FI123751B - METHOD FOR PROCESSING BIOLOGICAL MATERIAL - Google Patents

Description

METHOD FOR PROCESSING BIOLOGICAL MATERIAL

Field of the invention

The present invention relates to the field of environmental technology. In particular, 5 the embodiments of the present invention relate to biological processes and apparatuses for processing organic nitrogen rich material and for producing ammonia from organic raw materials.

Background of the invention

Ammonia (NH3) is one of the most produced chemical compounds in the world. The 10 global production reached 131M metric tons in 2010 (US Geological Survey 2012). Most of the produced ammonia is used in chemical fertilizers to provide the nitrogen crops need for growing. Ammonia has also been used to produce plastics, synthetic fibers and resins, explosives, and numerous other chemical compounds.

The challenge with ammonia is in its production, which is very resource consuming 15 and produces unwanted greenhouse gases. Synthetic ammonia is typically produced by converting cleaned methane derived from natural gas into gaseous hydrogen and the resulted hydrogen is then combined with nitrogen to produce ammonia. The most common industrial method for producing ammonia is the Haber-Bosch process, where hydrogen gas derived from methane originated from natural gas and nitrogen 20 gas react with each other and ammonia is formed in the presence of iron or ruthenium catalyst (Smil 2001).

The hydrogen needed for ammonia production can also be derived from water using o electrolysis, but this process consumes substantial amounts of energy. According to c\j chemical fertilizer industry, the average carbon dioxide emissions per metric ton of o 1 25 ammonia are 2 metric tons C02 with an average recovery rate of some 38 ^ percentage (IFA 2012). Before getting to the actual fertilizer product ammonia needs

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£ to be combined with other substances to produce known fertilizer compounds such >- as urea, ammonium nitrate, or ammonium phosphates.

co m ^ Currently, there is a growing need to reduce adverse environmental impacts of o 00 30 ammonia production using the current technology which relies on fossil fuels.

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The biochemical process of converting nitrogen existing in biological material into ammonia is called ammonification (Strock 2008) or mineralization. The final step in ammonification is the hydrolysis of protein amino groups. Released amines and amino acids react with heterotrophs (bacteria and fungi) and form ammonia (NH3) 5 which is as ammonium ions (NH4+) in a solution.

The kinetics of ammonification, its detailed biochemistry within cells and variation between organisms are not fully understood yet. Scientific literature on bacterial ammonification is based on spontaneous production obtained from tube scale laboratory studies and have involved at least 24 bacterial genera mainly from 10 ruminal origin (Bladen et al. 1960 and citations therein; Vince & Burridge 1980;

Chen 8i Russel 1988; Russel et al. 1988; Atwood et al. 1998; Rychlik 8i Russel 2000;

Eschenlauer et al. 2002; Whitehead & Cotta 2004) including gram positive and negative bacteria (Whitehead & Cotta 2004).

From the bacteria capable of ammonification with detectable amounts of NH4+, only 15 approximately 20 strains belonging to genera such as Clostridium, Eubacterium, Fusobacterium, Peptostreptococcus, and Pseudomonas from ruminal and swine manure origin have been reported to form ammonia (NH3) more than 40 mM (i.e. 681 mg NH3 / liter = about 730 mg NH4 / liter) per 24 h (Paster et al. 1993; Atwood et al. 1998; Russel et al. 1988; Chen & Russell 1989; Whitehead & Cotta 2004).

20 These bacteria have been called as hyper ammonia-producing (HAP; e.g. Attwood et al. 1998; Whitehead & Cotta 2004) and as hyper ammonia-producing bacteria (HAB) (Rychlik & Russel 2000).

The ammonia producing bacteria vary markedly in their preference of carbon source as well as amino acids and peptides of the substrate they use in ammonification 25 (Vince 8i Burridge 1980; Rychlik 8i Russel 2000; Whitehead 8i Cotta 2004). Highest o ^ production has been obtained in growth media containing peptides and amino acids o digested from the milk protein casein (e.g. tryptone and casamino acids) while on ^ intact casein, growth and production was detected only from 11 bacterial strains out ^ of 40 including only a single strain with a high production (47.6 mM NH3 per 24 hrs;

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30 Whitehead & Cotta 2004). Depending on the bacterial strain, the presence of glucose £§ or lactose increased, had no effect on, or decreased ammonia production (Vince & m £! Burridge 1980; Eschenlauer et al. 2002; Whitehead & Cotta 2004).

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Short description of the invention

The inventors have surprisingly developed an environmentally sustainable way to produce ammonia by utilizing various nitrogen rich organic raw materials and living microorganisms to convert nitrogen existing in the proteinaceous material of these 5 streams into ammonia.

