EP2582835A1 - Utilisation de la digestion anaérobique psychrophile dans un réacteur discontinu à séquençage pour la dégradation de prions - Google Patents

Utilisation de la digestion anaérobique psychrophile dans un réacteur discontinu à séquençage pour la dégradation de prions

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Publication number
EP2582835A1
EP2582835A1 EP10853040.3A EP10853040A EP2582835A1 EP 2582835 A1 EP2582835 A1 EP 2582835A1 EP 10853040 A EP10853040 A EP 10853040A EP 2582835 A1 EP2582835 A1 EP 2582835A1
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EP
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Prior art keywords
keratin
reactors
feather
sludge
sbr
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EP10853040.3A
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German (de)
English (en)
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EP2582835A4 (fr
EP2582835B1 (fr
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Daniel Y. Masse
Yun Xia
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Agriculture and Agri Food Canada AAFC
Bio Terre Systems Inc
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Agriculture and Agri Food Canada AAFC
Bio Terre Systems Inc
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/28—Anaerobic digestion processes
    • C02F3/282—Anaerobic digestion processes using anaerobic sequencing batch reactors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L11/00—Methods specially adapted for refuse
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C02F11/04—Anaerobic treatment; Production of methane by such processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/22—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/06—Controlling or monitoring parameters in water treatment pH
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/08—Chemical Oxygen Demand [COD]; Biological Oxygen Demand [BOD]
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/10—Solids, e.g. total solids [TS], total suspended solids [TSS] or volatile solids [VS]
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/12—Volatile Fatty Acids (VFAs)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/14—NH3-N
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/16—Total nitrogen (tkN-N)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/28—CH4
    • C02F2209/285—CH4 in the gas phase
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2301/00—General aspects of water treatment
    • C02F2301/10—Temperature conditions for biological treatment
    • C02F2301/103—Psychrophilic treatment

Definitions

  • the present invention relates to the use or process of use of a sequencing batch reactor for eliminating prion in specified risk materials.
  • Prions are proteins devoid of nucleic acids and cell membranes which are largely unaffected by standard methods of sterilisation. Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383). Inactivation of prions poses significant environmental and health issues both for the disposal of prions infected animals and in the preparation of materials of animal origin, such as animal feed. Slaughterhouse sludge and animal mortalities (carcasses) are an important source of pathogens or infectious prions proteins.
  • Prions are protein naturally found in animals. Cellular or normal form of prion proteins are constitutively expressed in the brains of healthy adult animals, but are highly regulated, both spatially and temporally, during development (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383). There are two isoforms of prion proteins. PrPC (Prion Protein Cellular isoform) is a cell surface, N-linked, a-helices-rich, globular soluble glycoprotein protein that may serve as a signal transduction protein (Mouillet-Richard et al., 2000, Science, 289: 1925-1928) and may play an essential role in the normal development of mammal brain. PrPC occurs both in healthy and diseased tissues.
  • PrPSc Primary Protein Scrapie agent
  • PrPSc Primary Protein Scrapie agent
  • PrPSc is a ⁇ -sheet-rich, fibrous, highly insoluble protein which is the causative agent of central nervous system diseases in mammals. It is thought that a higher content in ⁇ -sheet confer heat- and protease-resistance to PrPSc.
  • PrPC abnormal, pathological isoform
  • PrPSc pathological isoform
  • PrPC and PrPSc are identical in their primary structure. Differences occur in secondary structures: PrPC contains three a-helices (about 40% a-helix) and a short antiparallel ⁇ -sheet, whereas PrPSc is composed of two a-helices (about 30% a-helix) and four ⁇ -sheets (45% ⁇ -sheets) (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383)). This induces conformational changes in the tertiary structure of PrPSc, resulting in modified characteristics. Both PrP isoforms are devoid of nucleic acids (Prusiner, 1998, Proc Natl Aca Sci, 95: 13363-13383).
  • prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals. Sterilizing prions involves the denaturation of the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins.
  • prions are generally quite resistant to proteases, heat, radiation, and formalin treatments, although their infectivity can be reduced by such treatments.
  • Effective prion decontamination relies upon protein hydrolysis or reduction and/or destruction of protein tertiary structure. Examples include bleach, caustic soda, or strong acidic detergents.
  • TDE Transmissible Degenerative Encephalopathies
  • PrPc normal prion protein
  • PrPsc infective prion protein
  • TDE affect a wide variety of wild animals and livestock, as well as humans, and present in 3 ways, all of which involve modifications of the prion protein: heritably as a result of genetic mutations; sporadically by spontaneous conversion of the prion protein into a pathologic form via yet undefined mechanisms; by infection following exposure to the exogenous misfolded form of the prion protein.
