WO2021023564A1

WO2021023564A1 - Marinomonas ef1 and rhodococcus ef1 for the production of secondary metabolites

Info

Publication number: WO2021023564A1
Application number: PCT/EP2020/071193
Authority: WO
Inventors: Sandra PUCCIARELLI; Maria Sindhura JOHN; Alessio Mancini; Kesava Pryian RAMASAMY; Nagoth Joseph AMRUTHRAJ
Original assignee: Irides S.R.L.
Priority date: 2019-08-06
Filing date: 2020-07-28
Publication date: 2021-02-11
Also published as: KR20220041204A; EP4010480A1; IT201900014121A1

Abstract

Strains of bacteria Marinomonas and Rhodococcus are disclosed together with a process for the in vitro production of metabolites, in particular of Ag nanoparticles and fluorescent dyes is disclosed, and a process for the production of antiseptic textiles impregnated of Ag nanoparticles synthetized to be used for surfaces cleaning.

Description

MARINOMONAS EF1 AND RHODOCOCCUS EF1 FOR THE PRODUCTION OF SECONDARY

METABOLITES

Background of the Invention

The present invention refers to the field of microbiology since it relates to the characterization of a Marinomonas and a Rhodocossus species and their use in methods for the in vitro production of metabolites.

State of the art

It is known in the art the biological synthesis of silver nanoparticles by the strain Brevibacterium frigoritolerans DC2 (Singh et al., 2015, Biosynthesis, characterization, and antimicrobial applications of silver nanoparticles, Int. J. of Nanomedicine, 10, 2567-2577) and by the strain Ochrobactrum anhtropi (Roshmi et al., 2014, Antibacterial properties of silver nanoparticles synthesized by marine Ochrobactrum sp, Brazilian J. of Microbiology 45, 4, 1221-1227).

More in details silver nanoparticles are synthetized by the strain Ochrobactrum anhtropi by the method wherein the bacterial is in an Erlenmeyer flask containing 100 mL marine broth, than the flask is incubated in a rotating shaker at room temperature and agitated at 200 rpm for 48 hours; after incubation, the biomass and supernatant are obtained by centrifugation at 12000 rpm for 10 minutes and used separately for the synthesis of silver nanoparticles. For the extracellular production of silver nanoparticles, the supernatants are mixed with a filter sterilized AgN03 solution with a final concentration of 1 mM; for intracellular production; 2 grams of bacterial wet biomasses is re-suspended in 100 mL aqueous solution of 1 mM AgN03 in a 250 mL Erlenmeyer flask, then the mixtures are kept on rotating shaker set at 200 rpm for a period of 72 hours at room temperature in light, then the heat killed biomass and heat inactivated supernatant incubated with silver nitrate and silver nitrate solution alone were also maintained as control, the bio reduction of Ag+ ions was monitored by changes in colour, and also the optical characteristics of synthesized silver nanoparticles were measured using UV-visible spectrophotometer (Hitachi U5100) at 200- 800 nm range with control as reference (Roshmi et al., 2014, Antibacterial properties of silver nanoparticles synthesized by marine Ochrobactrum sp, Brazilian J. of Microbiology 45, 4, 1221- 1227).

Silver nanoparticles are produced by the strain Brevibacterium frigoritolerans DC2 by a method wherein the bacterial are inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile Tryptic soy broth, the cultured flasks are then incubated in a rotating shaker set at 37°C for 120 rotations per minute (rpm) for 24 hours, after the incubation time, the culture are centrifuged at 8,000 rpm for 10 minutes to remove the bacterial pellet; the obtained supernatant is mixed with a filter-sterilized AgN03 solution, with a final concentration of 1 mM, for the extracellular production of silver nanoparticles. Furthermore, the culture supernatant with 1 mM AgN03 is incubated in an orbital shaker at 200 rpm and 25°C, and the synthesis of silver nanoparticles monitored by visual inspection for a change in the colour of the culture medium. After the completion of the incubation period, the mixture is first centrifuged at 2,000 rpm for 5 minutes to remove any medium components, and then the silver nanoparticles are collected by high speed centrifugation at 16,000 rpm for 20 minutes; the obtained product is washed several times by centrifugation and re-dispersed in water to remove the unconverted silver ions and any medium components.

Is known in the art the reduction of aqueous silver ions to silver nanoparticles by biomass of B. cereus and Bacillus sp. (Babu G, Gunasekaran P (2009) Production and structural characterization of crystalline silver nanoparticles from Bacillus cereus isolate. Colloids Surf B 74:191-195; Das VL, Thomas R, Varghese RT, Soniya EV, Mathew J, Radhakrishnan EK (2013). Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area. 3 Biotech 4: 121- 126). The process known in the art do not provide really pure nanoparticles.

Chinese patent N. CN105002117 discloses the micro-ecological preparation of silver pomfret wherein a culture medium sequentially inoculated with 25-30% by weight of lactobacillus plantarum, 20-25% by weight of saccharomycetes, 25-30% by weight of denitrified marinomonas sp and 10-15% by weight of bacillus subtilis, is fermented, and the so obtained bacterium liquid is mixed with a carrier material to obtain the micro-ecological preparation special for the silver pomfret.

Korean patent n. KR20150070896 discloses strains of Marinomonas arctica PT-1, a proteolytic composition including the strains and the culture product, a proteolytic kit including the composition, and a proteolytic method to break down the casein, gelatin, and protein substances contained in animal waste or contaminated seawater to be used to purify animal waste or contaminated seawater.

US patent application n. US2002/0072108 discloses a process for preparing an anti-freeze peptide and to the peptides obtained from bacteria from an aqueous low-temperature environment, such as Marinomonas protea and a novel Pseudomonas species, wherein said anti-freeze peptides can be incorporated in frozen food products.

Us patent application n. US2017/0204317 DISCLOSES anti-freeze proteins produced by the bacteria Marinomonas primoryensis which are used in additive compositions, fluids compositions and methods for reducing freezing point of fluids compositions.

Phenazines are used as colorimetric redox indicators and are used in the development of sensors and in nanotechnology, for example, a phenazine derivative was used to develop a luminescence-based pH sensor (O.A. Ryazanova, I.M. Voloshin, V.L. Makitruk, V.N. Zozulya, V.A. Karachevtsev,pH-Induced changes in electronic absorption and fluorescence spectra of phenazine derivatives,Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,Volume 66, Issues 4- 5, 2007,Pages 849-859,) and to develope an amperometric sensor for hydrogen peroxide determination utilizing neutral red attached to multi-walled carbon nanotubes (D R Shobha Jeykumari and S Sriman Narayanan. Covalent modification of multiwalled carbon nanotubes with neutral red for the fabrication of an amperometric hydrogen peroxide sensor. Nanotechnology. 2007 Volume=18, number=12,125501. Phenazines conjugated to other compounds are components of organic light emitting devices (OLED), such as a phenanthroline-fused phenazine (JP Chen and CL Xiao-Chang 2004,0rganis light emitting device having phenanthroline-fused phenazine . US patent 6313781).

