| PATENT ASSIGNEE'S COUNTRY | France |
| UPDATE | 02.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 15.02.00 |
| PATENT TITLE |
Combinations of bacillus and lactobacillus species for producing protein from waste |
| PATENT ABSTRACT |
Bacterial complexes are provided which may be used in the digestion and decomposition of residues of biological origin in the form of biomass, and the transformation of these residues into non-polluting organic compounds. Also provided are the applications of these bacterial complexes to the treatment of waste of biological origin such as excrement (pig, ruminent, equid, or poultry litter), liquid manures, corpses, and stagnant waters. In these applications, this waste is converted into compost or other stable, biodegradable, and non-polluting nitrogenous compounds. The bacterial complexes essentially contain at least one non-pathogenic Bacillus and at least one non-pathogenic Lactobacillus. These bacterial complexes are capable of converting inorganic nitrogen, in particular aqueous ammonia, nitrates, nitrites, and organic nitrogen molecules (such as urea, urates, amino acids, nitrogenous bases, or any other nitrogenous compounds of low molecular weight) into organic nitrogen in the form of bacterial proteins. The bacterial complexes may also contain comprise Pediococcus. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 11.01.96 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
JAPIO, AN 84-013175, JP-A-59 013 175, Jan. 23, 1984. JAPIO, AN 93-078663, JP-A-05 078 663, Mar. 30, 1993. JAPIO, AN 85-012198, JP-A-60 012 198, Jan. 22, 1985. JAPIO, AN 89-211487, JP-A-01 211 487, Aug. 24, 1989. |
| PATENT CLAIMS |
I claim: 1. A process for converting an organic nitrogenous compound of low molecular weight or an inorganic nitrogenous compound into protein, comprising the step of contacting the organic nitrogenous compound or the inorganic nitrogenous compound with a viable bacterial complex comprising at least one viable non-pathogenic Bacillus species selected from the group consisting of Bacillus subtilis, Bacillus amyoliquefaciens, Bacillus megaterium, Bacillus licehniformis, and Bacillus circulans, and at least one viable non-pathogenic Lactobacillus species selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus acidophilus, wherein the complex has the characteristic of having the capacity to convert the organic nitrogenous compound or the inorganic nitrogenous compound into protein, thereby converting the organic nitrogenous compound or the inorganic nitrogenous compound into protein. 2. The process of claim 1, wherein the organic nitrogenous compound or the inorganic nitrogenous compound is from animal waste. 3. The process of claim 2, wherein the animal is monogastric. 4. The process of claim 3, wherein the animal is a poultry animal. 5. The process of claim 1 wherein the organic nitrogenous compound or the inorganic nitrogenous compound is from poultry litters. 6. The process of claim 1, wherein the viable bacterial complex is biologically pure. 7. A viable bacterial complex comprising at least one viable non-pathogenic Bacillus species selected from the group consisting of Bacillus subtilis, Bacillus amyoliquefaciens, Bacillus megaterium, Bacillus licheniformis, and Bacillus circulans, and at least one viable non-pathogenic Lactobacillus species selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus acidophilus, wherein the complex has the characteristic of having the capacity to convert an organic nitrogenous compound of low molecular weight or an inorganic nitrogenous compound into protein, and wherein the ratio of the Lactobacillus species to the Bacillus species is between 1 to 100 and 100 to 1. 8. The bacterial complex of claim 7, wherein the organic nitrogenous compound is selected from the group consisting of urea, urates, amino acids, and nitrogenous bases. 9. The bacterial complex of claim 7, wherein the inorganic nitrogenous compound is selected from the group consisting of ammonia, nitrates, and nitrites. 10. The bacterial complex of claim 7, wherein the complex has at least one enzymatic activity selected from the group consisting of cellulolytic activity, proteolytic activity, amylolytic activity, lipolytic activity, and pectinolytic activity. 11. The bacterial complex of claim 7, wherein the ratio of the Lactobacillus species to the Bacillus species is between 1 to 10 and 10 to 1. 12. The bacterial complex of claim 7, wherein the ratio of the Lactobacillus species to the Bacillus species is between 1 to 10 and 1 to 1. 13. The bacterial complex of claim 7, wherein the ratio of the Lactobacillus species to the Bacillus species is about 1 to 1. 14. The bacterial complex of claim 7, comprising more than one Bacillus species and more than one Lactobacillus species, wherein the ratio of one of the Bacillus species to another of the Bacillus species is between 1 to 1 and 1 to 100, and the ratio of one of the Lactobacillus species to another of the Lactobacillus species is between 1 to 1 and 1 to 100. 