Chemical ammonia (NH3) production is dependent on fossil fuels and has heavy energy consumption. The inventors have developed bacteria driven biological methods to produce ammonium and ammonia in a sustainable way from organic raw material and nitrogen waste streams. The method utilizes nitrogen and amine rich 10 waste sources, such as meat-and-bone meal (MBM), and ammonia producing bacteria. The ammonia production can be further enhanced by supplying with a carbohydrate source such as glucose, molasses and or waste vegetables, fruit, root vegetables, or their peels.

The biological production described herein is dependent on bacteria's capability to 15 mineralize proteins and produce ammonia. This method is less dependent of external energy production (e.g. crude oil) and thus it decreases dramatically carbon dioxide emissions caused by chemical ammonia production.

In certain embodiments the methods involve use of nitrogen rich (such as protein) and carbohydrate rich biological wastes in a sustainable way, avoiding this way also 20 other green-house gas emissions to enter to atmosphere.

In certain embodiments the method involves pretreatment or optimization of the protein rich substrate for increased mineralization by an enzymatic treatment of the ^ organic raw material to be used as substrate by the bacteria.

δ c\j ^ The produced ammonia can be collected from the bacterial culture as gas (NH3) and ° 25 can be e.g. dissolved into an acid solution with e.g. ammonia stripping method (Gustin & Marinsek-Logar 2011) in which ammonium is converted to ammonia by I high pH 11-14, high temperature (e.g. 60 °C) and then led as gas with the help of ,- aeration into nitric acid (HN03). Ammonium nitrate (NH4N03) is formed in a chemical co [g reaction of ammonia and nitric acid: NH3 + HN03 NH4N03. Other acids for c\j £ 30 ammonia to be reacted with are e.g.: sulphuric acid (H2S04) in a reaction 2 NH3 + 00 H2S04 -> (NH4)2S04 (ammonium sulphate), hydrochloric acid (HCI) in a reaction NH3 + HCI -> NH4CI (ammonium chloride), and phosphoric acid (H3P04) in three alternative reactions: 3 NH3 + H3P04 (NH4)3P04 (ammonium phosphate), 3 NH3 + 4 H3P04 -> (ΝΗ4)2ΗΡ04 (diammonium hydrogen phosphate), or 3 NH3 + H3P04 -> NH4H2P04 (ammonium dihydrogen phosphate). Finally, the solution can be concentrated and or dehydrated. Alternatively, ammonia can be collected as ammonium ions (NH4+) using one of the precipitation methods that results in e.g. 5 struvite (MgNH4P04*6H20, MAP) in a reaction Mg2+ + NH4+ + P043' MgNH4P04 (Schulze-Rettmer et al. 2001; Nelson et al. 2003) or with a mechanical method such as size based ultra-membrane filtration.

Figure legends

Figure 1. Example of the nitrogen rich substrate such as MBM as compound of a 10 growth medium (later MBM medium), of the bacterial strains cultivated in Ductor's culture collection, and of incubation roll tubes used in the experiments in the examples. (A) Meat-and-bone meal (MBM). (B) Bacterial cultures of a single species. (C) Enzymatically hydrolyzed and (D) non-hydrolyzed meat-and-bone meal medium in a 10 ml loose cap roll tube and in 15 ml Hungate roll tubes.

15 Figure 2. Bacterial ammonium (NH4+) production from 179 g/l of non-hydrolyzed meat-and-bone meal (MBM) and of 0.2 % glucose (Example 1).

The NH4+ amount detected from culture medium without bacteria (background) was 150 mg/l and has been here extracted from bacterial production values. Biological sample number was 2 with identical result values. No bacterial ammonium (NH4+) 20 production was measured from non-hydrolyzed meat-and-bone meal medium when bacteria were incubated without glucose.

Figure 3. The effect of an added carbon source on bacterial ammonification on non- ^ hydrolyzed meat-and-bone meal (MBM) (Example 2). The final concentration of o added reducing sugars in apple peal and potato concentrates, molasses, and glucose cd 25 was approximately 0.2 %. Results are from 2 biological replicates with 3 technical o replicates in each. A-18-hr incubation with non-hydrolyzed MBM and an added carbohydrate enabled bacterial ammonification.

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,- Figure 4. The effect of enzymatic protein hydrolyzation of meat-and-bone meal

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(MBM) (Example 3). (A) The MBM medium was hydrolyzed with six different

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^ 30 concentrations (x-axis) of proteolytic Alcalase® enzyme (CLEA Technologies) at 50 00 °C for 4 hrs. The amount of free amino groups measured from MBM medium refer to the degree of MBM protein and peptide hydrolyzation. (B) The amounts of free amino groups were calculated with a help of a standard curve made of glycine dilution 5 series (a glycine molecule contains a single amino group). The amount of free amino groups in MBM medium increased significantly after enzymatic treatment. Furthermore, instead of a 4 hr incubation, a 24 hr incubation with 8 mUnit of Alcalase per ml of MBM medium increased the degree of MBM hydrolyzation over 30 5 % (data not shown).

