JP2013529969A - Use of low temperature anaerobic digestion in a continuous batch reactor for prion degradation - Google Patents

Abstract

The present invention relates to the use and process of using a continuous batch reactor to remove prions at specific risk sites, and to measuring the effectiveness of continuous batch reactors to degrade prion proteins at specific risk sites. is there.
[Selection] Figure 1

Description

(Cross-reference to related applications)
This application is the first application in the present invention.

  The present invention relates to the use or process of using a continuous batch reactor to remove prions at specific risk sites.

  Prions are proteins without nucleic acids and cell membranes and are hardly affected by standard sterilization methods. Prions are an unprecedented infectious pathogen that results in a group of neurodegenerative diseases that die without exception through a completely new mechanism (1). Inactivation of prions poses significant environmental and health problems both in the disposal of prion-infected animals and the preparation of animal-derived materials such as animal feed. Slaughterhouse sludge and animal death (carcasses) are important sources of pathogens or infectious prion proteins.

  Prions are proteins that are found naturally in animals. Cellular or normal prion protein is constitutively expressed in the brain of healthy mature animals, but is highly regulated spatially and temporally during development (Non-patent Document 1). There are two isoforms of prion protein. PrPC (normal prion protein) is a cell structure, N-linked, and is a globular soluble glycoprotein containing a lot of α-helix, acting as a signal transduction protein (Non-patent Document 2), and for normal development of the mammalian brain. It plays an important role. PrPC occurs in both healthy and diseased tissues. PrPSc (abnormal prion protein) is a fibrous, highly insoluble protein containing a large amount of β-sheet, and is a causative agent of mammalian central nervous system diseases. The fact that it contains a large amount of β-sheet is thought to bring heat resistance and protease resistance to PrPSc.

  The presence of an abnormal pathological isoform (PrPSc) is typical of a pathological condition. PrPC and PrPSc have the same primary structure, and a difference occurs in the secondary structure. PrPC contains three α helices (about 40% α helix) and one short antiparallel β sheet, and PrPSc consists of two α helices (about 30% α helix) and four β sheets (45% β helix). Sheet) (Non-patent Document 1). This induces a structural change in the tertiary structure of PrPSc, which is a modification property. Neither PrP isoform has a nucleic acid (Non-patent Document 1).

  Experimental data shows that prions can survive in the natural environment for a long time. For example, Brown and Gajdusek suggested that a prion buried in the garden soil could dissolve slightly deep into the soil and survive for three years to remain infectious ( Non-patent document 3).

  Recent experiments suggest that the primary method of infection of animals is ingestion. Prions are thought to accumulate in the environment through dead animal debris and urine, saliva, and other body fluids. The prion then combines with clay and other minerals to remain in the soil. Prion sterilization involves denaturing the protein to a state where the molecule can no longer cause abnormal folding of the normal protein. However, prions are generally very resistant to these treatments, although their infectivity is weakened by treatment with proteases, heat, radiation and formalin. Effective prion decontamination relies on protein hydrolysis or protein tertiary structure reduction and / or destruction. For example, bleach, caustic soda or strong acid detergent.

  Transmissible degenerative encephalopathy (TDE) forms a group of fatal neurodegenerative diseases by the accumulation of prions in the mammalian brain. In TDE, the normal prion protein (PrPC) of the host is modified to infectious prion protein (PrPSc) upon infection (Non-patent Document 4), and deposits are formed in infected tissues, particularly the central nervous system. Is unique. TDE affects many types of wildlife, livestock and humans and exists in three ways, all of which involve modification of the prion protein: genetic by gene mutation; Sporadic by spontaneous conversion of the prion protein to a pathological form by an undefined mechanism; infection by contact with an exogenous abnormally folded prion protein.

  Bovine Spongiform Encephalopathy (BSE) is the most famous prion disease. The International Trade Commission (ITC) reported that trade restrictions imposed by BSE caused $ 11 billion in damage to the livestock industry from 2004-2007.

  Thus, the management of animal fertilizers is not only harmful to the environment in many cases, but it also creates a foul odor, promotes fly breeding, induces weed problems and air, soil and water contamination, as well as humans. And represents a potential risk to animal health.

  According to the Canadian Food Inspection Agency, the estimated amount of specific dangerous parts (SRM) that occurred in Canada was 170,000 tons per year. Safe disposal of SRMs that may have been contaminated with prions is difficult because these TDE causative agents are compared to inactivation by physical or chemical treatments that are usually applied to microorganisms This is because there is mechanical resistance (Non-patent Document 5). Furthermore, some processes are costly. The physical and chemical methods of prion deactivation that efficiently and / or rapidly immobilize prions, including rapid heating with alcohol, aldehydes and steam, protect the prions from deactivation and thus the heat of the prions Stability is enhanced. Therefore, rather than immobilizing the protein structure, a process of destroying it is necessary when considering a process of SRM that may be contaminated with prions. Furthermore, treatments using chemicals such as denaturants, detergents, strong alkalis, etc. are unsuitable for inactivating prions in the SRM due to the possibility of touching the user on the farm and disposal issues on the farmland.

(Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383) Mouillet-Richard et al., Science, 289: 1925-1928 Brown and Gajdusek, 1991, J Infect Dis 153: 1145-1148 Carp et al., 1985, J Gen Virol, 66: 1357-1368. Taylor et al., 1994, Arch Virol, 39: 313-326

  Therefore, there is a need to develop an environmentally friendly biological treatment that degrades prions in the SRM.

  Therefore, it would be highly desirable to provide a process for removing specific risk site prion proteins from animals that is low cost, very stable, easy to operate and does not interfere with normal farm operations.

  According to the present disclosure, a process for degrading a prion protein at a specific risk site is provided. The process includes the steps of supplying a specific risk site (SRM) to a continuous batch reactor (SBR) containing a layer of conditioned anaerobic sludge, and reacting the specific risk site with sludge at 25 ° C. or less to produce prion protein. Disassembling.

  It also provides a process for measuring the effectiveness of a continuous batch reactor (SBR) that degrades prion protein at specific risk sites. The process includes supplying a specific risk site (SRM) to a continuous batch reactor (SBR) containing a layer of conditioned anaerobic sludge, adding a model protein to the SBR, a specific risk site and a model protein, Reacting with sludge at a temperature of 5 ° C. to 25 ° C., and the degradation of the model protein shows the effectiveness of SBR to degrade the prion protein in the SRM.

  In a preferred embodiment, the specific risk area also includes an animal carcass.

  In another embodiment, the anaerobic sludge is from pig manure, livestock manure and / or slaughterhouse sludge.

  In a preferred embodiment, the specific hazardous area is reacted with sludge at a temperature of 5 ° C to 25 ° C, preferably at a temperature of 20 ° C to 25 ° C, more preferably at a temperature of 20 ° C.

  In another embodiment, the process for degrading a specific risk site prion protein described herein also includes adding keratin to the SBR. Keratin can be derived from feather keratin or hoof keratin, which correspond to β-keratin or α-keratin. Suitably, the feather keratin is derived from chicken feathers and the hoof keratin is derived from cow hooves.

  In another embodiment, the SBR is an anaerobic digestion continuous batch reactor or a medium temperature anaerobic digestion continuous batch reactor.

  In another embodiment, the model protein described herein can be at least one of perchlorate soluble protein, collagen, elastin and keratin.