15. The bacterial complex of claim 7, wherein the total concentration of the Bacillus species and the Lactobacillus species is between 1.times.10.sup.2 cfu/g and 1.times.10.sup.8 cfu/g. 16. The bacterial complex of claim 7, wherein the concentration of the Bacillus species is between 1.times.10.sup.2 cfu/g and 1.times.10.sup.7 cfu/g and the concentration of the Lactobacillus species is between 1.times.10.sup.2 cfu/g and 1.times.10.sup.8 cfu/g. 17. The bacterial complex of claim 16, wherein the concentration of the Lactobacillus species is between 1.times.10.sup.3 cfu/g and 1.times.10.sup.8 cfu/g. 18. The bacterial complex of claim 17, comprising Bacillus subtilis and Bacillus megaterium. 19. The bacterial complex of claim 16, wherein the Bacillus is Bacillus subtilis. 20. The bacterial complex of claim 19, wherein the concentration of the Bacillus species is at least 1.times.10.sup.3 cfu/g. 21. The bacterial complex of claim 20, wherein urates are the organic nitrogenous compound. 22. The bacterial complex of claim 7, comprising Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, Bacillus circulans, Lactobacillus rhammosus, Lactobacillus paracasei, Lactobacillus fermentum, and Lactobacillus acidophilus. 23. The bacterial complex of claim 7, further comprising a non-pathogenic Pediococcus. 24. The bacterial complex of claim 7, further comprising at least one neutral diluent. 25. The bacterial complex of claim 7, further comprising a chemical tracer or a microbiological tracer. 26. The viable bacterial complex of claim 7, which is biologically pure. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to bacterial complexes which may be used in the digestion, the decomposition and the transformation of residues of biological origin in the form of biomass and of stable, non-polluting organic compounds and to their applications to the treatment of waste of biological origin, such as excrement (pig, ruminant, equid or poultry litter or human excrement, liquid manures) or liquid manures, corpses, stagnant waters and their conversion into compost or other stable, biodegradable and non-polluting nitrogenous compounds. 2. Description of the Background Heterotrophic organisms use nitrogenous compounds, including plant or animal proteins, as nutrient sources and return the nitrogen to the soil via their excreta or via post-mortem decomposition products, mainly in the form of aqueous ammonia or urea which are converted into nitrites and nitrates by nitrifying bacteria present in the soil, such as Nitrosomonas or Nitrobacter. However, substantial production of nitrogenous compounds in gaseous or liquid-solid form, such as ammonia gas, aqueous ammonia, nitrites and nitrates, constitutes an important cause of atmospheric pollution and of pollution of soils, water courses and water tables. All of these processes of destruction lead to the formation of compounds with particularly nauseating odours (NH.sub.3 and H.sub.2 S in particular, etc.). Processes are known for the treatment of liquid manures, litters or waste waters, which involve a bacterial culture and enzymes or a bacterial culture, enzymes and yeasts, in order especially to reduce the evolution of ammonia and odours, particularly at the time of handling and during spreading (liquid manure). Such compositions contain in particular, as bacterium: Bacillus subtilis, as enzymes: batinase and amylase, and as yeasts: Saccharomyces cerevisiae (French patent application 2,658,077). However, the compositions known for treating excrement do not allow the conversion of inorganic nitrogen (NH.sub.4, NO.sub.2 and NO.sub.3) and urates into amino acids and proteins (organic nitrogen) but merely reduce the evolution of ammonia, by adsorption or solubilization. Such conversions do not lead to a reorganization of the nitrogen, by its use at the level of the nitrogenous metabolism of microorganisms. Consequently, an effective treatment of products of biological origin is necessary and crucial in order to allow the conversion of inorganic nitrogen (NH.sub.4, NO.sub.2 and NO.sub.3) and urates into organic nitrogen (amino acids and proteins), in order to prevent pollution and to return the waste into the circuit of anabolism. SUMMARY OF THE INVENTION In order to overcome this problem, the Applicant has selected bacterial complexes, which essentially allow the conversion of inorganic nitrogen into organic nitrogen, in the form of bacterial proteins (stabilization of the nitrogen and increase in the biomass), that is to say which allow the conversion of excrement into nitrogenous compounds (stable nitrogenous compounds and/or compost) and, particularly for waste having a sufficient C/N ratio (in relation to the level of solids content), into non-polluting compounds rich in fulvic acid and humic acid, by digestion and conversion of excrements, while at the same time removing the associated pathogenic germs, in particular Clostridium, Bacteroides, colibacilli, Listeria, salmonellae and staphylococci. The subject of the present invention is a bacterial complex, characterized in that it essentially comprises at least one non-pathogenic Bacillus and at least one non-pathogenic Lactobacillus, in that it uses as nitrogen source essentially inorganic nitrogen, in particular aqueous ammonia, nitrates, nitrites and organic nitrogen molecules such as urea, urates, amino acids, nitrogenous bases or any other nitrogenous compound of low molecular weight, and in that it is capable of converting the said nitrogen into organic nitrogen, in the form of bacterial proteins. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1-8: results of the API 50 CH galeries giving the fermentation profiles of sugars in Example 1; FIGS. 9-20: results of the API 50 CH galeries giving the fermentation profiles of sugars in Example 2; FIG. 21: results from Example 13; FIG. 22: results from Example 13; FIG. 23: results from Example 13; FIG. 24: the change in the nitrate content of manueres in Example 13; FIG. 25: the change in soluble nitrogen from Example 13; FIGS. 26-28: the comparative change in and distribution of nitrogen from Example 13; and FIG. 29: results of API 50 CH galeries giving the fermentaion profiles of sugars in Example 3. DETAILED DESCRIPTION OF THE INVENTION In accordance with the invention, the said bacterial complex has at least the following enzymatic activities: cellulolytic activity, proteolytic activity, amylolytic activity, lipolytic activity, pectinolytic activity. Unexpectedly, the Bacillus/Lactobacillus combination present in the bacterial complex according to the invention acts in synergy in order to: use both inorganic nitrogen and organic nitrogen, resynthesize bacterial proteins, and remove the pathogenic microorganisms associated with the products to be treated, such as Clostridium, Bacteroides, colibacilli, Listeria, salmonellae and staphylococci. Such a bacterial complex thus effectively makes it possible to return the waste into the circuit of anabolism (organization in the trophic chain). According to an advantageous embodiment of the said bacterial complex, it comprises at least one Bacillus selected from the group consisting of B. subtilis, B. amylolicuefaciens, B. megaterium, B. licheniformis and B. circulans and at least one Lactobacillus selected from the group consisting of L. rhamnosus, L. paracasei, L. fermentum and L. acidophilus. In accordance with the invention, the Lactobacillus/Bacillus proportions in the said complex are, depending on the case, either between 100/1 and 1/100, preferably between 10/1 and 1/10, or of 1/1. In accordance with the invention, the concentrations of bacteria are between 10.sup.2 and 10.sup.8 cfu/g. The said bacterial complex advantageously comprises at least one Bacillus, at a concentration between 10.sup.2 and 10.sup.7 cfu/g and at least one Lactobacillus, at a concentration between 10.sup.3 and 10.sup.8 cfu/g. Such a bacterial complex preferably finds application in the treatment of ruminant, equid or pig litter. According to an advantageous arrangement of the said embodiment, the said bacterial complex comprises at least B. subtilis at a concentration between 10.sup.2 and 10.sup.7 cfu/g and a Lactobacillus selected from the group consisting of L. rhamnosus, L. paracasei, L. fermentum, and L. acidophilus, at a concentration between 10.sup.3 and 10.sup.8 cfu/g. According to another advantageous arrangement of the said complex, it comprises the following 5 Bacilli: B. subtilis, B. amyloliquefaciens, B. megaterium, B. licheniformis and B. circulans, each at a concentration of between 10.sup.2 and 10.sup.7 cfu/g and the following 4 Lactobacilli: L. rhamnosus, L. paracasei, L. fermentum, L. acidophilus, each at a concentration of between 10.sup.3 and 10.sup.8 cfu/g. Advantageously, when the said bacterial complex comprises several Bacilli and/or several Lactobacilli, the various strains within the same genus (Bacillus or Lactobacillus) are in a ratio of between 1:1 and 1:100. In another embodiment of the said bacterial complex, it preferably comprises at least one Bacillus using urates as nitrogen source, in particular B. subtilis at a minimum concentration of 10.sup.3 cfu/g, optionally another Bacillus, selected from the group consisting of B. amyloliquefaciens, B. megaterium, B. licheniformis and B. circulans and at least one Lactobacillus selected from the group consisting of L. rhamnosus, L. paracasei, L. fermentum and L. acidophilus at a concentration of between 10.sup.2 and 10.sup.8 cfu/g. Such a bacterial complex is particularly well suited to the treatment of poultry litters or of the litters of other monogastric animals (with the exception of pigs). In another embodiment of the said bacterial complex, it preferably comprises at least one Lactobacillus selected from the group consisting of L. rhamnosus, L. paracasei, L. fermentum and L. acidophilus and at least one Bacillus selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. megaterium, B. licheniformis and B. circulans, the Lactobacillus/Bacillus ratio being between 1/1 and 1/10. Such a bacterial complex is particularly well suited to the treatment of septic tanks, lagoons or liquid manures. In another embodiment of the said bacterial complex, it preferably comprises the following two