Figure 5. The effect of protein hydrolyzation of meat-and-bone meal (MBM) on bacterial ammonification (Example 4). (A) MBM medium was hydrolyzed with proteolytic Alcalase® enzyme (CLEA Technologies) at 50 °C for 4 hrs prior bacterial inoculation (Aeromonas, Citrobacter, Clostridium, and Enterococcus). The control is 10 untreated MBM medium with bacteria. Error bars refer to standard deviation of 2-4 experiments with three technical sample replicates. (B) Ammonium was produced with hydrolyzed MBM with and without various carbon sources for 18 hrs. Error bars refer to standard deviation of 3 experiments with 3 technical sample replicates in each. Hydrolyzation of MBM prior bacteria inoculation (Clostridium and Aeromonas) 15 results in effective ammonia production. Moreover, the incubation with non-hydrolyzed MBM and a carbon source resulted in less ammonium than incubation with hydrolyzed MBM without an added carbon source (compare to Fig. 3). However, enzymatic protein hydrolyzation may have a positive co-effect with an added carbon source on bacterial ammonia production.

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Definitions

Unless otherwise specified, the terms used herein have the meaning commonly used in the art. Some terms, however, may be used to describe the present invention in a somewhat different manner and some terms benefit from additional explanation to 25 be correctly interpreted for understanding the present invention. Therefore some of ° the terms are explained in more detail below.

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Organic raw material as used herein refers to any carbon and nitrogen rich material of biological origin which can be used in the processes described herein as a

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£ substrate for mineralization and ammonia producing microorganisms. Examples of T- 30 such material include amine containing material, proteinaceous material, meat-and-co (g bone meal (MBM), slaughterhouse waste, whey, municipal waste, fish meal, food C\l ^ industry waste streams such as animal and plant by-products such as meals of

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meat-and-bone, fish, and feather as well as beet root, legumes, fruit, and sugar industry waste.

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Ammonia producing and microorganisms as used herein include any microorganisms capable of ammonification or mineralization. Such organisms include bacteria and fungi that are able to convert organic nitrogen into ammonia. In particular embodiments, bacteria belonging to the taxonomic genera Aeromonas, Citrobacter, 5 Clostridium, Enterobacter, Enterococcus, Klebsiella, Pseudomonas, or Staphylococcus are preferred.

Hydrolytic enzymes as used herein refer to any enzyme that is able to catalyze hydrolysis of a chemical bond and that can be used in the inventive process. Preferred examples of hydrolytic enzymes are enzymes that catalyze the hydrolysis 10 of a chemical bond of a macromolecular compound present in the organic raw material to be fermented, such as proteins, peptides, nucleic acids, starch, fats, phosphate esters, and other macromolecular substances. Preferred hydrolytic enzymes comprise serine and other proteases and peptidases (such as alkalases, collagenases, keratinases, and pepsin). By action of these enzymes proteinaceous 15 substrate can be hydrolyzed into peptides and amino acids which can be utilized by fermenting bacteria. Other hydrolytic enzymes to be used in certain embodiments comprise amylases (such as a-amylases, β-amylases and glycoamylases), cellulases (such as endoglucanases, cellobiohydrolases and β-glucosidases), and hemicellulases (such as xylanases and mannanases, and side-chain cleaving enzymes, such as a-20 glucuronidases, acetyl xylan esterases, α-arabinofuranosidases, and a-galactosidases). By action of these enzymes the starch, cellulose, and hemicellulose polymers present in the fermentation can be hydrolyzed into monomeric hexose and pentose sugars, which may be fermented by the microorganisms. Different enzyme mixtures for hydrolyzing proteins and starch, cellulose, and hemicellulose polymers, 25 depending on the material to be hydrolyzed, are commercially available.