  • Bovine Spongiform Encephalopathy is among the most notable prion disease.
  • the International Trade Commission (ITC) released a report estimating that trade restrictions resulting from Bovine BSE cost the cattle industry $ billion from 2004 to 2007.
  • animal manure management practices are often detrimental to the environment, they also represent a potential hazard to human and animal health, in addition in producing strong odours, encourage fly breeding, induce weed problems and pollute air, soil and water.
  • a process for degrading a prion protein in a specified risk material comprising the steps of feeding the specified risk material (SRM) to a sequencing batch reactor (SBR) containing a layer of acclimatized anaerobic sludge; and allowing the specified risk material to react with the sludge at a temperature below 25°C so as to allow degradation of the prion protein.
  • SRM specified risk material
  • SBR sequencing batch reactor
  • a process of measuring the efficacy of a sequencing batch reactor (SBR) to degrade prion proteins in a specified risk material comprising the steps of feeding the specified risk material (SRM) to the sequencing batch reactor (SBR) containing a layer of acclimatized anaerobic sludge; adding a model protein to the SBR; and allowing the specified risk materials and model protein to react with the sludge at a temperature between 5°C to 25°C, wherein degradation of the model protein is indicative of the efficiency of the SBR to degrade prion proteins in the SRM.
  • SRM specified risk material
  • SBR sequencing batch reactor
  • the specified risk material comprises animal carcasses.
  • the anaerobic sludge is derived from swine manure, dairy manure and/or slaughterhouse sludge.
  • the specified risk material reacts with the sludge at a temperature between 5°C to 25°C; preferably at a temperature between 20°C to 25°C; more preferably at a temperature of 20°C.
  • the process of degrading a prion protein in a specified risk material as described herein also comprises the step of adding keratin to the SBR.
  • the keratin can be from feather keratin or hoof keratin, and correspond to ⁇ -keratin or a-keratin.
  • the feather keratin is from chicken feather and the hoof keratin is from bovine hoof.
  • the SBR is an anaerobic digestion sequencing batch reactor or a mesophilic anaerobic digestion sequencing batch reactor.
  • the model protein described herein can be at least one of perchloric acid-soluble protein, collagen, elastin and keratin.
  • a "specified risk material” is intended to mean any material that may contain prions concentrate therein, such as tissues of ruminant animals. Such material can include the brain, skull, eyes, trigeminal ganglia, spinal cord, vertebral column, dorsal root ganglia, and the tonsils and distal ileum of the small intestine for example.
  • FIG. 1 illustrates a schematic representation of a laboratory scale sequencing batch reactor.
  • FIG. 2A and B illustrate a graphic representation of feather keratin decrease in bags in reactors supplied with feather keratin.
  • FIG. 3 illustrates the gas production measured in reactors, and more specifically in (A) the cumulative gas production and in (B) the daily gas production in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 4 illustrates in (A) the cumulative methane production, (B) acetic acid concentrations, (C) propionic acid concentrations and (D) propionic acid profiles measured in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 5A illustrates the isobutyric acid concentration measured in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 5B illustrates the soluble chemical oxygen demand (SCOD) measured in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 6 illustrates in (A) the pH profiles and in (B) the alkalinity profiles measured in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 7 illustrates in (A) the total solids (TS), (B) volatile solids (VS) and (C) volatile suspended solids (VSS) profiles measured in reactors with (reactors 34 and 35) and without feather keratin (reactors 13 and 14).
  • Fig. 8 illustrates in (A) the amount of keratin hydrolyzing organisms (KHOs) present in the mixed liquor of control reactor 13 and reactor 34 with feather keratin and in (B) the correlation measured between the degradation of feather keratin and the number of KHOs.
  • KHOs keratin hydrolyzing organisms
  • FIG. 9 illustrates in (A) the cumulative gas production and in (B) the daily gas production measured in reactors with (reactors 15 and 16) and without feather keratin (reactors 13A and 13B).
  • Fig. 10 illustrates in (A) the cumulative methane production, (B) acetic acid concentrations, (C) propionic acid concentrations and (D) isobutyric acid concentrations measured in reactors with (reactors 15 and 16) and without feather keratin (reactors 13A and 13B).
  • Fig. 1 1 illustrates the soluble chemical oxygen demand (SCOD) measured in reactors with (reactors 15 and 16) and without feather keratin (reactors 13A and 13B).
  • Fig. 12 illustrates in (A) the pH profiles and in (B) the alkalinity profiles measured in reactors with (reactors 15 and 16) and without feather keratin (reactors 13A and 13B).