  By “specific risk site” is meant any site that may contain a prion concentrate, such as ruminant cellular tissue. Such sites include the brain, skull, eye, trigeminal ganglion, spinal cord, spinal column, dorsal root ganglion, tonsils and the distal ileum of the small intestine, for example.

  The following description is made with reference to the accompanying drawings.

1 is a schematic diagram of a laboratory scale continuous batch reactor. FIG. It is a graph which shows the reduction | decrease of the feather keratin in the bag of the reaction tank which supplied the feather keratin. It is a graph which shows the reduction | decrease of the feather keratin in the bag of the reaction tank which supplied the feather keratin. It shows the gas generation measured in the reaction tank, and more specifically, the cumulative gas generation in the reaction tanks (reaction tanks 34 and 35) containing feather keratin and the reaction tanks (reaction tanks 13 and 14) not containing them. It is shown. It shows the gas production measured in the reaction tank, more specifically, per day in the reaction tank (reaction tanks 34, 35) containing feather keratin and the reaction tank (reaction tanks 13, 14) not containing This shows the gas generation. It shows the cumulative methane production measured in the reaction tanks (reaction tanks 34, 35) containing feather keratin and the reaction tanks (reaction tanks 13, 14) not containing feather keratin. The acetic acid concentration measured by the reaction tank (reaction tanks 34 and 35) containing feather keratin and the reaction tank (reaction tanks 13 and 14) not containing them is shown. It shows the propionic acid concentration measured in a reaction tank (reaction tanks 34 and 35) containing feather keratin and a reaction tank not containing (reaction tanks 13 and 14). The propionic acid profile measured by the reaction tank (reaction tanks 34 and 35) containing feather keratin and the reaction tank (reaction tanks 13 and 14) which do not contain is shown. It shows the concentration of isobutyric acid measured in a reaction vessel containing feather keratin (reaction vessels 34 and 35) and a reaction vessel not containing them (reaction vessels 13 and 14). It shows the soluble chemical oxygen demand (SCOD) measured in the reaction tank (reaction tanks 34 and 35) containing feather keratin and the reaction tank (reaction tanks 13 and 14) not containing. The pH profile measured by the reaction tank (reaction tanks 34 and 35) containing feather keratin and the reaction tank (reaction tanks 13 and 14) which do not contain is shown. The alkalinity profile measured by the reaction tank (reaction tanks 34 and 35) containing feather keratin and the reaction tank (reaction tanks 13 and 14) which do not contain is shown. It shows the evaporation residue (TS) measured in a reaction tank (reaction tanks 34, 35) containing feather keratin and a reaction tank (reaction tanks 13, 14) not containing feather keratin. It shows the ignition loss (VS) measured in a reaction tank (reaction tanks 34, 35) containing feather keratin and a reaction tank (reaction tanks 13, 14) not containing feather keratin. The organic suspended solids (VSS) profile measured with the reaction tank (reaction tanks 34 and 35) and the reaction tank (reaction tanks 13 and 14) which do not contain feather keratin is shown. It shows the amount of keratin hydrolyzate (KHO) present in the mixed liquid of the management reaction tank 13 and the reaction tank 34 containing feather keratin. Figure 2 shows a measured correlation between feather keratin degradation and KHO number. The accumulated gas production | generation measured by the reaction tank (reaction tanks 15 and 16) and the reaction tank (reaction tanks 13A and 13B) which do not contain feather keratin is shown. It shows the gas production per day measured in a reaction tank (reaction tanks 15 and 16) containing feather keratin and a reaction tank (reaction tanks 13A and 13B) not containing feather keratin. It shows the cumulative methane production measured in the reaction tanks (reaction tanks 15 and 16) containing feather keratin and the reaction tanks (reaction tanks 13A and 13B) not containing feather keratin. The acetic acid concentration measured by the reaction tank (reaction tanks 15 and 16) containing feather keratin and the reaction tank (reaction tanks 13A and 13B) not containing feather keratin is shown. It shows the propionic acid concentration measured in a reaction tank (reaction tanks 15 and 16) containing feather keratin and a reaction tank (reaction tanks 13A and 13B) not containing feather keratin. It shows the isobutyric acid concentration measured in a reaction tank (reaction tanks 15 and 16) containing feather keratin and a reaction tank (reaction tanks 13A and 13B) not containing feather keratin. It shows a soluble chemical oxygen demand (SCOD) measured in a reaction tank (reaction tanks 15 and 16) containing feather keratin and a reaction tank not containing (reaction tanks 13A and 13B). The pH profile measured by the reaction tank (reaction tanks 15 and 16) containing feather keratin and the reaction tank (reaction tanks 13A and 13B) not containing is shown. The alkalinity profile measured by the reaction tank (reaction tanks 15 and 16) containing feather keratin and the reaction tank (reaction tanks 13A and 13B) not containing them is shown. It shows the evaporation residue (TS) measured in a reaction tank (reaction tanks 15 and 16) containing feather keratin and a reaction tank (reaction tanks 13A and 13B) not containing feather keratin. The ignition loss (VS) measured by the reaction tank (reaction tanks 15 and 16) containing feather keratin and the reaction tank (reaction tanks 13A and 13B) not containing feather keratin is shown. The volatile suspended solids (VSS) measured in the reaction tank (reaction tanks 15 and 16) containing feather keratin and the reaction tanks (reaction tanks 13A and 13B) not containing feather keratin are shown. It shows microbial degradation of feather keratin in PAD planted with pig manure (Experiment 2). FIG. 2 shows microbial degradation of hoof keratin in a PAD planted with pig manure.

  Low temperature anaerobic digestion batch reactor (PADSBR) and medium temperature anaerobic digestion series to remove specific risk site (SRM) prions and reduce biological contamination of fauna, soil, groundwater and surface water Provides use of batch reactor (MADSBR) technology.

  Currently, incineration is the primary method of SRM processing in Europe. Experimental results indicate that prions in the SRM are unlikely to survive incineration (Taylor et al., 1996, Neuropath Appl Neuro, 22: 256-258). However, this process is expensive and any value derived from SRM is eliminated. In addition, incineration is not a practical method in many regions due to the geographical dispersion of the herd because it involves the additional charges and unacceptable contamination risks associated with transporting prion-contaminated SRMs.

  The very good resistance of prions to dry heat is found in lyophilized samples, ie samples dried on glass or steel surfaces. Initial experiments show prion removal with a gravity displacement autoclave performed at a temperature of 134 ° C. for 1 hour. However, experiments conducted in porous loaded autoclaves showed inconsistent results. According to the data, the thermal stability of the prion increased with increasing autoclave temperature (from 134 ° C to 138 ° C) (Taylor, 1999, Vet Microbiol, 67, 13-16). This indicates that increased temperature and increased exposure time of SRM contaminated with prions to a porous loaded autoclave is not effective as a reliable decontamination.

  Ionization, UV irradiation and microwave irradiation have little effect on prions (Taylor and Diprose, 1996, Neuropath Appl Neuro, Vol. 22, pages 256-258). In general, it is recognized that the inactivation effect of microwaves on microorganisms is due to the generated heat.

  While strong sodium hypochlorite solution or hot solution of sodium hydroxide is considered to be the only way to completely inactivate prions, concentrated formic acid reduces the infection level of histologically fixed tissues. Decrease significantly. Prions are not completely inactivated by exposure to autoclave or sodium hydroxide solution, but inactivation is achieved by combining these two treatments. However, chemicals added to the SRM can adversely affect the fertilizer value of the SRM, so chemical treatments that inactivate prions are useful for large disposal strategies on cattle slaughterhouse sludge or carcass farms. Is not practical.

