Classifications

• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
  • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
  • B09C—RECLAMATION OF CONTAMINATED SOIL
  • B09C1/00—Reclamation of contaminated soil
  • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
• • 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
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the present invention relates to a new bacterial strain
• Novosphingobium puteolanum PPlY (DSM 19530), isolated from seawater samples collected at the sea surface inside the harbour of Pozzuoli (NA).
• This strain shows the ability to use as the sole carbon and energy source several mono- and poly-cyclic aromatic hydrocarbons both in pure form and dissolved in oils as paraffin oils and silicone oils.
• the strain PPlY is able to use as the sole source of carbon and energy the aromatic fractions of several fuel oils as gasoline, kerosene and diesel oil.
• the strain produces extracellular material, prevalently composed by proteins, with emulsifying and gelatinizing activity which stabilizes the emulsion of the oil phase in water/oil biphasic systems.
• the metabolic abilities of this strain could be used for the bioremediation of complex mixtures of mono- and poly-cyclic aromatic hydrocarbons even when dissolved in oil phases. Moreover the strain could be used as source of molecules with emulsifying and gelatinizing activity and of enzymatic catalysts for the biosynthesis of compounds with high added value.
• the present invention relates to a new bacterial strain able to degrade mono- and poly-cyclic aromatic hydrocarbons dissolved in oil phases and to synthesise molecules with emulsifying and gelatinizing activity.
• PAH poly-cyclic aromatic hydrocarbons
• benzene, benz[a]anthracene, benzo[a]pyrene and chrysene are known carcinogenic compounds.
• the high chemical stability, the low biodegradability and the lipophylicity favour their accumulation in the environment and inside living organisms (biomagnification).
• Resistance to degradation, hydrophobicity and hence ability to adhere to substrates (soil, mud, sand etc.) increase with the number of methyl groups and aromatic rings.
• the main source of aromatic hydrocarbons released into the environment are crude oil and all the oil fuels derived from crude oil distillation. Even if the composition of crude oil varies depending on the oilfield, it can contain up to 40-45% aromatic hydrocarbons, from the simple benzene molecule to complex PAHs containing ten or more rings. Gasoline can contain up to 20-25% monocyclic aromatic hydrocarbons, including benzene, and 5-10% PAHs. Kerosene and diesel oil for cars contain up to 15-20% PAHs and 5-10% monocyclic aromatic hydrocarbons. Heavier fractions of crude oil contain even higher percentages of PAHs, for example diesel oil used in boats contains higher concentrations of PAHs than diesel oil used in cars.
• Polycyclic aromatic hydrocarbons including the known carcinogenic molecules benz[a]anthracene, benzo[a]pyrene and chrysene, are also generated by all the combustion reactions of organic matter. For example incinerators of municipal wastes and forest fires are important source of PAHs.
• Residual oils become enriched by the less biodegradable hydrocarbons as o-xylene, trimethylbenzenes, PAHs and highly branched saturated hydrocarbons. Due to the surfactants and to the degradation of saturated hydrocarbons, aromatic hydrocarbons can be dispersed into the environment rather than degraded. Moreover the surfactants themselves can persist into the environment contributing to increase the chemical pollution.
• the efficacy of the bioremediation interventions could be increased by enriching the endogenous micro-flora with strains able to degrade preferentially recalcitrant aromatic compounds as PAHs - thus producing oils with a low content of aromatic hydrocarbons - and to produce "biosurfactants" which would avoid or reduce the necessity to use synthetic surfactants.
• the present invention provides a new bacterial strain, Novosphingobium puteolanum PPlY, able to degrade a very wide spectrum of mono- and polycyclic aromatic compounds provided both separately and in complex mixtures.
• the bacterial strain PP lY is able to degrade aromatic compounds and the mixtures of aromatic compounds provided both in pure form or dissolved in hydrophobic or oil phases such as, but not limited to, gasoline, diesel oil, crude oil, paraffins, isoparaffins, and inert oils such as silicone oils.
