TECHNICAL FIELD

The invention relates to the use of a composition comprising a rod-shaped, aerobic, Gram-positive microorganism for the degradation of liquid polyether-based polyurethane, preferably a microorganism of Corynebacterium sp. or Bacillus sp, for the degradation of polyether-based polyurethane, as well as the production of amino acids using polyurethane as carbon source. Thus, the present invention relates to the degradation of polyurethane and more specifically, to the biodegradation of polyurethane and the production of amino acids as a consequence of said biodegradation.

BACKGROUND ART

The global market of polyurethane (PU) foams is estimated in 9.5 million tons, growing at 3.3% CAGR until 2024. The leading consumer region is Asia Pacific with a market share of 45%, with demand from Chinese and Indian economies rapidly expanding this manufacturing sector. In terms of product segments, 55% of global volume corresponds to flexible foams, used mostly by transportation and furniture. Rigid PU foams are also used massively as heat insulators, e.g., for refrigerators, because of their thermal transfer properties. And lastly, PU is also commonly utilized as a constituent material in many products including Coatings, Adhesives, Sealants and Elastomers (CASE).

Altogether, this strong PU demand for a broad range of end-user applications has led to a serious waste disposal problem. At present, PU waste is either incinerated or disposed in landfills as noncombustible garbage. Incineration is disadvantageous as it generates air polluting emissions that accelerate global warming, while landfill disposal suffers also from problems such as the lack of landfills and environmental pollution.

While microbial biodegradation can be presented as a preferred disposal technique from the viewpoint of protection and conservation of the natural environment, a problem exists in that polyether-based polyurethane is not prone to biodegradation. Actually, it has been estimated that it takes hundreds of years for polyether-based polyurethane foams to be fully decomposed. Structurally, PU are formed by reacting a polyol (a polymer compound with more than two reactive hydroxyl groups per molecule) with a diisocyanate or a polymeric isocyanate in the presence of suitable catalysts and additives. As a consequence, intramolecular urethanes bonds (carbamate bond, -NH-(C=0)-0-) are constituted. Depending on what type of polyols is used, PUs can be classified in polyester (PS) or polyether (PE) polyurethanes. Thus, PU contains urethane bonds together with ester or ether bonds in its molecule, and biodegradation proceeds through cleavage of these bonds. Three types of PU biodegradation have been identified in literature: bacterial, fungal, and enzymatic biodegradation.

There are some reports of ester bonds in polyol units being cleaved by fungi and/or bacteria. Darby et al. 1968 (Appl. Microbiol., 16: 900-905) performed fungal degradation tests on various PU. They reported that ester-based PUs are more sensitive to degradation than ether-based PUs, and that degradation profiles vary depending on the type of isocyanate and/or polyol. Kay et al. 1991 (Int. Biodeterio., 27(2): 205-222) investigated the ability of 16 bacterial strains to degrade ester-based PU and found a species of Corynebacterium and Pseudomonas aeruginosa (ATCC 13388) among the very few to be capable of some degradation.

Still related to solid PU-degrading bacteria, the following strains are also known to degrade polyester-based PUs: Paenibacillus amylolyticus strain TB-13 (Japanese Patent Application No. 2002-334162) and Comamonas acidovorans strain TB-35 (T. Nakajima- Kambe, F. Onuma, N. Kimpara and T. Nakahara, 1995. FEMS Microbiology Letters, 129: 39-42). However, while these strains do actually degrade ester bonds in PU, they do not substantially degrade ether bonds.

On the other hand, there is almost no knowledge or information about degradation of ether-based polyurethanes. Jansen, B. et al. 1991 (Zbl. Bakt. 276: 36-45) reported that survival of Staphylococcus epidermidis strain KH11 in the presence of synthetic polyether- based polyurethane films (Biomer and Tuftane) was prolonged 5 days more in comparison to control experiments performed in the absence of any nutrients. Investigations of the bacteria after contact with the polymers revealed changes in their surface properties and metabolism, in particular a significant induction of urease activity. However, neither weight changes in both polyether-based polyurethane films nor ether bond breakage could be detected. More recently, Rafiemanzelat, F., Jafari, M., and Emtiazi, G. 2015 (Appl. Biochem Biotechnol, 177(4): 842-60) disclosed the degrading activity of Bacillus amyloliquefaciens M3 on a new synthesized polymer poly(ether-urethane-urea) (PEUU), wherein they changed the standard formulation of polyether-based polyurethane in the lab by adding L-leucine anhydride cyclopeptide (LACP), a known degradable monomer, to allow biodegradation. The synthesized degradable PEUU was used in a powder state. Unfortunately, PEUU is not commercially available and, therefore, it does not account for any of the environmental problems associated to PU waste.

Regarding fungal degradation of polyether-based PU, there are also very few examples reported. Matsumiya et al. 2010 (Journal of Applied Microbiology 108: 1946-1953) isolated a fungus identified as Alternaria alternata PURDK2 capable of degrading solid ether-PU (up to 27.5% weight loss after 70 days). In addition, Alvarez-Barragan et al. 2016 (Applied and Environmental Microbiology 82, 17: 5225-5235) found four different species of Cladosporium cladosporioides complex to efficiently degrade solid polyether- based PU. After 21 days of incubation, foams inoculated with these fungi changed their shape, size, and weight (up to 65% loss) compared to the similarly treated but non- inoculated pieces.

And finally, the last approach to degrade polyurethane relates to the use of polyurethane- degrading enzymes. Although there are only a few reports on microorganisms capable of degrading polyether-PU, little is known about the degradation enzymes or the genes involved, and much of what is known pertains to only polyester-based polyurethane degraders. As an example, Nomura et al. 1998 (Journal of Fermentation and Bioengineering, 86: 339-345) reported that Comamonas acidovorans TB35 has two types of extracellular esterases. Meanwhile, Howard et al. 1999 (International Biodeterioration and Biodegradation, 43: 23-30) found that the supernatant of a liquid culture of Pseudomonas chlororaphis had three different types of PU activity and a culture of Alicycliphilus sp. showed esterase activity when degrading PU (Oceguera-Cervantes et al. 2007, Applied and Environmental Microbiology, 73(19): 6214-6223). Loredo-Treviho et al. 201 1 (Advances in Bioscience and Biotechnology, 2: 52-58) tested fungal ability to grow on PU as sole carbon and nitrogen source and detected similar enzymatic activities as in bacteria. Among the enzymatic activities detected, the most common was the urease activity (95% of the strains) followed by protease (86%), esterase (50%), and laccase (36%). Interestingly, and to our knowledge, no etherase activity has ever been detected neither in fungi nor in bacteria. Thereof, there is a necessity on the state of the art to provide new ways of degrading PU, mainly, polyether-based PU which will help us to develop new bioremediation technology for PU waste recycling.

BRIEF DESCRIPTION OF THE INVENTION

The authors of the present invention have discovered that a rod-shaped, aerobic, Gram- positive microorganism, preferably a bacteria belonging to Corynebacterium sp. or to Bacillus sp. are capable of, surprisingly, breaking remarkably the highly recalcitrant ether bond in liquefied polyether-based polyurethane (PE-PU). This is an innovative approach for the development of biotechnological processes for polyurethane degradation and recycling, thus reducing potential negative effects of waste polyurethane materials on the environment. Additionally, the inventors observed that when the microorganism is degrading polyether-based polyurethane, the production of amino acids is unexpectedly increased in comparison with the production of amino acids using sugar as carbon source. Thus, the present invention provides a new way for degrading liquid polyurethane, specifically, polyether-based polyurethane, and taking advantage of the products resulting from the polyether-based polyurethane foams in the production of high value chemicals, such as amino acids.

In order to arrive to the above discovering, the inventors tested microorganisms by their capacity to grow up in the presence of liquefied polyether-based polyurethane (see Example 1 ). By this way, rod-shaped, aerobic, Gram-positive microorganisms belonging to Corynebacterium sp. or to Bacillus sp. were identified. Next, a range of strains belonging to Corynebacterium and Bacillus genera were further evaluated in liquefied polyether-based polyurethane cultures. The amount of degradation in polyether-based polyurethane was quantified as the disappearance of liquefied polyether-based polyurethane characteristic functional groups and correlated to microbial growth (Example 2).

Subsequently, experiments were conducted to identify metabolite production in PE-PU growing cultures. Significant synthesis of amino acids (e.g. phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), methionine (Met), etc.) was detected in a range of Bacillus and Corynebacteria species (see Example 3). Finally, amino acid production was compared in sugar versus liquefied polyether-based polyurethane grown cultures, finding an induction effect of liquefied polyether-based polyurethane substrate on amino acid production. Thus, in one aspect, the present invention relates to the use of a composition comprising a rod-shaped, aerobic, Gram-positive microorganism for the degradation of liquid polyether-based polyurethane.

In another aspect, the invention relates to a composition obtained from the degradation of liquefied polyether-based polyurethane, by the use of a composition comprising a rod- shaped, aerobic, Gram-positive microorganism.

In another aspect, the invention relates to the use of a composition comprising a rod- shaped, aerobic, Gram-positive microorganism for producing amino acids using polyurethane.

Based on the above discovering, a set of inventive aspects have been developed, as well as particular embodiments thereof, which will be disclosed in detailed below.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, the present invention provides a new use for rod-shaped, aerobic, Gram-positive microorganisms.

Thus, in an aspect, the present invention relates to the use of a composition comprising a rod-shaped, aerobic, Gram-positive microorganism for the degradation of liquid polyether- based polyurethane, hereinafter“first use of the invention”.

In the present invention the term "composition" refers to a composition comprising at least a rod-shaped, aerobic, Gram-positive microorganism having the ability to degrade liquid polyether-based polyurethane, hereinafter“microorganism of the invention”.

As used herein, the term “a rod-shaped microorganism” refers to a rod, bacillus or bacilliform bacterium which usually divide in the same plane and are solitary, although may combine to form diplobacilli (two bacilli arranged side by side with each other) or streptobacilli (bacilli arranged in chains).

As used herein, the term“aerobic microorganism” refers to a microorganism which can survive and grow in an oxygenated environment. Depending on the capacities to grow in the presence of oxygen, they are classified in (1 ) obligate aerobes (these microorganisms need oxygen to grow since, in a process known as cellular respiration, they use oxygen to oxidize substrates and generate energy), (2) facultative anaerobes (these microorganisms use oxygen if it is available, but also have anaerobic methods of energy production), (3) microaerophiles (these microorganisms require oxygen for energy production, but are harmed by atmospheric concentrations of oxygen (21% 02)), or (4) aerotolerant anaerobes (these microorganisms do not use oxygen but are not harmed by it).

As used herein, the term“Gram-positive microorganism” refers to a microorganism that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell wall. Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through a microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the decolorization stage of the test (Gram-negative bacteria cannot retain the violet stain after the decolorization step). In general, the following characteristics are present in Gram- positive bacteria: (1 ) Cytoplasmic lipid membrane, (2) Thick peptidoglycan layer, (3) Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence, (4) Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase, and/or (5) a much smaller volume of periplasm than that in Gram-negative bacteria.

The“microorganism of the invention”, may be any newly screened microorganisms. By way of example, screening of microorganisms having the ability to degrade polyether- based polyurethane may be accomplished by growing the microorganism to be tested in liquid polyether-based polyurethane as shown in Example 1 of the present description.

In a particular embodiment of the first used of the invention, the microorganism is selected from the list consisting of Corynebacterium sp., and Bacillus sp.

