WO2023274937A1 - Production of biomaterials from food waste products by antarctic bacterial strains - Google Patents

Production of biomaterials from food waste products by antarctic bacterial strains Download PDF

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WO2023274937A1
WO2023274937A1 PCT/EP2022/067531 EP2022067531W WO2023274937A1 WO 2023274937 A1 WO2023274937 A1 WO 2023274937A1 EP 2022067531 W EP2022067531 W EP 2022067531W WO 2023274937 A1 WO2023274937 A1 WO 2023274937A1
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rscic
dps
access
food waste
production
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PCT/EP2022/067531
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French (fr)
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Sandra PUCCIARELLI
Rita GIOVANNETTI
Marco ZANNOTTI
Alessio Mancini
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Irides S.R.L.
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Publication of WO2023274937A1 publication Critical patent/WO2023274937A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present application concerns the field of microbiology and in particular a process for the production of biomaterials using non-genetically modified isolated and verified bacterial strains starting from food waste products.
  • Polyhydroxyalkanoates are known to be produced by bacteria, including the genera Bacillus, Rhodococcus and Pseudomonas.
  • Polyhydroxyalkanoates (PHA) are thermoplastic polyester polymers synthesised by bacteria through the fermentation of sugars or lipids. These materials are biodegradable and are used in the production of bioplastics.
  • PhaC and PhaR genes for the production of bioplastics are further known (Gabriel J. McCool, Maura C. Cannon. PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium Journal of Bacteriology Jul 2001, 183 (14) 4235-4243; DOI: 10.1128/JB.183.14.4235-4243.2001), in which the product remains trapped in the bacterium.
  • Italian patent application No. 102019000014121 describes an in vitro process for the preparation of metabolites by Marinomonas ef1 or Rhodococcus, in particular silver nanoparticles and fluorescent dyes.
  • Italian patent application No. 102020000031769 describes the use of bacterial strains Bacillus ef1 and Brevundimonas ef1 for the production of biocellulose.
  • US Patent Application No. 2012210745A1 describes the application of enzymes for the repair of plastic products and the bacterial synthesis of bioplastics.
  • US Patent No. US6022729 describes the gene isolated from Rhodococcus ruber involved in PHA biosynthesis and a method for producing PHA granules in bacteria and binding them to a protein.
  • US Patent Application No. US2014234944A1 describes the use of engineered Rhodococcus to produce PHA starting from ethanol.
  • Acyclic carotenoids with a C30 aglycone the xylosilesters of diapolycopenedioc acid A-C and methyl 5-glucosyl-5,6-dihydro- apo-4,4'-lycopenoate were isolated from the new Gram-negative bacterium Rubritalea squalenifaciens, belonging to the phylum Verrucomicrobia, as well as from the Gram-positive bacterium Planococcus maritimus belonging to the class Bacilli, phylum Firmicutes (Shindo K. and Misawa N., Mar. Drugs 2014, 12).
  • the production of hydrocarbons by bacteria is known in the art (Wackett L.P. (2010) Aliphatic Hydrocarbon Producers.
  • a method for the production of beta-carotene by said bacterial strain wherein the carbon source is carbohydrates such as glucose, sucrose, fructose, lactose, starches, purified or in mixtures containing said carbohydrates, such as corn liquor and whey, edible oils, vegetable oils such as olive oil, soybean oil, rapeseed oil, palm oil, peanut oil, canola oil and also glycerol and lipids is thus described.
  • carbohydrates such as glucose, sucrose, fructose, lactose, starches, purified or in mixtures containing said carbohydrates, such as corn liquor and whey, edible oils, vegetable oils such as olive oil, soybean oil, rapeseed oil, palm oil, peanut oil, canola oil and also glycerol and lipids
  • the US patent application, publication no. US2015/272835 describes a method for producing a carotenoid-containing composition that is a needle crystal or needle crystal having a maximum length of 5 ⁇ m or more and whose astaxanthin content based on the total composition is 40% by mass or more, wherein the method comprises: an extraction step wherein a culture obtained by culturing a carotenoid-producing microorganism including astaxanthin is extracted using one to three-carbon atom alcohol, an extract separation step wherein an extract is obtained by separating the microorganism and the extract, a concentration step wherein a concentrate is obtained by concentrating the extract 1.25 to 20 times in terms of amount of liquid, a crystallization step wherein crystals of the carotenoid-containing composition are produced in the concentrate, and a crystal separation step wherein the crystals are separated and obtained from the mother liquor, wherein the microorganism may belong to the genera Agrobacterium, Brevibacterium, Paracoccus, Brevundimonas and Erythrobacter
  • Marinomonas ef1 Bacillus ef1 and Brevundimonas ef1 are deposited at the Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna "Bruno Ubertini”, in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure, 28 April 1977.
  • the above technical problem is solved by providing the use of at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of biomaterials from food waste products, with the provision that it is not and which therefore is not an object of the present invention, Brevundimonas ef1 Access No. DPS RE RSCIC 24, for the production of carotenoids from soybean oil.
  • It is another object of the present invention a process for the production of biomaterials comprising culturing at least one strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4 Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature in the presence of culture medium added with food waste materials until the final product is obtained in the culture medium.
  • the above technical problem is further solved by providing the use of Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the simultaneous production of cellulose and carotenoids from food waste products.