• the bacterial strain of the present invention is a Gram negative bacterium with short rod shaped cells which can bring flagellums.
• Non-flagellated cells can be isolated cells or can be gathered in rows and bunches hold together by an extracellular matrix which can be stained by methylene blue.
• the strain PPlY forms spontaneously biofilm on solid hydrophobic surfaces, such as plastic surfaces, or on liquid hydrophobic surfaces as for example drops of silicone oils, paraffin and diesel oil.
• the strain PPlY secretes extracellular material - prevalently composed by proteins - which is able to stabilize water/oil emulsion and to increase the viscosity of the culture broths.
• the strain PPlY determines the fragmentation of the oil phase in drops with diameter lower than 1 mm. These drops are covered by a layer of bacterial biofilm.
• the bacterial strain of the present invention as been isolated by means of a standard enrichment procedure starting from seawater samples collected at the sea surface in different point inside the harbour of Pozzuoli (Naples) and is able to grow in a wide range of salt concentrations (0-40 g/1 NaCl).
• strain PPlY Due to its biochemical and microbiological features the strain PPlY is particularly well suited for the use in in-situ and ex-situ bioremediation strategies for the decontamination of waters and solid substrates such as but not limited to sand, mud, and sediments produced by dredging polluted by aromatic hydrocarbons bound to the substrate or dissolved in oil phases.
• the enrichment of the endogenous micro-flora of polluted waters and soils with the bacterial strain of the present invention could be used to reduce or eliminate aromatic hydrocarbons and to stimulate the degradative activity of the endogenous micro-flora by reducing the concentration of toxic aromatic compounds and emulsifying the oil phases thus increasing the accessibility of non-aromatic hydrocarbons to the endogenous micro-flora.
• the ability of the strain PP lY to grow in the absence of NaCl makes it suitable for the use both in seawater and brackish water.
• FIGURES is a picture of a Petri plate containing agar with phenanthrene microcrystals as the sole source of carbon and energy. The picture shows that each colony of the strain PP lY is surrounded by a clear halo created by the degradation of phenanthrene crystals.
• Figure 2 is the 16S rDNA sequence of the strain PPlY (SEQ ID 1).
• Figure 3 A is a graphic showing the growth of the stain PPlY (measured spectrophotometrically at 600 nm) as function of time at different NaCl concentrations.
• Figure 3B is a graphic showing the growth of the stain PPlY measured at 18 hours from the beginning of the incubation (measured spectrophotometrically at 600 nm) as function of the NaCl concentration.
• Figures 4A and 4B are pictures, obtained using an optical microscope, of cell aggregates (flocks) of the stain PPlY after growth in a biphasic system constituted by an aqueous saline solution and a dodecane phase containing o-xylene (20 ⁇ l/ml of dodecane) as the sole source of carbon and energy.
• the sample shown in figure 4A is not fixed and not stained.
• the sample in figure 4B is fixed by drying and stained by methylene blue.
• Figures 5A and 5B are pictures, obtained using an optical microscope, of a water/diesel oil emulsion produced by the PPlY strain after 3 and 14 days of incubation at 28°C.
• Figure 6A is a graphic showing the growth of the stain PPlY at 28 0 C (measured spectrophotometrically at 600 nm) as function of the pH at 20 hours from the beginning of the incubation (exponential phase) and at 30 hours from the beginning of the incubation (stationary phase).
• Figure 6B is a graphic showing the growth of the stain PPlY at 22°C (measured spectrophotometrically at 600 nm) as function of the pH at 20 hours from the beginning of the incubation (exponential phase) and at 30 hours from the beginning of the incubation (stationary phase).
• Figure 7 is a graphic showing the growth of the stain PPlY (measured spectrophotometrically at 600 nm) as function of time in a saline medium containing gasoline or diesel oil (5 ml of fuel oil per litre of saline solution) as the sole source of carbon and energy.
