Patent Description:
It is known that numerous microorganisms, in particular bacteria, are capable of metabolizing a large number of polluting substances which can be present in a body of water due to the spillage of various kinds of chemical substances, in particular hydrocarbon substances of a petroleum origin. These microorganisms degrade these substances through metabolic processes of the oxidative type until water and carbon dioxide are obtained. Processes for the remediation of polluted waters, known as bioremediation, are based on this natural effect.

Bioremediation, however, is often quite ineffective, mainly due to the low quantity of microorganisms present in the environment that are capable of causing degradation in an acceptable time or under the environmental conditions of the site to be remediated, not optimal for bacterial growth.

In some cases, it is therefore advantageous to effect a so-called biostimulation of the site to be remediated, which comprises applying stimulation techniques of the growth rates of natural microbial communities having biodegradation capabilities by the addition of nutrients, in organic and/or inorganic form.

In a marine environment, the bacterial growth is generally limited by the low concentration of nutrients, normally represented by nitrogen and phosphorous compounds. Marine ecosystems are in fact, for biotic and abiotic reasons, generally lacking in these substances, which can undergo a strong "uptake" on the part of microorganisms that do not degrade crude oil (also including phytoplankton).

In order to support the growth of autochthonous populations of bacteria capable of degrading hydrocarbons, one of the techniques most widely adopted during bioremediation processes is the use of fertilizers as nutritional source, for example soluble nitrogen-based fertilizers, slow-release fertilizers (SFRs) or oleophilic fertilizers. Another supply of nutrients can be provided by the introduction of water-soluble nutrients such as mineral salts (for example KNO<NUM>, NaNO<NUM>, NH<NUM>NO<NUM>, K<NUM>HPO<NUM>, MgNH<NUM>PO<NUM>) and commercial inorganic fertilizers.

If compared with other nutrients (for example oleophilic nutrients), water-soluble nutrients are more readily available for the microbial metabolism. Due to their soluble nature, however, they have the main drawback of being more readily diluted and dispersed by the action of waves and tides.

In order to enhance the bioremediation process, it is also possible to effect a so-called bio-augmentation, which consists in adding to the system to be remediated, large densities of bacterial populations (single bacteria or microbial consortia) with particular catabolic abilities, to integrate the indigenous population in order to accelerate or activate the degradation of polluting substances. According to some studies, bioaugmentation has proved to be extremely effective for the remediation of polycyclic aromatic hydrocarbons (IPA) in sediments with little or no potential for intrinsic degradation, whereas other studies have demonstrated that this technique does not significantly improve what may be natural attenuation.

A problem observed in the application of bioaugmentation is that of guaranteeing the survival and activity of the organisms introduced into the environment. Furthermore, the bioaugmentation can be inhibited by various factors, among which the pH and the presence of products with a high redox potential and toxic pollutants, the concentration and bio-availability of contaminants or the absence of specific substrates. The key factor for considering for the success of this technique, however, is definitely the choice of the strain and/or bacterial consortium, which must take into account the type of community present in the environment considered.

Bioaugmentation strategies can prove to be effective above all in the remediation of contaminants of an anthropic origin, where specialized bacteria with the appropriate catabolic pathways may not be present in the contaminated environment. The selection of bioaugmentation as remediation strategy becomes important if the limiting factor of natural biodegradation processes is the absence of specific catabolic genes in the indigenous microbial community. This lack of genetic information will be therefore completed by the strain introduced.

With respect to the microorganisms present in the environment, which are capable of degrading hydrocarbons, these are normally bacteria which are known as hydrocarbon-degrading bacteria or oil-eating bacteria (BICs). A single bacterial species is capable of degrading only a limited number of oil compounds, whereas a consortium composed of various bacterial species (with different enzymatic features) can develop a metabolic syntropy which can lead to a complete mineralization of the hydrocarbons up to the production of CO<NUM> and H<NUM>O. The capacity of degrading oil hydrocarbons is not restricted to a few microorganisms: over <NUM> kinds of marine bacteria have been identified and distributed in different (sub)phyla (α-, β-, γ-Proteobacteria; Gram positive; Flexibacter-Cytophaga-Bacteroides). Among the most important types (based on the frequency of isolation) the following can be mentioned: Pseudomonas, Achromobacter, Nocardia, Micrococcus, Vibrio, Acinetobacter, Brevibacter, Flavobacterium.

