IT202100017333A1 - PRODUCTION OF BIOMATERIALS FROM FOOD WASTE PRODUCTS BY ANTARCTIC BACTERIAL STRAIN - Google Patents

Description

Description of the application for an industrial invention entitled: PRODUCTION OF BIOMATERIALS FROM FOOD WASTE PRODUCTS BY ANTARCTIC BACTERIAL STRAIN

Background of the invention

The present application relates to the field of microbiology and in particular to a process for the production of biomaterials through the use of isolated and verified non-genetically modified bacterial strains, starting from food waste products.

State of art

AND? note the production of polyhydroxyalkanoates by bacteria, including the genera Bacillus, Rhodococcus and Pseudomonas. Polyhydroxyalkanoates (PHA) are thermoplastic polyester polymers synthesized by bacteria through the fermentation of sugars or lipids. These materials are biodegradable and are used in the production of bioplastics.

The metabolic pathways that lead to the bacterial production of PHA are known, which, like other bio materials, remain trapped inside the bacterium and therefore require purification processes to obtain the final product

Further known are Bacillus strains genetically modified with the PhaC and PhaR genes for the production of bioplastics ( 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.

The Italian patent application n. 102019000014121 discloses an in vitro process for the preparation of metabolites by Marinomonas ef1 or Rhodococcus, in particular silver nanoparticles and fluorescent dyes.

The Italian patent application n. 102019000024493 describes the use of Marinomonas ef1 bacterial strains for the bioremediation of waters polluted by contaminants, such as fuels, toxic post-transition metals and/or toxic transition metals.

The Italian patent application n.102020000031769 describes the use of bacterial strains Bacillus ef1 and Brevundimonas ef1 for the production of biocellulose.

US Patent Application No. 2012210745A1 discloses the application of enzymes for the repair of plastic products and the bacterial synthesis of bioplastics.

The international patent application n. 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 discloses 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.

The international patent applications n. WO2007017270, WO0011188 and WO2016040653 and describe the production of PHA from engineered Rhodococcus.

United States patent application no. US2014234944A1 describes the use of engineered Rhodococcus to produce PHA starting from ethanol.

AND? The production of carotenoids by Antarctic bacteria such as Planococcus sp.ANT_H30 and Rhodococcus sp. has been described. ANT_H53B ( Molecules 2020, 25, 4357; doi:10.3390/molecules25194357).

Acyclic carotenoids with a C30 aglycone, the xylosilesters of diapolicopenedioc acid A?C and methyl 5-glucosyl-5,6-dihydro-apo-4,4?-lycopenoate, have been 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 Bacilli class, phylum Firmicutes ( Mar. Drugs 2014, 12).

AND? 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 from different microorganisms has been described ( Lu Xuefeng, 2013, Microbial Synthesis of Alka(e)nes, Frontiers in Bioengineering and Biotechnology, 1, 1-10, DOI=10.3389/fbioe.2013.00010), however it is not? The production of solid hydrocarbons, such as paraffins and waxes, by bacteria has been reported in the literature.

Technical problem

In consideration of the data reported in the state of the art, the inventors of the present invention have verified and tested the possibility by the Antarctic bacterial strains isolated from them, which are not genetically modified, and which have been duly deposited in accordance with the Treaty of Budapest, to produce biomaterials in the presence of organic food waste material, as well as? to simultaneously produce biocellulose and carotenoids, of which it is not? simultaneous synthesis 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.

Treaty of Budapest

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 .n. DPS RE RSCIC 4 of 08/05/2019

Bacillus ef1 Access .n. DPS RE RSCIC 23 of 11/25/2020

Brevundimonas ef1 Access n. DPS RE RSCIC 24 of 11/25/2020 Object of the invention

The aforementioned technical problem ? resolved by providing the 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.

AND? Still object of the present invention is a process for the production of biomaterials which provides for the cultivation of at least one strain selected from the group consisting of Marinomonas ef1 Access n. DPS RE RSCIC 4 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 added with food waste materials until the final product is obtained in the culture medium.

The aforementioned technical problem ? further resolved by providing the 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.

Further object of the present invention ? a process for the simultaneous synthesis of biocellulose and carotenoids comprising the following steps:

a) growth of 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 stage 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 obtaining the final product carotenoids and cellulose

Further features of the present invention will be apparent from the following detailed description with reference to the experimental data presented.