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g The term unified form, as used herein in the context of recovery of ammonium and c\j ^ ammonia from the fermentation products, refers to conversion of ammonium ions ? into another chemical form such as ammonia (NH3). Ammonium and ammonia occur in certain equilibrium dependent on temperature and pH, e.g. the higher I 30 temperature and pH the higher proportion of ammonium ions are as ammonia.

co Detailed description m C\l ^ The following description and examples refer to particular embodiments of the present invention. It shall be understood that the presently disclosed embodiments are to be considered in all respects solely as illustrative and not restricting the scope 7 of the invention. The scope of the invention is indicated by the appended claims rather than by the following description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

5 Ammonium and ammonia can be produced biologically from a nitrogen rich (e.g. protein rich) substrate such as meat-and-bone meal (MBM) (Fig. la), feather and fish meal, as well as biological waste from food industry, households, and groceries in the presence of carbohydrate such as glucose, fructose, lactose, and/or starch in a process called mineralization or ammonification. In mineralization (or 10 ammonification) heterotrophs, such as bacteria, convert the organic nitrogen within organic material back into inorganic ammonia (NH3) that under ammonificative conditions will occur as ammonium ions (NH4+).

The bacterial ammonia production can be fastened and increased, and processed with or without added carbohydrates by hydrolyzing the protein or nitrogen rich 15 substrate. The hydrolyzation may be performed e.g. chemically, thermally, by using proteolytic enzymes prior bacterial fermentation, or with bacteria or with their secretes that contain these enzymes. Degrading enzymes such as proteases or any other can be incubated with MBM at 40 - 70 °C for 4-24 hrs. (10 hrs. is efficient). Bacteria can produce ammonium from 50 mg or more of the MBM per medium liter, 20 at least 600 mg being a desirable amount for 1 I of culture medium containing 179 g of MBM during 6-18 hrs. Depending on the bacterial taxon, their ammonia production occurs under mesophilic and thermophilic conditions, and in most cases well at 37 °C.

Carbohydrates such as glucose and lactose can be used to induce and maintain 25 growth of certain bacteria. In an embodiment the carbohydrates are provided as e.g. o ^ apple peal and potato concentrates, and molasses which are rich sources of co o carbohydrates. In further embodiments, vitamins and minerals may be provided for ^ the mineralization microorganisms. In certain embodiments the carbohydrate can be ^ obtained from waste sources such as sugar cane, sugar beet, fruit, and from all

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30 other root vegetables from industry, groceries, and households. Starch degrading cd enzymes or bacteria which produce these enzymes such as amylase can be used for m £! degradation of starch rich plant material, o C\l

Embodiment 1. In an embodiment the present invention provides a method of biologically processing organic raw material, characterized in that the method 8 comprises the steps of: providing a substrate comprising organic raw material and capable of being ammonified into ammonia (NH3) by ammonia producing microorganisms; and fermenting at least partly the substrate with at least one ammonia producing microorganism to obtain a fermentation product comprising 5 ammonium, dissolved ammonia and water.

Embodiment 2. In another embodiment the present invention provides the method according to embodiment 1, characterized in that the method further comprises providing a carbohydrate source to obtain a carbohydrate enriched substrate promoting bacterial growth.

10 Embodiment 3. In another embodiment the present invention provides the method according to embodiment 1 or 2, characterized in that the method comprises a hydrolyzing step for at least partly hydrolyzing the substrate with hydrolytic enzymes.

Embodiment 4. In another embodiment the present invention provides the 15 method according to any one of embodiments 1-3, characterized in that at least one ammonification microorganism is selected from the group of microorganisms that are capable of anaerobic ammonification in both presence and absence of monosaccharides and, optionally, disaccharides.

Embodiment 5. In another embodiment the present invention provides the 20 method according to any one of embodiments 1-4, characterized in that the ammonia producing microorganism is selected from bacteria belonging to the taxonomic genus Aeromonas, Citrobacter, Clostridium, Enterobacter, Enterococcus, Klebsiella, Pseudomonas, or Staphylococcus.

Embodiment 6. In another embodiment the present invention provides the 25 process according to any one of embodiments 1-5, characterized in that the 5 ammonia producing microorganism is selected from bacteria belonging to the

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^ taxonomic genus Aeromonas, Citrobacter, Clostridium or Enterococcus.

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Embodiment 7. In another embodiment the present invention provides the x process according to any one of embodiments 1-6, characterized in that the cc 30 ammonia producing microorganism is genetically modified to produce at least one 5 recombinant protein, preferably an enzyme hydrolyzing proteins or carbohydrates of co ^ the substrate, preferably an enzyme selected form the group consisting of amylase, o cellulase, protease and phytase.

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Embodiment 8. In another embodiment the present invention provides the method according to any one of embodiments 1-7, characterized in that the method comprises recovering ammonia from the fermentation product.

Embodiment 9. In another embodiment the present invention provides the 5 method according to any one of embodiments 1-8, characterized in that ammonia is recovered mechanically or precipitated.

Embodiment 10. In another embodiment the present invention provides the method according to any one of embodiments 1-9, characterized in that method comprises: dehydrating at least partly the fermentation product to form dry matter 10 of said fermentation product; vaporizing at least partly the dissolved ammonia and water from the fermentation product; collecting at least partly the vaporized ammonia-water or gas mixture; and recovering at least partly the ammonium produced in the method.