In addition to these heterotrophic bacteria (i.e. capable of using alternative carbon sources in addition to hydrocarbon sources), a new series of hydrocarbon-degrading marine bacteria have been isolated through different culture methods containing hydrocarbons as sole carbon source and subsequent taxonomic and physiological analysis, characterized by a slow growth under oligotrophic conditions, which have proved to be competent in using exclusively petroleum hydrocarbons with the sole source of carbon and energy.

An analysis of the gene sequence of <NUM> rRNA reveals that these BICs often prove to be correlated with Marinomonas vaga, Oceanospirillum linum and Halomonas elongate belonging to the group of γ-Proteobacteria.

With reference to their metabolic properties, these can be subdivided into two groups, those that degrade aliphatic hydrocarbons and those that degrade aromatic hydrocarbons. Alcanivorax borkumensis (isolated from the North Sea), Alcanivorax sp. ST1 (sea of Japan), Marinobacter hydrocarbonoclasticus (Mediterranean sea) and Marinobacter sp. CAB (Mediterranean sea) degrade linear or branched aliphatic chains, whereas bacteria such as Cycloclasticus oligotrophus, C. pugetii and Psychroserpens burtonensis use aromatic hydrocarbons such as toluene, naphthalene, phenanthrene and anthracene as sole carbon source.

BICs occupy a unique trophic niche among heterotrophic bacteria that participate in the global carbon cycle, as they preferably consume aliphatic and aromatic hydrocarbons which are relatively difficult to use for normal autotrophic and heterotrophic microbial flora present in the environment. As these bacteria have unusual physiological features, they also have few rRNA operons (<NUM> or <NUM>), few cytoplasmic proteins (not more than <NUM>) and a small genome (<NUM>-<NUM> Mbp). Furthermore, the number of membrane proteins is <NUM>-<NUM> times lower than other heterotrophic bacteria such as E. coli or Pseudomonas, and this can probably be explained by the fact that the cells can only use some substrates.

<CIT> relates to a moving bed biofilm carrier (MBCC) having the shape of a Kaldnes-like carrier, characterized in that it is inoculated with one or more microbial strain(s) and is manufactured from bioplastic.

<CIT> relates to a method for purifying soil contaminated with an oil by using a porous body of a certain shape, which is made from a biodegradable material, specifically a PHA. The foamed body may be produced by a foam molding process. The contaminating oil may be selected from a wide range of products, including crude oil, gasoline, light oil, kerosene, heavy oil, etc..

<NPL>, is a scientific paper relating to the use of β-hydroxy-butyrate as a slow release electron donor for the microbial reductive dechlorination of trichloroethene (TCE). The experiments were carried out in anaerobic conditions. <CIT>discloses a known prior art.

The Applicant has considered the problem of increasing the effectiveness of bioremediation processes through the supply of substances that can in some way favour the development of aerobic microorganisms capable of metabolizing hydrocarbons, without supplying non-biodegradable materials which would have to be removed after the treatment, making the process complex and expensive and not without risks from an environmental point of view.

This problem and others which will be described in greater detail hereunder, have been solved by putting waters contaminated with hydrocarbons in contact with a poly-hydroxyalkanoate (PHA), a highly biodegradable polymeric material which the Applicant has verified as being surprisingly capable of stimulating, alone, without the addition of other substances, the metabolic activity of aerobic microorganisms capable of metabolizing hydrocarbons. By allowing these microorganisms to act on hydrocarbons under an aerobic condition, a significant reduction in environmental pollution is obtained in relatively short times, without introducing extraneous non-biodegradable materials into the environment.