  Concentrated formic acid solubilizes the protein. As with irradiation, prions have little effect after exposure to ethylene oxide, glutaraldehyde, formalin, β-propiolactone or acetylethyleneimine.

  The only detergent that has some effect on prions is sodium dodecyl sulfate (SDS), but reports that SDS must be used only for contaminated fluids (Taylor et al., 1999, Vet. Vet Microbiol, 67: 13-16). Thus, it seems impractical to treat SRM with SDS or other detergents.

  Industrial solvent extraction processes using ethanol, ether, acetone, 5% chloroform, 4% phenol, general phenolic disinfectants and industrial solvents using hexane, heptane, perchlorethylene or petroleum reduce prion infectivity The effect is weak.

  Scrapie pathogens exposed to 3% hydrogen peroxide, chlorine dioxide, or peracetic acid were either inactivated or only slightly inactivated.

  All of these known treatments are costly and may protect prions from inactivation, thus increasing the thermal stability of the prions and destroying the protein structure rather than immobilizing it on the farm. It is not suitable for inactivation of prions in the SRM because it can be exposed and has disposal problems on farmland.

  Thus, environmentally friendly biological treatments that degrade prions in the SRM represent an alternative means, as described herein.

  Anaerobic digestion of organic matter is a process used for organic waste treatment and energy generation. In an anaerobic bioreactor, organic matter is removed from wastewater by the cooperation of various microbial groups. The activity of microbial species involved in mineralization of organic matter is extremely important for obtaining efficient decomposition of organic matter in a biological reactor. During anaerobic digestion, organic matter is converted into hydrolyzable / fermentable acid-producing bacteria, acid-oxidizing bacteria and acid-oxidizing bacteria through acetic acid, short-chain volatile fatty acids (propionate, butyric acid, isobutyric acid, etc.) and hydrogen dioxide / carbon dioxide. It is converted to methane by the activity of a complex microorganism consisting of methanogenic archaea. Four large groups of microorganisms with different functions have been identified throughout the degradation process. The first group is hydrolyzing and fermenting microorganisms, which first attack the polymers and monomers found in waste to produce mainly acetate and hydrogen, and propionate, but propionic acid Volatile fatty acids (VFA) such as butyric acid and some alcohols are also produced in different amounts; the second group are hydrogen-producing acetic acid producers, which convert propionic acid and butyric acid into acetic acid and hydrogen; Two groups of methane producing archaea (3 and 4) produce methane from acetic acid or hydrogen, respectively.

  Cattle slaughterhouse sludge or carcass is a substrate of readily biodegradable organic matter that can be converted to high quality biogas that can be processed into heat and electrical energy. Slaughterhouse waste is suitable for anaerobic treatment and removal of many solid waste is achieved. The concentration of effluent in heavy metals (cadmium, cobalt, nickel, copper, chromium) is below the detectable limit. Nutrients such as calcium, magnesium, sulfur and ions are at concentrations suitable for biological treatment of wastewater. Slaughterhouse effluent is generally coagulated and agglomerated to remove colloidal substances and suspended solids responsible for color and turbidity (Al-Mutairi, 2006, Ecotoxicology and Environmental Safety, No. 1) 65: 74-83). Flock formers such as lime, alum, sodium aluminate, ferric chloride, and ferrous sulfide are used to remove suspended solids.

  The generated sludge is mainly composed of blood, rumen contents, stomach contents, undigested food, urine, free meat, soluble protein, fat, fecal matter and the like. The proportion of these elements in the sludge varies from slaughterhouse. Al-Mutairi (2006, Ecotoxicology and Environmental Safety, 65: 74-83) describes the alum and polymer used to solidify and agglomerate solids from slaughterhouse effluent. Point out that it may be toxic to the microflora of the next ecological treatment process. When the concentration of the coagulant in the precipitation tank exceeds 100 mg / l, the waste water becomes toxic to microorganisms. The precipitated sludge is more contaminated than the supernatant of the precipitation tank and is toxic.

  Hygiene is important when bringing external ingredients to livestock farmers for mixed digestion. Wastewater from poultry and pork processing plants can be contaminated with biological contaminants. Pathogen removal prior to land return is important to prevent reintroduction of the pollution cycle into livestock production systems.

  The fate of prions in anaerobic treatment has never been reported before (Kirchmayr et al., 2006, Wat Sci Technol, 53, 91-98). There was no reduction in prion titer at moderate temperatures (Kirchmayr et al., 2006, Wat Sci Technol, 53, 91-98).

  FIG. 1 is a schematic diagram of a laboratory scale continuous batch reactor (SBR) used in the present disclosure, as described in Canadian Patent 2,138,091, A reference number is attached. Each reference number is as follows.

  In the SBR shown in FIG. 1, manure is loaded on the supply system 14 and supplied to the biological reaction tank 1 through the supply line 7. Manure is supplied through the bottom of the biological reaction tank 1 inoculated with anaerobic sludge 2 in advance. The biogas in the biological reaction tank 1 can be recirculated to the gas space 4 through the biogas recirculation line 5 using the biogas recirculation pump 6.

  Described herein is the use of SBR to decontaminate animal brains, remove prions, and reduce the risk of biological contamination of soil, groundwater and surface water.

  Experiments performed with infectious isoforms of prion protein (PrPSc) require a biosafety level 3 laboratory. Proteins that are similar to PrPSc in structure (number of amino acids, high percentage of glycine residues, high content of high β sheet structure, high aggregation) and properties (protease resistance, heat resistance, weak solubility) Selected to confirm the effectiveness of SBR against prion protein in livestock manure (Arai et al., 1996, J Appl Polym Sci, 60: 169- 179; Caughey and Raymond, 1991, J Biol Chem, 266: 18217-18223; Pan et al., 1993, US Science Proc Natl Acad Sci USA, 90: 10962-10966; Prusser et al., 1988, Cell, 93 : 337-348; Weissmann, 1999, J Biol Chem, 274, 3-6). Non-limiting examples of such suitable model proteins added to an anaerobic digestion batch reactor (AD-SRB) for reproduction of the fate of prions in these biological reactors and for experimentation are disclosed below. To do.

  Perchlorate soluble protein (PSP) (14.149 kDa protein with 137 amino acid residues) was isolated from rat kidney, brain and lung and rat, pig and chicken liver (Hui et al., 2004). Biochem Biophys Res Commun, Vol. 321: 45-50). The secondary structure of the protein (2α helix and 6β sheet) is similar to that of PrPSc (2α helix and 4β sheet). PSP, like PrPSc, is thermostable and resistant to proteinase K. This suggests that PSP is a suitable model protein for experiments of PrPSc fate by AD-SBR.

  Collagen is the most abundant protein in mammals and represents 25% of the total protein. Collagen is the primary, fibrous, insoluble extracellular protein of skin, tendons, cartilage, bones and teeth and serves to bind cells.

  Elastin is an extracellular matrix fibrous cross-linking protein that provides elasticity to many tissues, which allows the cells to stretch many times and return to their original state when tension is released. Elastin is abundant in blood vessel walls and ligaments, especially in the neck of herbivores.

