EP4267720A1

EP4267720A1 - Bacterial strains for biocellulose production

Info

Publication number: EP4267720A1
Application number: EP21824606.4A
Authority: EP
Inventors: Sandra PUCCIARELLI; Rita GIOVANNETTI; Marco ZANNOTTI; Alessio Mancini; Maria Sindhura JOHN; Joseph Amruthraj NAGOTH
Original assignee: Irides SRL
Current assignee: Irides SRL
Priority date: 2020-12-22
Filing date: 2021-12-20
Publication date: 2023-11-01
Also published as: IT202000031769A1; KR20230124919A; EP4267721A1; KR20230124918A; WO2022136277A1; WO2022136246A1

Abstract

A bacterial strain belonging to the genus Bacillus and the use thereof for biocellulose production, as well as a process for biocellulose production and the biocellulose obtained with said process are described.

Description

Bacterial strains for biocellulose production

★ ★ ★ ★ ★

Background of the invention

The present application relates to the field of microbiology and in particular isolated and verified bacterial strains for biocellulose production .

State of the Art

A bacterial cultivation system for the production of cellulose gel (HSBC) containing Gluconacetobacter xylinus (G. xylinus) in an interface system between an aqueous and an oily phase in the presence of glucose and mannitol at 30°C is known (Horu Hoshia, Kazuyoshi Yamazakia, Yuki Satob, Takaya Shidaa, Takao Aoyagia, Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation, Heliyon 4, 2018, e00873) .

Cellulose production by Acetobacter xylinum strains with incubation at 30 degrees is further known (Yang Hu and Jeffrey M. Catchmark, Formation and Characterization of Spherelike Bacterial Cellulose Particles Produced by Acetobacter xylinum JCM 9730 Strain, iomacromolecules 2010, 11, 1727-1734) .

Cellulose production by Gluconacetobacter xylinus with incubation in Schramm and Hestrin medium composed of 20 g/1 glucose, 5 g/1 bactopeptone, 5 g/1 yeast extract, 2.7 g/1 disodiumpohosphate and 1.15 g/1 citric acid first at 25°C under stirring for 24 hours at 150 rpm and then static cultivation at 30°C for 9 days has been described (Richard Auta, Grazyna Adamus, Michal Kwiecien, Iza Radecka, Paul Hooley, Production and characterization of bacterial cellulose before and after enzymatic hydrolysis, African Journal of Biotechnology, Vol. 16(10) , pages 470-482, 2017) .

The synthesis of cellulose by Pseudomonas strains in equine serum medium containing 2% date molasses, 1% yeast extract at pH 5 and incubation at 30°C for one week in a stirrer incubator has been described . A method for producing cellulose from glucose using strain ZTB2 of Bacillus lichenif ormis is known (Bagewadi Zabin K et al., 2020, "Statistical optimization and characterization of bacterial cellulose produced by isolated thermophilic Bacillus lichenif ormis strain ZBT2", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 491) and also from other bacteria (Lucie Bacakova et al., 2019, "Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing", NANOMATERIALS, vol. 9, no. 2) .

The production of cellulose in spherical drops produced by Gluconacetobacter xylinus and used as a vehicle for drugs is also known (Hoshi Toru et al. , 2018, "Production of hollow-type spherical bacterial cellulose as a controlled release device by newly designed floating cultivation", HELIYON, vol. 4, no. 10) .

Technical problem

In consideration of the data reported in the state of the art, the inventors of the present invention have isolated new bacterial strains belonging to the genus Bacillus and Brevundimonas capable of producing biocellulose.

Said strains have peculiar characteristics compared to strains known in the art. In particular, they are able to synthesise biocellulose under room temperature conditions, i.e. without energy expenditure, compared to known processes which take place at a temperature of around 30°C. In the process, no specific saline solutions are used or prepared, but plain water is used, and the culture medium is not a limiting factor and different types can be used in the bacterial culture without affecting the success of the process for biocellulose production. In contrast to what is known in the art in which the culture media are added with glucose and mannitol, the former at concentrations of at least 3%, in the culture of the present invention glucose or fructose at 1.5% is added.