According to a first aspect, the present invention therefore relates to a method for the bioremediation of waters contaminated with hydrocarbons, which comprises:.

Said PHA is dispersed in the contaminated waters in the form of particles, in particular in the form of powder or microgranules.

Without the intention of being bound to an interpretative theory of the present invention, the fact that PHA is surprisingly capable of stimulating, alone, without the addition of other substances, the metabolic activity of microorganisms capable of metabolizing hydrocarbons, can be due to the highly biodegradable nature of PHA itself, which is produced through a fermentation process of organic substrates and is thus akin to microorganisms in general, in particular to hydrocarbon-degrading bacteria and/or to oil-eating bacteria (BICs).

Poly-hydroxyalkanoates (PHAs) are polymers produced by microorganisms isolated from natural environments or also by genetically modified microorganisms, which act as carbon and energy reserves and which are accumulated by various species of bacteria under unfavourable growth conditions and in the presence of an excess carbon source. PHAs are synthesized and accumulated by about <NUM> different microbial species, included within more than <NUM> kinds of Gram-positive and Gram-negative bacteria, such as, for example, Bacillus, Rhodococcus, Rhodospirillum, Pseudomonas, Alcaligenes, Azotobacter, Rhizobium. In cells, PHAs are stored in the form of microgranules, whose size and number per cell varies in the different bacterial species. In general, PHAs are polymers containing repetitive units having the formula.

Preferably, R<NUM> is methyl or ethyl, and n is <NUM> or <NUM>.

PHAs can be either homopolymers or copolymers or terpolymers. In the case of copolymers or terpolymers, these can consist of different repetitive units having formula (I), or at least one repetitive unit having formula (I) in combination with at least one repetitive unit deriving from co-monomers capable of co-polymerizing with hydroxy-alkanoates, for example lactones or lactams. In the latter case, the repetitive units having formula (I) are present in a quantity equal to at least <NUM>% by moles with respect to the total moles of the repetitive units.

Particularly preferred repetitive units having formula (I) are those deriving from: <NUM>-hydroxybutyrate, <NUM>-hydroxyvalerate, <NUM>-hydroxyhexanoate, <NUM>-hydroxyoctanoate, <NUM>-hydroxyundec-<NUM>-enoate, <NUM>-hydroxyvalerate.

PHAs can be divided into three groups, in relation to the number of carbon atoms forming the monomeric unit: PHAscls (short chain length) are composed of monomeric units having from <NUM> to <NUM> carbon atoms, PHAmcls (medium chain length) are composed of monomeric units having from <NUM> to <NUM> carbon atoms, whereas PHAlcls (long chain length) are composed of monomeric units having more than <NUM> carbon atoms. PHAscls have a high degree of crystallinity, whereas PHAmcls and PHAlcls are elastomers with a low crystallinity and have a low melting point.

Particularly preferred PHAs are: poly-<NUM>-hydroxybutyrate (PHB), poly-<NUM>-hydroxyvalerate (PHV), poly-<NUM>-hydroxyhexanoate (PHH), poly-<NUM>-hydroxyoctanoate (PHO), poly(<NUM>-hydroxybutyrate-co-<NUM>-hydroxyvalerate) (PHBV), poly(<NUM>-hydroxybutyrate-co-<NUM>-hydroxyhexanoate) (PHBH), poly(<NUM>-hydroxybutyrate-co-<NUM>-hydroxybutyrate), poly(<NUM>-hydroxyoctanoate-co-<NUM>-hydroxyundecen-<NUM>-enoate) (PHOU), poly(<NUM>-hydroxybutyrate-co-<NUM>-hydroxyvalerate-co-<NUM>-hydroxyvalerate (PHBVV), or mixtures thereof.

PHAs preferably have a weight average molecular weight (Mw) ranging from <NUM>,<NUM> to <NUM>,<NUM>,<NUM> Da, more preferably from <NUM>,<NUM> to <NUM>,<NUM>,<NUM> Da. The weight average molecular weight can be determined according to known techniques, in particular by means of GPC (Gel Permeation Chromatography) analysis.