Corynebacterium sp. is genus of bacteria that are Gram-positive and aerobic or facultatively anaerobic. They are bacilli (rod-shaped), and in some phases of life they are, more particularly, club-shaped, which inspired the genus name. They are widely distributed in nature in the microbiota of animals (including the human microbiota) and are mostly innocuous. They are Gram-positive, catalase-positive, nonspore-forming, nonmotile, rod-shaped bacteria that are straight or slightly curved. Their size falls between 2 and 6 pm in length and about 0.5 pm in diameter. Examples of microorganisms belonging to the genus Corynebacterium and having the ability to degrade polyether- based polyurethane including, but not limiting to, C. camporealensis, C. striatum, C. xerosis, C. glutamicum , C. ammoniagenes, C. flavescens , C. afermentans , C. ammoniagenes, C. amycolatum, C. callunae, C. efficiens, C. flavescens, Corynebacterium hansenii and C. humireducens. Thus, in a particular embodiment, the microorganism of Corynebacterium sp. is selected from the list consisting of

- C. camporealensis, preferably, C. camporealensis strain CECT4897 (deposit number under Budapest Treaty CECT 9947),

- C. striatum, preferably, C. striatum strain LMG19648 (deposit number under Budapest Treaty CECT 9948),

- C. glutamicum, preferably, C. glutamicum strain CECT79 (deposit number under Budapest Treaty CECT 9946),

- C. ammoniagenes and

- C. flavescens.

C. camporealensis is a Gram-positive bacterium. The scientific classification of C. camporealensis is: Kingdom: Bacteria, Phyilum: Actinobacteria, Class: Actinobaceria, Subclass: Actinobacteridae, Order: Actinomycetales, Suborder: Corynebacterineae, Family: Corynebacteriaceae, genus: Corynebacterium, Species: Corynebacterium camporealensis.

In a more particular embodiment, C. camporealensis is C. camporealensis strain CECT4897. C. camporealensis strain CECT 4897 (also called BPL202 by the depositor) was isolated from sheep milk. This strain was deposited by ADM-BIOPOLIS (Parc Cientific Univ. Valencia, Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on July 18, 2019, under the Budapest Treaty in the Coleccion Espaiiola de Cultivos Tipo (CECT) as an International Depositary Authority (based in Building 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino, 9, 46980 Paterna, Valencia, SPAIN). The deposit number assigned was CECT 9947. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application. C. striatum is a Gram-positive bacterium. The scientific classification of C. striatum is: Kingdom: Bacteria, Phylum: Actinobacteria, Class: Actinobaceria, Subclass: Actinobacteridae, Order: Actinomycetales, Suborder: Corynebacterineae, Family: Corynebacteriaceae, genus: Corynebacterium, Species: Corynebacterium striatum.

In a more particular embodiment, C. striatum is C. striatum strain LMG19648 (from unknown origin), also called BPL203 by the depositor. This strain was deposited by ADM- BIOPOLIS (Parc Cientific Univ. Valencia, Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on July 18, 2019, under the Budapest Treaty in the Coleccion Espahola de Cultivos Tipo (CECT) as an International Depositary Authority (based in Building 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino, 9, 46980 Paterna, Valencia, SPAIN). The deposit number assigned was CECT 9948. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application.

C. glutamicum is a Gram-positive bacterium. The scientific classification of C. glutamicum is: Kingdom: Bacteria, Phylum: Actinobacteria, Class: Actinobaceria, Subclass: Actinobacteridae, Order: Actinomycetales, Suborder: Corynebacterineae, Family: Corynebacteriaceae, genus: Corynebacterium, Species: Corynebacterium glutamicum.

In a more particular embodiment, C. glutamicum is C. glutamicum strain CECT 79 (from unknow origin), also called BPL201 by the depositor. This strain was deposited by ADM- BIOPOLIS (Parc Cientific Univ. Valencia, Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on July 18, 2019, under the Budapest Treaty in the Coleccion Espahola de Cultivos Tipo (CECT) as an International Depositary Authority (based in Building 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino, 9, 46980 Paterna, Valencia, SPAIN). The deposit number assigned was CECT 9946. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application. Bacillus sp. is a genus of Gram-positive, rod-shaped bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes (oxygen depending), or facultative anaerobes (having the ability to be aerobic or anaerobic). They will test positive for the enzyme catalase when there has been oxygen used or present. Ubiquitous in nature, Bacillus includes both free-living (nonparasitic) and parasitic pathogenic species. Under stressful environmental conditions, the bacteria can produce oval endospores that are not true 'spores', but to which the bacteria can reduce themselves and remain in a dormant state for very long periods. Examples of microorganisms belonging to the genus Bacillus and having the ability to degrade polyether-based polyurethane including, but not limiting to, B. subtilis, B. licheniformis, B. amyloliquefaciens, B. amylolyticus, B. ciriculans, B. clausii, B. coagulans, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. stearothermophilus, Bacillus subtilis, Bacillus thuringiensis. In a particular embodiment of the first used of the invention, the microorganism of Bacillus sp. is selected from the list consisting of

- B. subtilis, preferably, B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887) or B. subtilis strain BPL83 (deposit number under Budapest treaty DSM 32886),

- B. licheniformis, preferably, B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888) and

- B. amyloliquefaciens.

B. subtilis is a Gram-positive bacterium. The scientific classification of B. subtilis is\ Kingdom: Bacteria, Phylum: Firmicutes, Class: Bacilli, Order: Bacillales, Family: Bacillaceae, genus: Bacillus, Species: Bacillus subtilis.

In a more particular embodiment, B. subtilis is B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM32887) or B. subtilis strain BPL83 (deposit number under Budapest Treaty DSM32886).

B. subtilis BPL84 was isolated from pig faeces. This strain was deposited by Biopolis S.L. (address: Catedratico Agustin Escardino, 9, 46980 Paterna, Valencia, Spain) on August 7, 2018, under the Budapest Treaty in the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures as an International Depositary Authority (Inhoffenst^e 7B, 38124 Braunschweig, Germany). The deposit number assigned was DSM 32887. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application.

B. subtilis BPL83 was isolated from liquid polyether-based polyurethane growing bioreactor. This strain was deposited by Biopolis S.L. (address: Catedratico Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on August 7, 2018, under the Budapest Treaty in the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures as an International Depositary Authority (IhIioIΐbhbΐGBbb 7B, 38124

Braunschweig, Germany). The deposit number assigned was DSM 32886. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application.

B. licheniformis is a Gram-positive bacterium. The scientific classification of B. licheniformis is: Kingdom: Bacteria, Phylum: Firmicutes, Class: Bacilli, Order: Bacillales, Family: Bacillaceae, genus: Bacillus, Species: Bacillus licheniformis.

In a more particular embodiment, B. licheniformis is B. licheniformis strain BPL89 (deposited number under Budapest Treaty DSM 32888). B. licheniformis strain BPL89 was isolated from pig faeces. This strain was deposited by Biopolis S.L. (address: Catedratico Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on August 7, 2018, under the Budapest Treaty in the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures as an International Depositary Authority (Inhoffenst^e 7B, 38124 Braunschweig, GERMANY). The deposit number assigned was DSM 32888. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application.

B. amyloliquefaciens is a Gram-positive soil bacteria closely related to the species B. subtilis. The two species share many homologous genes and appear so similar it is not possible to visually separate the two species. B. amyloliquefaciens are Gram-positive rods with peritrichous flagella allowing motility. The cells often appear as long chains unlike many other Bacillus species that form as single cells. The optimal temperature for cellular growth is between 30 and 40 degrees Celsius.

The microorganism of the invention may be either a wild-type or mutant strain as long as it has the ability to degrade liquid polyether-based polyurethane. Mutant strains may be obtained by mutagenesis with ethylmethanesulfonic acid, a conventionally commonly used mutagen), treatment with other chemical substances (e.g., nitrosoguanidine, methylmethanesulfonic acid), ultraviolet irradiation, or so-called spontaneous mutation without using any mutagen.

In a more particular embodiment, the mutant strain with the ability to degrade liquid polyether-based polyurethane is B. subtilis strain BPL200. B. subtilis strain BPL200 was obtained from B. subtilis BPL83 (deposit number under the Budapest Treaty DSM 32886) by ethyl methanesulfonate (EMS) random mutagenesis. This mutant strain was deposited by AMD-Biopolis (address: Parc Cientific Univ. Valencia, Agustin Escardino 9, 46980 Paterna, Valencia, Spain) on July 18, 2019, under the Budapest Treaty in the Coleccion Espahola de Cultivos Tipo (CECT) as an International Depositary Authority (based in Building 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino, 9, 46980 Paterna, Valencia, SPAIN). The deposit number assigned was CECT 9945. The depositor authorizes the applicant (REPSOL S.A.) to refer to the aforementioned deposited biological material in the present patent application and gives his unreserved and irrevocable consent to the deposited material being made available to the public as from the date of filing, or if priority has been claimed, from priority date, of the present patent application. Furthermore, the mutant strain B. subtilis CECT 9945 produces 50% more methionine than the wild type strain (B. subtilis DSM 32886), around 300mg/L.

Additionally, the microorganism of the invention may be genetically modified in order to obtain a recombinant microorganism with improved features.

The composition comprising the microorganism of the invention may be in a liquid, solid, semisolid state, and may comprise different substances or compounds for culturing the microorganisms, such as vitamins, nitrogen and/or carbon source. Any medium can be used without particular limitation in culturing rod-shaped, aerobic, Gram-positive microorganisms as long as it allows growth of rod-shaped, aerobic, Gram-positive microorganisms. Examples include, but are not limited to, LB medium (1% tryptone, 0.5% yeast extract, 1% NaCI), NB medium (0.5% peptone, 0.3% meat extract), Corynebacterium medium (1 % casein peptone, 0.5% yeast extract, 0.5% glucose, 0.5% NaCI).

More specifically, the medium used for growing the microorganism of the present invention may contain a carbon source (e.g., glucose) assimilable by the microorganism of the present invention and a nitrogen source assimilable by the microorganism of the present invention. Such a nitrogen source includes an organic nitrogen source such as peptone, meat extract, yeast extract, urea or corn steep liquor, as well as an inorganic nitrogen source such as ammonium sulfate or ammonium chloride. If desired, the medium may further contain salts composed of cations (e.g., sodium ion, potassium ion, calcium ion, magnesium ion) and anions (e.g., sulfate ion, chlorine ion, phosphate ion). Moreover, the medium may also be supplemented with trace components such as vitamins and nucleic acids.

In a particular embodiment of the first use of the invention, the composition further comprises combination of two or more microorganisms selected from the group consisting of C. camporealensis, C. striatum, C. xerosis, C. glutamicum, C. ammoniagenes, C. fiavescens, C. afermentans, C. ammoniagenes, C. amycolatum, C. callunae, C. efficiens, C. fiavescens, Corynebacterium hansenii, C. humireducens, B. subtil is, B. licheniformis, B. amyloliquefaciens, B. amylolyticus, B. ciriculans, B. clausii, B. coagulans, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. stearothermophilus, Bacillus subtilis, Bacillus thuringiensis.

Preferably, the microorganisms of Corynebacterium sp. are selected from the list consisting of

- C. camporealensis, preferably, C. camporealensis strain CECT4897 (deposit number under Budapest Treaty CECT 9947),

- C. striatum, preferably, C. striatum strain LMG19648 (deposit number under Budapest Treaty CECT 9948),

- C. glutamicum, preferably, C. glutamicum strain CECT79 (deposit number under Budapest Treaty CECT 9946),

- C. ammoniagenes and

- C. fiavescens·, and/or

the microorganisms of Bacillus sp. are selected from the list consisting of - B. subtilis, preferably, B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887) or B. subtilis strain BPL83 (deposit number under Budapest treaty DSM 32886),

- B. licheniformis, preferably, B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888) and

- B. amyloliquefaciens.

Within the context of the present invention, it is also encompassed a composition comprising any combination of the above-mentioned microorganisms. In a more particular embodiment, the composition comprises the microorganisms Corynebacterium sp. and Bacillus sp. as those disclosed above simultaneously.