Phenazines are associated with antitumor activities (Laursen JB, Nielsen J. Phenazine natural products: Biosynthesis, synthetic analogues, and biological activity. Chemical Reviews. 2004;104:1663- 1685; Mavrodi DV, Blankenfeldt W, Thomashow LS, Mentel M. Phenazine compounds in fluorescent Pseudomonas spp biosynthesis and regulation. Annual review of Phytopathology. 2006;44:417-445), cells that are actively respiring, such as tumor cells, appear to be more susceptible to respiratory interference and ROS generation caused by phenazine compounds. Additionally, phenazine derivatives are known to interfere with topoisomerase I and II activities in eukaryotic cells have been identified. Cancer cells, having high levels of both topoisomerases, are more susceptible to this interference.

Cadmium and tellurite resistant Antarctic bacteria belonging to the genera Pseudomonas, Psychrobacter and Shewanella cultured in LB Medium are capable of synthesizing CdS and CdTe quantum dots when exposed to these oxidizing heavy metal (D. O. Plaza, C. Gallardo, Y. D. Straub, D. Bravo and J. M. Perez-Donoso, Biological synthesis of fluorescent nanoparticles by cadmium and tellurite resistant Antarctic bacteria: exploring novel natural nanofactories, Plaza et al. Microb Cell Fact (2016) 15:76).

Potential pathogenicy is a drawback of producing metabolites from bacteria, as reported, for example, for Ochrobactrum anthropi (Adrian Kettaneh, Francois-Xavior Weill, Isabelle Poilane, Oliver Fran, Michel Thomas, Jean-Louis Herrmann, Laurent Hocqueloux. Septic Shock Caused by Ochrobactrum anthropi in an Otherwise Healthy Host. J. Clin Microbiol 2003. doi: 10.1128/JCM.41.3.1339-1341.2003), and for strains that belong to the genus Pseudomonas (Jin-Hyung Lee, Yong-Guy Kim, Moo Hwan Cho, Jintae Lee, ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production, Microbiological Research, Volume 169, Issue 12, 2014, Pages 888-896).

Another drawback of producing metabolites from bacteria is that the optimum temperature for the growth of the strains is only between 20 and 37^°C (B. Holmes, M. Popoff, M. Kiredjian, M. Kersters. Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd. International Journal of Systematic and Evolutionary Microbiology 2012. doi: 10.1099/00207713-38-4-406).

Moreover the secondary metabolites produced by said strains are usually active to a narrow range of virulent organisms.

Another limiting factor is that the marine broth usually used for bacteria culturing and the production of nanoparticles is quite expensive.

The only known and characterised secondary metabolite produced by Marinomonas is Marinocine produced by Marinomonas mediterranea (Lucas-Elio P, Gomez D, Solano F, Sanchez-Amat A. The Antimicrobial Activity of Marinocine, Synthesized by Marinomonas mediterranea, Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity. Journal of Bacteriology. 2006;188(7):2493-2501. doi:10.1128/JB.188.7.2493-2501.2006).

It is of interest the environmental impact of the silver nanoparticles (AgNP) and their application in consumer products including textiles, medical products, domestic appliances, food containers, cosmetics, paints and nano-functionalised plastics (McGillicuddy E., et al, Silver nanoparticles in the environment: Sources, detection and ecotoxicology, Sci Total Environ. 2017 Jan 1;575:231-246). It is of particular interest in the medical field the application of the silver nanoparticles (AgNP) as antimicrobial against human pathogenic microbes that are multi-resistance to antibiotics (MDR) (Rai M.K. et al., Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria, J Appl Microbiol. 2012 May;112(5):841-52).

The Korean patent n. KR20130077122 discloses the recovery of silver (Ag) from waste water and a method to generate electricity by a microbial fuel cell.

Silver nanoparticles (Ag-NPs) can be synthesised by cyanobacteria and micro-algae (Vijay Patel et al., Screening of cyanobacteria and microalgae for their ability to synthesize silver nanoparticles with antibacterial activity, Biotechnol Rep (Amst). 2015 Mar; 5: 112- 119).

The Chinese patent n. CN101475930 discloses the use of superparamagnetic nanoparticles, in situ adsorption and the separation of immobilized Rhodococcus for catalytic desulfation.

The Chinese patent n. CN107777838 discloses a method for water purification for aquaculture by a water purifier containing Rhodococcus.

The Chinese patent n. CN107988096 discloses a complex substance for pollutants degradation that includes among the other substances microorganisms as Rhodococcus.

The Czech patent n. CZ307392 discloses polyamide nanofibres supports containing microorganisms including Rhodococcus.

The French patent n. FR2873386 discloses the preparation of a vaccine against Rhodococcus equi.

The Russian patent n. RU2011151802 discloses a method for water purification from hydrocarbons with microorganisms including Rhodococcus. The international patent application publication n. W02018/107291 discloses a method for the production of tellurium nanostructure using Rhodococcus aetherivorans (BCP1) in a medium containing tellurium, in aerobic conditions.

The German patent n. DE102005031711 discloses a process for the preparation of antiseptic textiles by diffusion of a water solution containing Ag nanoparticles on textiles, incubation and drying and their use for surfaces cleaning.

The Chinese patent n. CN107893038 discloses a process in vitro for metabolites productions by Isoptericola SYSUZL-3.

Technical problem

The inventors of the present invention, in view of the findings of the prior art, characterized a new species of Marinomonas and Rhodococcus and investigated its role in the in vitro production of useful metabolites.

The same inventors unexpectedly found that the specific Marinomonas and Rhodococcus strains grow in a wide range of temperatures, from 4° to 30 °C, and is able to produce pure silver nanoparticles that are active to a wide range of virulent pathogens, by mean of a protocol which is simple, quick and avoid the use of toxic chemicals.

More in details, the inventors investigated the protocol disclosed by Singh et al., 2015, Biosynthesis, characterization, and antimicrobial applications of silver nanoparticles, Int. J. of Nanomedicine, 10, 2567-2577 reporting that the isolate bacteria were inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile Tryptic soy broth, then cultured flasks were incubated in a rotating shaker set at 37°C for 120 rotations per minute (rpm) for 24 hours and, after the incubation time, the culture was centrifuged at 8,000 rpm for 10 minutes to remove the bacterial pellet. The supernatant was obtained and was mixed with a filter sterilized AgN03 solution (1 mM final concentration) for the extracellular production of silver nanoparticles. Furthermore, the culture supernatant with 1 mM AgN03 was incubated in an orbital shaker at 200 rpm and 25 °C. The synthesis of silver nanoparticles was monitored by visual inspection for a change in the color of the culture medium. After the completion of the incubation period, the mixture was first centrifuged at 2,000 rpm for 5 minutes to remove any medium components, and then the silver nanoparticles were collected by high speed centrifugation at 16,000 rpm for 20 minutes. The obtained product was washed several times by centrifugation and was re-dispersed in water to remove the unconverted silver ions and any medium components. Finally, the silver nanoparticles were collected in the form of a pellet and were used for characterization.

