METHOD FOR RECOVERING

PHOSPHORUS FROM ORGANIC MATERIALS

TECHNICAL FIELD

The present invention relates generally to new processes for producing or recovering phosphorous compounds, such as phosphates, from organic raw materials by a process employing microbial fermentation and acid solubilization.

BACKGROUND OF THE INVENTION

Bone consists of inorganic minerals and organic substances. The inorganic minerals are formed from hydroxyapatite, Caio(P0 ₄ )6(OH)2. The organic substances consist mainly of proteins, and the proteins consist mainly of collagen. To release phosphorus containing compounds, e.g., phosphates, from bone, bone materials can be treated with acid in an aqueous medium, which solubilizes hydroxyapatite, resulting in the release of calcium (Ca ²⁺ ) and phosphate (P0 ₄ ^" ) ions into the aqueous medium (reaction equation 1). The presence of the bone protein matrix increases the amount of acid required, which increases both the size of the process equipment and the cost of mineral extraction, according to the following general reaction scheme.

Caio(P0 ₄ )6(OH) ₂ + 8 H ⁺ 10 Ca ²⁺ + 2 H ₂ 0 + 6 HP0 ₄ ^2" (1)

Phosphorus containing compounds such as phosphates are important, for example, for producing agricultural fertilizers. Currently the manufacture of phosphorous fertilizer requires manufacturing of an acid and then reacting the acid with certain bases in order to generate a fertlizer salt, such as ammonium phosphate, potassium phosphate etc.

European patent application EP1964828B 1 discloses a method of using certain bacteria to generate reduced forms of phosphorus, such as phosphites and/or hyposphosphites from inorganic materials, such as rock phosphate and soils. US patent No. 6,464,875 describes fermenting animal by-products to produce ammonia and solid materials for fertilizer production. CN 102020508 A relates to preparing a fertilizer by fermenting with "phosphorus- solubilizing bacterium" e.g. donkey bone and other organic material. The process includes a pre-fermenting step wherein the organic material is hydrolysed with a strong base. US patent No. 6,776,816 describes production of magnesium ammonium phosphate by fermenting manure.

Nevertheless, there remains a longstanding need in the art for improved processes for phosphate production from food production byproducts and agricultural waste.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a process for producing phosphates from an organic material, the method comprising:

(a) fermenting an organic material in a medium in the presence of at least one microorganism, wherein the fermenting is under conditions, and for a sufficient period of time, to produce a fermentation product;

(b) treating solids of the fermentation product with acid, in a medium to solubilize phosphates from the fermentation product;

wherein the organic material, and thus the solids of the fermentation product, comprises bone suitable for extraction of phosphates.

In a preferred embodiment, the medium is an aqueous medium.

In one embodiment, the fermentation process is conducted with an organic material ranging in density from 10 to 50 g/lOOml (wt/vol) of aqueous medium or more preferably, with an organic material ranging in density from 20 to 40, g/lOOml (wt/vol) of aqueous medium.

In an optional embodiment, the at least one microorganism is a bacteria capable of ammonification, and the process includes co-producing ammonia or ammonium during fermenting step (a). Preferably, the at least one at least one microorganism is a mixed bacterial population that has a correlation coefficient that is substantially similar to a mixed bacterial population of S I, FIl or F02. More preferably, the mixed bacterial population has a correlation coefficient of at least 0.90 relative to a mixed bacterial population of S I, FIl or F02, or alternatively, the mixed bacterial population has a correlation coefficient of at least 0.95, relative to a mixed bacterial population of S I, FIl or F02.

In a preferred embodiment, the acid is an organic acid or an inorganic acid, for example, tartaric acid, malic acid, citric acid, sulfuric acid and/or combinations thereof.

The inventive process optionally further includes the steps of:

separating a liquid phase from the acid treated fermentation product, admixing a reagent comprising NH ₄ ⁺ and/or a reagent comprising Mg ²⁺ ions into the separated liquid phase in a sufficient amount and for a period of time sufficient to precipitate solubilized phosphates from the separated liquid phase, and

recovering the precipitated phosphate.

Preferably, the precipitated phosphate is struvite or magnesium ammonium phosphate, formed by the reaction of Mg ²⁺ + NH ₄ ⁺ + P0 ₄ ^3" + 6H ₂ 0 MgNH ₄ P0 ₄ -6H ₂ 0.

Preferably, the fermenting step (a) is conducted with an inoculum of microorganisms ranging in dose from 1 to 20 (vol-%) or more preferably with an inoculum of microorganisms ranging in dose from 5 to 10 (vol-%.) Preferably, the fermenting step (a) is conducted at a temperature ranging from 30 to 60 °C or more preferably the fermenting step is conducted at a temperature ranging from 40 to 55 °C. Preferably, the fermenting step (a) is conducted for a time ranging from 10 hours to 7 days, or more preferably, the fermenting step is conducted for a time ranging from 12 hours to 18 hours.

Preferably, the acid solubilizing step (b) is conducted at a pH ranging from 1 to 6 or more preferably from a pH ranging from 2 to 3. Preferably, the acid solubilizing step (b) is conducted for a time ranging from 15 min to 14 days, or more preferably, for a time ranging from 7h to 48h. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of experiments to determine the effects of the fermentation of crushed bone on the phosphorous compounds solubilized from crushed bone by citric acid. The amount of citric acid was 117 g per 1 kg crushed bone. The results are the averages of three parallel experiments. The yield of the dissolved phosphorus was determined as a mass percent of the phosphorus content of the crushed bone. FIG. 1 is generated from Example 3.

FIG. 2 illustrates the results of experiments to determine the effects of the fermentation of crushed bone on the phosphorous compounds solubilized from crushed bone by sulfuric acid. The amount of the acid was 117 g per 1 kg crushed bone. The results are the averages of three parallel experiments. The yield of the dissolved phosphorus was determined as a mass percent of the phosphorus content of the crushed bone. FIG. 2 is generated from Example 3.

FIG. 3 illustrates treatment of fermented crushed bone with 5%, 7% and 10% tartaric acid concentrations (calculated as mass percentages in the whole reaction mixture) at room temperature. FIG. 3 is generated from Table XXVII in Example 7.

FIG. 4 illustrates an example embodiment according to present disclosure.

FIG. 5 Illustrates phosphate recovery as g/1 with different times (3, 6, 10, 12, 14, 16h) fermented crushed bone when sulfuric acid is used as a dissolving acid. FIG. 5 is generated from Table XXI in Example 4.

DETAILED DESCRIPTION

Accordingly, the present invention provides improved methods for the solubilization and extraction of phosphorous compounds, such as phosphates, from animal by-products, farm and food processing wastes. Typically, slaughterhouse waste, for example, bovine and porcine by-products, contain a high proportion of organic matter (e.g., connective tissue, muscle, blood and fat) and a minor proportion of bones. The organic matter needs to be removed in order to facilitate solubilization of phosphates from the bone fraction by means of acid solubilization.

The two-step process according to present invention starts with a fermentation step and thereafter continues with a post-fermenting step of adding acid to the product of the fermentation step. The acid lowers the pH and solubilizes phosphates. The fermentation step is conducted in order to remove and/or break up the organic material, thus facilitating the acid solubilization step. For example, the organic material present in bone containing material is primarily a protein matrix that interferes with the acid solubilization step by reacting with the acid and producing potentially explosive compounds. In addition, removal of the organic material from the bone itself reduces the amount of acid needed for the acid solubilization step. The fermentation process is also optionally utilized to produce or co-produce ammonia for commercial applications or production of biofertilizers.

In order to more clearly appreciate the invention, the following terms are defined. The terms listed below, unless otherwise indicated, will be used, and are intended to be defined as indicated. Definitions for other terms can occur throughout the specification. It is intended that all singular terms also encompass the plural, active tense and past tense forms of a term, unless otherwise indicated.

The term "phosphorus" refers to the chemical element phosphorus. The term "phosphorous" is an adjective meaning containing phosphorus. It refers to phosphorous compounds, such as phosphates, e.g., calcium phosphates, suitable for use as fertilizer or conversion to other useful compounds.

The term "MBM" or " meat- and-b one meal" as employed herein is defined by European Union Commission Regulation No. 142/2011 "meat-and- bone meal means animal protein derived from the processing of Category 1 or Category 2 materials in accordance with one of the processing methods set out in Chapter III of Annex IV" of European Union Commission Regulation No. 142/2011, incorporated by reference herein in its entirety.

In order to induce the mixed bacterial populations preferably employed in the fermentation stage of the inventive process, MBMs (designated infra as MBM1 and MBM2, respectively, were produced from animal by-products according to methods described in EU Commission Regulation 142/2011, and consisted of category 3 (EC Regulation 1069/2009) low infection risk material. In particular, MBM1 was obtained from Findest Protein Oy, Finland, and MBM2 was obtained from SARIA Bio-Industries AG & Co. KG, Germany. The term, "crushed bone" refers to bone waste from agriculture, food processing, restaurants and the like, that has been prepared by crushing.

The terms "fermentation" or "fermenting" refer to a process where organic molecules serve as both electron donors and acceptors. It differs from respiration, where electrons derived from nutrient molecules are donated to oxygen (aerobic respiration) or other inorganic molecules/ions such as nitrate, sulfate, carbon dioxide or ferric iron (anaerobic respiration). In fermentation, nutrient molecules are reduced to small organic molecules such as volatile fatty acids and alcohols.

The term "ammonification" refers to the mineralization of nitrogen in organic macromolecules, i.e., conversion of organic nitrogen to ammonium or ammonia, is called ammonification. It is performed by ammonifying bacteria and consists of enzymatic hydrolysis of proteins to amino acids, and release of nitrogen as ammonium/ammonia through deamination and elimination reactions. Carbon backbones of amino acids are fermented to organic acids

The term "ammonia" refers to the compound H ₃ found in gaseous form or dissolved in a non-ionized form in a medium e.g., an aqueous medium. The term "ammonium" refers to the ion NH ₄ ⁺ which is the ionic form of NH ₃ found in e.g., aqueous solution. In aqueous solution, ammonium and ammonia occur in an equilibrium that is dependent on temperature and pH, e.g. the higher the temperature and the pH, the greater the proportion that is in the form of ammonia. For this reason, reference to "ammonia" herein with regard to the inventive process and/or ammonification microorganisms and products thereof should be understood to include reference to both NH ₃ and NH ₄ ⁺ forms of this compound, unless otherwise indicated. For example, discussion of ammonification microorganisms as "ammonia producing" or "ammonium producing" is understood to include production of H ₃ and/or NH ₄ ⁺ according to the NH ₃ /NH ₄ ⁺ equilibrium found in the particular medium.