  • Fig. 13 illustrates in (A) the total solids (TS), (B) volatile solids (VS) and (C) volatile suspended solids (VSS) profiles measured in reactors with (reactors 15 and 16) and without feather keratin (reactors 13A and 13B).
  • Fig. 14 illustrates the biodegradation of feather keratin in a PAD seeded with swine manure (run 2).
  • Fig. 15 illustrates the biodegradation of hoof keratin in a PAD seeded with swine manure.
  • PADSBR psychrophilic anaerobic digestion sequencing batch reactor
  • MADSBR mesophilic anaerobic digestion sequencing batch reactor
  • Incineration is currently the primary method by which SRM are disposed in Europe. Laboratory results suggested that prions in SRM are unlikely to survive incineration (Taylor et al., 1996, Neuropath Appl Neuro, 22: 256-258). However, this treatment is expensive and precludes any value to be derived from the SRM. Furthermore, incineration is impractical in many regions due to the geographical dispersal of the cattle populations, which involves supplementary costs and unacceptable contamination risks associated to the transportation of prion-contaminated SRM.
  • SDS sodium dodecyl sulfate
  • Anaerobic digestion of organic matter is a process used for treatment of organic waste and production of energy.
  • organic matter is removed from the wastewater by the concerted action of various groups of microorganisms.
  • the activity of the microbial species participating in mineralization of organic matter is of crucial importance for obtaining an efficient degradation of organic material in bioreactors.
  • the organic matter is converted via acetate, short chained volatile fatty acids (like propionate, butyrate and iso-butyrate) and hydrogen/carbon dioxide to methane by the activity of a complex microbial consortium consisting of hydrolytic/fermentative acidogenic bacteria, acid-oxidizing bacteria and methanogenic archaea.
  • the first group represent the hydrolyzing and fermenting microorganisms which are responsible for the initial attack on polymers and monomers found in the waste material and produce mainly acetate and hydrogen, but also varying amounts of volatile fatty acids (VFA) such as propionate and butyrate as well as some alcohols;
  • group 2 represent the hydrogen-producing acetogenic bacteria which convert propionate and butyrate into acetate and hydrogen;
  • two groups (3 and 4) of methanogenic Archaea produce methane from acetate or hydrogen, respectively.
  • Bovine slaughterhouse sludge and carcasses are substrates composed of readily biodegradable organic matter that can be converted into a high quality biogas which can be processed into thermal and electrical energy. Slaughterhouse wastes are well suited to anaerobic treatment and high solids removal is achieved. The effluent concentrations in heavy metal (cadmium, cobalt, nickel, copper, chromium) are below detectable limit. Nutrients such as calcium, magnesium, sulphur and iron are in concentration adequate for biological treatment of the wastewater.
  • Slaughterhouse effluents are generally coagulated and flocculated to remove colloidal material and suspended solids responsible for the color and turbidity (Al-Mutairi, 2006, Ecotoxicology and Environmental Safety, 65: 74-83).
  • Floc-forming compounds such as lime, alum, sodium aluminates, ferric chloride and ferrous sulphate are used to remove suspended solids.
  • the sludge produced are mainly composed of blood, paunch, stomach contents, undigested food, urine, loose meat, soluble proteins, fat, fecal matters, etc.
  • the proportions of these elements in the sludge vary between slaughterhouses.
  • Al-Mutairi 2006, Ecotoxicology and Environmental Safety, 65: 74-83 indicated that alum and polymer used to coagulate and flocculate solids from slaughterhouse effluents can be toxic to the microflora of a subsequent biological treatment process.
  • concentrations of a coagulant exceed 100 mg/l in the sedimentation tank, the effluent is toxic to micro-organisms. Settled sludges are more contaminated and toxic than the sedimentation tank supernatant.
  • Hygienic aspects are important when an external feedstock is brought on a livestock farm for co-digestion. Effluent from poultry and swine abattoir can be contaminated with biological contaminants. Pathogens removal prior to land application is important to prevent reintroduction of contamination cycles in livestock production systems.
  • FIG. 1 is a schematic illustration of a laboratory scale sequencing batch reactor (SBR) as used in the present disclosure and as described in Canadian patent No. 2, 138,091 , the content of which is enclosed herewith by reference.
  • SBR laboratory scale sequencing batch reactor
  • thermocouple
  • the manure is loaded into the feeding system 14 and fed to the bioreactor 1 through the feeding line 7.
  • the manure is fed through the bottom of the bioreactor 1 which has been pre-inoculated with an anaerobic sludge 2.