Detailed description of the invention

Definitions

In the context of the present invention, food waste materials or waste from the food process mean any edible substance, raw or cooked, which is discarded, or is intended to be discarded or ? necessary to discard, as defined by the European Commission in the 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 does not fall under the definition of "agricultural material". (https://web.archive.org/web/20091009041034/http://www.ciwmb.c a.gov/Regulations/Title14/ch31.htm).

In the context of the present invention by biocellulose is meant cellulose of natural, microbial origin characterized by fibers having variable diameters.

In the context of the present invention, bioplastics and biopolymers mean 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.aspx).

Within the scope of the present invention, culture medium suitable for bacterial growth means solid or liquid solutions containing nutrients on which possible to cultivate bacterial colonies. For example, the culture medium ? a nutrient broth or lysogenic broth that contains ingredients such as yeast extract, peptides derived from enzymatic digestion such as hydrolyzed 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 resulting from the partial hydrolysis of proteins, such as tryptone which is ? an assortment of peptides formed by the digestion of casein by the protease trypsin, or the proteous peptone. Additives can also be used to supplement culture media containing peptides deriving from enzymatic digestion such as glycerol, yeast extract, NaCl, K2HPO4, MgSO4. For example, the minimum terrain (minimum medium) ? a culture medium containing only growth support ingredients, in other words the minimum of nutrients possible for the growth of the colony, typically including salts, essential elements such as magnesium, nitrogen, phosphorus, sulfur and water to which then ? added a carbon source. Or, the LB Medium culture medium? a lysogenic broth containing casein peptides and peptones, vitamins, trace elements, and minerals, sometimes containing sodium chloride or tryptone.

The present invention consists in the 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.

The inventors of the present invention have verified that the Marinomonas ef1 Access.n. DPS RE RSCIC 4 possesses Fab pathway genes involved in PHA synthesis and genes involved in 2-pyrone 4,6-dicarboxylic acid synthesis.

SEQ. ID. NO. 1: PROKKA_02018 3-oxoacyl-[acyl-carrier-protein] synthase 3 (fabH)

MISSVVISGVGLWTPAYSISNEELVDSYNAWVEQFNAKNADAIEKGQIEAKPLSSAAFIEKA SGIKSRYIYSKDGALDINRMRPILEIRNDEQISHQAEMAIAAAKKALD

AANKKASDIDMVIVSCAYTQRSYPAIAIEVQSALGIEGFGFDMLVACSAATFGLQRAVDAIK AGSSKGVLVINPELTSPQVNYMDRDSHFIFGDVATAVVVESSETCQSA

HSFDVLSSKCITSYSNNIRSNFGYTTRVTDEDPYSADMLFHQNGRKVFKEVCPMAADHIENH LKSQGIQVSQLKRWWLHQANINMNLLISKRLLGRDATLEEAPVVLDEY

ANTASAGSIIAFAKHHKDLAAGDIGVICSFGAGYSIGSLILRKR

SEQ. ID. NO. 2: PROKKA_00698 3-oxoacyl-[acyl-carrier-protein] reductase FabG

MSFDLELNGRIALVTGGTKGLGAAVVESLHGAGVSVIAVARSVPKEVTEGISYIKGDLLSAV GCTAVVNTVLDKFGGVDIVVNVLGGSSAPSGGFAALDEQEWENALSQN

LLSAVRIDRGLLPSMITKGSGVIVHVTSIQNKLPLPESTTAYAAAKAALSTYSKSLSKEVAP KGIRVVRVSPGWIETEASVRLAERLADHAGTDYEGGKKIIMEALGGIP

LGRPAQPSEVADLITFLVSARASSITGTEYIIDGGTVPTA

SEQ. ID. NO. 3: PROKKA_00804 Malonyl CoA-acyl carrier protein transacylase (FabD)