Embodiment 11. In another embodiment the present invention provides the 15 method according to any one of embodiments 1-10, characterized in that the method further comprises: a conversion step for converting ammonia or ammonium to a unified form in the mixture; a recovery step for recovering the unified form; and a conversion step for at least partly converting ammonia into at least one compound selected from the group consisting of: ammonium nitrate, ammonium sulphate, 20 ammonium chloride, ammonium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, or some other compound by reacting with nitric acid, sulphuric acid, hydrochloric acid, or phosphoric acid, or some other compound, respectively.

Embodiment 12. In another embodiment the present invention provides the 25 method according to any one of embodiment 1-11, characterized in that organic raw material comprises meat-and-bone meal (MBM). o

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^ Embodiment 13. In another embodiment the present invention provides the ° method according to any one of embodiments 1-12, characterized in that fermentation is conducted at pH 2-14 at 20-70 °C.

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30 Embodiment 14. In another embodiment the present invention provides a 5 fermentation product, characterized in that the product is produced using the co method according to any one of claims 1-13.

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Examples

Example 1. Bacterial ammonification in liquid medium made from meat-and-bone meal (MBM) and glucose.

The sterilized and milled MBM (Fig. 1A) has a total protein concentration of 5 approximately 56 %, which corresponds to 8 % of nitrogen of the MBM mass. The MBM has also other important nutrients, such as phosphorous (6 %) and calcium (10 %). Bacterial isolates (Fig. IB) from different origins and belonging to three different bacterial genera according to their 16S rRNA gene sequence (Aeromonas, Clostridium, and Enterococcus) were Ductor's own isolates. Bacteria were 10 anaerobically grown as two replicates in 10 ml loose cap (LC) roll tubes (Fig. ID) in 5 ml of an autoclaved liquid medium [179 g of MBM per liter, 0.2 % of D(+)-Glucose (Merck), RO water, pH 7.4] at + 37 °C for 24 hrs from which 100 pi of the culture was added to anaerobic Hungate culture tubes (Hungate 1969) containing 14 ml of the same, autoclaved MBM medium (Fig. 1C-1D). The controls contained media 15 without bacteria. All these were incubated again at + 37 °C for 24 hrs. 1 ml of the sample was collected and centrifuged at 16,000 x g (Eppendorf) for one minute. 500 μΙ of the supernatant was used for ammonium detection. The ammonium was measured with Ammonium Test (colorimetric with test strips; Merck) according to manufacturer's instructions. The same experiment was conducted also with bacteria 20 from genera Aeromonas and Clostridium with MBM medium lacking glucose. In this experiment without glucose, no ammonium could be detected.

To conclude, bacterial cultures identified to belong to three different taxonomic genera (Aeromonas, Clostridium, and Enterococcus) were tested to be capable of mineralizing protein-rich meat-and-bone meal (MBM) and producing ammonia in the 25 presence of glucose. Without added glucose, bacteria produced no detectable o 00 amounts of ammonia.

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o- Example 2. Positive effect of various carbohydrate sources on bacterial ammonia production on non-hydrolyzed meat-and-bone meal (MBM) co m C\l £ 30 Carbohydrate sources used in this study were apple peals, molasses, and 00 D(+)glucose (Merck). Concentration for reducing sugars of apple peals and molasses was measured with DNS-method (dinitrosalicylic acid; Miller 1959) as follows. Apple peal crush after boiling in small amount of water and molasses-water mix were 11 sterile filtered and further diluted to 1:10 and 1:100. Samples including standard dilution series were incubated with 600 pi of DNS solution (1 % dinitrosalicylic acid, 0.2 % phenol, 0.05 % Na-sulphate) at 90 °C for 10 min. 200 μΙ of 40 % KNa tartrate solution was added to terminate the color reaction. Absorbance was measured at 5 575 nm by using a spectrophotometric plate reader Synergy HI Hybrid Reader (Biotek). Results were calculated from the standard curve made with dilution series of D(+)-Glucose. Based on the results the concentration of reducing sugars for each sample in the experiment was chosen to be approximately 0.2 %. Bacteria were incubated with and without the carbohydrate sources as in the example 4 except for 10 the difference that the MBM medium lacked glucose and hydrolyzation. Ammonium production by bacteria incubated in non-hydrolyzed MBM supplied with a fruit concentrate or molasses indicate that some bacteria including Clostridium are able to use also other than purified glucose as their carbon source (Table 1; Fig. 3).