The composition comprising the microorganism of the invention, or the microorganism per se, may be lyophilized in a routine manner to give a powder, and may further be blended with various vitamins, minerals and necessary nutrient sources (e.g., yeast extract, casamino acid, peptone) for formulation into solid preparations including tablets. Lyophilization is a method of sublimation in which all liquid is removed from the microorganism to produce a freeze-dried product in pellet form, which is more stable than a live culture with the advantage of allowing longer storage time and easier shipping.

As explained above, the microorganism of the invention (or the composition comprising it) can be used for degrading liquid polyether-based polyurethane.

As used herein, the term “degradation” or“degrading” refers to the disruption of the chemical linkages that form a polymeric blend matrix from a number of one or more different monomeric subunits. Therefore, degradation may be a result of chemical activities such as desorption, dissociation, hydrolysis, dissolution, oxidation, reduction, photolysis, etc. as well as physical activities that may erode a polymeric blend matrix such as diffusion, abrasion, cracking, peeling, mechanical breakage, spinodal decomposition, etc. or any combination of these chemical and physical activities. In the context of the present invention, the terms“degradation” and“decomposition” are equivalents. When any of the above-mentioned chemical processes responsible for the degradation of polyether-based polyurethane is consequence of the action of microorganisms, the degradation is called “biodegradation”, “biological degradation” or “microbiological degradation”. A way of seeing polyol consumption/degradation in the samples is to see a decrease in the signal corresponding to the ether bond by Fourier-transform infrared spectroscopy (FTIR) (see Example 3).

The term "polyurethane" is a generic name for high molecular compounds comprising urethane bonds (-NHCOO-) in their molecule and it means a polymer comprising groups such as ester, ether, amide, urea and/or carbamate, which is obtained by reaction between a multifunctional isocyanate and a hydroxyl group-containing compound. When varying the functionality of hydroxyl or isocyanate groups, it is possible to prepare a wide variety of branched or crosslinked polymers. They can be broadly divided into ester-based and ether-based polyurethanes depending on the type of polyol used. Because of their good properties such as easy processability, resistance to putrefaction, resistance to spoilage and low density, polyurethanes have a wide range of uses including elastic materials, foamed materials, adhesives, coating materials, fibers and synthetic leather, and are also widely used as automobile components. There is no particular limitation on the number average molecular weight of polyurethane resins which can be treated by the composition of the present invention. The polyurethane used in the present invention is in liquid state.

Additionally, this liquid polyurethane does not need to be modified or to incorporate labile and hydrolyzable moieties into the polymer backbone, for example a cyclopeptide moiety, in order to be biodegraded by the microorganism of the invention. That is, the polyurethane of the present invention can be non-modified polyurethane. This is an important advantage of the present invention in relation to the prior art. For example, Rafiemanzelat et al. 2015. (Appl. Biochem Biotechnol, 177(4):842-60) discloses a modified polyurethane with a cyclopeptide moiety in order to be degraded.

The microorganism of the invention is capable of degrading liquid polyether-based polyurethane. As used herein, the term“liquid polyether-based polyurethane” refers to polyurethane in liquid state comprising ether groups in their molecule. In the context of the present invention, the terms “polyether-based polyurethane”, “poly(ether-urethane)”, “ether-based polyurethane” and“polyether-polyurethane” are equivalent and can be used interchangeably throughout the present description. The liquid polyether-based polyurethane can be incorporated in the formulation of coatings, adhesives, sealants and elastomers for what are called CASE applications/marketplace (CASE is the acronym of Coatings, Adhesives, Sealants and Elastomers). Thus, in the context of the present invention, the term“liquid polyether-based polyurethane” also encompasses the above- mentioned formulations. Thus, in a particular embodiment, the liquid polyether-based polyurethane is a liquid polyether-based polyurethane used as a component for polyurethane CASE applications, such as polyether-based polyurethane used in varnishes, coatings, adhesives, sealants and elastomers.

In a particular embodiment, the liquid polyether-based polyurethane is liquefied polyether- based polyurethane. As used herein, the term“liquefied polyether-based polyurethane” refers to the resulting liquid after a chemical depolymerisation treatment (chemolysis) has been applied for recycling material containing polyether-based polyurethanes. Water (hydrolysis), glycols (glycolysis), acids (acidolysis) and amines (aminolysis) typically serve as reagents to break the urethane bonds. The acidolysis and glycolysis processes are widely known in the state of the art and are routine practice for the skilled person. More details about the acidolysis and glycolysis processes can be found in Simon, D., et al. 2018, Recycling of polyurethanes from laboratory to industry, a journey towards the sustainability. Waste Management, https://doi.Org/10.1016/j.wasman.2018.03.041 ; Sottysmski, M. et al. Polymery 2018, 63(3): 234-238; Motokucho, S et al. J. APPL. POLYM. SCI. 2017, 2017. DOI: 10.1002/APP.45897; and Datta, J. & Kopczyhska, P., 2016, Critical Reviews in Environmental Science and Technology, 46 (10): 905-946.

The resulting liquid can be used as such, or the individual components separated to feed the microorganism of the invention. The inventors have found that the liquefied polyether- based polyurethane comprises a non-volatile polymeric fraction and a volatile polymeric fraction. In a particular embodiment, the liquid polyether-based polyurethane is liquefied polyether-based polyurethane foam (rigid or flexible foams).

Thus, as used herein, the term“liquefied polyether-based polyurethane foam” refers to the resulting liquid after a chemical depolymerisation treatment (chemolysis) has been applied for recycling polyether-based polyurethanes foams. Water (hydrolysis), glycols (glycolysis), acids (acidolysis) and amines (aminolysis) typically serve as reagents to break the urethane bonds. Examples of liquefied polyurethane foam include, without limiting to, polyether-based polyurethane for and/or from mattresses (such as POL-3, POL-5, POL-6 and POL-8 disclosed in the examples of the present description), polyether-based polyurethane for and/or from car seats (such as POL-4 and POL-7 disclosed in the examples of the present description), and combinations thereof. Thus, in a more particular embodiment, the liquefied polyether-based polyurethane foam is polyether-based polyurethane for and/or from mattresses, polyether-based polyurethane for and/from car seats, flexible polyether-based polyurethane.

Additionally, the liquid polyether-based polyurethane of the invention can be non-modified liquid polyether-based polyurethane. In a particular embodiment, the liquid polyether- based polyurethane is not a polyether-based polyurethane containing cyclopeptide moiety and PEG when the microorganism used in the degradation thereof is Bacillus amyloliquefaciens, M3.

It should be noted that the amount of polyether-based polyurethane compounds added to the medium is desirably 0.01 % to 10% by weight, preferably, 1 % by weight. Microorganisms of the invention may be added in a very small amount; and it is preferable to use them in an amount of at least 0.1% by weight (wet weight) relative to polyether- based polyurethane compounds in consideration of degradation efficiency. Polyether- based polyurethane compounds to be degraded may be provided either alone or in combination.

In an embodiment based on a phenomenon in which ether bonds are degraded and therefore the liquid polyether-based polyurethane compounds are consumed as a nutrient source during growth of microorganisms (see Table 4 from the Examples of the present description), polyether-based polyurethane compounds may be provided as a carbon source, as a sole carbon source or as a sole carbon and nitrogen source, or together with other carbon and/or nitrogen sources. The medium available for use may contain polyether-based polyurethane compound(s) or glucose or the like as a carbon source, as well as an assimilable nitrogen source by the microorganism of the present invention, including an organic nitrogen source (e.g., peptone, meat extract, yeast extract, corn steep liquor, yeast nitrogen base, urea, etc.) or an inorganic nitrogen source (e.g., ammonium sulfate, ammonium chloride, etc.). If desired, the medium may further contain salts composed of cations (e.g., sodium ion, potassium ion, calcium ion, magnesium ion, etc.) and anions (e.g., sulfate ion, chlorine ion, phosphate ion, etc.). Moreover, the medium may also be supplemented with trace components such as vitamins

In an embodiment, the medium may be a buffer, which may further be supplemented with nitrogen sources, inorganic salts, vitamins, etc. as cited above. Examples of a buffer include phosphate buffer. The time required for degradation of polyether-based polyurethane compounds will vary depending on the type, composition, shape and amount of polyether-based polyurethane compounds to be degraded, the type and amount (relative to urethane compounds) of microorganisms used, as well as culture conditions, etc.

In the present invention, the degradation of polyether-based polyurethane compounds can be observed when static culture, shaking culture or aeration culture is performed on the microorganisms of the invention under aerobic or anaerobic conditions. Preferred is rotary shaking culture, a rotation speed of which may be in the range of 30 to 250 rotations per minute. In relation to culture conditions, the culture temperature may be 10°C to 50°C, particularly preferably around 30, 31 , 32, 33, 34, 35, 36, or 37°C. The pH of the medium may be in the range of 4 to 10, preferably around 7.

The degradation reaction can be carried out in any kind of device that allows the growth of microorganisms and the degradation of polyether-based polyurethane and/or amino acid production. Example of bioreactor includes, without limiting to, those that support Batch, Fed-batch, free cell continuous fermentation, immobilized cell continuous reactors and membrane cell recycle reactors.

A) Batch, Fed-batch, and Free Cell Continuous Fermentation

The fermenter is filled with the culture medium and then the microorganisms are added. The fermentation takes place under controlled pH and aeration, and the products remain in the fermenter until completion. Air or oxygen can be supplied to meet oxygen consumption requirements.

Fed-batch fermentation is an industrial technique, which is applied to processes where a high substrate concentration is toxic to the culture. In such cases, the reactor is initiated in a batch mode with a low substrate concentration (non-inhibitory to the culture) and a low medium volume, usually less than half the volume of the bioreactor.

As the substrate (liquid polyether-based polyurethane) is used by the microorganism of the invention, it is replaced by adding a concentrated substrate solution at a slow rate, thereby keeping the substrate concentration in the bioreactor below the toxic level for the microorganism. In this type of system, the culture volume increases in the reactor over time. The culture is harvested when the liquid volume is approximately 75% of the volume of the bioreactor.

The continuous culture technique can be used to improve bioreactor productivity and to study the physiology of the culture in a steady state. In such systems, the reactor is initiated in a batch mode and cell growth is allowed until the cells are in the exponential phase. While the cells are in the exponential phase, the reactor is fed continuously with the substrate (liquid polyether-based polyurethane) and a product stream is withdrawn at the same flow rate as the feed, thus keeping a constant volume in the bioreactor. Running the process in this manner eliminates downtime, thus improving bioreactor productivity. Additionally, the process runs much longer than in a typical batch process.

B) Immobilized cell continuous reactors

High cell concentrations result in high bioreactor productivity. Such systems are continuous where feed is introduced into a tubular bioreactor at the bottom with product escaping at the top. These systems are often non-mixing reactors where product inhibition is significantly reduced. To improve bioreactor productivity, cells may be immobilized onto clay brick particles by adsorption and achieve higher bioreactor productivity, resulting in economic advantage.

C) Membrane Cell Recycle Reactors

Membrane cell recycle reactors are another option for improving bioreactor productivity. In such systems, the reactor is initiated in a batch mode and cell growth is allowed. Before reaching the stationary phase, the fermentation broth is circulated through the membrane. The membrane allows the aqueous product solution to pass while retaining the cells. The bioreactor feed and product (permeate) removal are continuous and a constant volume is maintained in the reactor. In such cell recycle systems, cell concentrations of over 100 g/L can be achieved. However, to keep the cells productive, a small bleed should be withdrawn (<10% of dilution rate) from the bioreactor.

Degradation of liquid polyether-based polyurethane compounds in the medium can be confirmed, e.g., by measuring the weight loss of polyether-based polyurethane compounds provided for degradation, by measuring the amount of residual polyether- based polyurethane compounds by high performance liquid chromatography (HPLC), by measuring the generation of diamine compounds (urethane bond hydrolysis products) or by measuring the vibration of ether bond by attenuated total reflection coupled to Fourier- transform infrared spectroscopy (ATR-FTIR). The generation of diamine compounds can be confirmed, e.g., by thin-layer chromatography using, as standard substances, diamine compounds expected to be generated, or by gas chromatography.