As far as the production of PHAs is concerned, this is preferably obtained by microbial fermentation of an organic substrate (for example, carbohydrates or other fermentable substrates, such as glycerol) by means of a strain of microorganisms capable of producing PHAs, and the subsequent recovery of the PHAs from the cell mass. For further details, reference should be made, for example, to patent applications <CIT>, <CIT> and <CIT>. Substrates suitable for the production of PHAs via fermentation can be obtained in particular from the processing of vegetables, for example juices, molasses, pulp from sugar beet processing, sugar cane. These substrates generally contain, in addition to sucrose and other carbohydrates, organic growth factors, nitrogen, phosphorous and/or other minerals useful as nutrients for cell growth. An alternative consists of glycerol, a low-cost organic carbon source, as it is a by-product of the production of biodiesel (see for example patent <CIT>).

For the implementation of the present invention, the PHA is used in the form of particles, so as to increase the exchange surface with the environment and therefore the bioremediation effect. The particles have an average size ranging from <NUM> to <NUM>,<NUM>, preferably from <NUM> to <NUM>. These dimensions can be determined according to techniques well known in the art, such as particle-size detection systems in suspension with laser detectors, known as Dynamic Light Scattering (DLS) techniques (see the standard ISO <NUM>-<NUM>). As an alternative, electron microscope images can be used (SEM) which are processed by means of digital analysis.

For the preparation of the composition according to the present invention, it is advantageous to use the aqueous suspension of PHA obtained directly from the bacterial fermentation process which produces PHA itself, without having to precipitate and dry it. The aqueous suspension obtained directly from the production process has optimal characteristics in terms of homogeneity, dispersion and particle size of PHA. The aqueous suspension of PHA obtained from the fermentation process is in any case preferably previously subjected to a purification and whitening step, in order to eliminate residues and substances present in the fermentation broth.

As far as the quantity of PHA to be added and dispersed in the contaminated waters is concerned, this is mainly pre-determined in relation to the type and entity of the pollution to be treated, and can therefore vary within wide limits. The quantity of PHA added to the waters to be bioremediated is generally such as to obtain a concentration preferably ranging from <NUM> to <NUM>,<NUM>, more preferably from <NUM> to <NUM> per litre of contaminated water.

The following embodiment examples are provided for purely illustrative purposes of the present invention and should not be considered as limiting the protection scope defined by the enclosed claims.

A suspension of polyhydroxybutyrate (PHB) in water was collected directly from the purification process of the culture broth in which the polymer had been produced by means of bacterial fermentation on sugar beet molasses. The weight average molecular weight of PHB (determined via GPC) was about <NUM> kDa. The suspension contained <NUM> of PHB per litre of suspension.

The PHA suspension was subjected to a drying process by means of spray-drying at a temperature of <NUM>.

The final product was a powder of PHB with an apparent density of <NUM>÷<NUM>/L and an average particle size equal to <NUM>-<NUM>. The moisture content was lower than <NUM>%. The product was ready for bagging and direct use.

A mixture of nutritive substances was added to the PHA suspension, consisting of an aqueous solution of mineral salts thus composed:
ammonium chloride (NH<NUM>Cl) <NUM>/L, potassium dihydrogenphosphate (KH<NUM>PO<NUM>) <NUM>/L, sodium nitrate (NaNO<NUM>) <NUM>/L.

The PHA suspension containing the above mixture was subjected to a drying process by means of spray-drying at a temperature of <NUM>.

The final product was a powder containing PHB and mineral salts, with an apparent density of <NUM>÷<NUM>/L and an average particle size equal to <NUM>-<NUM>. The moisture content was lower than <NUM>%. The product was ready for bagging and direct use.

