EP2771474A1

EP2771474A1 - Method for anaerobic fermentation and biogas production

Info

Publication number: EP2771474A1
Application number: EP12790786.3A
Authority: EP
Inventors: Frank Ulrik ROSAGER; Anders Peter JENSEN; Stefan Borre-Gude
Original assignee: XERGI NIX TECHNOLOGY AS
Current assignee: NGF Nature Energy Biogas AS
Priority date: 2011-10-28
Filing date: 2012-10-28
Publication date: 2014-09-03
Also published as: WO2013060338A1

Abstract

The present invention relates to a method for biomass processing, anaerobic fermentation of the processed biomass, and the production biogas. In particular, the invention relates to a system and method for generating biogas from anaerobic fermentation of processed organic material that comprises solid and liquid parts.

Description

ņigure Summary of the Invention

The present invention facilitates efficient biomass processing and an increased production of renewable energy from processing and anaerobic fermentation of a wide variety of organic materials.

Many types of organic materials have a high energy potential which can be exploited by processing the organic material. One form of processing an organic material is by performing an anaerobic fermentation resulting in the production of biogas. This process represents a conversion of an energy potential to a readily usable energy source.

Pre-treatment of biomasses - including lime pressure cooking - and partial stripping of ammonia N prior to performing a biogas fermentation, is not always sufficient to preclude an undesirable inhibition of biogas producing bacteria by ammonia released from organic bound N not stripped during the pre-treatment step.

Hence, there is a need for novel and innovative methods for removal of organic bound N from different biomasses. In particular, there is a need for improving the removal of ammonia N from AD (anaerobic digestion) processes in which e.g. poultry manures and other organic materials having a high organic bound N content are used for biogas production.

According to one aspect of the invention there is provided a plant for generating biogas from an anaerobic fermentation of processed organic material, including solid and liquid parts, is disclosed. The plant includes i) a lime pressure cooker for hydrolysing a first organic material comprising one or more sources of nitrogen; ii) an absorption unit for absorbing and

condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker when said first organic material is subjected to lime pressure cooking; iii) a mixing tank for mixing lime pressure cooked organic material with a further organic material prior to diverting the mixed, organic materials to one or more fermenters; iv) one or more fermenters for

anaerobically fermenting said organic materials diverted to said one or more fermenters from said mixing tank, wherein said fermentation results in the generation of biogas; v) a separation unit for separating organic materials diverted to the separation unit from said one or more fermenters, wherein said separation of said organic materials results in the generation of a solid organic material fraction and a liquid fraction comprising one or more sources of nitrogen; and vi) a means for diverting said liquid fraction comprising one or more sources of nitrogen to the lime pressure cooker, wherein said liquid fraction is mixed in the lime pressure cooker with first organic material comprising one or more sources of nitrogen. According to another aspect of the invention there is provided a method for generating biogas from an anaerobic fermentation of processed organic material, including solid and liquid parts, is disclosed. The method includes i) diverting a first organic material comprising one or more sources of nitrogen to a lime pressure cooker; ii) subjecting said first organic material to a lime pressure cooking step resulting in at least partly hydrolysing said first organic material comprising one or more sources of nitrogen, wherein said lime pressure cooking step results in the formation of ammonia fluids; iii) diverting said ammonia fluids formed in the lime pressure cooker to an absorption unit; iv) absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker; v) diverting lime pressure cooked organic material from said lime pressure cooker to a mixing tank; vi) mixing lime pressure cooked organic material with a further organic material in the mixing tank; vii) diverting the mixed, organic materials to one or more fermenters; viii) fermenting under anaerobic fermentation conditions said organic materials in said one or more fermenters, ix) generating biogas from said anaerobic fermentation of said organic materials; x) diverting said organic materials from said one or more fermenters to a separation unit; xi) separating organic materials into a solid organic material fraction and a liquid fraction comprising one or more sources of nitrogen; and xii) diverting said liquid fraction comprising one or more sources of nitrogen to the lime pressure cooker, xiii) optionally mixing said liquid fraction comprising one or more sources of nitrogen with a first organic material and/or a further organic material comprising one or more sources of nitrogen, and xiv) stripping ammonia from said liquid fraction, or from the mixture of liquid fraction and said first and/or further organic material obtained in step xiii).

Brief Description of the Accompanying Figures

The embodiments of the invention, together with its advantages, may be best understood from the following detailed description taken in conjunction with the accompanying figures in which:

Figure 1 illustrates a biogas plant where a lime pressure cooker is used to hydrolyze organic material prior to anaerobic fermentation and biogas production, according to an embodiment of the invention. Figure 2 illustrates a pre-treatment system according to another embodiment of the invention.

Figure 3 illustrates an energy system according to an embodiment of the invention.

Figure 4 illustrates a mode of operation of the biogas plant according to an embodiment of the invention. Figure 5 illustrates a mode of operation of the biogas plant according to another embodiment of the invention.

Figure 6 illustrates a mode of operation of the biogas plant according to yet another embodiment of the invention.

Figure 7A-7C illustrates a biogas reactor fitted with a service facility allowing access to interior of the biogas reactor according to an embodiment of the invention. Figure 8A illustrates methane yield from batch bottles with untreated Chicken Litter and Nix treated Chicken Litter with or without an extended thermal treatment at 70 °C according to an embodiment of the invention, Figure 8B illustrates which illustrates methane yield from batch bottles with untreated Chicken Litter and Nix treated Chicken Litter in the absence of saturated lime according to an embodiment of the invention, and Figure 8C illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated Chicken Litter according to an embodiment of the invention.

Figure 9A illustrates methane yield from batch bottles with untreated Cow Deep Litter and Nix treated Cow Deep Litter with or without an extended thermal treatment at 70 °C. Methane yields have been normalized according to an internal standard; Figure 9B illustrates effect of saturated lime in Nix treatment Cow Deep Litter and Nix treated in the absence of Ca(OH)₂ according to an embodiment of the invention, and Figure 9C illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated Chicken Litter according to an embodiment of the invention.

Figure 10A illustrates methane yield from batch bottles with untreated and NiX treated pig fibres with or without an extended thermal treatment at 70 °C according to an embodiment of the invention, Figure 10B illustrates methane yield from batch bottles with untreated pig manure and Nix treated pig manure in the absence of saturated lime according to an embodiment of the invention, and Figure 10C illustrates graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated pig fibres according to an embodiment of the invention.

Figure 1 1 A illustrates methane yield from batch bottles with treated and untreated AD fibre according to an embodiment of the invention; and Figure 1 1 B illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated AD fibre according to an embodiment of the invention. Figure 12A illustrates methane yield from batch bottles with treated and untreated hen litter according to an embodiment of the invention, Figure 12b illustrates a representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated hen litter according to an embodiment of the invention.

Figure 13A illustrates a process configuration during thermophile operation without NiX treatment according to an embodiment of the invention; and Figure 13B illustrates a process configuration during thermophile operation with NiX treatment according to an embodiment of the invention.

Figure 14 illustrates a process configuration during phase 2 - mesophile operation with recirculation and NiX treatment according to an embodiment of the invention.

Figure 15 illustrates effect of NiX treatment on NH₄ ⁺-N concentration in the digester according to an embodiment of the invention.

Figure 16 illustrates removal of NH₄ ⁺-N with NiX treatment vs. loss caused by high rate of pressure reduction according to an embodiment of the invention. Figure 17 illustrates the nitrogen flow in the system is expressed in g of nitrogen per day according to an embodiment of the invention.

Figure 18 illustrates nitrogen flow around the NiX treatment unit (data are in g of nitrogen at a specific NiX treatment according to an embodiment of the invention.

Figure 19 illustrates Hydraulic Retention Time (HRT) and Solid Retention Time (SRT) according to an embodiment of the invention.

Figure 20 illustrates methane yield during phase 2 (mesophile operation) according to an embodiment of the invention.

Figure 21 illustrates feed flow rate, biogas yield (main vertical axis), NH₄ ⁺-N concentration in the digester (secondary vertical axis) according to an embodiment of the invention. Figure 22 illustrates Total Solids (TS) content of the digester and of the liquid after separation with centrifuge according to an embodiment of the invention. Figure 23 illustrates the mass balance around the NiX treatment unit for a specific treatment according to an embodiment of the invention. Figure 24 illustrates Nitrogen flow in the system where the data represents flow rates in g of nitrogen per day, according to an embodiment of the invention;

Figure 25 illustrates one embodiment of the processes and the plant for bioenergy generation according to the present invention. Organic biomass and re-circulated liquid from the anaerobic digester (biogas fermenter(s)) are diverted to a lime pressure cooking step (NiX treatment) and lime (CaO / Ca(OH)₂ is added to create - at a temperature of more than 100°C and a pressure of more than 1 bar - a pH suitable for converting ammonium N (NH₄ ⁺) to gaseous ammonia (NH₃) - which can be stripped, collected e.g. in an ammonia scrubber, and converted into ammonium sulphate - which can subsequently be used as a fertilizer (cf. Figs. 1 , 2 and 4). Following ammonia stripping in the lime pressure cooker, the lime pressure cooked biomass is diverted to a buffer tank (as also illustrated in Figs. 1 and 2 (i.e. "dosing module") - prior to being diverted into one, or a series of at least two, connected anaerobe digesters for the production of biogas (cf. "primary digester" and "secondary digester" as illustrated in Fig. 4). Additional biomass may be mixed with the lime pressure cooked biomass in the buffer tank (as also illustrated in Figs. 1 and 4). A conversion of organic nitrogen to ammonium N (NH₄ ⁺) may take place in the buffer tank - and the generated ammonium N (NH₄ ⁺) may subsequently be converted to gaseous ammonia (NH₃) under suitable conditions. Following anaerobic digestion and biogas production, a separation of fiber (i.e. solid fraction, "spent biomass", also known as the digestate) and liquid phase takes place and the liquid fraction is diverted (i.e. re-circulated) back to the lime pressure cooker, as also illustrated in Fig. 4.

Figure 26 illustrates a plant and a process as described above for Fig. 25 - with the addition that a pre-incubation step is being inserted prior to Nix treatment. Under suitable conditions, the biomass is being converted into more basic constituents, i.e. fx peptides, saccharides and fatty acids, chemically and/or biologically under anaerobic and/or aerobic conditions, and organic N - i.e. organic nitrogen bound in and forming part of the biomass, is subsequently released as or further converted into ammonium N (NH₄ ⁺) - which in turn - again under suitable conditions can be converted into gaseous ammonia (NH₃). Depending on the retention time and reactions conditions in the pre-incubation tank and in the buffer tank, respectively, these to supplementary pre-treatment steps may aid significantly the conversion of organic nitrogen to inorganic nitrogen - i.e. what is also termed N-mineralization. Figure 27 illustrates the addition of lime to the pre-incubation tank in order to increase the conversion of organic N to ammonium N (NH₄ ⁺) - and to shift the equilibrium between ammonium N (NH₄ ⁺) and gaseous ammonia (NH₃) in the direction of gaseous ammonia (NH₃) - with a view to stripping gaseous ammonia (NH₃) also during the pre-incubation. Additional like may be added during the lime pressure cooking step (not shown).

Figure 28 illustrates an embodiment in which ammonia is also stripped from the buffer tank following NiX treatment - in addition to being stripped during the lime pressure cooking step (i.e. nitrogen extraction - NiX treatment).

Figure 29 illustrates an embodiment in which gaseous ammonia (NH₃) is stripped from each and all of the pre-incubation tank, the lime pressure cooker (NiX treatment) and the buffer tank. It is illustrated that lime (CaO) is added to the pre-incubation tank, but additional lime may be added to the lime pressure cooker, if needed. The operational conditions as well as the chemical reaction conditions are different for the pre-incubation tank and for the lime pressure cooker.

Figure 30 illustrates TAN generated during pre-incubation as function of %TS. %TS varies from 15 % to 47% (hen manure).

Figure 31 illustrates the development of TAN and a comparison between water and AD liquid used as re-circulated suspension liquid for hen litter. Darker bars: TAN concentration at day 0; Lighter bars: TAN concentration after 17 days incubation according to an embodiment of the invention.

Figure 32 illustrates development of TAN for seven TS levels according to an embodiment of the invention.

Figure 33A illustrates development of TAN (TS = 20%. RT = Room temperature in the research facility), and Figure 33B illustrates development of TAN at physiological

temperatures. TS = 20%, oxygen level normal according to an embodiment of the invention.

Figure 34 illustrates effect of seeding on the mineralization process. TAN from the seeding material has been subtracted according to an embodiment of the invention.

Figure 35 illustrates content of uric acid bound nitrogen and protein bound nitrogen before and after mineralization according to an embodiment of the invention. Figure 36A illustrates dry matter loss in hen litter during incubation, and Figure 36B illustrates Total solids loss in hen litter during incubation according to an embodiment of the invention.

Figure 37 illustrates Net VFA production during incubation according to an embodiment of the invention.

Figure 38 illustrates specific methane production in the incubated samples according to an embodiment of the invention. Figure 39 illustrates contribution of nitrogen mineralization to VS loss in hen litter under aerobic and anaerobic conditions according to an embodiment of the invention.

Figure 40A illustrates TS content of incubated samples, Figure 40B illustrates VS content of incubated samples, wherein darker bars represent Day 0 and lighter bars represent Day 22 according to an embodiment of the invention.

Figure 41 illustrates development of pH and TAN during incubation with chicken litter inoculated with pre-tank material (Figure 41 A), with AD liquid - 'neutral' (Figure 41 B), with AD liquid - alkaline (Figure 41 C), with AD liquid - acidic (Figure 41 D), where cTAN data depicted on the right Y-axis and pH on the left Y-axis, according to an embodiment of the invention.

Figure 42 illustrates TAN, TKN and OrgN levels according to an embodiment of the invention, where Figure 42A illustrates TAN levels before and after incubation, where the increase after 22 days is shown above the lighter bar, Figure 42B illustrates TKN levels before and after incubation, where the decrease after 22 days is shows above the lighter bar, Figure 42C illustrates OrgN levels before and after incubation, where the decrease after 22 days is shown above the lighter bar, Figure 42D illustrates OrgN levels before and after incubation for chicken litter alone, where the decrease after 22 days is shown above the lighter bar, Figure 42E illustrates TAN levels before and after incubation for chicken alone, where the increase after 22 days is shown above the lighter bar.

Figure 43 illustrates uric acid levels before and after incubation, where the decrease after 22 days is shown above the lighter bar, according to an embodiment of the invention. Figure 44 illustrates VFA levels according to an embodiment of the invention, where Figure 44A illustrates VFA levels in substrates, Figure 44B illustrates VFA levels in samples, Figure 44C illustrates change in VFAs during incubation as seen by subtracting substrate VFA levels from the sample VFA levels, and Figure 44D illustrates total VFA before and after incubation. Figure 45 illustrates TAN in substrate materials according to an embodiment of the invention.

Figure 46 illustrates development of VFA levels in substrates from beginning to the end of the experiment included in Example 4 according to an embodiment of the invention.

Detailed description of the Invention

There are four key biological and chemical stages of an anaerobic fermentation: Hydrolysis;

acidogenesis; acetogenesis; and methanogenesis. In order for bacteria under anaerobic conditions to exploit the energy potential of the organic materials used as substrates,

macromolecules present in the organic materials must initially be broken down into their smaller constituent parts - unless this has already occurred at an earlier processing step, such as e.g. lime pressure cooking. Lime pressure cooking is an example of a pre-treatment processing step resulting in nitrogen extraction - a technical term often abbreviated "NiX" (cf. Figures 25 to 29). Lime pressure cooking is in principle conducted at temperatures above 100°C and at a pressure of above 1 bar. Lime pressure cooking results in the conversion of inorganic ammonia N (MH₄ ⁺) to gaseous ammonia (NH₃) as illustrated in Figures 25 to 29. The term pre-treatment signifies that this processing step occurs prior to the step of anaerobic digestion and the production of biogas.

An additional pre-treatment processing step, which occurs prior to the step of lime pressure cooking, is that of pre-incubation of the biomass to be lime pressure cooked and later subjected to anaerobic digestion. The pre-incubation step also serves to improve biogas production by converting a biomass into smaller constituent parts by chemical hydrolysis, or by biological conversion, using one or more microbial populations and/or one or more enzymes.

The initial process of breaking down macromolecular structures of a substrate in an anaerobic fermentation involves a hydrolysis of macromolecular structures, such as proteins, carbohydrates and organic acids. The constituent parts, or monomers, such as amino acid residues, sugars and fatty acids can be readily metabolized by microbial organisms. Accordingly, hydrolysis of macromolecular components of organic materials represents an initial step in an anaerobic fermentation. Anaerobic fermentations are sensitive to high levels of ammonia as the ammonia inhibits the bacteria which are responsible for the methanogenesis. Hence, when the bacteria are inhibited by high levels of ammonia, reduced amounts of biogas are being produced. In order to prevent ammonia inhibition, or to reduce the problem of ammonia inhibition during an anaerobic fermentation resulting in a reduced production of biogas, it is necessary to remove significant parts of the nitrogen which is present in an organic biomass.

The pre-incubation steps of the methods of the present invention are aimed at increasing the removal of nitrogen sources from an organic biomass. In combination with the subsequent lime pressure cooking step, during which ammonia is stripped, the pre-incubation step serves to effectively prevent ammonia inhibition during anaerobic fermentation and biogas production.

Nitrogen can be present in an organic biomass either as organic nitrogen - fx nitrogen present in proteins and organic acids - or as inorganic nitrogen - in the form of ammonium. In order to strip gaseous ammonia from the lime pressure cooker, the organic bound nitrogen will have to initially be converted into inorganic ammonium, which is then stripped in the form gaseous ammonia. This is performed under suitable conditions - primarily involving a high pH and an increased temperature.

However, it has been observed that organic nitrogen cannot - or only to a very limited extent - be converted into inorganic nitrogen during a lime pressure cooking step. Hence, the ammonium available for stripping in the lime pressure cooker is determined by the amount of inorganic nitrogen which is entered into the lime pressure cooker for ammonia stripping.

Essentially no conversion of organic nitrogen into inorganic nitrogen is expected to take place during lime pressure cooking.

This poses a significant challenge to anaerobic biogas fermentations - as the organic material will contain significant amounts of organic nitrogen - which is not converted into removable ammonia during lime pressure cooking.

In order to increase the amount of inorganic nitrogen available for ammonia stripping in the lime pressure cooking step, a further pre-treatment step in the form of a pre-incubation of an organic biomass is introduced. The pre-incubation step comprises one or both of a pre- fermentation step and/or a chemical hydrolysis step. Pre-fermentation can result in a hydrolysis and/or further break-down of e.g. proteins, carbohydrates and other

macromolecules present in an organic biomass. Hence, hydrolysis of macromolecules can also be obtained by microbial means - and not exclusively by chemical means. According to one presently preferred hypothesis, the pre-incubation step comprises a microbial fermentation resulting in the decomposition of organic macromolecules present in the organic material which is to be subsequently subjected to anaerobic fermentation and biogas fermentation.

The microbial fermentation and/or hydrolysis of macromolecules present in an organic material which takes place during the pre-incubation step will thus contribute to an increased N- mineralization process during pre-incubation.

Accordingly, the conversion of organic N to inorganic N which takes place at the pre-incubation step is at least facilitated by biological and enzymatic processes catalyzed by microbial organisms present in the biomass comprising the organic bound N. Without being bound by theory, and according to one presently preferred hypothesis, enzymes synthesized by microbial organisms residing in the organic material to be processed are largely responsible for the conversion of organic N to inorganic N during pre-incubation - and such enzymes will not be allowed to exert their action during a lime pressure cooking step - which is a thermo-chemical process. Hence, it is likely that this represents one explanation as to why organic bound N can be converted into inorganic N when being subjected to enzymatic action during pre-incubation, whereas no, or only a very modest part, of the organic bound N is converted to inorganic N during the thermo-chemical lime pressure cooking step under conditions, when no enzyme is active. It has been observed that the microbial organisms capable of contributing to the conversion of organic N to inorganic N during the pre-incubation step are apparently not involved in biogas production - as essentially no methanogenesis takes place during pre-incubation.

Accordingly, in view of the above observation, the methods of the present invention can be characterized as a two-step fermentation method in which the individual steps are separated by a thermo-chemical processing step - i.e. lime pressure cooking - performed at an elevated temperature and pressure, and under alkaline pH conditions.

The first fermentation step - preceding the thermo-chemical processing step - is a facultative anaerobic fermentation reaction during which, in one embodiment of the present invention, essentially no biogas is produced - as the organic material can be expected to undergo initial fermentation stages during the first fermentation step, but not, or only to a limited extent, methanogenesis. Methanogenesis constitutes one of the latter stages of an anaerobic fermentation - i.e. a stage which is reached only after prior stages, such as e.g. acidogenesis and acetogenesis.

The second fermentation step, which takes place after the thermo-chemical processing step - is a strictly anaerobic methanogenesis. Accordingly, the second fermentation step is aimed at producing biogas by using the pre-fermented and thermo-chemically treated organic material as a substrate.

Accordingly, there is provided in accordance with the present invention a dual fermentation method for generating biogas from anaerobic fermentation of processed organic material comprising solid and liquid parts, said method comprising the steps of i) subjecting an organic material comprising one or more sources of nitrogen to a facultative anaerobic fermentation resulting in at least partial hydrolysis of the organic material and at least partial conversion of organic N (nitrogen) present in the organic material to inorganic N, and wherein essentially no biogas is produced; ii) subjecting the organic material fermented in step i) to lime pressure cooking, wherein the lime-pressure cooking results in a further hydrolysis of said organic material, and wherein said lime pressure cooking step results in the conversion of inorganic N to ammonia fluids; iii) subjecting the organic material fermented in step i) and subjected to lime

pressure cooking in step ii) to a strictly anaerobic fermentation resulting in the production of biogas.

Accordingly, when the first microbial fermentation reaction results in a conversion of organic bound N to inorganic N - an increased amount of biogas can be produced in the second microbial fermentation reaction (i.e. the methanogenesis) - as more inorganic N enters the lime pressure cooking step - where the inorganic N is converted to gaseous ammonia which is stripped. When more nitrogen is stripped during lime pressure cooking, less nitrogen will remain and cause potential problems with ammonia inhibition during the subsequent methanogenesis.