  • the biogas in the bioreactor 1 can be recirculated in the gas spaced 4 through the biogas recirculation line 5 using the biogas recirculation pump 6.
  • PSP Perchloric acid-soluble protein
  • Collagen is the most abundant protein in mammals, representing 25% of total proteins. Collagen is a major, fibrous, insoluble, extracellular protein of skin, tendon, cartilage, bone and teeth, and serves to hold cells together.
  • Elastin is a fibrous cross-linked protein in the extracellular matrix that provides elasticity for many tissues which can stretch to several times their length and return to their original state when the tension is released. Elastin is found in large amounts in the walls of blood vessels and in ligaments, particularly in the neck of grazing animals.
  • Keratins forms one of the most diverse classes of fibrous proteins. There are various types of keratins, each composed of a complex mixture of proteins. Keratins are major constituents of structures that grow from the skin. a-Keratins are found in the hair (including wool), horns, nails, claws and hooves of mammals. A hoof is the tip of a toe of an ungulate mammal, strengthened by a thick horny (keratin) covering. The hoof consists of a hard or rubbery sole, and a hard wall formed by a thick nail rolled around the tip of the toe.
  • a-Keratins are the most prevalent forms and are composed largely of polypeptide chains in the a-helix conformation.
  • the harder ⁇ -keratins are alternative forms that exist in the claws, scales and shells of reptiles, and in the claws, scales, beaks and feathers of birds (Zubay, 1988, Biochemistry, New York, NY, USA: Macmillan Publishing Company).
  • ⁇ -Keratins are composed of stacked and folded ⁇ -sheets, and better represent PrPSc.
  • ⁇ -keratins The amino acid sequences of ⁇ -keratins from various bird species are highly similar and, like collagen and elastin, contain a high proportion of glycine (Harrap and Woods, 1964, Biochem J, 92: 19-26). Glycine-rich repeats are found in some, but not all, ⁇ -keratins. ⁇ -keratins are shorter (98 to 155 amino acids) than PrP proteins (256 to 264 amino acids).
  • ⁇ -Keratins are composed of extremely long a-helical polypeptide chains that are arranged side by side to form fibers in a spiral fashion. The packing of a-helices is optimized and strengthened by wrapping of individual helices around each other. Many forms of a-keratins include (covalent) disulfide bonds formed between cysteine residues of adjacent polypeptide chains, which confers permanent, thermally-stable cross linking. The amount of disulfide bridges modulates strength and rigidity in keratins. Approximately one quarter to one third of feather keratins have a ⁇ -conformation, mostly concentrated in the central, hydrophobic portion of the molecule. The rest of the protein forms an irregular matrix. Closely related ⁇ -keratins are the main constituent of bird feathers and account for about 90% of the feather rachis.
  • Slaughterhouse inoculums was used herein, collected from a semi- industrial scale anaerobic sequence bench bioreactor (AD-SBR).
  • the SBR reactor is running at 20°C and fed every week with fresh sludge from a commercial cattle slaughterhouse (Colbex, Levinoff, Quebec) that processes about 1000 cattle per day. Every week about 50 kg of slaughterhouse sludge was collected and a representative sub-sample (1 L) was stored at 20°C for later analysis and what was left was used to feed the semi-industrial SBR reactor.
  • the swine manure inoculums also used herein was collected from a semi industrial scale anaerobic sequence bench bioreactor (AD-SBR). Similarly, it is kept at 20°C and fed every week with fresh sludge from a commercial pig farm (1944 ch. Tremblay. Ste-Edwidge, Quebec) that processes about 4000 pigs per year. Every week about 50 kg swine manure was collected and a representative sub-sample (1 L) was stored at 20°C for later analysis and what was left was used to feed the semi-industrial SBR.
  • AD-SBR semi industrial scale anaerobic sequence bench bioreactor
  • Feather is one of the large solid wastes in North America. Feathers are mainly composed of keratins. Keratins are insoluble and show high mechanical stability and resistance to proteolysis. Feather keratins are a ⁇ - keratin composed of mainly stacked and folded ⁇ -sheets; therefore, feather keratins are poorly digested by common digestive enzymes, such as trypsin and pepsin because of the high degree of cross-linking by disulphide bonds, hydrogen bonding and hydrophobic interactions (Papadopoulos, 1986, Animal Feed Science and Technology, 14: 279-290). A similar structure is found in pathagenous protein (PrPsc).
  • chicken feathers are used as model proteins of PrPsc due to feather keratins having a number of amino acids, high proportion of glycine residues, high content of ⁇ -sheet structures, high degree of aggregation and properties (protease and heat resistance, weak solubility) similar to prion proteins.