MTQVFTFPGQGSQRAHMLQNLPDHPNVTLIKQQAEDILGVKLNALDSQEALTDTVNVQLALL ICGVAWGQYLQNVGIKPDYVLGLSIGAYPAAVVAGSLDFGDALRLVKA

RADSMKSAYPSGYGMLAITGAEFKHVQAAVNLLQEKGEKVYLANLNGEYQFVLAGEKRVLKN AAEQVRSQGIGASVFLDVTVPSHCPLLKEQSEKLLAMFQAITLKTSNS

LYVSASKARVLRKPDDIKEDLALNMAHQLHWYDSCQMLAERGVNKALEMPPGSTLTGLFRKV LNGGECYSIENINLKTIL

SEQ. ID. NO. 4: PROKKA_01991 Malonyl CoA-acyl carrier protein transacylase (FabD)

MSTKLAFVFPGQGSQQLGMLADLAEKHEIIKQTFAEASDVLGYDLWDLVQNDAERLSQTDKT QPALLTASVALWRLWEQQGGDKPAYVAGHSLGEYSALVCAGVIAFTDA

VELVKLRGEYMQQAVPAGDGAMAAIIGLDDDKVVAACAAAPGIVSAVNFNSPGQVVIAGHVA AVEAAMVNAKEAGAKRALPLPVSVPSHCELMIPAGAKLAEKLETIVFK

SPSCTLVQNVTAQAVSDPSVIKANLVSQLSEPVLWTQSVVLLSELGVTSTVECGPGKVLSGL NKRIVKGLETSSLGDLAGFEAALSA

SEQ. ID. NO. 5: PROKKA_01992 3-oxoacyl-[acyl-carrier-protein] reductase FabG

MGLEGKIALVTGATRGIGKAIAVALVEQGATVIGTATSESGAQSITEYLGANGKGWVLDVSS SESVDAVIKEITAEFGAPTVLVNNAGITRDNLMMRMKEDEWDQVINTN

LTSVFRVTKACLRGMTKAKFGRVISISSVVGSMGNGGQTNYSAAKAGLEGFSRSLAAEIASR GITVNCVAPGFIETDMTKVLSEEHKAKLVEKVPSSRLGQPEEIAAAVA FLASNGAAYITGETLHVNGGMYMS

SEQ. ID. NO. 6: PROKKA_03165 3-hydroxyacyl-[acyl-carrierprotein] dehydratase FabZ