In an exemplary embodiment, provided that the pre-incubation step achieves a convertion of 75 % of the orgN to TAN, then, for a typical poultry litter substrate having 16 g OrgN/kg and 6 g TAN/kg, this would result in increasing the TAN-pool by an additional 75 % of 16 g/kg = 12 g/kg, thereby increasing the removable Nitrogen to 18 g/kg. With Nitrogen stripping of at least 65 % of the TAN-pool during the NiX-treatment, without pre-incubation, 65 % of 6 g TAN/kg = 3.9 g TAN/kg would be removed by stripping, whereas with the pre-incubation step present, the stripped amount increases to 65 % of 18 g TAN/kg = 1 1 .7 g TAN/kg. The overall nitrogen removal per kg of substrate added is therefore in this scenario increased from 3.9 g TAN/kg to 1 1 .7 g TAN/kg. The pre-incubation thus reduces overall the TAN-concentration in the anaerobic digester (biogas fermenter).

The facultative anaerobic bacteria according to the methods of the present invention have a temperature optimum in the range of from approx. 30°C to 37°C. Accordingly, the bacteria can be termed "mesophilic" because of this temperature optimum.

It has also been observed that seeding of the pre-incubation is important for the pre- fermentation which takes place - and approx. 10 to 20 % (w/w) of the contents of a pre- fermentation tank is preferably retained and re-circularized to the next batch pre-fermentation. Accordingly, several interconnected pre-fermenters can be present - so that one can seed approx. 10 to 20 % (w/w) of the contents of one pre-fermenter into a connected pre-fermenter.

Any suitable number, such as fx 2, 3, 4, 5 or 6 interconnected pre-fermentation tanks can be operated as individual, but connected pre-fermentation "batch" fermentations at different stages of the pre-fermentation can be present. Each "batch" pre-fermenter is connected to the lime-pressure cooker and pre-fermented biomass can be diverted from any pre-fermenter to the lime pressure cooker. In this way, one will be able to operate the methods of the present invention as a continuous fermentation process for pre-fermentation and biogas production.

The maximum TS (total dry matter) content of the biomass subjected to pre-fermentation is preferably approx. 30%, such as at the most 25 % (w/w).

The pH optimum for the pre-fermentation is broad and ranges from a pH value of approx. 6,5 to a pH value of approx. 8,5. pH values following a pre-fermentation are preferably in the range of from approx. 6,0 to approx. 7,5.

The duration of the pre-fermentation will depend on the reaction conditions, including temperature, pH, total dry matter content, and the like. It is preferred that the pre-fermentation is at the most approx. 96 hours, such as at the most 72 hours, for example at the most 60 hours, such as at the most 50 hours, for example 40 hours. However, both longer and shorter durations can be employed. It is possible to obtain, following a pre-fermentation step according to the present invention, a conversion of organic bound N to inorganic N of up to 70 to 80 %. However, depending on the reaction conditions and the content of organic N, lower values can also be obtained - such as for example conversion rates of approx. 35%, approx. 40%, approx. 45%, approx. 50%, approx. 55%, approx. 60%, and approx. 65%. This will also depend on the reaction conditions employed for the pre-fermentation.

Importantly, in one embodiment, at least 80%, such as at least 85%, for example at least 90%, such as at least 95% or more of all nitrogen containing organic acids, such as e.g. uric acid, are converted to ammonia N during a pre-fermentation step operated under the conditions disclosed e.g. herein above.

It is preferred that a minimum of 30%, such as a minimum of 40%, for example a minimum of 50%, such as a minimum of 60% of the organic bound nitrogen originating from protein is converted into inorganic N during a pre-fermentation.

The anaerobic fermentation can in principle be conducted either i) as a pre-incubation step, prior to a nitrogen extraction step, and/or ii) in the form of an anaerobic fermentation and biogas production (methanogenesis, cf. above) conducted following a nitrogen extraction step. This is illustrated in figures 26 to 29. Depending of the retention time of the nitrogen extracted biomass in the buffer tank illustrated in Figures 26 to 29, and the reaction conditions, it is in principle also possible to regard as an anaerobic fermentation the process which takes place while the nitrogen extracted biomass resides in the buffer tank. The aim of the buffer tank of to adjust the

temperature and the pH of the nitrogen extracted (i.e. lime pressure cooked) biomass prior to entry of this biomass into the biogas producing plant.

Acetate and hydrogen produced in the first stages of an anaerobic fermentation can be used directly by methanogens. Other molecules, such as volatile fatty acids (VFAs) with a chain length that is greater than that of acetate, must first be catabolised into compounds that can be directly metabolised by methanogens.

The biological process of acidogenesis is one wherein there is further breakdown of the remaining components by acidogenic (fermentative) bacteria. Here, VFAs are created along with ammonia, carbon dioxide, and hydrogen sulphide, as well as other by-products.

The third stage of an anaerobic fermentation is acetogenesis. Here, simple molecules created through the acidogenesis phase are further digested by acetogens to produce largely acetic acid as well as carbon dioxide and hydrogen. The final stage of an anaerobic fermentation is that of methanogenesis. Methanogens metabolise intermediate compounds formed during the preceding stages of the anaerobic fermentation, and these compounds are metabolised into methane, carbon dioxide, and water. The afore-mentioned compounds are the major constituents of a biogas.

Methanogenesis is sensitive to both high and low pHs - and methanogenesis generally occurs between pH 6.5 and pH 8. Remaining, non-digestible organic material that the microbes present in the biogas fermenter cannot metabolise, along with any dead bacterial remains, constitutes what is termed the digestate from the fermentation.

Apart from having a high energy potential, many organic materials also have a high content of nitrogen (N) - in the form of inorganic N (calculated as TAN - total amount of N (NH₃ and NH₄ ⁺)) and organic N e.g. present in proteins, uric acid and other organic sources of N.

When such organic materials are used as substrates for converting organic materials into bio energy in a bio energy plant, in particular biogas in a biogas plant, organic N and / or protein will gradually be converted to ammonia e.g. during an anaerobic fermentation resulting in the production of biogas.

The formation of ammonia in a bio energy plant - especially at high levels - represents a problem as many biogas producing bacteria are sensitive to high levels of ammonia - and high ammonia levels in a biogas fermenter will thus reduce or inhibit the production of methane.

Ultimately, the formation of high levels of ammonia, i.e. above a certain threshold level, cf. below, will kill biogas producing bacteria and inhibit any further biogas formation. The inhibitory levels of ammonia in a biogas fermenter depend on the conditions used. Under thermophilic fermentation conditions, approx. 3,0 to 4,2 kg ammonia per ton of biomass is considered inhibitory, while under mesophilic fermentation conditions the figure is approx. 5,0 to 7,0 kg ammonia per ton of biomass - depending of the pH value in the digester. The biogas generating fermentation process can be expected to be completely inhibited at ammonia levels of approx. 7,0 kg to 7,5 kg ammonia per ton of biomass. Accordingly, at this high level of ammonia, fermentation of organic materials by biogas producing bacteria no longer takes place. It is ammonia (NH₃) which is inhibitory to the biogas production. The equilibrium between ammonia and ammonium (NH₄ ⁺) salts will depend on e.g. pH and temperature. The higher the pH and the higher the temperature, the more the equilibrium is shifted towards the ammonia.

Stripping of ammonia will result in a decreased pH value in the fermenter and it is preferred that the pH value of an anaerobic biogas fermentation shall be below a pH value of approx. 8,5.

The above-cited ammonia inhibition threshold values are generally taken into consideration when operating commercial biogas plants using conventional organic materials as substrates for the biogas producing bacteria. Many such plants are operated according to a two step strategy initially adopting thermophile digestion conditions in a first fermentation step and mesophile digestion conditions in a separate and subsequent, second fermentation step. The conversion of organic N to ammonia N progresses during an anaerobic fermentation process - i.e. during the process of generating biogas by anaerobic fermentation - and a conversion of as much as approx. 50 % to 70 % of organic N to ammonia N can be expected in accordance with the present invention. Particular challenges arise when it is desirable to process organic materials having a particularly high N content - as inhibitory levels of ammonia during biogas fermentation can be expected to occur relatively early on in the fermentation process due to the high levels of organic N and protein in the organic material to be processed. When employing e.g. poultry manure it is possible to strip up to about 65 % of the ammonia N present in this form of manure. Approximately 30% of all N in poultry manure is in the form of ammonium N. Poultry manure is rich in uric acid and uric acid is not converted - or only converted very inefficiently - to ammonia N during e.g. lime pressure cooking. Hence, there is also a need for devising a strategy for increasing the conversion of organic dry matter, such as uric acid, into ammonium N.

Accordingly, there is a general need for improved methods for organic material processing involving an increased conversion of both total solids (TS) and volatile solids (VS) into ammonium N, as well as securing a biogas production capable of solving the problem of ammonia inhibition of biogas producing bacteria during the production of biogas in a bio energy plant - in particular when more complex types of organic waste products are used - such as e.g. solid manures e.g. from poultry, which have a high organic bound N content. The present invention thus also provides a technical solution to the problem of how to improve biogas production in a commercial biogas plant. The solution involves novel and inventive methods for reducing organic N contents in an organic material further comprising at least one carbon (C) source during or after the progress of performing an anaerobic fermentation resulting in the production of biogas.

The anaerobic fermentation resulting in the production of biogas may be preceded by one or more initial processing steps aimed at stripping ammonia N from the organic material prior to the biogas production.

One such initial processing step is a pre-incubation step - performed prior to lime pressure cooking - wherein organic N forming part of the biomass to be processed is converted to inorganic N by chemical hydrolysis or by microbial action. The pre-incubation step takes place in a preincubation tank, as illustrated e.g. in Figs. 26 to 29.

Another initial processing step is that of lime pressure cooking - a step which subjects the optionally pre-incubated organic material, cf. herein above, to an initial hydrolysis under alkaline conditions at an elevated pressure - i.e. more than 1 bar - and at a temperature of more than 100°C.

Prior to anaerobic digestion and biogas production, yet another pre-treatment step may be used for increasing the conversion of organic bound N in an organic biomass. The lime pressure cooked organic material can be diverted to a buffer tank and the retention time in this buffer tank determines the result of this pre-treatment step. The pH is adjusted to a pH value of less than 8,5, such as less than 8,0, for example less than 7,0, and preferably not less than 6,0. A further conversion of organic bound N in an organic biomass can be allowed to occur in the buffer tank and - optionally - ammonia can also be stripped from the buffer tank.

Ammonia N stripped from the organic material e.g. under a lime pressure cooking step - and/or during pre-incubation, and/or during buffer tank treatment - can initially be diverted to a stripper and sanitation tank - or alternatively diverted directly to an absorption column for absorption of the stripped ammonia N. The stripper and sanitation tank will also be connected to an absorption column for absorption of the stripped ammonia N. According to one presently preferred hypothesis, no organic bound N is converted to ammonia during the lime pressure cooking step. However, organic bound N is converted to ammonia both during the pre-incubation step and during the subsequent anaerobic fermentation resulting in the production of biogas - as illustrated e.g. in Figs. 26 to 29. Ammonia N stripped from the organic material prior to, during, or after the lime pressure cooking step can be diverted to a stripper and sanitation tank for further incubation under conditions resulting in further conversion of organic N to inorganic N - or, alternatively, diverted directly to an absorption column for absorption of the stripped ammonia N. The absorption column can also be connected to the stripper and sanitation tank so that any ammonia N stripped from the stripper tank can be diverted to the absorption column.

The lime pressure cooked and, at least partly, ammonia N stripped organic material is

subsequently diverted to a biogas fermenter and subjected to anaerobic fermentation conditions resulting in the production of biogas. Preferably, the lime pressure cooked organic material is initially diverted to a mixing tank prior to being diverted to the biogas fermenter. Mixing of lime pressure cooked material with further organic materials, for which there is no need for performing a lime pressure cooking step, can take place in a mixing tank prior to diverting the mixture to the biogas fermenter for anaerobic fermentation.

According to this aspect of the present invention, anaerobically fermented organic material is separated into a solid and a liquid fraction. The liquid fraction comprising ammonia N is diverted, or re-cycled, to the lime pressure cooker for stripping of ammonia.

One principle for large scale stripping of ammonia from e.g. a biomass is to increase the pH in combination with aerating and/or heating of the biomass. Ca(OH)₂ or CaO, collectively referred to as lime, can be used to increase the pH in a lime pressure cooking step. Lime is used on an industrial scale by for instance the cement industry and is therefore cheap and readily available as a bulk ware. Other bases may also be employed, such as e.g. NaOH or KOH.

When the stripped ammonia is absorbed and an ammonia concentrate is produced, one can divert stripped ammonia to e.g. sulphuric acid present in an absorption column. Sulphuric acid is an industrial bulk ware and it is available in a technical quality appropriate for use in

absorption columns stripping ammonia from slurry and other waste waters (e.g. Sacuk et al. 1994).

It is often preferred to strip ammonia by performing a thermal and chemical hydrolysis of a biomass at temperatures of e.g. around or less than 100°C - and at a pressure of about 1 atm. Thermal and chemical hydrolysis of a biomass represents one way to increase the availability of organic material for biogas generation. Complex carbohydrates, such as e.g. cellulose, hemicelluloses and lignin, are not completely hydrolysed by such treatments. In particular, fibres from straw, maize and other crops are not made available as suitable substrates for methane formation by such treatments (Bjerre et al 1996; Schmidt and Thomsen 1998; Thomsen and Schmidt 1999; Sirohi and Rai 1998).

Accordingly, higher temperatures and a higher pressure will have to be used - and a combination of temperature and pressure will have to be selected depending on the nature of the biomaterial.

In preferred aspects, the present invention concerns methods for performing an anaerobic digestion of a biomass, such as e.g. organic materials comprising one or more of animal manures, energy crops, category 2 waste materials, and similar biomaterials.

Further exemplary biomasses capable of being used as an "input biomass" and subsequently processed in accordance with the methods of the present invention are disclosed herein below in more detail. The biomasses can comprise e.g. solid manure waste products from e.g. animal farms, poultry farms, dairies, slaughterhouses, marine fish farms, fish and meat industries as wells as energy crops and or other plants.

The input biomass or feedstock can also comprise liquid manure, dry litter, such as cattle, poultry, offal from cattle, poultry, mink, vegetable oil and glycerin, sludge, whey and the like, corn silage, fish category 2 waste, and industrial waste, including category 3 waste materials.

Biomass can also be any material that comes from plants. Some plants, like sugar cane and sugar beets, store the energy as simple sugars. These are mostly used for food. Other plants store the energy as more complex sugars, called starches. These plants include grains like corn and are also used for food.

Another type of plant matter, called cellulosic biomass, is made up of very complex sugar polymers, and is not generally used as a food source. This type of biomass is under consideration as a feedstock for bioethanol production. Specific feed stocks under

consideration include: 1 . Agricultural residues (leftover material from crops, such as the stalks, leaves, and husks of corn plants)

2. Forestry wastes (chips and sawdust from lumber mills, dead trees, and tree branches)

3. Municipal solid waste (household garbage and paper products)

4. Food processing and other industrial wastes (black liquor, a paper manufacturing by- product) 5. Energy crops (fast-growing trees and grasses) developed just for this purpose

The main components of these types of biomass are:

• Cellulose is the most common form of carbon in biomass, accounting for 40%-60% by weight of the biomass, depending on the biomass source. It is a complex sugar polymer, or polysaccharide, made from the six-carbon sugar, glucose. Its crystalline structure makes it resistant to hydrolysis, the chemical reaction that releases simple, fermentable sugars from a polysaccharide.

• Hemicellulose is also a major source of carbon in biomass, at levels of between 20% and 40% by weight. It is a complex polysaccharide made from a variety of five- and six-carbon sugars. It is relatively easy to hydrolyze into simple sugars but the sugars are difficult to ferment to ethanol.

• Lignin is a complex polymer, which provides structural integrity in plants. It makes up 10% to 24% by weight of biomass. It remains as residual material after the sugars in the biomass have been converted to ethanol. It contains a lot of energy and can be burned to produce steam and electricity for the biomass-to-ethanol process.

The percentages cited herein below are weight percentages - i.e. (weight / weight), or (mass / mass). The input biomass has in one aspect of the present invention a carbon content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a protein content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals. The input biomass has in one aspect of the present invention a fat or lipid content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a nitrogen or ammonia content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a fiber content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals. The input biomass has in one aspect of the present invention a sugar content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a polysaccharide content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a monosaccharide content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a lignin content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals. The input biomass has in one aspect of the present invention a hemicellulose content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a starch content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a sugar polymer content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a cellulose content of from 5% to 90% by weight, such as from 5% to 10%, for example from 10% to 15%, such as from 15% to 20%, for example from 20% to 25%, such as from 25% to 30%, for example from 30% to 35%, such as from 35% to 40%, for example from 40% to 45%, such as from 45% to 50%, for example from 50% to 55%, such as from 55% to 60%, for example from 60% to 65%, such as from 65% to 70%, for example from 70% to 75%, such as from 75% to 80%, for example from 80% to 85%, such as from 85% to 90% or any combination of these intervals.

The input biomass has in one aspect of the present invention a pH between 0 and 14, such as from 0 to 1 , for example from 1 to 2, such as from 2 to 3, for example from 3 to 4, such as from 4 to 5, for example from 5 to 6, such as from 6 to 7, for example from 7 to 8, such as from 8 to 9, for example from 9 to 10, such as from 10 to 1 1 , for example from 1 1 to 12, such as from 12 to 13, for example from 13 to 14, or any combination of these intervals.

Using virtually any fermentable, organic material as an "input biomass", including the "input biomasses" cited herein above, the methods of the present invention are capable of producing increased amounts of renewable energy while at the same time refining several nutrients comprised in the digested biomass to fertilizers of commercial quality.

In one embodiment, ammonia stripping results in lowering of the ammonia concentration by more than 10%, such by more than 20%, such as by more than 30%, such as by more than 40%, such as by more than 50%, such as by more than 60%, such as by more than 70%, such as by more than 80%, such as by more than 90%, such as by more than 95% or such as more than 99%.

The level of ammonia and/or nitrogen can be measured before and after the ammonia stripping step and the lowering of the ammonia concentration can be determined.

The ammonia stripping can result in an ammonium concentration of less than 50 g dm^"3, such as less than 40 g dm^"3, such as less than 30 g dm^"3, such as less than 20 g dm^"3, such as less than 15 g dm^"3,such as less than 10 g dm^"3,such as less than 8 g dm^"3, such as less than 6 g dm^"3, such as less than 2 g dm^"3, such as less than 1 g dm^"3, such as less than 0.5 g dm^"3,or such as less than 0.1 g dm^"3. Anaerobic digestion as used herein shall denote any breakdown of organic matter by bacteria in the absence of oxygen. The terms anaerobic digestion and anaerobic fermentation are used interchangeably herein.

There is provided, in accordance with this aspect of the invention, a method for generating biogas from an anaerobic fermentation of processed organic material comprising solid and liquid parts, said method comprising the steps of i) diverting a first organic material comprising one or more sources of nitrogen to a lime pressure cooker; ii) subjecting said first organic material to a lime pressure cooking step resulting in at least partly hydrolysing said first organic material comprising one or more sources of nitrogen, wherein said lime pressure cooking step results in the formation of ammonia fluids; iii) diverting said ammonia fluids formed in the lime pressure cooker to an

absorption unit; iv) absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker; v) diverting lime pressure cooked organic material from said lime pressure cooker to a mixing tank; vi) mixing lime pressure cooked organic material with a further organic material in the mixing tank; vii) diverting the mixed, organic materials to one or more fermenters; viii) fermenting under anaerobic fermentation conditions said organic materials in said one or more fermenters, ix) generating biogas from said anaerobic fermentation of said organic materials; x) diverting said organic materials from said one or more fermenters to a

separation unit; xi) separating organic materials into a solid organic material fraction and a liquid fraction comprising one or more sources of nitrogen; and xii) diverting said liquid fraction comprising one or more sources of nitrogen to the lime pressure cooker, xiii) optionally mixing said liquid fraction comprising one or more sources of nitrogen with a first organic material and/or a further organic material comprising one or more sources of nitrogen, and xiv) stripping ammonia from said liquid fraction, or from the mixture of liquid fraction and said first and/or further organic material obtained in step xiii).

The one or more sources of nitrogen in the liquid fraction preferably comprise inorganic nitrogen sources, such as ammonium salts.

Solid organic material is preferably diverted to the lime pressure cooker from a reception station suitable for receiving solid organic material, and liquid organic material is preferably diverted to the lime pressure cooker from a reception tank suitable for receiving liquid organic material. Lime is preferably diverted to the lime pressure cooker from a lime storage tank suitable for diverting lime directly to the lime pressure cooker.

Solid and/or liquid organic materials for which there is no need for lime pressure cooking can be diverted directly to the mixing tank and mixed with lime pressure cooked organic material in the mixing tank. It is possible in one embodiment to divert ammonia fluids from the mixing tank to the absorption unit, prior to diverting said mixed organic materials stripped of ammonia from the mixing tank to one or more fermenters suitable for the production of biogas.

The mixed organic materials are preferably fermented initially in a first fermenter under a first set of fermentation conditions, and subsequently diverted to a second or further fermenter and fermented under a second or further set of fermentation conditions. Preferably, the organic materials are initially fermented under thermophile fermentation conditions and subsequently, in a separate fermentation step, the organic materials are fermented under mesophilic fermentation conditions. The biogas produced by thermophilic and/or mesophilic fermentation is preferably diverted to a gas storage facility operably connected to the one or more fermenters. Biogas as used herein denotes a renewable, gaseous fuel derived from biological materials that can be used as an energy source instead of fossil fuels, typically to replace conventional natural gas, propane, heating fuel oil, diesel fuel, or gasoline. Raw biogas is composed of a mixture of combustible gases (principally methane, but also including hydrogen and light hydrocarbons, such as e.g. carbon monoxide, ethane, etc.), and various inert gases and impurities, such as carbon dioxide and hydrogen sulfide. Methane is a combustible gas with the chemical formula CH₄ that can come from fossil or renewable processes. The present invention can be used for producing increased amounts of biogas from a wide range of organic substrates, including all types of animal manures, energy crops, crops residues and other organic waste materials, including category 2 waste materials.

The present invention is also directed to an optimized waste-to-energy process based on bio- gasification using anaerobic digestion and wet fermentation for increasing the yield of energy obtained e.g. per ton of biomass.

The above-cited method can include a subsequent slurry separation step, i.e. one or more steps resulting in the refinement of selected nutrients, such as phosphor (P) and/or potassium (K) contained in e.g. animal manures. The invention may be applied to separate the main nutrients nitrogen (N) and/or phosphorus (P) from animal manures and refine the nutrients to fertilizer products of commercial quality.