The inventors found that the above method is not well explained and impossible to reproduce and do not produce real pure nanoparticles. Furthermore, salts are used in the purification steps, which may interfere with further experiments.

In addition, it was also found by the same inventors that also the method disclosed by Roshmi et al., 2014 do not produce real pure nanoparticles.

The inventors also investigated the protocol disclosed by Roshmi et al., 2014, Antibacterial properties of silver nanoparticles synthesized by marine Ochrobactrum sp, Brazilian J. of Microbiology 45, 4, 1221-1227 which reports: for nanoparticles synthesis the isolate bacteria were freshly inoculated in an Erlenmeyer flask containing 100 mL marine broth. The flask was incubated in a rotating shaker at room temperature and agitated at 200 rpm for 48 h. After incubation, the biomass and supernatant were obtained by centrifugation at 12000 rpm for 10 minutes and were used separately for the synthesis of silver nanoparticles. For the extracellular production of silver nanoparticles, supernatants were mixed with filter sterilized AgN03 solution with a final concentration of 1 mM. For intracellular production; 2 g of bacterial wet biomasses were re-suspended in 100 mL aqueous solution of 1 mM AgN03 in a 250 mL Erlenmeyer flask. Then the mixtures were kept on rotating shaker set at 200 rpm for a period of 72 h at room temperature in light. The heat killed biomass and heat inactivated supernatant incubated with silver nitrate and silver nitrate solution alone were also maintained as control. The bio-reduction of Ag+ ions was monitored by changes in colour. Also the optical characteristics of synthesized silver nanoparticles were measured using UV-visible spectrophotometer (Hitachi U5100) at 200-800 nm range with control as reference. For purification, the bacterial pellets were collected by centrifugation at 8,000 rpm for 10 minutes, re-suspended in 0.9% NaCl solution, and centrifuged again at 8,000 rpm for 10 minutes. The washing process was repeated three times to ensure removal of any undesirable materials. The pellets were then transferred to a test tube and were disrupted by ultrasound sonicator. This was then re-suspended and centrifuged (12,000 rpm) in a solution containing 0.5 M NaCl and 0.5 M sucrose and was finally re-suspended in 35 mL of complete salt solution [NaCl (17.5 g/L), KC1 (0.74 g/L), MgS04- 7H20 (12.3 g/L), Tris HC1 (0.15 g/L), and pH 7.5]. For the lysis of remaining cells, this was then treated with 20 mg of egg white lysozyme and incubated at 22 °C for 18 h. Then the nanoparticles were separated from the lysed mixture by wash with the complete salt solution and for further characterization and application, the cleaned nanoparticles were re-suspended in deionized water and stored under ambient condition.

The same inventors verified that by the above protocol the nanoparticles are not pure.

In view of the drawbacks of the prior art the same of the present invention characterized the species Marinomonas efl Marinomonas sp and Rhodococcus efl Rhodococcus sp., and investigated its properties, unexpectedly finding that can be efficiently used for the in vitro preparation of useful metabolites.

The technical problem is therefore to provide a methods for the preparation of useful metabolites, and in particular of Ag nanoparticles, which overcomes the drawbacks of the protocols disclosed by the prior art.

Budapest treaty

Marinomonas efl E Rhodococcus efl have been deposited c/o the Istituto Zooprofilattico Sperimentale della Lombardia of Emilia- Romagna "Bruno Ubertini", in conformity with Budapest treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, 28 April 1977.

Marinomonas efl Access n. DPS RE RSCIC 4 of 08/05/2019 Rhodococus efl Access n. DPS RE RSCIC 17 of 08/05/2019

Object of the invention

The above technical problem is solved by providing an in vitro method for the preparation of metabolites by Marinomonas efl or Rhodococcus efl comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising peptides deriving from enzymatic digestion suitably supplemented with additives depending on the metabolites to be prepared; b)Collecting a sample of marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 5 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising a precursor of the metabolites to be prepared until the metabolite is obtained; e) Purification of the metabolite as obtained in step d).

Are also object of the present invention the metabolites as obtained by the above process. A further object of the present invention is a method for the preparation of Ag nanoparticles comprising the following steps: a)Coulturing Marinomonas efl or Rhodococcus efl in a culture medium comprising tryptone, yeast extract and NaCl; b)Collecting a sample of marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b); d) Re-suspending biomass as obtained in step c) in a aqueous solution comprising AgNO₃, until Ag nanoparticles are obtained; e) Purification of Ag nanoparticles as obtained in step d).

Are also object of the present invention the Ag nanoparticles as obtained by the above process.

A further object of the present invention is a process for the preparation of antiseptic textiles comprising the following steps: a)Coulturing Marinomonas efl or Rhodococcus efl in a culture medium comprising tryptone, yeast extract and NaCl; b)Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separating biomass and supernatant as obtained in step b); d) Re-suspending biomass as obtained in step c) in a aqueous solution comprising AgNO₃; e) Spreading the acqueous solution as obtained in step d) on textiles, and incubation at 22 °C for 24 hours; f) Drying the textiles obtained in step e) at 37 °C for 2 hours, wherein the obtained antiseptic textiles are impregnated of Ag nanoparticles which are synthesized at the end of step d). Is another object of the present invention the use of the antiseptic textiles obtained by the above process for surface cleaning.

A further object of the present invention is a method for the preparation of fluorescent dyes comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising proteose-peptone, K₂HPO_4, MgSO₄, glycerol and distilled water; b) centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b); d) Re-suspending biomass as obtained in step c) in a culture medium in the presence of CaCl₂ until the synthesis of the fluorescent dye is completed; e) Separation of the fluorescent dye as obtained in step d).

Alternatively in step a) the medium comprising proteose-peptone, K₂HPO₄, MgSO₄, glycerol and distilled water is replaced by a medium containing glutammic acid sodium salt, K₂HPO₄, MgSO₄, NaCl and distilled water.

Are also object of the present invention the fluorescent dyes as obtained by the above process.

Brief Description of the Drawings

Figure 1 shows the growth curves of Marinomonas sp. efl in LB medium at 4°C, 10°C, 22°C, and 30°C. Data represent mean + standard error of three replicates.

Figure 2 shows the phylogenetic analysis by Maximum Likelihood method of Marinomonas species based on 16S rRNA gene sequences.

Figure 3 shows the UV-Vis spectra of the bio synthesized AgNPs by Marinomonas sp. efl of various concentrations Figure 4 shows the analysis of the mean dimensions of silver nanoparticles produced by Rhodococcus efl estimated by the light dynamic diffusion technique.

Detailed Description of the Invention

Definitions

Within the meaning of the present invention Marinomonas sp. efl is a member of the bacterial consortium associated to the psychrophilic ciliate E. focardii endemic of the Antarctic coastal seawaters. The genoma of Marinomonas sp. efl is known and disclosed in uniprot. Accession N. 2005043. It is filed c/o the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna "Bruno Ubertini" in accordance of the Budapest treaty. Access .n. DPS RE RSCIC 4 of 08/05/2019.

