1. A mixture with high carbon contents characterised in that said mixture comprises a deaminated solution of hydrolysates from protein waste subjected to acidic, basic, thermal or enzymatic hydrolysis.
2. Mixture according to claim 1, wherein the protein waste is the tannery waste.
3. Mixture according to claim 1, wherein the protein waste is waste from a chicken farm.
4. Mixture according to any of the claims 1-3, wherein the C:N ratio of the mixture is in the range of 100:10 to 100:1 , preferably of 100:5 to 100:2.
5. A method for preparing a mixture with high carbon contents wherein said method comprises steps of obtaining the hydrolysate solution from protein waste subjected to acidic, basic, thermal or enzymatic hydrolysis.
6. The method according to claim 5, wherein said method comprises steps of soaking the wastes, preferably wet-blue wastes from a tannery, in aqueous base solution, preferably NaOH, in an amount of 5%-35% in relation to the amount of dry leather and in water, mixing the leather and lye suspension for 1-5 hours to obtain a liquid solution of products of protein hydrolysis and a suspension of chromium hydroxide (III) Cr(OH) particles, separating the precipitate of chromium hydroxide from the hydrolysate solution, and following said separation step, obtaining a clear hydrolysate solution.
7. The method according to claim 5, wherein said method comprises the steps of adding the waste, in particular chicken waste, to an autoclave and pouring water, followed by heating under pressure in the range of 1 to 10 atm and at temperature in the range of 100-180°C and maintaining temperature in this range under mild mixing, until the hydrolysis is completed.
8. The method according to any of claims 5-7, wherein the method comprises then the step of deamination using aqueous solution of nitric(III) acid.
9. The method according to claim 8, wherein the method comprises the step of carrying out the deamination process with simultaneous generation of nitric(III) acid by mixing the reagents in the hydrolysate solution.
10. The method according to claim 9, wherein the mineral acid solutions and solutions of nitric(III) acid salts are used to generate nitric(III) acid.
11. The method according to claim 10, wherein sulphuric, hydrochloric and/or orthophosphoric acid or their mixtures are used as the mineral acid, and sodium, potassium, ammonium, calcium or magnesium salt, or their mixtures as the salt of nitric(IIl) acid.
12. The method according to any of claims 5-11 , wherein the method comprises the steps of acidifying the hydrolysate solution with acid and cooling in ice, then adding
13. The method according to any of claims 5-12, wherein carrying out the deamination at temperature 10°C or lower.
14. The method according to any of claims 5-13, wherein the method comprises the steps of heating the alkaline solution of protein hydrolysate to boiling at pH of at least 10 for 8-15 hours under atmospheric pressure, at about 100°C under reflux, and controlling the pH value using NaOH, adding sulphuric acid to pH value of 4 and cooling in ice, then adding slowly cold NaN02 solution into the mixture, in such amount that the amount of NaN02 used is at least half of the initial protein mass in the sample, and maintain the obtained mixture at low temperature by adding ice, then heating the mixture up to 40°C and cooling it down to room temperature.
15. Use of mixture with high carbon contents according to any of the claims 1 -4 for bacterial growth media.
16. Use of claim 15 for adjustment of high-nitrogen waste composition in an activated sludge process.
Biological methods of waste treatment in an activated sludge process are based on culturing of a specific group of microorganisms that use for their growth biogenic compounds contained in waste water. These microorganisms are able to absorb compounds present in waste water and to increase their mass as a result of this process. The suspension of microorganisms formed is easily separated mechanically from the treated water. A part of the obtained microbial activated sludge is returned to the process where they come into contact with a fresh part of waste water, and so called excessive activated sludge comprises final waste directed to further processing, often after additional concentration and dehydration. Exact proportion of key structural elements such as carbon, nitrogen, phosphorus, oxygen and hydrogen in treated waste ensures proper growth of the activated sludge, which is actually a living organism. It is assumed that proper activated sludge process can be achieved with the C:N:P ratio of 100: 10: 1 to 100:5:1. In case of domestic sewage, however, this ratio should be 100:20:5. Waste composition could be corrected using various methods. Increasing the carbon content in regards to nitrogen can be achieved by adding an alcohol, e.g. methanol, or molasses. This results in additional costs. Moreover, use of methanol requires implementation of special safety precautions, because of its harmful and flammable properties. Tanning industry is a source of protein waste, including wastes from tanned leather. Tanned leather waste, so called„wet blue", can be processed in order to recover chromium. Treating such leather with bases leads to chromium release and its transformation into relatively easily separated chromium (III) hydroxide, insoluble in water. However, the process of chromium separation by hydrolysis leads to large amounts of protein hydrolysates obtained from collagen being the main ingredient of leather. Large amounts of hydrolysates generated in these methods formed a new type of waste that has to be reclaimed. Lack of waste management method for such hydrolysates makes this simple method of chromium separation for leather waste not profitable.