The organic material to be pre-incubated and/or lime pressure cooked can comprise a maximum of 50% solid parts, such as a maximum of 40% solid parts, for example a maximum of 30% solid parts, such as a maximum of 20% solid parts. The organic material may be in the form of a liquid fraction comprising a maximum of 10% solid parts, or the organic material to be pre-incubated or lime pressure cooked may be mixed with such a liquid fraction.

The lime pressure cooking of the organic material can be performed at a temperature of from more than l OCO to preferably less than 250 °C, at a pressure of from preferably 2 to preferably less than 20 bar, and with an addition of lime sufficient to reach a pH value of from about 9 to preferably less than 12, and with an operation time of from at least 10 minutes to preferably less than 60 minutes.

The method may include the step of adding lime (CaO) in an amount of from about 2 to preferably less than 80 g per kg dry matter organic material, such as from about 5 to preferably less than 60 g per kg dry matter. The methods of the present invention may comprise the step of diverting an organic material to a first fermenter, under a first set of fermentation conditions, and subsequently diverting said fermented, organic material to a second, or further, fermenter, and fermenting said organic material under a second, or further, set of fermentation conditions. The conditions can be thermophile fermentation conditions and/or mesophile fermentation conditions.

The method may include performing the one or more biogas fermentation step(s) at a temperature of from about ^~\ 5°C to preferably less than about 65 °C, such as at a temperature of from about 25^ to preferably less than about 55°C, for example at a temperature of from about 35 °C to preferably less than about 45 °C. The fermentation may be allowed to occur over a time of from about 5 days to preferably less than 15 days. The biogas production is achieved by bacterial anaerobic fermentation of the organic material, and the fermentation method may initially performing the biogas production in the first of two plants by anaerobic bacterial fermentation of the organic material, initially by fermentation with thermophilic bacteria in the first plant, followed by diverting the thermophilicly fermented organic material to a second plant, wherein a fermentation with mesophilic bacteria can take place.

Thermophilic reaction conditions include a reaction temperature ranging from 45°C to 75°C, such as a reaction temperature ranging from 55 °C to 60 °C, for example a reaction temperature ranging from 20°C to 45 Ό, such as from 30°C to 35°C. In one embodiment, the methods of the present invention results in a biogas production output in Nm³ per 1 ,000 tons biomass input of more than 70,000 Nm³, such as more than 80,000 Nm³ per 1 ,000 tons input, for example more than 90,000 Nm³ per 1 ,000 tons input, such as more than 100,000 Nm³ per 1 ,000 tons input, for example more than 1 10,000 Nm³ per 1 ,000 tons input, such as more than 120,000 Nm³ per 1 ,000 tons input, for example more than 130,000 Nm³ per 1 ,000 tons input, such as more than 140,000 Nm³ per 1 ,000 tons input, for example more than 150,000 Nm³ per 1 ,000 tons input, such as more than 160,000 Nm³ per 1 ,000 tons input, for example more than 170,000 Nm³ per 1 ,000 tons input, such as more than 180,000 Nm³ per 1 ,000 tons input, for example more than 190,000 Nm³ per 1 ,000 tons input, such as more than 200,000 Nm³ per 1 ,000 tons input, for example more than 250,000 Nm3 per 1 ,000 tons input, such as more than 300,000 Nm³ per 1 ,000 tons input, for example more than

400,000 Nm³ per 1 ,000 tons input, such as more than 500,000 Nm³ per 1 ,000 tons input, for example more than 1 ,000,000 Nm³ per 1 ,000 tons input, such as more than 75,000 Nm³ per 1 ,000 tons input, or for example more than 80,000 Nm³ per 1 ,000 tons input. In another embodiment, methods of the present invention results in an electricity output in KWh per 1 ,000 tons biomass input of more than 200, such as more than 220 KWh per 1 ,000 tons input, for example more than 240 KWh per 1 ,000 tons input, such as more than 260 KWh per 1 ,000 tons input, for example more than 280 KWh per 1 ,000 tons input, such as more than 300 KWh per 1 ,000 tons input, for example more than 320 KWh per 1 ,000 tons input, such as more than 340 KWh per 1 ,000 tons input, for example more than 360 KWh per 1 ,000 tons input, such as more than 380 KWh per 1 ,000 tons input, for example more than 400 KWh per 1 ,000 tons input, such as more than 450 KWh per 1 ,000 tons input, for example more than 500 KWh per 1 ,000 tons input, such as more than 600 KWh per 1 ,000 tons input, for example more than 700 KWh per 1 ,000 tons input, such as more than 800 KWh per 1 ,000 tons input, or for example more than 1 ,000 KWh per 1 ,000 tons input.

In a further embodiment, methods of the present invention results in a heat output in MWh per 1 ,000 tons biomass input of more than 200 MWh per 1 ,000 tons input, such as more than 220 MWh per 1 ,000 tons input, for example more than 240 MWh per 1 ,000 tons input, such as more than 260 MWh per 1 ,000 tons input, for example more than 280 MWh per 1 ,000 tons input, such as more than 300 MWh per 1 ,000 tons input, for example more than 320 MWh per 1 ,000 tons input, such as more than 340 MWh per 1 ,000 tons input, for example more than 360 MWh per 1 ,000 tons input, such as more than 380 MWh per 1 ,000 tons input, for example more than 400 MWh per 1 ,000 tons input, such as more than 450 MWh per 1 ,000 tons input, for example more than 500 MWh per 1 ,000 tons input, such as more than 600 MWh per 1 ,000 tons input, for example more than 700 MWh per 1 ,000 tons input, such as more than 800 MWh per 1 ,000 tons input, or for example more than 1 ,000 MWh per 1 ,000 tons input. In an even further embodiment, methods of the present invention results in a steam output in MWh per 1 ,000 tons biomass input of more than 40 MWh per 1 ,000 tons input, such as more than 50 MWh per 1 ,000 tons input, for example more than 60 MWh per 1 ,000 tons input, such as more than 70 MWh per 1 ,000 tons input, for example more than 80 MWh per 1 ,000 tons input, such as more than 90 MWh per 1 ,000 tons input, for example more than 100 MWh per 1 ,000 tons input, such as more than 105 MWh per 1 ,000 tons input, for example more than

1 10 MWh per 1 ,000 tons input, such as more than 1 15 MWh per 1 ,000 tons input, for example more than 120 MWh per 1 ,000 tons input, such as more than 125 MWh per 1 ,000 tons input, for example more than 130 MWh per 1 ,000 tons input, such as more than 150 MWh per 1 ,000 tons input, for example more than 200 MWh per 1 ,000 tons input, such as more than 300 MWh per 1 ,000 tons input, or for example more than 500 MWh per 1 ,000 tons input. Bioenerqy plant for producing bioenerqy - including biogas

In another aspect of the present invention there is provided a plant for generating biogas from an anaerobic fermentation of processed organic material comprising solid and liquid parts, said plant comprising

i) a lime pressure cooker for hydrolysing a first organic material comprising one or more sources of nitrogen; ii) an absorption unit for absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker when said first organic material is subjected to lime pressure cooking; iii) a mixing tank for mixing lime pressure cooked organic material with a further organic material prior to diverting the mixed, organic materials to one or more fermenters; iv) one or more fermenters for anaerobically fermenting said organic materials diverted to said one or more fermenters from said mixing tank, wherein said fermentation results in the generation of biogas; v) a separation unit for separating organic materials diverted to the separation unit from said one or more fermenters, wherein said separation of said organic materials results in the generation of a solid organic material fraction and a liquid fraction comprising one or more sources of nitrogen; and vi) means for diverting said liquid fraction comprising one or more sources of

nitrogen to the lime pressure cooker, wherein said liquid fraction is mixed in the lime pressure cooker with first organic material comprising one or more sources of nitrogen.

The lime pressure cooker for hydrolysing said first organic material comprising one or more sources of nitrogen is preferably operably connected to a reception station suitable for receiving solid organic material and/or to a reception tank suitable for receiving liquid organic material. The lime pressure cooker is also in one embodiment operably connected to a lime storage tank suitable for diverting lime directly to the lime pressure cooker. The absorption unit for absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker preferably comprises a steam condenser and a scrubber.

The mixing tank is operably connected to a reception station suitable for receiving solid organic material and/or operably connected to a reception tank suitable for receiving liquid organic material. Also, the mixing tank is in one embodiment further operably connected to the absorption unit for absorbing and condensing ammonia fluids, wherein said connection allows ammonia to be stripped in the mixing tank and diverted to the absorption unit. The plant according to the second aspect of the invention in one embodiment further comprises a silage tank for storage of energy crops.

The plant comprises one, or more than one, fermenter for anaerobically fermenting said organic materials, wherein said one or more than one fermenter are serially connected so that organic material having been fermented in a first fermenter under a first set of fermentation conditions can be diverted to a second or further fermenter and fermented under a second or further set of fermentation conditions.

In one embodiment the plant according to the present invention does not contain a stripper and sanitation tank connected to the lime pressure cooker and an absorption unit for absorbing ammonia N. Accordingly, the lime pressure cooker is connected directly to the absorption unit and ammonia formed in the lime pressure cooker during operation thereof under practical circumstances is diverted directly to the absorption unit. In this embodiment, the lime pressure cooker is also connected to a mixing tank which is connected to the lime pressure cooker and which is not connected to the absorption unit. The mixing tank receives lime pressure cooked biomass from the lime pressure cooker and optionally also biomass which has not been processed in the lime pressure cooker. The mixing tank is connected to one or more biogas reactors. Furthermore, the lime pressure cooker is connected to the one or more biogas reactors and receives from said one or more biogas reactors a liquid fraction comprising ammonia N. The liquid fraction is obtained by removing digestate from liquid biomass removed from the one or more biogas reactors. This can be achieved in a number of ways according to state-of-the-art methods. The obtained liquid fraction can be diverted or recycled back to the lime pressure cooker where the liquid fraction is stripped for ammonia N without mixing the liquid fraction with a further biomass. Alternatively, the liquid fraction is mixed with a further biomass which enters the lime pressure cooker prior to processing and stripping of ammonia N. Once stripped at least partly for ammonia N, the liquid fraction can be diverted to the mixing tank and mixed with biomass entering this mixing tank directly and without having been subjected to an initial lime pressure cooking step. In one embodiment, stripping of ammonia N from the liquid fraction takes place only by lime pressure cooking and not by any other means.

It is preferred that the more than one fermenter comprises at least one primary fermenter suitable for thermophilic fermentation and at least one secondary fermenter suitable for mesophilic fermentation. A gas storage facility is operably connected to the one or more than one fermenters.

In one embodiment, the bioenergy plant according to the present invention further comprises biogas fermenters comprising one or more service facilities, or maintenance shafts. The terms service facility and maintenance shaft are used interchangeably herein.

In one embodiment, the service facility is a movable sealing facility comprising of at least one sealing wall made out of a flexible, gas tight fabric, film tube or similar. The sealing facility can be converted from a resting to a sealing position wherein a separation of the service area from the gas compartment is achieved in the sealing position. In the sealing position the sealing facility has a canal-like shape. The sealing facility can be extended telescope-like and is for example comprised of slightly coned cylinder segments which can be extended. For that the sealing body does not have to be of cylindrical shape but can also have a rectangular or multi- angular or some other shape. Preferably there is a guidance element for example a rope or chain with which the sealing facility can be converted at least from the resting to the sealing position. To retract or extend the guidance element preferably a crank is attached to the guidance element. Altogether the sealing facility forms a gas curtain which separates the biogas area in the fermenting container from the service area. The actual maintenance of the stirring device can be carried out inside the container or also on the roof or above the roof of the fermenting container roof. In the regular operation of the biogas plant the sealing facility with the sealing wall is retracted. For maintenance purposes of the stirring device the stirring device can be swung to the outer wall and can be winded upwards via the height adjuster. At the same time or afterwards the sealing wall can be lowered through the guidance element until it reaches the fermenting substrate and can dip into it slightly or even more if necessary. Subsequently the service opening can be opened and the remaining gas in the service area can be let out. Afterwards the stirring device can be wound up completely to be maintained. The service facility can also comprise a maintenance shaft capable of sealing the interior of the biogas fermenter from an external, aerobic environment. During maintenance, no air would be allowed into the fermenting area - which would lead to a decrease in the gas production by anaerobic bacteria. Furthermore, the maintenance shaft prevents the loss of biogas. The maintenance room can be separated from the fermenting room through a rectangular maintenance shaft, which at normal filling level extends into the liquid. 10 cm of the shaft should at least dip into the liquid, but preferably not more than 15 cm. A rigid shaft can be used for ferments with a low solid amount and for high amounts of solids a shaft with a vertically movable lower part. When having a movable lower shaft part a sealing device has to be fitted between the shaft parts to prevent gas leaking into the maintenance area. This is achieved through a sealing canal at the rigid shaft part's end and a fitting collar at the movable part's top. The canal can be filled with water to support the sealing function. The gas production is only disrupted at the maintenance shaft's area. A winch system enables the lifting of the rotary stirrer above the container's surface. For rigid shafts a pipe for connection with the fermenting area is needed. A window pane can be installed into the maintenance hatch if it has not been installed somewhere else. To prevent the stirrer blades from getting damaged while lifting the stirrer a safety frame is to be installed.

The plant may further comprise a lime pressure cooker for hydrolysing said first organic material comprising one or more sources of nitrogen is operably connected to a reception tank suitable for receiving liquid organic material.

The plant may further comprise a lime pressure cooker for hydrolysing said first organic material comprising one or more sources of nitrogen is operably connected to a lime storage tank suitable for diverting lime directly to the lime pressure cooker.

The plant may further comprise an absorption unit for absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker comprises a steam condenser and a scrubber.

The plant may further comprise a pre-incubation tank is operably connected to a reception station suitable for receiving solid organic material, wherein the mixing tank is also operably connected to a reception tank suitable for receiving liquid organic material. The pre-incubation tank can further be operably connected to the absorption unit for absorbing and condensing ammonia fluids, wherein said connection allows ammonia to be stripped in the pre-incubation tank and diverted to the absorption unit. The plant may further comprise more than one fermenter for anaerobically fermenting said organic materials, wherein said more than one fermenters are serially connected so that organic material having been fermented in a first fermenter under a first set of fermentation conditions can be diverted to a second or further fermenter and fermented under a second or further set of fermentation conditions. The more than one fermenter preferably comprises at least one primary fermenter suitable for thermophilic fermentation and, serially connected thereto, at least one secondary fermenter suitable for mesophilic fermentation. The plant may further comprise a gas storage facility operably connected to the afore-mentioned, one or more biogas fermenters.

Brief Description of the Figures

Figure 1 discloses a biogas plant 100 in which a lime pressure cooker is used to hydrolyze organic material prior to anaerobic fermentation and biogas production. The input biomass can comprise organic industrial waste and/or animal manure and/or energy crops. The biomass is added to one or more reception facilities 105 and subsequently transferred to one or more dosing systems (part of pre-treatment system, refer Figure 2, 215). The biomass is transferred from the one or more dosing systems to one or more primary digesters 1 10. After the primary digestion in the one or more primary digesters, biogas and/or organic material biomass can be transferred to one or more secondary digesters 1 15. Biogas is transferred from the one or more secondary digesters 1 15 to a Combined Heat and Power plant (the CHP unit) 120 which generates power and heat. In one embodiment the generated heat is used for heating of the one or more dosing systems. The digestate generated in the one or more secondary digesters 1 15 is transferred to one or more post processing facilities 125 such as one or more decanters. The output from the decanter is a liquid fraction and a solid fraction. In one preferred embodiment, the liquid fraction comprising ammonia N is diverted back to the lime pressure cooker and the ammonia N is stripped.

Figure 2 discloses a pre-treatment system 200 according to the second aspect of the present invention. Biomass comprising solid biomasses and/or liquid biomasses that needs pre- treatment is added to one or more pre-treatment systems 205. In one embodiment steam and/or burnt lime is added to the pretreatment system. NH₃ and steam is transferred from the pre-treatment system 205 to one or more steam condensers 210. The one or more steam condensers result in generation of heat and a liquid fraction. Biomass from the pre-treatment system 205 and optionally liquid biomasses that do not need pre-treatment is transferred to one or more dosing modules 215 and subsequently to one or more digesters 220. In one embodiment, free ammonia is transferred from the steam condenser 210 to an ammonia scrubber 225 to which sulphuric acid has been or will be added. (NH₄)₂S0₄ is generated in the ammonia scrubber. (NH₄)₂S0₄ can be used as a fertilizer. Figure 3 discloses an energy system 300 according to the present invention. In one

embodiment of the present invention, biogas is transferred to one or more gas engines 305 and/or one or more steam boilers 310. The output from the gas engine is transferred to one or more exhaust gas boilers 315 comprising water. The output from the one or more steam boilers 310 and one or more exhaust gas boilers 315 can be transferred to one or more steam storages 320. The steam is finally transferred to one or more pre-treatment systems/batch cookers 325.

Figure 4 discloses the principles of one mode of operation of a biogas plant 400 according to the second aspect of the present invention. Biomass comprising organic materials is transferred to a lime pressure cooker 405 and ammonia N is stripped 410. Lime pressure cooked organic material is mixed in a mixing tank 415 with further organic materials which have not been subjected to lime pressure cooking. The mixed, organic materials are transferred to one or more biogas reactors 420 and subjected to anaerobic fermentation and biogas production. Following separation 425, a liquid fraction comprising inorganic nitrogen is diverted / recycled to the lime pressure cooker and ammonia N is stripped.

Figure 5 discloses one preferred embodiment of the present invention. In this embodiment, manure, preferably in the form of a slurry, generated in a house or stable (1 ) for the rearing of animals, including domestic animals, such as pigs, cattle, horses, goats, sheep; and/or poultry, including chickens, turkeys, ducks, geese, and the like, is transferred to either one or both of a first pre-treatment tank (2) and/or a second pre-treatment tank (3).

The working principles are that the manure, preferably in the form of a slurry including, in one embodiment, water such as reject water used for cleaning the house or stable, is diverted to the first pre-treatment tank comprising a stripper tank, where ammonia is stripped by means of addition to the stripper tank of e.g. CaO and/or Ca(OH)₂. However, addition of CaO and/or Ca(OH)₂ to the slurry may also take place prior to the entry of the slurry into the first treatment tank or stripper tank.

At the same time as the addition of CaO and/or Ca(OH)₂, or at a later stage, the pre-treatment tank comprising the stripper tank is subjected to stripping and/or heating, and the stripped N or ammonia is preferably absorbed prior to being stored in a separate tank (1 1 ). The stripped N including ammonia is preferably absorbed to a column in the stripper tank comprised in the first treatment tank before being directed to the separate tank for storage.

Organic materials difficult to digest by microbial organisms during anaerobic fermentation are preferably pre-treated in a second pre-treatment tank (3) prior to being directed to the first pre- treatment tank (2) comprising the stripper tank as described herein above. Such organic materials typically comprise a significant amounts of e. g. cellulose and/or hemicellulose and/or lignin, e. g. preferably more than 50% (w/w) cellulose and/or hemicellulose and/or lignin per dry weight organic material, such as straws, crops, including corn, crop wastes, and other solid, organic materials. N including ammonia is subsequently stripped from the pre-treated organic material.

In both the first and the second pre-treatment tank, the slurry is subjected to a thermal and alkali hydrolysis. However, the temperature and/or the pressure is significantly higher in the second pre-treatment tank (3), which is therefore preferably designed as a closed system capable of sustaining high pressures. Finally, the slurry having been subjected to a pre-treatment as described herein above is preferably diverted to at least one thermophile reactor (6) and/or at least one mesophile biogas reactor (6). The slurry is subsequently digested anaerobically in the reactors concomitantly with the production of biogas, i. e. gas consisting of mainly methane optionally comprising a smaller fraction of carbon dioxide. The biogas reactor (6) preferably forms part of an energy plant for improved production of energy from the organic material substrate.

Liquid organic material comprising ammonia N can be diverted from the one or more biogas reactors and to either one or both of the first (2) and/or second pre-treatment tanks (3). This is indicated by reference numerals 14 and 15.

The biogas can be diverted to a gas engine, and the energy generated from this engine can be used to heat the stripper tank. However, the biogas can also be diverted into a commercial biogas pipeline system supplying household and industrial customers.

The remains from the anaerobic fermentation, still in the form of a slurry comprising solids and liquids, is preferably diverted, in a preferred embodiment, to at least decanter centrifuge (7) for separating solids and fluids. One result of this separation is an at least semi-solid fraction comprising almost exclusively P (phosphor), such as an at least semi-solid fraction preferably comprising more than 50% (w/w) P (12). In the same step (7), or in another decanter centrifuge separation step (8), an at least semi-solid fraction preferably comprising almost exclusively K (potassium), such an at least semi-solid fraction preferably comprising more than 50% (w/w) K (13) is preferably also obtained. These fractions, preferably in the form of granulates obtained after a drying step, including a spray drying step or a slurry drying step, preferably comprise P and/or K in commercially acceptable purities readily usable for commercial fertilisers (10). Such fertilisers may be spread onto crops or agricultural fields. The liquids (9) also resulting from the decanter centrifuge separation step, such as reject water, can also be diverted to agricultural fields, they can be diverted back to the stable or animal house, or into a sewage treatment system.

In a further embodiment, the first pre-treatment tank may be supplied with organic material originating from silage tanks (4) comprising fermentable organic materials.

The diversion of such organic materials to the first pre-treatment tank may comprise a step involving an anaerobic fermentation such as e.g. thermophile fermentation tank capable of removing gasses from the silage. Additionally, straws and e.g. crop wastes originating from agricultural fields (5) may also be diverted to stables or animal houses and later to the first and/or second pre-treatment tank.

Figure 6 illustrates an embodiment essentially as described in Fig. 5, but with the difference that only phosphor (P) is collected following decanter centrifuge separation, and water in the form of reject water is collected in a separate tank for further purification, including further removal of N, removal of odours, and the majority of the remaining solids. This may be done e. g. by aerobic fermentation. Potassium (K) may also be separated from the liquids at this stage. Numeral indication in this figure is essentially same as those in Figure 5, therefore indicating the same element/ step.

Fig. 7 A; B and C illustrate a biogas reactor fitted with a service facility (700) allowing access to the interior of the biogas reactor. The service facility can comprise a shaft providing a limited exposure to biogas during service of the biogas reactor. The service facility extends from the top of the biogas plant to below the surface of the liquid contents of the reactor. The door in the service facility can be opened e.g. when an internally located stirrer has to be maintained or replaced. The service facility can comprise one or more port holes for easy inspection of the interior of the service facility. EXAMPLES

Example 1 NiX (Nitrogen Extraction) Thermo-chemical treatment of animal manure for biogas production

1 Summary

The thermo-chemical pre-treatment method, NiX, was performed with respect to pressure, temperature and base addition, and tested on different types of biomass in batch (part 1 ) and continuous experiments (part 2).