15 Table 1. Ammonium production in non-hydrolyzed MBM with different sugars (0,2 %) added in a 18-hr fermentation. Results are from 2 experiments with 3 technical sample replicates in each.

apple molasses glucose no sugar __NH4+ mg/l NH4+ mg/l NH4+ mg/l NH4+ mg/l

Clostridium 390 ± 32 279 ± 14 315 ± 38 133 ± 41

Aeromonas 142 ± 15 146 ±29 118 ± 40 142 ± 32 MBM 106 ± 41 79 ± 40 87 ± 29 76 ± 22 20

Example 3. Degrading effect of the proteolytic alcalase enzyme on meat-and-bone meal (MBM)

The MBM medium [179 g of MBM per liter RO water, 0.2 % of D(+)-Glucose (Merck),

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^ 50 mM MOPS, pH 7.5] was hydrolyzed with six different concentrations (up to ^ 25 769 mU/ml) of proteolytic Alcalase® enzyme (CLEA Technologies) at 50 °C for 4 hrs.

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9 The proteolytic enzyme was inactivated with 50 μΙ of 20 % SDS per 1.3 ml reaction >- volume and by incubation at 75 °C for 15 min. The hydrolyzation of MBM was I verified by quantifying free amino groups from the MBM medium with and without protease treatment using TNBS-method (trinitrobenzene sulfonic acid; Navarrete 8c co co 30 Garcia-Carreno 2002). Standard dilution series was made of glycine and treated ^ similarly as the MBM containing samples. 100 μΙ of the hydrolyzed MBM medium, o 00 controls, and standards were each mixed with 900 μΙ of 0.1 M Na-bicarbonate buffer (pH 8.5), then 100 μΙ of those were mixed with 50 μΙ of 0.01 % (w/v) of TNBS reagent [picrylsulfonic acid 5 % (w/v) in H20 (Sigma) and Na-bicarbonate buffer 12 pH 8.5] in wells of a clear 96-well plate and incubated in the dark at 37 °C for 2 hrs. Reaction was terminated by adding 50 pi of 10 % SDS and 25 pi of 1 M HCI. Absorbance of cooled samples was measured at 335 nm by using a spectrophotometric plate reader Synergy HI Hybrid Reader (Biotek). Results 5 (Fig. 4A) were calculated from the standard curve made with dilution series of glycine (Fig. 4B). The amount of free amino groups in MBM medium increased significantly after enzymatic treatment. With 19 mUnit of Alcalase per ml of MBM medium, hydrolyzation of MBM was doubled compared to an untreated sample and with 769 mUnit of Alcalase per ml of MBM medium, hydrolyzation of MBM was tripled 10 (Fig. 4A). Furthermore instead of a 4 hr incubation, a 24 hr incubation with 8 mUnit of Alcalase per ml of MBM medium increased the degree of MBM hydrolyzation over 30 % (data not shown).

Example 4. Positive effect of meat-and-bone meal (MBM) protein hydrolyzation on 15 bacterial mineralization

The MBM medium [179 g of MBM per liter RO water, 0.2 % of D(+)-Glucose (Merck), 50 mM MOPS, pH 7.5] was hydrolyzed with 385 mU of Alcalase® enzyme (CLEA Technologies) per ml at 50 °C for 4 hrs. The enzyme was inactivated in the MBM medium by boiling for 5 min. In Example 3 this concentration of Alcalase® enzyme 20 (CLEA Technologies) was shown to cause significant proteolysis of MBM (see Fig 4A of example 3). The extent of MBM hydrolyzation was quantified by using the TNBS-method (Navarrete & Garcia-Carreno 2002) described in Example 3.

Bacterial isolates from four different genera (Aeromonas, Citrobacter, Clostridium, c\i and Enterococcus) were cultivated anaerobically in autoclaved Brain-Heart-Infusion ^ 25 (BHI) Broth (Oxoid) (37 g BHI per liter of RO water) at 37 °C for 16-24 hrs. From

§ these, 70 μΙ of the culture was inoculated to hydrolyzed and nontreated liquid MBM

medium [179 g MBM, 0.2 % D(+)Glucose (Merck), RO water, 50 mM MOPS, pH 7.5] x and incubated at 37 °C for 18 hrs. In addition, bacterial isolates of Aeromonas and