  • a further object of the present invention is a process for the simultaneous synthesis of biocellulose and carotenoids including the following steps: a) culturing Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 in culture medium, or nutrient broth; b) incubation of the bacteria obtained at the end of step a) at room temperature under stirring; c) dilution of the solution obtained at the end of step b) and addition of the food waste product and incubation until the final carotenoid and cellulose product is obtained.
  • food waste materials or waste from the food process is understood to be any edible substance, either raw or cooked, that is discarded, or is intended to be discarded or needs to be discarded, as defined by the European Commission in EC Directive 2006/12/EC. Or they can be defined as any material acquired for animal or human consumption that is separated from the municipal solid waste stream and which does not fall within the definition of "agricultural material",
  • biocellulose means cellulose of natural, microbial origin characterised by fibres having variable diameter.
  • bioplastics and biopolymers are understood to be biodegradable and/or bio-based plastics and polymers (EUBIO Admin, Bioplastics, su European Bioplastics e.V; https://www.cen.eu/work/areas/chemical/biobased/Pages/default.as px).
  • culture medium suitable for bacterial growth means solid or liquid solutions containing nutrients on which it is possible to cultivate bacterial colonies.
  • the culture medium is a nutrient broth or lysogenic broth containing ingredients such as yeast extract, peptides derived from enzymatic digestion such as hydrolysed casein, which consists of a mixture of different chemical components in unknown proportions.
  • peptides derived from enzymatic digestion are a mixture of water-soluble polypeptides and amino acids derived from the partial hydrolysis of proteins, such as tryptone which is an assortment of peptides formed by the digestion of casein from trypsin protease, or proteose peptone.
  • Additives may also be employed to supplement culture media containing peptides derived from enzymatic digestion such as glycerol, yeast extract, NaCl, K 2 HPO 4 , MgSO 4 .
  • the minimum medium is a culture medium containing only the growth-supporting ingredients, in other words the minimum possible nutrients for colony growth, typically including salts, essential elements such as magnesium, nitrogen, phosphorous, sulphur and water to which a carbon source is then added.
  • the LB Medium culture medium is a lysogenic broth containing casein peptides and peptones, vitamins, trace elements and minerals, sometimes containing sodium chloride or tryptone.
  • the present invention consists of the use of at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of biomaterials from food waste products, with the provision that it is not and which therefore is not an object of the present invention, Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of carotenoids from soybean oil.
  • the inventors of the present invention have verified that the strain of Marinomonas ef1 Access No. DPS RE RSCIC 4 possesses the Fab metabolic pathway genes involved in the synthesis of PHA and genes involved in the synthesis of 2-pyrone 4,6-dicarboxylic acid.
  • SEQ. ID. NO. 1 PROKKA_02018 3-oxoacyl-[acyl-carrier-protein] synthase 3 (fabH)
  • SEQ. ID. NO. 2 PROKKA_00698 3-oxoacyl-[acyl-carrier-protein] reductase FabG
  • SEQ. ID. NO. 3 PROKKA_00804 Malonyl CoA-acyl carrier protein transacylase (FabD)
  • SEQ. ID. NO. 4 PROKKA_01991 Malonyl CoA-acyl carrier protein transacylase (FabD)
  • SEQ. ID. NO. 5 PROKKA_01992 3-oxoacyl-[acyl-carrier-protein] reductase FabG
  • SEQ. ID. NO. 6 PROKKA_03165 3-hydroxyacyl-[acyl-carrier- protein] dehydratase FabZ
  • SEQ. ID. NO. 7 PROKKA_02218 protocatechuate 4,5- dioxygenase beta chain
  • SEQ. ID. NO. 8 pROKKA protein_02219
  • SEQ. ID. NO. 9 PROKKA_02220 2-hydroxymuconate semialdehyde hydrolase
  • SEQ. ID. NO. 10 PROKKA_02288 protocatechuate 4,5- dioxygenase alpha chain
  • SEQ. ID. NO. 11 PROKKA_02289 protocatechuate 4,5- dioxygenase beta chain
  • SEQ. ID. NO. 12 PROKKA_022903-oxoadipate enol-lactonase2
  • SEQ. ID. NO. 14 PROKKA_03276 protocatechuate 4,5-dioxygenase alpha chain
  • SEQ. ID. NO. 15 PROKKA_03277 protocatechuate 4,5- dioxygenase beta chain
  • SEQ. ID. NO. 17 WP 100636466.1 bifunctional 3-hydroxydecanoyl- ACP dehydratase/trans-2-decenoyl-ACP isomerase
  • SEQ. ID. NO. 18 PROKKA_03927 3-oxoacyl-[acyl-carrier-protein] synthase 1
  • SEQ. ID. NO. 20 PROKKA_03165 3-hydroxyacyl-[acyl-carrier- protein] dehydratase FabZ
  • SEQ. ID. NO. 21 PROKKA_025414'-phosphopantetheinyl transferase psf-1
  • the bacterial strain is Marinomonas ef1 Access No. DPS RE RSCIC 4.
  • the biomaterials are selected from the group consisting of: biopolymers, bioplastics, carotenoids. More preferably the biopolymers may be paraffin-like polyunsaturated hydrocarbon polymers.
  • food waste materials or waste from the food process are food waste products containing sugars, such as for example glucose and/or fructose, food waste products waste oil comprising soybean oil.
  • a further object of the present invention is a process for the production of biomaterials comprising culturing at least one strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4 Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature in the presence of culture medium containing food waste materials until the final product is obtained in the culture medium.