  Keratin forms one of the most diverse types of fibrous proteins. There are various types of keratin, each consisting of a complex mixture of proteins. Keratin is a major component of the structure that grows from the skin. Alpha keratin is found in mammalian hair (including wool), horns, nails, claws and hoofs. The hoof is the toe of the ungulate and is covered with a thick cornea (keratin). The hoof consists of a hard or elastic sole, with a hard wall formed by thick nails wrapped around the toes.

  Alpha keratin is the most common form and consists mostly of polypeptide chains in helix conformation. Harder β-keratin is an alternative form present in reptile claws, scales and shells, and bird claws, scales, wings and feathers (Mr. Zubay, 1988, Biochemistry, New York, NY, USA): Macmillan Publishing Company). β-keratin is composed of stacked and folded β-sheets and is more contained in PrPSc. The amino acid sequences of various avian β-keratins are as similar as collagen and elastin and contain a high proportion of glycine (Harrap and Woods, 1964, Biochem Jay ( Biochem J), 92: 19-26). Glycine-rich repeats are found in some of the β-keratins, but not all. β-keratin is shorter (98-155 amino acids) than PrP protein (256-264 amino acids).

  Alpha keratin is composed of extremely long alpha helix polypeptide chains that are arranged side by side to form a helical fiber. The alpha helix packing is optimized and enhanced by wrapping individual helices together. Many forms of alpha keratin contain (covalent) disulfide bonds formed between cysteine residues of adjacent polypeptide chains, thereby providing permanent thermostable crosslinking. The amount and strength of keratin is controlled by the amount of disulfide bridges. About one-quarter to one-third of feather keratin has a β-conformation, most of which is concentrated in the central hydrophobic part of the molecule. The remaining proteins form an irregular matrix. Closely related β-keratin is the main constituent of feathers and constitutes about 90% of the wing shaft.

  Slaughterhouse inoculum recovered from a semi-industrial scale anaerobic array bench bioreactor (AD-SBR) was used here. The SBR reactor was operated at 20 ° C. and was supplied weekly with fresh sludge from a commercial cattle slaughterhouse (Colbex, Levinoff, Quebec) that processed about 1000 cattle per day. Approximately 50 kg of slaughterhouse sludge was collected weekly, a representative subsample (1 L) was stored at 20 ° C. for later analysis, and the remainder was used to feed the semi-industrial SBR reactor.

  Again, the pig manure inoculum used was recovered from a semi-industrial scale anaerobic array bench bioreactor (AD-SBR). Similarly, this was maintained at 20 ° C. and fresh sludge was supplied weekly from a commercial pig farm (1944 ch. Tremblay. Ste-Edwidge, Quebec) that processes approximately 4000 pigs per year. Approximately 50 kg of pig manure was collected weekly, a representative subsample (1 L) was stored at 20 ° C. for later analysis, and the remainder was used to feed the semi-industrial SBR reactor.

  Also described is the use of acclimatized anaerobic sludge as a starting material. Bacterial sludge requires time to adapt to the new substrate when there are significant changes in the feed components of the bioreactor. New substrates in the feed may control the sludge microflora. Usually, bacterial performance increases with time. Anaerobic sludge adapts when the sludge reaches maximum specific methane production (1 L of methane per gram of feed) and this maximum performance is maintained over time.

  In North America, feathers are one of the largest solid wastes. The feathers are mainly made of keratin. Keratin is insoluble and exhibits high mechanical stability and resistance to proteolysis. Feather keratin is a beta keratin that consists mainly of stacked and folded beta sheets, so feather keratin is commonly used for trypsin and pepsin because of its high degree of cross-linking through disulfide bonds, hydrogen bonds and hydrophobic interactions. Incompletely digested by various digestive enzymes (Papadopoulos, 1986, Animal Feed Science and Technology, 14: 279-290). A similar structure is found in infectious protein (PrPSc). Thus, feather keratin has many amino acids, a high proportion of glycine residues, a high β-sheet structure content, a high degree of aggregation, and properties similar to prion proteins (protease resistance, heat resistance, weak solubility) For this reason, chicken feathers are used as model proteins.

  Similarly, hoofs also have high hard keratin content, insolubility, and resistance to degradation by common digestive enzymes such as trypsin, pepsin and papain due to advanced crosslinking by disulfide bonds, hydrogen bonds and hydrophobic interactions. Used as a model protein of PrPSc.

  Degradation of feather keratin in AD-SBR is described herein by way of example (see Table 1 for exemplary conditions). A decrease in feather keratin in the bag was seen in the reaction vessel, as shown in FIGS. 2A and 2B. Feather keratin in each reaction vessel decreased almost linearly from 31 g at the start of the SBR experiment, to 5 g after 120 days. On the other hand, there was no or only slight decrease in feather keratin in the reaction vessel bag supplied with water containing antibiotics. The addition of feather keratin had an effect on gas generation in the reaction vessel. As shown in FIG. 3A, gas production in all reactors with or without feather keratin increased dramatically over the first 15 days. After 15 days, gas production in the control reactor gradually increased until the experiment was completed. No gas production was seen for a certain period. After some time, gas production began again in the reactor at a faster rate than the controlled reactor. On average, the gas production in the reactor containing feather keratin was 1.2 times higher than the gas production in the management reactor containing no feather keratin. Addition of feather keratin stimulated gas generation.

  The effect of feather keratin on gas production was also observed in the daily gas production rate. As shown in FIG. 3B, the gas production rate in all reactors fluctuated between 3-23 L per day in the first 13 days. Thereafter, the gas production rate in the control reaction tank gradually decreased, and became 1 L or less per day on the last day of the experiment. However, the gas production rate in the reaction vessel containing feather keratin fluctuated between 0-7.7 L per day even after 13 days, and the same trend continued until the last day of the experiment. The average rate measured in the reaction vessel was 1.6 times higher than that measured in the control reaction vessel.

  The effect of feather keratin addition on gas generation can also be seen in the cumulative methanogenesis profile (FIG. 4A). Methane production in all reactors increased rapidly in the first 15-20 days. Thereafter, methane production in the controlled reactor gradually decreased until the experiment was completed. On average, the reaction vessel containing feather keratin produced 1.4 times more keratin than the control reaction vessel. The concentration of volatile fatty acids (VFA) including acetic acid, propionic acid and isobutyric acid in the mixture of all reactors was also observed during SBR operation. The profiles of these VFAs are shown in FIGS. 4B, 4C, and 4D, respectively. During the first 10-15 days, the concentration of the three organic acids was significantly higher. This would have been due to the presence of fermentable organic substrates in the initial sludge. After their consumption, the concentration of each VFA varied within a certain range. Acetic acid varied between 23 and 97 mg / L. There was no apparent difference in acetic acid concentration between the reactor containing feather keratin and the control reactor for the first 60 days. The average acetic acid concentration in the reaction vessel containing feather keratin was 93 mg / L and 97 mg / L, respectively, higher than 75 mg / L and 68 mg / L in the control reaction vessel. This suggests that the addition of feather keratin stimulated the production of acetic acid.