1.1 Part 1 : Batch experiments

The major conclusions from the batch experiments are as follows:

NiX treatment is able to create long-term improvements in biomethane potential in a number of diverse and commercially interesting biomasses. The biomasses tested in batch assays were anaerobically digested fibre, (obtained from Morso Bioenergi), cow deep litter, dewatered pig manure, chicken litter and hen litter. Biomethane potential improvements were 34%, 33% 27%, 22% and 2% respectively compared to the untreated biomass.

There exists an inverse relationship between relative improvement and absolute methane yields, in that the biomasses producing the lowest amounts of methane without NiX treatment also showed the highest relative improvements. This indicates that the more difficult a substrate is to degrade the higher the effect of the NiX treatment.

In addition to the effect on biomethane potential, NiX treatment consistently removes approximately two-thirds of the inorganic nitrogen, thus facilitating the use of nitrogen rich substrates for biogas production. The combination of NiX treatment with sudden pressure release and extended thermal treatment was also investigated, however the effect hereof was found to be insignificant.

1.2 Part 2: Continuous experiments

The major conclusions from the continuous pilot-scale experiment are as follows:

A CSTR biogas process with thermo-chemical treatment NiX and with chicken litter as mono- substrate was run for four months at thermophilic conditions and for five months at mesophilic conditions. The thermophilic CSTR biogas process could run steadily when the substrate was a mixture of raw chicken litter and water at a ratio of 1 :4.17 (1 kg wet weight of chicken litter and 4.17 kg of water). Applying NiX treatment to the thermophilic CSTR biogas process decreased the NH₄ ⁺- N (ammonium nitrogen) concentration in the digester by 15%.

Liquid re-circulation and NiX treatment were applied to the mesophilic CSTR biogas process. Extra water was added to the process only via steam condensation during NiX treatment (0.05 kg-H₂0 / kg WW chicken litter). The mesophilic process did not result in stable conditions because of lack of water. As a consequence, the TS (total solids) and NH₄ ⁺-N concentration in the digester increased.

Addition of extra water (0.42 kg-H₂0/kg-chicken litter, including 0.05 kg H₂0 / kg WW chicken litter as steam during NiX treatment) is needed to run stable biogas processes based on chicken litter as mono-substrate. The amount of water needed may change depending on the re-circulation ratio and on the organic loading rate (OLR).

During mesophilic operation, the methane yield was stable at 297 ± 35 mL CH₄ / g VS (where VS is the organic matter content of the chicken litter) and decreased when the NH₄ ⁺-N concentration in the digester increased beyond 6.4 g / kg WW. NiX treatment improved by 13% the methane yield of the chicken litter (measured in batches) and removed 47% of the NH₄ ⁺-N from the substrate.

The results obtained during the pilot-scale experiments were lower than the results obtained during Part 1 of the project (65% NH₄ ⁺-N removal, 22% methane yield improvement) mainly because of differences in the raw chicken litter and because of the different scale of the experiments.

Nitrogen measurements showed that the concentration of organic nitrogen does not change during NiX treatment. Approximately 30% of the organic nitrogen is converted into NH₄ ⁺-N during the storage of the NiX-treated mixture in the substrate tank/ mixing tank (see buffer tank, Figures 25-29). The biogas process resulted in 45% conversion of organic nitrogen into NH₄ ⁺-N. Preliminary investigations on uric acid degradation showed that the degradation of uric acid follows the same pattern as the degradation of the overall organic nitrogen. 2 Abbreviations

AD Anaerobically digested

BMP Biomethane potential

CSTR Continously stirred tank reactor

GEA GEA westphalia mobile decanter

HRT Hydraulic retention time

N Nitrogen

NmL Normal mL (milliliter at 1 bar and 273 K)

Org-N Organic Nitrogen

PC Pressure cooking

RSD Relative standard deviation

SD Standard deviation

TAN Total Ammonium Nitrogen

TKN Total Kjeldahl Nitrogen

TS Total Solids

VS Volatile Solids

w/w% Weight percentage 3 Introduction

The major contributing factor to the power production of a biogas plant is the quality of the biomass input. Certain substrates, such as glycerol, oil and energy crops, are energy rich and/or easily degradable and are therefore desirable as substrates for biogas production. Other biomass types, such as pig and cow slurry, deep litter and poultry litter, are low in energy yield, hard to degrade and/or contain compounds inhibitory to the biogas process. Energy rich substrates are more expensive and harder to come by, and technologies that may improve on the biogas yield of the inexpensive, low energy type biomasses, are increasingly more interesting.

The major reason for the poor degradability of biomass types such as straw and manure, is their high content of lignin. Lignin is a macromolecule, which serves to uphold the rigid structure of the plant as well as offer protection against fungi, bacteria and vira. Lignin encompasses the easily degradable cellulose and hemi-cellulose in a compound called lignocellulose, and hence creates a physical barrier for the microorganisms in the anaerobic digestion process. In order to create long term improvements in biogas yield it is thus essential to attack the ligno-cellulosic structure. The NiX technology is a registered trademark. It refers to a pre-treatment technology developed to attempt to overcome the problems related to lignified biomasses. NiX combines heat and alkalinity in a pressure cooking vessel to attack and dissolve the ligno-cellulosic structure as well as create an environment for removal of the inhibitory nitrogen in the process. The NiX technology comprises treatments of a biomass with saturated steam until reaching working temperatures between 100 °C and 220 °C in combination with a base.

The following sections describe the results obtained in batch and CSTR.

4 Batch investigations

4.1 Purpose

The purpose was to investigate the influence of parameters such as e.g.

temperature/pressure, base concentration, base choice, biomass choice, aeration, and sudden pressure drop (Flash) on NiX treatment with respect to biomethane potential (BMP) enhancement and nitrogen removal.

4.2 Introduction

The parameters that are of greatest importance in the process include are:

1 . Temperature/Pressure

2. Alkaline concentration

3. Choice of base

4. Biomass type

5. Post-pressure cooking aeration and thermal treatment

6. Flash

Treatment of a biomass at elevated temperature/pressure in combination with a strong base has several potential effects. The ligno-cellulosic structure is altered and parts of it are dissolved in a manner which likely increases the amount of degradable substrate available, hence increasing the biomethane potential of the biomass. However, by doing this, there is a chance of releasing small molecules which function as inhibitors of the anaerobic digestion (AD) process.

Temperature and base also affects the removal of ammonium nitrogen. At higher temperatures and base concentrations the nitrogen equilibrium shifts from dissolved ammonium nitrogen (NH₄ ⁺ _(aq)) towards gaseous ammonium nitrogen (NH_3(g)). NH₃ is volatile and may be removed from the system via the airspace above the liquid. The manner in which pressure is released from the system also influences the removal of nitrogen. Actively releasing steam from a pressurized system causes boiling of the liquid. This in turn causes continuous replacement of the airspace volume with steam, and aids in transferring NH₃ from the liquid to the headspace. Aeration (i.e. bubbling of air through the treated sample) is another way of facilitating the removal of ammonia. Finally, sudden pressure decreases (Flash) may contribute in altering ligno-cellulosic structure resulting in higher BMP levels.

It is clear that the parameters mentioned above interlink and influence each other. However, they also exert individual effects and they may thus be addressed individually.

The batch investigations used the following experimental approach to attempt to elucidate the influence of the above mentioned parameters on BMP and nitrogen content: 4.2.1 Phase A: Pressure and base optimization

Parameters 1 , 2 and 3 were addressed first. Determination of the optimal combination of temperature, base concentration and base choice on a few biomasses yielded a "standard" NiX pressure cooking method which is useable when addressing the remaining parameters. 4.2.2 Phase B: Biomass treatments and overall optimization

Parameters 4, 5 and 6 were addressed second. Determination of the robustness of the treatment using the "standard" NiX treatment on a variety of biomasses. Determination of the additive effect of Flash and extended treatment.

4.3 Materials and Methods 4.3.1 Biomass Characterization

Total solids (TS) contents were determined by heating the sample to 105 °C for a minimum of 24 hours. Volatile solids (VS) contents were determined by burning the sample at 550 °C for 3- 4 hours. The substrates tested were selected based on their commercial potential (availability, cost, etc.) and composition (such that the tested substrates represented a wide variety of biomasses). 4.3.2 Nix (Nitrogen Extraction) treatment

The Nix technology consists of a thermo-chemical treatment of the substrate. Substrate is mixed with or without saturated lime (Ca(OH)₂) and water to the desired concentration. The treatment is performed in a pilot scale pressure cooker in which saturated steam is used to raise the pressure and temperature. After treatment pressure may be decreased either by passive cooling or by actively releasing vapor from the treatment. Samples were collected and processed in accordance with the process having following treatment and analysis flow.

Subst ate ^Ca (€>l% - I¾C>

Prwts s!

X ' Homomgksii n

SMS* A fys Ufavgm Aml^$i&

Overview of treatment and analysis flow

4.3.3 Biomethane potential analysis

All BMP assays were carried out according to the German standard VDI4630 with certain alterations. Batches were prepared in 500 ml infusion glass bottles. The inoculum was taken from a thermophilic main digester at Foulum biogas plant and incubated at 52 ± 1 °C for 10 days before substrate addition in order to minimize the relative contribution from the inoculum to the total gas production. 200 ml of inoculum was used per bottle.

Each batch bottle was prepared by addition of approx. 1 g VS followed by addition of 200 ml_ inoculum (VS ~ 1 %). Resulting substrate VS concentration in each of the substrate batch bottles was 5 g substrate VS/L inoculum. From each of the independent Nix treatments 3-6 replicates were incubated.

For examination of inoculum quality, 3-6 bottles containing 0.5 g cellulose per 200 ml inoculum were incubated (positive controls). For determination of CH₄ production from the inoculum during substrate digestion, 3-6 replicate control batches of 200 ml inoculum were also incubated (blanks). After addition of inoculum and substrate all bottles were flushed with N₂, mixed and closed with gas tight rubber stoppers and aluminum screw lids before incubation at 52 ± 1 °C in heat cabinet for the duration of the batch test. 4.3.4 Measurements and Analysis

The CH₄ content in the headspace of the batch bottles was measured by GC (Perkin Elmer, Auto-analyzer XL) equipped with a capillary column (wax 0.53 mm ID, 30 m) and a FID detector.

By means of a standard curve created by injection of various volumes of 100 % pure CH₄ the number of CH₄ molecules in the headspace could be determined at regular intervals. Based on this the volumetric CH₄ production could be calculated during the test period. The biogas produced by the batches was released several times during the experiment in order to maintain low pressure in the bottles. The specific methane yield (mL methane per gram substrate VS added) was calculated by subtraction of background and normalizing according to VS concentration.

4.3.5 Nitrogen content analysis

Samples were analyzed for Total Ammonium Nitrogen (TAN) and Total Kjeldahl Nitrogen (TKN) according to the Kjeldahl method. Destruction of samples were performed on a

Tecator™ Digestion Unit Auto Lift 20 and distillations were performed on a Buchi K355 distillation unit 4.4 Phase A: Pressure and base optimization

4.4.1 Experimental considerations and setup

Temperature/pressure and alkaline addition are the parameters expected to have the greatest influence on BMP and nitrogen removal. A set of experiments were set up to determine the optimal temperature and base concentration. The initial experiments were performed on one biomass with varying temperatures and base concentrations, as per the following table.

Parameter choice

Alkaline addition

Table 1 - Experimental matrix showing the combination of parameters tested to elucidate the optimal combination of temperature and base concentration. Pressures tested were 0 barg, 4 barg and 9 barg. Base concentrations were 0 wt%, 0,25 wt%, 0,5 wt%, 1 wt%, and 2 wt% Ca(OH)₂

The experimental matrix consisted of 5 base concentrations, 3 temperatures/pressures and 3 replicates, making up a total of 45 different treatments to be tested. Since each pressure cooking cycle takes 3-4 hours, it was unfeasible to carry out each experimental combination individually. The experiments were thus performed without active pressure release to avoid boiling of the samples, and thus allow more than one treatment per pressure cooking cycle. In other words, the pressure cooker was allowed to cool down without opening any vents to release the pressure. This passive cool down method resulted in very little boiling of the liquid. More than one treatment per pressure cooking cycle is made possible by placing 500 ml_ beakers with chicken manure, wherein each beaker contains varying amounts of Ca(OH)₂ corresponding to either 0%, 0,25%, 0,5%, 1 % or 2% (w/w). As a consequence, nitrogen release from this setup only occurs to a limited degree, and the chemical equilibrium will not be allowed to reach its natural levels.

4.4.2 Results and discussion

The results of Phase A are briefly presented below as they are relevant only for the following phases.

• No significant differences were found between two different bases (NaOH and Ca(OH)₂) and all following experiments were continued using the cheaper alternative Ca(OH)₂.

• Nitrogen removal optimum occurs at 1 %-2% Ca(OH₂) addition.

• BMP is slightly improved in pressure cooking of chicken litter at 4 barg without base

addition.

• Base addition seems to have a negative effect on BMP.

Ca(OH)₂ has a low solubility, and it was therefore a possibility that the very soluble NaOH, may behave differently. However, no observable difference was found between the two compounds with respect to nitrogen removal, which may indicate that the non-dissolved Ca(OH)_2(s) functions as a buffer and the equilibrium settles so fast that it is not a limiting factor.

It was unexpected that the addition of base to the samples seemed to lower the BMP both at 4 barg and 9 barg. This was not expected as previous preliminary result had indicated an increase in BMP levels with addition of base. The results may however be explained with consideration to the equilibrium equation of ammonia. When ammonium turns into ammonia and leaves the system it utilizes a hydroxyl molecule. Both ammonia and hydroxyl are both potentially inhibitory to the AD process. It was considered possible that a different result might be obtainable when carrying out the experiments with active pressure release. Based on the nitrogen removal optimum a concentration of 2% was chosen for the following experiments using active pressure release.

4.5 Phase B: Biomass treatments and overall optimization 4.5.1 Experimental considerations and setup

To establish the effect of the parameter settings determined in phase A, as well as evaluate the influence of biomass type and aeration on the Nix treatment, a number of biomasses were treated and analyzed.

The following table shows the biomasses that were examined and the parameter settings used. More detailed descriptions of the individual biomasses may be seen under the relevant subsections.

Table 2 - Parameter settings for the individual biomasses.

The results from NiX treatment of the different biomasses are presented below. Each section may be read individually. 4.5.2 Chicken litter

Summary and Conclusion

Chicken Litter was treated with Xergi Nix (Nitrogen Extraction) technology and analyzed for biomethane potential and nitrogen content before and after treatment. Results from untreated Chicken Litter are compared with Nix treated Chicken Litter to evaluate the effect of the treatment. Several variations with respect to the general Nix treatment were included in the investigation of Chicken Litter:

1 . Nix treatment in the absence of Ca(OH)₂ to elucidate the effect of saturated lime on the biological methane potential (BMP)..

2. Incubation at 70 °C subsequent to Nix treatment to investigate the potential effect of an extended thermal treatment on BMP (with no aeration).

BMP assays on Nix treated Chicken Litter were initiated on October 19^th 2010 and were allowed to run for 48 days, with valid data available upto 27 days is considered.. BMP assays on Chicken Litter treated with Nix in the absence of saturated lime were initiated on January 17^th 201 1 and run for 81 days.

Table 3 - Overview of key results associated with the analysis of Chicken Litter. NiX treatment of Chicken Litter resulted in a 22% BMP increase compared to untreated substrate.

Over 90% of the final BMP yield in the treated sample was obtained after 7 days of incubation. The untreated sample realized over 90% of its final BMP value after 10 days of incubation. An extended thermal treatment at 70 °C for 3 days showed no effect on BMP.

NiX treatment with Flash showed no improvement (data not shown).

Treating Chicken Litter with Nix technology has a significant effect on BMP, and saturated lime is necessary to obtain optimal improvements.

Nix treatment has no effect on removal of organic nitrogen, however total ammonium nitrogen levels are decreased by -65%.

Total nitrogen levels are reduced by 15% due to Nix treatment Results

TS and VS analysis

TS and VS contents of the Chicken Litter were determined in triplicate prior to Nix treatment.

Table 4 - VS and TS levels of Chicken Litter BMP analysis

Specific methane yields obtained during the BMP analysis of treated and untreated Chicken Litter are shown in Figure 8A and Figure 8B. All curves show a steady increase in methane yield until day 15. Error bars represent ±1 standard deviation. Figure 8A illustrates Methane yield from batch bottles with untreated Chicken Litter and Nix treated Chicken Litter with or without an extended thermal treatment at 70 °C.

After 27 days BMP levels have reached 277 (± 19) NmL CH₄/g VS for the untreated Chicken Litter and 338 (± 23) NmL CH₄/g VS for treated Chicken Litter without extended thermal treatment, and 347 (± 22) NmL CH₄/g VS with extended thermal treatment. There is a clear effect of Nix treatment on Chicken Litter, whereas there is no additional effect of a subsequent prolonged incubation at 70 °C. More than 90% of the final BMP yield in the treated sample was obtained after 7 days of incubation. The untreated sample attained over 90% of its final BMP yield after 10 days of incubation. Technical problems precluded the analysis after 27 days. It should be noted that other similar experiments show that although the improvements obtained in the Nix treatments are gradually reduced over time, they are usually still significant after 50-80 days of thermophillic digestion.

Based on a kinetic analysis of the gas production the expected yield in a full scale continuous AD plant with a 20 days thermophilic primary digestion and a 10 days mesophilic secondary digestion will be approx. 325 NmL CH4/g VS equal to 94% of the obtained batch yield.

To investigate the effect of saturated lime in Nix treatment Chicken Litter was Nix treated in the absence of Ca(OH)₂ (see Figure 8B, which illustrates methane yield from batch bottles with untreated Chicken Litter and Nix treated Chicken Litter in the absence of saturated lime. ¹ This BMP assay was run for 81 days and also shows a significant effect of the treatment. The course of the graphs show a slight peak around day 15, after which the curves drop and continue on a slight increase. This type of behaviour is often observed in BMP assays with low substrate concentrations, and is initial overproduction of methane from the inoculum VS is the expected cause, but this evens out over time as the inoculum controls catch up. The highest improvements are obtained between 10 and 20 days, after which the effect is very gradually reduced.

Nitrogen analysis

The nitrogen content of treated and untreated Chicken Litter are shown in Figure 8C, which illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated Chicken Litter. Org-N is calculated from TKN and TAN levels (Org-N = TKN - TAN). PC = Pressure Cooked. Specific values and the resulting nitrogen reductions as a result of Nix treatment are shown in

Table 5.

Inorganic nitrogen constitutes about a quarter of the entire nitrogen pool (-24%). Organic nitrogen constitutes the remaining -76%. Using Nix technology inorganic nitrogen is removed to an absolute level of 5,0 g N/kg VS, which constitutes a 65% reduction in TAN. Organic nitrogen is not removed in this treatment. Subsequent prolonged thermal treatment does not remove further nitrogen.

¹ Methane yields have been normalized according to an internal standard Total ammonium TAN SD RSD% Reductio Reductio nitrogen content (g N/k g (g N/kg VS) n n in

VS) (g N/kg percent

VS)

Untreated Chicken Litter 14.5 1 ,3 III IB!!!

Treated Chicken Litter 5.0 0,2 Ill 9111111 9,5 65%

+ 70 °C for 3 days 5.7 0,4 III III!!! 8,8 61 %

Table 5 - Values of TAN, TKN and Org-N with standard deviations (SD), relative standard deviations (RSD) and reductions as a consequence of Nix treatment. Negative reduction values reflect a value that is higher than the untreated organic nitrogen pool. Note that the extended thermal treatment is without aeration.

4.5.3 Cow deep litter

Summary and Conclusion

Cow Deep Litter was treated with Xergi Nix (Nitrogen Extraction) technology and analyzed for biomethane potential and nitrogen content before and after treatment. Results from untreated Cow Deep Litter is compared with Nix treated Cow Deep Litter to evaluate the effect of the treatment.

Several variations with respect to the general Nix treatment were included in the investigation of Cow Deep Litter:

1 . Nix treatment in the absence of Ca(OH)₂ to elucidate the effect of saturated lime on the biological methane potential (BMP).

BMP assays on Nix treated Cow Deep Litter were initiated on October 19^th 2010 and were allowed to run for 48 days. However, due to technical problems only data up to day 27 are valid. BMP assays on Cow Deep Litter treated with Nix in the absence of saturated lime were initiated on January 17^th 201 1 and run for 80 days.

Table 6 - Overview of key results associated with the analysis of Cow Deep Litter.

NiX treatment of Cow deep litter resulted in a 33% BMP increase compared to untreated substrate.

Close to final BMP yields were obtained in the treated sample after 10 days of incubation. The untreated sample realized most of its final BMP yield after 20 days of incubation. An extended thermal treatment at 70 °C for 3 days showed no effect on BMP.

Treating Cow Deep Litter with Nix technology has a significant effect on BMP.

Nix treatment has no effect on removal of organic nitrogen, however total ammonium nitrogen levels are decreased by -64%, corresponding to a reduction in total nitrogen of 12%.

Measured TKN levels before and after show a reduction in TKN of 3%. However, Cow Deep Litter is a very inhomogeneous substrate, from which it is much easier to measure TAN than TKN, and it is thus likely that the measured TKN level after treatment is a result of the heterogenic nature of the substrate. This is corroborated by TKN results on Nix treated Cow Deep Litter, showing a total TKN reduction of 15%.

Results

TS and VS analysis

TS and VS contents of the Cow Deep Litter were determined in triplicate prior to Nix treatment.

Table 7 - VS and TS levels of Cow Deep Litter BMP analysis

Specific methane yields obtained during the BMP analysis of treated and untreated Cow Deep

Litter are shown in Figure 9A - Methane yield from batch bottles with untreated Cow Deep

Litter and Nix treated Cow Deep Litter with or without an extended thermal treatment at 70 °C.

Methane yields have been normalized according to an internal standard.

Figure 9B illustrates methane yield from batch bottles with untreated Cow Deep Litter and Nix treated Cow Deep Litter in the absence of saturated lime. All curves show a steady increase in methane yield until day 15. Error bars represent ±1 standard deviation.

After 27 days BMP levels have reached 252 (± 24) NmL CH₄/g VS for the untreated Cow Deep

Litter and 334 (± 18) NmL CH₄/g VS for treated Cow Deep Litter without extended thermal treatment, and 315 (± 13) NmL CH₄/g VS with extended thermal treatment (see Figure 9A).