Within the meaning of the present invention full length of 16S rRNA gene sequence of Marinomonas sp. efl is SEQ. ID. NO. 1:

Within the meaning of the present invention Rhodococus efl belongs to Rhodococcus genus, aerobic Gram-positive bacteria, non corporative, non motile. It is a member of the bacterial consortium associated with the Antarctic ciliate Euplotes focardii.

It is filed c/o the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna "Bruno Ubertini" in accordance of the Budapest treaty. Access .n DPS RE RSCIC 17 of 08/05/2019

Within the meaning of the present invention metabolites produced by Marinomonas sp. efl or Rhodococcus efl are selected from the group consisting of: Ag nanoparticles, fluorescent dyes.

Within the meaning of the present invention culture medium means a culture medium is a nutrient broth or a lysogeny broth being a d medium comprising complex ingredients, such as yeast extract, peptides from enzymatic digestion such as casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions.

Within the meaning of the present invention peptides from enzymatic digestion are water-soluble mixtures of polypeptides and amino acids formed by the partial hydrolysis of protein, such as tryptone which is an assortment of peptides formed by the digestion of casein by the protease trypsin, or proteose-peptone.

Within the meaning of the present invention the additives used to supplement the culture medium comprising peptides from enzymatic digestion are selected from the group consisting of: glycerol, yeast extract, NaCl, K₂HPO_4, MgSO₄. Within the meaning of the present invention precursor of the metabolites means any chemical which should be added to the culture medium of Marinomonas efl or Rhodococcus efl comprising in order to induce the production by said bacteria of the desired metabolite, for example the precursor of the metabolites can be selected from the group consisting of: CaCl₂, AgNO₃.

Object of the present invention is a method for the preparation of metabolites by Marinomonas efl comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium containing peptides from enzymatic digestion suitably supplemented with additives depending on the metabolites to be prepared; b)Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising a precursor of the metabolites to be prepared until the metabolite is obtained; e) Purification of the metabolite as obtained in step d).

Are also object of the present invention the metabolites as obtained by the above process.

When the metabolite to be prepared is Ag nanoparticles, the method for the preparation of Ag nanoparticles comprises the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising tryptone, yeast extract and NaCl; b)Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 5 minutes; c) Separation of biomass and supernatant as obtained in step b); d) Re-suspending biomass as obtained in step c) in a aqueous solution comprising AgNO₃, until Ag nanoparticles are obtained; e) Purification of Ag nanoparticles as obtained in step d).

A further object of the present invention is a process for the preparation of antiseptic textiles comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising tryptone, yeast extract and NaCl; b)Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separating biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution comprising AgNO₃, e) Spreading the aqueous solution as obtained in step d) on textiles, incubation at 22 °C for 24 hours f) Drying the textiles obtained in step e) at 37 °C for 2 hours.

Another object of the present is the use of the present invention antiseptic textiles as obtained by said process for surfaces cleaning.

Textiles may be for example tissue or sponges for surfaces cleaning, in particular hospitals cleaning.

When the metabolite is the fluorescent dye, the method for the preparation of the fluorescent dye comprises the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising proteose-peptone, K₂HPO₄, MgSO₄, glycerol e distilled water; b) centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 10000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separating biomass and supernatant as obtained in step b); d) Re-suspending biomass as obtained in step c) in a aqueous solution comprising CaCl₂ until the synthesis of the fluorescent dye is completed; e) separation of the fluorescent dye obtained in d).

Are also object of the present invention the fluorescent dye directly obtained by said process.

Alternatively, in step a) the culture medium comprising proteose- peptone, K₂HPO₄, MgSO₄, glycerol e distilled water is replaced by a medium comprising sodium salt of glutamic acid, K₂HPO₄, MgSO₄, NaCl and distilled water.

Preferably in step b) the temperature is comprised between 0°C and 22 °C.

Preferably in step b) the temperature is room temperature.

Preferably in step b) frequency of rotation is 16000 rpm.

Preferably in step b) centrifugation is for a period of 2 minutes.

Preferably in step e) purification of metabolites is carried out with a method selected from the group consisting of filtration, chromatography, centrifugation.

Preferably in the method for the preparation of Ag nanoparticles in step a) the culture medium consisting of 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL of deionized water at pH 7.

Preferably, in the method for the preparation of Ag nanoparticles in step d) AgNO₃ is in a concentration comprised between 0.8 and 6 nM. Preferably in the method for the preparation of fluorescent dyes in step a) the culture medium consisting of a total of 500 ml, distilled water 500 ml, proteose-peptone 10g, K₂HPO₄ 0,75 g, MgSO₄.7H200,75 g, glycerol 5 ml, AGAR 7,5.

Preferably in the method for the preparation of fluorescent dyes in step d) CaCl₂ is added in a concentration between 20 and 30 nM.

More preferably, in the method for the preparation of fluorescent dyes in step d) CaCl₂ is added in a concentration of 20 nM.

More preferably the fluorescent dye is phenazine.

Examples

Example 1 Marinomonas sp. efl characterization

Materials and methods: Marinomonas sp. efl bacterial strain was isolated from the cold-adapted ciliate E. focardii, maintained in laboratory at 4 °C and fed with the green algae Dunaliella tertiolecta. The logarithmically growing cultures were harvested by centrifugation at 3000 rpm for lOmin and the pellet was resuspended with sterile sea water. The suspension was sonicated for 5-10 seconds at a pulse rate of 6V. The total cell extract was then inoculated directly into Luria Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with agar (1,5%) and incubated at 4 °C for one week. The pure single colonies were obtained and sub cultured routinely in LB agar plate. The isolated strains were stored in 30% glycerol at -20 °C for further use. Mean optimal growth temperature of the bacterial strain in LB medium was estimated by incubating the cells at 4 °C, 10 C, 22 °C and 30 °C, and monitoring the increasing of the absorbance at 600 nm (OD600) using a spectrophotometer. Growth data were fitted with a logistic standard model (Fujikawa H, Morozumi S. 2005. Modeling surface growth of Escherichia coli on agar plates. Appl Environ Microbiol 71:7920-6.). Total genomic DNA were extracted from fresh overnight culture at 22 °C using PureLinkTM Genomic DNA mini kit (Invitrogen) according to manufacturer's instructions. The quantity and quality of the extracted DNA were determined using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and by agarose (1% concentration) gel electrophoresis. The 16S rRNA gene (SEQ. ID. NO. 1) was amplified by PCR using bacterial universal degenerated primers 27F:

SEQ ID NO. 2 FORWARD PRIMER (5'- agagtttgatcmtggctcag 3')

SEQ ID NO. 3 REVERS PRIMER 1492R (5’-tacggytaccttgttacgactt 3’

The amplification was performed in a Biometra Thermal Cycler (Biometra Ltd., Kent, UK) and was composed by an initial denaturation of 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for lmin, annealing at 60 °C for lmin, and extension at 72 °C for lmin. A final extension step was performed at 72 °C for 5 min. The sequencing of 16S rDNA amplicon was performed by BMR Genomics (Padova, Italy).