• Figure 8 is a graphic showing the degradation of a mixture of ethylbenzene, biphenyl, and phenanthrene dissolved in dodecane (0.3 mg of each aromatic hydrocarbon per ml of dodecane).
• Figure 9 is a picture of a sodium dodecylsulphate/polyacrylamide gel of the total proteins produced by the strain PPlY.
• Lane 1 shows protein markers of known molecular weight (molecular weights in kDa are shown on the left of the lane).
• Lane 2 shows the total cell-proteins of cells precipitated by centrifugation at 600Og.
• Lane 3 shows the total extracellular proteins precipitated by trichloroacetic acid from the culture broth after the removal of the cells.
• the present invention provides a new bacterial strain for the degradation of aromatic hydrocarbons isolated from samples of surface seawater collected inside the harbour of Pozzuoli (Naples).
• Isolation of the strain was performed by a standard enrichment procedure using crystals of naphthalene, phenanthrene and anthracene as growth substrates.
• the samples which showed the appearance of turbidity or yellow/brown colour were used to inoculate samples of saline solution at pH 7 containing naphthalene, phenanthrene and anthracene (20 mg/ml) separately or in mixtures.
• the sample which showed the appearance of turbidity or yellow/brown colour were spread on agar plates with phenanthrene microcrystals as the sole source of carbon and energy. On some plates the growth of yellow colonies surrounded by clear halos indicated the degradation of phenanthrene crystals as shown in figure 1.
• the strain of the present invention was purified by one of the yellow colonies by repeated plating on agar plates with phenanthrene microcrystals as the sole source of carbon and energy followed by 4 cycles of plating on rich medium (Luria Bertani agar) to ascertain the homogeneity of the strain.
• the strain PPlY (DSM 19530) was identified as a strain belonging to the Novosphingobium genus on the basis of the sequence of the 16S rDNA shown in figure 2.
• the strain PPlY unlike the strain US6-1 T , is able to use as the sole carbon and energy source monocyclic hydrocarbons and biphenyl too.
• the aromatic hydrocarbons which can be degraded and used as the sole carbon and energy source by the strain PPlY include toluene, o-xylene, m-xylene, p-xylene, 1-ethyltoluene, 2-ethyltoluene, 3-ethyltoluene, 4-ethyltoluene, propylbenzene, isopropylbenzene, butylbenzene, 1,2,4-trimethylbenzene, and 1,3,5-trimethylbenzene.
• the PAHs which can be degraded and used as the sole carbon and energy source include biphenyl, naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, dimethylnaphthalenes, phenanthrene, anthracene, pyrene, chrysene, benz[a]anthracene, fluoranthene, acenaphthene, fluorene, tetralin (tetrahydronaphthalene).
• Aromatic hydrocarbon which can be degraded but not used as the sole carbon source include benzene and 1,2,3-trimethylbenzene.
• aromatic compounds which can be degraded and used as the sole carbon and energy source include dibenzofuran, dibenzothiophene, carbazole, anisole (methoxybenzene) and benzyldimethylamine.
• the strain PPlY degrades aromatic hydrocarbons dissolved in oil phases of paraffines or silicone oils more effectively than the pure aromatic hydrocarbons provided in aqueous phase.
• the stain PPlY is able to grow in solution without NaCl as shown by the growth curve in figure 3.
• the maximal growth rate is observed at NaCl concentrations between 0.5% and 1.5%.
• the strain tolerates NaCl concentrations up to 4%. Growth is very slow at NaCl concentration above 5%.
• the strain PPlY is positive for ⁇ -galactosidase, urease, arginine-dihydrolase and citrate assimilation. In the same tests the strain US6-1 T is negative. Moreover the positive results for urease and arginine-dihydrolase differentiate the strain PPlY from the other known strains belonging to the Novosphingobium genus.
• the strain PPlY is Gram(-) and the cells, which are short rods, can be mobile and not mobile.
• the not mobile cells can be free or form flocks i.e. macroscopic aggregates 1-10 mm long.