The inventive process provides methods for producing or recovering phosphorous compounds, e.g., phosphates, from an organic material. Broadly, the method includes, but is not limited to, two steps, which may be conducted separately and/or in combination. The method includes, e.g.,

(a) fermenting an organic material in a medium in the presence of at least one microorganism, wherein the fermenting is under conditions, and for a sufficient period of time, to produce a fermentation product;

(b) treating the solid fermentation product with acid to solubilize phosphates from the fermentation product;

wherein the organic material, and thus the solid fermentation product, comprises bone suitable for extraction of phosphates.

While not wishing to be limited to any theory or hypothesis as to the operation of the invention, it is believed that the fermentation process removes the bulk of the non-mineral component from the organic material. For example, when the organic material is MBM or crushed bone, it is believed that the fermentation process removes the bulk of muscle and/or connective tissue proteins, thus increasing the exposure of the residual bone mineral to dissolution and/or solubilization by contact with aqueous media having an acid pH.

The fermentation process can be conducted with any suitable microbial organisms, under anaerobic or aerobic conditions in a suitable reaction chamber or vessel for a time period and in a temperature range effective for efficient reduction of the organic material. The fermentation process is optionally conducted with at least one microorganism that is capable of ammonification. The advantage of fermenting the organic material with an ammonification microorganism is that ammonia or ammonium can be co- produced, while the instant invention provides for producing and/or recovering phosphorous compounds from a mineral component of the processed organic material.

The fermentation process is generally conducted with an organic material ranging in density from 10 to 50, g/ 100ml (wt/vol) of aqueous medium. Preferably, fermentation process is conducted with an organic material ranging in density from 20 to 40, g/ 100ml (wt/vol) of aqueous medium.

The fermentation process is generally conducted with an inoculum of fermentation microorganisms ranging in dose from 1 to 20 (vol-%) determined. Preferably, the fermentation process is conducted with an inoculum of fermentation microorganisms ranging in density from 5 to 10 (vol-%.)

The fermentation step of the process is generally conducted at a temperature ranging from 30 to 60 °C. Preferably, the process is conducted at a temperature ranging from 40 to 55 °C of aqueous medium.

The fermentation step of the process is generally conducted for a time ranging from 1 to 7 days. Preferably, the process is conducted for a time ranging from 2 to 3 days.

Processes and microorganisms for efficiently producing ammonia or ammonium by fermenting organic material, such as MBM, are disclosed, for instance, by co-owned U.S. Patent Appl. Ser. No. 13/722,228, filed on December 20, 2012, claiming the benefit of U.S. Provisional Appl. Ser. No. 61/659,647, filed on June 14, 2012, and by co-owned U.S. Patent Appl. Ser. No. 14/066,089, filed on October 29, 2013, the contents of both of which are incorporated by reference herein in their entireties.

Exemplary single-strain bacteria for fermenting organic material according to the present invention and producing ammonia include, for example, strains taught by co-owned U.S. Patent Appl. Ser. No. 13/722,228. These include, for example, "Strain 385" that belongs to the Clostridium genus, beijerinckii or butyricum species, and that was deposited on 17 December 2012 as VTT E- 123273 (VTT collection of Industrial Microorganisms, Finland) and "Strain 393" that belongs to Clostridium genus, perfringens species, and was deposited on 17 December 2012 as VTT E- 123272 (VTT collection of Industrial Microorganisms, Finland) under the terms of the Budapest Treaty, in support of co-owned U.S. Patent Appl. Ser. No. 13/722,228.

Exemplary populations of bacteria for fermenting organic material according to the present invention and producing ammonia include, for example, defined mixed bacterial populations S I, FI1 and F02 as detailed hereinbelow and produced by methods described in the above-noted co-owned U.S. Patent Appl. Ser. No. 14/066,089, filed on October 29, 2013. In brief, mixed bacterial populations designated as S I, FI1 and F02 were obtained as follows.

The S I mixed bacterial population was created by mixing non-sterile MBM2 with cold tap water in a proportion of 180 g MBM per liter of water. MBM2 was cultured without aeration at 50 °C until NH ₃ concentration leveled out, and stationary growth phase was reached as explained hereinbelow.

The S I population has been deposited as a patent deposit, under the terms of the Budapest treaty, in support of co-owned U.S. Application Ser. No. 14/066,089 (as above), in the Centraalbureau voor Schimmelcultures (CBS), located at Uppsalalaan 8 3584 CT Utrecht, The Netherlands, as (CBS Accession No. 136063) on August 22, 2013.

The FI1 population was created by mixing non- sterile field (FI) soil with cold tap water in a proportion of 180 g soil per liter of water. The mixture was cultured without aeration at 50 °C.

The F02 population was created by mixing non-sterile forest (FO) soil with boiling tap water in a proportion of 180 g of soil per liter of water. The mixture was let cool to room temperature. Both mixtures were incubated without aeration at 50 °C. After 7 days incubation, 5 ml of each culture was inoculated in 100 ml of sterile MBM1 medium [180 g meat-and-bone meal 1 (MBM1) per liter of water]. The cultures were incubated at 50 °C for 7 days. Growth of the mixed bacterial populations was monitored by measuring the ammonium production of the populations. A maximal ammonia level of about 8-10 g/1 was repeatedly determined for culture growth under the conditions described above. Therefore, when the ammonia concentration reached this level, it was interpreted as transition to stationary phase of growth. The diverse nature of the populations restricted the use of culture based methods for cell counting, and opacity of the MBM medium prevented the use of optical density measurement for estimation of cell densities. In all the following, "inocula of mixed bacterial populations" refer to bacterial cultures, which have reached stationary growth phase.

All populations were maintained by storing the liquid culture at +4 °C.

As noted in co-owned U.S. Application Ser. No. 14/066,089 (as above), the optimal temperature range for ammonification (via fermentation) by S I mixed bacterial population is 37-60 °C. However, S I retained some of its ammonification efficiency even at room temperature (RT, 23 °C) and 70 °C. In addition, the optimal pH range for ammonification (via fermentation) by S 1 is about pH 6 to about pH 9.

Thus, the working range for ammonification by fermentation of proteinacious materials with S I mixed bacterial population is as follows: Temperatures from 23°C to 70 °C, more particularly from 37-60 °C, and pH 6-9 were the best for bacterial ammonification with S 1 population described here. Ammonification by fermentation works in anaerobic, microaerobic, and aerobic conditions using the mixed S 1 bacterial population.

Additional mixed bacterial populations are also disclosed by co-owned U.S. Application Ser. No. 14/066,089 (as above), and are contemplated to be employed in the inventive process of the present invention. These additional mixed bacterial populations were obtained from MBM2 (Al, CI, HI, and PI), broiler chicken by-product (CBP-M), porcine/bovine by-product (PB-M), chicken feather (FE-M), fish by-product (MF-M), crushed porcine/bovine bone (CB-M), field soil mixed with boiling water (FI2) and forest soil mixed with cold water (FOl). In certain embodiments, the process of the invention can be conducted wherein the fermentation step is conducted at a different location and the fermentation product is acid treated at a later time.

The process of the invention is contemplated to be conducted by extracting phosphorous compounds from the fermentation product with any suitable inorganic or organic acid. Simply by way of example, suitable acids include, e.g. citric acid, glutaric acid, maleic acid, malic acid, malonic acid, oxalic acid, tartaric acid, acetic acid, formic acid, hydrochloric acid, lactic acid, sulfuric acid, mixtures thereof, e.g., mixtures of acetic and citric acids. Preferably, the acid is an organic acid such as tartaric acid, malic acid and/or citric acid. Alternatively the acid is an inorganic acid, such as hydrochloric acid or sulfuric acid. Of the later, sulfuric acid is preferred.

The acid solubilization step of the process is generally conducted at a pH ranging from 1 to 6. Preferably, the process is conducted at a pH ranging from 2 to 3 and for a time ranging from 15 min to 14 days, or more preferably for a time ranging from 7h to 48h.

EXAMPLE 1

ACID SOLUBILIZATION OF PHOSPHORUS COMPOUNDS FROM MEAT BONE MEAL (MBM)

Different acids, including organic and inorganic acids, were tested to determine which acids are the most effective in solubilizing phosphorus compounds from meat bone meal (MBM). The tested acids were boric acid (manufacturer VWR LLC, Belgium), citric acid (YA pharmacy, Finland), maleic acid (Sigma-Aldrich, Austria), DL-malic acid (Sigma-Aldrich, Germany), malonic acid (Sigma-Aldrich, China), oxalic acid (Sigma-Aldrich, USA), tannic acid (Sigma-Aldrich, Belgium), L-(+)-tartaric acid (Sigma- Aldrich, Italy), tartaric acid (YA pharmacy, Finland), acetic acid (Sigma- Aldrich, Germany), formic acid 98% (Sigma-Aldrich, Germany), hydrochloric acid 37% (VWR LLC, France), L-(+)-lactic acid 85% (Sigma-Aldrich, Netherlands) and sulfuric acid 95% or 95-98% (VWR LLC or Sigma-Aldrich, Germany). Organic acids were interesting because they have a biological origin, whereas inorganic acids are derived from mineral sources. In addition, some of the organic acids can be produced biotechnically which makes them a more environmentally sustainable option. Nitric acid was excluded from the experiments due to its higher price compared to another inorganic acid, sulfuric acid. The bone feedstocks were meat bone meal (MBM).

Tested Acids

The experiment was conducted to test the properties of different acids to solubilize phosphorus compounds from MBM at room temperature (RT). The phosphorus content of the MBM employed in the experiments was 6.2 %, and the moisture content was 3.4 %. The acids tested in the experiments are presented in Table I. Some of the acids were purchased as solid crystallized powders and some of them were purchased as liquids. The acids were chosen based on their pK _a values and solubilities in water. The pK _a value reflects how strongly the acid retains a proton or how widely the acid is protolyzed in water. If the pH of a water solution is the same as the pK _a value of the acid, then 50 % of the acid is in the anionic form and 50 % is in the undissociated form. Most of the tested acids had small pK _a values (high acidities) and good solubilities in water. In addition, some weaker and less soluble acids were tested to determine the differences between the solubilizing properties of the different acids.