There is a clear effect of Nix treatment on Cow Deep Litter, whereas there is no significant additional effect of a subsequent prolonged incubation at 70 °C. In the treated sample the final BMP yield was obtained after approximately 10 days incubation. The untreated sample required 20 days of incubation to attain its final BMP. Technical problems precluded the analysis after 27 days. It should be noted that other similar

experiments show that, although the improvements obtained in the Nix treatments are gradually reduced over time, they are usually still significant after 30-80 days of thermophillic digestion.

Based on a kinetic analysis of the gas production the expected yield in a full scale continuous AD plant with a 20 days thermophilic primary digestion and a 10 days mesophilic secondary digestion will be approx. 317 NmL CH4/g VS equal to 92% of the obtained batch yield.

To investigate the effect of saturated lime in Nix treatment Cow Deep Litter was Nix treated in the absence of Ca(OH)₂ (see Figure 9B). This BMP assay was run for 80 days and also shows a significant effect of the treatment. The highest improvements are obtained between 15 and 20 days, after which the effect is gradually reduced. After 80 days there is no longer any significant effect of the treatment.

Nitrogen analysis

The nitrogen content of treated and untreated Cow Deep Litter are shownFigure 9C, which illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated Cow Deep Litter. Org-N is calculated from TKN and TAN levels (Org-N = TKN - TAN and PC = Pressure Cooked). Specific values and the resulting nitrogen reductions as a result of Nix treatment are shown in

Table 8.

Inorganic nitrogen constitutes about one fifth of the entire nitrogen pool (-19%). Organic nitrogen constitutes the remaining -81 %. Using Nix technology inorganic nitrogen is removed to an absolute level of 3,6 g N/kg VS, which constitutes a 64% reduction in TAN. Such a reduction in TAN alone should lead to a TKN reduction of 12%. However, measured TKN levels before and after only show a reduction in TKN of 3%. Cow Deep Litter is a very inhomogeneous substrate, from which it is much easier to measure TAN than TKN, and it is likely that the measured TKN level after treatment is a result of the heterogeneous nature of the substrate. This is corroborated by TKN results on Nix treated Cow Deep Litter, showing a total TKN reduction of 15%(see Figure 9C). From this it is concluded that organic nitrogen is not removed in this treatment.

When exposing Cow Deep Litter to a prolonged thermal treatment subsequent to Nix treatment there seems to be a small effect on TAN removal. Total ammonium TAN SD RSD Reducti Reducti nitrogen content (g N/k g (g N/kc I % on on in

VS) VS) (g N/kg percent

VS)

Untreated Cow Deep Litter 3.6 0.1 III WSi

Treated Cow Deep Litter 1 .3 0.1 1111 li!l 2,3 64%

+ 70 °C for 3 days 0.8 0.0 III !lilll 2,8 77%

Table 8 - Values of TAN, TKN and Org-N with standard deviations (SD), relative standard deviations (RSD) and reductions as a consequence of Nix treatment. Negative reduction values reflect a value that is higher than the untreated organic nitrogen pool. Note that the extended thermal treatment is without aeration. 4.5.4 GEA Dewatered Pig Manure

Summary and Conclusion

GEA Dewatered Pig Manure is the fiber fraction after dewatering using a GEA decanter and will be referred to as "pig fibers" in the following. The pig fibers were treated with Xergi Nix (Nitrogen Extraction) technology and analyzed for biomethane potential and nitrogen content before and after treatment. Results from untreated pig fibers are compared with Nix treated fibers to evaluate the effect of the treatment.

Several variations with respect to the general Nix treatment were included in the investigation of pig fibers:

1 . Nix treatment in the absence of Ca(OH)₂to elucidate the effect of saturated lime on the biological methane potential (BMP)..

2. Incubation at 70 °C subsequent to Nix treatment to investigate the potential effect of an extended thermal treatment on BMP.

3. Incubation at 70 °C and aeration subsequent to Nix treatment to investigate the potential effect on removal of nitrogen.

BMP assays on Nix treated pig fibers were initiated on October 19^th 201 1 and were allowed to run for 48 days. However, due to technical problems only data up to day 27 are valid. BMP assays on pig fibers treated with Nix in the absence of saturated lime were initiated on January 17^th 201 1 and run for 81 days.

Table 9 - Overview of key results associated with the analysis of pig fibers

² After 27 days

³ With aeration and extended thermal treatment for 3 days TAN is reduced to 4,0 (±1 ,4) g N/kg VS NiX treatment of pig fibers resulted in a 27% BMP increase compared to untreated substrate. Final BMP levels are reached after 20 days of incubation in both treated and untreated samples.

An extended thermal treatment at 70 °C for 3 days showed no effect on BMP.

Treating pig fibers with Nix technology has a significant effect on BMP, and saturated lime seems to be necessary to obtain optimal improvements. The beneficial effect of Nix treatment is gradually diminished over time in the samples treated without saturated lime. However, after 27 days the improvement on BMP shows no tendency to fade.

Nix treatment has no effect on removal of organic nitrogen, however total ammonium nitrogen levels are decreased by -64%.

Total nitrogen levels are reduced by 30% due to Nix treatment

Results

TS and VS analysis

TS and VS contents of the pig fibers were determined in triplicate prior to Nix treatment.

Table 10 - VS and TS levels of GEA dewatered PIG MANURE BMP analysis

Specific methane yields obtained during the BMP analysis of treated and untreated pig fibers PIG MANURE are shown in Figure 10A and Figure 10B. Figure 10A illustrates methane yield from batch bottles with untreated and NiX treated pig fibers with or without an extended thermal treatment at 70 °C. Methane yields have been normalized according to an internal standard. All curves show a steady increase in methane yield until day 15. Error bars represent ±1 standard deviation.

After 27 days BMP levels have reached 260 (± 19) NmL CH₄/g VS for the untreated pig manure and 331 (± 17) NmL CH₄/g VS for treated pig manure without extended thermal treatment, and 329 (± 30) NmL CH₄/g VS with extended thermal treatment (see OA). There is a clear effect of Nix treatment on pig fibers, whereas there is no additional effect of a subsequent prolonged incubation at 70 °C. Final BMP yields are reached before day 20 in both treated and untreated samples. Other similar experiments show that, although the improvements obtained in the Nix treatments are gradually reduced over time, they are usually still significant after 50-80 days of thermophillic digestion.

Based on a kinetic analysis of the gas production the expected yield in a full scale continuous AD plant with a 20 days thermophilic primary digestion and a 10 days mesophilic secondary digestion will be approx. 305 NmL CH4/g VS equal to 91 % of the obtained batch yield.

To investigate the effect of saturated lime in Nix treatment pig fibers were Nix treated in the absence of Ca(OH)_2. Figure 10B illustrates methane yield from batch bottles with untreated pig MANURE and Nix treated pig manure in the absence of saturated lime. Methane yields have been normalized according to an internal standard. This BMP assay was run for 81 days and shows a significant effect of the treatment. The course of the graphs show a peak around day 15, after which the curves drop and stabilise at the current level. This type of behaviour is often observed in BMP assays with low substrate concentrations, and is believed to be caused by an initial overproduction of methane from the inoculum VS, which evens out over time as the inoculum controls catch up.

The highest improvements are obtained between 15 and 20 days, after which the effect is gradually reduced. After 36-49 days BMP levels have reached 259 (± 23) NmL CH₄/g VS for the untreated pig manure and 288 (± 42) NmL CH₄/g VS for treated pig manure without added Ca(OH)₂ (see Figure 10B). After 81 days there is no effect of the treatment.

Nitrogen analysis

The nitrogen content of treated and untreated pig fibers are shown in Figure 10C, which illustrates graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated pig fibers. Org-N is calculated from TKN and TAN levels (Org-N = TKN - TAN and PC = Pressure Cooked. Specific values and the resulting nitrogen reductions as a result of Nix treatment are shown

Table 1 1 .

Inorganic nitrogen constitutes a little less than half of the entire nitrogen pool (-46%). Organic nitrogen constitutes the remaining -54%. Using Nix technology inorganic nitrogen is removed to an absolute level of 6,7 g N/kg VS, which constitutes a 64% reduction in TAN. Organic nitrogen is not removed in this treatment. When exposing the Nix treated pig manure to aeration at elevated temperatures TAN is further reduced to an absolute level of 4 g N/kg VS corresponding to a 78% reduction. However, high standard deviations preclude a formal conclusion on this result.

+ Aeration at RT for 3 days 21 9 8% 0 5 -2%

+ Aeration at 70 °C for 1 day 22.4 1 .2 5% 0 9 -4%

+ Aeration at 70 °C for 3 21 8 7% 0 3 -1 % days

Table 11 - Values of TAN, TKN and Org-N with standard deviations (SD), relative standard deviations (RSD) and reductions as a consequence of Nix treatment. Negative reduction values reflect a value that is higher than the untreated organic nitrogen pool.

4.5.5 Morso Biogas AD Fibers Summary and Conclusion

Anaerobically digested (AD) fibers from Morso biogas plant was treated with Xergi Nix

(Nitrogen Extraction) technology and analyzed for biomethane potential and nitrogen content before and after treatment. Results from untreated AD fibers are compared with Nix treated AD fibers to evaluate the effect of the treatment.

BMP assays were initiated December 10^th 2010 and run for 88 days.

Table 12 - Overview of key results associated with the analysis of AD fibers.

- NiX treatment AD fibers resulted in a 34% BMP increase compared to untreated substrate. - Approximately 90% of the final BMP yield in the treated sample was obtained after 33 days of incubation. The untreated sample had only reached 86% of its final BMP value after 53 days of incubation.

Treating the AD fibers with Nix technology has a significant and lasting effect on BMP - Nix treatment has no effect on removal of organic nitrogen, however ammonium/ammonia levels are decreased by >60%.

- Total nitrogen levels are reduced by 40% due to Nix treatment.

Results

TS and VS analysis

TS and VS contents of the AD fibers were determined in triplicate prior to Nix treatment.

Table 13 - VS and TS levels of AD fibers. BMP analysis

Specific methane yields obtained during the BMP analysis of treated and untreated AD fibers are shown in Figure 1 1 A, which illustrates methane yield from batch bottles with treated and untreated AD fiber. All curves show a steady increase in gas yield until day 88. Error bars represent ±1 standard deviation.

After 88 days BMP levels have reached 268 (± 18) NmL CH₄/g VS for the untreated AD fiber and 325 (± 38) NmL CH₄/g VS for treated AD fiber. On day 33 app. 90% of the final yield had been obtained in the treated sample. The same relative level was obtained after 53 days in the untreated sample. The effect of the Nix treatment on BMP seems to decrease after prolonged incubation. However since HRT values typically are lower than 88 days, this is unlikely to have any significant practical consequence. Both treated and untreated samples continue producing gas even after 88 days, which is in contrast to most substrates which reach their maximal BMP value after 4-6 weeks incubation. However, this may be explained by the fact that the Morso Bioenergi AD fiber has been both anaerobically digested and dewatered prior to Nix treatment and analysis. The content of easily digestible organic compounds is thus likely to be very low, with longer digestion times as a result. Based on a kinetic analysis of the gas production the expected yield in a full scale continuous AD plant with a 20 days thermophilic primary digestion and a 10 days mesophilic secondary digestion will be approx. 245 NmL CH4/g VS equal to 78% of the obtained batch yield.

Nitrogen analysis

The nitrogen content of treated and untreated AD fibers are shown in Figure 1 1 B, which illustrates a graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated AD fiber where Org-N is calculated from TKN and TAN levels (Org-N = TKN - TAN) and PC = Pressure Cooked. Specific values and the resulting nitrogen reductions as a result of Nix treatment are shown in

Table 14.

Table 14 - Values of TAN, TKN and Org-N with standard deviations (SD), relative standard deviations (RSD) and reductions as a consequence of Nix treatment. Negative reduction values reflect a value that is higher than the untreated organic nitrogen pool. Inorganic nitrogen constitutes a significant amount of the entire nitrogen pool (-73%). Organic nitrogen constitutes the remaining -27%. With Nix technology inorganic nitrogen is removed to an absolute level of 13 g N/kg VS, which constitutes a 63% reduction. Since Nix technology is able to reduce TAN levels to 4-6 g N/kg VS in most substrates (with TAN levels of up to -20 g N/kg VS in untreated substrate), it is likely that an optimization of the procedure will lead to even higher removal levels. This is corroborated by the pH after treatmentwhich indicates that most of the added base is utilised in the removal of ammonium. 4.5.6 Glenrath Hen Litter

Summary and Conclusion

Hen litter from Glenrath was treated with Xergi Nix (Nitrogen Extraction) technology and analyzed for biomethane potential and nitrogen content before and after treatment. Results from untreated hen litter is compared with Nix treated litter to evaluate the effect of the treatment.

BMP assays were initiated January 24^th 201 1 and run for 79 days.

Table 15 - Overview of key results associated with the analysis of hen litter.

Treating the Hen litter with Nix technology had minor effect on BMP.

More than 90% of the final BMP yield was obtained after 14 days of incubation. Nix treatment had no effect on removal of organic nitrogen, however ammonium/ammonia levels are decreased by 40-50%.

- Total nitrogen levels were decreased by 7% due to Nix treatment. Results

TS and VS analysis

TS and VS contents of the hen litter were determined in triplicate prior to Nix treatment.

Table 16 - VS and TS levels of hen litter BMP analysis

Specific methane yields obtained during the BMP analysis of treated and untreated hen litter are shown in Figure 12A, which illustrates methane yield from batch bottles with treated and untreated hen litter. All curves show a steady increase in gas yield until a maximum yield is obtained on day 23 after which the substrates no longer contribute to methane production. Error bars represent ±1 standard deviation. After 79 days data for maximum yield show BMP values of 322 (± 5) NmL CH₄/g VS for the untreated hen litter and 329 (± 9) NmL CH₄/g VS for treated hen litter. On day 14 more than 90% of the final yield had been obtained.

Based on a kinetic analysis of the gas production the expected yield in a full scale continuous AD plant with a 20 days thermophilic primary digestion and a 10 days mesophilic secondary digestion will be approx. 290 NmL CH4/g VS equal to 90% of the obtained batch yield.

Nitrogen analysis

The nitrogen content of treated and untreated hen litter are shown in Figure 12B, which illustrates Graphical representation of TKN, TAN and Organic nitrogen (Org-N) levels in untreated and treated hen litter. Org-N is calculated from TKN and TAN levels (Org-N = TKN - TAN). Specific values and the resulting nitrogen reductions as a result of Nix treatment are shown in Table 17. Total ammonium TAN SD RSD Reduction Reduction nitrogen content (g N/kg VS) (g N/k g % (g N/kg VS) in percent

VS)

Untreated hen litter 9,8 0.3 3%

from Glenrath

Treated hen litter 5,5 2.3 43% !!!!! Μβ ≡≡ 44%

Table 17 - Values of TAN, TKN and Org-N with standard deviations (SD), relative standard deviations (RSD) and reductions as a consequence of Nix treatment.

Inorganic nitrogen constitutets a rather small amount of the entire nitrogen pool (-12%).

Organic nitrogen constitutes the remaining -88%. The measured removal of inorganic nitogen is therefore somewhat uncertain, as a small release of nitrogen from the organic N pool after the Nix treatment potentially could blur the measurement significantly. Generally the removal of inorganic nitrogen from other biomasses is 60 - 70 % at the described treatment conditions. 5 CSTR investigations

5.1 Introduction The project was run at the experimental facilities of Xergi in Foulum (Denmark) and it included two parts:

Part 1 : lab-scale tests (batch);

Part 2: pilot-scale tests (CSTR);

This part of the example focuses on Part 2, the pilot-scale CSTR tests. The tests lasted for 9 months, from September 2010 to May 201 1 :

Sep. 2010 - Dec. 2010: thermophilic operation (phase 1 A, phase 1 B)

Jan. 201 1 - May 201 1 : mesophilic operation (phase 2)

5.1.1 Objective of the project

The objective of the project was to run a stable biogas process fed with chicken litter mono- substrate and with minimal water addition. Also, the ammonia removal with NiX treatment had to be determined.

Nitrogen is required by the microorganisms and often the nitrogen source is ammonia

NH₃. However, NH₃ can be toxic. Chicken litter contains high amount of nitrogen (mainly organic nitrogen) and this results in high NH₄ ⁺-N concentration in the biogas digester

(ammonium nitrogen, sum of ammonia NH₃ and ammonium ions NH₄ ⁺). Also, chicken litter has high TS content and to be pumped it requires dilution.

5.1.2 Choice of thermo-chemical treatment (NiX)

The treatment conditions used for the thermo-chemical treatment was based on the Nitrogen Extraction NiX treatment and on the results described in the first part of the example (Part 1 ). Details of the treatment conditions used during Part 2 (described in this part of the example) are given in Appendix A: NiX treatment. 5.2 Procedure

5.2.1 CSTR experiments

Xergi's pilot-scale plant was used for the experiments. A 1 -step CSTR biogas process was run at thermophilic conditions (52 °C) during the first period of the experiment until December 2010 (Phase 1 A, Phase 1 B) and at mesophilic conditions (37 °C) from January 201 1 until May 201 1 (Phase 2). Chicken litter was the substrate used.

Sept 2010 - Oct 2010: phase 1 A, thermophilic operation no NiX

Nov - Dec 2010: phase 1 B, thermophilic operation with NiX

Jan 201 1 - May 201 1 : phase 2, mesophilic operation

Mesophilic conditions were chosen to minimize the negative effect of high NH₄ ⁺-N

concentration in the digester.

5.2.2 Phase 1 : Thermophilic conditions 5.2.2.1 Substrate

The substrate was a mixture of raw chicken litter and water at a ratio of 1 :4.17 (1 kg wet weight of chicken litter and 4.17 kg of water). The chicken litter was obtained from a local farm (Tjele, Denmark) breeding chickens (broilers). Chicken litter was collected on August 24, 2010, distributed into barrels (approximately 60 kg each) and frozen at -10 °C. The chicken litter contained approximately 2.3% of sphagnum wet weight. The chickens were 42 days old when the chicken litter was collected.

The raw chicken litter contained TS 62.8 ± 1 .8%, VS 51 .2 ± 2.7% (average of 1 1

measurements), NH₄ ⁺-N 7.0 ± 0.7 g/kg (average of 15 measurements), TKN 28.8 ± 1 .4 g/kg (average of 9 measurements). 5.2.2.2 Phase 1 A: thermophilic, without NiX treatment

Raw chicken litter and water in the proportions above were used as substrate (Figure 13A, which illustrates a process configuration during thermophilic operation without NiX treatment ) from September 2010 until November 1 2010. The organic loading rate (OLR) was maintained at 6.0 ± 1 .0 kg VS / (m³ d), where VS is the organic matter content of the raw chicken litter. The hydraulic retention time (HRT) was 14.6 ± 2.0 d. 5.2.2.3 Phase 1 B: thermophilic, with NiX treatment

Between November 2 2010 and December 2010, the substrate was made of raw chicken litter and water in the proportions above treated with NiX (Figure 13B, which illustrates a process configuration during thermophilic operation with NiX treatment). Details of NiX treatment are given in Appendix A: NiX treatment. The organic loading rate (OLR) was maintained at 6.0 ± 1 .0 kg-VS / (m³ d), where VS is the organic matter content of the raw chicken litter. The hydraulic retention time (HRT) was 14.6 ± 2.0 d.

5.2.3 Phase 2: mesophilic conditions

5.2.3.1 Substrate

The substrate was a mixture of chicken litter and liquid fraction separated from the effluent of the biogas process. The mixture was treated with NiX before being used as substrate. No water was added to the substrate (aside from steam added during NiX treatment,

approximately 0.05 kg-H₂0 / kg WW chicken litter).

5.2.3.2 Mesophilic, re-circulation, no water addition

Re-circulation of the liquid fraction of the effluent was applied (Figure 14, which illustrates a process configuration during phase 2 (mesophilic operation with re-circulation and NiX treatment). The OLR was 4.4 ± 0.3 kg VS / (m3 d).

5.2.4 Batch tests

The substrates for the batch tests were sampled during the CSTR experiments. Details about the preparation of the batches are given in Appendix B: batch experiments.

5.3 Results

Treatment with NiX removed 47% of the NH₄ ⁺-N from the substrate. With NH₄ ⁺-N concentration higher than 6.4 g / kg WW in the digester, the methane yield showed clear signs of decrease. Addition of water was required for stable operation of the CSTR. Batch tests resulted in 13% higher methane yield from NiX-treated chicken litter compared to raw chicken litter. 5.3.1 Effect of NiX treatment

5.3.1.1 Effect of NiX treatment on NH₄ ⁺-N concentration in the digester

The effect of NiX treatment on the NH₄ ⁺-N concentration in the digester was tested during thermophilic operation. The NH₄ ⁺-N concentration in the digester decreased from 3.30 ± 0.14 to 2.79 ± 0.24 g / kg WW (Figure 15, which illustrates effect of NiX treatment on NH₄ ⁺-N concentration in the digester).

5.3.1.2 Effect of NiX treatment on NH₄ ⁺-N concentration in the substrate

The effect of NiX treatment on the concentration of NH₄ ⁺-N in the substrate was studied during mesophilic operation with accurate mass balances.

NiX treatment removed 47% of the NH₄ ⁺-N from the substrate. The rate of pressure reduction after NiX treatment could be regulated by adjusting the opening of the valve for pressure release. This affected the amount of loss, because increasing the rate of pressure reduction increased the amount of material that leaved the NiX treatment unit and thereby was "lost". The optimal rate of pressure release had to maximize NH₄ ⁺-N removal and the amount of treated substrate collected after the treatment (i.e. the loss had to be minimized). Also, the time needed for the NiX treatment had to be minimized.

Avoiding losses, 47% NH₄ ⁺-N removal was obtained. Increasing the rate of pressure release and allowing part of the treated material to be lost (4 kg WW), removed 60% NH₄ ⁺-N (corrected for the mass loss) as shown in Figure 16, which illustrates removal of NH₄ ⁺-N with NiX treatment vs. loss caused by high rate of pressure reduction

Based on the results shown in the Figure 16, it was chosen to operate the NiX treatment unit avoiding losses to minimize the amount of material to be treated with NiX and to make the mass balance easier.

5.3.1.3 Effect of NiX treatment on yield

The effect of NiX treatment on the yield of chicken litter was measured with batch tests. The substrates used for the batch tests were the same as those used for the CSTR experiments. The batch tests resulted in 303 ± 5 mL CH₄ / g VS (where VS is the organic matter content of the raw chicken litter) and showed that NiX treatment increased by 13% the methane yield of chicken litter (on VS basis, contribution from the re-circulated liquid is subtracted). Yield chicken litter L CH₄ / kg VS

Without NiX 303

treatment

With NiX treatment 343

increase iimiiiiiiiiiiiiiiiiiiiii

Table 18 shows effect of NiX on the methane yield of the chicken litter.