  • hooves are also used as model proteins of PrPsc due to thei high contant in hard keratins, insoluble and resistant to degradation by common proteolytic enzymes, such as trypsin, pepsin and papain because of their high degree of cross-linking by disulfide bonds, hydrogen bonding and hydrophobic interaction.
  • Propionic acid concentration was also affected by adding feather keratin. As shown in Fig. 4C, propionic acid concentration in starting sludge is high. It gradually decreased in the first 15 days. After that, it fluctuated from 0 to 26 mg/L (see Fig. 4D). As observed in acetic acid profile, no significant difference in propionic acid production could be found between reactors with feather keratin and their control reactors in the first 62 days. After 72 days values of propionic acid concentration measured in reactors with feather keratin are mostly higher than those in control reactors. Addition of feather keratin did not significantly affect isobutyric acid concentration (see Fig. 5A).
  • Feather keratin also affects the concentration of soluble COD. As shown in Fig. 5B, SCOD in all reactors decreased gradually in the same trend in the first 60 days. Then, SCOD in control reactors kept decreasing to the end of experiment. SCOD in reactors with feather keratin, however, increased gradually to the end of the experiment. The average SCOD concentration in reactors with feather keratin were of 8757 mg/L, which is 1 .1 times higher than that (8171 mg/L) measured in control reactors.
  • Feather keratin also affected mixed liquor pH in SBR reactors. As shown in Fig. 6A, pH in all reactors gradually decreased from 8.0-8.1 to 7.8 in the first 50 days. After that, all the pH values measured in reactors with feather keratin are 0.05-0.10 higher than those measured in control reactors. Feather keratin significantly affected alkalinity. As shown in Fig. 6B, in the first 60 days alkalinity in all reactors mostly fluctuated between 25000 and 27000 mg/L. After that, alkalinity measured in control reactors still kept the same trend to the end of the experiment. However, alkalinity in reactors with feather keratin gradually increased up to 28839 and 28171 mg/L.
  • Feather keratin in addition, also affected the TS profiles. As shown in Fig. 7A, TS in all control reactors gradually decreased in the first 80 days then fluctuated within a certain range. Generally, TS in reactors with feather keratin decreased at a faster speed than in control reactors. TS values measured in reactors with feather keratin are mostly lower than those measured in control reactors. In average, TS value in control reactors is 1 .1 times higher than in reactors with feather keratin. Similar profiles observed in TS also exist in VS and VSS profiles. VS and VSS in all reactors gradually decreased in the first 60- 80 days then fluctuated within a certain range. The significant difference is that VS and VSS in reactors with feather keratin decreased to a lower level than in control reactors. VS and VSS values measured in reactors with feather keratin are mostly lower than in reactors with feather keratin.
  • Feather keratin in bags decreased in AD-SBRs fed with swine manure. The decrease observed is most due to microbial activity, i.e. keratin hydrolyzing microorganisms excreting keratinase and hydrolyzing the feather keratin in bags. Keratin added in AD-SBRs fed with swine manure did not affect negatively the performance of AD-SBRs. It improved the biogas production and reduced biomass production of the ADSBRs.
  • BODIPY fluorescence exoenzyme staining methods were used to label and visualize keratin hydrolytic microorganisms (KHOs).
  • KHOs keratin hydrolytic microorganisms
  • BODIPY FL casein was first applied to samples of feather bags in bioreactors. After 30 min of staining, fluorescence on bacteria with a morphotype of rod was observed on a weak fluorescent background. No other morphotype was observed during 180 min staining. Sampling and microscopic examination were carried out every 15 min. The background fluorescence, however, increased with time. The same staining was carried out on samples from all the reactors with or without feather keratins added.
  • the individual inhibitors were added at concentrations at which the energy metabolisms of all the microorganisms in AD-SBRs were effectively inhibited (Xia et al., 2008, supra). In the presence of the inhibitors, all fluorescing rods observed previously were still observed; the difference being the number of KHOs remarkably reduced after inhibition. Therefore, the bacteria positively stained are putative KHOs responsible for the degradation of feather keratin observed in these AD-SBRs. [0080] The relative numbers of KHOs in mixed liquor of AD-SBRs were estimated by keeping all the procedure including sampling, sample preparation, microscopic examination, image capture and image analysis constant. The results are listed in Table 1. As shown in Fig.
  • the numbers of KHOs in control reactor gradually decreased from 150 at day 0 to 56 at day 1 13.
  • the number of KHOs in reactor with feather keratin slightly decreased in the first 20 days from 142 to 131 , and then gradually increased up to 175 at day 85 before decreasing to 121 at day 1 13.