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“ Clostridium were incubated with hydrolyzed MBM and with various sugar sources and 5 30 with no sugar (see methods in the example 2). Ammonium (NH4+) was measured by c3 using a quantitative, enzymatic determination kit of ammonia (Ammonia Assay Kit ^ AA0100; Sigma-Aldrich) for biological samples according to manufacturer's instructions. Before measuring, samples were diluted with Phosphate Buffer Solution, Medicago (0.14 M NaCI, 0.003 M KCI, 0.01 M Phosphate buffer, pH 7.4) as 1:50 and 13 the controls without bacteria as 1:10. Ammonia Standard Solution (10 pg/ml) was diluted to contain 2, 4, 6, and 8 pg/ml of ammonia to act as a standard curve. The absorbance was measured at 340 nm by using a spectrophotometer Synergy HI Hybrid Reader (Biotek). Hydrolyzed MBM medium resulted in even 6 to 65 times 5 more of bacterial NH4+ production (about 473 mg and 457 mg per liter per 18 hrs) than non-hydrolyzed (i.e. unprocessed) that resulted in about 79 mg and 7 mg of NH4+ per 18 hrs from Clostridium and Aeromonas, respectively (Table 2; Fig. 5A). The enzymatic treatment of MBM prior bacterial fermentation increased significantly bacterial ammonium production and the magnitude of the effect was dependent on 10 the bacteria (Table 2; Fig. 5A-B).

Table 2. Ammonium (NH4+) production with bacteria in unprocessed and hydrolyzed MBM in a 18 hr fermentation. Results are from 2-4 experiments with 3 technical replicates in each.

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unprocessed MBM hydrolyzed MBM

__NH4+ mg/1__NH4+ mg/1

Clostridium 192 ± 75 649 ± 83

Enterococcus 135 ± 15 292 ± 49

Citrobacter 169 ± 23 273 ± 44

Aeromonas 130 ± 46 633 ± 39

No bacteria 113 ± 45 176 ± 62

Table 3. Ammonium production in hydrolyzed MBM with different sugars (with concentration of reducing sugars 0.2 %) added in a 18 hr fermentation. Results are from 3 experiments with 3 technical replicates in each.

20 _____ apple molasses glucose no sugar __NH4+ mg/l NH4+ mg/l NH4+ mg/l NH4+ mg/l

Clostridium 944 ± 64 779 ± 120 895 ± 179 634 ± 86 £! Aeromonas 371 ± 33 403 ± 38 479 ± 77 627 ± 43

No bacteria 148 ± 30 150 ± 19 176 ± 81 158 ± 62 co o jc While bacteria from genera Aeromonas, Clostridium, and Shigella produced ammonium from non-hydrolyzed MBM only small amounts or none without added £§ glucose (see examples 2 and 3), the same bacteria from genera Clostridium and ln £! 25 Aeromonas did not need additions of carbohydrate for their ammonia production ° when the MBM was enzymatically hydrolyzed (see Table 3; Fig. 5B) even though apple peal crush, molasses, and glucose increased ammonia production in Clostridium (Table 3; Fig. 5B).

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To conclude, a treatment of MBM with proteolytic enzyme prior bacterial fermentation increases significantly bacterial ammonia production. Furthermore, the treatment obviates the necessity of an added carbohydrate in some cases. Carbohydrate addition has an enhancing co-effect with hydrolyzation on bacterial 5 ammonia production with certain bacteria.

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References

Bladen, H. A., M. P. Bryant, and R. N. Doetsch. 1961. A study of bacterial species from the rumen which produce ammonia from protein hydrolysate. Appi. Microbiol. 9:175-180.

5 Chen, G., and J. B. Russell. 1988. Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus. Appi. Environ. Microbiol. 54:2742-2749.

Eschenlauer, S. C. P., N. McKain, N. D. Walker, N. R. McEwan, C. J. Newbold, and R. J. Wallace. 2002. Ammonia production by rumen microorganisms and enumeration, 10 isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appi. Environ. Microbiol. 68:4925-4931.

Gustin S., Marinsek-Logar R. 2011. Effect of pH, temperature and air flow rate on the continuous ammonia stripping of the anaerobic digestion effluent. Process Safety and Environmental Protection 89: 61-66 15 Hungate R.E. 1969. A Roll Tube Method For Cultivation of Strict Anaerobes" in Methods of Microbiology, Vol. lllpps. 117-132, Academic Press, London.

IFA 2012. Technical Committee of International Fertilizer Industry Association.

http://www.fertilizer.org/ifa/HomePage/ABOUT-IFA/IFA-s-structure/IFA-s-

committees#PIT

20 Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Biochem. 31: 426-428.

δ c\j ^ Navarrete del Toro MA, Garcia-Carreno FL. 2002. Evaluation of the Progress of ? Protein Hydrolysis. Current Protocols in Food Analytical Chemistry B2.2.1-B2.2.14.

Nelson, N.O., Mikkelsen, R.L. & Hesterberg, D. L. 2003. Struvite precipitation in

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25 anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate c§ constant. Bioresource Technology 89: 229-236.