MMDVNEIRQYLPHRYPFLLVDRIVEINLNDSIIAYKNVTINEPFFNGHFPNHPVMPGVLIIE AMAQAAGVLGFKTMGKKPEDGSIYYFVGVDNARFKRPVVPGDRLQLEA

KIIAEKRGIWKFECRATVDGQLACSATIMCADRKI

SEQ. ID. NO. 7: PROKKA_02218 protocatechuate 4,5- dioxygenase beta chain

MATIVSGFLVPHDPIMFVAPDAPAKDIADKAWGAYETCAVHLADLDVDTVIIIGNDHYMLFG TTCLPQYCIATGDLDGPLDQMPGLKRQLIPNNEALATHIATHGHANGF

DWAVARSFTTDHSFSIPFQKIVQRASEIKGKEIKAIPVYLASAVDPFLSMQRSQQLGQAIAK AVKAFKGDERIAVIGSGGISHWVGTKESGTVNEDFDREILTFCLSLDE

ASIMALSDEKILSEGGNGAMELRNFVCAMAAVEADSSELIEYQPVPEWVTGLGFVELKKNPT KS

SEQ. ID. NO. 8: PROKKA_02219 protein

MKRDVIERVLWNLSVDRFSKVKYKEDPNKFLSRFALDKSDIEKILTFDVKALQEMGVNPMLT MGYWVEMSPDNSMQTYNQKLGSNGVHSASIKG

SEQ. ID. NO. 9: PROKKA_02220 2-hydroxymuconate semialdehyde hydrolase

MKELDRKFVMAAGHKTAYFECGSGEPLLLLHGSGPGVSGWTNWNGVMAELGEHFHVFVPDIA GFGFTEFKEDTQYDIKFWVNHLVEFMNAVGIEKASLVGNSFGGAVGVG

LALFSPDRLNKLALLGTPAGTFVQTPGLAGAWRYEPSENNMRELMALFPYDKSLITDELVKS RYEASARSGSQEALRKLIPKPNDDGETLVKGFPEGALQKITAPTLVLH

GREDAVVPLECGQRLVNNIPNADLYVFGQCGHWVQSEQRSKFMNVVTAFVGG

SEQ. ID. NO. 10: PROKKA_02288 protocatechuate 4,5- dioxygenase alpha chain

MNNNNKPYSDIPGTTVFDGDMARIGFHLNQFCMSLMQESNRTAFKQNERAYLDKWPMTEAQK KAVIARDFSQLIALGGNIYYLVKISSSDGLSVAAAVSTMTNLSVDEYI

DMMRRGGRSPEGNRYVVSNTSEEKQ

SEQ. ID. NO. 11: PROKKA_02289 protocatechuate 4,5- dioxygenase beta chain

MAKITCGLGTSHIPLLGHIIDKGDSGDEKWAPIFDGYQFTKKWIREQKPDVVILVYNDHATA FDMKVIPTFAIGCGEEYHPADEGYGARPVPTVKGAPKLAWHITESLIK

QGFDMTIVNEMDVDHGLTVPLSMVFGEVEEWPCQVIPLAVNTVVYPSPTGDRCLALGKAIRA AVEQFPEDLNVQVWGTGGMSHQLLGERAGLINQEWDLAFLDDLKSDYE

KLGAISQVEYIRESGTEGSEMVMWLIMRGALNHEVNEIHRHYHVPVSNTALGHLIYENVDKK GE

SEQ. ID. NO. 12: PROKKA_02290 3-oxoadipate enol-lactonase2 MNVRITRPKLVCLAGLLCDQRMWQKVAAQLENDADVEIISFAGFDDLTGMARSVLSSIEGDF ALAGHSMGGRVALEIYRLAPQRVTRLALLNTGVHPKSDKEIPGRMRLI

EDARTRGISVLATQWLIPMMSPKGLNNANLMADLAKMVESYSVEDFEKQIHALINRPDAEAL LSQISVPTLLLSGSQDTWSPVAQHQAIQKHIIGSELVVIDDAGHMAPA

EDPENVAAALHSWLKKK

SEQ. ID. NO. 13:PROKKA_03275 2-pyrrone-4,6-dicarboxylate hydrolase

MMDKDYIPFHPAPSKPAFKAPAGAVDAHCHVFGPADKFPYHPKRKYTPCDASKEQLFALRDH LGFSRNVIVQASCHSTDNSALIDALETAGDLARGVAFVDDSITHEEIE

KMHAVGIRGVRFNFVKRLVDSTPREVFFSIADKIRPFGWHIVVYFEAADLEDLIPFLKELNT TIVVDHMGTPNVANGVDHPDFQRFVNFMADNDNVWCKVSCPERLTQHA

PDYSDVYPFARTLVEKFSDRVLWGTDWPHPNMKVEAPNDGHLVDVIPHIAPTPELQQALLVD NPMRLYWK

SEQ. ID. NO. 14: PROKKA_03276 rotocatechuate 4,5- dioxygenase alpha chain

MASNYTDRQLQGTYLFDGQMAKKGYGLNKMCYSFNDQVARDEFNADPEAYCEKFKLTEAQKE AIRNRDVIGLLREGGSIYYLAKFTGLLKLNMQDIGGLQTGMSTDEFKA

MLEKNGRGEV

SEQ. ID. NO. 15: >PROKKA_03277 rotocatechuate 4,5- dioxygenase beta chain

MANIIGAITTSHIPAIANAMANGLEQTPYWKPFFDAYPPVRDWLNEKKPDVIILFYNDHGLE FFIDKKPTFAIGVADEYHNSDEGWGIPTIPPVTGESELSWHIANSIVD

EGFDMTICQEMRVDHGLTVPLNLMWPGHQYGHVKVIPVCINCEQYPMAQPWRTFELGRAIGR AVASYEEDVNVVVFGTGGMSHQLDGERAGFINRAFDIDCLDKLVSDPE

ALTKYTNLDLVEQGGAQAVELNMHIAMRGTLLGKVTELHRNYHAPISNTGSGLMLLEHEMPK

The inventors further verified that the Marinomonas ef1 Access .n. DPS RE RSCIC 4 possesses genes involved in hydrocarbon synthesis.