  Propionic acid concentration was also affected by the addition of feather keratin. As shown in FIG. 4C, the propionic acid concentration was high in the initial sludge. This decreased gradually over the first 15 days. Thereafter, it varied between 0 to 26 mg / L (see FIG. 4D). As seen in the acetic acid profile, during the first 62 days, there was no significant difference in propionic acid production between the reactor containing feather keratin and the control reactor. After 72 days, the concentration of propionic acid measured in the reaction vessel containing feather keratin was almost higher than the concentration in the control reaction vessel. Addition of feather keratin did not significantly affect the concentration of isobutyric acid (see FIG. 5A). After 90 days, most of the isobutyric acid concentrations measured in the reactor containing feather keratin were higher than those in the control reactor. The average concentration in the reaction tank containing feather keratin was 1.9 times the average concentration in the control reaction tank, indicating that the addition of feather keratin had a positive effect.

  Feather keratin also affects the concentration of soluble COD. As shown in FIG. 5B, in the first 60 days, the SCOD of all reactors gradually decreased with the same trend. Thereafter, the SCOD in the control reactor continued to decrease until the end of the experiment. However, the SCOD in the reaction vessel containing feather keratin gradually increased until the end of the experiment. The average SCOD concentration in the reaction vessel containing feather keratin was 8757 mg / L, 1.1 times that measured in the control reaction vessel (8171 mg / L).

  Feather keratin also affected the pH of the mixture in the SBR reactor. As shown in FIG. 6A, the pH of all reactors decreased from 8.0 to 8.1 to 7.8 in the first 50 days. Thereafter, all pH values measured in the reaction vessel containing feather keratin were 0.05-0.10 higher than those measured in the control reaction vessel. Feather keratin significantly affected alkalinity. As shown in FIG. 6B, in the first 60 days, the alkalinity of all reactors varied between 25000-27000 mg / L mostly. Thereafter, the alkalinity measured in the control reactor maintained the same tendency until the experiment was completed. However, the alkalinity of the reactor containing feather keratin gradually increased to 28839 and 28171 mg / L.

  Furthermore, feather keratin also affected the TS profile. As shown in FIG. 7A, TS in all control reactors gradually decreased in the first 80 days and then fluctuated within a certain range. In general, the TS of the reaction vessel containing feather keratin decreased at a faster rate than in the case of the control reaction vessel. The TS values measured in the reaction tank containing feather keratin were almost lower than those measured in the control reaction tank. On average, the TS value in the control reaction tank was 1.1 times higher than in the reaction tank containing feather keratin. Similar profiles to those seen in TS are also found in VS and VSS profiles. VS and VSS in all reactors gradually decreased over the first 60-80 days and then fluctuated within a certain range. The major difference is that the VS and VSS of the reactor containing feather keratin decreased to a lower level than that in the control reactor. The VS and VSS values measured in the reaction vessel containing feather keratin were almost lower than those measured in the reaction vessel not containing feather keratin.

  Feather keratin in the bag decreased in AD-SBR fed pig manure. The observed decrease was mostly due to microbial activity, ie keratin hydrolyzing microorganisms excreted keratinase in the bag and hydrolyzed feather keratin. Keratin added to AD-SBR fed porcine manure did not negatively affect the performance of AD-SBR. Keratin improved biogas production and reduced AD-SBR biomass production.

  Fluorescence labeling and visualization of keratin hydrolyzate (KHO) was performed using the BODIPY® fluorescent exoenzyme staining method. BODIPY® FL casein was first applied to a sample of feather bags in the bioreactor. Thirty minutes after the start of staining, fluorescence was seen on bacteria with a rod-like morphology on a weak fluorescence background. No other morphology was seen during the 180 minute staining. Sampling and microscopy were performed every 15 minutes. However, background fluorescence increased with time. The same staining was performed on samples from all reactors with and without feather keratin. When samples from different AD-SBRs (feather bag fluid or mixture) were heated at 100 ° C. for 10 minutes (as a negative control), no fluorescence (including background fluorescence) was seen, which was observed It shows that the fluorescence is due to the activity of microorganisms. A set of inhibitors for identifying and labeling KHO in AD-SBR and not labeling consuming microorganisms of the labeled hydrolyzate that may have been stained by BODIPY® FL staining (Xia et al., 2008, FEMS Microbiology Ecology, 66: 462-471) was added to all staining cultures. To inhibit glycolysis, TCA cycle and electron transport chain, iodoacetic acid, fluoroacetic acid and azide were added, respectively. Each inhibitor was added at a concentration that effectively inhibits energy metabolism of all microorganisms in AD-SBR (Xia et al., 2008, supra). In the presence of the inhibitor, all previously observed rod-like fluorescence was continuously observed. The difference is that the number of KHO significantly decreased after inhibition. Thus, positively stained bacteria are KHOs that are presumed to perform the degradation of feather keratin observed in these AD-SBRs.

  The relative number of KHO in the AD-SBR mixture was measured while keeping all processes including sampling, sample preparation, microscopy, image capture and image analysis constant. The results are shown in Table 1. As shown in FIG. 8A, the number of KHO in the control reaction tank gradually decreased from 150 on the 0th day to 56 on the 113th day. The number of KHO in the reactor containing feather keratin decreased slightly from 142 to 131 in the first 20 days, increased to 175 on day 85, and decreased to 121 on day 113.

  As the KHO number increased and the accompanying KHO activity increased in the feather bag, a considerable number of soluble keratin was released into the mixture. Thereafter, KHO in the mixed solution began to grow, propagated, became large (101) at the end of the third month, and then decreased. However, it remained high (81) at the end of the fourth month. This is because when a bag was once taken out from the SBR, a new feather bag was installed, so that soluble keratin or a hydrolyzate thereof continued to be released into the mixed solution. The relative number of KHO in the feather keratin bag of the reaction vessel was also counted. Measure the number of bacterial cells obtained after centrifugation at 800 g, 1000 g, 1500 g, 2000 g, 2500 g, or 3000 g to effectively collect KHO in feather bags for BODIPY® FL casein staining did. The obtained bacteria were stained with DAPI, and the number thereof was measured with a microscope. The results are shown in Table 1. A positive correlation was observed between the number of KHO in the feather bag and the degradation of feather keratin (see FIG. 8B). It was confirmed that the degradation of feather keratin seen in the bag was due to the visualized activity of KHO and not other physiological and scientific reactions. The highest number of KHOs (101 cells) was found at month 3 (day 85), which corresponds to the maximum degradation rate of feather keratin (Table 1: 71% of reaction vessel 34, 73% of reaction vessel 35). . Both the maximum number of KHO and the maximum degradation rate were achieved at 3 months. A slight decrease in KHO numbers was seen on day 113. All results showed that the rate of hydrolysis was a step that limited the degradation of feather keratin in AD-SBR fed with pig manure. These evidences confirmed that the degradation of feather keratin was due to exoenzymes secreted by KHO rather than chemical and physical activity in AD-SBR fed with pig manure.

  BODIPY® FL casein staining was also performed on initial sludge samples from commercial pig farms and cattle slaughterhouses. The results are shown in Table 2.

  No positive signal was seen. There was no KHO observed in AD-SBR. BODIPY® FL enzyme staining was also used to detect potential KHO present in anaerobic buckets in which feather bags were incubated with distilled water under the same conditions as other AD-SBR. There was no KHO in the bag or the mixture (Table 2).