  • the process for the production of biomaterials comprises the following steps: a) culturing Marinomonas ef1 at room temperature in the presence of 2% Peptone under stirring until growth to the plateau step; b) dilution of the obtained culture at the end of step a) with sea water and addition of the waste food product and subsequent incubation at room temperature under stirring until the biomaterial is obtained in the culture medium; c) collection of the product obtained at the end of step c) and washing at room temperature; d) hot washing to remove any oily residue; e) drying.
  • steps c) and d) may be repeated.
  • a step f) may follow wherein the product is dispersed in a polar solvent and subsequently centrifuged to recover the supernatant which is in turn centrifuged and dried under nitrogen atmosphere to obtain the product in powder form.
  • SEQ. ID. NO. 22 phytoene synthase
  • SEQ. ID. NO. 24 phytoene synthase
  • SEQ. ID. NO. 25 lycopene cyclase
  • a further object of the present invention is the use of Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the simultaneous production of cellulose and carotenoids from food waste products.
  • a further object of the present invention is a process for the simultaneous synthesis of biocellulose and carotenoids comprising the following steps: a) culturing Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 in culture medium, or nutrient broth; b) b) incubation of the bacteria obtained at the end of step a) at room temperature under stirring c) dilution of the solution obtained at the end of step b) and addition of the food waste product and incubation until the final carotenoid and cellulose product is obtained.
  • the nutrient broth is composed of bovine extract (1g/L), peptone (5g/L), yeast extract 2g/L, sodium chloride 5g/L).
  • the final carotenoid product is presented as a yellow/orange pigment, which is recovered by two steps of centrifugation, the first step in order to sediment the bacteria at centrifugation, and to collect the supernatant containing the pigment, preferably at lOOOxg, the second step, in order to remove the particulate preferably at lOOOOxg.
  • Cellulose is produced at a later time and is in the form of flakes or spherules of biocellulose in the culture medium.
  • the process for the production of biocellulose and carotenoids comprises: incubation of Bacillus ef1 Access no. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature under stirring in the presence of culture medium suitable for bacterial growth until biocellulose is obtained by the bacterial culture, both in static culture condition and under stirring, the second preferably at 100 rpm; then biocellulose is collected, for example with spatulas, and washed with sterile double-distilled water, then biocellulose is incubated at 80 °C to eliminate bacteria, the washing in sterile double-distilled water is repeated.
  • a biomaterial was produced with the process set out below: a) culture of Marinomonas ef1 at room temperature in the presence of 2% Peptone under stirring until plateau growth; b) dilution of the culture obtained at the end of step a) with sea water (50% vol/vol) and addition of soybean oil in an amount equal to 1% of the total and subsequent incubation at room temperature under stirring until white ovoid aggregates are obtained in the culture medium; c) the latter is collected with a spatula and transferred into a centrifuge tube for washing with sterile distilled water; d) to remove oil residues, the sample is washed with hot distilled water (approx. 80 °C) for about 5 minutes.
  • the liquid is then removed and the sample washed with new distilled water at 80°C; this process was repeated three times; e) the solid sample, separated from the aqueous phase, is subsequently dried under an inert nitrogen atmosphere; f) the dry sample is dispersed in absolute ethanol (approx. 40 ml) and subjected to ultrasonication until a homogeneous dispersion is obtained, then centrifuged at 10000g in a HERMLE - Z 323 K centrifuge and the solid precipitate, which is white in colour, is recovered; g) the latter is then dried under an inert nitrogen atmosphere.
  • the base structure of the new biomaterial is composed of at least 22 carbon atoms.
  • the signal at 35 ppm is attributable to the carbon bonded to the bromine atom, the signals in the range 127-131 ppm are attributable to the sp2 carbons and the other carbons are those within the chain; the signal at 23 ppm is related to the deuterated chloroform CDC13.
  • the HNMR analysis revealed the presence of signals at 5.4 ppm that are attributable to sp2 carbons, while the signal at 2.8 ppm is related to the proton of the carbon near a bromine atom.
  • the SEM analysis showed a lamellar structure with overlapping leaflets.step
  • the agar diffusion test showed antibiotic properties against a Pseudomonasstrain: 5 mg of the material produces an inhibition halo of about 5 mm.
  • the product obtained after various analyses turns out to be a polyunsaturated hydrocarbon similar to paraffin.
  • the nutrient broth had the following composition:
  • Beef extract and peptone provide amino acids, nitrogen, carbon, vitamins and minerals for the growth of the organisms.
  • Yeast extract is a source of vitamins, particularly B group vitamins. Sodium chloride maintains the osmotic balance of the medium.
  • bacteria incubated at 22°C at 2000 rpm rotation. Once the optical density of about 2 OD at 550 nanometres is reached, the culture is diluted with sea H20 (50% of the final volume) and added with 1.5-2% fructose, and further incubated under stirring at 22°C.
  • a yellow/orange pigment is produced after 24 hours.
  • the pigment is recovered by two centrifugation steps: Centrifugation at 1000g to sediment the bacteria and collect the supernatant containing the pigment
  • the aqueous part containing the pigment is dried under nitrogen atmosphere, redispersed in absolute Ethanol (about 40 ml) and analysed by UV-Vis and MS.
  • the pigment was found to be a mixture of B-cryptoxanthin and lycopene/B-carotene.