As a consequence of degrading liquid polyether-based polyurethane by the microorganism of the invention (or the composition comprising it), a higher amount of amino acids is produced in comparison to the amount of amino acids detected when the same microorganism is fed with only glucose (see Table 6, Example 5). Thus, in a particular embodiment of the use of the invention, the degradation of liquid polyether- based polyurethane by the microorganism of the invention gives rise to the production of amino acids, specifically, to an increase in the production of amino acids with respect to the degradation of sugar by the microorganism. Any amino acid, or their derivatives, can be produced by the microorganism of the invention. Nevertheless, in a more particular embodiment, the amino acids are selected from the group consisting of phenylalanine, tyrosine, tryptophan, methionine, pyroglutamic acid, lysine and any combination thereof. In a particular embodiment, the amino acids produced by the microorganism of the invention are phenylalanine, tyrosine, tryptophan, and methionine.

As the skilled person in the art understands, analogously to the above use, the present invention also provides a method for degrading liquid polyether-based polyurethane comprising putting into contact a composition comprising a rod-shaped, aerobic, Gram- positive microorganism (or the composition comprising it) with liquid polyether-based polyurethane. All the definitions and particular embodiments disclosed above for the first use of the invention apply to the present method.

The present invention also encompasses the composition obtained by using the microorganism of the invention for degrading liquid polyether-based polyurethane.

Thus, in another aspect, the present invention relates to

(i) a composition obtained from the degradation of liquid polyether-based polyurethane by a rod-shaped, aerobic, Gram-positive microorganism (or by the composition comprising it), or to (ii) a composition obtained by a method comprising culturing a composition comprising a rod-shaped, aerobic, Gram-positive microorganism in the presence of liquid polyether- based polyurethane.

Hereinafter, the composition obtained from the degradation of liquid polyether-based polyurethane by using a rod-shaped, aerobic, Gram-positive microorganism (microorganism of the invention), or by using a composition comprising it, is called“final composition of the invention”. The final composition of the invention comprises, further to the microorganism of the invention (biomass), the products resulting from the degradation of liquid polyether-based polyurethane. Examples of said products include, without limiting to, amino acids, nucleotides, vitamins, and organic acids. In a particular embodiment, the final composition of the invention comprises phenylalanine, tyrosine, tryptophan, and methionine.

Thus, in a particular embodiment, the composition is Biomass comprising a rod-shaped, aerobic, Gram-positive microorganism. In a more particular embodiment, the biomass coming from the final composition of the invention comprises - C. camporealensis, preferably, C. camporealensis strain CECT4897 (deposit number under Budapest Treaty CECT 9947),

- C. striatum, preferably, C. striatum strain LMG19648 (deposit number under Budapest Treaty CECT 9948),

- C. glutamicum, preferably, C. glutamicum strain CECT79 (deposit number under Budapest Treaty CECT 9946),

- C. ammoniagenes,

- C. flavescens,

- B. subtilis, preferably, B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887) or B. subtilis strain BPL83 (deposit number under Budapest treaty DSM 32886),

- B. licheniformis, preferably, B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888) and/or

- B. amyloliquefaciens.

Furthermore, the biomass coming from the final composition of the invention can be used as animal feeding after washing it with any polar solvent non-toxic for animals, such as water or ethanol 70% (v/v) at room temperature. The final composition of the invention may be obtained by culturing the composition comprising the microorganism of the invention and the liquid polyether-based polyurethane in a bioreactor. The degradation reaction of polyether-based polyurethane can be carried out in the different bioreactors suitable for the amino acid production. Examples of bioreactors have been disclosed previously in the present description.

As explained at the beginning of the present invention, as a consequence of the degradation of liquid polyurethane by the microorganism of the invention, the amount of amino acids produced by the microorganism is increased. This fact is applicable to any kind of liquid polyurethane, i.e. both polyether-based polyurethane and polyester-based polyurethane. Therefore, in another aspect, the present invention relates to the use of a composition comprising the microorganism of the invention for producing amino acids using liquid polyurethane, hereinafter“second use of the invention”.

The terms“composition”,“microorganism of the invention” and“polyurethane” have been explained in previous paragraphs of the present description regarding the first use of the invention, and they are applicable to the second use of the invention.

In a particular embodiment of the second use of the invention, the microorganism of the invention is selected from the list consisting of Corynebacterium sp. and Bacillus sp., more preferably, the microorganism of Corynebacterium sp. is selected from the list consisting of C. camporealensis, C. striatum, C. xerosis, C. glutamicum, C. ammoniagenes, and C. flavescens, and the microorganism of Bacillus sp. is selected from the list consisting of B. subtiiis, B. licheniformis and B. amyloliquefaciens.

Even in a more particular embodiment, C. camporealensis is C. camporealensis strain CECT4897 (deposit number under Budapest Treaty CECT 9947); C. striatum is C. striatum strain LMG19648 (deposit number under Budapest Treaty CECT 9948), C. glutamicum is C. glutamicum strain CECT79 (deposit number under Budapest Treaty CECT 9946), B. subtiiis is B. subtiiis strain BPL84 (deposit number under Budapest treaty DSM32887) or B. subtiiis strain BPL83 (deposit number under Budapest Treaty DSM32886) and B. licheniformis is B. licheniformis strain BPL89 (deposit number under Budapest treaty DSM32888).

The composition may comprise other microorganisms, apart from the microorganism of the invention, useful for degrading liquid polyurethane or other compounds which can be together with the polyurethane. In this case, the composition may comprise a consortium of microorganisms.

In a particular embodiment of the second use of the invention, the composition further comprises the combination of two or more microorganisms selected from the group consisting of C. camporealensis, C. striatum, C. xerosis, C. glutamicum, C. ammoniagenes, C. fiavescens, C. afermentans, C. ammoniagenes, C. amycolatum, C. callunae, C. efficiens, C. fiavescens, Corynebacterium hansenii, C. humireducens, B. subtilis, B. licheniformis, B. amyloliquefaciens, B. amylolyticus, B. ciriculans, B. clausii, B. coagulans, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. stearothermophilus, Bacillus subtilis, Bacillus thuringiensis.

Preferably, the microorganisms of Corynebacterium sp. are selected from the list consisting of

- C. camporealensis, preferably, C. camporealensis strain CECT4897 (deposit number under Budapest Treaty CECT 9947),

- C. striatum, preferably, C. striatum strain LMG19648 (deposit number under Budapest Treaty CECT 9948),

- C. glutamicum, preferably, C. glutamicum strain CECT79 (deposit number under Budapest Treaty CECT 9946),

- C. ammoniagenes and

- C. fiavescens·, and/or

the microorganisms of Bacillus sp. are selected from the list consisting of

- B. subtilis, preferably, B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887) or B. subtilis strain BPL83 (deposit number under Budapest treaty DSM 32886),

- B. licheniformis, preferably, B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888) and

- B. amyloliquefaciens.

Within the context of the present invention, it is also encompassed a composition comprising any combination of the above-mentioned microorganisms. In a more particular embodiment, the composition simultaneously comprises the microorganisms Corynebacterium sp. and Bacillus sp. as those disclosed above. In order to produce amino acids, the microorganism of the invention, or the composition of the invention as defined above, can use both liquid polyether-based polyurethane and liquid polyester-based polyurethane. Thus, in a particular embodiment of the second use of the invention, the polyurethane is selected from liquid polyether-based polyurethane and liquid polyester-based polyurethane.

In another more particular embodiment, the liquid polyether-based polyurethane is liquefied polyether-based polyurethane, more preferably, liquefied polyether-based polyurethane foam. The terms liquid polyether-based polyurethane, liquefied polyether- based polyurethane and liquefied polyether-based polyurethane polyurethane foam, have been defined in previous paragraphs. The inventors have found that the liquefied polyether-based polyurethane comprises a non-volatile polymeric fraction and a volatile polymeric fraction, and that the volatile polymeric fraction comprises mainly monopropylene glycol (MPG). Without intending to be linked to any theory, it is believed that the microorganisms are capable of using the volatile polymeric fraction alone, particularly MPG, for producing amino acids.

As use herein, the term“liquid polyester-based polyurethane” refers to polyurethane in liquid state comprising ester groups in their molecule. In the context of the present invention, the terms“ester-based polyurethane”,“poly(ester-urethane)”, and“ester-based polyurethane” are equivalent and can be used interchangeably throughout the present description. The liquid polyester-based polyurethane is incorporated in the formulation of coatings, adhesives, sealants and elastomers for what are called CASE applications/marketplace. Thus, in the context of the present invention, the term“liquid polyester-based polyurethane” also encompasses the above-mentioned formulations.

In another particular embodiment, the liquid polyester-based polyurethane is liquefied polyester-based polyurethane. In another particular embodiment, the liquid polyester- based polyurethane is used as a component for polyurethane applications, such as polyester-based polyurethane varnishes, coatings, adhesives, sealants and elastomers.

As used herein, the term“liquefied polyester-based polyurethane” refers to the resulting liquid after a chemical depolymerisation treatment (chemolysis) has been applied for recycling material containing polyester-based polyurethanes. Water (hydrolysis), glycols (glycolysis), acids (acidolysis) and amines (aminolysis) typically serve as reagents to break the urethane bonds. The resulting liquid can be used as such, or the individual components separated to feed the microorganisms of this invention.

In another more particular embodiment, the liquefied polyester-based polyurethane is liquefied polyester-based polyurethane foam (rigid or flexible foams).

As used herein, the term “liquefied polyester-based polyurethane foam” refers to the resulting liquid after a chemical depolymerisation treatment (chemolysis) has been applied for recycling polyester-based polyurethanes foams. Water (hydrolysis), glycols (glycolysis), acids (acidolysis) and amines (aminolysis) typically serve as reagents to break the urethane bonds. The resulting liquid can be used as such, or the individual components separated to feed the microorganisms of this invention. The inventors have found that the liquefied polyester-based polyurethane comprises a non-volatile polymeric fraction and a volatile polymeric fraction, and that the volatile polymeric fraction comprises mainly monopropylene glycol (MPG). Without intending to be linked to any theory, it is believed that the microorganisms are capable of using the volatile polymeric fraction alone, particularly MPG, for producing amino acids.

Resulting from the action of the microorganism of the invention, high amount of amino acids is produced. Thus, in another particular embodiment, the amino acids are selected from the group consisting of phenylalanine, tyrosine, tryptophan, methionine, pyroglutamic acid, lysine and any combination thereof. For the quantification of amino acids, several analytical methods can be used. Examples of analytical methods include, without being limited to, HPLC-ELSD without derivatization; Bridge-it(R) L-Methionine Fluorescence Assay Kit, colorimetric test, HPLC with samples derivatized with Edman reagent. Preferably, the method is HPLC with samples derivatized with Edman reagent.

When putting into practice the present invention, the composition which is going to be used for degrading polyurethane (i.e. composition comprising a rod-shaped, aerobic, Gram-positive microorganism as defined in previous paragraphs throughout the present description) may be forming part of a kit. Thus, in a particular embodiment, the invention relates to the use of a composition comprising a rod-shaped, aerobic, Gram-positive microorganism for the degradation of liquid polyether-based polyurethane or for producing amino acids using polyurethane, wherein the composition is comprised in a kit. As used herein, the term “kit” refers to a product containing the different reagents necessary to carry out the invention packaged allowing transport and storage. Suitable materials for packaging kit components include glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like. Additionally, kits of the invention may contain instructions for the use of the different components found in the kit use. Such instructions may be in the form of printed material or in the form of an electronic device capable of storing instructions so that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD- ROM, DVD) and the like. In addition or alternatively, the media can contain Internet addresses that provide such instructions.