MX2013007213 discloses a method of chromium separation comprising an effective processing method for wastes generated during mechanical leather tanning. Efficiency of this method lies in chromiums separation from protein hydrolysates obtained during hydrolysis of chromium-tanned leather and the possibility of using products obtained as a result of ingredient separation. The process is performed using basic hydrolysis facilitated by proteolytic enzymes and by cyclohexylamine. The protein hydrolysate comprising a mixture of amino acids is useful in production of diet supplements for birds or in production of cosmetics. Contrary to conventional methods, the core of the method according to the invention is use of cyclohexylamine, leading to high purity hydrolysate which is substantially chromium-free.
BG60136 presents a method of hydrolysate production in food, pharmaceutical and cosmetics industry. Protein hydrolysate was obtained as a result of enzymatic hydrolysis, using basic protease of subtilisin, under mild conditions, followed by addition of calcium chloride.
Waste streams of many types of protein materials are also generated in many other industry sectors. They are present i.e. in food industry or in meat processing industry.
Patent RU2352134 discloses a method of hydrolysate production from salmon milts. Salmon milts are mixed with water, heated at pH 7.6-8.0, chloroform is added, followed by filtration. The mixture is cooled down to 48-50°C and alkalised using Na 2 C0 3 in the presence of phenolphtalein up to pH 8.2. Extract from bovine thymus and pancreas and 2% chloroform were added. The mixture was maintained in a heat chamber at 35-37°C and incubated for 4-5 days and on the sixth day pepsine was added. The mixture was mixed and stored in the heat chamber for 9-10 days in 35-37°C. Once the reheating was finished, IN HC1 solution was added to pH 4.1 and the mixture was boiled for 9-10 minutes. The obtained hydrolysate was neutralised using 20% NaOH solution to pH 7.0, 2% chloroform was added and the mixture was stored at 2-8°C.
Similarly, RU2344618 discloses amino acid production using enzymatic processing of hydrolysate obtained from fish waste. The reaction mixture was obtained using ground raw fish waste with water and enzymatic preparation. The enzymatic system was inactivated by heating the reaction mixture. The reaction mixture is separated by extraction, yielding the hydrolysate, which has its fat content removed and dried in the next steps. The enzymatic treatment is performed using catholyte in an amount equal to 30% of raw material mass, at 55°C.
Still, there is a need for profitable methods enabling management of such hydrolysates, however, which posed an obstacle in application of methods of chromium separation from wet-blue.
Surprisingly, it was found that it is possible to use entire amount protein hydrolysates. A method of use of hydrolysates as a correcting substrate for constituent element ratios (C N) in wastewater subjected to biological treatment was developed. The processed protein hydrolysates are a natural and inexpensive additive, they stimulate development of microorganisms of the active activated sludge, increase effectiveness of the waste treatment process, lower waste treatment costs and at the same time ensure an effective reclaim method for waste hydrolysates.
Hydrolysates used in the art contain approx. 14% nitrogen in dry mass, which makes them inappropriate as a carbon source in the growth medium because of excessive nitrogen content. A need exists for development of a carbon source of type of methanol, comprising essentially no nitrogen. The disclosed method enables carbon product with very low nitrogen content to be obtained whilst using industrial waste, which makes the component of the growth medium inexpensive and the method beneficial for the environment. The subject of the present invention is a mixture with high carbon contents comprising a deaminated solution of hydrolysates from protein waste subjected to acidic, basic, thermal or enzymatic hydrolysis. Preferably, the protein waste includes waste from a tannery or from a chicken farm. The C:N ratio in the preferred mixture is in the range of 100: 10 to 100:1, preferably of 100:5 to 100:2.
The presented invention also includes a method of obtaining a mixture with high carbon contents including stages in which a hydrolysate solution is obtained from protein waste subjected to acidic, basic, thermal or enzymatic hydrolysis.