A suspension was prepared of poly-(<NUM>-hydroxybutyrate-co-<NUM>-hydroxyvalerate-co-<NUM>-hydroxyvalerate (PHBVV) in water starting from the polymer in powder form, having a weight average molecular weight (determined by GPC) of about <NUM> kDa. The suspension contained <NUM> of PHBVV per litre of suspension.

A mixture of bacteria consisting of Alcanivorax sp. , Marinobacter sp. , Sphingongomonas sp. , Rhodococcus sp. , Bacillus sp. was added to the PHBVV suspension. The various bacterial species, in spore, vegetative and/or quiescence form, were inserted at a concentration of about <NUM> cell bodies per gram of PHBVV present in suspension.

The suspension of PHBVV containing the above mixture was subjected to an orthogonal filtration process, obtaining a cake having <NUM>% of moisture. The cake thus obtained was subjected to a drying process using a bed-dryer at a temperature of <NUM>.

The product thus obtained, containing PHBVV and the bacterial mixture, was in powder form with an apparent density of <NUM>÷<NUM>/L. The moisture content was lower than <NUM>%. The product was ready for bagging and subsequent direct use.

In order to verify the effectiveness of the materials prepared according to Examples <NUM> and <NUM> in a bioremediation process, a microscale experiment was carried out on a volume of seawater to which a volume of oil was added as described hereunder.

The following products were introduced into a tank having dimensions of <NUM> x <NUM> x <NUM> (total volumetric capacity equal to <NUM>):.

The content of the tank was kept in motion by means of an internal pump, with recycling equal to <NUM>/hr, which allowed a non-turbulent stirring to be maintained. The system also included an "overflow" system and a continuous charge of seawater (<NUM>/hr) in order to guarantee continuous replacement and simulate the conditions present in a marine environment.

After the oil had been introduced, <NUM> of PHB, prepared according to Example <NUM>, were dispersed in the tank. The powder was distributed homogeneously on the surface in correspondence with and on the oil stain. The PHB powder showed a marked tendency to adhere to the oil, forming lumps which partially tended to precipitate. The recirculating system, however, allowed the lumps of PHB to remain in suspension.

A representative sample was collected at regular time intervals, and the following parameters were measured:.

The results are indicated in the graphs of <FIG> and <FIG>. <FIG> also shows the value of the microbial abundance present in seawater as such (NSW, natural seawater).

As can be seen in these graphs, with respect to the time zero of the experiment, starting from the fourth day, an increase was observed in the quantitative values (abundance) of the natural microbial population, presumably due to the presence of PHB. At the same time, a significant reduction in the quantity of hydrocarbons was observed, correlated with the beginning of the biodegradation processes attributed to the metabolic activity of the hydrocarbon-degrading bacterial flora. This activity continued until the end of the experimentation period (<NUM> days), when the total abatement proved to be equal to about <NUM>%, whereas the degradation peak (about <NUM>%) was observed on the <NUM>Th day of experimentation (<FIG>).

Treatment with PHB and nutritive substances (OIL-PHA-MIX1) (not according to the invention).

The experiment was carried out according to the same operative procedures described above, using, instead of PHB alone as in Example <NUM>, a composition consisting of PHB and nutritive substances prepared according to Example <NUM>, which was added in a quantity of <NUM>.

The results are indicated in <FIG> and <FIG>, in which a trend of the DAPI count and abatement of hydrocarbons substantially analogous to the OIL-PHA case, can be observed, with slightly improved values (hydrocarbon abatement equal to about <NUM>% already after <NUM> days).

Claim 1:
A method for the bioremediation of waters contaminated with hydrocarbons, which comprises:
- putting said contaminated waters in contact with at least one polyhydroxyalkanoate (PHA);
- allowing the microorganisms present in said contaminated waters and capable of metabolizing hydrocarbons, to develop and degrade the hydrocarbons under an aerobic condition;
wherein said PHA is dispersed in the contaminated waters in the form of particles having an average size ranging from <NUM> to <NUM>,<NUM>;
wherein said PHA is dispersed in the contaminated waters alone.