Details about the preparation of the batches are given in Appendix B: batch experiments.

The comparison of the results described in this example (Part 2 of the project) to the results in the first part of the report (Part 1 of the project) shows that a higher yield improvement was achieved in Part 1 (22% improvement). The difference can be explained with differences in the raw chicken litter used during the two parts of the example. The raw chicken litter used for the experiments in Part 1 was not from the same batch used for the experiments in Part 2, therefore the effect on Nix may have been different. The fact that the raw chicken litter of Part 1 was different from the one of Part 2 is confirmed by the different methane yields: 277 L CH₄ / kg VS (Phase 1 ) and 303 L CH₄ / kg VS (Phase 2). Also, the smaller-scale of the experiments of Part 1 resulted in higher NH₄ ⁺-N removal (65% NH₄ ⁺-N removal). This shows that the amount of raw material to be treated affects the effect of NiX. With low amounts, raw samples are homogenous and have low mass compared to the headspace and the effect of NiX can be maximised. Finally, the experiments of Phase 1 were made at 10% TS, while the substrate treated during Phase 2 contained approximately 22% TS.

5.3.1.4 Effect on nitrogen degradation Nitrogen flow and degradation in the system

Nitrogen measurements (NH₄ ⁺-N and TKN) were made once per week during the mesophilic operation (Phase 2). The concentration of organic nitrogen was calculated as the difference between TKN and NH₄ ⁺-N.

Uric acid was measured once on May 10, 201 1 . Because the results from uric acid

measurements in this report are preliminary, they are described only in Appendix C: uric acid. Figure 17 illustrates the nitrogen flow in the system is expressed in g of nitrogen per day, where:

CL: chicken litten raw

Liquid - liquid fraction of effluent digester after separation with centrifuge

NiX - NiX treatment material collected immediately after NiX treatment

Loss - loss after NiX treatment during quick pressure release

ST - substrate tank

AD - biogas digester (anaerobic digester)

Solid - solid fraction of effluent digester after separation with centrifuge

This flow is calculated from the measured concentrations (g nitrogen per kg wet weight, measured once per week) and the measured flow rates (kg wet weight per day, measured every day). The averages of two or three nitrogen measurements and of four or five flow rate measurements have been used to calculate the nitrogen flow.

Because the flows in Figure 17 are calculated from measurements, measurement errors and uncertainties have to be taken into account. For example, the balance around the NiX treatment unit shows that approximately 6% of organic nitrogen is missing:

58 + 15 = 73 g N / d

73 = 94% of 78 g N / d

This difference is probably due to measurement uncertainties.

Also, the system was not at steady state when the nitrogen flow was made. Therefore, exact balance between the nitrogen entering and leaving the system should not be expected.

Nitrogen balance substrate tank The balance around the substrate tank is closed, as 77 g N / d (as TKN) enter the substrate tank and 76 g N / d leave it.

In the substrate tank, approximately 30% of the organic nitrogen was converted into NH₄ ⁺-N. This was probably due to microbial activity in the substrate tank (although the substrate tank was maintained at 10-15 °C), as revealed by dedicated experiments. As a consequence of the 30% conversion of organic nitrogen into NH₄ ⁺-N, the substrate entering the biogas digester had higher NH₄ ⁺-N concentration compared to the material entering the substrate tank. This contributed to increase the NH₄ ⁺-N concentration in the digester.

A better understanding of the nitrogen degradation in the substrate tank is needed to allow development of methods to reduce the conversion of organic nitrogen into NH₄ ⁺-N or to remove the NH₄ ⁺-N from the substrate tank. This would result in lower NH -N concentration in the biogas digester.

Nitrogen balance biogas digester The TKN balance around the biogas digester shows that 17% TKN is missing: 76 g N / d enter the biogas digester with the substrate and only 63 g N / d leave it with the effluent. The main reason for this is the transient state of the biogas digester at the time of the measurements.

In the biogas digester, approximately 45% of the total organic nitrogen entering the digester was converted into NH₄ ⁺-N. Approximately 80% of the total organic nitrogen entering the digester was from the raw chicken litter, while the remaining 20% was from the re-circulated liquid used to prepare the mixture treated with NiX.

Nitrogen balance separation liquid-solid A centrifuge was used to separate the effluent of the biogas digester into the liquid and the solid fraction. The TKN balance around the separation was accurate at 10%, which can be considered satisfactory.

On wet weight basis, approximately 70% of the TKN of the effluent was collected in the liquid fraction. More than 75% of the NH₄ ⁺-N of the effluent is collected in the liquid fraction.

Balance NiX treatment unit

The TKN and NH₄ ⁺-N in the fraction leaving the NiX treatment unit as gas during pressure release was not measured. The balance is based on the assumption that only steam and NH₄ ⁺-N were in this fraction (see Figure 18, which illustrates nitrogen flow around the NiX treatment unit (data are in g of nitrogen at a specific NiX treatment).

Correcting for the mass loss on VS basis, NiX treatment removed 47% of the NH₄ ⁺-N, in average (Figure 16, above). Organic nitrogen was not affected by the NiX treatment or only a very small part of it was converted into NH₄ ⁺-N. Only 7% of the organic nitrogen was missing from the balance and this was within the accuracy of the measurements. 5.3.2 Effect of re-circulation

Re-circulation had the overall effect of increasing the TS content of the digester at the rate of 0.4% per week and increasing the NH₄ ⁺-N concentration of the digester at a rate of 0.5 g / kg WW per week. This was due to the lack of water (no water was added to the system, aside from steam during NiX treatment). In the substrate, the VS content was approximately constantly 68% of the TS (therefore the ashes were 32% of the TS). The VS fraction of the TS of the digester increased from 44% in February 201 1 to 55% at the end of May 201 1 , indicating that the conversion of VS into biogas was decreasing. The increase was

approximately linear. A similar increase was observed in the liquid fraction. 5.3.2.1 HRT and SRT

The HRT was different from the SRT (solids retention time, retention time of the alive microorganisms), Figure 19 illustrates Hydraulic Retention Time (HRT) and Solid Retention Time (SRT). The HRT was calculated from the flow rate of material leaving the system. The HRT was approximately 145 d. This is the average time that solids and water spend in the system (the system includes the re-circulation). The HRT did not depend on the flow rate of the

recirculation. The SRT was calculated as the average time that the living microorganisms spend in the system. The SRT was 29 ± 5 d. This is acceptable for a mesophilic biogas process. The liquid fraction (re-circulate) contains microorganisms and these are killed during NiX treatment.

Therefore, the retention time of the microorganisms (SRT) had to be calculated from the flow rate of the material that leaved the digester. The SRT depends on the flow rate of the recirculation.

5.3.2.2 Effect of recirculation on t -N and yield

During mesophilic operation, the yield reached the constant 297 ± 35 mL CH₄ / g VS (where VS is the organic matter content of the raw chicken litter).

The system showed clear signs of decreasing efficiency of conversion of substrate into methane starting from May 10 (Figure 20). The yield decreased strongly from 297 ± 35 mL CH₄ / g VS with a rate of approximately 8 ml. CH₄ / g VS per day. This was due to high NH₄ ⁺-N concentration in the digester (Figure 20, Figure 21 , and Figure 22). Figure 20 illustrates methane yield during phase 2 (mesophilic operation), Figure 21 illustrates feed flow rate, biogas yield (main vertical axis), NH₄ ⁺-N concentration in the digester (secondary vertical axis), and Figure 22 illustrates TS content of the digester and of the liquid after separation with centrifuge.

On May 10, the NH₄ ⁺-N concentration in the digester was 6.4 g / kg WW and this can be considered the threshold level that caused decreased conversion efficiency or inhibition. On May 10, the concentration of CH₄ in the biogas was 55% and the tot VFA in the digester was 2000 mg/L. On May 23, the tot VFA in the digester was 2400 mg/L, just slightly higher than on 10 May. The methane yield was decreasing, but the tot VFA concentration did not show any clear or sharp increase. The methane yield decrease of March 3 - March 7 was due to a feeding pump stop. In the period April 13 - May 7 the yield showed large variations (the points in Figure 20 are scattered) because of technical problems with pumps and with the computer controlling the pilot plant. The TS and NH₄ ⁺-N concentration in the digester always showed increasing trends (Figure 21 , and Figure 22).

5.3.2.3 Effect of re-circulation on separation of liquid fraction

The effluent of the digester was separated into the liquid and the solid fraction. The liquid fraction was re-circulated and mixed with the raw chicken litter before being treated with NiX.

The separation was made by sedimentation until middle of March 201 1 and with a centrifuge from middle of March 201 1 and can be seen in Figure 22. The new separation method

(centrifugation) was introduced to decrease the TS% of the liquid fraction, because

sedimentation became not effective as the TS% of the material to be separated increased. The purpose was to decrease the TS% in the digester by decreasing the TS% of the separated liquid. However, lower TS% in the liquid obtained with centrifugation was not sufficient to lower the TS% in the digester.

Approximately 75% WW was collected in the liquid fraction. The TS content of the liquid fraction increased as the TS content of the digester effluent increased, i.e. the separation efficiency of the centrifuge used for separation depended on the TS content of the material to be centrifuged.

5.3.2.4 Effect of re-circulation on water requirements

A very low amount of water was added to the system. This was done adding steam during NiX treatment.

Steam was added to the substrate during NiX treatment and was released from it at the end of the treatment, when the pressure was released. The net amount of water added to the system during NiX treatment was the difference between the steam entering the substrate

(approximately 0.1 kg steam/kg-chicken litter, according to the steam flow meter) and the steam leaving the treated substrate. The net amount of water added during NiX treatment was measured as the difference of water in the untreated substrate (water in the raw chicken litter plus water in the re-circulated liquid) and the water in the treated substrate. On average, 0.05 kg of water (condensed steam) for each kg of raw chicken litter entered the system during NiX treatment.

Water was leaving the system via the solid fraction after separation. Mass balances and experimental data showed that the amount of water leaving the system was higher than the amount of water entering it.

5.3.3 Balances NiX treatment

Figure 23 shows the mass balance around the NiX treatment unit for a specific treatment. The data represented are per treatment. The nitrogen balance (NH₄ ⁺-N) showed that NiX treatment removed 55% of NH₄ ⁺-N from the substrate. The concentration of NH₄ ⁺-N in the treated substrate (after NiX treatment) was 3.2 g- NH₄ ⁺-N/kg-WW. These data are from one specific treatment considered. On average, NiX treatment removed 47% NH₄ ⁺-N.

5.4 Conclusions

NiX treatment removed 47% of the NH₄ ⁺-N from the substrate and decreased by 15% the NH₄ ⁺-N concentration in the biogas digester.

Re-circulation of liquid fraction separated from the effluent of the biogas digester caused increasing TS and NH₄ ⁺-N concentration in the digester because of lack of water. Addition of extra water (besides 0.05 kg H₂0 / kg WW chicken litter as steam during NiX treatment) is needed to run stable biogas processes based on chicken litter as monosubstrate. The methane yield was stable at 297 ± 35 mL CH₄ / g VS (where VS is the organic matter content of the chicken litter) and decreased probably because of NH₄ ⁺-N inhibition when the NH₄ ⁺-N concentration in the digester became higher than 6.4 g / kg WW. Nitrogen measurements showed that the concentration of organic nitrogen does not change during the 20-minute NiX treatment. Approximately 30% of the organic nitrogen is converted into NH₄ ⁺-N during the storage of the NiX treated mixture in the substrate tank. The biogas process resulted in 45% conversion of organic nitrogen into NH₄ ⁺-N.

5.5 Appendices

5.5.1 Appendix A: NiX treatment

NiX treatment was applied with Xergi's thermo-chemical treatment unit. The substrate for NiX treatment were the mixtures of raw chicken litter and water or of raw chicken litter and liquid separated from the digester's effluent.

Steam at 8-12 bar was injected into a chamber containing the substrate in the proportions given below. When the set-point relative pressure (4 bar, corresponding to approximately 140 °C inside the chamber) was reached, it was maintained for 20 minutes. The sample was mixed during the treatment.

Pressure is expressed as relative pressure above atmospheric pressure (gauge pressure). A pre-heating of the external walls of the chamber and injection of steam on these walls during NiX treatment was made to minimize steam condensation inside the chamber during the treatment. At the end of the treatment, the pressure was released in approximately 2 hours. The treatment conditions are summarized below: Thermophilic operation:

Sample: raw chicken litter

Water: 4.17 kg water per kg WW of raw chicken litter

Catalyst: 2% Ca(OH)₂ (percentage by wet weight of total mixture)

Treatment: 4 bar (140 °C), 20 min, mixing

Mesophilic operation:

Sample: raw chicken litter Water: no water added, aside from steam during steam treatment (net approximately 0.05 kg steam per kg WW chicken litter)

Liquid: liquid fraction from effluent biogas digester, 2.61 kg WW / kg WW raw chicken litter Catalyst: 2% Ca(OH)₂ (percentage by wet weight of total mixture)

Treatment: 4 bar (140 °C), 20 min, mixing

5.5.2 Appendix B: batch experiments

The methane potential was determined in batches, with infusion bottles of 543 mL total volume, at 52 °C. Anaerobic digested effluent from a thermophilic biogas plant (52 °C) was used as inoculum for the batch assays.

Inoculum (200 mL) and substrate were added into the bottle in the amounts given in Table 19. Control batches with pure cellulose as substrate (for examination of inoculum viability) and blank batches without substrate (to measure the methane production from the inoculum) were included. The assays were made in triplicates.

To calculate the effect of NiX on the methane yield of the chicken litter, the results from the batch tests were corrected for the ratio between raw chicken litter and total mixture and for the contribution to the methane yield from the liquid separated from the effluent. The results of the calculations are given in Table 19:

Table 19: Effect of NiX on the methane yield of the chicken litter

Liquid 16.41 6.3 3.4 121 ± 6

Substrate 6.80 21 .7 16.6 314 ± 18 collected

immediately after

NiX treatment

Table 20: Batch experiments: batch preparation and methane yield

5.5.3 Appendix C: uric acid

Uric acid was measured once as a preliminary test. Uric acid measurements were made to have a better understanding of the degradation of nitrogen in the system.

Uric acid is a non-protein organic nitrogen compound, main end product of purine metabolism and present mainly in urine.

On 10 May 201 1 , preliminary uric acid measurements were made on samples of:

Raw chicken litter

NiX-treated mixture of raw chicken litter and liquid fraction from effluent digester

- Material lost during pressure release from NiX treatment unit

- Substrate sampled from substrate tank

Digester effluent

Liquid fraction separated from digester effluent

- Solid fraction separated from digester effluent The preparation of the samples was made as described in:

B. Pekic, B. Slavica, Z. Zekovic

High-Performance Liquid Chromatographic Determination of Uric Acid in Feces of Egg- Laying Hens

Chromatographia Vol. 27, No. 9/10, May 1989.

The dried sample was extracted into a solution of Li₂C0₃ (extraction made in Xergi's laboratory) then this liquid fraction was injected into a HPLC (HPLC analysis made by Aalborg university) The results of the preliminary uric acid measurements are given in Table 21 (concentrations of TKN and NH₄ ⁺-N are given for comparison):

Table 21. Results from preliminary uric acid measurements (data are concentrations)

According to the preliminary measurements for raw chicken litter, uric acid is approximately 10% of the TKN. This result is lower than values from literature, which indicate that uric acid is approximately 60% of total N.

The reason for this difference may be an incomplete extraction of uric acid from the sample into the liquid fraction to be injected into the HPLC. Results from HPLC seem reliable, according to the calibration curve.

The flow and degradation of uric acid in the system were calculated with the concentrations obtained from the preliminary uric acid measurements. These are summarized in Figure 24, illustrating nitrogen flow in the system (data are flowrates, g of nitrogen per day), where

CL: chicken litten raw

Liquid - liquid fraction of effluent digester after separation with centrifuge

NiX - NiX treatment material collected immediately after NiX treatment

Loss - loss after NiX treatment during quick pressure release

SR - substrate tank (small reactor)

LR - biogas digester (large reactor)

Solid - solid fraction of effluent digester after separation with centrifuge

The data in Figure 24 are flowrates, g of nitrogen per day. These have been calculated from the measured concentrations (g of nitrogen per kg wet weight) and the measured flowrates (kg wet weight per day). Therefore, measurement errors and uncertainties have to be taken into account when considering the flows shown in Figure 24.

For example, the balance around the NiX treatment unit shows that approximately 6% of organic nitrogen is missing:

58 + 15 = 73 g N / d

73 = 94% of 78 g N / d

This difference is due to measurement uncertainties.

The degradation of uric acid was slightly higher than the degradation of organic nitrogen (Table 22), however a similar trend can be observed: lowest degradation during NiX treatment, higher in substrate tank and highest in biogas digester.

Table 22 Degradation of org-N and uric acid in the system

The results above have to be considered preliminary because of the suspicion of incomplete extraction of uric acid during measurements.

Example 2

Parameter values for pre-incubation step In the following, the meaning of "process" refers to degradation of organic nitrogen to ammonium.

1. Temperature

The best result obtained so far at 33°C. 46 °C and 59 °C yields significantly poorer effect. Optimum temperature is expected to be established around 37°C. Conclusion

Temperature optimum for uric acid degrading enzyme (urease) is approx. 60°C. Expected optimum temperature indicates that the process is biologically induced by mesophilic microorganisms.

2. Pressure

No external over pressure is applied and no significant pressure build up takes place. 3. Aerobic/anaerobic conditions

Three different levels of oxygen were tested: Low, Medium and High. "Low" is flushing with nitrogen to remove all oxygen in headspace and dissolved in the liquid, "Medium" is with atmospheric air in headspace, and "High" is flushing of headspace with oxygen. The process slows down in the "High" treatment, while the rate is the same both in "Low" and "Medium".

Conclusion

The process performs best at low oxidation state and indicates that the process is carried out by facultative anaerobic microorganisms.

4. Liquid for dilution

Three different types of liquid for dilution have been tested: Water, separated digester liquid (TS 6.8 %) and separated pre-tank liquid (TS 9.2 %). The two separated liquids originated from the pilot plant operated on NiX-treated chicken litter.

Separated liquid from pre-tank as well as from digester has a significant positive effect on the rate compared to water. Conclusion

Positive effect of separated liquid from pre-tank and digester, respectively, can be explained by presence of microorganisms and/or enzymes in these liquids or by supplying the process with favorable physical/chemical conditions. 5. Dry matter

Various dry matter (TS) contents from 10 to 47 % have been tested in mixtures of hen litter and water. Optimal effect has been obtained at TS 10 %. At TS 15 % the rate was reduced but the final effect after 7 days was the same as at TS 10 %. At TS 20 % the rate was further reduced but the final effect after 7 days was the same as at TS 10 %. At TS equal to or higher than 25 % the effect is almost absent even after 7 days

Conclusion

Using hen litter and water, increases TS levels to >10 %, which have a negative effect on process rate. TS >/= 25 % have negative effect on both rate and final effect. Optimal TS level will depend on biomass and liquid used for dilution.

6. pH

Experiments have been carried out with chicken litter and separated digester liquid where pH in the solution was manipulated up or down by adding lime (pH 10,5 in solution) or sulphuric acid (pH 6,5 in solution), respectively, compared to no pH manipulation (pH 8,5 in solution).

There was no significant effect on the process of the pH manipulation. In all cases the final pH value after 22 days stabilized within the range 6.4 - 7.2. The initial, relative pH levels were reflected in the final pH levels.

Conclusion

The process is not sensitive to different initial pH-levels in the range from slightly acidic to highly basic.

7. Time

Highest rate obtained at 33° C with full effect after approx. 84 hours. Rate expected to be increased at optimum temperature (see this). Further rate increase expected by retaining a small fraction (eg. 10 %) of each batch for subsequent seeding of next batch.

Conclusion

Process minimum cycle time is expected to be less than 84 hours by temperature optimization and kick-start by retention of small fraction of each for seeding.

8. Degradation of organic nitrogen

Best results obtained have shown 70 - 80 % degradation of organic nitrogen to ammonium. Uric acid is degraded completely, while protein-bound nitrogen is degraded by approximately 33 %.

Enzymatic treatment could potentially increase the degradation of proteins. 9. Loss of organic dry matter

Organic dry matter (VS) loss is in the range 5 - 10 %. Degradation of organic nitrogen can explain nearly all VS loss and even in a worst case scenario still more than 50 %.

10. Gas production

Approx. half of the VS loss can be attributed to methanogenesis. However, methane production does not seem to be a requirement for the obtained rates and extents of organic nitrogen degradation.

EXAMPLE 3

Mineralisation of organically bound nitrogen to TAN 1 Abbreviations

AD Anaerobic digestion

AD liquid - Liquid from an anaerobic digester

BMP Biological methane potential

cTAN Centrifuged TAN

N Nitrogen

NmL Normal mL (volume at 1 bar and 273 K)

OrgN Organic Nitrogen

PC Pressure cooking

RSD Relative standard deviation

RT Room temperature of the research facility (between 17 °C and 20 °C)

SD Standard deviation

TAN Total Ammoniacal Nitrogen

TKN Total Kjeldahl Nitrogen

TS Total Solids

VS Volatile Solids

w/w% Weight percentage

2 Summary and Conclusion

This example covers characterization and optimization of "mineralization", a process of shifting of organic fraction (OrgN) of the nitrogen pool in animal litter to the inorganic fraction (TAN). The mineralization process is described later in Example 4, where it is shown that the organic fraction (OrgN) of the nitrogen pool in chicken litter could be shifted to the inorganic the inorganic fraction (TAN).

The focus is on investigating substrates (chicken litter vs. hen litter), suspension liquid (AD liquid vs. water), pH, water content, temperature, and seeding. Material with an active microbiological flora from a previous incubation is added to the samples to "kick-start" the process.

As a result of a series of experiments, following questions are addressed in the example:

1 . Can mineralisation be performed on different a poultry substrates like chicken litter, hen litter?

2. Does mineralisation of the nitrogenous compounds occur due to chemical hydrolysis or biological metabolism? If the process is biological, does the microbial community facilitating the conversion stem from the AD liquid or the poultry substrate?

3. What is the extent of nitrogen mineralisation in protein bound nitrogen and uric acid bound nitrogen?

4. What is the level of VS degradation associated with mineralisation, and is VS degradation coupled to diminished energy potential of the substrate?