SEQ. ID. NO. 16: WP_100636658.1 malonyl-CoA decarboxylase MNFFTELFSSVFDRRYRKARSNEEDNRTVKAMCQDLLTRHGEVSGGALARNILDRYDQMSSD EKRELFEFMTHGLEVVTDEVITALTEYQKQPSKQTYKAFASACEPRRQ

ELIRRFNQVPGATPKLVKMREDLLKATKEDASLGILDVDFNHLFTSWFNRGFLVLRPINWDS PASILEKIIAYEAVHAIDSWDDLRQRLKPSDRRCYGFFHPSMADDPLI

FVEVALTKGTASSIQSLLTTDREIIEADEADTAVFYSISNCQPGLAGISFGNFLIKQVAANL ARDLDNLNTFVTLSPIPGLAKWANSAGMGSLGKGKDQALAAYYLLNAK

RPDGKPLDPVTRFHLGNGAMIQAVHADADISKNGLKQSNGAMVNYLYDLSQVSKNHEQFVTE QKVVASDAVHALSASIATRKKGE

SEQ. ID. NO. 17: WP_100636466.1 3-Hydroxydecanoyl-ACP dehydratase/trans-2-decenoyl-ACP bifunctional isomerase

MKFSNADIIAMSEGRFFGEGNAQLPSDQMLMIDEIIHIDSIGGKYNKGLLKASLAIQPDHWF FACHFKGDPVMPGCLGLDALWQLLGVFLGWSGLPGKGRALGVGKVRFT GQIYPDAKGIEYQVHIKRIIKKPLGMGIADGFVLLNGEVIYEATDLKVGLIKN

SEQ. ID. NO. 18: PROKKA_03927 3-oxoacyl-[acyl-carrier protein] synthase 1

MRRVVVTGLGIVSCLGNDKESVLNSLREGQSGIRFAEDYKEHGFRSHVCGRIDLDAESIDRK IRRFMGDAATYAYVSMQQAILDSGLTEDQVSNIRTGLVAGSGGASAKN

LVDSADTLREKGVKRVGPYRVTQTMGSTVSACLATPFKIKGVNYSITSACSTSAHCIGNAME LIQLGKQDIVFAGGGEEESWTQTCMFDAMGALSSKYNETPEKASRAYD

ANRDGFVIAGGGGMLVIEELEHAKARGAKIYAELVGYGATSDGYDMVAPSGEGAVRCMQMAM STIDGDIDYINTHGTSTPAGDMKELQAVNETFGDNIPLISSTKSLSGH

SLGAAGVHEAIYCLLMMENNFIAASANIDELDPAAGNIPVVTTRKDNVDLNMIMSNSFGFGG TNASLVFKKYTA

SEQ. ID. NO. 19: WP_100636855.1 oxidoreductase family SDR

MSFDLELNGRIALVTGGTKGLGAAVVESLHGAGVSVIAVARSVPKEVTEGISYIKGDLLSAV GCTAVVNTVLDKFGGVDIVVNVLGGSSAPSGGFAALDEQEWENALSQN

LLSAVRIDRGLLPSMITKGSGVIVHVTSIQNKLPLPESTTAYAAAKAALSTYSKSLSKEVAP KGIRVVRVSPGWIETEASVRLAERLADHAGTDYEGGKKIIMEALGGIP

LGRPAQPSEVADLITFLVSARASSITGTEYIIDGGTVPTA

SEQ. ID. NO. 20: PROKKA_03165 3-Hydroxyacyl-[acyl vector protein] dehydratase FabZ

MMDVNEIRQYLPHRYPFLLVDRIVEINLNDSIIAYKNVTINEPFFNGHFPNHPVMPGVLIIE AMAQAAGVLGFKTMGKKPEDGSIYYFVGVDNARFKRPVVPGDRLQLEA

KIIAEKRGIWKFECRATVDGQLACSATIMCADRKI

SEQ. ID. NO. 21: PROKKA_02541 4'-phosphopantetheynyl transferase psf-1

MDYNLGSSVVLTIAFSKKAFPIDCTLDDSEKKRASMFRFEHLREQYIFAHAFKRRILSHYFP FRKASEWYFAQTSSGKPFIKEGIAFNLSHSTSSVAVALVESTDKSAVG

VDIECFREMDDLESMIEMVCHSDEIQQLDRCHDRSKGFFKLWTAKEALLKACGSGLIDDLNH INCLPSLLSDQVYPLNWQGNQYCLQSISFDWGVITAAWSTDLPVSDIR

LDNWASGVPVSEAFCI

Preferably the bacterial strain ? Marinomonas ef1 Access .n. DPS RE RSCIC 4.