The full length of 16S rRNA gene sequence of Marinomonas sp. efl SEQ. ID. NO. 1 (acc. No: MF156139) was compared with homologous sequences published from other Marinomonas species in the Genbank database: Marinomonas polaris (AJ833000), Marinomonas hwangdonensis (JN087530), Marinomonas arctica (DQ492749), Marinomonas prymoriensis (AB074193), Marinomonas profundimaris (kC565667), Marinomonas ushuaiensis (AJ627909), Marinomonas rhizomae (EU188443), Marinomonas foliarum (EU188444), Marinomonas pontica (AY539835), Marinomonas alcarazii (EU188442), Marinomonas arenicola (AB467281), Marinomonas pollencensis (EU188441), Marinomonas dokdonensis (DQ011526), Marinomonas equiplantarum (EU188447), Marinomonas posidonica (CP002771), Marinomonas balearica (EU188448), Marinomonas mediterranea (CP002583), Marinomonas brasiliensis (GU929940), Marinomonas vaga (XP67025), Marinomonas communis (DQ011528), Marinomonas aquimarina (AJ843077), Marinomonas ostreistagni

(AB242868), Marinomonas sp. AN44 (JQ409370). The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model (Jukes TH, Cantor CR. 1969. Evolution of protein molecules, p 21-132. In Munro HN (ed), Mammalian Protein Metabolism. Academic Press, New York). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There was a total of 1544 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731-9.).

For fluorescent in situ hybridization (FISH), E. focardii cells were fixed with 100% methanol and hybridized with 20m1 of hybridization buffer composition containing 2m1 of both specific fluorescent probes. Applied probes were:

SEQ ID NO. 4 MAR707 Fluos (5'-gccactgatgttccttccta-3')

SEQ ID NO. 5 AR662 cy3: (5'-ggaaattccacacccctcta-3') and the slides were incubated at 46 °C for 3-4h in a humid chamber. The slides were incubated with 2XSSCP (lx SSCP = 0.15M NaCl, 0.015 M sodium citrate, 0.02 M sodium phosphate. pH 7.0) at 48 °C for 20 min. The slides were rinsed with filtered distilled water and the cover slide was mounted with glycerol. FISH results were visualized at the Nikon fluorescent microscope.

The genome was sequenced by Next Generation Sequencing (NGS) using a Whole Genome Shotgun (WGS) approach. Illumina reads were "de novo" assembled using SPAdes version 3.6 (Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-77). The genome was annotated using Prokka version 1.11 (Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-9) and Blast2GO (Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A. 2008. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420-35.). The annotation step was performed with default parameters. The multiple genome alignment was generated by Mauve software (Darling AC, Mau B, Blattner FR, Perna NT. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14:1394- 403.) to produce a set of genome islands found exclusively in Marinomonas sp. Ef1.

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession NC 009654 NZ AAVH01000000 NZ_AAVH01000001-NZ_AAVH01000052.

Gene Ontology (GO) analysis was performed by an enrichment evaluation using the Fisher's Exact Test available of the Blast2GO software (Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J,Conesa A. 2008. High- throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420-35). The distributions of Marinomonas sp. efl and M. polaris CK13 GO terms were compared considering all the GO categories (molecular function, biological process and cellular component) using a p-value threshold of 0.05 and a multiple testing correction of false discovery rate (FDR) as p-value filter mode (Benjamini Y, Hochberg Y. 1995. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 57:289-300).

Results: a preliminary characterization of the isolated strain was obtained by the analysis of the 16S rDNA gene sequence. The sequence of the 1450 bp 16S rDNA amplicon (acc. No: MF156139) was used as query for a Blastn search on the NCBI data bank (https://blast.nebi.nlm.nih.gov/Blast.cgi).

SEQ ID NO. 6: 1450 bp 16S rDNA amplicon

Highest similarities were found to the 16S rDNAs from strains of the genus Marinomonas, including Marinomonas Bswl0506 (EF437161, 99% identical, E-value = 0.0), isolated from Antarctic seawaters,

SEQ ID NO. 7: 16S rDNAs from Marinomonas Bswl0506

and the Subantarctic species M. polaris CK13 (AJ833000) (99% identical, E-value = 0.0).

Therefore, the newly isolated strain was named Marinomonas sp. efl. The maximal growth temperature of Marinomonas sp. efl in LB medium is 22 °C, with a significant drop at 30 °C , as shown in figure 1, indicating that it can be classified as psychrophile (De Maayer P, Anderson D, Cary C, Cowan DA. 2014. Some like it cold: understanding the survival strategies of psychrophiles. EMBO Rep 15:508-17).

A phylogenetic tree constructed with the obtained 16S rDNA sequence and closely related sequences confirmed the evolutionary relationship with other Antarctic Marinomonas species. Marinomonas sp. efl forms a clade with the Antarctic Marinomonas Bswl0506, the Subantarctic M. polaris CK13, and Marinomonas sp. CK16 (also isolated from Antarctic sea waters) supported by a high bootstrap value. FISH experiments using oligonucleotide probes specific to the characterized 16S rRNA, SEQ. ID.NO 4, named MAR707 Fluos and SEQ. ID.NO 5, named MAR662 cy3) showed that the fluorescence signals are localized mainly close to the cell membrane of E. focardii, suggesting that Marinomonas sp. efl is mainly localized at the surface of the host cells in a potential ectosymbiotic relationship, or is inside the E.focardii cells.

The whole genome sequence consists of 4,740,116 bp (SEQ. ID. NO: 9) the G+C content is 42.55%, falling in the range of other Marinomonas genomes (Liao L, Sun X, Yu Y, Chen B. 2015. Draft genome of

Marinomonas sp. BSi20584 from Arctic sea ice. Mar Genomics 23:23- 5.). The following table 1 reports the characteristics of the genome of the Subantarctic species M. polaris CK13, that is the closest relative to Marinomonas sp. efl, and Marinomonas sp. efl whole- genome sequence.

Table 1

To check the gene ontology (GO) terms distribution of Marinomonas sp. efl and to verify GO term changes with respect to M. polaris CK13, was performed an enrichment analysis using the Fisher's Exact Test available in the Blast2GO software (Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M,

Dopazo J, Conesa A. 2008. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420-35.). After the complete annotation of Marinomonas sp. efl and M. polaris CK13 genomes, the two distributions were compared considering all the GO categories: molecular function, biological process and cellular component, as shown in figure 2. The comparison shows that the only GO term that is less represented in the Marinomonas sp. efl is transposition DNA mediated. By contrast, all the other enriched terms are more represented in the Marinomonas sp. efl genome. To produce a set of genome islands found exclusively in Marinomonas sp. efl, the enriched terms were also compared with other phylogenetically related Marinomonas strains: M. acquimarina, M. mediterranea, Marinomonas MWYL1, M. profundimaris, M. ushuaiensis. We found two main categories of genes: genes exclusively present in Marinomonas sp. efl and genes that show a low percentage of identity with the homologous from other Marinomonas, The first category is represented by gene encoding the following molecules: Ankyrin repeats containing protein, 2-keto-4-pentenoate hydratase, and the alpha and beta subunits of the benzene 1,2-dioxygenase .