Budapest Treaty

The two strains named Bacillus efl and Brevundimonas efl were deposited at the "Bruno Ubertini" Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna (Istituto Zooprof ilattico Sperimentale della Lombardia e dell' Emilia-Romagna "Bruno Ubertini") , also named Biobank of Veterinary Resources (BVR) of the Experimental Zooprophylactic Institute of Lombardy and Emilia- Romagna (Istituto Zooprof ilattico Sperimentale della Lombardia e dell 'Emilia Romagna) . according to the Budapest Treaty.

Bacillus efl Access no. DPS RE RSCIC 23 of 24/11/2020

Brevundimonas efl Access no. DPS RE RSCIC 24 of 24/11/2020

Object of the invention

The above technical problem is solved by providing Bacillus efl Access no. DPS RE RSCIC 23.

A further object of the present invention is the use of Bacillus efl Access no. DPS RE RSCIC 23 for biocellulose production, optionally in combination with Brevundimonas efl Access no. DPS RE RSCIC 24.

Still another object of the present invention is a process for biocellulose production including the following steps: a) incubation of Bacillus efl Access no. DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b) dilution of the culture obtained at the end of step a) with water and addition of glucose or fructose in an amount ranging between 1.5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture.

A further object of the present invention is biocellulose obtained by means of the above process.

Brief description of the figures

Figure 1 shows a representative infrared spectroscopy (FTIR) spectrum of the materials obtained in static or stirring mode with both bacteria: the spectrum indicates that the material in question is cellulose.

3

RECTIFIED SHEET (RULE 91) ISA/EP Figure 2 shows a reference spectrum from Yim, S. M.; Song, J. E., Kim, H. R. Production and characterization of bacterial cellulose fabrics by nitrogen sources of tea and carbon sources of sugar, Process Biochemistry 2017, 59, 26-36

Detailed description of the invention

Definitions

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, culture medium suitable for bacterial growth means solid or liquid solutions containing nutrients on which bacterial colonies are possible.

The object of the present invention is Bacillus efl Access no. DPS RE RSCIC 23.

Still another object of the present invention is a process for biocellulose production including the following steps : a) incubation of Bacillus efl Access no. DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b) dilution of the culture obtained at the end of step a) with water, addition of glucose or fructose in an amount ranging between 1.5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture .

Optionally in step a) Bacillus efl Access no. DPS RE RSCIC 23 is in combination with Brevundimonas efl Access no. DPS RE RSCIC 24. A further obj ect of the present invention is biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Bacillus ef l Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture .

The obj ect of the present invention is also the use of biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Bacillus ef l Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture for the absorption of antibiotics and the subsequent slow release thereof into the environment .

A further obj ect of the present invention is the use of biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Bacillus efl Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture for the production of biocellulose filters.

Optionally in stage a) Bacillus efl Access no. DPS RE RSCIC 23 is in combination with Brevundimonas efl Access no. DPS RE RSCIC 24.

By way of example, the filters can be used in air conditioning or water filtration systems, for example in swimming pools and/or aquariums to block bacterial growth and maintain a healthy environment .

In one embodiment, the biocellulose spheres contain silver nanoparticles with antimicrobial activity.

Preferably, the bacterial culture medium can be selected from the group consisting of: Hestrin-Schramm, Yamanaka, Zhou, nutrient broth or yeast extract.

More preferably the yeast extract is yeast extract (0.5-1%) .

Preferably the water is seawater or deionised water.

More preferably the nutrient broth has 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

Preferably in step a) and step b) the bacteria are incubated at a temperature ranging from 22 and 25 °C.