Bacilli: B. subtilis and B. megaterium, each at a concentration of between 10.sup.2 and 10.sup.7 cfu/g and at least one Lactobacillus selected from the group consisting of L. rhamnosus, L. paracasei, L. fermentum and L. acidophilus, at a concentration of between 10.sup.3 and 10.sup.8 cfu/g. Such a bacterial complex is especially suitable for the treatment of animal corpses. Surprisingly, the combination of at least one Bacillus and at least one Lactobacillus, as are defined above, effectively makes it possible to obtain a bacterial complex which: converts faecal or urinary residues or other waste of biological origin into bacterial proteins via bacterial synthesis, that is to say using inorganic nitrogen orginating from the various wastes, either directly or by degradation; deodorizes manure; can denitrify various media and/or water; significantly accelerates composting, without destruction of the material and at low temperatures (below 45.degree. C.). Such a selection of strains does not, moreover, lead to a loss of nitrogen over time (stabilization of the level of nitrogen). In addition, the bacterial complex in accordance with the invention is stable over time. As a variant, the said bacterial complex comprises at least one non-pathogenic Bacillus, at least one non-pathogenic Lactobacillus and a non-pathogenic Pediococcus. When a bacterial complex in accordance with the invention comprises a Pediococcus, the latter is at the same concentrations as the Lactobacilli. Surprisingly, besides the properties outlined above, such a bacterial complex has an inhibitory activity with respect to Staphylococcus aureus. Another subject of the present invention is Gram+Bacillus strains which can be used in a bacterial complex as defined above, characterized: in that they are able to use, as a source of nitrogen, inorganic nitrogen, in particular aqueous ammonia, nitrates and nitrites, and organic nitrogen molecules such as urea, urate, amino acids, nitrogenous bases or other nitrogenous compounds of low molecular weight, in that they have at least one of the following activities: amylolytic activity, cellulolytic activity, lignocellulolytic activity, pectinolytic activity, lipolytic activity, proteolytic activity, keratolytic activity, bacteriocin-type activity or bacteriocin-like activity, in that they have at least the following biochemical characteristics: gelatinase+, catalase+, urease-, oxidase-, indole- and in that they are preferably facultative aerobes/anaerobes. Such Bacillus strains are preferably selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis and Bacillus circulans. The said Bacillus strains have been filed with the Collection Nationale de Cultures de Micro-organismes (CNCM) [National Collection of Microorganism Cultures], on Jun. 8, 1994, under the numbers I-1433, I-1438 and I-1440 as regards Bacillus subtilis, under the numbers I-1434 and I-1435 as regards Bacillus amyloliquefaciens, under the number I-1436 as regards Bacillus megaterium, under the number I-1437 as regards Bacillus licheniformis and under the number I-1439 as regards Bacillus circulars. Another subject of the present invention is Gram+Lactobacillus strains which can be used in a bacterial complex as defined above, characterized: in that they are able to use, as a source of nitrogen, inorganic nitrogen, in particular aqueous ammonia, nitrates and nitrites, and organic nitrogen molecules such as urea, urate, amino acids, nitrogenous bases or other nitrogenous compounds of low molecular weight, in that they have at least one bacteriocin-type activity or bacteriocin-like activity, in that they advantageously have at least the following biochemical characteristics: catalase-, oxidase-, and in that they are preferably facultative aerobes/anaerobes. Such Lactobacillus strains are preferably selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus fermentum. The said Lactobacillus strains have been filed with the Collection Nationale de Cultures de Micro-organismes (CNCM), on Jul. 28, 1994, under the numbers I-1450, I-1452, I-1453, I-1454, I-1455, I-1456 and I-1459 as regards Lactobacillus rhamnosus, under the numbers I-1451, I-1457 and I-1458 as regards Lactobacillus paracasei, under the number I-1460 as regards Lactobacillus acidophilus and under the number I-1461 as regards Lactobacillus fermentum. Another subject of the present invention is Gram-positive Pediococcus strains which can be used in a bacterial complex as defined above, characterized: in that they have a bacteriocin-type or bacteriocin-like activity at least with respect to Staphylococcus aureus, in that they advantageously have at least the following biochemical characteristics: catalase-, oxidase-, and in that they are preferably facultative aerobes/anaerobes. A Pediococcus strain corresponding to this definition which may be mentioned in particular is the strain Pediococcus pentosaceus, filed with the Collection Nationale de Cultures de Micro-organismes (CNCM), on Dec. 22, 1995, under the number I-1654. Unexpectedly, bacterial complexes comprising in combination at least one Bacillus