  The degradation of feather keratin in AD-SBR is due to microbial activity. Microorganisms capable of hydrolyzing feather keratin (keratin hydrolyzing microorganisms: KHO) are present in swine manure used for supply to the SBR reactor. When a bag containing feather keratin is installed in a reaction tank supplied with pig manure, KHO penetrates the bag together with the liquid and adheres to the feather particles. KHO then excretes extracellular keratinase and hydrolyzes crystal feather keratin into soluble keratin, oligopeptides and amino acids. Once amino acids and / or oligopeptides are formed, KHO grows and propagates until the number and activity reaches a certain level at which amino acid and oligopeptide accumulation begins. Even internal amino acids, oligopeptides and soluble keratins are driven out of the bag by chemical gradients, where they are used by microorganisms including hydrolyzing bacteria, fermenting bacteria, methanogens and sulfate reducing bacteria. This process takes at least 30-40 days. Therefore, during this period, there is a big difference in gas production, gas production rate, methane production, VFA, pH, alkalinity, TS, VS, VSS between the reaction tank containing feather keratin and the management reaction tank not containing feather keratin. There was no.

  As extra amino acids and oligopeptides are obtained, microorganisms in the mixture grow using them as a nitrogen source and / or microorganisms increase activity. Hydrolysis and fermentation activity in the reaction vessel is improved and therefore higher VFA (mainly acetic acid) concentrations are detected in the reaction vessel containing feather keratin. High VFA levels further stimulate the activity of methane bacteria. Methane bacteria convert most of the produced VFA to methane and carbon dioxide. As a result, as described above, more gas and methane are produced in the reaction vessel containing feather keratin than in the control reaction vessel, and a significant portion of the carbon source in the mixture is transferred to the gas, so low TS, VS and VSS values. Since more carbon dioxide is produced in the reaction vessels containing feather keratin, higher alkalinity levels are detected in these reaction vessels than in a management reaction vessel that does not contain feather keratin.

  Addition of feather keratin affected gas generation. As shown in FIG. 9A, the cumulative gas production in the reaction vessel containing feather keratin increased rapidly in the first 40 days (about 100 on the 40th day), while in the controlled reaction vessel, the gas production gradually Increased to 32 L on the 40th day. On average, the cumulative gas production in the reactor containing feather keratin was 6.7 times that produced in the control reactor.

  The effect of feather keratin on gas production could also be seen in the daily gas production rate. As shown in FIG. 9B, the gas production rate of all reactors fluctuated within 0-12 L per day in the first 60 days. Thereafter, the gas production in the control reaction tank gradually decreased to 1 L or less per day. However, the gas production rate in the reaction vessel containing feather keratin continued to fluctuate within 0-2.5 L per day after the first 60 days, and the same trend continued until the experiment was completed. The average rate measured in the reactor containing feather keratin was 3.1 times that observed in the control reactor.

  The effect of feather keratin addition on gas generation is also seen in the cumulative methanogenesis profile (FIG. 10A). Methane production in the reactor containing feather keratin increased at a much faster rate than the control reactor. The average cumulative methane production in the reactor containing feather keratin was 3.4 times that in the control reactor. As shown in FIG. 10B, the acetic acid concentration measured in all the reaction tanks with and without feather keratin varied between 18 and 70 mg / L throughout the experiment. However, for the first 15 days, a fairly high acetic acid concentration was detected in the reactor containing feather keratin. Similarly, there was no significant difference in propionic acid concentration (FIG. 10C) and isobutyric acid concentration (FIG. 10D) between the reaction vessel containing feather keratin and the control reaction vessel. The propionic acid concentration varied between 0 and 19 mg / L, and the isobutyric acid concentration varied between 0 and 16 mg / L. Therefore, the added feather keratin did not significantly affect the production of VFA.

  Addition of feather keratin did not significantly affect soluble COD. As shown in FIG. 11, SCOD decreased in the same trend in the first 30 days before it fluctuated between 1912 to 2185 mg / L in all reactors.

Similarly, the addition of feather keratin did not significantly affect the pH of the mixture (FIG. 12A). However, the addition of feather keratin had at least a slight effect on alkalinity. The CaCo ₃ measured in the reactor containing feather keratin (FIG. 12B) was slightly higher than that measured in the control reactor. The average CaCo ₃ concentration in the reaction vessel containing feather keratin was 15578 mg / L, higher than 13606 mg / L observed in the control reaction vessel.

  The TS profile in the reaction vessel containing feather keratin was also different from that in the control reaction vessel containing no feather keratin. As shown in FIG. 13A, TS in the two control reactors gradually decreased from the 0th day to the 20th day, and thereafter varied between 1.7 and 3.1%. However, TS in the reaction vessel containing feather keratin did not decrease and varied between 2.8 and 7.9%. On average, the TS value in the reactor containing feather keratin was 3.7%, which was 1.6 times the 2.3% measured in the control reactor. The observed VS (FIG. 13B) and VSS (FIG. 13C) profiles were similar to the TS profiles. The VS and VSS values measured in the reactor containing feather keratin were all higher than those measured in the control reactor, averaging 1.6 (VS) and 1.7 (VSS) times higher.

  The degradation of feather keratin in the PAD planted with pig manure and with a feather bag (feather PAD) was measured at a 99% effect level after 150 days (see Table 5, FIG. 14). It has been disclosed that keratinase is an inducible enzyme that can be induced in the presence of keratin. Thus, a suitable enrichment process increases the efficiency of keratin degradation in PAD.

  Cattle hoof keratin degradation in a PAD planted with pig manure and with a hoof bag was also disclosed. In all individual nylon bags, 86% of hoof keratin was degraded after 113 days (see Table 6, FIG. 15).

  Thus, it was demonstrated that feather keratin and cow hoof were hydrolyzed and degraded, at least in AD-SBR supplied with swine manure and slaughterhouse sludge. The addition of feather keratin improved the operation of AD-SBR and improved biogas production. Because feather keratin and / or cow hooves and prions are similar in structure, prions in contaminated carcasses can thus be processed in AD-SBR. Most importantly, the enzymatic degradation of prions aids regeneration for the use of animal meat as food.

  The present disclosure may be understood more readily by reference to the following examples, given for illustration of the embodiments, rather than limiting its scope.

Example 1: Feather pretreatment and characterization Feathers of freshly stripped white chickens are collected from a slaughterhouse (rue Principale, Saint-Anselme, Quebec) that processes 900,000 chickens per week and as soon as possible Moved to the laboratory. These chicken feathers were equally divided (2 kg) into a clean normal cotton bag and washed with a washing machine (delicate washing). The feather samples were then dried at 45 ° C. using a Unitern ™ dryer (Construction, CQLTD, UK) until constant weight. These were cut and ground with a mill through a 4 mm sieve. The feather samples were then divided equally into 50-μm pore-free nitrogen-containing polyester feed bags (ANKOM Technology, 2052 O'Neil Road Macedon, NY14502, USA) (each about 33 g), with plastic strings. And was washed again with a washing machine under the same conditions to remove as much dust as possible. The finally washed bag was dried again at a temperature of 45 ° C. until the weight was constant. A feather bag was then attached to the bar and prepared for installation in the bioreactor.