Optionally in step a) the bacteria are incubated statically or under stirring . When in step a ) the bacteria are incubated under stirring , they are preferably under stirring by means of a stirrer at a speed of about 150-200 rpm.

Preferably in step a ) the adequate bacterial growth is the achievement of an optical density of about 0 . 6 OD at 550 nanometres .

Preferably in step b ) if the water is seawater in an amount equal to 50% of the final volume .

Preferably in step b ) if the water is deionised water it is in an amount equal to 90 % of the final volume .

Preferably in step b ) glucose or fructose is present in an amount equal to 1 . 5% .

Preferably in stage b ) the temperature is 22 ° C .

Preferably the obtained biocellulose is in the form of a sheet when in step a ) the incubation is in static mode .

Preferably the obtained biocellulose is in the form of spheres when in step a ) the incubation is under stirring and preferably they have an average diameter ranging between 2 and 10 mm .

Examples

The first strain Bacillus ef l Access no . DPS RE RSCIC 23 was identified as belonging to the genus Bacillus on the basis of the 16S rDNA sequence , it is similar to Bacillus altitudinis , it is a gram-positive rod-shaped bacterium .

SEQ ID . NO . 1 PROKKA_03858 Bacillus 16S ribosomal RNA

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC

GGACAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAA

CCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGA

ACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCG

CATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAG

GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG

GAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTT

CGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTG ACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG

TGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTG ATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAG AAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCA GTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGA ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGT TTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCA AGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGG GCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGA GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTT GGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCA TCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGA GACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACT CGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTT CCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTG AGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGGCAGATGATTGGGGTGAAGTCGTAA CAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCT

It possesses two enzymes involved in cellulose biosynthesis , the sequences of which are given in the sequences SEQ . ID . NO 2 and SEQ . ID . NO 3 .

SEQ . ID . NO 2 cellulose enzyme 1

MSFITTYYPLFMLCMLSLLYMIYRIQPMASTVKKVI IVLCVLTNAAYICWRLFFTLPHEGTFNVVMGI LLVVCECIGFIQLLVFYTLVWKPSNRKQVMISDLERLPTVDI FIATYNEPIEVLKRTVAGCLNLSYPK ESVQIYLCDDGNREAVEQLASAFGVHYLTRTDNRFAKAGNLNHAMSQTDGELILTLDADMVPLPSFLE KTVPYFHDGATAFVQVPQAFYNEDPFQYNMFSKDRIPNEQDFFMQTLQAGKDRFNAVMYVGSNTVFRR TALDEIGGFATGVITEDMATGMLLQGKFKSVSVGEVLAVGLAPESWLDLLKQRDRWSRGNIQCARKFN PLKVRGLTLMQRVLYLDGIVYWFNGLFKMIYILTPILFLLFGIQSFWADFQSVFMFWLPAFFSSYLAF KLVSNQKRSMFWSHIYESSMAFHLAGVALSELFLKKRVEFLVTPKGIQTDKRHFHLKTMIPHLIFLLL SLLALVKIGYDVKVNGTMNADLMLINI FWVLYNGAGLFMALLVAFDRPRYRKSERFVIEKEGHFRSDH QDQSIACFLLDMSDSGARLSIPLDQASSLYQGQTQLFFQENESVSCDVVWSYPEGDKLMVGVAFTDTK KSEYLSLIRFLFIREHVAMTDREKKSHAVRTFLRFLRETEKVPKALRRKWMRRPLQGVTGTIAYEDRL EEKVPVI IHDISLSGCKIEFEDSIELNGKVLITIDTESLTDQPAIAVWTSQKRRRTMAGLKFVQPEIK