SEQ. ID. NO: 10 gene encoding Ankyrin repeats containing protein,

SEQ. ID. NO: 11 gene encoding 2-keto-4-pentenoate hydratase,

SEQ. ID. NO: 12 genes encoding and the alpha and beta subunits of the benzene 1,2-dioxygenase

SEQ. ID. NO: 13 Benzene 1,2-dioxygenase subunit beta

A phylogenetic tree was constructed a using these sequences and the most similar identified by Blast search (figure 3). All the Marinomonas sp. efl sequences cluster in clades that are different from those containing the other Marinomonas sequences. Three out of four sequences appear phylogenetically close to homologous genes from Nitrincola sp. suggesting that these genes might derive from a horizontal gene transfer event. Whereas it is difficult to give an interpretation on the biological role of the ankyrin repeats proteins which is found in different protein families), the role of 2-keto-4-pentenoate hydratase and benzene 1,2-dioxygenase alpha and beta subunit may be used to perform alternative metabolic pathways.

Example 2 silver nanoparticles preparation by Marinomonas efl and antibacterial properties

Materials and Methods: Marinomonas efl, Luria-Bertani (LB) medium [10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water ,pH 7], Mueller- Hinton Agar [2 g Beef extract, 17.50 g Acid hydrolysate of casein, 1.50 g Starch,17 g Agar, 1000 dist. Water], Silver Nitrate. Pathogenic bacteria used were Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus agalactie, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, Citrobacter koseri, Acinetobacter baumanii, Serratia marcescens, Candida albicans, Candida parapsilosis.

For nanoparticles synthesis studies, Marinomonas efl was freshly inoculated in an Erlenmeyer flask containing 100 mL LB broth. The flask was incubated in a rotating shaker at room temperature and agitated at 200 rpm for 48 h. After incubation, the biomass and supernatant were obtained by centrifugation at 8000 rpm for 10 min and were used separately for the synthesis of silver nanoparticles.

For intracellular production, bacterial wet biomass of 2 g were re suspended in 100 mL aqueous solution containing different concentrations of AgNO₃ i.e., 0.4mM, 0.8mM, ImM, 2mM, 3Mm, 4Mm and 6mM in a 250 mL Erlenmeyer flask. Then the mixtures were kept on rotating shaker set at 200 rpm for a period of 72 h at room temperature in light. The heat killed biomass incubated with silver nitrate solution maintained as control. The bio reduction of Ag+ ions was monitored by changes in colour [Ronald S. Oremland, Mitchell J. Herbel, Jodi Switzer Blum, Sean Langley, Terry J.Beveridge, Pulickel M. Ajayan, Thomas Sutto, Amanda V. Ellis, and Seamus Curran. Structural and Spectral Features of Selenium Nanospheres Produced by Se- Respiring Bacteria. Applied And Environmental Microbiology, Jan. 2004, Vol. 70, No. 1, p. 52-60).

Bacterial biomass were suspended in 1 ml of PBS buffer containing 1 mg/ml lysozyme and incubated at room temperature for 30 min. The cells were subjected to disruption by sonication (3 min with 30 s intervals), after centrifuging at 5,000_*g for 5 min. Afterwards, 1 ml of the solution was ultra centrifuged over a 40-60% sucrose cushion at 30,000_*g for 2 h [30.Monras JP, Diaz V, Bravo D, Montes RA, Chasteen TG, Osorio-Roman 10, et al. (2012) Enhanced Glutathione Content Allows the In Vivo Synthesis of Fluorescent CdTe Nanoparticles by Escherichia coli. PLoS ONE7 (11): e48657.]. To remove contaminating proteins the resulting solution was fitered using Sephadex G-50 Medium, a well established gel filtration medium for desalting and buffer exchange of biomolecules. Finally the resulting solution, highly enriched in NPs, was used in NPs characterization experiments.

Change in colour was observed in the silver nitrate solution incubated with the Marinomonas efl.The UV-visible spectra of this solution were recorded with UV- visible spectrophotometer. And aliquots of the solution to be tested was taken in a quartz cuvette and monitored for wavelength scanning from 300 to 700 nm [.D. Singh, V. Rathod, S. Ninganagou da, J. Hiremath, A.S. Kumar, J. Mathew Optimization and characterization of silver nanoparticle by endophytic fungi Penicillium sp. Isolated from Curcuma longa (Turmeric) and application studies against MDR E. coliand S. aureus Bioinorg. Chem. Appl., 2014 (2014), pp. 1-8). Based on the peak, optimum nanoparticles biosynthesis was identified.

The bacterial isolated were incubated at 30° C overnight, and were adjusted to 0.5 McFarland standard. Total of 10ml tube LB broth medium was prepared then each sample was inoculated aseptically with lml of the respective bacterial suspension (about 108 CFU/mL). Different concentrations of AgNPs (0.4mM, 0.8mM, ImM, 2mM,3mM, 4mM, 6mM) is tested and a negative control (without AgNPs) was used. Tests were performed in triplicates for each isolate. The inoculated sets were incubated at 30° C overnight. After incubation period, the visible turbidity in each tube was investigated. The lowest concentration with no turbidity is represented as the MIC for the tested strain. Tubes showed no turbidity were cultured on LB agar plates and incubated at 30°C overnight. Bacterial colonies growth was checked and the concentration that shows no growth is represented as the MBC (Clinical and Laboratory Standards Institute, CLSI, 2006).

MH agar was prepared according to the manufacturer's instructions and is dispensed in Petri dishes to achieve an even depth of 4.0 mm with a maximum variation of +0.5 mm

Morphologically similar colonies were taken from a overnight growth (16-24 h of incubation) on a LB medium with a sterile loop or a cotton swab and suspending them in sterile saline (0.85% NaCl w/v in water) to the density of a 0.5 McFarland standard.

The density of the suspension is compared visually to a 0.5 McFarland turbidity standard. The density is adjusted to McFarland 0.5 by addition of saline or more organisms. All inoculum suspensions is used within 15 min and always within 60 minutes of preparation. A sterile cotton swab is dipped into the inoculum suspension and the excess fluid removed by turning the swab against the inside of the tube to avoid over-inoculation of plates, particularly for Gram negative organisms. The inoculum is spread evenly over the entire surface of the agar plate by swabbing in three directions.

To the disks, 50m1 of AgNps were added and applied firmly on the agar surface within 15 minutes of inoculation of the plates. It is important that zone diameters can be reliably measured and the maximum number of disks on a plate depends on the size of the plate, the organism and the antimicrobial agents tested. The number of disks on a plate should be limited so that unacceptable overlapping of zones is avoided.

Within 15 min of application of disks, the plates were inverted and incubated at 28°C for 16-20 h. Small stacks of plates with a space between stacks were more likely to ensure uniform and rapid heating.