and at least one Lactobacillus and optionally a Pediococcus, having the characteristics specified above, act in synergy in order for the products treated in their presence to be rendered in a useful form and thereby reduce the drawbacks, nuisance or even pollution of the said products, to sanitationally acceptable levels for soils, crops and the food chain as a whole and thus participate favourably in waste treatment. The bacterial complex according to the invention is advantageously obtained from the said strains as follows: culturing and production of each strain separately, production of various cultures each having microorganism concentrations of the order of 10.sup.10 -10.sub.11 cfu/g, freeze-drying of each culture, dilution, to between 1/100-1/1,000,000, of each of the said freeze-dried strains, in the presence of a neutral diluent (plant or mineral diluent such as clay, corn lithothamne-grit, etc.), and mixing together the various strains thus diluted in order to obtain the desired bacterial complex. It may moreover comprise additives such as a chemical or microbiological tracer. Another subject of the present invention is a composition for the treatment of biological residues (dilute, ready-to-use product), characterized in that it comprises in combination a bacterial complex in accordance with the invention, at least one neutral diluent and at least one microparticle binder. Indeed, the bacterial complexes as defined above constitute a bacterial concentrate which may preferably be bound to a support in order to be used for the treatment of manures, liquid manures, composts, corpses, etc. Such compositions thus advantageously comprise a neutral diluent, which is identical to or different from that used in order to prepare the bacterial complex, and a microparticle binder, such as molasses, which acts both as an energetic element and provides an effect of adhesive bonding of the particles, starch derivatives or derivatives of other sugars (complex sugars which are degraded slowly) and a coating fat or oil. Preferably, the said composition essentially comprises 5-15% of bacterial complex, 80-89% of neutral diluent and 3-5% of microparticle binder, preferably 10% of bacterial complex, 87% of neutral diluent and 3% of particle binder. The said treatment composition has the same applications as the bacterial complex in accordance with the invention, at lower but effective concentrations. It may be used in particular, by direct incorporation into litters, liquid manures or any other organic product to be treated [manures, plant waste, organic waste (abattoir waste and corpses)], in proportions of 0.2 to 50 kg per ton of organic product. In accordance with the invention, the proportions of the said incorporation vary depending on the application, in particular: as regards litters: a bacterial complex or a treatment composition in accordance with the invention is incorporated into the litter at a rate of 10 kg/t of straw; weekly maintenance is recommended at a rate of 3 kg/t of straw; as regards liquid manures: a bacterial complex or a treatment composition in accordance with the invention is incorporated into the liquid manures at a rate of 100 g to 1 kg/t; weekly maintenance is recommended at a rate of 100 g to 3 kg/t; as regards manures to be composted: a bacterial complex or a treatment composition in accordance with the invention is incorporated into the manures to be composted at a rate of 1 to 5 kg/t, in a single addition; as regards corpses to be composted: a bacterial complex or a treatment composition in accordance with the invention is incorporated with the corpses to be composted at a rate of 1 to 10 kg/t, in a single addition; as regards lagoons, a bacterial complex or a treatment composition in accordance with the invention is incorporated into the lagoons at a rate of 500 g/m.sup.2, once every 3 months; as regards septic tanks: a bacterial complex or a treatment composition in accordance with the invention is incorporated into the septic tanks at a rate of 100 g to 1 kg/t; weekly maintenance is recommended at a rate of 100 g to 3 kg/t; as regards the disinfection of premises and in particular the disinfection of lavatories: a bacterial complex or a treatment composition in accordance with the invention is applied in the form of a layer (artificial biofilm), to the walls and floors of the said premises, at a rate of 10 g/l of water; the solution obtained is preferably sprayed onto the said walls. Another subject of the present invention is a process for the treatment of biological residues, such as excrement, manure, corpses or the like, characterized in that it comprises the placing in contact of a bacterial complex in accordance with the invention or of a composition as defined above with a biological residue to be treated. Besides the preceding arrangements, the invention also comprises other arrangements, which will emerge from the following description which refers to examples of implementation of the process forming the subject of the present invention. |
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