QIEEREGVVS

SEQ . ID . NO 3 cellulose enzyme 2 MKSLKWLIYILGLSLAIQPFFMAGQAMAQSNRIQVKDDWVQSSKESKTKTQHLSEDVVTLYGQEDRTE FTYQVDQEKTESSTLTFNIEASPLLISPSSFTVMIDGEIEKTIPVSGKNQKKSIQIKLNKAQLKKGTH RIQVTFYGVLKEGVCINQETPANWLKVYPESELTFKGIQTKDFTLDSFPSPFIQTGDQKEQTDIVIPN SPDAAELEAAIKVYRTLKNKDRQKEIKLIQEKDIKQVAHPTIAVGAKGSWDGRMKTIEQAAGIKTKSN HLTLAMRTLTAKKTEQPILFVTAEKPVTIAEKINVLTQSELTDQLTGTDLVLQKAAAQTSKPSHKIHL KDFGGDDVTVGTNKTASDHYYYPKALLANQKSGAKLNLSFKKSETAASKTERLTVMINDEPHDVPLTK LGSKDANGFYHVSIPVDSKILQKNEYIDLQFVTSGFKNMESCRHTDEEGWIFIDKNSSLQIPEGTTSE KPDLAAWPLPFTSKNTLII IPDQIKREIINQMAMLTESFSQPEVAQYHLIKASQVTNEQLKNHPLIFI GGIQTFSLLKEKAADLVVPVKKDQYDVSSFGMINETTARIVWTQPSVWNKEQTMTVFSGMTAAEANVS NKVMQFLQTNTEKATVAIESKNKGVFTNHQSVSSTSNSLKTSEKQSSSESWIYFVCIAALILFVLVMI VYFVRKNRKKTEF

The second strain Brevundimonas ef l Access no . DPS RE RSCIC 24 was identified as belonging to the genus Brevundimonas on the basis of the 16S rDNA sequence

SEQ . ID . NO 4 16s_rRNA-ctg_33-3

AGAGTTTGATCCTGGCTCAGAGCGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGAACTCTT CGGAGTTAGTGGCGGACGGGTGAGTAACACGTGGGAACGTGCCTTTAGGTTCGGAATAACTCAGGGAA ACTTGTGCTAATACCGAATGTGCCCTTCGGGGGAAAGATTTATCGCCTTTAGAGCGGCCCGCGTCTGA TTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGC CACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGC GAAAGCCTGACGCAGCCATGCCGCGTGAATGATGAAGGTCTTAGGATTGTAAAATTCTTTCACCGGGG ACGATAATGACGGTACCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAG GGGGCTAGCGTTGCTCGGAATTACTGGGCGTAAAGGGAGCGTAGGCGGACATTTAAGTCAGGGGTGAA ATCCCGGGGCTCAACCTCGGAATTGCCTTTGATACTGGGTGTCTTGAGTATGAGAGAGGTGTGTGGAA CTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGACACACTGGCT CATTACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGATTGCTAGTTGTCGGGATGCATGCATTTCGGTGACGCAGCTAACGCATTAAGCAATCCG CCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCACCTTTTGACATGCCTGGACCGCCAGAGAGAT CTGGCTTTCCCTTCGGGGACTAGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCATTAGTTGCCATCATTTAGTTGGGAACTCTAATG GGACTGCCGGTGCTAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACAGGGTGGG CTACACACGTGCTACAATGGCGACTACAGAGGGTTAATCCTTAAAAGTCGTCTCAGTTCGGATTGTCC TCTGCAACTCGAGGGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT TCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTCTACCCGAAGGCGCTGCGCTGAC CGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGG

GAACCTGCGGCTGGATCACCT

Brevundimonas efl Access no . DPS RE RSCIC 24 is s imilar to Brevundimonas ves icularis adapted to live in Martian conditions . Among the sequences s imilar to the 16S rRNA gene , there is one from the strain Brevundimonas sp . R-37024 isolated from a Lake Lundstrdm in Antarctica .