Preferably the biomaterials are selected from the group consisting of: biopolymers, bioplastics, carotenoids.

Pi? preferably the biopolymers may be polymers of paraffin-like polyunsaturated hydrocarbons.

Preferably food waste materials or wastes from the food process are sugar-containing food waste products, such as for example glucose and/or fructose, food waste products waste oil including soybean oil.

Preferably when the bacterial strain ? Marinomonas ef1 Access .n. DPS RE RSCIC 4.

Further object of the present invention ? a process for the production of biomaterials which provides for the cultivation of at least one strain selected from the group consisting of Marinomonas ef1 Access n. DPS RE RSCIC 4 Marinomonas ef1 Access n. DPS RE RSCIC 4, Bacillus ef1 Access .n. DPS RE RSCIC 23 and Brevundimonas ef1 Access n. DPS RE RSCIC 24at 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 producing biomaterials comprises the following steps:

a) culture of Marinomonas ef1 at room temperature in the presence of 2% Peptone under stirring until growth to the plateau phase;

b) dilution of the culture obtained at the end of step a) with sea water and addition of the waste food product and subsequent incubation at room temperature with stirring until obtaining the biomaterial in the culture medium;

c) collection of the product obtained at the end of stage c) and washing at room temperature;

d) hot washing to remove any oily residues; e) drying

Optionally steps c) and d) can be repeated.

Optionally after stage e) can? follow a stage f) in which the product ? dispersed in a polar solvent and subsequently centrifuged to recover the supernatant which is in turn centrifuged and dried in a 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

MVDVQTALKICEETIQTHSKTFYRAFSMLPKKKRQAVWAVYSFCRRADDIVDESPSPKEELA SFQEAFNRFLQGEVDRDDPMWVALEYTFQQFRMDESPFRDLLRGQEMD

LEQHRYETLDELLIYSYHVASTVGLMLLPIIAPRKKEQLKEAAISLGIGMQLTNILRDIGED KAERNRIYLPKQVMDQFGYTEQELQEGIVNQAFQHVWEYIAFEAEAYY

EEFFDHLHEFPLYSRIPVKAAAHFYKAILDKTRENEYRVFTKRSYISTQEKALILEDIT

SEQ. ID. NO. 23:, Diapolycopene oxygenase

MKKVIIIGGGLGGLSAAIRLQSRGFQVTILEKEASLGGKLQRYQASGYSFDLGPSTITMKAY FEEVFTSCHRRMEDYVTFYPIFPLTKNVFSDGHTVEFTPDIEQMESQI AVFSPEDAKQYRAFLQESKKLFQMAEDQFLNRLLLTWKDKADVKLMKALLAVKPFTSVQRLL RQYFRHPHTLAMFGRYATYIGSSPYEAPAIF NMMAYLEGEKGIYGIKG

GTYRLVEAFETLAKELGVQIHLNESVKKIHVNNKRIQGVETDQQLYEADQVIAGADALTVYR QLIDEEDRPSFGNQKLANIEPSLSGFVLLLGVPKVYEQLSHHNVFFPE

NYEQEFKDIFQKKQLPHDPTLYICYSGHSEPALSGGPGRSNLFVLANAPYVTKEQNWDMLKE TYGQHMKTKLKEKGLFDLDQLVEFEAVQTPLDLQQKTGAYHGAIYGMS

SNSFKQAFFRINNQSKDIEGLWFVGGTSHPGGGTPMVTKSGQLVAEAIIKHLT

The following molecules responsible for the synthesis of carotenoids are present in the brevundimonas genome:

SEQ. ID. NO. 24: phytoene synthase

MTDAVLDHSRQSMEQGSKSFAAAARLFPAAIRDDAWMLYSWCRHCDDEIDGQVLGHGAVGID PVLAGEKLDELRLRTAAALAGEPQTDPVFTAFQRVAMRHGIPADEAMD

LLQGFEMDVVGRRYDTLEDTLDYAYHVAGVVGVMMARIMGVDDAPTLRRAQDLGLAFQLTNI ARDVVEDAKGGRVYLPGAWLDEAGVPRDQVDQPQHRAAVARTAQRLVA

AAEPFYASARWGLRDLNPRSAWAIATARGVYRSIGRHVSRAGVSAWDGRTSVDKAGKLMLLG RGGLIALWCKTLDAWREPPPRPALWTHV

SEQ. ID. NO. 25: lycopene cyclase

MVGGGLANGLLALRLSQLRPDLDVRIVEAAQTLGGVHTWSFFDSDLTQAQRDWIAPLVVHRW PGYSVRFPQFKRALSTPYCSVTAERFAAVVEAALPGKVMLGAPVASVS PTEAVLADGRRLSAKAVIDGRGPTATPDLALGFQKFVGLEVRLAAPHGLTTPIVMDACVDQA GGYRFLYTLPFDDRTLLIEDTRYTDG DALDRAAFRQGVLDYARAQGWE

IETIFREEDGVLPVALDGDIGAHLSRMGPTALSGLRAGLFHPTTGYSLPDAVRLADRLVRDF EPATVAEDIRRHAHDVWAGRGFYRLLNRMLFRAARPDERYKVLERFYR

LPQPLVERFYAAGPTLADKARILSGKPPVPIGAALTCLVERGRA

SEQ. ID. NO. 26: Geranylgeranyl diphosphate synthase

MAIVGIRRQPPLADASCDPDDLRLQVQAHLAEVAPAHGGLLSTAAREALLGQGKRVRPVLAM LAAAHVGGDPEDALDYACALEMVHAASLVLDDLPCMDDASLRRGQPTL

HRRHGEDTAVLAAVALLNHATRLVLRTPAPAETRLAALDLLTQAIGFDGLSEGQMRDLRDDA RDRDETALRQINDLKTGALFVATVRGGGLLGGGDAQDLARLSIFGEAI

GFAFQLCDDLLDVCSTVDAVGKDVGQDDDTTTFVDLWGECRTRAAVRQSLAKAVEAVGHDSP LALYVLDLFQQAELGV

Object of the present invention ? also the 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.

Further object of the present invention ? a process for the simultaneous synthesis of biocellulose and carotenoids comprising the following steps:

d) growth of Bacillus ef1 Access .n. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access n. DPS RE RSCIC 24 in culture medium, or nutrient broth;

e) incubation of the bacteria obtained at the end of stage a) at room temperature under stirring

f) dilution of the solution obtained at the end of stage b) and addition of the food waste product and incubation until obtaining the final products carotenoids and cellulose

Preferably the nutritious broth ? composed of bovine extract (1g/L), peptone (5g/L), yeast extract 2g/L, sodium chloride 5g/L).

The final carotenoid product appears as a yellow/orange pigment, which is recovered by means of two centrifugation stages, the first stage to sediment the bacteria by centrifugation, and to collect the supernatant containing the pigment, preferably at 1000xg, the second stage, to eliminate the particulate preferably at 10000xg.

The cellulose is produced at a later stage and is presented in the form of small flakes or spherules of biocellulose, in the culture medium.

In a preferred embodiment, the process for the production of biocellulose and carotenoids provides:

a) incubation of Bacillus ef1 Access .n. DPS RE RSCIC 23 and/or Brevundimonas ef1 Access n. DPS RE RSCIC 24 at room temperature under stirring in the presence of culture medium suitable for bacterial growth until biocellulose is obtained from the bacterial culture, both under static culture conditions and under stirring, the latter preferably at 100 rpm; then the biocellulose is collected, for example with spatulas, and washed with sterile bidistilled water, subsequently the biocellulose is incubated at 80°C to eliminate the bacteria, the washing in sterile bidistilled water is repeated.