5. What is the effect of water content, temperature and oxygen?

6. What is the effect of seeding the process with biologically active material?

The primary conclusions from the experiments are:

1 . Mineralisation of OrgN to TAN was performed both on chicken litter and hen litter. The chicken litter substrate used in Example 4 experiment contained bedding as well as faeces, whereas the hen litter used in the subsequent experiments in this example only contained faeces. It therefore seems likely that this type of mineralisation could occur in most poultry litters, since the two types of poultry substrates are different both in terms of the animal source and the presence of bedding.

2. The mineralisation process is most likely due to a biological process. It is independent of the incubation liquid used and works equally well with AD liquid and water. No methane production is observed in the samples containing hen litter and water. The microbial community able to perform this type of mineralisation is thus inherently present in the poultry manure, and methanogenesis is not a prerequisite for mineralisation.

3. Nitrogen mineralisation levels range from 70%-80% conversion of the total organic nitrogen fraction. Uric acid nitrogen constitutes 50% of the OrgN fraction, and was completely degraded in all analysed samples. Degradation of the remaining OrgN pool (primarily protein bound nitrogen) ranged from 48-60%. 4. TS and VS values are not completely consistent and further accuracy could be obtained by re-analysis. However, losses average around 10% of the total VS pool. Mineralisation of uric acid and protein is estimated to account for at least 80% of this VS loss. The total methane potential loss from mineralisation is less than 4%.

5. Nitrogen mineralisation is very dependent on the water content of the samples.

Mineralisation becomes significantly slower with decreasing amounts of water. The temperature optimum for the process is in the range 33 °C - 37 °C. High oxygen levels were inhibitory to the reaction.

6. Seeding with up to 20% of material from an active culture significantly speeds up the

mineralisation process.

3 Materials and Methods

3.1 Analytical methods

3.1.1 Dry Matter

Total solids (TS) and volatile solids (VS = organic dry matter) were determined in triplicate according to Danish Standard⁴, TS were determined by heating the samples to 105°C for a minimum of 24 hours to constant weight. VS were determined by burning the samples at 550 °C for 3-4 hours.

The methods are modified to take into account the evaporation of volatile fatty acids during drying.

Table 23 - TS and VS levels of the substrate materials. Seeding materials vary in TS/VS depending on the setup.

3.1.2 Nitrogen content analysis

Samples were analysed for Total Ammonium Nitrogen (TAN) and Total Kjeldahl Nitrogen (TKN) according to the Kjeldahl method.

⁴ TS: DS/EN 12880. VS: DS/EN 12879. The centrifuged TAN (cTAN) method was developed to minimize the amount of sampling needed to monitor the shift in nitrogen during incubation. Samples were centrifuged at 3500 x g (rcf) for 12 minutes after which the supernatant was analysed for TAN. Removal of particulate material enabled the sampling of much smaller amounts while still maintaining reproducibility.

Destruction of samples was performed on a Tecator™ Digestion Unit Auto Lift 20 and distillations were performed on a Buchi K355 distillation unit. 3.1.3 Volatile fatty acid analysis

Volatile fatty acids (VFA) were analysed on an Agilent 7890A chromatograph using a 0.23 mm Wax capillary column.

3.1.4 Uric acid analysis

Uric acid was analysed using the method outlined in Pekic Chromatographica v.27 no. 9/10 p.467, 1989.

3.1.5 Methane measurements

The CH₄ content in the headspace of the batch bottles was measured by GC (Shimadzu, 2010) equipped with a capillary column (wax 0.53 mm ID, 30 m) and a FID detector.

By means of a standard curve created by injection of various volumes of 100 % pure CH₄, the number of CH₄ molecules in the headspace was determined at regular intervals. Based on this the volumetric CH₄ production could be calculated during the test period. The biogas produced by the batches was released several times during the experiment in order to maintain low pressure in the bottles.

The specific methane yield (Nml methane per gram substrate VS added) is calculated by subtraction of background, normalizing to standard pressure and temperature (STP) and relating the yield to the quantity of VS added.

3.2 Experimental setup

All substrate materials (Hen litter, Centrifugal liquid from AD, seeding material) were homogenized and mixed in a suitable container. Each container was thoroughly mixed, the final weight registered and covered with parafilm. Containers were mixed when appropriate. Before sampling, masses were registered and adjusted with water to compensate for evaporation. 4. Results

4.1 Introduction

Experiments were set up sequentially with the purpose of characterizing the mineralization process. The Experiments 1 investigated whether the process could be repeated in poultry substrates different from that used in Example 4 experiment and the effect of substituting AD liquid with water. Experiment 2 tested the effect of water content on the process. Experiment 3 made a rough determination of the temperature optimum, and investigated the effect of different oxygen levels. Experiment 4 made a more exact determination of the temperature optimum, and looked into the effect of seeding the process with an active culture. Experiment 1 was run for 17 days; experiments 2-4 were run for 7 days.

In the following sections the results from all 4 experiments are summarised. Before setting up the experiments substrate materials were analysed for TS/VS, TKN/TAN, cTAN, VFA and/or Uric Acid. From these data, starting concentrations of each experimental setup was calculated. During or after incubation, the samples were analysed for some or all of the above parameters. 4.2 Suspension liquid

In Example 4 experiment, we show that the mineralisation process worked equally well when chicken litter was suspended in AD liquid or liquid from a buffer tank used to feed the digester. In this experiment we investigated the difference between using water and AD liquid for suspension of hen litter. Hen litter was suspended in either AD liquid or water and the process was monitored for TAN concentration. The experiment was run at room temperature (RT) between 17 °C and 20 °C for 17 days with 15% TS.

Figure 31 illustrates Development of TAN. Comparison between water and AD liquid as suspension liquid for hen litter. Darker bars: TAN concentration at day 0; Lighter bars: TAN concentration after 17 days incubation according to an embodiment of the invention;

As may be seen from the columns in the Figure 31 , the TAN increase after 17 days incubation of hen litter with either water or AD liquid is almost identical in the two samples. Nitrogen mineralisation is thus not dependent on biological or chemical constituents in the AD liquid. It should be noted that pH and cTAN measurements showed that the process reached final yields after about 7 days, and that the samples containing AD liquid reacted a little faster than the samples containing water (data not shown). 4.3 Water content

To determine the influence of water content on mineralisation speed and final yield hen litter was suspended in varying amounts of water. The TS content was varied in discrete steps, and the reaction was allowed to run at RT for 7 days, while monitoring TAN levels. Refer Figure 32, which illustrates development of TAN for seven TS levels.

On day 3 the sample containing 15% TS begins to show significant mineralisation levels. The 20% TS sample reaches the same final yield after 7 days although at a slower rate. The remaining samples with less water content show no significant mineralisation levels within the 7 day test period.

4.4. Temperature and oxygen

The following experiments are all performed at 20% TS. The effect of temperature on the reaction speed and final mineralisation yield was investigated first in large temperature intervals and subsequently in smaller steps. In the first trial samples were set up at RT, 33 °C, 46 °C and 59 °C. Temperature intervals were based on the assumption that the bottle neck in the reaction might be the urease enzyme, which catalyses the conversion of urea to ammonia. Urease has an activity profile which increases steadily until it reaches an optimum around 68 °C after which it drops off sharply. Oxygen levels were investigated by either flushing the bottles with N₂ (low oxygen level), 0₂ (high oxygen level) or without flushing (normal oxygen level).

There was no difference between low and normal oxygen levels with respect to mineralisation speed and ultimate yield at all four temperatures. However, high oxygen levels significantly impaired mineralisation speeds (data not shown).

Figure 33A illustrates development of TAN at normal oxygen level. It is evident that 33 °C results in faster mineralisation speeds compared to the other temperatures tested.

The reason why the RT sample did not show the same TAN increase as shown in later Example 4 is possibly because the temperature in the research facility had been lower during this experiment. To further narrow down the optimal range, the temperatures surrounding 33 °C were investigated. Since the microbial community in the litter stems from the intestines of hens, the physiological temperatures 30 °C and 37 °C were chosen. The TAN development showed that the mineralisation process at both temperatures tested was comparable to 33 °C (see Figure 33B, which illustrates development of TAN at physiological temperatures). The fact that the process is inhibited at high oxygen levels and that it has a temperature optimum around 30 °C - 37 °C, is a strong indication that the mineralisation process is biological. Chemical reactions are not likely to be inhibited by increasing oxygen levels, and would be expected to increase its speed with increasing temperatures.

In these experiments it is not possible to conclude if the process is faster at temperatures between 37 °C and 46 °C. However, since the process is likely bacteriologically catalysed and most intestinal bacteria have temperature optimal around 37 °C, it is likely that the process will not increase significantly beyond this temperature.

4.5 Seeding

Seeding of the sample during incubation is carried out by adding material from a previous successful incubation. In the seeding material the bacterial cultures are actively growing and metabolising. If the mineralisation process, as extrapolated, is catalysed by bacteria then these are likely to be dormant due to the high TS content of the hen litter. A certain time interval will thus be required after hydration to activate their metabolism, causing a lag-phase before TAN development may be detected. Seeding the sample with an active bacterial population is therefore expected to shorten the lag-phase.

Seeding of the samples with 20% material from previous incubations resulted in significant increases in reaction speed at all temperatures (see Figure 34, which illustrates effect of seeding on the mineralization process). After 48 hours more than 90% of the final yield is obtained. The TAN development in this experiment is a little slower at 30 °C than at 33 °C and 37 °C. It should be noted that final TAN levels may be a little higher than the true value since it was assumed that the OrgN fraction from the seeding material was not mineralised further. 4.6 Organic nitrogen

Experiment 1 was analysed for organic nitrogen content before and after incubation. Organic nitrogen (OrgN) in poultry litter is bound in uric acid (UA) and proteins.

Figure 35 illustrates content of uric acid bound nitrogen and protein bound nitrogen before and after mineralization. From the figure, it can be seen that nitrogen mineralisation levels are not influenced by the type of suspension liquid. UA is 100% degraded while protein bound N is degraded by approximately 40%. On average total mineralisation levels range from 71 % - 80%. 4.7 TS and VS

Initial TS and VS levels of the mixtures were calculated from the initial amounts of substrate (see Table 23 above). After incubation TS and VS were determined by analysis.

Actual dry matter changes in these types of experiments are inherently difficult to determine. The reason for this is that the microbiological communities in the AD liquid and hen litter change when they are mixed. The VS degradation profiles may thus also change, which makes it difficult to compare controls with samples. Figure 36A and Figure 36B shows the dry matter loss (VS) and TS changes during incubation of the samples respectively. Samples were incubated for 17 days at RT.

In the samples containing AD liquid and hen litter the VS loss is 7%. However, the VS loss in the AD liquid control is higher than this, indicating that the entire VS degradation occurs in the AD liquid.

A more accurate estimate may come from the samples where hen litter is suspended in water. In these samples the VS loss is directly coupled to mineralisation. The VS losses in these samples are on average 10%.

4.9 Volatile Fatty Acids (VFA)

VFAs are breakdown products from hydrolytic action on carbohydrates, protein and fat.

Acetate is produced during anaerobic breakdown of UA. The VFA is monitored during the experiment and was also monitored in Example 4 experiment.

Figure 37 illustrates net VFA production during incubation. The figure shows that there is a significant production of VFA's during the mineralisation process. In particular acetate is increased during incubation but also propanoate and butanoate show significant increases. 4.10 Methane

Example 4 experiment showed that there was a significant VS loss during incubation. In order to investigate whether the VS was degraded to methane, headspace samples were analysed for methane content. Figure 38 illustrates specific methane production in the incubated samples. The figure shows that there is no methane production in the sample incubated with water. This was suspected as hen litter is not known to contain methanogenic microbes. In contrast, there is a small methane production in the samples containing AD liquid. However, the absolute production only amounts to 2-3% of the theoretical methane potential.

5 Discussion

Breakdown of simple compounds such as uric acid (see below) may be caused by chemical hydrolysis reaction or by microbial metabolism.

Chemical formula for Uric Acid

In the experiments described here, there are several strong indications that the process is biological. During the mineralisation process there is a significant production of VFAs, in particular acetate and butanoic acid. There is a temperature optimum around 33°C - 37°C, and the process is affected by varying oxygen amounts. Finally, seeding causes a significant increase in reaction speed. A positive correlation between water content and mineralisation speed would have been expected regardless of whether the reaction had been chemically or enzymatically catalysed (see Section 4.3 of this Example). However, the nature of the TAN development indicates that the reaction has a lag-phase that is dependent on the amount of water present. This is consistent with a microbiological community which needs to re-establish its metabolism and begin proliferating after being dormant, and also explains why seeding drastically shortens the lag-phase.

Since the mineralisation process is followed by anaerobic digestion the question arises whether the methane potential of the hen litter is diminished during mineralisation.

Mineralisation of nitrogen will inherently result in a VS loss since organic material is converted to inorganic compounds. However, VS loss is not necessarily coupled to loss in methane potential. In protein degradation one of the first steps is deamination of the amino groups. This occurs independently of whether the environment is aerobic or anaerobic. Since the mineralisation step takes place in both cases, it does not cause a loss in methane potential.

When uric acid is aerobically degraded, the end products formed are ammonia and carbon dioxide (see Equation below). Anaerobically, the process produces compounds such as acetate, formate and glycine in addition to ammonia and carbon dioxide. Aerobic breakdown thus leads to loss in methane potential, because of the difference in the reaction products compared with anaerobic degradation.

Aerobic

» C0₂ + NH₃ + H₂0

Uric Add Anaerobic

> C0₂ + NH₃ 4- Acetate + Formate + Glycine

Uric acid breakdown thus differs with respect to VS loss depending on the pathway used. Aerobic breakdown causes complete conversion into inorganic compounds, whereas anaerobic breakdown only causes 60% conversion into inorganic compounds (Diirre and Andreesen, 1982).

If uric acid is broken down aerobically the VS loss from the mineralisation process would be approx. 13%, whereas anaerobic breakdown of uric acid would result in 8% VS loss (see Figure 39, which illustrates contribution of nitrogen mineralisation to VS loss in hen litter under aerobic and anaerobic conditions). Comparing this to the observed 10% VS loss (see Section 4.7) this means that approximately half of the uric acid seems to be broken down aerobically and half anaerobically.

The methane potential loss depends on the loss associated with the part of uric acid that is broken down aerobically. Since uric acid has a theoretical methane potential of 100 NmL/g VS the hen litter loses approx. 5-6 NmL CH₄ per treated gram hen litter VS⁵ (corresponding to 2 NmL/g hen litter).

It should be noted that when adding AD liquid to the hen litter, 2% - 3% of the VS is lost during the mineralisation process due to methane production. If the gas is not collected this will also contribute to diminished methane potential once the hen litter enters the biogas reactor.

⁵ 5,5 g uric acid pr. 100 gram hen litter VS with a methane potential of 100 NmL CH4/g VS. 5 References:

Dijrre P, Andreesen JR; Anaerobic Degradation of Uric Acid via Pyrimidine Derivatives by Selenium-starved Cells of Clostridium purinolyticum. Arch Microbiol (1982); 131 :255-260

EXAMPLE 4

Degradation of organically bound nitrogen to TAN using pre-hydrolysis 1 Abbreviations

AD - Anaerobic digestion

BMP - Biological methane potential

cTAN - Centrifuged TAN

N - Nitrogen

NmL - Normal mL (volume at 1 bar and 273 K)

OrgN - Organic Nitrogen

PC - Pressure cooking

RSD - Relative standard deviation

SD - Standard deviation

TAN - Total Ammoniacal Nitrogen

TKN - Total Kjeldahl Nitrogen

TS - Total Solids

VS - Volatile Solids

w/w% - Weight percentage

2 Summary and Conclusion

In the CSTR experiment with chicken litter, an increase in TAN during pretank storage of the substrate was observed. The CSTR trial setup consisted of a one-stage mesophilic AD.

Effluent was separated and the liquid was used to suspend chicken litter prior to NiX treatment. The treated chicken litter was stored in a pretank from which daily portions were fed into the AD.

The current experiments summarised in this example answer the following questions -

1 . Can the increase in TAN be repeated/ confirmed under controlled conditions and is it correlated with a corresponding decrease in organically bound nitrogen (OrgN)?

2. Is it possible to shift the nitrogen from OrgN to TAN before NiX treatment to increase the pool of removable nitrogen (mineralization process) and to what extent?

3. Is the OrgN to TAN shift sensitive to pH? 4. Is uric acid more or less susceptible to degradation than other organic nitrogen containing compounds (proteins etc.)?

The conclusions from the experiments are:

1 . The TAN increase was repeated and correlated with a corresponding decrease in OrgN.

The cause for the shift is likely hydrolysis of uric acid and/or protein. A significant increase in VFA's during the incubation indicates that the process is microbiological.

2. It was possible to carry out the nitrogen hydrolysis (N-hydrolysis), using liquid from

separated AD effluent prior to NiX treatment. N-hydrolysis was only observed in the fresh chicken litter and not in the liquid fraction from the separated effluent. The amount of OrgN hydrolysed during this treatment ranged from 37-50%, thus increasing the amount of strippable nitrogen from 20% of the total nitrogen pool to 49-60%.

3. The N-hydrolysis was not affected significantly by pH shifts in either acidic or basic

direction.

4. Uric acid in the chicken litter is completely degraded and may account for up to 60% of the entire production of TAN during incubation.

5. There is a significant VS loss during incubation (1 1 -17%), most likely caused by anaerobic degradation. However, determination the contents of the gas produced requires

experimentation, and whether it may be avoided or collected and utilised for power production.

3 Materials and Methods

3.1 Analytical methods

3.1.1 Dry Matter

Total solids (TS) and volatile solids (VS = organic dry matter) were determined in triplicate by heating the samples to 105 °C for a minimum of 24 hours. VS were determined by burning the samples at 550 °C for 3 - 4 hours.

3.1.2 Nitrogen content analysis

The centrifuged TAN (cTAN) method was developed to minimize the amount of sampling needed to monitor the shift in nitrogen during incubation. Samples were centrifuged at 3500 x g (rcf) for 12 minutes after which the supernatant was analysed for TAN. Removal of particulate material enabled the sampling of much smaller amounts while still maintaining reproducability. Destruction of samples was performed on a Tecator™ Digestion Unit Auto Lift 20 and destinations were performed on a Buchi K355 distillation unit.

3.1.3 Volatile fatty acid analysis

3.1.4 Uric acid analysis

3.2 Experimental setup

All substrate materials (Chicken Litter, Centrifugal liquid from AD, Centrifugal liquid from pretank) were homogenized and added to a suitable container in the amounts shown in Table 24. Each container was thoroughly mixed, the final weight registered and covered with parafilm. Every day (except weekends) the containers were mixed. Before sampling the weights were registered and adjusted with water to compensate for evaporation. Controls consisted of centrifugal liquid from both AD and pretank stored under the same conditions as the mixed samples.

Chicken litter inoculatChicken litter inoculatChicken litter inocuChicken litter inoculated with AO liquid - ed with AD liquid - lated with AD liquid - ed with pretank mateacidic 'neutral' alkaline rial

1A IB 2A 2B 3A 3B 4A 48

Chicken Utter 211 211 211 ^' 211 ^"' 211 ^' 211 211 ^" 211

Centrifugal liquid from AD 704 704 704 704 704 704 0 0

Centrifugal liquid from pretank 0 0 0 0 0 0 704 704

Wate 85 85 8S 85 85 85 85 85

CaO 0 0 0 0 1.44 1.44 ^" 0

H2S04 To pH 6.5 To pH 6.5 _{. .} 0 0 0 :i S o

Table 24: Overview of experimental setup. All units are in grams. A and B are duplicate setups with identical conditions

4 Results

Before setting up the experiments as described in the preceding Section 3.2, all substrate materials were analysed for TS/VS, TKN/TAN, cTAN, VFA and Uric Acid. From these data, starting concentrations of each experimental setup was calculated. During the incubation period each of the setups were analysed for pH and cTAN to monitor the proces. 4.1 TS and VS

The results of the TS and VS analysis of the substrate materials are shown in Table 25, below.

Unit I Chicken Litter AD liquid j Pratank liquid

Total Soiids w/w% 68.1 ±0 1 8,8 ±0.01 9 2 ±0.01

Volatile Soiids w w% 4.0 ±0,1 i 5.9 ±0.1

Table 25: TS and VS levels of the substrate materials

TS and VS levels of the mixtures were calculated from the starting amounts used of each substrate (see Table 24). After incubation all samples were analysed for changes in solids content. Figure 40A illustrates TS content of incubated samples, Figure 40B illustrates VS content of incubated samples, where darker bars representing Day 0 and lighter bars Day 22 according to an embodiment of the invention;

There is a 5-17 % drop in TS during incubation (Refer Figure 40A). VS drops are in the order of 1 1 -17 % (Refer Figure 40B). For all samples, except the alkaline adjusted, there is a nice correlation between VS and TS loss, with VS losses being slightly higher or equal to TS losses. It is thus possible that the apparent lower TS loss in the alkaline adjusted is due to sampling errors. If the VS loss is due to aerobic, then C0₂ and H₂0 are produced. However, if the VS loss is caused by anaerobic degradation, CH₄ and/or H₂ are produced_.

4.2 pH and cTAN

The change in pH and cTAN may be seen from Figure 41 A - Figure 41 D. Figure 41 illustrates development of pH and cTAN during incubation with chicken litter inoculated with pretank material (Figure 41 A), with AD liquid - 'neutral' (Figure 41 B), with AD liquid - alkaline (Figure 41 C), with AD liquid - acidic (Figure 41 D), where cTAN data depicted on the right Y-axis and pH on the left Y-axis, according to an embodiment of the invention. In all cases there are significant pH drops during the incubation period. In the two setups where pH was not adjusted prior to incubation (Figure 41 A and Figure 41 B) the pH drop occurs quickly within 2 days. In the setup with added CaO the drop continues until app. day 8 (Figure 41 C). In the acidified sample (Figure 41 D) there seems to be an increase in pH that occurs during the first day, after which the pH development looks similar to the other setups. It seems that the final pH level settles between 6.5 and 7.5, independently of the starting pH. TAN increases for all setups during the first 8 days of incubation. However, as it is not known if the increase is linear or sigmoidal it cannot be concluded whether the maximal level is reached before the 8^th day. The increase in cTAN ranges from 40% for the alkaline setups to 50-56% for the 'neutral' and acidic setups and 70% for the pretank liquid setup.

The increase in cTAN is not due to a process occuring in the liquid substrate materials (see Figure 45, which illustrates cTAN in substrate materials according to an embodiment of the invention) and is thus a result of breakdown of nitrogen fractions in the chicken litter.