  • the culture is incubated at 80 °C to eliminate bacteria.
  • the material composed of flakes and spherules is collected and washed with sterile double-distilled water and then dried in an oven at 40°C.
  • a dry portion of the material is analysed with FTIR.
  • the material is biocellulose.
  • a dry portion of biocellulose is treated with absolute ethanol (approx. 40 ml) and the pigment is extracted for subsequent analysis by UV-Vis and MS.
  • the pigment was found to be a mixture of B-cryptoxanthin and lycopene/B-carotene.
  • Rale 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.

Abstract

The use of isolated and non-genetically modified bacterial strains belonging to the genera Marinomonas, Bacillus and Brevundimonas for the in vitro production of biomaterials from food waste products, wherein the biomaterials are found in the culture medium, is described.

Description

PRODUCTION OF BIOMATERIALS FROM FOOD WASTE PRODUCTS BY ANTARCTIC
BACTERIAL STRAINS
★ ★★ ★ ★
Background of the invention
The present application concerns the field of microbiology and in particular a process for the production of biomaterials using non-genetically modified isolated and verified bacterial strains starting from food waste products.
Background
Polyhydroxyalkanoates are known to be produced by bacteria, including the genera Bacillus, Rhodococcus and Pseudomonas. Polyhydroxyalkanoates (PHA) are thermoplastic polyester polymers synthesised by bacteria through the fermentation of sugars or lipids. These materials are biodegradable and are used in the production of bioplastics.
The metabolic pathways leading to the bacterial production of PHA are known, which, like other bio-materials, remain trapped within the bacterium and thus require purification processes to obtain the final product (Mozejko-Ciesielska & Kiewisz, 2016).
Bacillus strains genetically modified with PhaC and PhaR genes for the production of bioplastics are further known (Gabriel J. McCool, Maura C. Cannon. PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium Journal of Bacteriology Jul 2001, 183 (14) 4235-4243; DOI: 10.1128/JB.183.14.4235-4243.2001), in which the product remains trapped in the bacterium.
Italian patent application No. 102019000014121 describes an in vitro process for the preparation of metabolites by Marinomonas ef1 or Rhodococcus, in particular silver nanoparticles and fluorescent dyes.
Italian patent application No. 102019000024493 describes the use of Marinomonas ef1 bacterial strains for the bioremediation of water polluted by contaminants, such as fuels, toxic post transition metals and/or toxic transition metals.
Italian patent application No. 102020000031769 describes the use of bacterial strains Bacillus ef1 and Brevundimonas ef1 for the production of biocellulose.
US Patent Application No. 2012210745A1 describes the application of enzymes for the repair of plastic products and the bacterial synthesis of bioplastics.
International Patent Application No. WO2016191734A1, US Patent Application No. US2014004597A1 and US Patent No. US10421951B2 describe the biosynthesis of alcohol and biofuels by engineered bacteria.
US Patent No. US6022729 describes the gene isolated from Rhodococcus ruber involved in PHA biosynthesis and a method for producing PHA granules in bacteria and binding them to a protein.
International Patent Applications No. W02007017270, W00011188 and W02016040653 and describe the production of PHA from engineered Rhodococcus.
US Patent Application No. US2014234944A1 describes the use of engineered Rhodococcus to produce PHA starting from ethanol.
The production of carotenoids by Antarctic bacteria such as Planococcus sp.ANT_H30 and Rhodococcus sp. ANT H53B has been disclosed (Styczynski M. et al., Molecules 2020, 25, 4357; doi:10.3390/molecules25194357).
Acyclic carotenoids with a C30 aglycone, the xylosilesters of diapolycopenedioc acid A-C and methyl 5-glucosyl-5,6-dihydro- apo-4,4'-lycopenoate were isolated from the new Gram-negative bacterium Rubritalea squalenifaciens, belonging to the phylum Verrucomicrobia, as well as from the Gram-positive bacterium Planococcus maritimus belonging to the class Bacilli, phylum Firmicutes (Shindo K. and Misawa N., Mar. Drugs 2014, 12). The production of hydrocarbons by bacteria is known in the art (Wackett L.P. (2010) Aliphatic Hydrocarbon Producers. In: Timmis K.N. (eds) Handbook of Hydrocarbon and Lipid Microbiology. Springer, Berlin, Heidelberg, https://doi.org/10.1007/978-3-540- 77587-4 48); in particular, the synthesis of alkanes or alkenes by various microorganisms has been described (Wang Weihua, Lu Xuefeng, 2013, Microbial Synthesis of Alka(e)nes, Frontiers in Bioengineering and Biotechnology, 1, 1-10, DOI=10.3389/fbioe.2013.00010), however, the production of solid hydrocarbons, such as paraffins and waxes, by bacteria has not been reported in the literature.
Prior art closer to the invention
The international patent application, publication no. W02008/108674, describes naturally occurring bacterial strains that constitutively produce carotenoids, in particular beta- carotene using agricultural products and waste as raw material. Among the strains there is the genus Brevundimonas. However, only a natural strain, isolated from medium, of the genus Sphingomonas named Sphingomonas M63Y is exemplified.