Examples

AND? A biomaterial was produced with the process set out below:

a) Marinomonas ef1 culture 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 a quantity equal to 1% of the total and subsequent incubation at room temperature with stirring until obtaining white ovoid aggregates 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 (about 80 ?C) for about 5 minutes. Subsequently the liquid is eliminated and the sample washed with new distilled water at 80°C; this process ? been repeated three times.

e) the solid sample, separated from the aqueous phase, is subsequently dried in an inert nitrogen atmosphere;

f) the dry sample is dispersed in absolute ethanol (about 40 ml) and subjected to ultrasounds, until a homogeneous dispersion is obtained, then subsequently centrifuged at 10,000g in a HERMLE - Z 323 K centrifuge and the solid precipitate is recovered, which is colored white;

g) the latter is subsequently dried in an inert nitrogen atmosphere;

h) Part of the obtained solid is then analyzed by IR spectroscopy, elemental analysis and XRF spectroscopy, SEM electron microscopy.

i) Part of the solid obtained is then dissolved in chloroform (about 2 ml), the filtered solution, and subsequently analyzed by elemental analysis CNMR and HNMR

The elemental analysis has shown that the new biomaterial? composed of 72% from C and 10.5% from H, and in the remaining 17.5% ? present bromine highlighted by XRF analysis.

XRF analysis highlighted the signal relating to bromine at 13.9 Kev.

The IR analysis highlighted the presence of CH2, CH3 stretching from the bands between 2916 and 2850 cm-1, =C-H stretching from the band at 3010 cm-1, C=C stretching about 1600 cm-1, C-H scissoring at 1425 cm-1, and two bands at 718 and 695 cm-1 due to C-Br stretching.

The CNMR analysis has highlighted that the basic structure of the new biomaterial? composed of at least 22 carbon atoms. The 35ppm signal? attributable to the carbon bonded to the bromine atom, the signals in the 127-131 ppm range are attributable to the sp2 carbons and the other carbons are those within the chain; the signal at 23 ppm ? related to deuterated chloroform CDCl3.

The HNMR analysis highlighted the presence of signals at 5.4 ppm which are attributable to sp2 carbons, while the signal at 2.8 ppm ? related to the carbon proton next to a bromine atom.

The SEM analysis highlighted a lamellar structure with superimposed sheets.

The diffusion test on agar showed properties antibiotics against a Pseudomonas strain: 5 mg of the material produces an inhibition halo of about 5 mm.

The product obtained after various analyzes appears to be a polyunsaturated hydrocarbon similar to paraffin.

Two bacterial strains capable of producing biocellulose in the presence of fructose have been isolated: Brevundimonas ef1 and Bacillus ef1.

Two processes have been conducted to synthesize biocellulose, and in both cases the first stage 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 organisms.

Yeast extract? a source of vitamins, especially of group B. Sodium chloride maintains the osmotic balance of the medium.

In the second stage, the bacteria were incubated at 22°C with 2000 rpm rotation. Once the density is reached optical density of about 2 OD at 550 nanometres, the culture is diluted with sea water H20 (50% of the final volume) and added with 1.5-2% fructose, and further incubated under stirring at 22°C.

In the third stage after 24 hours the production of a yellow/orange pigment occurs. The pigment is recovered through two phases of centrifugation:

- Centrifugation at 1000g to sediment the bacteria and collect the supernatant containing the pigment

- Centrifugation at 10000 g to eliminate the particulate.

The aqueous part containing the pigment is dried in a nitrogen atmosphere, redispersed in absolute ethanol (about 40 ml) and analyzed by UV-Vis and MS.

AND? result that the pigment ? a mix of B-cryptoxanthin and lycopene/B-carotene.

After about 10 days, orange flakes or spherules are produced from the same culture in the culture medium.

The culture is incubated at 80?C to kill bacteria.

The material composed of flakes and spherules is collected and washed with sterile bidistilled water. and subsequently dried in an oven at 40°C.

A dry portion of the material is analyzed with FTIR.

AND? result that the material ? biocellulose.

A dry portion of biocellulose is treated with absolute ethanol (about 40 ml) and the pigment is extracted for subsequent analysis by UV-Vis and MS.

AND? result that the pigment ? a mix 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. Furthermore, the co-synthesis of carotenoids makes it possible to optimize a production process: starting from a single substrate, two products are obtained which can be used in a single application, such as, for example, beauty masks containing carotenoids.

Claims (6)

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 starting from food waste products. The 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. The use according to claim 1 wherein biomaterials are selected from the group consisting of biopolymers, biocellulose, carotenoids. 4. Process for the production of biomaterials which provides for a culture 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 at room temperature in the presence of culture medium added with food waste materials until the final product is obtained 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) growth of 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 stage 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 obtaining the final carotenoid and cellulose products