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Paster, B. J., J. B. Russell, C. M. J. Yang, J. M. Chow, C. R. Woese, and R. Tanner. 1993. Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus 16 anaerobius, Clostridium sticklandii and Clostridium aminophilum sp. nov. Int. J. Syst. Bacteriol. 43:107-110.

Russell, J. B., H. J. Strobel, and G. Chen. 1988. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Appi. 5 Environ. Microbiol. 54:872-877.

Rychlik, J. L, and J. B. Russell. 2000. Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production. FEMS Microbiol. Ecol. 32:121-128.

Schulze-Rettmer, R., von Fircks, R. 8i Simbach, B. 2001. MAP precipitation - pilot 10 plant investigation in Germany. Environmental Technology.

Smil, V. 2001. Enriching the Earth: Fritz Haber, Carl Bosch, and the Transformation of World Food Production. MIT Press, pp. 338. ISBN 0-262-19449-X. http ://books. google. fi/books/about/Enriching_the_Earth.html?id=G9FljcEASycC&redi r_esc=y 15 Strock, J.S. 2008. Ammonification, In: Editors-in-Chief: Sven Erik Jorgensen and Brian Fath, Editor(s)-in-Chief, Encyclopedia of Ecology, Academic Press, Oxford, Pages 162-165.

US Geological Survey. 2012. US Department of Interior, Mineral Commodity Summaries, US Geological Survey.

20 http://minerals.usgs.gov/minerals/pubs/commodity/nitrogen/mcs-2012-nitro.pdf

Vince AJ, Burridge SM. 1980. Ammonia production by intestinal bacteria: the effects ^ of lactose, lactulose and glucose. J Med Microbiol 13: 177-91.

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Claims (13)

A biological process for organic raw material, characterized in that the process comprises the steps of: providing a substrate comprising an organic raw material 5 which can be ammoniumated by ammonia-producing microorganisms (NH3); at least partially hydrolyzing the substrate with hydrolytic enzymes added to the substrate; and at least partially fermenting the substrate with at least one ammonia-producing microorganism to obtain a fermentation product comprising ammonia, dissolved ammonium and water. 2. The method of claim 1, further comprising providing a carbohydrate source to provide a carbohydrate-enriched substrate that promotes bacterial growth, and wherein the carbohydrate source is added to the substrate prior to fermentation. The method of claim 1 or 2, wherein the hydrolytic enzyme is a protease. Method according to any one of claims 1 to 3, characterized in that the at least one ammonification microorganism is selected from the group of microorganisms capable of anaerobic ammonification in the presence and absence of monosaccharides, and optionally disaccharides. C/O A method according to any one of claims 1 to 4, characterized in that the o ammonia producing microorganism is selected from bacteria belonging to the taxonomic genus Aeromonas, Citrobacter, Clostridium, Enterobacter, CVJ Enterococcus, Klebsiella, Pseudomonas or Staphylococcus. cc Method according to any one of claims 1 to 5, characterized in that the microorganism producing <g of ammonia is selected from bacteria belonging to the taxonomic genus Aeromonas or Citrobacter. δ 00 30 Method according to any one of claims 1 to 6, characterized in that the ammonia-producing microorganism is genetically engineered to produce at least one recombinant protein, preferably an enzyme selected from the group consisting of amylase, cellulase, protease and phytase. Process according to any one of claims 1 to 7, characterized in that the process comprises the recovery of ammonia from the fermentation product. Process according to any one of claims 1 to 8, characterized in that the ammonia is collected mechanically or precipitated. Process according to any one of claims 1 to 9, characterized in that the process comprises: at least partially dehydrating the fermentation product to form a dry substance 10 from said fermentation product; evaporating at least partially dissolved ammonia and water from the fermentation product; recovering, at least partially, the evaporated ammonia-water mixture or gas mixture, and recovering, at least partially, the ammonia produced by the process. Process according to any one of claims 1 to 10, characterized in that the process further comprises: a conversion step for converting the ammonia or ammonium to the combined form in the mixture; 20 collecting steps for recovering the combined form; and co to convert the ammonia of the conversion step at least in part to at least i § into one compound selected from the group consisting of ammonium nitrate, io ammonium sulfate, ammonium chloride, ammonium phosphate, x diammonium hydrogen phosphate, ammonium dihydrogen phosphate or another compound cc ' respectively with another compound. LO C \ 1 Process according to any one of claims 1 to 11, characterized in that the organic raw material comprises meat-and-bone meal. Process according to any one of claims 1 to 12, characterized in that the fermentation is carried out at a pH of 2 to 14, at 20 to 70 ° C. co δ c \ j i co o o C \ l X cc CL δ CD m CM δ CM