SEQ . ID . NO 5 FR691410 . 1 Brevundimonas sp . R-37024 partial 16S rRNA gene , strain R-37024

AGCGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGAACTCTTCGGAGTTAGTGGCGGACGGGTG AGTAACACGTGGGAACGTGCCTTTAGGTTCGGAATAACTCAGGGAAACTTGTGCTAATACCGAATGTGCC CTTCGGGGGAAAGATTTATCGCCTTTAGAGCGGCCCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCTC ACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAAC TCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGACGCAGCCATGCCGCGTGAAT GATGAAGGTCTTAGGATTGTAAAATTCTTTCACCGGGGACGATAATGACGGTACCCGGAGAAGAAGCCCC GGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATTACTGGGCGTAAA GGGAGCGTAGGCGGACATTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCTCGGAATTGCCTTTGATACT GGGTGTCTTGAGTATGAGAGAGGTGTGTGGAACTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAG AACACCAGTGGCGAAGGCGACACACTGGCTCATTACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATTGCTAGTTGTCGGGATGCATGCATTTCGGTG ACGCAGCTAACGCATTAAGCAATCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCACCTTTTGAC ATGCCTGGACCGCCAGAGAGATCTGGCTTTCCCTTCGGGGACTAGGACACAGGTGCTGCATGGCTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCATTAGTTGCCATCATT TAGTTGGGAACTCTAATGGGACTGCCGGTGCTAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATG GCCCTTACAGGGTGGGCTACACACGTGCTACAATGGCGACTACAGAGGGTTAATCCTTAAAAGTCGTCTC AGTTCGGATTGTCCTCTGCAACTCGAGGGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCG CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTCTACCCGAAGGCG CTGCGCTGACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAG CCGTAGGGGAACC

Brevundimonas ef l posses ses an enzyme involved in cellulose biosynthes is .

SEQ . ID . NO 6 cellulose enzyme 2 MIASRSRLFAASLTVLAVALAGHASAQDTTPPAPTPDTHPPAVILEPGQTAPGKGTPSQVPTQAAPDPHI I ITDT

PATPAI PAVWAPI PTNAEGRSAYGLYLAGKLALMQGEGATGSDYLAQAERLTPEQPRVREQAFTSALLTGDLDVA

AALAPTDATASPAFVEGGRLVRLVQDFVRGDARTPNAELARQPIGAPHARAGLLVAPWIAAAAGDWTRALQPVPT

AGDPLTLAFARINRAALLEKRRDYAEAEVELKTASEVAGVGALFKRPYGEFLERRGRRDDAVALYETAMAVQPVD

PGVARALQRLKDGGRPPALPDFREGAAQGLITAAAQASAERGNEFAAVYLRLAQNLHDDDETEYQLAQVLSRAGL

KSAARNALSRVGTADPKLYAAARAQLAVALEEDGQSQEALTELRRAAAASPDDRQIALVLAGQLMQLKQHDEALA

LLDGPLLNTADQGASVHFLRGAAYEALGRVPEAEAELWAALQTAPNDADMLNYLGYLWVDKGLRVQEGAEMIARA

HALEPDNGNIQDSLGWAQFKQGQYETAVNTLEEAVDKEPSNAEINDHLGDAYWKVGRQREAVWLWNRVLVLEPEP

ERRAEVERKIANGLDSALSAKGVAQ

Synthesis of biocellulose

The parameters of the process for biocellulose production from bacteria known in the art are shown in the following Table 1 .

Table 1

Two processes have been carried out to synthesise biocellulose : in both cases the first stage consists in growing the bacteria in culture medium, or nutrient broth or 1% yeast extract .

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.

The bacteria incubated at 22°C at 150-200 rpm of rotation.

Once the optical density of about 0.6 OD at 550 nanometres has been reached, the culture is diluted with seawater H₂0 (50 % of the final volume) or deionised water (90 % of the final volume) . and added with 1.5-2% glucose, and further incubated statically (without stirring) or with stirring at 22°C.