  The ground feather samples were physically and chemically characterized by the following method. Total chemical oxidation demand (TCOD), ash content and organic content can be determined using standard methods (APHA, 1992: Greenberg, AE, Clesceri, LS, Eaton, AD (editor). Standard Method for the Examination of Water and Wastewater: Determined according to American Public Health Association, Washington DC. The amount of Kjeldahl nitrogen (TKN) and the amount of ammoniacal nitrogen were determined by the macro Kjeldahl method using a Tecator 1030 Kjeltec autoanalyzer (Tecator AB, Hoganas, Sweden). The protein concentration was calculated by multiplying the difference between TKN and ammonia-N by 6.25 (AOAC, 1984: Williams, S, Baker, D (editor), official analytical method of the Certified Analytical Chemists Association. (Official Methods of Analysis of the Association of Official Analytical Chemists, Arlington, VA) Fat content is by Schrooyen et al. (2000, Journal of Agricultural and Food Chemistry, Vol. 48: 4326-). 4334) The chicken feather sample (30 g) was extracted with a Soxhlet extractor using petroleum ether for 12 hours (boiling range 40-60 ° C.) and the extracted fat was measured (Mr. Schrooyen Et al., 2000, supra).

The amino acid concentration was determined by the isotope dilution method by Calder et al. (1999, Rapid Communications in Mass Spectrometry, Vol. 13: 2080-2083). A raw feather sample was hydrolyzed with 50 ml of 6 mol / L phenolic hydrochloric acid at 110 ° C. for 24 hours, and the hydrolyzate was filtered. Thereafter, 2 g of the hydrolyzate was diluted with 3 g of ultrapure water. This 1g of solution labeled amino acids ^{(3 C} and ^{15 N} amino isotope standard, CDN Isotopes, Pointe-Claire, Quebec, Canada; Cambridge Isotope Laboratories, Inc. (Cambridge Isotope Laboratories Inc.), Massachusetts, USA Andover ) Was used as an internal standard. Samples of deproteinized plasma and hydrolysates were eluted through a poly-prep chromatography column (resin 100-200 mesh H, BIO RAD, Hercules, Calif.) And N- (tert-butyldimethylsilyl) -N methyltrifluoroacedo Derivatized in a 1: 1 ratio with amide (MTBSTFA) and dimethylformamide (DMF) (Sigma-Aldrich, Ontario, Canada). Measurement of [2-15N] lysine and amino acids in treated samples was performed using a gas chromatographic mass spectrometer (GC-MS, model CG6890-MS5973, Hewlett Packard Co., Wilmington, USA) Delaware).

Example 2: Attaching SBR Eight 42L plexiglass SBRs were used. FIG. 1 is a schematic view of these digesters. When loaded, the SBR consists of a solid, liquid and gas phase. Samples were taken with individual parts mounted on each phase of the SBR. The initial starting sludge volume for each cycle was 35L. All SBFs were operated in a room maintained at a temperature of 25 ° C. Both pig manure and slaughterhouse sludge had a sludge load of 1.5 g COD / L-d. The load factor of feather keratin was determined according to the limited space of the cylinder and the total oxygen demand (TCOD) concentration of the feather in AD-SBR. The loading rate of feather keratin (added to the bag or mixture) in all SBRs was kept the same (0.21 g COD / L-d). To load the SBR with sludge, 160 L of pig manure or slaughterhouse sludge was removed from the semi-industrial scale reactor and transferred to a 200 L container. While the sludge was evenly divided, the solid matter was kept floating using a paint mixer. A 5 L container was used for the sludge equalization. Finally, 35 L of sludge was transferred to each of the eight SBRs (four of which were supplied with pig manure and the other four were supplied with slaughterhouse sludge). When sampling for physiochemical analysis, biogas was circulated for 5 to 10 minutes and the reaction vessel was mixed before taking out the sample.

  All SBR experiments and their conditions are shown in Table 3. Two SBR experiments were performed. In Experiment 1, four SBR reactors were used to examine the degradation of chicken feathers in AD-SBR fed with pig manure. In Experiment 2, the degradation of chicken feathers in AD-SBR fed with slaughterhouse sludge was examined using another four SBR reactors. Chicken feathers were added in two ways. As shown in Table 3, in Experiment 1 the feather sample was left in the bag and in Experiment 2 it was mixed directly with slaughterhouse sludge. Feather bags were cultured in sealed buckets (6 bags each) using distilled water at a temperature of 25 ° C. as a negative control.

Example 3: Physicochemical and microbiological characterization All the physiochemical factors measured for AD-SBR and their sampling frequency are shown in Table 4.

  The pH, alkalinity, evaporation residue (TS), loss on ignition (VS) and organic suspended matter (VSS) were determined according to standard methods (APHA, 1992, supra). The pH value was measured using a pH meter. The alkalinity was adjusted to pH 4.38 by titration. TS content was determined by drying a 10 ml subsample at 105 ° C. for 24 hours. The dried solid was incinerated at 550 ° C. for 3 hours for VS measurement. Similarly, VSS was determined by using a centrifuged slurry and incineration at 550 ° C. for 3 hours. Soluble chemical oxygen demand (SCOD) was determined by analyzing the supernatant of the centrifuged slurry. Total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) were determined using the closed reflux colorimetric method (APHA, 1992, supra). The feather bag was taken out every month and weighed. When removing the feather bag, a new feather bag of the same weight was placed in the SBR. A wet tip gas meter is used to observe daily biogas production and its components (methane, carbon dioxide, hydrogen, sulfide and nitrogen) are analyzed for Hach Carle 400 AGC gas chromatograph (Hach) Weekly analysis using Love-land, Colorado. The column and heat detector were operated at 80 ° C. Total nitrogen (TKN) and ammonia-N were determined by the Macrokjeldahl method (APHA, 1992, supra) using a Tector 1030 Kjeltec autoanalyzer (Tecator AB, Hoganas, Sweden). Volatile fatty acids (VFA), including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, and Autosystem ™ gas chromatography (Perkin-Elmer) with a high resolution megabore column. Elmer Corporation); 06859 Norwalk, Connecticut, USA, and analyzed by connecting it to a flame ionization detector (Masse et al., 2000, Bioresource Technology, 75: 205- 211; Masse et al., 2008, BioResource Technology, 99: 7307-7311).

Example 4: Biodegradation of chicken feather keratin (β-keratin) in a low temperature anaerobic digestion continuous batch reactor (PAD) inoculated with swine manure sludge Peeled white chicken feathers were slaughtered (rue Principale, Saint- From Anselme Quebec) and transferred to the laboratory within 4 hours. These chicken feathers were aliquoted into clean cotton bags (2 Kg each) and washed with a washing machine (Frigidaire, Martinez, Georgia, USA) using tap water (delicate washing). Subsequently, the feather samples were dried at 45 ° C. using a Unithern ™ dryer until constant weight (approximately 8 weeks). These samples are ground in a mill (Thomas-Wiley Laboratory Mill) through a 4 mm sieve, and then the feather samples are fed into a nitrogen-free polyester feed bag (ANKOM) with a pore size of 50 μm. Technology), 2052 O'Neil Road Macedon, NY14502, United States of America). These bags were sealed with plastic string-like wrapping paper and again washed in a washing machine under the same conditions to remove as much dust as possible. The finally washed feather bag was dried at 45 ° C. until the weight was constant. When required, 12 dry feather bags (each containing 31 g of powdered feathers) were attached to the steel bar and inserted into the PAD.

