) or from the anaerobic digestion pre- process tank in step f) to anaerobic digestion in a digestion tank.

7. Method according to any of claims 3, 5, or 6, wherein the temperature is 40 - 50 °C in the anaerobic digestion pre-process tank in step f).

8. Method according to any of claims 3, 5, 6, or 7, wherein the pH is about 6 in the anaerobic digestion pre- process tank in step f). Method according to claim 3, 4, 6, 7, or 8, comprising the following additional step: i) adding water selected from process water, process water obtained from step h), such as reject water, water from an external water source, or any combinations thereof to the reactor in step a) and/or b).

10. Method according to claim 3, 4, 6, 7, 8, or 9, comprising the following additional step: j) adding the hydrogen obtained from step c) and/or step g) into the digestion tank in step h).

11. Method according to any of the previous claims, further comprising: k) use of the hydrogen collected in one or both of step c) and step g) for the production of ammonia or as an additive in an anaerobic digestion process for increasing the production of methane.

12. Method according to any of the previous claims, wherein the MSW has a dry matter content above 20%, such as 20 - 50%.

13. Method according to any of the previous claims, wherein the pH of the water in step a) is between 5.5 and 6.

14. Method according to any of the previous claims wherein the temperature in step b) is between 30°C and 65°C.

15. Method according to any of the previous claims wherein said water in in step a) is selected from process water and water from external sources and any combinations thereof.

16. Method according to any of the previous claims wherein said water in step a) comprises reject water from an anaerobic digestion process and/or water from other sources such as process water other than reject water, water from external sources, and any combinations thereof.