In another aspect, the present invention relates to the use of a kit comprising a composition comprising the microorganism of the invention for degrading liquid polyether- based polyurethane, particularly, the liquid polyether-based polyurethane is liquefied polyurethane foam or liquid polyether-based polyurethane used as a component for CASE applications/marketplace.

In another aspect, the present invention relates to the use of a kit comprising a composition comprising the microorganism of the invention for producing amino acids from polyurethane, particularly, the polyurethane is liquid polyether-based polyurethane or liquid polyester-based polyurethane, more particularly, liquid polyether-based polyurethane or the polyester-based polyurethane is liquefied polyurethane foam or liquid polyether-based polyurethane used as a component for CASE applications/marketplace.

All the particular embodiments disclosed for previous inventive aspects are applicable to the present aspect, as well as all the definitions of the terms.

In another aspect, the present invention relates to a method for degrading of liquid polyether-based polyurethane comprising putting into contact a composition comprising a rod-shaped, aerobic, Gram-positive microorganism with liquid polyether-based polyurethane, more preferably, liquefied polyurethane foam or liquid polyether-based polyurethane used as a component for CASE applications/marketplace.

In another aspect, the present invention relates to a method for producing amino acids comprising putting into contact a composition comprising a rod-shaped, aerobic, Gram- positive microorganism with polyurethane, preferably, the polyurethane is liquid polyether- based polyurethane or liquid polyester-based polyurethane. More preferable, the liquid polyether-based polyurethane of the liquid polyester-based polyurethane is liquefied polyurethane foam or liquid polyether-based polyurethane used as a component for CASE applications/marketplace.

All the terms used in these inventive aspects, as well as additional particular embodiments, have been explained in previous paragraphs and are applicable to the present inventive aspects.

In another aspect, the present invention relates to an isolated rod-shaped, aerobic, Gram- positive microorganism selected from B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887), B. subtilis strain BPL83 (deposit number under Budapest Treaty DSM 32886), B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888), C. camporealensis strain CECT 4897 (deposit number under Budapest Treaty CECT 9947), C. striatum strain LMG 19648 (deposit number under Budapest Treaty CECT 9948), C. glutamicum strain CECT 79 (deposit number under Budapest Treaty CECT 9946) and a mutant strain thereof.

In a particular embodiment, the isolated rod-shaped, aerobic, Gram-positive microorganism mutant strain is B. subtilis strain CECT 9945.

All the terms used in these inventive aspects, as well as additional particular embodiments, have been explained in previous paragraphs and are applicable to the present inventive aspects.

In another aspect, the invention also relates to a composition comprising an isolated rod- shaped, aerobic, Gram-positive microorganism selected from B. subtilis strain BPL84 (deposit number under Budapest Treaty DSM 32887), B. subtilis strain BPL83 (deposit number under Budapest Treaty DSM 32886), B. licheniformis strain BPL89 (deposit number under Budapest Treaty DSM 32888), C. camporealensis strain CECT 4897 (deposit number under Budapest Treaty CECT 9947), C. striatum strain LMG 19648 (deposit number under Budapest Treaty CECT 9948), C. glutamicum strain CECT 79 (deposit number under Budapest Treaty CECT 9946), a mutant strain thereof, and combinations thereof, together with a carrier and/or excipient. The use of this microorganism or composition for degrading polyether-based polyurethane and/or producing amino acids has been disclosed above in previous inventive aspects. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - GPC Chromatogram of the mattress sample (POL-3).

Figure 2 - GPC Chromatogram of the POL-8 sample.

Figure 3 - Overlapping chromatogram of acydolisis liquefied polyether-based polyurethane samples PW 18171 and PC 18172.

Figure 4 - FTIR spectrum of the mattress sample (POL-3).

Figure 5 - FTIR spectrum of the sample POL-8.

Figure 6 - Comparison of the spectra of both samples: POL-3 and POL-8 sample.

Figure 7 - Spectra obtained from samples PW 18171 and PC 18172.

Figure 8 - Comparison of strains of the genus Corynebacterium.

Figure 9 - Comparison of the strains of the genus Bacillus.

Figure 10 - Production of glutamic acid with C. glutamicum ATCC13032 in the control experiment. Figure 11 - Production of glutamic acid with C. glutamicum ATCC13032 with the addition of polyol (10 g/L at 24 hours).

Figure 12 - Production of lysine with C. glutamicum ATCC 21253 in the control experiment.

Figure 13 - Production of glutamic acid with C. glutamicum ATCC13032 with the addition of polyol.

Figure 14 - Mutagenesis and selection strategy.

Figure 15 - Comparison of the mutants obtained in the first round of mutagenesis, in multiwell plate assays. The ratio of methionine produced with respect to wild (BPL83) is shown.

Figure 16 - Production of methionine by selected mutants of the first generation of B. subtilis BPL83 mutants.

Figure 17 - Viability curves at OD30.

Figure 18 - Viability curves at OD20.

Figure 19 - Viability curves at OD10.

Figure 20 - Experimental design for testing the feed intake in C. elegans.

Figure 21 - Feed Intake values obtained in C. elegans fed with B. subtilis.

Figure 22 - Relative percentage of Feed Intake obtained in C. elegans fed with B. subtilis versus a control feed (E. coli OP50).

Figure 23 - HPLC-GPC analysis of samples from an experiment for the consumption of PW 18171 liquefied polyether-based polyurethane. Experiments were carried out in fully controlled fermenters with B. subtilis strain CECT 9945. Cultures were carried out in fed- batch mode with 20 g/L glucose and 6 g/L liquefied PUR. The supernatant (Figure 23A) and precipitate (Figure 23B) fractions were analyzed separately as shown in the Figures. Dotted line represents the initial state of liquefied polyether-based polyurethane. Solid line represents the final state of the polyether-based polyurethane. The peak eluting at 1 1 minutes corresponds to the polystyrene (Mw 597,500 g/mol) internal standard added to each sample. The percentage of degradation of liquefied polyether-based polyurethane in each fraction was calculated by means of the reduction in the integration area of the rest of the peaks detected. The result is shown in the Table 15.

Figure 24 - HPLC-GPC analysis of samples from an experiment for the consumption of PC 18172 liquefied polyether-based polyurethane. Experiments were carried out in fully controlled fermenters with B. subtilis strain CECT 9945. Cultures were carried out in fed- batch mode with 20 g/L glucose and 6 g/L liquefied PUR. The supernatant (Figure 24A) and precipitate (Figure 24B) fractions were analyzed separately as shown in the Figures. Dotted line represents the initial state of liquefied polyether-based polyurethane. Solid line represents the final state of the polyether-based polyurethane. The peak eluting at 1 1 minutes corresponds to the polystyrene (Mw 597,500 g/mol) internal standard added to each sample. The percentage of degradation of liquefied polyether-based polyurethane in each fraction was calculated by means of the reduction in the integration area of the rest of the peaks detected. The result is shown in the Table 15.

EXAMPLES

Example 1. Screening of microorganisms growing on liquefied polyether-based polyurethane as single carbon source

The objective of this example is to identify microorganisms capable of growing on liquefied PU as single carbon source.

Materials and methods

A range of representative liquefied polyether-based polyurethane (PU-ether) foams was selected. These raw materials were chosen attending to their origin, processing method and availability (Table 1 ).

Table 1. List of liquefied polyether-based polyurethane foams used for microbial growth

Name Origin Process

POL-1 POL-3, POL-4, POL-5, POL-6, POL-7, POL-8 Mixture POL-2 POL-3, POL-8 (PU-ether mattresses) Mixture (1 :1 v/v)

POL-3 PU-ether foam from landfill mattresses Glycolysis (DEG 1 :1.2v/v)

POL-4 PU-ether car seat foam from pilot plant Glycolysis (DEG 1 :1.2 v/v) POL-5 PU-ether foam block from reactor Glycolysis (DEG 1 :1.2 v/v)

POL-6 PU-ether foam block from chemical laboratory Glycolysis (DEG 1 :1.3 v/v) POL-7 PU-ether car seat foam from reactor Glycolysis (DEG 1 :1.2 v/v) POL-8 PU-ether foam from mattresses Acidolysis (DEG 1 :1 v/v)

All the compositions (POL-1 to POL-8) comprised DEG (diethyleneglycol), recycled polyol, amines, and pigments in different rates. POL-4 and POL-7 further comprised waxes. In POL-5 and POL-6, the pigments rate was higher than in the rest of foams.

The compositions POL-3 to POL-7 were obtained liquefying grinded PU-ether from different origins, and subjecting them to a glycolysis process with diethyleneglycol (DEG) in different amounts, as shown in Table 1. The composition POL-3 was additionally vacuum distilled and press filtered to obtain the liquefied product.

• POL-3: Glycosylated of PU-ether obtained from landfill mattresses in a rate 1 :1.2 (DEG: grinded landfill mattresses PU-ether).

• POL-4: Glycosylated of PU-ether obtained from 2 kg of grinded car seats PU-ether in a 5 L pilot plant reactor in a rate 1 :1.2 (DEG: PU-ether).

• POL-5: Glycosylated of PU-ether obtained from 900 kg of grinded flexible foam in a 2,500 L reactor in a rate 1 :1.2 (DEG: grinded PU-ether block foam). · POL-6: Glycosylated of PU-ether obtained from 500 g of grinded PU-ether foam in a 1

L chemical laboratory reactor in a rate 1 :1.3 (DEG: grinded PU-ether block foam).

• POL-7: Glycosylated of PU-ether obtained from 900 kg of grinded car seats PU-ether in a 2,500 L reactor, in a rate 1 :1.2 (DEG: grinded PU-ether car seats foam).

• POL-8 is poly (ether-ester alcohols) and polyureas based on PU flexible foams, having the following composition: Polyurethane-polyurea oligomer (35-65%), polyether polyol (20-40%) and diethyleneglycol (1-10%), and was obtained by subjecting a sample of PU-ether mattress to an acidolysis process in the presence of basic polyol and a mixture of carboxylic acids.

The elemental CHNS analysis of two of the compositions are shown in Table 2.

Table 2. CHNS analysis of POL-3 and POL-8 samples (%).

Note: each sample analysis was done in triplicates.

The acidolysis and glycolysis processes are widely known in the state of the art and are routine practice for the skilled person. More details about the acidolysis and glycolysis processes can be found in Simon, D., et al. 2018, Recycling of polyurethanes from laboratory to industry, a journey towards the sustainability. Waste Management, https://doi.Org/10.1016/j.wasman.2018.03.041 ; Sottysihski, M. et al. Polymery 2018, 63(3): 234-238; Motokucho, S et al. J. APPL. POLYM. SCI. 2017, 2017. DOI: 10.1002/APP.45897; and Datta, J. & Kopczyhska, P., 2016, Critical Reviews in Environmental Science and Technology, 46 (10): 905-946.

A collection of microbial cultures was assembled to evaluate the capacity of selected microorganisms to grow on the different liquefied polyurethane mixtures shown above as single carbon sources (Table 3 below). The microorganisms were activated in a reference medium and grown under optimum temperature and oxygenation conditions for each particular species. With these cultures, the working cell bank was prepared and stored in glycerine stocks at -80°C.