After incubation, inhibition zones were read at the point where no obvious growth is detected by the unaided eye when the plate is held about 30 cm from the eye. The inhibition zone diameters were measured to the nearest millimetre with a ruler, calliper or an automated zone reader.

Results: the synthesis of AgNPs by the isolated marinomonas efl was monitored by visible observation of changes in colour of biomass in the presence of various concentrations of AgNO₃.

In a study the synthesis of AgNPs from the isolate CB2 was monitored by visible observation of changes in colour of biomass and supernatant in the presence of 1 mM AgNO₃. Observations indicated that intracellular components of the strain reduced Ag+ ions as observed by change in colour of the samples from pale yellow to brown within 24 h of incubation in the presence of light. The excitation of surface plasmon resonance of silver nanoparticles is responsible for colour change (Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R (2003) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf B 28:313-318; Kalimuthu K, Babu RS, Venkataraman D, Bilal M, Gurunathan S (2008) Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B 65:150-153).

The colour change is due to the oscillation of electron in the silver nanoparticles ([N. Duran, P.D. Marcato, G. Souza G, O.L. Alves, E. Esposito, "Antibacterial Effect of Silver Nanoparticles Produced by Fungal Process on Textile Fabrics and Their Effuent Treatment" ,Journal of Biomedical Nanotechnology, vol. 3, pp. 203- 208, 2007). The intensity of the brown colour increased up to 24 h and was maintained throughout the 72 hours period of observation. This finding indicates that the reduction of the Ag+ ions takes place intracellularly under visible-light irradiation. Visible light irradiation is known to have significant increasing effect on the biosynthetic rate of silver nanoparticles formation (37.Mokhtari N, Daneshpajouh S, Seyedbagheri S, Atashdehghan R, Abdi K, Sarkar S, Minaian S, Shaverdi HR, Shaverdi AR (2009) Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia: The effects of visible-light irradiation and the liquid mixing process. Mater Res Bull 44:1415-1421; Wei X, Luo M, Li W, Yang L, Liang X, Xu L, Kong P, Liu H (2012) Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgN03, Bioresour Technol 103:273-278; Thomas R, Viswan A, Mathew J, Radhakrishnan EK (2012 b) Evaluation of antibacterial activity of silver nanoparticles synthesized by a novel strain of marine Pseudomonas sp. Nano Biomed Eng 4:139-143). In addition, in presence of silver nitrate experimental control, heat killed biomass showed no colour changes. Thus, this visual observation of colour changes in samples containing bacterial biomass can be taken as an indication of intracellular synthesis of silver nanoparticles.

UV-vis spectral analysis is carried out to monitor the formation and stability of the reduced silver nanoparticles in the colloidal solution. The UV-vis spectra of silver nanoparticles synthesized by using Marinomonas efl was shown in figure 3. It was confirmed by sharp peaks observed by the absorption spectrum of this solution by using UV-Vis spectrophotometer. The surface Plasmon resonance (SPR) band of silver occurs at 419.50nm, 420nm, 420.50nm, 427nm, 421nm, 415nm and 422nm for 0.4mM, 0.8mM, 1mM, 2mM, 3mM, 4mM, and 6mM respectively. AgNO₃ reduced to silver nanoparticles and settles down at the bottom of the conical flask. As the size of the silver nanoparticles increases, the colour of the solution varied from yellow to brown colour precipitation. (S. Rajeshkumar and C. Malarkodi, In Vitro Antibacterial Activity and Mechanism of Silver Nanoparticles against Foodborne Pathogens, Bioinorganic Chemistry and Applications Volume 2014. 2014). Due to its surface plasmon excitation, the absorption spectra of silver nanoparticles exhibits an intense absorption peak range. It is due to collective excitation of conduction electron in metal. Presence of such peak assigned to a surface plasmon resonance of silver nanoparticles was previously reported in the case of Chlorella vulgaris and the Chaetoceros calcitrans, Fusarium species, C. albicans, Myxococcus virescens .[ 43. P. Karthikeyan, D. Mohan, G. Abishek, R. Priya. Synthesis of silver nanoparticles using Phytoplankton and its characteristics. International Journal of Fisheries and Aquatic Studies 2015; 2(6): 398-401; Swapnil C. Gaikwad; Sonal S. Birla; Avinash P. Ingle; Aniket K. Gade, ; Priscyla D. Marcato; Mahendra Rai; Nelson Duran. Screening of different Fusarium species to select potential species for the synthesis of silver nanoparticles. J. Braz. Chem. Soc. vol.24 no.12 Sao Paulo Dec. 2013; Ghasem Rahimi, Fahimeh Alizadeh and Alireza Khodavandi, Mycosynthesis of Silver Nanoparticles from Candida albicans and its Antibacterial Activity against Escherichia coli and Staphylococcus aureus. Tropical Journal of Pharmaceutical Research February 2016; 15 (2): 371-375; 46. Wioletta Wrbtniak -Drzewiecka, Swapnil Gaikwad, Dariusz Laskowski, Hanna Dahm , Janusz Niedojadlo, Aniket Gade and Mahendra Rai. Novel Approach towards Synthesis of Silver Nanoparticles from Myxococcus virescens and their Lethality on Pathogenic Bacterial Cells. Austin J Biotechnol Bioeng - Volume 1 Issue 1 -2014):

The following table 2 shows that the MIC of AgNPs ranged from 0.8 mM to 3mM and the MBC ranged from 2Mm to 4mM. Whereas Proteus mirabilis and Serratia marcescens showed the highest sensitivity.

Silver nanoparticles which were synthesized from Marinomonas efl having good zone of inhibitions are shown in the following table 3.

The antibacterial activity was conducted against the pathogenic bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactie, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, Citrobacter koseri, Acinetobacter baumanii, Serratia marcescens, Candida albicans, Candida parapsilosis. Different concentration were used to kill the bacteria: 0.4Mm, 0.8mM, ImM, 2mM, 3mM, 4mM and 6Mm. 50m1 of Agnps and AgNo3 is used against the isolates. Bacterial synthesized AgNPs were involved in the antibacterial activity against all the pathogenic isolated.

Increased concentrations of AgNPs increased the zone of inhibition and showed strong antibacterial effect than the silver nitrate used as control.

Maximum zone of inhibition of 21mm appeared against Staphylococcus aureus, Escherichia coli, Citrobacter koseri.

Example 3 - Comparative example with Marinomonas sp. efl

Nanoparticles were prepared by using the protocol disclosed by Singh et al., 2015 and Roshmi et al.. The nanoparticles so obtained did not gave any good results in the experiments performed for their characterization. In particular, diffractograms experiments revealed that the preparation was very poor in silver nanoparticles, when compared with samples obtained by the protocol of the present invention.

Moreover the antimicrobial activity of the silver nanoparticles was compared.

The results are reported in the following table 4. Table 4

Example 4- Production of fluorescent dyes from Marinomonas sp. efl

Production of fluorescent dyes- Marinomonas efl is also able to produce fluorescent molecules, that is excited by UV light. The phenomenon is particularly evident when the strain is grown in Kings medium supplemented with glycerol.