  • BODIPY FL casein staining was also carried out on original sludge samples from a commercial pig farm and a cattle slaughter house. The results are also shown in Table 2.
  • KHOs When bags containing feather keratin are put into the reactors fed with swine manure, KHOs penetrate with liquid into the bags and attach on the feather particles. Then, the KHOs excrete extracellular keratinase and hydrolyze crystal feather keratin into soluble keratin, oligopeptides and amino acids. Once the amino acids and/or oligopeptides are available, KHOs grow and multiply until their number and activity reach a certain level, when amino acids and oligopeptides start to accumulate.
  • the amino acids, oligopeptides and even soluble keratin inside are driven outside of the bags by chemical gradient, where they are used by microorganisms including hydrolyzers, fermenters, methanogenes and sulfate reducing bacteria. This process take at least 30- 40 days. During this period, therefore, no significance difference in gas production, gas production rate, methane production, VFAs, pH, alkalinity, TS, VS and VSS is observed between the reactors with feather keratin and the control reactors without feather keratin.
  • TS profiles in reactors with feather keratin are also different from those in control reactors without feather keratin.
  • TS in the two control reactors gradually decreased from day 0 to day 20 then fluctuated within 1.7-3.1 %.
  • TS in reactors with feather keratin did not decrease and fluctuated between 2.8-7.9%.
  • TS value in reactors with feather keratin is 3.7%, being 1.6 times higher than 2.3% measured in control reactors.
  • the VS (Fig. 13B) and VSS (Fig. 13C) profiles observed is similar to the TS profiles.
  • VS and VSS values measured in reactors with feather keratin are all higher than those measured in control reactors, in average being 1.6 (VS) and 1 .7 (VSS) times, respectively, higher than those measured in control reactors.
  • Bovine hoof keratin degradation in the PAD seeded with swine manure and added with hoof bags is also disclosed. In all individual nylon bags, 86% of the hoof keratin was degraded after 113 days (see Table 6, Fig. 15).
  • feather keratin and bovine hoof can be hydrolyzed and degraded in AD-SBRs fed with at least swine manure and slaughterhouse sludge. Addition of feather keratin improves the operation of AD-SBRs improving the biogas production. Prions in contaminated carcasses can thus be treated in AD-SBRs, since feather keratin and/or bovine hoof and prions are similar in their structure. The enzymatic breakdown of prions would most importantly help revive the use of animal meal as feed. [0095]
  • the present disclosure will be more readily understood by referring to the following examples which are given to illustrate embodiments rather than to limit its scope.
  • Freshly plucked white chicken feathers were collected from a slaughterhouse (rue Principale, Saint-Anselme, Quebec) that processes 900,000 chickens per week; and transferred to laboratory as soon as possible.
  • the chicken feathers were aliquot (2 Kg) into clean normal cotton bags and washed in a washing machine (delicate wash).
  • the feather samples were subsequently dried at 45°C in a UnithernTM drier (Construction, CQLTD, England) until a constant weight was reached. They were cut and ground in a mill through a 4 mm screen.
  • the feather samples were aliquot (around 33 g each) into nitrogen-free polyester forage bags with a pore size of 50 microns (ANKOM Technology, 2052 O'Neil Road Cincinnati, NY14502, USA), sealed with plastic tie wraps and washed again in washing machine using the same condition to get rid of as much dusts as possible. Finally, the washed feather bags were dried again at 45°C until their weights was constant. Feather bags were then attached into a steel stick and ready to be put into the bioreactors.
  • TCOD total chemical oxidation demands
  • ash content ash content
  • organic matter content were determined according to the standard methods (APHA, 1992, In: Greenberg, A.E., Clesceri, L.S., Eaton, A.D. (Eds.), Standard Methods for the Examination of water and wastewater. American Public Health Association, Washington DC).
  • Total Kjeldahl nitrogen (TKN) and ammonia-nitrogen were determined with a Tecator 1030 Kjeltec auto-analyser (Tecator AB, Hoganas, Sweden) following macro-Kjeldahl method described standard methods.
  • Protein concentration was calculated by multiplying the difference between TKN and ammonia-N with 6.25 (AOAC, 1984, In: Williams, S., Baker, D. (Eds.), Official Methods of Analysis of the Association of Official Analytical Chemists, Arlington, VA.). Fat content was determined according to Schrooyen et al. (Schrooyen et al., 2000, Journal of Agricultural and Food Chemistry, 48: 4326-4334). Chicken feather samples (30 g) were Soxhlet extracted for approximately 12 h with petroleum ether (boiling range 40-60°C) and the fat extracted was measured (Schrooyen et al., 2000, supra).