A method for the production of beta-carotene by said bacterial strain wherein the carbon source is carbohydrates such as glucose, sucrose, fructose, lactose, starches, purified or in mixtures containing said carbohydrates, such as corn liquor and whey, edible oils, vegetable oils such as olive oil, soybean oil, rapeseed oil, palm oil, peanut oil, canola oil and also glycerol and lipids is thus described. As a source of nitrogen, soybean hulls, soybean flour, corn flour, yeast extract, cotton flour, peptons, casein, amino acids, ammonium sulphate, ammonium chloride, ammonium nitrate, ammonium hydroxide are envisaged. And mineral salts such as phosphates, sulphates, chlorides or molybdates of cations such as, but not limited to, sodium, potassium, ammonium, calcium, copper, iron, manganese, magnesium or zinc.
The US patent application, publication no. US2015/272835 describes a method for producing a carotenoid-containing composition that is a needle crystal or needle crystal having a maximum length of 5 μm or more and whose astaxanthin content based on the total composition is 40% by mass or more, wherein the method comprises: an extraction step wherein a culture obtained by culturing a carotenoid-producing microorganism including astaxanthin is extracted using one to three-carbon atom alcohol, an extract separation step wherein an extract is obtained by separating the microorganism and the extract, a concentration step wherein a concentrate is obtained by concentrating the extract 1.25 to 20 times in terms of amount of liquid, a crystallization step wherein crystals of the carotenoid-containing composition are produced in the concentrate, and a crystal separation step wherein the crystals are separated and obtained from the mother liquor, wherein the microorganism may belong to the genera Agrobacterium, Brevibacterium, Paracoccus, Brevundimonas and Erythrobacter, and algae belonging to the genus Haematococcus, yeasts belonging to the genus Phaffia and the like. Actually, only strain E-396 belonging to the genus Paracoccus is exemplified and specifically described.
Technical problem
In view of the data reported in the state of the art, and given that the art closest to the invention relates exclusively to the production of carotenoids, the inventors of the present invention have verified and tested the possibility by Antarctic bacterial strains isolated from themselves, which are not genetically modified and which have been duly deposited in accordance with the Budapest Treaty, to produce biomaterials in the presence of organic food waste material, as well as to simultaneously produce biocellulose and carotenoids, the simultaneous synthesis of which by the same bacterium has never been reported in the literature. The end products are released into the culture medium and are not retained within the bacterial cells.
Budapest Treaty
Marinomonas ef1, Bacillus ef1 and Brevundimonas ef1 are deposited at the Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna "Bruno Ubertini", in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure, 28 April 1977.
Marinomonas ef1 Access No. DPS RE RSCIC 4 of 08/05/2019
Bacillus ef1 Access no. DPS RE RSCIC 23 of 24/11/2020
Brevundimonas ef1 Access no. DPS RE RSCIC 24 of 24/11/2020
The filing was carried out by Sandra Pucciarelli, as inventor and as President of IRIDES SRL, who also authorised the Applicant IRIDES S.R.L. at Via Gentile III da Varano 1, 62032 Camerino (MC), Italy to refer to the deposited biological material in the application and has given his unreserved and irrevocable consent to the deposited material being available to the public in accordance with Rule 33 EPC.
Subject of the invention
The above technical problem is solved by providing the use of at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of biomaterials from food waste products, with the provision that it is not and which therefore is not an object of the present invention, Brevundimonas ef1 Access No. DPS RE RSCIC 24, for the production of carotenoids from soybean oil.
It is another object of the present invention a process for the production of biomaterials comprising culturing at least one strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4 Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature in the presence of culture medium added with food waste materials until the final product is obtained in the culture medium. The above technical problem is further solved by providing the use of Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the simultaneous production of cellulose and carotenoids from food waste products.
A further object of the present invention is a process for the simultaneous synthesis of biocellulose and carotenoids including the following steps: a) culturing Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 in culture medium, or nutrient broth; b) incubation of the bacteria obtained at the end of step a) at room temperature under stirring; c) dilution of the solution obtained at the end of step b) and addition of the food waste product and incubation until the final carotenoid and cellulose product is obtained.
Further features of the present invention will be clear from the following detailed description with reference to the submitted experimental data. Detailed description of the invention
Definitions
Within the meaning of the present invention, food waste materials or waste from the food process is understood to be any edible substance, either raw or cooked, that is discarded, or is intended to be discarded or needs to be discarded, as defined by the European Commission in EC Directive 2006/12/EC. Or they can be defined as any material acquired for animal or human consumption that is separated from the municipal solid waste stream and which does not fall within the definition of "agricultural material",
(https://web.archive.org/web/20091009041034/http://www.ciwmb.ca. gov/Regulations/Titlel4/ch31.htm). Within the meaning of the present invention, biocellulose means cellulose of natural, microbial origin characterised by fibres having variable diameter.
Within the meaning of the present invention, bioplastics and biopolymers are understood to be biodegradable and/or bio-based plastics and polymers (EUBIO Admin, Bioplastics, su European Bioplastics e.V; https://www.cen.eu/work/areas/chemical/biobased/Pages/default.as px).