Table I The tested acids in the initial experiments and their pK _a values and water-solubilities. Water-solubilities of the acids that are liquid at room temperature are not presented.

Solubility in water, g/1 (20

Acid pK _a -values

°C)

Boric acid 9.23 40 - 50

Citric acid 3.13, 4.77, 6.39 750

Glutaric acid 4.31, 5.41 430

Maleic acid 1.83, 6.07 790 (25 °C)

DL-Malic acid 3.4, 5.11 558

Malonic acid 2.83 1400

Oxalic acid 1.23, 4.19 90 Tannic acid circa 10 250

L-(+)-Tartaric acid 2.98, 4.34 1390

Acetic acid 4.76 miscible

Formic acid 3.75 miscible

Hydrochloric acid -8 miscible

L-(+) -Lactic acid 3.08 miscible

Sulfuric acid -3 miscible

Mixture of acetic

and citric acids miscible

Initial experiments were conducted on a test tube scale

The first part of the initial determination of phosphorus solubility was conducted on a test tube scale and by using MBM medium, which was made by adding 1 liter of RO (reverse osmosis) water to 180 g meat bone meal. The concentration of MBM in this medium was 161 g/1. First, the MBM medium and the acid solution were mixed according to Table II. The mixture was shaken to properly mix the MBM and the acid. Then the mixture stayed at room temperature without any mixing. As the acid treatment continued, timed 1 ml samples were taken and centrifuged at 20,000 x g (Eppendorf) for three minutes. The separated liquid, supernatant, was collected and was diluted with water with a dilution ratio of 1:20 (50 μΐ (microliter) supernatant + 950 μΐ milli-Q water (water purified using a Millipore Milli-Q lab water system)) to stop the reaction between the acid and MBM. The diluted supernatant was used for determination of dissolved phosphates. The phosphates were measured spectropho tome trie ally (Synergy HI Reader) using a Malachite green phosphate assay kit (POMG-25H, BioAssay Systems) according to the manufacturer's instructions. The results are presented in Table III.

Table II The initial experiments performed in a test tube scale. Meat bone meal (MBM) medium contained 161 g/1 meat bone meal. First the acid solution was made in milli-Q water after which it was added to MBM medium. Volume of Volume of

Number of Time of acid

MBM Acid solution acid solution,

experiment treatment, h medium, ml ml

1 0.2

4 50 g/1 Boric acid 20 2 2

3 0.2

4 0.4

5 0.6

4 480 g/1 Citric acid 20 6 0.8

7 1

8 2

9 4 0.5

10 3 1.5

90 g/1 Oxalic acid 24 11 2 2.5

12 2 4

13 0.2

4 125 g/1 Tannic acid 20 14 2

15 0.2

16 0.4

17 4 80 % Acetic acid 0.6 20

18 0.8

19 1

20 0.215

4 98 % Formic acid 24 21 1.026

22 0.4

37 % Hydrochloric

23 4 0.6 20 acid

24 0.8

25 85 % L-(+)-Lactic 0.25

4 24

26 acid 1.231

27 95 % Sulfuric acid 0.2

4 20

28 (VWR) 0.4

29 0.1 + 0.1

30 0.2 + 0.2

48 % Acetic acid +

31 4 0.3 + 0.3 20

48 % citric acid

32 0.4 + 0.4

33 0.5 + 0.5

Table III The results of the initial experiments performed in a test tube scale. The amount of the used acid is presented as a mass per 1 kg MBM. The amount of the dissolved phosphorus is presented as concentrations of dissolved phosphates and dissolved phosphorus and also as a yield of dissolved phosphorus from total phosphorus content of MBM.

Initial experiments in a bottle scale The second part of the initial experiments for phosphorus solubilization was done in a bottle scale and by using MBM as a feedstock. First the acid solution was made and then added to the weighed MBM according to Tables IV and V. The experiments were conducted according to the same procedure as the experiments in a test tube scale. The results are presented in Tables VI and VII.

Table IV The initial experiments performed in a bottle scale with the solid acids. First the acid was weighed in a measuring bottle which was then filled with milli-Q water until the total volume given was reached. After making the acid solution it was added to MBM. The samples from the bottles were taken after acid treatments of 24 h and 1 week.

Table V The initial experiments performed in a bottle scale with liquid acids. After making the acid solution in milli-Q water it was added to MBM. The samples from the bottles were taken after acid treatments of 24 h and 1 week.

Number of Mass Acid Volume of Volume Total volume

Table VI The results of the initial experiments performed in a bottle scale after acid treatment of 24 h. The amount of the used acid is presented as a mass per 1 kg MBM. The amount of the dissolved phosphorus is presented as concentrations of dissolved phosphates and dissolved phosphorus and also as a yield of dissolved phosphorus from phosphorus content of MBM.

Table VII The results of the initial experiments performed in a bottle scale after acid treatment of 1 week. The amount of the used acid is presented as a mass per 1 kg meat bone meal (MBM). The amount of the dissolved phosphorus is presented as concentrations of dissolved phosphates and dissolved phosphorus and also as a yield of dissolved phosphorus from phosphorus content of MBM.

Boric acid, tannic acid and acetic acid were found to be the weakest acid to dissolve phosphorus from bone.

EXAMPLE 2

COMPARING ACIDS FOR SOLUBILIZING PHOSPHORUS COMPOUNDS FROM CRUSHED BONE The acids were selected based on the results of Example 1. In practice the least efficient acids in solubilizing phosphorus compounds from MBM were excluded from the later experiments based on the initial experiments.

Hydrochloric acid also was excluded due to its higher price compared to another inorganic acid, sulfuric acid. The bone feedstock that was employed was crushed bone (a/k/a "bone crush," or "BC," manufactured by Oy Musch

Ltd., Pietarsaari, Finland), which contained bovine and pork bones. The crushed bone composition that was tested is presented in Table VIII.

Table VIII Composition of the used crushed bone matrix (analysed by Novalab, Karkkila, Finland).

Phosphorus, % 2.2 - 2.7

Nitrogen, % 2.6 - 2.9

Organic matter in dry matter, % 68

Moisture, % 53.8 - 60.7

The crushed bone used in these experiments contained 67% organic matter and 33% of inorganic matter/material in dry matter. The total amount of phosphorus was 2.7 % of the material (crushed bone). The bone material contained 28.9 % nitrogen and 53.8 % moisture. The solid acids were citric acid, maleic acid, DL-malic acid, malonic acid, oxalic acid, L-(+)-tartaric acid and tartaric acid (purchased from pharmacy). The acid solutions from the solid acids were made as presented in Tables IX and X. The liquid acids were formic acid, L-(+)-lactic acid and sulfuric acid (VWR). The acid solutions from the liquid acids were made as presented in Table XI. The concentrations of the acid solutions were determined based on their mass percent in the reaction mixture consisting of crushed bone and acid solution. 80 g crushed bone was weighed in glass bottles and each of the made 200 ml acid solutions was added to one bottle. Though the moisture content of the crushed bone was high, 53.8 - 60.7 %, it was observed that the volume of the added acid solution was not changed due to the moisture of the bone feedstock. The experiments were conducted at room temperature according to the same procedure as the experiments in Example 1. The results are presented in Table XII. Table IX Making the acid solutions of the solid acids which were citric acid, oxalic acid, L-(+) -tartaric acid and tartaric acid (purchased from pharmacy). The total volume of each acid solution was 200 ml and the solutions were made in milli-Q water.

Mass percent of

Amount of Concentration of acid in reaction

acid, g acid solution, g/1 mixture, %

2 5.6 28

5 14.0 70

7 19.6 98

10 28.0 140

Table X Making the acid solutions of the solid acids which were maleic acid, DL-malic acid and malonic acid. The solutions were made in milli-Q water.

Mass percent of

Amount of Concentration of Volume of acid acid in reaction

acid, g acid solution, g/1 solution, ml mixture, %

1.7 5.6 22.4 250

4.2 14.0 56 250

5.9 19.6 78.4 250

8.5 28.0 112 250

10 28.0 140 200

Table XI Making the acid solutions of the liquid acids in milli-Q water. The total volume of each acid solution was 200 ml. The density of 98 % formic acid was 1.22 kg/1, the density of 85 % L-(+)-lactic acid was 1.206 kg/1 and the density of 95 % sulfuric acid was 1.837 kg/1.

Mass percent

Amount Volume of Volume Concentration of acid in

Acid of 100 % used acid, of water, of acid reaction

acid, g ml ml solution, g/1 mixture, %

2 5.6 4.7 195.3 28

98 % 5 14.0 11.7 188.3 70

Formic acid 7 19.6 16.4 183.6 98

10 28.0 23.4 176.6 140

2 5.6 5.5 194.5 28

85 % L- 5 14.0 13.7 186.3 70 (+)-Lactic

7 19.6 19.1 180.9 98 acid

10 28.0 27.3 172.7 140 5.6 3.2 196.8 28

95 %

14.0 8.0 192.0 70 Sulfuric

188.8 98 acid 19.6 11.2

28.0 16.0 184.0 140

Table XII The best results in different acid concentrations of the best acids in the solubilization of phosphorus. The experiments were conducted at room tem erature.

2 70 5h 5.9 17.7 5 L-(+)- 175 24h 27.0 80.5

Lactic

7 245 7d 22.0 65.7 acid

10 350 3d 27.6 82.8

2 70 7h 12.0 35.9

5 Formic 175 24h 23.0 69.1

7 acid 245 3d 22.7 80.8

10 350 8d 25.8 96.5

2 70 7h 15.2 45.6

5 Sulfuric 175 7d 40.3 84.6

7 acid 245 7d 43.7 92.0

10 350 6d 40.9 85.6

Conclusions on the Example 2: Based on experiments it appears that phosphorus can be dissolved from crushed bone if sufficient time is allowed for solubilization.

EXAMPLE 3

POSITIVE EFFECT OF FERMENTATION STEP TO PROCESS OF SOLUBILIZING PHOSPHORUS COMPOUNDS FROM CRUSHED BONE.

In this Example, crushed bone was first fermented and then subjected to solubilization using a selected set of acids.

The fermentation step was performed in the following manner: First the crushed bone was weighed in a bucket, then tap water was added to the crushed bone and stirred with a suitable spatula. The mixture was preheated to 50°C in an incubator. Then a mixed population of bacterial inoculum, typically 5 vol-% (volume percent), capable of ammonification (S I population as defined by Table XIII(a) below) was inoculated into the mixture.