Preferably, waste from a wet-blue tannery are soaked in an aqueous solution of a base, preferably NaOH, in an amount of 5%-35% related to the amount of dry leather and in water, the leather-lye suspension is mixed for 1-5 hours in order to obtain a liquid solution of protein hydrolysis products and a suspension of chromium hydroxide (III) Cr(OH) 3 particles, the precipitate of chromium hydroxide is separated from the hydrolysate solution, said separation yielding a clear hydrolysate solution.
In a preferred embodiment of the method, in particular chicken waste is placed in an autoclave and soaked in water, followed by heating under pressure in the range of 1 to 10 atm and in temperature in the range of 100-180°C and maintained in this temperature under mild mixing, until the hydrolysis is complete. Preferably, the hydrolysis is followed by deamination process using aqueous solution of nitric(III) acid.
The deamination process takes place with simultaneous production of nitric(III) acid by mixing the reagents in the mixture of hydrolysates, whereby a solution of a mineral acid and a nitric(III) acid salt is used in order to generate nitric(III) acid. Sulphuric, hydrochloric and/or orthophosphoric acid or their mixtures are used as the mineral acid, and the salt of nitric(III) acid used is - sodium, potassium, ammonium, calcium or magnesium salt, or their mixtures.
In the preferred embodiment of the method, the hydrolysate solution is acidified with acid and cooled in ice, followed by addition of cold aqueous solution containing the salt of nitric(III) acid.
Preferably, the deamination process is performed at temperature not higher than 10°C. In another preferred embodiment of the method, an alkaline solution of protein hydrolysate is heated to boiling at pH of at least 10 for 8-15 hours under atmospheric pressure, at ca. 100°C under a reflux condenser, the pH is controlled using NaOH, followed by sulphuric acid addition to pH=4 and cooling in ice. Next, cold NaN0 2 solution is gradually introduced into the mixture, in such an amount that the amount of NaN0 2 used comprises at least half of the initial protein mass in the sample, and the obtained mixture is kept at low temperature by adding ice, followed by heating the mixture up to 40°C and cooling it down to room temperature.
The presented invention also includes use of mixture with high carbon contents according to the invention in media for microbial cultures, preferably in order to correct the composition of high-nitrogen waste in the activated sludge process.
During studies related to management of wet-blue wastes and separation of chromium salts used during leather tanning, the process of chromium separation by leather waste hydrolysis was studied. Depending on the degree of the performed hydrolysis, a mixture of polypeptides and peptides is obtained, or finally, of free amino acids comprising constituents of collagen. In general, large amounts of protein hydrolysates were obtained in the process. These hydrolysates comprise ca. 95 %wt. of the entire waste mass. They are present in the form of yellow, aqueous solutions with specific, mild protein-like odour. Chemical composition of the hydrolysate may be variable, depending on the type of tanned leather. The value of chromium separated from the leather does not cover the recycling costs. A method employing protein hydrolysates had to be developed in order to ensure profitability of tanned leather waste management process. It turned out that large amounts of waste hydrolysates many be used in a completely different field, such as biological waste treatment facilities. An aerobic culture of specific bacterial microflora is actually provided in the activated sludge process, using biogenic material contained in waste in its metabolic processes. Correct ration of constituent elements in the waste supplied to the waste treatment facility is necessary for correct growth of the microbial flora. Such wastes very often show a carbon deficit. This should be understood as carbon present in various chemical compounds found in the waste. Various compounds, such as methanol, ethanol, etc. are added during this stage of waste treatment in order to decrease said deficit and improve the ratio of carbon to other elements. Such additives ensure correct growth of microbial flora and efficient waste treatment. Economic balance of the waste treatment facility includes, however, additional costs of purchase of said carbon additives. Finding inexpensive substitutes of carbon additives could lower the costs of waste treatment facility operation and provide a flexible approach to the waste treatment process. It turned out that protein hydrolysates may be used as additives in the process of biological waste treatment as well as be used in order to correct the ratio of constituent elements in waste. This process would open a large hydrolysate market, simultaneously creating options enabling these wastes to be properly managed. On the other hand, it creates the possibility of recycling for various protein waste, in particular wet-blue waste which has been unprofitable because of i.e. problems related to management of large amounts of obtained hydrolysates. Thanks to the use of processed hydrolysates, efficiency and effectiveness of biological waste treatment is also improved.