4.3 TAN, TKN and OrgN

Centrifuged TAN and pH provide an insight into the development of the inorganic nitrogen fraction during incubation. However, cTAN may only be used as a relative measure of the development, as the majority of particular matter has been removed. The absolute nitrogen levels may be seen in Figure 42A - Figure 42E. Figure 42 illustrates TAN, TKN and OrgN levels according to an embodiment of the invention, where Figure 42A illustrates TAN levels before and after incubation, where the increase after 22 days is shown above the lighter bar, Figure 42B illustrates TKN levels before and after incubation, where the decrease after 22 days is shows above the lighter bar, Figure 42C illustrates OrgN levels before and after incubation, where the decrease after 22 days is shown above the lighter bar, Figure 42D illustrates OrgN levels before and after incubation for chicken litter alone, where the decrease after 22 days is shown above the lighter bar, Figure 42E illustrates TANIevels before and after incubation for chicken alone, where the increase after 22 days is shown above the lighter bar. TAN is increased in all samples during treatment (see Figure 42A) ranging from 36% - 48% in the samples inoculated with AD material, up to 56% in the sample with inoculated with pretank material. There seems to be a larger TAN increase in the acidic than in the neutral and alkaline sample. However, it cannot be concluded if this is caused by a more efficient N-hydrolysis or due to ammonia evaporation from the neutral and alkaline samples as a result of higher pH.

Total ammonia (TKN) levels only change slightly during the test (see Figure 42B). The TKN loss observed is likely due to either sampling (for cTAN analysis), loss during mixing of the samples, or ammonia evaporation, and is considered insignificant in the following calculations. The change in OrgN is calculated as the difference between TAN and TKN, and may be seen from Figure 42C. The decrease in OrgN in the mixture ranges from 27%-37%. From the substrate controls it can be seen that there is no TAN increase in the inoculum liquids from pretank and AD (Figure 45). The entire TAN increase/ OrgN decrease can thus only be attributed to N-hydrolysis in the added chicken litter. When the nitrogen change is correlated to the OrgN from chicken litter only, degradation levels range from 37% to 50% in the organic nitrogen fraction of the chicken litter (see Figure 42D).

When the TAN increase is correlated to chicken litter alone, the amount of strippable nitrogen is seen to increase by a factor 2.5-3 (see Figure 42E). The potential for removing nitrogen prior to an-aerobic digestion is thus increased significantly.

4.4 Uric acid

The change in Uric acid during the incubation period may be seen from Figure 43, which illustrates uric acid levels before and after incubation, where the decrease after 22 days is shown above the lighter bar, according to an embodiment of the invention.

The vast majority of uric acid is removed during the 22 day incubation period. In absolute amounts the removal of uric acid corresponds to app. 1 ,1 mg/g sample, while the TAN increase is between 1 ,8 - 2,4 mg/g sample. 4.6 VFA

Figure 44 illustrates VFA levels according to an embodiment of the invention, where Figure 44A illustrates VFA levels in substrates, Figure 44B illustrates VFA levels in samples, Figure 44C illustrates change in VFAs during incubation as seen by subtracting substrate VFA levels from the sample VFA levels, and Figure 44D illustrates total VFA before and after incubation.

VFA levels were measured in the substrates prior to mixing (see Figure 44A). After incubation (on day 22) substrate controls and all samples were analysed for VFA composition. Absolute VFA levels may be seen in Figure 44B. When compared to the initial VFA composition in the substrates, all samples show a significant change in the composition of volatile fatty acids, There is a clear trend that production of acetic acid and butanoic acid takes place in all samples containing AD liquid, and the amount of propionic acid is decreased. The ratio between acetic acid and butanoic acid becomes higher with increasing starting pH, as does the decrease in propionic acid. The reason for this is not yet known. In the sample containing pretank liquid, the total amount of VFA's are only slightly increased. There is a significant increase in butanoic acid only, and a slight decrease in acetic and propionic acid.

In the substrate controls there is a decrease in acetic acid and propionic acid (see Figure 46, which illustrates development of VFA levels in substrates from beginning to the end of the experiment included here in Example 4). It is not possible to compare the samples and the substrate controls directly since the substrate controls have been incubated without addition of substrate. The microbiological environments change according to organic loading and it is therefore not certain if the decrease of inherent VFA's in the controls also occurs in the samples. However, if that is the case the VFA increase observed in the incubated samples is even higher than what is shown in Figure 44C. There is a significant drop in acetic acid and propionic acid in the liquid substrates during incubation. This does not necessarily indicate that a corresponding drop takes place in the samples which contain both AD/ pretank liquid and chicken litter, since the high organic load in these samples may have caused inhibition of the VFA breakdown pathway. If it does occur, however, the net amount of VFA produced due to hydrolysis is higher than indicated, since the contribution of the starting materials to the total VFA level would have decreased.

Total VFA levels increase by a factor 2 with respect to the starting conditions as shown in Figure 44D.

EXAMPLE 5

NiX treatment of high-solids & high-nitrogen biomasses for continuous biogas production

Pilot-scale experiments

The pilot-scale experiments were performed at 37°C in continuously stirred tank reactors with twice daily feedings of NiX-treated chicken litter mixed with recycled liquid from the AD process after separation of non-dissolved fibers. NiX-treatments were carried out in a pilot-scale pressure cooker with addition of 1 .5 % CaO relative to the total batch weight. Each experiment had a duration of more than 6 months during which the effectiveness of the NiX-treatment method was investigated while the process stability and biogas (methane) yield of the AD process was monitored.

Experiment 1

In Experiment 1 the chicken litter (62-64 % dry matter) was mixed with recycled liquid at a weight ratio of 1 :2.61 . Extra water was added to the process only via steam condensation during NiX treatment (0.05 kg-H₂0 / kg WW chicken litter).

A model calculation had shown that stable and non-inhibiting process conditions with respect to dry matter and nitrogen could be obtained with 2.5 % dry matter (TS) in the recycled liquid and with a 65 % efficiency of ammonia during NiX-treatment of the mixed chicken litter and recycled liquid.

However, the dry matter in the recycled liquid could not be maintained at 2.5 % but rose to more than 6 % during the experiment due to inefficient separation equipment. Also, the ammonia removal during NiX-treatment was only 47 % on average due to lack of capacity in the pressure cooker forcing the batch size up at the expense of the necessary headspace volume for ammonia gas release As a consequence no stable dry matter or ammonium level was obtained in the digester.

Ammonium increased to a concentration of more than 7 g/L and already at 6.4 g/L caused a significant drop in the methane yield. Before ammonium inhibition occurred the methane yield was stable for more than two months at an average 297 mL CH₄ / g organic dry matter (VS). Batch testing showed 13-22 % improvement of the methane yield compared no untreated chicken litter.

Nitrogen measurements showed that the concentration of organic nitrogen does not change during NiX treatment. Subsequently, approximately 30% of the organic nitrogen is converted into ammonium during the storage of the NiX-treated mixture in the substrate tank. The biogas process resulted in 45% conversion of organic nitrogen into ammonium.

Experiment 2

Model calculations made on the results from Experiment 1 showed that when using chicken litter with a very high dry matter content (62-64 % TS) addition of extra water is needed to run a stable biogas process based on chicken litter as mono-substrate. The amount of water needed will vary with varying dry matter contents in the biomass used and may also change as a function of the re-circulation ratio and on the organic loading rate (OLR).

For Experiment 2 it was calculated that 0.42 kg water was needed for each kg of chicken litter, including 0.05 kg water as steam during NiX treatment. Apart from the water addition

Experiment 2 was carried out in the same way as Experiment 1 , but with more focus on tracking the concentration of nitrogen species during the process steps and the pH-dependent toxicity of the ammonium. The water addition ensured a stabilisation of the dry matter content in the digester at 13 %, which is high compared to normal practice in a full-scale system, but it did not cause problems with mixing or pumping. Ammonium removal during NiX-treatment reached 65 % on average but the ammonium concentration in the digester reached 7.5 g/L before stabilizing. As in Experiment 1 , this high ammonium level had a very negative effect on the methane yield, which dropped from an average of 310 mL CH₄ / g organic dry matter to less than half of this level. Gas yield was twice restored to the original high level again by water dilution bringing the ammonium down to the less than 6 g/L. The pH-level was within the range from 8.0 to 8.2 during almost the entire experiment but reached more than 8.5 for a shorter period. The pH-dependent free ammonia fraction (NH3) of the ammonium pool increased to more than 1 g/L during the experiment and peaked at 2,1 during the high pH period.

The observed degradation of organic nitrogen after the NiX treatment in Experiment 1 was confirmed in Experiment 2.

Conclusions

The following conclusion can be drawn from the results obtained during Experiment 1 and 2:

1 . For biomasses with high dry matter content water addition can be necessary for

reaching an acceptable, stable dry matter level in the digester. The potential need for water addition is a combined function of the dry matter content in the biomass, the extent of degradation of dry matter during the AD process and the water loss during separation of the digested effluent.

2. A stable and high methane yield can be obtained from chicken litter as a mono- substrate with recycling of digester liquid, but the yield is highly sensitive to the concentration of ammonium in the digester.

3. Changes in pH affect the fraction of toxic free ammonia drastically.

4. Organic nitrogen makes up 70-80 % of the total nitrogen pool in chicken litter and is not affected by the NiX treatment. Approximately 50 % of the organic nitrogen is uric acid while the other half is protein-bound. After NiX treatment more than half of the organic nitrogen is released during storage in the pre-tank and during the AD process itself.

It is important to note that Figures 1 to 46 illustrate specific applications and embodiments of the invention, and it is not intended to limit the scope of the present disclosure or claims to that which is presented therein. Throughout the foregoing description, for the purpose of explanation, numerous specific details, were set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without some of these specific details and by employing different embodiments in combination with one another. The underlying principles of the invention may be employed using a large number of different combinations.

Claims

A method for generating biogas from an anaerobic fermentation of processed organic material comprising solid and liquid parts, said method comprising the steps of i) diverting a first organic material comprising one or more sources of nitrogen to a pre-incubation tank and subjecting said organic material to a hydrolysis by chemical and/or biological means, wherein the hydrolysis results in the conversion of organic N (nitrogen) present in the organic material to inorganic N; ii) diverting the pre-incubated first organic material to a lime pressure

cooker and subjecting said first pre-incubated organic material to lime pressure cooking, wherein the lime-pressure cooking results in a further hydrolysis of said first organic material, and wherein said lime pressure cooking step results in the formation of ammonia fluids; iii) diverting said ammonia fluids formed in the lime pressure cooker to an absorption unit, and absorbing and condensing the ammonia fluids diverted to the absorption unit from the lime pressure cooker; iv) diverting pre-incubated and lime pressure cooked organic material from said lime pressure cooker to a mixing tank for adjusting the pH of the lime pressure cooked material, and optionally mixing in the mixing tank the lime pressure cooked organic material with a further organic material; wherein the further organic material has preferably not been subjected to lime pressure cooking prior to the mixing with the lime pressure cooked organic material; v) diverting the optionally mixed, organic materiai(s) from the mixing tank to at least one, anaerobic biogas fermenter suitable for conducting an anaerobic, bacterial digestion of the organic material, wherein the anaerobic, bacterial digestion results in the generation of biogas; and vi) fermenting, under anaerobic fermentation conditions, the organic material diverted to the anaerobic biogas fermenter, and collecting the biogas resulting from said anaerobic fermentation of said organic material.

2. The method of claim 1 comprising the further steps of vii) diverting part or all of the organic material from the anaerobic biogas fermenter to a separation unit, and separating the organic material into a solid organic material fraction and a liquid organic material fraction comprising one or more sources of nitrogen selected from organic nitrogen sources and inorganic nitrogen sources; viii) diverting said liquid organic material fraction comprising one or more sources of nitrogen to the pre-incubation tank and/or to the lime pressure cooker, ix) mixing, in the pre-incubation tank and/or in the lime pressure cooker, said liquid fraction comprising one or more sources of nitrogen with a second organic material comprising one or more sources of nitrogen selected from organic nitrogen sources and inorganic nitrogen sources, and x) hydrolysing the mixture of second organic material and liquid fraction by chemical and/or biological means, when the liquid fraction comprising one or more sources of nitrogen is diverted to the pre-incubation tank and mixed with the second organic material in the pre-incubation tank, wherein the hydrolysis results in a conversion of organic N (nitrogen) present in the second organic material to inorganic N; and/or stripping ammonia from the mixture of second organic material and liquid fraction, when the liquid fraction comprising one or more sources of nitrogen is diverted to the lime pressure cooker and mixed with the second organic material in the lime pressure cooker.

The method of any of claims 1 and 2, wherein, during the pre-incubation step, if any biogas is produced, the amount of biogas constitutes less than 5 %, such as less than 4 %, for example less than 3 %, such as less than 2 %, for example less than 1 % of the amount of biogas produced in steps v) and vi).

The method of any of claims 1 and 2, wherein percentage conversion of organic N into inorganic N in the per-incubation tank is around at least 35 % of the total organic fraction, such as at least 40%, preferably at least 45%, at least 50%, more preferably at least 55%, at least 60%, even more preferably at least 65%.

The method of any of claims 1 and 2, wherein the percentage conversion of organic N into inorgnanic N in the incubation tank is around 70% to 80% of the total organic fraction.

The method of any of claims 1 and 2, wherein, in the pre-incubation tank, nitrogen containing organic acids, such as uric acid, is converted to inorganic N in an amount of 80% or more of the organic acid fraction, such as 90% or more, preferably 95% or more, even more preferably around 100% or more of the organic acid fraction.

The method of any claims 1 and 2, wherein, in the pre-incubation tank, organic bound nitrogen originating from protein is converted to inorganic N by a minimum of 30% of the organic bound N originating from protein fraction, such as by a minimum of 40%, by a minimum of 50%, by a minimum of 60% of the organic bound N originating from protein fraction.

The method of any of claims 1 and 2, wherein conversion of the organic N to inorganic N in the pre-incubation tank is performed at a temperature in the range of approximately 30 to 37 deg C, preferably around 33 to 37 deg C, and more preferably around 37 deg C.

9. The method of any of claims 1 and 2, wherein, when total solid (TS) is less than approx. 25%, and preferably less than around 30%, the rate of conversion of organic N to inorganic N in the pre-incubation tank is proportional to the amount of water present in the pre-incubation tank.

10. The method of any of claims 1 and 2, wherein, for oxygen levels greater than

normal oxygen levels in the pre-incubation tank, the rate of conversion of the organic N into inorganic N in the pre-incubation tank is inversely proportional to oxygen level.

11. The method of any of claims 1 and 2, wherein, for oxygen levels equal to or lower than the normal oxygen levels in the pre-incubation tank, the rate of conversion of the organic N to inorganic N in the pre-incubation tank is substantially same.

12. The method of any of claims 1 and 2, wherein the rate of conversion of the organic N to inorganic N in the pre-incubation tank is directly proportional to the amount of seeding organic material used, wherein the seeding organic material used involves adding material from an active fraction in an amount of approximately 10% to 30% w/w of the active fraction, preferably 10% to 25% w/w, more preferably around 10% to 20% w/w, wherein the active fraction comprises a separate pre-incubated organic material.

13. The method according to claim 12, wherein the seeding is selected from

adding material by retaining the amount of the active fraction of a first pre- fermentation for subsequent pre-fermentation in the pre-incubation tank; and/ or adding material by receiving, in a first pre-incubation tank, the amount of the active fraction from a second pre-incubation tank.

14. The method of any of claims 1 and 2, wherein the step in the pre-incubation tank comprises an operating pH in the pre-incubation tank in the range of around 6.0 to 8.5, typically around 6.4 to 7.5, more typically around 6.5 to 7.2.

15. The method of any of claims 1 and 2, wherein the process in the pre-incubation tank comprises an anaerobic facultative microbial fermentation carried out by microbial organisms present in the organic material and optionally also in the seeding material diverted to the pre-incubation tank.

16. The method of any of claims 1 and 2, wherein the hydrolysis in the pre-incubation tank is performed for around 96 hours or less, such as 72 hours or less, 60 hours or less, 50 hours or less.

17. The method of any of claims 1 and 2, wherein the one or more sources of nitrogen in the liquid fraction is an inorganic nitrogen source, such as ammonium.

18. The method of any of the claims 1 and 2, wherein the organic material comprises a maximum of 40% solid parts, such as a maximum of 30% solid parts for example a maximum of 25% solid parts such as a maximum of 20% solid parts.

19. The method of any of the claims 1 and 2, wherein the percentage of organic N with respect to the total N in the organic material is more than 30%, such as more than 40% w/w, more than 45%, more than 50%, more than 55%, more than 60%, more than 70%, more than 80%.

20. The method of any of the claims 1 and 2, wherein the organic material comprises deep litter or manure from animals selected from cattle, pigs and poultry.

21. The method of any of the claims 1 and 2, wherein at least part of the uric acid

which is present in the organic material is converted into ammonium during the preincubation, and optionally further converted into ammonia, which is optionally collected.

22. The method of any of the claims 1 and 2, wherein lime comprises or essentially consists of CaO or Ca(OH)₂.

23. The method of any of the claims 1 and 2, wherein the amount of added CaO used for lime pressure cooking is from about 2 to about 80 g per kg dry matter, such as from about 5 to about 60 g per kg dry matter.

24. The method of any of the claims 1 and 2, wherein the lime pressure cooking of the organic material is performed at a temperature of from about 100°C to preferably less than 250°C, under a pressure of from 2 to preferably less than 20 bar, with addition of lime sufficient to reach a pH value of from about 9 to preferably less than 12, and with an operation time of from at least one 10 minutes to preferably about less than 60 minutes.

25. The method of any of claims 1 and 2, wherein said mixed organic materials are fermented in a first, anaerobic fermenter under a first set of fermentation conditions, and subsequently diverted to a second, or further, anaerobic fermenter and fermented under a second or further set of fermentation conditions.

26. The method of any of the claims 1 and 2, wherein said organic materials are

fermented under thermophile fermentation conditions and/or mesophile

fermentation conditions.

27. The method of any of the claims 1 and 2, wherein said organic materials are initially fermented under thermophile fermentation conditions and subsequently under mesophile fermentation conditions.

28. The method of any of the claims 26 and 27, wherein biogas produced by

thermophile and/or mesophile fermentation conditions is diverted to a gas storage facility operably connected to the one or more fermenters.

29. The method of any of the claims 1 and 2, wherein the one or more biogas

fermentation step(s) is/are performed at a temperature of from about 20°C to preferably less than about 65°C.

30. The method of any of the claims 29, wherein the thermophilic reaction conditions include a reaction temperature ranging from 45°C to 65°C.

31. The method of any of the claims 29, wherein the thermophilic reaction conditions include a reaction temperature ranging from 55°C to 60°C.

32 The method of any of the claims 29, wherein the mesophilic reaction conditions include a reaction temperature ranging from 20°C to 45°C.

33 The method of any of the claims 29, wherein the mesophilic reaction conditions include a reaction temperature ranging from 30°C to 35°C.

34. The method of any of the claims 26 to 33, wherein the thermophilic reaction is performed for about 5 to15 days, such as for about 7 to 10 days.

35. The method of any of the claims 26 to 33, wherein the mesophilic reaction is

performed for about 5 to 15 days, such as for about 7 to 10 days.

36. The method of any of the preceding claims, wherein the nitrogen removal in form of removed inorganic nitrogen with respect to the total nitrogen of the organic material is at least 65%.

37. The method of any of the preceding claims, further comprising optionally feeding the anaerobic biogas fermenter with a third organic material for initiating the fermentation of the organic material diverted to the anaerobic fermenter.

38 A method for generating biogas from an anaerobic fermentation of processed

organic material comprising solid and liquid parts, said method comprising the steps of

a pre-incubation step comprising receiving in a pre-incubation tank an organic material having a first part removable inorganic N and organic N, and increasing amount of the removable inorganic N by converting the organic N into a second part of the removable inorganic N using first fermentation, wherein the preincubation step being free or substantially free from generation of biogas;

a nitrogen stripping step comprising stripping the removable inorganic N, comprising the first part and the second part, from the pre-incubated organic material using a lime-pressure cooker; and

a second fermentation step comprising anaerobically fermenting, in an anaerobic biogas fermenter, the pre-incubated organic material that is mixed with a further organic material for the generation of biogas.

39. A plant for generating biogas from an anaerobic fermentation of processed organic material comprising solid and liquid parts, said plant comprising i) a pre-incubation tank for hydrolysing an organic material by chemical or biological means; said pre-incubation tank being operably connected to a lime pressure cooker; ii) a lime pressure cooker for hydrolysing a first organic material comprising one or more sources of nitrogen; iii) an absorption unit for absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker when said first organic material is subjected to lime pressure cooking; iv) a mixing tank for mixing lime pressure cooked organic material with a further organic material prior to diverting the mixed, organic materials to one or more fermenters; v) one or more fermenters for anaerobically fermenting said organic materials diverted to said one or more fermenters from said mixing tank, wherein said fermentation results in the generation of biogas.

40. The plant according to claim 39, wherein the plant further comprises vi) a separation unit for separating organic materials diverted to the separation unit from said one or more fermenters, wherein said separation of said organic materials results in the generation of a solid organic material fraction and a liquid fraction comprising one or more sources of nitrogen; and means for diverting said liquid fraction comprising one or more sources of nitrogen to the lime pressure cooker, wherein said liquid fraction is mixed in the lime pressure cooker with first organic material comprising one or more sources of nitrogen.

41. The plant according to any of the preceding claims, wherein the absorption unit for absorbing and condensing ammonia fluids diverted to the absorption unit from the lime pressure cooker comprises a steam condenser and a scrubber.

42. The plant according to any of the preceding claims, wherein the mixing tank is operably connected to a reception tank or a reception station suitable for receiving liquid and solid organic material, respectively.

43. The plant according to any of the preceding claims, wherein the mixing tank is further operably connected to the absorption unit for absorbing and condensing ammonia fluids, wherein said connection allows ammonia to be stripped in the mixing tank and diverted to the absorption unit.

44. The plant according to any of the preceding claims comprising more than one

fermenter for anaerobically fermenting said organic materials, wherein said more than one fermenters are serially connected so that organic material having been fermented in a first fermenter under a first set of fermentation conditions can be diverted to a second or further fermenter and fermented under a second or further set of fermentation conditions.

45. The plant according to any of the preceding claims, wherein said more than one fermenter comprises at least one primary fermenter suitable for thermophilic fermentation and at least one secondary fermenter suitable for mesophilic fermentation.

46. The plant according to any of the preceding claims further comprising a gas storage facility operably connected to the one or more fermenters.