TABLE XIII(a). Bacterial diversity analysis results: genera and species in populations SI, FIl and F02. Cells from all cultures were harvested for DNA extraction at the age of four days i.e. 96 h after inoculating sterile MBM1 medium with the population and incubating at 50 °C. The results are expressed as percentage of total population. Species si Fll F02

Alicyclobacillus contaminans 0.23

Bacillus beijingensis 0.02

Bacillus benzoevorans 0.02

Bacillus coagulans 8.71

Bacillus ginsengi 0.02

Bacillus nealsonii 0.06

Bacillus pichinotyi 0.02

Bacillus smithii 0.02

Bacillus sp. 0.40 29.82

Bacillus thermoamylovorans 0.09

Bacillus vireti 0.04

Caldicoprobacter oshimai 0.09

Caloramator sp. 5.20 0.75

Carnobacterium divergens 0.06

Clostridium beijerinckii 0.04

Clostridium botulinum 4.63

Clostridium cochlearium 8.50 1.80 0.06

Clostridium oceanicum 0.06

Clostridium pasteurianum 0.45

Clostridium purinilyticum 0.03

Clostridium sp. 0.57 6.34 16.16

Clostridium sporogenes 0.48 3.68 0.06

Clostridium tyrobutyricum 0.06

Clostridium ultunense 1.25 2.82

Clostridium xylanovorans 0.08

Empedobacter brevis 0.02

Enterobacter cloacae 0.02

Enterococcus azikeevi 0.04

En terococcus faecal is 0.30

Enterococcus faecium 1.06

Enterococcus hirae 0.05 0.04

Enterococcus raffinosus 0.02

Enterococcus sp. 0.02

Faecalibacterium prausnitzii 0.02

Faecalibacterium sp. 0.03

Garciella sp. 0.03

Halobacillus trueperi 0.04

Klebsiella oxytoca 0.04

Lactobacillus pontis 0.02

Lactococcus garvieae 0.13

Lactococcus raffinolactis 0.06 Lactococcus sp. 0.13

Mahella australiensis 0.43 0.35

Pantoea sp. 0.02

Pediococcus acidilactici 4.80

Peptostreptococcus sp. 0.19

Petrobacter succinatimandens 0.04

Propionibacterium sp. 0.03

Pseudobutyrivibrio ruminis 0.03

Schlegelella thermodepolymerans 0.13

Shigella flexneri 0.02

Soehngenia sp. 0.03

Sporanaerobacter acetigenes 75.87 77.63 0.04

Sporolactobacillus inulinus 0.08

Streptococcus alactolyticus 0.02

Streptococcus mitis 0.03

Subdoligranulum variabile 0.06

Tepidanaerobacter sp. 0.68 1.29

Thermoanaerobacterium aciditolerans 0.89

Thermoanaerobacterium aotearoense 19.97

Thermoanaerobacterium sp. 0.28

Thermoanaerobacterium

15.65 thermosaccharolyticum

Tissierella sp. 1.68 4.97 0.02

Tuberibacillus calidus 0.02

The fermentation broth contained 40 % crushed bone, 55 % water and 5 % bacterial inoculum. It was in an incubator at temperature of 50 °C for three days. After a vacuum filtration step and washing with water the remained solid matter was the fermented crushed bone (FBC). Composition of the fermented crushed bone matrix is presented in Table XIII(b), below.

Table XIII(b) Composition of the fermented crushed bone matrix (analysed by Novalab, Karkkila, Finland). The fermented crushed bone was obtained after fermenting, vacuum filtrating and water washing of crushed bone.

Phosphorus, % 6.8 - - 6.9

Nitrogen, % 2.6- 2.7

Organic matter in dry matter, % 34 - - 39

Moisture, % 42.1 - - 42.6 The crushed bone used in Example 3 contained 68% organic matter and 32% of inorganic matter/material, before fermentation. Total amount of phosphorus was 2.2 % of the material (crushed bone). The crushed bone material contained 25.9 % nitrogen and 60.7 % moisture.

120 g of crushed bone was weighed in six buckets, 165 ml of tap water was added to each bucket and mixed. 15 ml bacterial inoculum was added to each mixture and the suspension was mixed properly. The fermentation step was conducted as described earlier. As six 120 g crushed bone batches had been fermented, vacuum filtrated and washed 37.7 - 40.5 % of the solid crushed bone remained. Each of six batches of the fermented crushed bone was mixed in a glass bottle with one of the acid solutions that were made according to Tables XII and XIII. 120 g crushed bone was weighed in six bottles and the acid solution was added to each bottle also according to Tables XIV and XV. Three parallel experiments were performed. The experiments were conducted at room temperature according to the same procedure as described in Example 1. The results are presented in Tables XVI and XVII and in FIGs. 1 and 2 respectively.

The ammonifying mixed bacterial populations include populations S I, FI1 and F02 and variations thereon, and they are described hereinbelow in detail.

Bacterial community analysis of mixed populations S I, FI1 and F02 was performed on DNA obtained by phenol-chloroform-isoamyl alcohol extraction from bacterial cultures where cells had been disrupted by bead beating. Populations had been cultured for four days at 50 °C in sterile MBMl medium [180 g meat-and-bone meal 1 (MBMl) per liter of water]. Bacterial 16S gene assay by tag-encoded FLX amplicon pyro sequencing (bTEFAP) and bacterial diversity data analysis were performed by the Research and Testing Lab (Lubbock, Texas, USA) as described by Dowd et al. 2008a and Wolcott et al. 2009. Primers 28F ' GAGTTTGATCNTGGCTC AG' (SEQ ID NO: 1) and 519R ' GTNTT ACNGCGGCKGCTG' (SEQ ID NO: 2) were used for amplification of 16S variable regions Vl-3 (wherein "N" is A, T/U, G or C) and wherein K is T/U or G).

Bacterial diversity analysis revealed the presence of bacteria belonging to 33 different genera (TABLE XIII(a)). Of the total of 64 results, 50 were identified at the species level and 14 at the genus level. TABLE XIII(c), hereinbelow, presents the predominant bacterial genera and species in each population. Bacteria belonging to 6-7 genera form the majority of all populations. Clostridium spp. and Sporanaerobacter acetigenes are predominant in populations S I and FI1. F02 differs from S I and FI1 in consisting predominantly of bacteria belonging to genera Bacillus, Thermoanaerobacterium and Clostridium.

TABLE XIII(c). Predominant bacterial genera and species in populations S I, FI1 and F02. The results are expressed as percentage of total population.

Correlation coefficients [TABLE XIII(d), hereinbelow] were calculated from data presented in TABLE XIII(a) using equation [1], where X and Yrefer to two matrices, e.g. S I and Fll, between which the correlation is calculated, x and y are single values within a matrix, and x and y are the means of all values within a matrix. Species not present in the population [empty cells in TABLE XIII(a)] were assigned a value 0. Corre X, Y) = [Equation 1]

V∑0-x) ² ∑(y-y) ²

TABLE XIII(d) Correlation coefficients between bacterial diversities of mixed populations calculated from data presented in TABLE XIII(a) using equation [1].

The term "substantially similar" with respect to a bacterial population as disclosed herein, means that a bacterial population has a correlation coefficient of at least 0.8 when compared to one or more of the bacterial populations defined by TABLE XIII(a). Preferably, a substantially similar bacterial population has a correlation coefficient of at least 0.9, and more preferably, a substantially similar bacterial population has a correlation coefficient of at least 0.95 when compared to one or more of the bacterial populations defined by TABLE XIII(a). Other statistical methods for comparing populations can be used as well.

TABLE XIII(c) shows a very high similarity between all populations at the age of 4 days. The majority of all populations comprises of only a few species and genera, remaining very similar under all conditions tested and outcompeting innate populations present in animal-origin materials.

Bacterial diversity analyses based on sequencing molecular methods are biased due to e.g. primer specificity and universality (Dowd et al. 2008b). Therefore, the method described hereinabove must be used as a standard when comparisons to the mixed populations presented herein are performed. Table XIV The experiments performed with the crushed bone and the fermented crushed bone to test the influence of the fermentation on phosphorus solubilization using citric acid. The fermented crushed bone was obtained after fermenting, vacuum filtrating and washing of 120 g crushed bone. After making the acid solutions in milli-Q water each of the solutions was mixed with the crushed bone or the fermented crushed bone.

Total volume

Amount of

Acid Amount of of acid

Bone material parallel solution acid, g solution,

experiments ml

120 g crushed bone Citric acid 14 200 3

Fermented crushed

bone derived from Citric acid 14 200 3

120 g crushed bone

Table XV The experiments performed with the crushed bone and the fermented crushed bone to test the influence of the fermentation on phosphorus solubilization using sulfuric acid (Sigma- Aldrich). The fermented crushed bone was obtained after fermenting, vacuum filtrating and washing of 120 g crushed bone. After making the acid solutions in milli-Q water each of the solutions was mixed with the crushed bone or the fermented crushed bone.

bone

Table XVI The results of the experiments to test the influence of the fermentation on phosphorus solubilization using citric acid. The amount of the acid was 117 g per 1 kg crushed bone. Each result is the average of three parallel experiments. The yield of the solubilized phosphorus was determined as a mass percent of the phosphorus content of the crushed bone. Crushed bone Fermented crushed bone

Time Yield of Concentration Yield of

Concentration

of acid dissolved of dissolved dissolved of dissolved

treatphosphorus, phosphate, phosphorus, phosphate, g/1

ment % g i %

7 h 8.13 20.1 12.15 30.0

24 h 15.29 37.8 22.95 56.7

30 h 24.52 60.6 33.84 83.6

2 days 27.82 68.7 35.36 87.4

3 days 23.60 58.3 32.32 79.8

Table XVII The results of the experiments to test the influence of the fermentation on phosphorus solubilization using sulfuric acid. The amount of the acid was 117 g per 1 kg crushed bone. Each result is the average of three parallel experiments. The yield of the dissolved phosphorus was determined as a mass percent of the phosphorus content of the crushed bone.