• Figure 4A shows a picture of the edge of a flock obtained using an optical microscope. The analysis performed using an optical microscope also showed that in the flocks the cells are embedded in a matrix which can be stained using methylene blue.
• the flocks adhere spontaneously to hydrophobic surfaces such as plastic surfaces and absorb and concentrate hydrophobic molecules such as hydrophobic stains (for example Sudan III) and PAHs.
• the strain PPlY forms spontaneously biofilm on hydrophobic surfaces both solid and liquid.
• strain PPlY Other microbiological and biochemical properties of the strain PPlY are described below.
• the strain PPlY is able to grow in a wide range of temperature values (25°C-38°C). Optimal temperature is 32-34°C. At temperature values below 25°C the growth is slower.
• the strain PPlY is able to grow at pH values between 5 and 7.5.
• the maximal growth rate was registered at pH 7 for temperatures 28-32°C and at pH 6 for temperatures below 25°C. Growth is slow at pH 7.2 and no growth was observed at pH above 8.
• the strain is able to grow in oil/water biphasic systems containing a wide variety of oils and high ratios between the oil and the water phases.
• the oil phase is diesel oil, paraffin or silicone oil, containing PAH up to 200 mg/ml
• the strain PPlY is able to grow even at hydrophobic phase: water phase ratios above 1 :1 (volume: volume).
• the strain PPlY is able to grow at gasoline:water phase ratios not above 0.02: 1 (volume:volume).
• the strain PPlY can be prepared at high cell density (3-4 O.D. ⁇ OOnm) using 20 mM potassium Phosphate pH 7 containing 100 mM NaCl and using as carbon source 1% triptone or 0.5% triptone and 0.25% yeast extract.
• a saturated culture (>3,5 O.D. 6 oon m ) can be obtained in about 24 hours inoculating 1 ml of a saturated culture in 1 1 of fresh medium and incubating at 30 0 C shacking vigorously.
• the strain PPlY can grow using as the sole carbon and energy sources petroleum and its derivatives (gasoline, kerosene, diesel oil).
• a saturated culture (>3 O.D. 600nm ) can be obtained in about 200 hours inoculating 1 ml of a saturated culture in 1 1 of saline medium containing 5 ml of gasoline, kerosene, diesel oil and incubating at 30 0 C shacking vigorously (figure 7).
• the strain PPlY can degrade mono- and poly-cyclic aromatic hydrocarbons dissolved in oil phases such as paraffins, isoparaffins, and inert oils as silicone oils.
• oil phases such as paraffins, isoparaffins, and inert oils as silicone oils.
• figure 8 shows the degradation of a mixtures of ethylbenzene, phenanthrene and biphenyl in dodecane (each 0.03 mg per ml of dodecane).
• the biphasic system contained water/dodecane in a 10: 1 (v:v) ratio. 200 ⁇ l of a saturated PPlY culture grown in Luria-Bertani medium were inoculated into the aqueous phase was incubated at 28°C in orbital shaking.
• the residual concentration of the aromatic hydrocarbons was measured by normal phase HPLC using cyanopropyl-silica as stationary phase and hexane as mobile phase. The concentration of the aromatic hydrocarbon was calculated as percentage of the concentration in a control sample not inoculated with the PPlY strain.
• Figure 8 shows that more than 95% of ethylbenzene and phenanthrene and about 50% of biphenyl present at the beginning of the incubation were degraded in less than five days.
• the strain PPlY produces bright yellow colonies due to the production of a yellow pigment identified as the carotenoid nostoxanthin by comparing the UV- visible spectrum of the PPlY pigment extracted by acetone and the UV-visible spectrum of nostoxanthin reported in the literature.
• the strain PPlY harbours a 48 kbp plasmid (an extracromosomal DNA molecule capable to replicate autonomously inside the bacterial cells).
• This plasmid can be extracted and purified by the standard methodologies for the isolation of bacterial plasmids.
• the 48 kbp plasmids could be used for the preparation cloning and expression vectors for the insertion of foreign genes in the cells of the strain PPlY.

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