Within the meaning of the present invention, culture medium suitable for bacterial growth means solid or liquid solutions containing nutrients on which it is possible to cultivate bacterial colonies. For example, the culture medium is a nutrient broth or lysogenic broth containing ingredients such as yeast extract, peptides derived from enzymatic digestion such as hydrolysed casein, which consists of a mixture of different chemical components in unknown proportions. Where peptides derived from enzymatic digestion are a mixture of water-soluble polypeptides and amino acids derived from the partial hydrolysis of proteins, such as tryptone which is an assortment of peptides formed by the digestion of casein from trypsin protease, or proteose peptone. Additives may also be employed to supplement culture media containing peptides derived from enzymatic digestion such as glycerol, yeast extract, NaCl, K2HPO4, MgSO4. For example, the minimum medium is a culture medium containing only the growth-supporting ingredients, in other words the minimum possible nutrients for colony growth, typically including salts, essential elements such as magnesium, nitrogen, phosphorous, sulphur and water to which a carbon source is then added. Or, the LB Medium culture medium is a lysogenic broth containing casein peptides and peptones, vitamins, trace elements and minerals, sometimes containing sodium chloride or tryptone.
The present invention consists of the use of at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of biomaterials from food waste products, with the provision that it is not and which therefore is not an object of the present invention, Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the production of carotenoids from soybean oil.
The inventors of the present invention have verified that the strain of Marinomonas ef1 Access No. DPS RE RSCIC 4 possesses the Fab metabolic pathway genes involved in the synthesis of PHA and genes involved in the synthesis of 2-pyrone 4,6-dicarboxylic acid.
SEQ. ID. NO. 1: PROKKA_02018 3-oxoacyl-[acyl-carrier-protein] synthase 3 (fabH)
Figure imgf000009_0001
SEQ. ID. NO. 2: PROKKA_00698 3-oxoacyl-[acyl-carrier-protein] reductase FabG
Figure imgf000009_0002
SEQ. ID. NO. 3: PROKKA_00804 Malonyl CoA-acyl carrier protein transacylase (FabD)
Figure imgf000009_0003
Figure imgf000010_0001
SEQ. ID. NO. 4: PROKKA_01991 Malonyl CoA-acyl carrier protein transacylase (FabD)
Figure imgf000010_0002
SEQ. ID. NO. 5: PROKKA_01992 3-oxoacyl-[acyl-carrier-protein] reductase FabG
Figure imgf000010_0003
SEQ. ID. NO. 6: PROKKA_03165 3-hydroxyacyl-[acyl-carrier- protein] dehydratase FabZ
Figure imgf000010_0004
SEQ. ID. NO. 7: PROKKA_02218 protocatechuate 4,5- dioxygenase beta chain
Figure imgf000010_0005
Figure imgf000011_0001
SEQ. ID. NO. 8: pROKKA protein_02219
Figure imgf000011_0002
SEQ. ID. NO. 9: PROKKA_02220 2-hydroxymuconate semialdehyde hydrolase
Figure imgf000011_0003
SEQ. ID. NO. 10: PROKKA_02288 protocatechuate 4,5- dioxygenase alpha chain
Figure imgf000011_0004
SEQ. ID. NO. 11: PROKKA_02289 protocatechuate 4,5- dioxygenase beta chain
Figure imgf000011_0005
SEQ. ID. NO. 12: PROKKA_022903-oxoadipate enol-lactonase2
Figure imgf000012_0001
SEQ. ID. NO. 13:PROKKA_03275 2-pyrone-4,6-dicarboxylate hydrolase
Figure imgf000012_0002
SEQ. ID. NO. 14: PROKKA_03276 protocatechuate 4,5-dioxygenase alpha chain
Figure imgf000012_0003
SEQ. ID. NO. 15: PROKKA_03277 protocatechuate 4,5- dioxygenase beta chain
Figure imgf000012_0004
The inventors further verified that the strain of Marinomonas ef1 Access No. DPS RE RSCIC 4 have genes involved in hydrocarbon synthesis. SEQ. ID. NO. 16: WP 100636658.1 malonyl-CoA decarboxylase
Figure imgf000013_0001
SEQ. ID. NO. 17: WP 100636466.1 bifunctional 3-hydroxydecanoyl- ACP dehydratase/trans-2-decenoyl-ACP isomerase
Figure imgf000013_0002
SEQ. ID. NO. 18: PROKKA_03927 3-oxoacyl-[acyl-carrier-protein] synthase 1
Figure imgf000013_0003
SEQ. ID. NO. 19: WP 100636855.1 oxidoreductase SDR family
Figure imgf000013_0004
Figure imgf000014_0001
SEQ. ID. NO. 20: PROKKA_03165 3-hydroxyacyl-[acyl-carrier- protein] dehydratase FabZ
Figure imgf000014_0002
SEQ. ID. NO. 21: PROKKA_025414'-phosphopantetheinyl transferase psf-1
Figure imgf000014_0003
Preferably the bacterial strain is Marinomonas ef1 Access No. DPS RE RSCIC 4.
Preferably the biomaterials are selected from the group consisting of: biopolymers, bioplastics, carotenoids. More preferably the biopolymers may be paraffin-like polyunsaturated hydrocarbon polymers.
Preferably food waste materials or waste from the food process are food waste products containing sugars, such as for example glucose and/or fructose, food waste products waste oil comprising soybean oil.