After about 5 days, either a biocellulose sheet is produced (static mode) , or biocellulose spheres are produced (stirring mode) by Bacillus efl Access no. DPS RE RSCIC 23 in nutrient broth diluted with 50% seawater and added with 1.5-2%.

After about 5 days, either a biocellulose sheet is produced (static mode) , or biocellulose spheres are produced (stirring mode) by Brevundimonas efl Access no. DPS RE RSCIC 24 of 25/11/2020 both cultivated in nutrient broth and in yeast extract occurs .

By weighing the biocellulose spheres before and after drying at 80°C, the ability to absorb a quantity of liquid equal to its own dry weight was highlighted.

The ability of the biocellulose spheres to absorb external substances as previously produced was further tested.

The ability of the biocellulose spheres to absorb methylene blue was highlighted. Methylene blue is partially released into the culture medium: after 18 hours, about l/20th of the initial amount absorbed is released. Table 2 reports the production methods ( rotation speed, medium volume , flask size ) , yield and average size of the biocellulose spheres synthesised by Bacillus ef l and Brevundimonas efl after 5 days of culture . Table 2

The biocellulose in the form of spheres was also soaked in a solution containing silver nanoparticles ( 20 mg/ml ) having antimicrobial activity, as described in the application incorporated herein for Italian patent reference no . 102019000014121 by the same inventors , 24 hours and subj ected to UV ray sterilisation for 10 minutes . Then the biocellulose spheres were placed in a plate with Muller hinton agar where a strain of Candida albicans was inoculated . The growth inhibitory effect of Candida was visible with a halo of about 4-5 mm even after 72 hours at 22 ° C .

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 identified at the bottom of this page

University of Camerino

Italy i 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. Bacillus efl Access no. DPS RE RSCIC 23.

2. Use of Bacillus efl Access no. DPS RE RSCIC 23 for biocellulose production .

3. Use according to claim 2 wherein Bacillus efl Access no. DPS RE RSCIC 23 is in combination with Brevundimonas efl Access no. DPS RE RSCIC 24.

4. Process for biocellulose production that includes the following steps : a) incubation of Bacillus efl Access no. DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b) dilution of the culture obtained at the end of step a) with water and addition of glucose or fructose in an amount ranging between 1.5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture.

5. Process according to claim 4 wherein in stage a) Bacillus efl Access no. DPS RE RSCIC 23 is in combination with Brevundimonas efl Access no. DPS RE RSCIC 24.

6. Process according to claim 4 or 5 wherein in step b) the water is seawater or deionised water.

7. Biocellulose of bacterial origin obtained by a process including the following steps: a) incubation of Bacillus efl Access no. DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b) dilution of the culture obtained at the end of step a) with water and addition of glucose or fructose in an amount ranging between 1.5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture.

8. Biocellulose according to claim 7 wherein in step a) Bacillus efl Access no. DPS RE RSCIC 23 is in combination with Brevundimonas efl Access no. DPS RE RSCIC 24. Biocellulose according to claim 7 or 8 wherein in step b ) the water is seawater or deionised water . Use of biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Bacillus ef l Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture according to any one of claims 7 to 9 for absorption of antibiotics and subsequent slow release thereof into the environment . Use of biocellulose of bacterial origin obtained by a process including the following steps : a ) incubation of Bacillus efl Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water and addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture according to any one of claims 7 to 9 for producing biocellulose filters . Biocellulose of bacterial origin obtained by a process comprising the following steps : a ) incubation of Bacillus efl Access no . DPS RE RSCIC 23 at room temperature in the presence of culture medium suitable for bacterial growth until obtaining adequate bacterial growth; b ) dilution of the culture obtained at the end of step a ) with water, addition of glucose or fructose in an amount ranging between 1 . 5 and 2% of the total and subsequent incubation at room temperature until production of biocellulose by the bacterial culture according to any one of claims 7 to 9 in the form of spheres containing silver nanoparticles with antimicrobial activity .

18