Kings medium contains, for a total of 500 ml, distilled water 500 ml, proteose peptone lOg, K₂HPO₄0,75 g, MgSO₄.7H₂₀0,75 g, glycerol 5 ml, AGAR 7,5 g which is prepared by firstly mixing peptone and water and bringing mixture to boiling, then adding the other ingredients and heating until complete dissolution.

The experiments reported for the first time the production of fluorescent pigment by Marinomonas. Kings medium is the only reported broth that induces fluorescence in different bacteria. Therefore, as a first step we verified that Marinomonas becomes fluorescent when grown in Kings medium. We obtained a positive response. However, fluorescence induction depended on the peptone used in preparing the medium. This means that the experimental conditions used for the production of the fluorescent molecule was not reproducible. Furthermore, it was not possible to identify what characteristic of the Kings medium induces the fluorescent molecule in this bacteria strain and in the others as well.

For this reason we tried to induce fluorescence in Marinomonas by using standard LB medium, that it is also easier to prepare and cheeper and additional molecules. We tried with differ salts, and we obtained a positive results by using 10 and 20 mM CaCl₂.

Therefore, we demonstrated for the first time that the presence of CaCl₂ (20-30 mM) in LB medium induce the synthesis of the dye in Mrinomonas. Most probably the fluorescent molecule so produced is phenazine, because the spectrum obtained overlap with the one reported in P Devnath , Md. K. Uddin, F. Ahamed , Md. T. Hossain and M. Abul Manchur, 2017 Extraction, purification and characterization of pyocyanin produced by Pseudomonas aeruginosa and evaluation for its antimicrobial activity, International Research Journal of Biological Sciences, Vol. 6(5), 1-9.

The gene responsible for the synthesis of phenazine has been found in the genome from Marinomonas.

Example 5: silver nanoparticles synthesis by Rhodococcus efl

Rhodococus efl has been isolated from a bacterial consortium associated with the Antarctic ciliate Euplotes focardii using selective conditions as for example minimal medium containing 1% of ethanol as carbon source at 4 ° C as described in (Pucciarelli S,

Devaraj RR, Mancini A, Ballarini P, Castelli M, Schrallhammer M, Petroni G, Miceli C. Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from

Genomic Sequences. Microb Ecol. 2015 Aug;70(2):484-97.).

Rhodococus efl 16S rDNa gene has been sequenced:

SEQ ID NO. 14 Rhodococus efl ribosomal 16S RNA

The sequence SEQ. ID. 14 shows 100% of identity with the 16S rDNA of Rhodococcus sp. NJ-530, isolated from Antarctic ice, which sequence in deposited under UNIPROT database under the acc. n CP034152.1 and reported hereafter:

SEQ. ID. NO. 15: CP034152.1:568549-570070 Rhodococcus sp. NJ-53016S rDNA

Rhodococcus was grown in LB medium containing AgN03 ImM. The culture was incubated at 22°C in continuous shaking for 48 hours. Samples were then centrifuged at 5000 rpm for 15 min. After centrifugation, the supernatant and pellet were separated. The supernatant was purified by centrifugation at 12000 rpm for 20 min at 4°C. The mean dimension of the biosynthesized AgNP was estimated with the light dynamic diffusion technique and it was 117,0 nm, as shown in Figure 4 .

Example 6 antibacterial activity of Rhodococcus efl silver nanoparticles

The disc diffusion test has been used for the analysis of the antimicrobial activity of AgNP produced in the example 5. All pathogenic bacteria were cultivated over night in LB medium. The suspension was check to reach the turbidity standard of 0,5 OD using the spectrophotometer at 600 nm. 100 pL of pathogens cultures ere spread by a sterile loop on agar plates containing Mueller-Hinton (MHA) medium. Sterile paper disks (6 mm) were soaked with the silver nanoparticles (50 mΐ) and air dried to allow the disk to adsorb silver nanoparticles and dry. Finally , the dry AgNP containing disks were layed on the agar plates inoculated with the pathogenic bacteria and incubated at 37 ° C for 24 hours. The control is represented by AgN031 mM 50 mL. The results are shown in the following table 5.

Table 5

Claims

1)An in vitro process for the preparation of metabolites by

Marinomonas efl or Rhodococcus efl comprising the following steps: a) Culturing Marinomonas efl or Rhodococcus efl in a culture medium containing peptides from enzymatic digestion suitably supplemented with additives depending on the metabolites to be prepared; b) Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 5000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising a precursor of the metabolites to be prepared until the metabolite is obtained; e) Purification of the metabolite as obtained in step d).

2) Process of claim 1 wherein the metabolite is Ag nanoparticles comprising the following steps: a) Culturing Marinomonas efl or Rhodococcus efl in a culture medium containing peptides from enzymatic digestion (tryptone), yeast extract and NaCl b) Collecting a sample of marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 5000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising AgNO₃ until Ag nanoparticles are obtained; e) Purification of Ag nanoparticles as obtained in step d). 3) Process for the preparation of antiseptic textiles comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium containing peptides from enzymatic digestion (tryptone), yeast extract and NaCl b)Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 5000 to 16000 rpm for a time comprised between 2 to 15 min; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising AgNO₃ e) Spreading the aqueous solution as obtained in step d) on textiles, incubation at 22 °C for 24 hours f) Drying the textiles obtained in step e) at 37 °C for 2 hours, wherein the obtained antiseptic textiles are impregnated of Ag nanoparticles which are synthetized at the end of step d).

4)Use of the antiseptic textiles directly obtained by the process comprising the following steps: a)Culturing Marinomonas efl or Rhodococcus efl in a culture medium containing tryptone, yeast extract and NaCl b) Collecting a sample of Marinomonas efl or Rhodococcus efl as obtained in step a) and centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 5000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a aqueous solution or culture medium comprising AgNO₃ e) Spreading the aqueous solution as obtained in step d) on the textile, incubation at 22 °C for 24 hours f) Drying the textiles as obtained in step e) at 37 °C for 2 hours, wherein the obtained antiseptic textiles are impregnated of Ag nanoparticles synthetized at the end of step d) for surfaces cleaning.

5) Process for the preparation of fluorescent dyes comprising the following steps: a) culturing Marinomonas efl or Rhodococcus efl in a culture medium comprising proteose-peptone, K₂HPO_4, MgSO₄, glycerol and distilled water; b) centrifuging at a temperature comprised between 0 °C and 25 °C at a frequency of rotation comprised between 5000 to 16000 rpm for a time comprised between 2 to 15 minutes; c) Separation of biomass and supernatant as obtained in step b) d) Re-suspending biomass as obtained in step c) in a culture medium in the presence of CaCl₂ until the synthesis of the fluorescent dye is completed; e) Separation of the fluorescent dye as obtained in step d).

6) Process for the preparation of fluorescent dyes according to claim 5 wherein alternatively in step a) the culture medium comprising proteose-peptone, K₂HPO_4, MgSO₄, glycerol and distilled water is replaced by a medium comprising sodium salt od glutamic acid, K₂HPO_4, MgSO_4, NaCl and distilled water.