The use of hydrolysate solutions in the activated sludge process in order to correct the C:N ratio is preceded by reaction of maximum protein hydrolysis execution performed on the raw material. Such hydrolysis may be performed as a basic process (basic hydrolysis) or acidic process (acidic hydrolysis), as well as an enzymatic hydrolysis process. Regardless of the technique of hydrolysis completion used, the initial processing should yield a solution of amino acids (a) or their forms combined with the base used, e.g. NaOH (b) or the acid used, e.g. HC1 (c):
(a) R-CH(NH 2 )-COOH
(b) R-CH(NH 2 )-COO " Na +
(c) R-CH(NH 3 + )-COOH CI "
Thus prepared solution of amino acids and/or of their derivatives is subjected to a deamination reaction. Amino acids deamination is performed efficiently and with high yield using nitric(III) acid. Nitric(III) acid causes the amino acids to undergo deamination reaction, the products of which include molecular nitrogen and a respective hydroxyacid. Nitric(III) acid is easily obtained in the reaction medium by introduction of nitric(III) acid salt into the acidic medium of the solution. An example of such reaction is given by equation (1): 2 NaN0 2 + H 2 S0 4 → 2 HN0 2 + Na 2 S0 4 (1)
Deamination of an a-amino acid generally occurs according to the general reaction (2), presented below:
H-C— COOH + HN0 2 *-H— C—COOH + N 2 + H 2 0
NH 2 OH
This reaction proceeds quantitatively and results in removal of nitrogen from amino acid molecules. Products of this reaction include chemically inert nitrogen hydroxyacid. Generally, primary amines undergo the deamination process.
Protein hydrolysates from wet blue wastes in the tanning industry accompanied the process of alkaline wet blue dissolution. Wet blue waste was soaked in an aqueous solution of strong base, e.g. NaOH in an amount of ca. 10%-20% in relation to the mass of dry leather, and in such an amount of water which enabled mixing which accelerated the process. Heating the leather suspension in a base for ca. 3 hours yielded a liquid solution of protein hydrolysis products, and a suspension of chromium(III) hydroxide particles, Cr(OH) 3 . The precipitate of chromium hydroxide may be separated from the hydrolysate solution relatively easily and it can be subjected to chromium separation in another technological path. A clear hydrolysate solution with a concentration of ca. 5%-35%, depending on the amount of water used per unit of leather mass, earlier being nothing more than an inconvenient waste, became a raw material for preparation of a corrective mixture for waste treatment involving the activated sludge process.
Basic solution of protein hydrolysates obtained in the process in Example 1 a, containing polypeptides, peptides and amino acids as their sodium salts, with dry mass content of 7.6% was gently boiled in alkaline medium with pH of at least 10 for 11 hours under atmospheric pressure, at ca. 100°C, under a reflux condenser. The pH level was controlled by NaOH addition. Piotrowski test for presence of peptide bonds was performed after the aforementioned time. The test was negative, proving that the protein material underwent complete hydrolysis. The solution of amino acids was acidified to pH=4 using sulphuric acid, and then cooled in ice. Cold NaN0 2 solution was gradually introduced into the cooled mixture, in such an amount that the NaN0 2 amount used comprised at least half of the initial protein mass in the sample. The mixture was cooled in ice, evolution of nitrogen bubbles was observed. The NaN0 2 solution was added over the course of 3 hours. Then the reaction mixture was heated up to 40°C followed by cooling to room temperature. Elemental analysis showed a C:N ratio of 100:4.1 in the product.
The obtained solution was successfully used in order to correct the composition of high-nitrogen waste in activated sludge process, replacing previously used methanol.
Basic hydrolysis of chicken feather waste obtained from a poultry slaughterhouse was performed. 350 kg of chicken feathers was placed in an autoclave and 1,500 kg of water was added, followed by heating under pressure to 155C and maintaining in this temperature applying mild mixing until hydrolysis was complete. Thus obtained amino acid solution from chicken feathers subjected to deamination. The hydrolysate solution was acidified using 230 kg of sulphuric acid and cooled in ice. Cold solution containing 320 kg NaN0 2 was introduced to this solution during 4 hours. Evolution of gas bubbles was observed over this period of time. After further 2 hours, the mixture was heated to almost 50°C to final degassing of the solution and then cooled. A high-carbon solution was obtained. Elemental analysis showed C:N ratio of 100: 2.8.