Preferably when the bacterial strain is Marinomonas ef1 Access No. DPS RE RSCIC 4. A further object of the present invention is a process for the production of biomaterials comprising culturing at least one strain selected from the group consisting of Marinomonas ef1 Access No. DPS RE RSCIC 4 Marinomonas ef1 Access No. DPS RE RSCIC 4, Bacillus ef1 Access No. DPS RE RSCIC 23 and Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature in the presence of culture medium containing food waste materials until the final product is obtained in the culture medium. In a preferred embodiment the process for the production of biomaterials comprises the following steps: a) culturing Marinomonas ef1 at room temperature in the presence of 2% Peptone under stirring until growth to the plateau step; b) dilution of the obtained culture at the end of step a) with sea water and addition of the waste food product and subsequent incubation at room temperature under stirring until the biomaterial is obtained in the culture medium; c) collection of the product obtained at the end of step c) and washing at room temperature; d) hot washing to remove any oily residue; e) drying.
Optionally steps c) and d) may be repeated.
Optionally, after step e), a step f) may follow wherein the product is dispersed in a polar solvent and subsequently centrifuged to recover the supernatant which is in turn centrifuged and dried under nitrogen atmosphere to obtain the product in powder form.
The following molecules responsible for the synthesis of carotenoids are present in the Bacillus genome: SEQ. ID. NO. 22: phytoene synthase
Figure imgf000015_0001
Figure imgf000016_0001
SEQ. ID. NO. 23: Diapolycopene oxygenase
Figure imgf000016_0002
The following molecules responsible for the synthesis of carotenoids are present in the brevundimonas genome:
SEQ. ID. NO. 24: phytoene synthase
Figure imgf000016_0003
SEQ. ID. NO. 25: lycopene cyclase
Figure imgf000016_0004
Figure imgf000017_0001
SEQ. ID. NO. 26: Geranylgeranyl diphosphate synthase
Figure imgf000017_0002
A further object of the present invention is the use of Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 for the simultaneous production of cellulose and carotenoids from food waste products.
A further object of the present invention is a process for the simultaneous synthesis of biocellulose and carotenoids comprising the following steps: a) culturing Bacillus ef1 Access No. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 in culture medium, or nutrient broth; b) b) incubation of the bacteria obtained at the end of step a) at room temperature under stirring c) dilution of the solution obtained at the end of step b) and addition of the food waste product and incubation until the final carotenoid and cellulose product is obtained.
Preferably the nutrient broth is composed of bovine extract (1g/L), peptone (5g/L), yeast extract 2g/L, sodium chloride 5g/L).
The final carotenoid product is presented as a yellow/orange pigment, which is recovered by two steps of centrifugation, the first step in order to sediment the bacteria at centrifugation, and to collect the supernatant containing the pigment, preferably at lOOOxg, the second step, in order to remove the particulate preferably at lOOOOxg.
Cellulose is produced at a later time and is in the form of flakes or spherules of biocellulose in the culture medium.
In a preferred embodiment the process for the production of biocellulose and carotenoids comprises: incubation of Bacillus ef1 Access no. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access No. DPS RE RSCIC 24 at room temperature under stirring in the presence of culture medium suitable for bacterial growth until biocellulose is obtained by the bacterial culture, both in static culture condition and under stirring, the second preferably at 100 rpm; then biocellulose is collected, for example with spatulas, and washed with sterile double-distilled water, then biocellulose is incubated at 80 °C to eliminate bacteria, the washing in sterile double-distilled water is repeated.
Examples
A biomaterial was produced with the process set out below: a) culture of Marinomonas ef1 at room temperature in the presence of 2% Peptone under stirring until plateau growth; b) dilution of the culture obtained at the end of step a) with sea water (50% vol/vol) and addition of soybean oil in an amount equal to 1% of the total and subsequent incubation at room temperature under stirring until white ovoid aggregates are obtained in the culture medium; c) the latter is collected with a spatula and transferred into a centrifuge tube for washing with sterile distilled water; d) to remove oil residues, the sample is washed with hot distilled water (approx. 80 °C) for about 5 minutes. The liquid is then removed and the sample washed with new distilled water at 80°C; this process was repeated three times; e) the solid sample, separated from the aqueous phase, is subsequently dried under an inert nitrogen atmosphere; f) the dry sample is dispersed in absolute ethanol (approx. 40 ml) and subjected to ultrasonication until a homogeneous dispersion is obtained, then centrifuged at 10000g in a HERMLE - Z 323 K centrifuge and the solid precipitate, which is white in colour, is recovered; g) the latter is then dried under an inert nitrogen atmosphere. h) Part of the solid obtained is then analysed by IR spectroscopy, elemental analysis and XRF spectroscopy, SEM electron microscopy. i) Part of the solid obtained is then dissolved in chloroform (approximately 2 ml), the filtered solution, and subsequently analysed by elemental analysis CNMR and HNMR.
The elemental analysis showed that the new biomaterial is composed of 72% C and 10.5% H, and in the remaining 17.5% there is bromine as evidenced by XRF analysis.
XRF analysis showed the signal for bromine at 13.9 Kev.
IR analysis showed the presence of CH2, CH3 stretching from the bands present between 2916 and 2850 cm-1, =C-H stretching from the band at 3010 cm-1, C=C stretching at around 1600 cm-1, C-H scissoring at 1425 cm-1, and two bands at 718 and 695 cm-1 due to C-Br stretching.
CNMR analysis showed that the base structure of the new biomaterial is composed of at least 22 carbon atoms. The signal at 35 ppm is attributable to the carbon bonded to the bromine atom, the signals in the range 127-131 ppm are attributable to the sp2 carbons and the other carbons are those within the chain; the signal at 23 ppm is related to the deuterated chloroform CDC13.