The microorganisms were culture in solid minimal medium M9, using as substrate 1 g/L of the different liquefied polyurethane mixtures (disclosed in Table 1 above), and glucose, polyethylene glycol and polypropylene glycol as reference control substrates. M9 medium was prepared as follows: Glucose 1 g/L, Na ₂ HP0 ₄ 6.8 g/L, KH2PO4 3 g/L, NaCI 0.5 g/L, NH4CI 1 g/L, MgS0 ₄ 0.24 g/L, CaCh 0.01 1 g/L, 2.5 mL of trace element solution (FeCI ₃ -6H ₂ 0 27 g/L, ZnCI ₂ -4H ₂ 0 2 g/L, CoCI ₂ -6H ₂ 0 2 g/L, Na ₂ Mo0 ₄ -2H ₂ 0 2 g/L, CaCI ₂ -2H ₂ 0 1 g/L, CuCI ₂ -6H ₂ 0 1.3 g/L, H3BO3 0.5 g/l, HCI cone. 100 mL/L) and 100 pg/L biotin. Base medium was autoclaved for 20 minutes at 121°C to further add MgS0 ₄ , CaCh and trace element solution that were sterilized separately. Glucose was substituted by 1 g/L of substrates described above (Table 1 above) whenever the only carbon source used was the polyurethane glycosylates.

Seed cultures of different microorganisms were standardized by growing preinocula in Brain Heart Infusion media (BHI), inoculating the final culture at initial ODeoo _nm 0.1 and incubating at optimal temperature conditions for each species and 150 rpm with orbital shaking. Each strain was grown on solid media plates, prepared by streaking seed cultures onto media plates and incubating them in optimal conditions for each species. The strains showing consistent growth in the presence of each substrate, were selected for further analysis (T able 3 below).

Results

After 48 hours of incubation, growth was analyzed. Qualitative evaluation of growth is depicted as follows: (-) no growth observed, (+) few colonies observed, (++) colonies observed in more than 48 hours, (+++) colonies observed in less than 48 hours.

Table 3. Candidate strains for the degradation of liquefied polyether-based polyurethane mixtures

In the above table (Table 3) it can be seen that all the rod-shaped, aerobic, Gram-positive microorganisms were capable, in different degrees, of growing in the presence of polyurethane, while those microorganisms not showing said features did not grow. The Gram-negative bacteria analyzed in this experiment did not show growth in plates with the different liquefied polyurethane foams employed as carbon source. The species that showed the fastest growth, among Gram-positive bacteria, were B. subtilis and C. camporealensis. Other Bacillus and Corynebacterium species show a moderate rate of growth.

Example 2. Microbial degradation of liquefied polyether-based polyurethane mixtures

The objective of this experiment was to quantify the microbial degradation of the liquefied polyurethane mixtures. For that purpose, the fastest growing strains were selected and their capacity of degradation of liquefied PU-ether mixtures was further evaluated in liquid cultures

Materials and methods

Selected microorganisms belonging to the species Bacillus amyloliquefaciens, Bacillus

A representative mixture of liquefied polyurethane (POL-2) was assayed as a substrate, to evaluate the degradation capacity of these microorganisms. Strains were cultured in flasks, in 100 mL of minimal medium M9, formulated as previously explained (see Example 1 above), containing POL-2 as sole carbon source at 1 g/L concentration. Seed cultures of different microorganisms were standardized by growing preinocula in Brain Heart Infusion media (BHI), inoculating the final culture at initial OD ₆ oonm 0.1 and incubating at optimal temperature conditions for each species and 150 rpm with orbital shaking. Cultures were incubated for up to 10 days and samples were withdrawn periodically.

Different markers were evaluated to quantify substrate consumption. Degradation of the substrate was quantified as the disappearance of liquefied polyurethane characteristic functional groups, measured by means of Fourier transform infrared spectroscopy (FTIR) as ether bond characteristic stretching bands. Characteristic ether stretching bands at 1080, 1100, 1374, 1346, 1297 cm ¹ are present when analyzing all polyol-ether containing substrates and its intensity decreases with substrate consumption. 1374, 1346 and 1297 cm ¹ bands were chosen for the quantification of decrease of the mean intensity (area) of the peak corresponding to the ether bonds in the substrate associated to liquefied polyurethane degradation. Ether bonds were identified using a spectrometer Tensor 27 FTIR from Bruker (Kalrsruhe, Germany) equipped with detector DLaTGS and an ATR module for liquid samples DuraSampllR from Smiths Detections (London, UK). Spectra were obtained by accumulation of 50 scans at resolution of 4 cm ¹ and scan speed of 10 kHz from 4,000 to 550 cm-1. Instrumental control and process was done using OPUS 6.5 program from Bruker. Colony forming unit (CFU/mL) counting method is disclosed in Goldman, Emanuel; Green, Lorrence H, 2008 (Practical Handbook (Second ed.) USA: CRC Press, Taylor and Francis Group p. 864. ISBN 978-0-8493-9365-5).

Results

The results of the analysis of the performance of the strains are summarized in Table 4. Viable cell counts at final time point are shown.

Table 4. Microbial degradation of liquefied polyether-based polyurethane mixtures

All the selected strains show certain degree of ether bond degradation, with C. camporealensis CECT 4897 and B. subtilis BPL84 standing out as the best degraders. Bacillus species shows higher growth, according to CFU/mL counting.

Example 3 - Consumption of polyether-based polyurethane by strains of the genus Corynebacterium and Bacillus.

Analysis of raw materials

The following are the analyses carried out on the fermentation substrate which is a 50:50 mixture of polyether-based polyurethane shakes obtained by glycolysis, called POL- mattress (POL-3) (liquefied polyether-based polyurethane from mattress foam) and POL-8 (liquefied polyether-based polyurethane from used mattress foam). Subsequently the acidolysis liquefied polyether-based polyurethane substrates PW 18171 and PC 18172 were included. These substrates were obtained as follows:

First, polyether-based polyurethane waste was broken down into small pieces of about 5 cm in a slicer for the production of recycling polyols. These were continuously introduced into a depressurised reaction container, in which there are already process reagents as basic substances depending on the type of residual substances, namely polyol, glycol or carboxylic acid, as well as catalysts and deaminating agents. At temperatures of about 200 degrees centigrade and with constant stirring, the PUR molecular chains were split. After the completion of the reaction process (lasting about 7 hours), the resultant liquid, which is a mixture of polyols and low-molecule urethane, was filtered, obtaining liquefied polyether-based polyurethane.

After this, the analyses which were carried out are the following:

- Elemental analysis CHNS

- Gel Permeability Chromatography GPC - Fourier Transform Infrared Spectroscopy

- HPLC Analysis

A CE Instrument CHNS1100 Elemental Analyzer was used. The result is the percentage of each element by weight (triplicate analysis, Table 5):

Table 5. Elemental analysis (%) of acidolysis liquefied polyether-based polyurethane.

Note: each sample analysis was done in triplicates.

The rest of the content can be oxygen and other elements that are not seen with this technique.

Determination of molecular weight by GPC

For the study of the molecular weight distribution of the samples, high performance gel permeation chromatography (GPC) was used to obtain the mean molecular weight (Mw) and the polidispersity index (Mw/Mn).

[Mn is the numerical average molecular weight, it is the statistical average molecular weight of the polymer chains; Mw is the average molecular weight, it takes into account the contribution of the molecular weight of a chain to the average molecular weight].

A Jasco LC Net ll/ADC chromatograph equipped with a refractive index detector (RI-2031 Plus) was used. Two PolarGel-M series columns (300mm x 7.5mm) were used. The calibration was performed with polystyrene standards (Sigma-Aldrich) in the range of 62500-266 g/mol.

The results of the analyses are shown in Table 6. It can be seen that the POL-8 sample has a much lower average molecular weight than the mattress sample. However, the polydispersity of this sample has increased significantly. This demonstrates the existence of fractions which, although low in molecular weight, differ greatly from each other resulting in a much higher polidispersity index. Table 6. Molecular weight, average mass (Mw) and average number (Mn) and polidispersity index (Mw/Mn) of liquefied polyether-based polyurethane samples

Mw (g/mol) Mn (g/mol) Mw/Mn

POL-3 6076 4891 1.2422

POLS 2011 537 3.7451

Both samples share a first signal at a molecular weight of 6000 g/mol and only in the case of the POL-8 sample signals at lower molecular weights appear. This is reflected in the chromatograms shown in Figures 1 and 2. The analysis of the liquefied polyether-based polyurethane samples obtained by acidolysis is shown in Table 7 and Figure 3.

Table 7 shows the average molecular weights in weight and number (Mw, Mn) and the polidispersity index (Mw/Mn) of both polyether-based polyurethane acidolysis samples.

Sample M _n M _w M _w /M _n

Both samples have been found to be very similar to each other, although as shown by the data shown in Table 7, the average molecular weights in weight and number (Mw and Mn) as well as the polydispersity index (Mw/Mn) of both samples are slightly different, being higher for sample PC 18172. This can be observed in the chromatogram (Figure 3) where it can be seen that the retention time of sample PC 18172 is slightly lower than sample PW 18171.

Chemical structure analysis using FTIR.

The FTIR spectrographic analysis of the two separate samples was performed with a PerkinElmer Spectrum Two FT-IR spectrophotometer. The spectrum was recorded in the range of 4000 to 600 cm ¹ with 32 scanners and a resolution of 16 cm ¹ . The spectra of each compound are shown in Figures 4 and 5, and have been superimposed in Figure 6. The band assignment is shown in Table 8.

Table 8. Wave number and band assignment of the sample spectra

It can be observed that the spectra referring to both samples have many bands in common, so their chemical structure does not differ excessively. Among the common bands, the band at 1090 cm-1 , which could be attributed to the torsion of the N-H group, in a primary amine, should be highlighted. However, new bands appear in sample POL-8. The two most characteristic bands that appear in the POL-8 sample spectrum and that do not appear in the mattress sample are at 1722 and 717 cm ¹ . The first band could be assigned to the elongation of the C=0 group in aliphatic ketones while the second band could be due to the presence of a saturated aliphatic chain (presence of CH2). It should also be noted that the band at 3450 cm ¹ is much wider and more pronounced in the case of sample POL-8, indicating a higher proportion of the amine group.

Other differences, although much more subtle, are found in the 1525 and 1237 cm ¹ bands. The first band would correspond to N-0 vibration while the second could be due to aromatic C-0 elongation.

All these bands could vary and be assigned to other links and organic groups, since the same signal can be due to different structures. In this case, on the basis that the samples contained amines, the signals have been assigned to the groups and links shown in Table 8.

A way of seeing polyol consumption/degradation in the samples has been to see a decrease in the signal corresponding to the ether bond. The FTIR analysis of the liquefied polyether-based polyurethane samples obtained by acidolysis is shown below. Figure 7 and Table 9 show respectively the spectrum obtained from samples PC18171 and PW18172and the assignment and wave number corresponding to each band of the spectrogram.

Table 9. Wave number and assignment of the bands of the spectra of the sample

Band number Wave number (cm ¹ ) Assignment

No differences are observed between the spectra of sample acydolisis polyether-based polyurethane PW 18171 and PC 18172. The intensity of the bands at 1098 cm ¹ associated with the group (C-O) and those of the bands at a wavelength of 2971 cm ¹ and 2870 cm ¹ associated with the vibration of the methylene and methyl groups should be noted. Peaks 8 (1300 cm ¹⁾ and 9 (1251 cm ¹ ) indicate the presence of phosphorus compounds in the samples, while peaks 1 (3315 cm ^-1 ) and 5 (1523 cm ^-1 ) indicate the presence of nitrogen compounds.

Conversion of liquefied polyether-based polyurethane to methionine

The culture medium in general has been M9 (Table 10), on which modifications have been made, depending on the results found. The tests in this section have been carried out generally in flasks of 100-250 ml_. The results are summarized below.

Table 10. Composition of the basal medium M9 ^* . Trace element: FeCl ₃ -6H ₂ 0 27 g/L, ZnCI ₂ -4H ₂ 0 2 g/L, CoCI ₂ -6H ₂ 0 2 g/L, Na ₂ Mo0 ₄ -2H ₂ 0 2 g/L, CaCI ₂ -2H ₂ 0 1 g/L,

CUCI ₂ -6H ₂ 0 1.3 g/L, H3BO3 0.5 g/l, HCI concentration 100 mL/L.