  • Amino acid concentration was determined following the isotope dilution method described by Calder et al. (Calder et al., 1999, Rapid Communications in Mass Spectrometry, 13: 2080-2083). Briefly, raw feather samples were hydrolysed with 50 ml of 6 mol/L phenol-HCI at 110°C for 24 h and the hydrolysate was filtrated.
  • FIG. 1 is a schematic representation of these digesters. Once loaded, the SBRs consist of a solid, liquid and gas phase. Individual parts are set up on each phase of the SBRs to take samples. The starting sludge volume at the beginning of each cycle was 35 L. All SBRs were operated in a room kept at 25°C. The sludge load for both swine manure and slaughterhouse sludge is 1 .5 g COD/L-d. The loading rate of feather keratin was determined according to the limited space of cylinder of AD- SBRs and the total oxygen demand (TCOD) concentration of feathers.
  • TCOD total oxygen demand
  • the loading rate of feather keratin (adding in bags or adding in mixed liquor) in all SBRs was kept as same (0.21g COD/Ld).
  • 160 L swine manure or slaughterhouse sludge was withdrawn from the semi- industrial scale reactors and transferred into a 200 L barrel.
  • a paint mixer was used to keep solids suspended during sludge aliquot.
  • a 5 L container was used to aliquot sludge.
  • 35 L sludge was transferred into each of the 8 SBRs (4 fed with swine manure and the other 4 with slaughterhouse sludge, respectively.)
  • the reactors were mixed by circulating the biogas for 5-10 min before samples were withdrawn.
  • TS total solids
  • VS volatile solids
  • VSS volatile suspended solids
  • Total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) were determined using the closed reflux colorimetric method (APHA, 1992, supra). Feather bags were taken off and weighed in each month. A new feather bag of the same weight was put in a SBR when a feather bag was taken out. Biogas production was monitored daily using wet tip gas meters and its composition (methane, carbon dioxide, hydrogen sulfide, and nitrogen) was analyzed weekly using a Hach Carle 400 AGC gas chromatograph (Hach, Love-land, CO). The column and thermal conductivity detector were operated at 80°C.
  • Total nitrogen (TKN) and ammonia-N were determined using an auto- analyzer according to the macro-Kje1dah1 method (APHA, 1992, supra) with a Tecator 1030 Kjeltec auto-analyzer (Tecator AB, Hoganas, Sweden).
  • Volatile fatty acids including acetic, propionic, butyric, isobutyric, isovaleric, valeric and caproic acids
  • VFA Volatile fatty acids
  • acetic, propionic, butyric, isobutyric, isovaleric, valeric and caproic acids were analyzed using an AutoSystemTM gas chromatography equipped with a high resolution megabore column (Perkin- Elmer Corporation; Norwalk, CT 06859, USA) connected to a flame ionization detector (Masse et al., 2000, Bioresource Technology, 75: 205-211 ; Masse et al., 2008, Biorescource Technology, 99: 7307-731 1 ).
  • Freshly plucked white chicken feathers were collected from a slaughterhouse (Principale Street, Saint-Anselme, QC, Canada) and transferred to the laboratory within 4 hours.
  • the chicken feathers were divided into aliquot (2 kg each) parts in clean cotton bags and washed (delicate cycle) in a washing machine (Frigidaire, Martinez, GA, USA) with tap water.
  • the feather samples were then dried at 45°C in a Unithern dryer (Construction CQLTD, England) until a constant weight was reached (about eight weeks).
  • the samples were ground in a mill (Thomas-Wiley Laboratory Mill), screened through a 4-mm screen and then divided into aliquot (around 33 g each) parts in nitrogen-free polyester forage bags with a pore size of 50 ⁇ (ANKOM Technology, Cincinnati, NY, USA).
  • the bags were sealed with plastic tie wraps and washed in the washing machine again to remove as much dust as possible. Finally, the washed feather bags were dried at 45°C until their weights remained constant. When needed, 12 dried feather bags (each containing around 31 g ground feathers) were attached onto a steel stick and inserted into a PAD.
  • the third PAD was filled with deionized water containing antibiotics (ampicillin at a final concentration of 100 Mg/mL) and added with 12 feather bags to determine the physiochemical loss of feathers from the feather bags.
  • the volume of the inoculum and deionized water for each PAD was 35 L. All PADs were operated in a room kept at 25°C. In run 1 , every 30 days, three feather bags were taken out of each PAD, washed with deionized water, dried for 48 h at 45°C, and weighed. Run 2 was start when run 1 was processing in the third month, 12 new feather bags were added into bioreactor and following the same experimental procedure as run 1 , the weight deduction of run 2 was also recorded.