The HNMR analysis revealed the presence of signals at 5.4 ppm that are attributable to sp2 carbons, while the signal at 2.8 ppm is related to the proton of the carbon near a bromine atom.
The SEM analysis showed a lamellar structure with overlapping leaflets.step The agar diffusion test showed antibiotic properties against a Pseudomonasstrain: 5 mg of the material produces an inhibition halo of about 5 mm.
The product obtained after various analyses turns out to be a polyunsaturated hydrocarbon similar to paraffin.
Two bacterial strains capable of producing biocellulose in the presence of fructose were isolated: Brevundimonas ef1 and Bacillus ef1.
Two processes have been carried out to synthesise biocellulose: in both cases the first step consists in growing the bacteria in culture medium, or nutrient broth.
The nutrient broth had the following composition:
Meat extract 1.0 (g/L)
Peptone 5.0 (g/L)
Yeast extract 2.0 (g/L)
Sodium chloride 5.0 (g/L) final pH 6.8 + 0.2 at 25°C
Beef extract and peptone provide amino acids, nitrogen, carbon, vitamins and minerals for the growth of the organisms.
Yeast extract is a source of vitamins, particularly B group vitamins. Sodium chloride maintains the osmotic balance of the medium.
In the second step, bacteria incubated at 22°C at 2000 rpm rotation. Once the optical density of about 2 OD at 550 nanometres is reached, the culture is diluted with sea H20 (50% of the final volume) and added with 1.5-2% fructose, and further incubated under stirring at 22°C.
In the third step, a yellow/orange pigment is produced after 24 hours. The pigment is recovered by two centrifugation steps: Centrifugation at 1000g to sediment the bacteria and collect the supernatant containing the pigment
Centrifugation at 10000 g to remove particulate matter.
The aqueous part containing the pigment is dried under nitrogen atmosphere, redispersed in absolute Ethanol (about 40 ml) and analysed by UV-Vis and MS.
The pigment was found to be a mixture of B-cryptoxanthin and lycopene/B-carotene.
After about 10 days, from the same culture, the production of orange-coloured flakes or spherules occurs in the same culture medium.
The culture is incubated at 80 °C to eliminate bacteria.
The material composed of flakes and spherules is collected and washed with sterile double-distilled water and then dried in an oven at 40°C.
A dry portion of the material is analysed with FTIR.
It was found that the material is biocellulose.
A dry portion of biocellulose is treated with absolute ethanol (approx. 40 ml) and the pigment is extracted for subsequent analysis by UV-Vis and MS.
The pigment was found to be a mixture of B-cryptoxanthin and lycopene/B-carotene.
The synthesis of biocellulose from fructose and biopolymers from soybean oil paves the way for the recycling of food waste, including fruit and oils. In addition, the co-synthesis of carotenoids makes it possible to optimise a production process: starting from a single substrate, two products are obtained that can be used in a single application, such as beauty masks containing carotenoids. BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL
FORM
TO Sandra Pucciarelli PhD RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant
School of Biosciences and Veterinary to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY Medicine-Univerity of Camerino identified at the bottom of this page Italy
Figure imgf000022_0001
Figure imgf000023_0001
1 Where Rale 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page)
BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL
FORM
TO Sandra Pucciarelli, PhD RECEIPT GN THE CASE OF AN ORIGINAL DEPOSIT Issued pursuant
School of Biosciences and Veterinary to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY
Medicine identified at the bottom of this page
University of Camerino Italy
Figure imgf000024_0001
Figure imgf000025_0001
1 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page)
BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL
FORM
TO Sandra Pucciarelli, PhD RECEIPT GN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant
School of Biosciences and Veterinary to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY
Medicine identified at the bottom of this page
University of Camerino Italy
Figure imgf000026_0001
Figure imgf000027_0001
1 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page)

Claims

1. Use of at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access n. DPS RE RSCIC 4, Bacillus ef1 Access .n. DPS RE RSCIC 23 and Brevundimonas ef1 Access n. DPS RE RSCIC 24 for the production of biomaterials from food waste products with the proviso that is not Brevundimonas ef1 Access n. DPS RE RSCIC 24 for the production of carotenoids starting from soybean oil.
2. Use according to claim 1 wherein the food waste products are selected from the group consisting of: food waste products containing sugars, food waste products, waste oil including soybean oil.
3. Use according to claim 1 wherein biomaterials are selected from the group consisting of biopolymers, biocellulose, carotenoids.
4. Process for the production of biomaterials comprising culturing at least one bacterial strain selected from the group consisting of Marinomonas ef1 Access n. DPS RE RSCIC 4, Bacillus ef1 Access .n. DPS RE RSCIC 23 and Brevundimonas ef1 Access n. DPS RE RSCIC 24 at room temperature in the presence of culture medium containing peptone and sea water to which waste soybean oil is added to obtain the final product in the culture medium.
5. Use of Bacillus ef1 Access .n. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access n. DPS RE RSCIC 24 for the simultaneous production of cellulose and carotenoids from food waste products.
6. Process for the simultaneous synthesis of biocellulose and carotenoids comprising the following steps: a) culturing Bacillus ef1 Access .n. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access n. DPS RE RSCIC 24 in culture medium, or nutrient broth; b) incubation of the bacteria obtained at the end of step a) at room temperature under stirring c) dilution of the solution obtained at the end of step b) and addition of the food waste product and incubation until the final carotenoid and cellulose product is obtained.
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