For the quantification of methionine and other amino acids several analytical methods were evaluated and developed:

^■ HPLC-ELSD without derivatization: discarded due to low sensitivity to methionine ■ Bridge-lt® L-Methionine (L-Met) Fluorescence Assay Kit, 96-well format

(Mediomics). It is based on the increased affinity of the MetJ repressor to its DNA binding site in the presence of S-adenosyl methionine (SAM). When the complex is formed it releases fluorescence, proportional to the amount of SAM. L-Met is converted to SAM by S-adenosyl methionine synthetase in the presence of ATP and Mg2+. It is discarded because it is limited in the number of samples and only methionine is measured.

^■ Colorimetric test: Based on the formation of a coloured iron-methionine complex when it reacts with sodium nitroprusside after acidification. It was discarded because the polyol culture medium interfered.

■ HPLC with samples derivatized with Edman reagent. PDA Detector. It is the selected method, since it has been possible to transfer to UPLC, decreasing the analysis time and improving the resolution of several amino acids (methionine, tyrosine, phenylalanine, lysine, threonine and tryptophan). The result is shown in Figure 8 for Corynebacterium and Figure 9 for Bacillus. In this case, the strains were grown with M9 and 1 g/L of liquefied polyether-based polyurethane. C. glutamicum CECT79 (deposit number under Budapest Treaty CECT 9946) and B. licheniformis 3C3 (renamed BPL89, deposit number under Budapest Treaty DSM 32888) and B. subtilis (RP1801 , renamed BPL83, deposit number under Budapest treaty DSM 32886) were selected as the best producers. Also noteworthy is the result obtained with C. striatum xerosis 44363 and C. striatum xerosis 20170.

Substrate degradation was quantified as the disappearance of functional groups characteristic of liquefied polyurethane, measured by Fourier Transform Infrared Spectroscopy (FTIR) as stretching bands characteristic of the ether bond. The characteristic stretching bands of ether at 1080, 1 100, 1374, 1346, 1297 cm ¹ are present when analysing all substrates containing polyether and their intensity decreases with substrate consumption. The bands 1374, 1346 and 1297 cm ¹ were chosen for quantification of the decrease in mean peak intensity (area) of the ether bonds in the substrate associated with the degradation of liquefied polyurethane. The ether bonds were identified using a Bruker Tensor 27 FTIR spectrometer (Kalrsruhe, Germany) equipped with a DLaTGS detector and an ATR module for DuraSampllR liquid samples from Smiths Detections (London, UK). Spectra were obtained by accumulating 50 scans with a resolution of 4 cnr1 and a scanning speed of 10 kHz of 4,000 to 550 cm ¹ . Bruker's OPUS 6.5 program was used for monitoring and instrument processing. Counting of colony forming unit is performed by plating dilutions of the culture in BHI medium. Dilutions are made in NaCI 0.9% (w/v) solution. After 24 hours incubation colony forming units in at the most suitable dilution are counted.

The results of the strain behavior analysis are summarized in Table 1 1. Viable cell counts at the endpoint are shown.

Table 11. Percentage of degradation of liquefied PUR-ether. ( ) ^* Deposit number under Budapest Treaty.

All the selected strains show some degree of degradation of the ether bond, with C. camporealensis CECT 4897 (deposit number under Budapest Treaty CECT 9947) and B. subtilis BPL84 (deposit number under Budapest Treaty DSM 32887) standing out as the best degraders. Bacillus species show higher growth according to CFU/mL count.

Example 4. Production of amino acids by liquefied polyurethane degrading strains

The strains showing highest percentages of degradation were selected to evaluate the metabolites resulting from substrate consumption.

Materials and methods

Seed cultures of different microorganisms were standardized by growing preinocula in Brain Heart Infusion media (BHI), inoculating the final culture at initial ODeoo _nm 0.1 and incubating at optimal temperature conditions for each species and 150 rpm with orbital shaking. Seed cultures were used to inoculate 100 ml. of minimal medium M9, formulated as previously explained (see Example 1 ), containing POL-2 as sole carbon source at 1 g/L concentration. Cultures were incubated in optimal temperature conditions for each species and samples were withdrawn periodically. After 72 hours, whole cultures were lysed mechanically in cycles of 10 minutes in a laboratory sonifier. The resulting cell extract was filtered by 0.24 pm and amino acids were evaluated by gas chromatography-mass spectrometry (GC-MS).

For quantification of amino acids, samples were mixed with 3 pL of internal standard (0.2 mg/ml ribitol in water) and reduced to dryness in a speed-vac. Residues following reduction were redissolved in methoxyamine hydrochloride in pyridine and derivatized for 90 minutes at 37 °C, followed by a 30 minutes treatment with 60 pL MSTFA (N-methyl-N- [trimethylsilyl]trifluoroacetamide) at 37°C. After derivatization, samples were injected in a splitless mode in a 6890 N gas chromatograph (Agilent Technologies) coupled to a Pegasus 4D TOF mass spectrometer (LECO). Gas chromatography was performed on a BPX35 (30 m ^c 0.32 mm ^c 0.25 mm) column (SGE Analytical Science Pty Ltd., Australia) with helium as carrier gas, constant flow 2 mL/minute. The liner was set at 230 °C. Oven program was 85 °C for 2 minutes, 8 °C/minute ramp until 360 °C. Mass spectra were collected at 6.25 spectra s-1 in the m/z range 35-900 and ionization energy of 70 eV. All results are given in ppm.

Results

Several amino acids were detected in the samples, among others, methionine, tyrosine, phenylalanine and tryptophan were quantified (Table 12).

Table 12. Production of certain amino acids by several liquefied polyether-based polyurethane degrading strains. Results in ppm. n.d. = not detected. () ^* Deposit number under Budapest Treaty.

Example 5. Production of amino acids from liquefied polyether-based polyurethane or glucose as substrate. The strains showing higher amounts of amino acids were selected to compare the levels of production using liquefied polyether-based polyurethane or glucose as a substrate.

Materials and Methods

As described previously, seed cultures of selected microorganisms were standardized by growing preinocula in Brain Heart Infusion media (BHI), inoculating the final culture at initial ODeoo _nm 0.1 and incubating at optimal temperature conditions for each species and 150 rpm with orbital shaking. Seed cultures were used to inoculate 100 ml. of minimal medium M9, formulated as previously explained (see Example 1 ), containing POL-2 as sole carbon source at 0.1 %, 0.5% and 1% concentration.

Cultures were incubated in optimal temperature conditions for each species and samples were withdrawn periodically. After 72 hours, whole cultures were lysed mechanically in cycles of 10 minutes in a laboratory sonicator. The resulting cell extract was filtered by 0.24 pm and amino acids were evaluated by GC-MS as previously described (see Example 3).

Results

Results are shown in Table 13. In most cases, there is a significative increase of amino acids when the microorganisms are grown in polyether-based polyurethane as the only carbon source compared to glucose. This effect is particularly clear in the case of methionine, particularly for C. glutamicum CECT79 (deposit number under Budapest Treaty CECT 9946) and B. subtilis BPL83 (deposit number under Budapest Treaty DSM 32886). Additionally, as shown in Example 1 , there is not sulphur molecules in the polyether-based polyurethane used as carbon source (POL-2 = POL-3+POL-8, see Table 1 above), which means that all the methionine produced is due to the action of the microorganisms and not to external contamination.

Table 13. Production of amino acids by several PE-PU degrading strains. Amino acid concentrations in ppm. () ^* deposit number under Budapest Treaty.

Example 6. Evaluation of liquefied polyether-based polyurethane on reference producers of amino acids. Evaluation of polyether-based polyurethane as substrate.

The objective of example is to evaluate the effect of liquefied polyether-based polyurethane in different species known as amino acid producers. The following producers were purchased from international culture collections:

- C. glutamicum ATCC 13032, glutamic acid producer

- C. glutamicum ATCC 21253, lysine producer

^■ Production of glutamic acid with C. glutamicum ATCC 13032 This strain is described in patent US5705370. The fermentation medium and protocol have been performed according to Example 4. The inoculum is an overnight culture in BHI at 30°C. The fermentation medium is composed of: 100 g/L glucose, 10 g/L (NH ₄ )2S0 ₄ , 2 g/L KH ₂ P0 ₄ , 1 g/L MgS0 ₄ , g/L FeS04, 0.01 g/L MnS0 ₄ , 500 pg/L thiamine hydrochloride, 5 pg/L biotin, 2 g/L soy peptone. The fermentation conditions are 31.5 °C, 1 ,200 rpm, 0.5 vvm, pH 7.2 (control with NH40H). At 24 hours, a pulse of 10 g/L of acydolysis liquefied polyether-based polyurethane PW 18171 was added. A control fermentation was run in parallel without liquefied polyether-based polyurethane. The analysis of the supernatant was performed by CG-FID using EX:faast derivatization and analysis system, as it was not possible to analyze this amino acid by UPLC. Figure 10 shows the production of glutamic acid with the strain in a control experiment. The fermentation with the addition of PW 18171 is shown in Figure 1 1.

The maximum glutamic acid production is similar in both examples, and it is independent of the acydolysis liquefied polyether-based polyurethane addition, as the maximum value is achieved at 16 hours, before the liquefied polyether-based polyurethane pulse. When liquefied polyether-based polyurethane is added the strain is still growing in the absence of glucose, and the degradation of glutamic acid is reduced. Further optimization of the fermentation would be required in order to study the full potential of acydolysis liquefied polyether-based polyurethane addition for the production of this amino acid.

^■ Production of Lysine with C. glutamicum ATCC 21253

The production of lysine with this strain is described in patent US579379. The culture medium and protocol was prepared according to Example 1. Inoculum was prepared as an overnight culture with 4% glucose, 2% polypeptone, 0.15% KH ₂ R0 ₄ , 0.05% K ₂ HPC>4, 0.05% MgS0 ₄ -7H ₂ 0, 50 pg/L biotin, 0.3% urea, 0.5% yeast extract, at 30°C. The fermentation medium is composed of: 10% glucose, 2% soy peptone, 0.07% KH ₂ P0 ₄ , 0.05% MgS0 ₄ , 0.3% urea, 0.5% (NH ₄ )2S0 ₄ , pH 7.5.

The batch is performed at 30°C, 1200 rpm, 0.5 vvm, pH 7.5 (NH ₄ OH control), for 48 hours. A pulse of 10 g/L of acydolisis liquefied polyether-based polyurethane PW 18171 was added at 24 hours, and the fermentation was stopped at 48 hours. A control assay was run without the addition of liquefied polyether-based polyurethane. Figure 12 shows the production of lysine with the strain in a control experiment. The fermentation with the addition of PW 18171 is shown in Figure 13.

The effect of the addition of polyether-based polyurethane for the production of lysine is similar to what happened for glutamic acid. Maximum lysine concentration is similar in both fermentations (above 50 g/L). After the addition of liquefied polyether-based polyurethane the strain keeps growing, whereas in the control experiment growth stops earlier. Example 7. Mutant strains capable of degrading liquid polyether-based polyurethane and producing methionine

Mutagenesis and selection strategy

Resistance to the methionine analogue, methionine sulfoxide, for which the minimum inhibitory concentration of about 8 g/L was found, was used. The mutagenesis was performed with an alkylating agent (EMS, ethyl methanesulphonate), and selection was made on M9 plates with a acydolysis liquefied polyether-based polyurethane concentration of 5 g/L and methionine sulfoxide of 32 g/L (to be sure to find resistant strains). The mutagen dose was set at 25 pg/mL to achieve 99.9% death after incubation of cells 1 hour at 37 °C. The strategy followed is reflected in Figure 14. About 4000 mutants were tested, of which 44 were selected for further evaluation. From a first analysis in multiwell plates, mutants 3, 4, 5, 17, 26 and 37 stood out (Figure 15), which were validated in flask and bioreactor assays.