Crushed bone Fermented crushed bone

Time Yield of Concentration Yield of

Concentration

of acid dissolved of dissolved dissolved of dissolved

treatphosphorus, phosphate, phosphorus, phosphate, g/1

ment % g i %

7 h 18.32 45.3 22.79 56.3

24 h 15.95 39.4 20.89 51.6

30 h 19.69 48.6 25.83 63.8

2 days 29.46 72.8 36.60 90.4

3 days 31.42 77.6 38.91 96.1

EXAMPLE 4

TESTING EFFECT OF FERMENTATION TIME ON THE PROCESS

Crushed bone with a phosphorus content of 2.2 % and organic matter content of 27 % was used in these experiments. Crushed bone was fermented with S I bacteria population for 3, 5, 7 and 10 days at 50 °C according to the procedure described in Example 3, above Fermentation broths were filtered as a warm suspension by suction filtration with a 140 μιη (micrometer) wire mesh. Fermented crushed bone was washed with warm water (60 °C) until the washing liquor remained clear. After fermentation and washing procedure, 32 - 34 w-% (weight percent) of the starting material remained, which indicates removal of organic matter up to 73.4 %. Table XVIII shows the organic matter contents and phosphorus contents for fermented crushed bone for each fermentation time period. Practically no phosphorus was lost during the fermentation step. This was confirmed by spectrophotometric analysis of the phosphorus content of the fermentation liquor, according to the procedure described in Example 1, and in addition, by analyzing the phosphorus content after fermentation according to Table XVIII. The results indicate that longer fermentation times (5, 7 and 10 days) do not further diminish the amount of organic matter.

Table XVIII Organic matter content and phosphorus content of the fermented crushed bone after different fermentation times. *)Calculated from wet material. The composition of crushed bone and fermented crushed bone were analysed by Novalab, Karkkila, Finland.

Phosphorus content

Fermentation time, Removed organic matter

after fermentation, days after fermentation, %

%*

3 70.9 6.8

5 73.4 5.5

7 69.3 5.8

10 69.1 6.8

Fermentation results were further examined with shorter fermentation times. Fermentation was performed with S I bacteria inoculate according to procedure described in Example 3. Crushed bone was fermented for different times, and then subjected to solubilization using citric and sulfuric acid. The Fermentation step was performed according to the procedure described in Example 3, above, with following quantities. 240 g of crushed bone was weighed in six bottles, 330 ml tap water was added to each bottle and mixed. 30 ml of bacterial inoculum was added to each mixture and was mixed. 260 g of crushed bone was weighed in six bottles, 357.5 ml of tap water was added to each bottle and the mixtures were shaken. 32.5 ml of bacterial inoculum was added to each mixture. The fermentation times (the incubation time) were

3, 6, 10, 12, 14, 16, 18, 24, 30, 48, 54 and 72 hours. After the desired incubation time had elapsed, two of the bottles (240 g and 260 g of crushed bone) were filtered and washed with warm tap water (60 °C) until the washing water was clear. The composition of the fermented crushed bone matrix is presented in Table XIII(b). Total amount of phosphorus was 2.2 % of the material (crushed bone). One organic and one inorganic acid were chosen for these solubilization tests based on the above previous results. The acids used were citric and sulfuric acid. The preparation of these two acids is shown in a table below.

Table XIX Making the citric acid solutions in milli-Q water.

Mass of acid per 1 Amount of Volume of acid

kg of solids, g acid, g solution, ml

150 7.5 100

Table XX Making the sulfuric acid solutions in milli-Q water.

Mass of acid per 1 Amount of Volume of used Volume of Volume of water, kg of solids, g 100 % acid, g acid, ml water, ml ml

150 7.5 4.29 95.71 100

Summary selection of acid dissolving with citric acid and sulfuric acid after different fermentation times is presented in Table XXI.

Table XXI. Concentration of dissolved phosphates in g/1 from different fermented crushed bone (FBC) using citric and sulfuric acid as dissolving acids.

Citric 100 3 14.3+0.3 4 h acid 6 14.3+0.5

(150g/l) 10 20.7+0.7

12 28.1+2.8

14 33.6+1.6

16 32.5+3.6

18 20.7+0.8

24 22.0+0.8

30 25.8+1.0

48 23.9+1.3

54 27.2+.2.2

72 35.4+1.5

Citric 100 3 17.5+0.2 7 h acid 6 19.0+0.2

(150g/l) 10 26.2+1.1

12 37.1+3.2

14 33.5+0.3

16 35.5+0.2

18 41.7+2.5

24 41.6+2.6

30 44.8+2.2

48 37.6+5.1

54 42.0+4.8

72 44.6+0.6

Citric 100 3 27.9+0.8 24 h acid 6 27.4+1.3

(150g/l) 10 39.6+0.7

12 52.4+2.4

14 54.3+4.0

16 45.1+6.5

18 72.1+6.4

24 53.3+3.2

30 68.5+5.7

48 62.5+6.1

54 66.4+5.7

72 71.6+3.3 Citric 100 3 44.1+1.9 2 d acid 6 34.9+3.8

(150g/l) 10 55.3+7.8

12 61.1+2.7

14 44.8+0.4

16 56.2+0.5

18 54.8+7.0

24 62.8 +6.1

30 83.0+7.7

48 88.8+4.5

54 79.0+2.6

72 83.1+4.8

Sulfuric 100 3 22.4+0.8 4h acid 6 25.2+1.3

(150g/l) 10 43.4+0.1

12 46.6+2.3

14 56.0+3.9

16 48.3+3.8

18 49.6+2.0

24 53.8+5.7

30 44.2+3.2

48 44.0+6.6

54 47.5+5.5

72 50.1+3.9

Sulfuric 100 3 23.6+0.9 7h acid 6 25.0+0.9

(150g/l) 10 42.5+1.8

12 45.5+4.3

14 49.6+0.9

16 55.4+0.7

18 57.7+2.7

24 57.5+8.1

30 59.8+4.3

48 53.4+4.6

54 69.8+3.6

72 64.3+1.2

Sulfuric 100 3 26.3+1.7 24h acid 6 27.3+0.3

(150g/l) 10 50.4+2.0

12 45.1+1.4

14 49.6+4.6 16 64.0+5.5

18 73.9+9.4

24 70.6+1.1

30 70.9+4.9

48 69.9+1.9

54 77.1+2.8

72 74.4+2.8

Sulfuric 100 3 33.7+1.3 2d

acid 6 35.9+1.6

(150g/l) 10 67.6+3.2

12 64.5+4.0

14 52.1+1.6

16 57.4+3.6

18 68.0+4.5

24 64.1+7.3

30 70.9+1.2

48 72.9+1.0

54 68.0+3.1

72 67.5+8.3

Sulfuric 100 3 36.4+0.2 7d

acid 6 40.9+0.3

(150g/l) 10 -

12 68.1+6.4

14 80.0+11.6

16 80.7+10.5

18 80.0+4.3

24 73.3+5.6

30 -

48 71.9+5.1

54 80.8+13.1

72 87.4+4.2

*BC is crushed bone

The above results clearly show that the recovery of phosphorus increases after fermentation times of over 10 hours, compared to shorter fermentation times. In addition, the amount of phosphorus recovered remained at the same level with fermentation times of 10-72 hours, which indicated that a sufficient time for fermentation can be as low as 10 hours with selected acid and bone containing material. The amount of organic matter present in crushed bone materials that were fermented for five different time periods (6, 18, 24 48, 72h) was analyzed at Novalab (Karkkila, Finland). Analysis indicated that all samples contained 35-39% of organic matter, in dry weight, that indicated similar removal of organic matter with all fermentation times of 18h, 24, 48h and 72 hours. This proves that a sufficient fermentation time for removal of organic matter is 18 hours or less. Table XXII shows the results of solids removal during different fermentation times, and also gives analysis data for organic matter and phosphorus content. FIG. 5 also clearly shows that shorter fermentation times of 3 and 6 hours produces a lower yield of dissolved phosphate. According to the results, sufficient time for the fermentation step is 12-18 hours.

Table XXII Removal of solids during fermentation with S I bacteria population. In addition, analysis data obtained from Novalab (Karkkila, Finland) in regards of organic matter and phosphorus content

Organic Removed Phosphorus

Crushed matter in organic content bone dry weight matter

Fermentation remained after time, h after % during fermentation, fermentation, fermentation,

% %

%

3 79.8 - - -

6 78.1 56 15.6 2.5

10 38.2 - - -

12 33.1 36 63,6 6.4

14 30.7 - - -

16 33.2 - - -

18 33.2 36 63.3 6.1

24 35.4 37 57.4 5.6

30 32.7

48 33.9 35 62.7 5.8

54 33.1

72 31.9 39 70.9 5.8 EXAMPLE 5

SOLUBILIZATION OF NONFERMENTED CRUSHED BONE IN ACID AT TEMPERATURES OF 50 °C AND 95 °C

Solubilization of phosphorus was tested at two different temperatures in Example 5. The crushed bone used in these experiments contained 2.7 % phosphorus, 28.9 % nitrogen, 31 % organic matter and 53.8 % moisture. The used acids were chosen based on the previous experiments and based on the prices of the acids. The acids included in these experiments were citric, L-(+)- lactic and formic acid. The acid concentrations were 2 % (w/w) and 5 % (w/w) of acid in the reaction mixture (w/w refers to weight/weight). The temperatures tested were 50 °C and 95 °C. The experiments were conducted in the following manner: 80 g of crushed bone was weighed in glass bottles. Different acid solutions of 200 ml were made according to Tables XXIII and XXIV. Acids were preheated to the temperatures 50°C and 95°C before adding each acid solution to one of the bottles containing crushed bone. The experiments were conducted according to the same procedure as the experiments in Example 1. The results are presented in Table XXV. Based on the results it seems that high temperatures do not bring benefits for the process, therefore lower temperatures can be used.

Table XXIII Making the citric acid solutions in milli-Q water. The total volume of the acid solution was 200 ml.

Mass percent of

Amount of Concentration of acid in reaction

acid, g acid solution, g/1 mixture, %

2 5.6 28

5 14.0 70

Table XXIV Making the acid solutions of the liquid acids in milli-Q water. The total volume of each acid solution was 200 ml. The density of 98 % formic acid was 1.22 kg/1 and the density of 85 % L-(+)-lactic acid was 1.206 kg/1. Mass percent

Amount Volume of Volume Concentration of acid in

Acid of 100 % used acid, of water, of acid reaction

acid, g ml ml solution, g/1 mixture, %

98 % 2 5.6 4.7 195.3 28

Formic acid 5 14.0 11.7 188.3 70

85 % L- 2 5.6 5.5 194.5 28

(+)-Lactic

5 14.0 13.7 186.3 70 acid

Table XXV The results of solubilization of phosphorus from crushed bone at elevated temperatures.