  • Bovine hooves (a -keratin) were collected from a local slaughterhouse (Colbex, Levinoff, Quebec) that processes about 1000 cattle per day and transferred to the laboratory within 4 hours. The bovine hooves were then washed with deionized water and dried at 45°C in a Unithern dryer (Construction CQLTD, England) until a constant weight was reached (about one week). Subsequently, the bovine hooves were manually cut into pieces of 3- 5cm in diameter with a drill and ground in a mill (Thosmas-Wiley, Laboratory Mill) through a 2-mm screen.
  • the third PAD was filled with deionized water containing antibiotics (ampicillin at a final concentration of 100 pg/mL) and added with 12 hoof bags to determine the physiochemical loss of bovine hooves from the hoof bags.
  • the volume of the inoculum for each PAD was 35 L. All PADs were operated in a room kept at 25°C. Every 30 days, three hoof bags were taken out of each PAD, washed with deionized water, dried for 48 h at 45°C, and weighed. The weight deduction was recorded.

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Abstract

Cette invention concerne l'emploi d'un processus d'utilisation d'un réacteur discontinu à séquençage pour l'élimination de prions dans des matériaux à risque spécifiés et de mesure de l'efficacité du processus pour la dégradation de protéines prions dans des matériaux à risques spécifiés.
EP10853040.3A 2010-06-17 2010-06-17 Procédé pour la détermination de l'efficacité d'un réacteur discontinu à séquençage anaérobique psychrophile pour la dégradation des prions Withdrawn - After Issue EP2582835B1 (fr)

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JP6567859B2 (ja) * 2015-04-21 2019-08-28 国立大学法人 琉球大学 プリオン不活化方法
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WO2002015945A1 (fr) * 2000-08-22 2002-02-28 Green Farm Energy A/S Technique de separation des boues et de production de biogaz
WO2002049460A1 (fr) * 2000-12-20 2002-06-27 Fritz Kortschack Procede de modification ciblee de la structure proteique de prions prp?sc¿
WO2003040047A1 (fr) * 2001-11-09 2003-05-15 United Utilites Plc Traitement d'incubation des boues permettant de reduire les agents pathogenes avant la digestion
US20060004237A1 (en) * 2003-03-28 2006-01-05 Appel Brian S Process for conversion of organic, waste, or low-value materials into useful products
WO2009090476A2 (fr) * 2007-12-21 2009-07-23 Highmark Renewables Research Corp. Installation de bio-digestion incorporée

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CA2138091C (fr) * 1994-12-14 2001-04-10 Daniel I. Masse Traitement anaerobie des eaux usees a la temperature ambiante
JP4568835B2 (ja) * 2004-03-31 2010-10-27 国立大学法人九州大学 木材腐朽担子菌を用いたプリオンの分解方法
JP4070781B2 (ja) * 2004-07-01 2008-04-02 独立行政法人農業・食品産業技術総合研究機構 Ｂｓｅの発症原因となる異常プリオン蛋白質を含む特定危険部位の亜臨界水処理方法
JP4904086B2 (ja) * 2005-05-25 2012-03-28 公益財団法人微生物化学研究会 クラゲ類の分解廃液の処理装置及び処理方法、並びに微生物
AU2009262515A1 (en) * 2008-06-27 2009-12-30 Bayer Cropscience Ag Thiadiazolyloxyphenylamidines and use thereof as fungicides
US20100297740A1 (en) * 2009-05-21 2010-11-25 Xiaomei Li Use of Anaerobic Digestion to Destroy Biohazards and to Enhance Biogas Production

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002015945A1 (fr) * 2000-08-22 2002-02-28 Green Farm Energy A/S Technique de separation des boues et de production de biogaz
WO2002049460A1 (fr) * 2000-12-20 2002-06-27 Fritz Kortschack Procede de modification ciblee de la structure proteique de prions prp?sc¿
WO2003040047A1 (fr) * 2001-11-09 2003-05-15 United Utilites Plc Traitement d'incubation des boues permettant de reduire les agents pathogenes avant la digestion
US20060004237A1 (en) * 2003-03-28 2006-01-05 Appel Brian S Process for conversion of organic, waste, or low-value materials into useful products
WO2009090476A2 (fr) * 2007-12-21 2009-07-23 Highmark Renewables Research Corp. Installation de bio-digestion incorporée

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* Cited by examiner, † Cited by third party
Title
See also references of WO2011156885A1 *

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JP2013529969A (ja) 2013-07-25

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