EP4010480A1 - Marinomonas ef1 and rhodococcus ef1 for the production of secondary metabolites - Google Patents

Marinomonas ef1 and rhodococcus ef1 for the production of secondary metabolites

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Publication number
EP4010480A1
EP4010480A1 EP20754650.8A EP20754650A EP4010480A1 EP 4010480 A1 EP4010480 A1 EP 4010480A1 EP 20754650 A EP20754650 A EP 20754650A EP 4010480 A1 EP4010480 A1 EP 4010480A1
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EP
European Patent Office
Prior art keywords
efl
marinomonas
rhodococcus
culture medium
biomass
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EP20754650.8A
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German (de)
English (en)
French (fr)
Inventor
Sandra PUCCIARELLI
Maria Sindhura JOHN
Alessio Mancini
Kesava Pryian RAMASAMY
Nagoth Joseph AMRUTHRAJ
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Irides SRL
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Irides SRL
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Publication of EP4010480A1 publication Critical patent/EP4010480A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
    • D06M11/83—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with metals; with metal-generating compounds, e.g. metal carbonyls; Reduction of metal compounds on textiles
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms

Definitions

  • 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.
  • 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.
  • 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 Ochro
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • OLED organic light emitting devices
  • 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).
  • the secondary metabolites produced by said strains are usually active to a narrow range of virulent organisms.
  • Marinomonas mediterranea 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 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 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.
  • 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.
  • 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 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 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.
  • the optical characteristics of synthesized silver nanoparticles were measured using UV-visible spectrophotometer (Hitachi U5100) at 200-800 nm range with control as reference.
  • UV-visible spectrophotometer Hagaku U5100
  • 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.
  • 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.
  • 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 AgNO 3 , 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)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 AgNO 3 ; 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
  • 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, K 2 HPO 4, MgSO 4 , 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 CaCl 2 until the synthesis of the fluorescent dye is completed; e) Separation of the fluorescent dye as obtained in step d).
  • step a) the medium comprising proteose-peptone, K 2 HPO 4 , MgSO 4 , glycerol and distilled water is replaced by a medium containing glutammic acid sodium salt, K 2 HPO 4 , MgSO 4 , NaCl and distilled water.
  • 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.
  • 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 08/01/2019.
  • 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.
  • metabolites produced by Marinomonas sp. efl or Rhodococcus efl are selected from the group consisting of: Ag nanoparticles, fluorescent dyes.
  • 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.
  • 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.
  • the additives used to supplement the culture medium comprising peptides from enzymatic digestion are selected from the group consisting of: glycerol, yeast extract, NaCl, K 2 HPO 4, MgSO 4 .
  • 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: CaCl 2 , AgNO 3 .
  • 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) Pur
  • 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 AgNO 3 , 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 AgNO 3 , 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
  • 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.
  • 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, K 2 HPO 4 , MgSO 4 , 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 CaCl 2 until the synthesis of the fluorescent dye is completed; e) separation of the fluorescent dye obtained in d).
  • step a) the culture medium comprising proteose- peptone, K 2 HPO 4 , MgSO 4 , glycerol e distilled water is replaced by a medium comprising sodium salt of glutamic acid, K 2 HPO 4 , MgSO 4 , NaCl and distilled water.
  • the temperature is comprised between 0°C and 22 °C.
  • the temperature is room temperature.
  • step b) frequency of rotation is 16000 rpm.
  • centrifugation is for a period of 2 minutes.
  • step e) purification of metabolites is carried out with a method selected from the group consisting of filtration, chromatography, centrifugation.
  • 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.
  • AgNO 3 is in a concentration comprised between 0.8 and 6 nM.
  • the culture medium consisting of a total of 500 ml, distilled water 500 ml, proteose-peptone 10g, K 2 HPO 4 0,75 g, MgSO 4 .7H200,75 g, glycerol 5 ml, AGAR 7,5.
  • CaCl 2 is added in a concentration between 20 and 30 nM.
  • CaCl 2 is added in a concentration of 20 nM.
  • the fluorescent dye is phenazine.
  • 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.
  • LB Luria Bertani
  • 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:
  • 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).
  • FISH fluorescent in situ hybridization
  • 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 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.
  • 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).
  • FDR false discovery rate
  • 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).
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • bacterial wet biomass of 2 g were re suspended in 100 mL aqueous solution containing different concentrations of AgNO 3 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Kings medium contains, for a total of 500 ml, distilled water 500 ml, proteose peptone lOg, K 2 HPO 4 0,75 g, MgSO 4 .7H 20 0,75 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.
  • 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,
  • Rhodococus efl 16S rDNa gene has been sequenced:
  • 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:
  • 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 .
  • 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 m ⁇ ) 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.

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