In this experiment, three 42 L Plexiglas PADs were used. Two of the PADs were inoculated with anaerobic sludge adapted to pig manure. PAD inoculum equivalent to 100% of the volume is from the Agriculture and Agri-Food Canada's Dairy and Swine Research and Development Center (Lennoxville, Quebec, Canada) ) but came directly from semi-industrial anaerobic biological reactor of the installed 7m ³ to one in which manure processing two years pigs was performed at 25 ° C.. Twelve feather bags (starting load 4 g / g VS sludge) were added to one of them, and the one without the feather bag was used as a negative control. The third PAD was filled with deionized water containing antibiotics (final concentration 100 mg / mL ampicillin) and 12 feather bags were added to determine the physiochemical loss of feathers from the feather bags. The amount of inoculum and deionized water for each PAD was 35L. All PADs were operated in a room maintained at a temperature of 25 ° C. In Experiment 1, three feather bags were removed from each PAD every 30 days, washed with deionized water, dried at 45 ° C. for 48 hours and weighed. Experiment 2 started when Experiment 1 was in the third month process, 12 new feather bags were added to the bioreactor, and the weight loss of Experiment 2 was recorded using the same experimental procedure as Experiment 1.

  The degradation of feather keratin was recorded in PAD (feather PAD) in which pig manure was planted and feather bags were added. In Experiment 1, after 113 days, 86% of the feather keratin was degraded (31 ± 0.3 g at the start of the experiment and 5 ± 1.6 g at the end). Conversely, feather keratin degradation in PAD containing water containing antibiotics was minimal (less than 3%) (data not shown). In Experiment 2, after 150 days, 99% of feather keratin (31 ± 0.1 g at the start of the experiment and 0.14 ± 0.08 g at the end) was decomposed (Table 5, FIG. 14). The feather decomposition rate in Experiment 2 is higher than that in Experiment 1, and Experiment 1 shows that keratin-degrading microorganisms (KDO) have increased steadily. All the results found in this experiment demonstrate that keratinase is an inducible enzyme that can be induced in the presence of keratin. Thus, an appropriate growth process is preferred for efficient keratin degradation with PAD.

Example 5: Cattle biodegradation in a low temperature anaerobic digestion batch reactor (PAD) Preparation of cow hoof Cattle hoof (alpha keratin) is processed in a local slaughterhouse (Colbex) that processes about 1000 cattle per day , Levinoff, Quebec) and transferred to the laboratory within 4 hours. The hoofs of these cows were washed with deionized water and dried at 45 ° C. with a Unitern ™ dryer until constant weight (about 1 week). These cow hooves were then cut manually into 3-5 cm diameter pieces using a drill and ground in a mill (Thomas-Wiley Laboratory Mill) through a 2 mm sieve. The obtained sample was further crushed with a blender (Vita-Mix 5200, Vitamix Corporation) and passed through a sieve having a pore size of 500 μm. The 27 g of homogenized cow hoof is then equally divided into nitrogen-free polyester feed bags (ANKOM Technology, 2052 O'Neil Road Macedon, NY14502, USA) with a pore size of 50μ and plastic. And was cleaned with a delicate wash in a washing machine (Frigidaire, Martinez, Georgia, USA) to remove as much dust as possible. The washed hoof bag was dried again at 45 ° C. until a constant weight was obtained. Finally, hoof bags (about 27 g each) were attached to the steel bar and installed in the bioreactor.

Installation of PAD
In this experiment, three 42L Plexiglas PADs were used. Two PADs were inoculated with anaerobic sludge adapted to pig manure. The inoculum of the PAD, which corresponds to 100% of capacity, which has come directly from the semi-industrial anaerobic biological reaction tank of 7m ^3, which is installed in the dairy, pig Research and Development Center of Agriculture and Agri-Food Canada, which is 25 Pig manure treatment was performed for 2 years at ℃. Twelve cow hoof bags were added to one of them, and the other without the cow hoof bag was used as a negative control. The third PAD is filled with deionized water containing antibiotics (final concentration 100 pg / mL ampicillin), and 12 cow hoof bags are added to determine the physiochemical loss of cow hoof from the hoof bag did. The amount of inoculum for each PAD was 35L. All PADs were run in a room maintained at a temperature of 25 ° C. Every 30 days, three hoof bags were removed from each PAD, washed with deionized water, dried at 45 ° C. for 48 hours, and weighed. The decrease in weight was recorded.

  The keratin degradation of the cow hoof in the PAD with the pig manure planted and the cow hoof bag added was recorded. In all individual nylon bags, after 113 days, 86% of cowshoe keratin was degraded (27.6 ± 0.1 g at the start of the experiment and 3.9 ± 0.2 g at the end) (Table 6). , FIG. 15). In contrast, in PAD containing water containing antibiotics, the degradation of hoof keratin was minimal (less than 10%).

  Although the invention has been described in connection with specific embodiments, it should be understood that further modifications are possible. This application is also generally in accordance with the principles of the invention and within the scope of known and common practice in the technical scope to which the invention pertains, as applied to the basic features set forth above, and in accordance with the appended claims. It is intended to encompass any modification, use or adaptation of the present invention, including departures from the present disclosure.

DESCRIPTION OF SYMBOLS 1 Bioreaction tank 2 Sludge bed area 3 Embankment area 4 Gas space 5 Biogas recirculation line 6 Biogas recirculation pump 7 Supply line 8 Discharge port 9 Sludge sampling port 10 Mixed liquid or floating substance sampling port 11 Gas outlet 12 Gas meter 13 Thermocouple 14 supply system

Claims (20)

A process of degrading a prion protein at a specific risk site,
(A) supplying a specific risk site (SRM) to a continuous batch reactor (SBR) containing a layer of habitually anaerobic sludge;
(B) reacting the specific risk site with the sludge at 25 ° C. or lower to decompose prion protein.   The process according to claim 1, wherein the specific risk site comprises an animal carcass.   The process according to claim 1, wherein the anaerobic sludge is derived from pig manure or livestock manure.   The process according to claim 3, wherein the livestock manure is derived from slaughterhouse sludge.   The process according to claim 1, wherein the specific danger site reacts with the sludge at a temperature of 5 ° C. to 25 ° C.   The process according to claim 5, wherein the specific dangerous part reacts with the sludge at a temperature of 20 ° C. to 25 ° C.   The process according to claim 5, wherein the specific danger site reacts with the sludge at a temperature of 20 ° C.   The process of claim 1, further comprising adding keratin to the SBR.   9. The process of claim 8, wherein the keratin is feather or hoof derived keratin.   10. The process of claim 9, wherein the keratin is beta keratin or alpha keratin.   10. The process of claim 9, wherein the feather keratin is derived from chicken feathers.   The process according to claim 9, wherein the hoof keratin is derived from bovine hoof.   The process according to claim 1, wherein the SBR is an anaerobic digestion continuous batch reactor.   The process according to claim 1, wherein the SBR is a medium temperature anaerobic digestion continuous batch reactor. A process for measuring the effectiveness of a continuous batch reactor (SBR) that degrades prion protein at a specific risk site,
(A) supplying a specific risk site (SRM) to a continuous batch reactor (SBR) containing a layer of conditioned anaerobic sludge;
(B) adding a model protein to the SBR;
(C) reacting the specific risk site and the model protein with the sludge at a temperature of 5 ° C. to 25 ° C., wherein the degradation of the model protein comprises SBR that degrades the prion protein in the SRM. The process of showing the effectiveness of   The process according to claim 15, wherein the model protein is at least one of perchloric acid soluble protein, collagen, elastin and keratin.   17. The process of claim 16, wherein the keratin is beta keratin or alpha keratin.   17. The process of claim 16, wherein the keratin is feather or hoof derived keratin.   The process according to claim 18, wherein the feather keratin is derived from chicken feathers. The process according to claim 18, wherein the hoof keratin is derived from bovine hoof.

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