Mass

percent Amount Yield of

Conditions Concentration

of acid in of acid dissolved

Acid of acid of dissolved

reaction per 1 kg phosphorus treatment phosphate g/1

mixture, BC, g %

%

2 70 50°C, 7h 6.26 18.9

Citric 5 175 50°C, 7h 11.78 35.6 acid 2 70 95°C, 7h 5.19 15.7

5 175 95°C, 7h 14.18 42.8

2 70 50°C, 4h 3.85 11.1

L-(+)- 5 175 50°C, 7h 11.08 33.5 Lactic

2 70 95°C, 4h 1.51 4.5 acid

5 175 95°C, 4h 5.30 16.0

2 70 50°C, 2h 7.90 23.8

Formic 5 175 50°C, 7h 14.98 45.2 acid 2 70 95°C, 2h 3.60 10.9

5 175 95°C, 7h 8.47 25.6

EXAMPLE 6

SOLUBILIZATION OF FERMENTED CRUSHED BONE IN ACID WITH DIFFERENT FERMENTATION TIMES AT TEMPERATURES OF 37 °C AND 70 °C

Crushed bone materials, fermented with S I bacteria population for 3, 5, 7 and 10 days, as described in Example 3, were further treated with 3 % and 5 % citric, formic and sulfuric acid solutions at 37 °C and 70 °C. Fermented crushed bone of 40 g and 100 ml of each acid solution were added to 24 different 250 ml glass bottles. The amount of acid was calculated as a percentage based on the whole reaction mixture. The bottles were shaken vigorously at start and placed in a water bath or an incubator. Samples (1 ml) were taken after 4, 7, 24 and 30 h, and in some cases experiments were continued for several days. Before each sampling, the reaction mixtures were thoroughly shaken. Samples were centrifuged and the separated liquid supernatant was spectrophoto metrically analyzed for phosphate(Synergy HI Reader) according to the procedure described in Example 1, above. The results are presented in Table XXVI. The phosphorus solubilization ability of the tested acids diminished in the following order: sulfuric acid > citric acid > formic acid. The highest recovery percent was observed after 4 and 7 h. The percentage of phosphorus recovery with sulfuric acid was reduced after 4 h treatment. A fermentation time of 3 days proved to be sufficient. Longer fermentation times did not yield better phosphorus recovery. High temperature treatment did not improve the recovery of phosphorus. The highest phosphorus recovery was observed by treating with 5 % sulfuric acid for 30 h at 37 °C and for 4 h at 70 °C, yielding phosphorus recovery percentages of 49 % and 39 %, respectively.

EXAMPLE 7

PHOSPHORUS DISSOLUTION BY DIFFERENT ACIDS AT 10°C AND ROOM TEMPERATURE FOR FERMENTED CRUSHED BONE

Crushed bone was fermented for 3 days at 50°C in an incubator according to Example 3. Fermented bone was treated with 3% and 5% citric, formic, DL-malic and sulfuric acid solutions according to procedure described in Example 2. The results are presented in Table XXII. More concentrated acid solutions of 5% gave up to 50% higher recovery rates than 3% acid solutions, at both room temperature (RT) and at 10°C. 24 hour treatment with 5% sulfuric acid and 5% DL-Malic acid at RT gave yields of 57% and 45%, respectively. A summary of the results is presented in the Table XXII. Table XXVI Phosphorus recovery (%) with acid treatments of sulfuric, citric, formic, DL-malic and L-(+)-lactic acid at different temperatures.

Acid Temperature

Sulfuric acid 3% 10°C RT 37°C 70°C

4h 23.3 24.7 23.5 22.4

7h 25.3 27.4 24.3 21.2

24h 19.3 21.6 17.9 15.9

30h 18.3 17.5 18.1 18.2

48h 19.0 16.1 14.9 15.9

3d 14.5 14.2 - -

6d 17.4 14.3 - -

Sulfuric acid 5% 10°C RT 37°C 70°C

4h 33.9 28.4 38.3 40.3

7h 49.7 47.9 47.2 38.7

24h 43.8 56.8 44.2 31.3

30h 44.7 48.1 49.1 33.8

48h 45.4 52.1 44.4 29.0

3d 41.4 53.4 - -

6d 32.5 37.1 - -

Citric acid 3% 10°C RT 37°C 70°C

4h 6.2 11.1 10.5 12.9

7h 13.8 - 12.9 13.8

24h 17.2 15.3 14.5 -

30h 13.9 19.8 17.5 15.6

48h 19.7 14.5 15.6 16.9

3d 18.3 13.0 - -

6d 19.7 11.6 - -

Citric acid 5% 10°C RT 37°C 70°C

4h 20.4 24.4 20.6 22.7

7h 27.6 27.1 20.6 25.4

24h 37.9 30.4 34.5 17.0

30h 34.9 38.2 29.7 16.7

48h 38.7 28.8 22.9 15.1

3d 29.7 22.9 - -

6d 23.0 21.1 - -

Formic acid 3% 10°C RT 37°C 70°C

4h 9.1 14.0 26.3 16.5

7h 20.7 16.3 22.4 13.4

24h 7.8 4.9 7.3 4.0

30h 5.0 4.6 8.5 4.6

48h 3.1 3.0 5.8 5.9 3d 2.8 3.8 - -

6d 1.8 3.1 - -

Formic acid 5% 10°C RT 37°C 70°C

4h 23.9 27.1 17.5 9.6

7h 25.8 30.0 14.3 9.3

24h 15.2 11.8 3.2 4.0

30h 12.3 9.9 3.3 5.5

48h 10.0 8.3 2.4 5.2

3d 14.7 7.7 - -

6d 8.6 11.0 - -

DL-Malic acid 3% 10°C RT 37°C 70°C

4h 21.3 20.8 - -

7h 10.9 14.0 - -

24h 13.8 16.3 - -

30h 13.9 16.8 - -

48h 18.8 21.4 - -

DL-Malic acid 5% 10°C RT 37oC 70oC

4h 29.2 28.4 - -

7h 27.2 26.3 - -

24h 31.1 45 - -

30h 32.7 37.1 - -

48h 34.3 24.8 - -

L-(+)-Lactic acid 3% 10°C RT 37°C 70°C

4h 5.4 7.6 - -

7h 7.0 6.3 - -

24h 7.9 6.6 - -

30h 8.0 6.2 - -

48h 5.2 4.0 - -

L-(+)-Lactic acid 5% 10°C RT 37°C 70°C

4h 18.0 19.7 - -

7h 17.4 22.4 - -

24h 9.1 13.2 - -

30h 7.0 9.1 - -

48h 3.8 5.8 - -

Comparing acid concentrations at room temperature

Crushed bone was fermented similarly according to the Example 3. 5%, 7% and 10% acid solutions of citric, tartaric, DL-malic, and sulfuric acid were used to acid dissolution experiments. Results presented in Table XXVII. Effect of different concentration (5%, 7%, 10%) of tartaric acid at room temperature are presented in FIG. 3 for clarity purposes to illustrate effect of acid concentration to the yield.

Table XXVII Recovery of phosphorus with different acid concentrations.

Recovery of Phosphorus

(%)

4h 7h 24h 30h

Citric acid 5% 25.3 25.7 30.0 42.6

Citric acid 7% 19.1 54.3 39.3 58.1

Citric acid 10% 29.7 41.3 76.4 75.2

Tartaric acid 5% 27 33.9 33.9 36.0

Tartaric acid 7% 39.3 48.2 55.0 62.7

Tartaric acid 10% 47.5 84.3 86.2 99.9

DL-Malic acid 5% 20.6 24.3 37.6 42.3

DL-Malic acid 7% 26.9 32.3 59.0 57.7

DL-Malic acid 10 % 32.1 44.9 62.7 75.6

Sulphuric acid 5% 31.0 48.0 53.9 40.2

Sulphuric acid 7% 29.5 49.0 67.6 66.4

Sulphuric acid 10% 48.9 60.6 86.5 86.4

Further tests were conducted with citric acid, malic acid and mixtures thereof, according to the procedure described in Example 2, above, at room temperature. First, the crushed bone was subjected to fermentation according to Example 3, above with S I bacteria population. Acid concentrations of 8.5 and 10% were used, and each sample had a biological replicate. The acid dissolving stage was continued for 7 days and the samples were analyzed for released phosphate after 4, 7, 24, 30h and 3d, 4d and 7d. The results are presented in Table XXVIII. The result is an average of two biological replicates, and the error bars show the standard deviations of three replicate spectrophotometry phosphate measurement.

Table XXVIII Recovery of phosphorus with citric acid, malic acid and their mixture.

Citric acid gave over 80% yields with both acid concentrations of 8.5% and 10%. Malic acid gave also high yields of dissolved phosphorus after 30h treatment time. An acid concentrations of 8.5% and 10% correlates to 120g/l and 140 g/1 acid solutions, respectively. However, mixture of citric acid and malic acid gave much lower recovery yields as seen in Table XXVIII. Results show that citric acid and malic acid with concentrations of 8.5-10%(w/w) are excellent options to dissolve phosphorus from bone containing material.

EXAMPLE 8

OPTIMIZING THE RATIO OF THE BONE FEEDSTOCK AND WATER IN THE ACID SOLUBILIZATION OF PHOSPHORUS

The mass of the fermented crushed bone and the amount of the acid were the same in each experiment, but the volume of the water where the acid was dissolved was changed in the different experiments. The crushed bone used in these experiments contained 2.2 % phosphorus, 25.9 % nitrogen, 27 % organic matter and 60.7 % moisture. 1.8 kg of crushed bone was weighed in a bucket, 2.475 1 tap water was added to the bucket, followed by mixing. The resulting mixture was preheated to 50 °C in an incubator. Then a 225 ml S 1 bacterial inoculum was added to the mixture, followed by mixing. The fermentation step was conducted as described in Example 3, above. After the 1.8 kg of crushed bone had been fermented, vacuum filtrated and washed, only 36.1 % of the solid crushed bone remained. 40 g of the fermented crushed bone was weighed into 14 glass bottles, and each of the 14 samples was mixed with the acid solutions, prepared according to Tables XXIX and XXX. The experiments were conducted at room temperature according to the same procedure as detailed by Example 1, above. The results are presented in Tables XXXI and XXXII.

Table XXIX Making the citric acid solutions for the experiments to optimize the ratio of the fermented crushed bone and the amount of water where the acid is dissolved. After making the acid solutions in milli-Q water each of the solutions was mixed with 40 g of the fermented crushed bone.