Analysis of the mutants

After the EMS random mutagenesis on the B. subtilis BPL83 strain (deposit number under Budapest Treaty DSM 32886), the 6 most promising mutants were selected and tested under process conditions optimized at flask scale for the wild strain (Figure 16). The selected conditions are: urea enhanced medium M9, glucose 20 g/L, sequential addition of 3 g/L of polyether-based polyurethane at 24 hours, MgS0 ₄ 4 mM. The results shown in Figure 16 were confirmed with bioreactor assays, and therefore, mutant 37 was selected to continue with new rounds of mutagenesis and intensify the process. This mutant, M1- 37, was deposited under Budapest Treaty (deposit number CECT 9945).

Example 8. Biomass characterization and evaluation as animal feed.

The objective of this experiment is to know the biomass composition and evaluate if the B. subtilis M1-37 (BPL200, deposit number under Budapest Treaty CECT 9945) biomass has a positive effect in“feed intake” in the in vivo model of Cahenorhabditis elegans.

^■ Method for obtaining liquefied polyether-based polyurethane PW 18171 and PC 18172.

First, polyether-based polyurethane waste was broken down into small pieces of about 5 cm in a slicer for the production of recycling polyols. These were continuously introduced into a depressurised reaction container, in which there are already process reagents as basic substances depending on the type of residual substances, namely polyol, glycol or carboxylic acid, as well as catalysts and deaminating agents. At temperatures of about 200 degrees centigrade and with constant stirring, the PUR molecular chains were split. After the completion of the reaction process (lasting about 7 hours), the resultant liquid, which is a mixture of polyols and low-molecule urethane, was filtered, obtaining liquefied polyether-based polyurethane.

^■ Fermentation conditions

The medium used in this case is based on that described in Chen et ai., 2013. Bioprocess. Biosyst. Eng. 36: 1851-1859. The inoculum is prepared with 20 g/L glucose, 5 g/L yeast extract, 0.2 g/L MgS04, 2.5 g/L NaCI. The batch fermentation medium is composed of 40 g/L glucose, 15 g/L (NH ₄ )2SC> ₄ , 15 g/L urea, 3 g/L KH ₂ PO ₄ , 0.5 g/L MgS0 ₄ -7H ₂ 0, 0.1 g/L MnS0 ₄ -H ₂ 0. A glucose pulse was added with 30 g/L, 5 g/L of polyether-based polyurethane and 6 mM of MgS0 ₄ . The pH control is done at 6.8 with NH ₄ OH and HCI. The p0 ₂ was set at 10%, regulated in cascade. It was performed at 35°C.

^■ The compositional analysis was performed on B. subtilis M1-37 biomass, after fermentation with the conditions described above, with 10 g/L of liquefied polyether- based polyurethane PW 18171. Biomass was recovered by centrifugation and washed with ethanol 70% v/v in order to remove residual acidolysis liquefied polyether-based polyurethane. The final washing was performed with water. Biomass composition is shown in Table 14:

Table 14. Bacillus subtilis M1-37 biomass composition

^■ Study of the toxicity of B. subtilis in the model of C. elegans.

The assay used biomass from a culture of B. subtilis M1-37 grown with glucose 20 g/L and in the presence of acidolysis liquefied polyether-based polyurethane PC 18172 10 g/L. Part of the biomass obtained was washed to remove the acidolysis liquefied polyether-based polyurethane. The objective was to analyze the potential toxicity of the original and clean culture (free of acidolysis liquefied polyether-based polyurethanel, washed with hexane and later evaporation) in the model organism of C. elegans. This nematode feeds on bacteria, so it is routinely kept in the laboratory in a culture medium containing a lawn of E. coli OP50 bacteria.

The toxicity test was conducted in a robotic system called WorMotel. It is a system based on artificial image that allows quantifying the mobility of populations of worms cultivated in 96-well plates. The plate was prepared by adding 250 pl_ of medium NGM (peptone, MgS0 ₄ (1 M), Kpi buffer pH6 1 M, 1 M CaCh and low melting agar), to which cholesterol and FUDR (avoidance of progeny) were added. Once solidified, 5 mI_ of E. coli (OP50) was inoculated at DO:30 on the surface of all the wells to ensure correct feeding of the worms. The software was designed to analyze 4 quadrants of each multiwell plate independently, so the plate was divided into one quadrant for control condition (E. coli OP50) and three quadrants for treatment with B. subtilis at a defined dose (tripled). A total of 6 plates were therefore prepared, 3 for the original culture and 3 for the clean culture without acidolysis liquefied polyether-based polyurethane. Three different concentrations were tested for each type of culture: Subsequently, using a COPAS flow cytometer, a defined number of adult worms per well (previously synchronized in age) was distributed. The plates were sealed with parafilm and placed in the WorMotel storage system. For 15 days, the robot made daily image captures of the surface of the plates, and by means of an analysis software the integrity of the movement by well and condition was obtained. Finally, viability curves were obtained based on the mobility of C. elegans both in control conditions and in the different treatments with B. subtilis.

The following graphs (Figures 17 to 19) show the results obtained at the different doses. The control without the addition of Bacillus biomass is C. elegans growing with E. coli ( NG line). It is observed that at the different ODs tested (tripled), the condition of unwashed biomass affects the mobility of the nematode, and that it is proportional to the dose used. The effect of washed biomass is much less than that of unwashed biomass, and practically negligible at the lowest dose (OD10). This would indicate a possible toxic effect of acidolysis liquefied polyether-based polyurethane and that it would be necessary to include a washing stage to use the residual biomass of the process as an ingredient.

Test of Feed Intake in C. elegans with "clean" B. subtilis cells. The objective of this subtask is to evaluate the effect of B. subtilis biomass in the feed intake in the biological model of C. elegans, so that it can be considered a potential ingredient for animal feeding.

Age-synchronized worms were obtained from pregnant adults, collecting the embryos in plates in medium NGM at a temperature of 20°C. In a 96-well plate, 150 pl_ of half complete S + E. coli OP50 was added to each well. When the worms reached larval stage L1 , a total of 10 worms/well were distributed by COPAS cytometer in all rows of the plate, except for the last row containing only the medium. When the worms reached larval stage L4, FUdR (200 mM) was added to all wells to avoid offspring.

Once the worms reached the age of the young adult worm, B. subtilis (grown with glucose and acidolysis liquefied polyether-based polyurethane PW 18171 , and washed with EtOH and water) cells were added at a dose of 10 ⁴ cells/mL following the design in Figure 20. Absorbance was then measured at 595 nm for day 1. After 3 days of incubation, the absorbance measurement 595 nm for day 4 was taken. After obtaining these data, the food intake value of each well was calculated:

Food intake = ((OD600day1 - OD600day4)-Basal decay)/number of worms where the "basal decay" is calculated with the difference of AOD600 = OD600day1 - OD600day4 of the wells that do not contain worms (Figure 20).

Figure 21 shows Feed Intake values in worm populations under control conditions and treated with B. subtilis biomass. As can be seen, under control conditions an average value of 0.0102 was obtained, while with treatment with B. subtilis the value is very similar, 0.0097. Figure 22 shows the same results but represented as a relative percentage to the control condition. It can therefore be concluded that treatment with the B. subtilis strain does not produce any change in the feeding behavior of C. elegans.

Example 9: Quantification of the degradation of the polymeric fraction of liquefied polyether-based polyurethane in batch and fed-batch cultures.

Fermentation experiments were carried out in order to confirm and quantify the degradation of the polymeric components of liquefied polyether-based polyurethane by bacteria. Two samples of liquefied polyether-based polyurethane were used: PW 18171 and PC 18172.

Method for obtaining liquefied polyether-based polyurethane PW 18171 and PC 18172. First, polyether-based polyurethane waste was broken down into small pieces of about 5 cm in a slicer for the production of recycling polyols. These were continuously introduced into a depressurized reaction container, in which there are already process reagents as basic substances depending on the type of residual substances, namely polyol, glycol or carboxylic acid, as well as catalysts and deaminating agents. At temperatures of about 200 degrees centigrade and with constant stirring, the polyurethane molecular chains were split. After the completion of the reaction process (lasting about 7 hours), the resultant liquid, which is a mixture of polyols and low-molecule urethane, was filtered, obtaining liquefied polyether-based polyurethane.

Culture of microorganisms with liquefied polyether-based polyurethane PW 18171 and PC 18172.

The experiments were carried out in minimal growth medium supplemented with liquefied polyurethane as carbon source. In some experiments, glucose was also used as carbon source in order to promote the growth of the microorganisms.

Cultures were inoculated with 5% (v/v) of late exponential phase cultures of Bacillus sp. strains ( B . subtilis strain CECT 9945) grown in M9 minimal medium with glucose and liquefied polyurethane as carbon sources.

Samples were withdrawn at the end of the run from fermentation experiments. Whole samples consisted of a turbid mixture of exhausted fermentation broth, suspended bacteria and the non-consumed liquefied polyether-based polyurethane. In order to identify the effect of microorganism on the liquefied polyurethane, control samples mimicking the initial composition of the fermentation broth and liquefied polyether-based polyurethane mixtures were run in parallel.

Liquefied polyether-based polyurethane contains a mixture of diverse molecular weights components, including high molecular weight components (polymeric) and low molecular weight components. At the working concentration of experiments (6-10 g/L), liquefied polyurethane is only partially soluble in the aqueous fermentation broth. Polymeric components of the liquefied polyurethane are present both in the liquid and solid phase. For analysis, the solid and liquid phases were separated by centrifugation in a bench centrifuge at 4500 rpm, 10 minutes at 4°C.

Therefore, two phases were considered, processed and analyzed independently:

• Solid phase (precipitate): consisting of non-soluble components of the liquefied polyether-based polyurethane and microorganisms.

• Liquid phase (supernatant): consisting of soluble components of the liquefied polyurethane, components of the fermentation broth and metabolic products formed as a result of microbial activity.

Following separation of precipitate and supernatants, water from both sample types was removed by lyophilization. The resulting dry powder was resuspended with the help of sonication into an adequate volume of tetrahydrofurane (THF). A standard solution of polystyrene (Mw 597,500 g/mol, 1 1.09 mg/mL) was added to be used as internal standard. Samples were filtered through PTFE 0.45 pm filters to remove any insoluble particles and subsequently analyzed by HPLC-GPC.

Gel permeation chromatography (GPC) is a liquid chromatography technique in which analytes are separated based on their molecular size by means of the interaction with a porous stationary phase. This technique is used for the analysis of polymeric materials.

Chromatographic conditions were as follow:

• Columns: two consecutive molecular size exclusion columns PLGel Mixed C were used.

• Detection: refraction index detector at 35°C.

• Mobile phase: tetrahydrofuran (THF), isocratic elution.

Areas of the chromatographic peaks detected were integrated. The amount of polymer in each of the peaks detected was estimated by comparison to the peak area of the polystyrene internal standard. For estimation of molecular parameters of the peaks detected, a molecular weight standard curve including 11 standards ranging from 685 to 575500 g/mol was used.

Chromatograms were analyzed in order to quantify degradation of liquefied polyether- based polyurethane. The following conclusions can be withdrawn: • Most liquefied polyether-based polyurethane is present in the insoluble fraction in the samples analyzed.

• By comparing the chromatograms of initial and end-point samples, the degradation of polymeric components of the liquefied polyurethanes is shown to be very relevant.

• The degradation occurs both in the supernatant (Figure 23A y 24A) and precipitate (Figure 23B y 24B) fractions of liquefied polyurethane.

• Degradation results observed in each fraction from different experiments with varying experimental conditions are shown in Table 15. Table 15. Degradation of liquefied polyether-based polyurethane polymeric components as analyzed by HPLC-GPC of supernatant and precipitate samples. Experiments were carried out in fully controlled fermenters with various Bacillus sp. strains and conditions as shown in the table.