Amount Total volume of

Acid

of acid, g acid solution, ml

40

60

Citric 80

14

acid 120

140

160

Table XXX Making the sulfuric acid solutions for the experiments to optimize the ratio of the fermented crushed bone fermented with S 1 bacteria population and the amount of water where the acid is dissolved. After the acid solutions were prepared in milli-Q water, each of the solutions was mixed with 40 g of the fermented crushed bone. Amount of Volume Total volume

Volume of 95 %

Acid 100 % acid, of water, of acid

acid solution, ml

g ml solution, ml

31.991 40

51.991 60

95 %

71.991 80

Sulfuric 14 8.009

111.991 120 acid

131.991 140

151.991 160

Table XXXI The results of the experiments to optimize the ratio of the fermented crushed bone and the amount of water where the citric acid is dissolved. 126 g of citric acid was added per 1 kg of crushed bone. The yield of the dissolved phosphorus was determined as a mass percent of the phosphorus content of the crushed bone.

Total

Time of Yield of

volume of Concentration

acid dissolved

acid of dissolved

treatment, phosphorus, solution, phosphate, g/1

h %

ml

4 61.38 32.9

40 7 89.56 47.9

24 144.54 77.4

4 43.84 35.2

60 7 61.33 49.2

24 107.89 86.6

4 38.41 41.1

80 7 58.64 62.8

24 81.36 87.1

4 22.63 29.7

100 7 31.47 41.3

24 58.24 76.4

4 25.14 40.4

120 7 32.81 52.7

24 50.15 80.5

4 21.08 39.5

140 7 30.22 56.6

24 45.25 84.8

4 18.94 40.6

160 7 21.48 46.0

24 32.63 69.9 Table XXXII The results of the experiments to optimize the ratio of the fermented crushed bone and the amount of water, where the sulfuric acid is dissolved. The amount of the sulfuric acid was 126 g per 1 kg crushed bone. The yield of the dissolved phosphorus was determined as a mass percent of the phosphorus content of the crushed bone.

Total

Time of Yield of

volume of Concentration

acid dissolved

acid of dissolved

treatment, phosphorus, solution, phosphate, g/1

h %

ml

4 52.11 27.9

40 7 107.45 57.5

24 124.20 66.5

4 - -

60 7 72.88 58.5

24 91.86 73.8

4 - -

80 7 53.42 57.2

24 84.60 90.6

4 37.27 48.9

100 7 46.17 60.6

24 65.91 86.5

4 40.46 65.0

120 7 40.36 64.8

24 44.86 72.0

4 35.08 65.7

140 7 35.36 66.2

24 43.56 81.6

4 24.38 52.2

160 7 29.25 62.6

24 32.59 69.8

Based on the above results, an 80 ml acid solution per 40 g of fermented crushed bone gave the highest recovery. EXAMPLE 9

SOLUBILIZATION OF FERMENTED CRUSHED BONE USING CRUSHED LEMON

Solubilization of phosphorus from fermented crushed bone (fermented with S 1 bacteria population) using crushed lemon was tested in two different temperatures. Room temperature (RT) was chosen based on previous results showing that higher temperatures did not give any advantages in solubilization of phosphorus. A temperature of 10°C was chosen to prove that lower temperatures will not affect the rate of solubilization of phosphorus from fermented crushed bone. The experiments were done in the following manner: Lemon was crushed in a mixer, and mixtures containing 5% and 30% (w/w%) of lemon were made according to Table XXXIIII. The mixture of lemon and water used at lower temperature was cooled first to 10°C. 40 g of fermented crushed bone was weighed in glass bottles. Each lemon mixture was added to one bottle containing fermented crushed bone. The results are presented in Table XXIV.

Table XXXIII Making the lemon mixtures in milli-Q water. The total volume of each mixture was 100 ml.

Concentration of

Number of The mass of Concentration of lemon in reaction

experiment lemon, g lemon mixture, g/1 mixture, %

1

5 7 70

2

3

30 42 420 4

Table XXXIV The results of solubilization of phosphorus from fermented crushed bone usin crushed lemon.

3 62.7 RT, 6d 4.38 5.7

4 375.9 10°C, 6d 8.18 10.6

Based on the results of these experiments, the ability of lemon to dissolve phosphorus was found to be limited.

EXAMPLE 10

OTHER WASTE MATERIALS

Phosphorous containing materials were tested without fermentation and after fermentation with S I bacteria inoculate according to a procedure described in Example 3. The tested materials were: minced food waste, fish by-product, minced chicken by-product, minced broiler by-products, feather meal, and bovine skin material. The phosphorus contents are presented in Table XXXV.

Table XXXV. Phosphorus content of different materials analyzed by Novalab (Karkkila, Finland)

Phosphorus content of different waste

materials (%)

Fish by-product 0.7

Broiler by-product 0.75

Minced chicken by-product 1.6

Ground feathers 0.33

Minced food waste 0.5

Unfermented waste material. Two biological replicate samples of minced food waste (50g) were weighed in a glass bottle and 100 ml of citric acid solution and 100 ml of sulfuric acid solution were added to the parallel samples. Acid solutions contained 15 g of acid per 100ml solution. This means 300g of acid/ 1 kg of waste material. The same procedure was repeated using different waste materials. The phosphates were measured spectrophotometrically (Synergy HI Reader) using a Malachite green phosphate assay kit (POMG-25H, BioAssay Systems) according to the manufacturer's instructions. Phosphate amounts were measured spectrophotometrically after Id, 2d and 1 week. Bovine skin gave a very small recovery of phosphorus. Recovery percentages of phosphorus from the different materials are presented in Table XXXVI. The result is an average of two biological replicates and the error bars show the standard deviations of three replicate spectrophotometric phosphate measurement.

It was clear that fish and chicken based waste materials gave the highest phosphorus yields. These materials were chosen for further examinations.

Table XXXVI

Phosphorus recovery

from different waste

material P Yield after 2d (%)

Citric acid 300g/lkg waste Sulfuric acid 300g/lkg waste material material

Minced food waste 8.5+1.3 9.3+0.4

Fish by-product 60.9+1.3 68.6+1.4

Broiler by-product 72.2+1.2 76.9+2.0

Minced chicken byproduct 78.1+1.0 79.7+1.0

Ground feather 19.5+0.7 12.8+0.5

Fermented waste material. Two biological replicate samples of minced chicken by-products were weighed (120g) to glass bottles. 165 ml of tap water and 15 ml of S I bacterial inoculate was added to the mixture. The mixtures were incubated at 50°C for 18 hours. After incubation time, the bottles were opened in a laminar flow hood to release the formed gases. Further, the bottles were heat-treated at 95°C for an hour to inactivate the added bacteria inoculate. Thereafter, the mixture was filtered with 100 μιη wire mesh filter and solids were washed with warm water (60°C). After fermentation and washing procedure, 28% of solids were recovered from minced chicken byproduct. Gained solids were subjected to acid dissolving stage with sulfuric acid. Acid solutions contained 14 g of acid per 100ml solution (140g/l). Acid amount was 117g of acid/ 1 kg of the original waste material and 350g of acid per fermented waste material. Phosphate amounts were measured (kit) after 4,7 24h and 2d. Results are presented in Table XXXVII. The result is an average of two biological replicates and the error bars show the standard deviations of three replicate spectrophotometric phosphate measurement.

Table XXXVII Phosphorus recovery from unfermented and fermented minced chicken by-product

P yield

Acid Sample Time % P g/1

Citric acid Minced 4h 31.3+1.0 6.0+0.6

(140g/l) Chicken 7h 32.8+4.9 6.3+2.9

by-product 24h 44.6+4.8 8.6+2.8

(unfermented) 2d 47.0+8.7 7.5+2.2

Citric acid Fermented 4h 56.3+5.2 10.8+3.1

(140g/l) minced 7h 57.4+5.8 11.0+3.4

Chicken 24h 67.5+9.6 13.0+3.9

by-product

(fermented with

S 1 bacteria

population) 2d 79.8+8.7 15.3+3.9

According to the results phosphorus can be recovered from bone containing chicken based waste materials. Phosphorus recovery from fermented chicken waste material was 79.8% that means 15.3g per liter of phosphorus. However, unfermented chicken material gave only 47% yield (7.5g/l P). EXAMPLE 11

PRODUCTION OF BIOFERTILIZER

Ammonia as a gas phase was directed from ammonia containing liquor, produced from the fermentation step, into a bottle containing a liquid derived from solubilization of crushed bone by sulfuric acid. Ammonia was removed as a gas from our bioreactor by increasing the pH and temperature, and by aerating the bioreactor contents, which converts the ammonium-ions into ammonia gas (the ammonia was stripped from the liquor).

Crushed bone was first fermented for three days at 50 °C, after which the solids were separated from the liquid by filtration. The solids, i.e., fermented and separated crushed bone, was solubilized for 17 hours using sulfuric acid, after which the solids were separated from the liquid by filtration. The filtered liquid was then used as "a trap liquid" which absorbed the gaseous ammonia. The trap liquid contained 0.62 g/kg ammonium nitrogen (NH ₄ -N) before stripping and 5.3 g/kg ammonium nitrogen (NH ₄ -N) after stripping/absorbing. The nitrogen content increased almost nine fold during the stripping stage. As a result, a biofertilizer liquor containing both ammonium and phosphates was formed. It was also observed that valuable micronutrients, in addition to phosphorus, were extracted from the organic waste material during the acid treatment stage. . Analysis (Novalab) of the obtained biofertilizer liquor showed that liquor typically contains small amounts of calcium, magnesium, boron, chlorine, copper, iron, manganese, molybdenum and zinc.

EXAMPLE 12

COMPARATIVE FERMENTATIONS

Comparative fermentations with water and two different bacteria inoculates (S I and soil population) were performed. Composition of the S I mixed bacterial population is presented in table XIII(a) and the soil mixed bacterial population was created by mixing non- sterile forest soil with boiling tap water

in a proportion of 180 g of soil per liter of water. Crushed bone (crushed

bovine and porcine bones) were used as a fresh waste material in this

experiment. Crushed bone samples of 120 grams were weighed, and tap water

(or tap water and bacteria inoculum), was added to samples. 180 ml of water

was added to zero (0) samples (controls) and 165 ml of water and 15 ml of

bacteria inoculate 5% (v/v) to inoculated samples. Samples were treated at

room temperature (RT) or 50°C for a period of 16 hours. After incubation

time, the samples were hygienized at 95°C water bath for an hour (this is

sufficient time and temperature to kill vegetative bacteria). Solids were

filtered through 140μιη wire mesh and washed with hot water (60°C).

Ammonia yields were measured from filtrates using enzymatic determination

kit for ammonia (Ammonia Assay Kit AA0100; Sigma- Aldrich). Washed

solids were subjected to an acid dissolving stage with 100 ml of citric acid

(140g/l) per each sample during 24 hours. Results of the experiment are

presented in table XXXVIII.

Table XXXVIII Crushed bone treated with or without bacteria inoculum at

RT and 50°C.

Mass

removed

Bacteria Ammonia during P yield Dissolved

Sample inoculate Temp, yield fermentation (%) phosphorus

(mg/1) after after 24 after 24 h

(°C) 16 h (%) h (s/i)

BC RT 89.8+44 23.3 59.6+0.8 15.7+2.8

BC S I RT 471.9+191 28.6 58.6+6.2 15.5+4.1

BC - 50 118.6+87 39.2 51.8+3.4 13.6+4.4

BC S I 50 3625.5+345 68.6 91.8+1.8 24.4+4.5

BC

(sterilized) - 50 101.4+24 28.3 38.7+2.7 10.2+2.6

BC

(sterilized) S I 50 3419.9+225 75.1 47.2+3.3 12.5+1.9

BC FI1 50 766.5+33 63.2 58.2+4.5 15.4+1.2

BC is bone crush or crushed bone Results showed that S 1 gives much higher yields of ammonia than plain water or the forest soil inoculum FI1 after 16 hours of treatment at 50°C. In addition, the amount of dissolved phosphates was 24.4g/l (91.8% yield) after a 24 hour treatment time with SI mixed bacteria population. However, samples treated with tap water or forest soil bacteria inoculum provided dissolved phosphate yields of 15.5g/ and 15.4g/l, respectively.

Discussion

Based on the results presented herein, it is clear that there are advantages provided by fermenting bone material before phosphorus solubilization. The advantages of fermenting bone material before phosphorus solubilization with acid treatment include a) the reduced consumption of acid used for the solubilization reaction and b) the removal of bone's organic matter which could potentially form explosive compounds with the solubilizing acid. Table XXXIV illustrates selected results from the various examples.

Acid treatment experiments of fermented crushed bone have clearly showed that phosphorus can be recovered in high yields from bone with several organic acids, such as tartaric acid, malic acid, citric acid, as well as with an inorganic acid, such as sulfuric acid. Nearly 100% recovery yield of phosphorus could be recovered with 10% tartaric acid after 30 hours at room temperature. In addition, 10% sulfuric acid yielded 86% after 30h at room temperature. Tartaric acid is a highly interesting option for the phosphorus solubilization stage of the process, since it is produced as a by-product of wine industry. Also, citric acid, malic acid and lactic acid are interesting and sustainable options for phosphorus solubilization since they can be produced biotechnically.

Room temperature proved to be a suitable reaction temperature. Elevated temperatures did not improve the yields obtained from phosphorus dissolution from bone. Interestingly, the selection of solubilization acid was bone material dependent. For example, for MBM, a different set of acids might be better than for bone crush (BC) and fermented BC. In addition, dissolution times were found to be function of material under treatment.

Based on embodiments phosphorus dissolution consists of two steps 1) fermentation of crushed bone material to remove organic matter and thereafter 2) treatment of fermented crushed bone with acid at different conditions to dissolve phosphorus into soluble phosphates. This two-step process results in a dissolved phosphate solution that can be used, after optional neutralization, as a biofertilizer. In general, phosphate containing crushed bone is fermented up to three (3) days and thereafter treated with organic acid, such as citric, tartaric, malic, lactic acid or with inorganic acid such as sulfuric acid.

According to an embodiment 3-10% acid solution, such as tartaric acid, citric acid, sulfuric acid, can be used as a solubilization acid. Preferably, 7-10% acid concentration is used. Fermented crushed bone could be treated at 10-70 °C temperatures for 4 hours to several days depending on the temperature and solubilization acid. Preferably, room temperature is used at the acid solubilization stage.

Both organic and inorganic acids can be used to solubilize phosphorus from bone material. However, organic acids are less corrosive than strong mineral acids. The solubilization reaction may be accelerated by higher temperatures, however, side reactions also start to occur at higher temperatures. For example, solubilized phosphate also solubilizes calcium ions into the acid solution and these can start to react with acids forming soluble or insoluble precipitates depending on the used acid. Table XXXIX Summary. The highest yields with different acids and concentrations.

Amount

Yield of

Acid of acid Concentration Concentration

Treatment dissolved

Acid concentration per 1 kg of dissolved of dissolved

conditions phosphorus

% crushed phosphate g/1 phosphorus g/1

% bone

3 35.8 RT, 30h 15.3 5.0 19.8

Citric 5 59.7 RT, 30h 29.5 9.6 38.2 acid 7 83.5 RT, 30h 44.3 14.5 58.1

10 119.3 RT,30h 57.3 18.7 75.1

3 35.8 RT, 7h 21.2 6.9 27.4

Sulfuric 5 59.7 RT, 24h 44.8 14.6 57.9 acid 7 83.5 RT, 24h 51.5 16.8 67.6

10 119.3 RT, 24h 65.9 21.5 86.5

Formic 3 35.8 37°C, 4h 12.6 4.1 16.3 acid 5 59.5 RT, 7h 23.4 7.6 30.3

L-(+)- 3 37.6 10°C, 24h 6.1 2.0 7.9

Lactic

5 62.7 10°C, 7h 17.4 5.7 22.4 acid

5 62.0 RT, 30h 27.4 8.9 36.0

Tartaric

7 86.7 RT, 30h 47.8 15.6 62.7 acid

10 123.9 RT, 30h 76.2 24.8 100.0

DL- 5 62.0 RT, 30h 32.2 10.5 42.3 malic 7 86.7 RT, 24h 45.0 14.7 59.0 acid 10 123.9 RT, 30h 57.6 18.8 75.6

An example environment 100 of a phosphorus recovery /harvesting/collection process from bone containing material is shown in FIG. 4. The bone containing material is stored in a container 106. The bones are preferably crushed to approximately 0-5mm particle size. The crushed bone in the container 106 is fed to a bioreactor 104. Water is added to the bioreactor 104 from a water source 102. Bacteria or mixed bacterial population (inoculum) is added to the bioreactor 104 from a source 116. The type of inoculum is selected preferably from group consisting of ammonification capable microorganisms. Examples of such bacteria are disclosed in co-owned U.S. Patent Appl. Ser. No. 13/722,228, filed on December 20, 2012, incorporated herein by reference in its entirety. Preferably the inoculum is from a mixed population of bacteria as disclosed by co-owned U.S. Application Ser. No. 14/066,089, filed on October 29, 2013, the contents of which are incorporated by reference herein its entirety.

In a preferred aspect, the bioreactor includes mixing / stirring means 105 and heating/temperature controlling means 110. According to certain embodiments, heating element 110 is used to heat the content of the bioreactor 104 to about 50 degrees Celsius, depending on the selected microorganisms. If needed, the pH can be controlled by adding a base (such as NaOH) to the bioreactor 104 to keep the pH at levels of over 6. Fermentation time is preferably about 3 days.

During the fermentation process using mixed bacterial population ammonium / ammonia is released to fermentation liquid. Samples of the liquid from the bioreactor 104 can be taken from time to time to follow the progress of the process. A parameter to follow is ammonia/ammonium concentration within the liquid. The fermentation process is complete or sufficient when change of the concentration between two consequence samples does not demonstrate significant increase.

The fermentation product of solids and liquid are treated with separate processes according to following procedures. All or some of the liquid from the bioreactor 104 can be led to stripping phase 108 where ammonium/ammonia is extracted as ammonia (NH ₃ ) from the liquid. The ammonia can be stored in a container 112 for future use such as a part of fertilizer production. Alternatively some or all of the liquid can be led to container 113 and used directly as fertilizer. Alternatively, some or all of the liquid can be used in a precipitation process as indicated with dashed line.

After removing all or some of the liquid the remaining solids can be collected in a container 114. Water from source 1140 and acids from source 1142 are added to the container. Alternatively, a ready acid solution can be used. The acid solution (acid / water mixture) results in dissolution of phosphorous from the solids to the liquid. Depending on the usage the liquid that contains phosphorous as phosphates can be collected to container 1144 and used as fertilizer either directly or after adjusting pH level. Alternatively, or additionally, the liquid can be collected to container 1146 for precipitation purposes. Precipitation can be done, for example, by adding an NH ₄ ⁺ containing liquid from 1148 and Mg ²⁺ ions containing solution to the container 1146.

Precipitation refers to the formation of a solid in a solution during a chemical reaction. When the reaction occurs in a liquid, the solid formed is called the precipitate. The chemical that causes the solid to form is called the precipitant. In case of forming phosphorus (P) containing precipitate NH ₄ ⁺ containing liquid and Mg ²⁺ ions containing solution are used as precipitants.

INCORPORATION BY REFERENCE

Numerous references are cited throughout this application, each of is incorporated by reference herein in its entirety.

REFERENCES

CN 102020508 A, Preparation and application method of water-soluble humic acid inorganic compound fertilizer, 2011

Dowd, S.E., Wolcott, R.D., Sun, Y., McKeehan, T., Smith, E., Rhoads, D. 2008a. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyro sequencing (bTEFAP). PLoS ONE 3(10): e3326.

Dowd, S.E., Sun, Y., Secor, P.R., Rhoads, D.D., Wolcott, B.M., James, G.A., Wolcott, R.D. 2008b. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosomes shotgun sequencing. BMC Microbiology 8: 43.

Grech, Nigel, M., EP 1964828B 1, Method of generating phosphorus fertilizers through the utilization of microbial fermentation technology, 2010

Ringelberg, David, B., US6,776,816 B l, Methods for accelerating production of magnesium ammonium phosphate while attaining higher yields thereof and a slow-release fertilizer produced therefrom, 2004

Wolcott, R., Gontcharova, V., Sun, Y., Dowd, S.E. 2009. Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches. BMC Microbiology 9: 226.

Woodruff, Steven R., US 6,464,875 B l, Food, animal, vegetable and food preparation byproduct treatment apparatus and process, 2002