Methods for bioremediation of waters contaminated with hydrocarbons

Abstract

A method for bioremediation of waters contaminated with hydrocarbons may include: putting the contaminated waters in contact with at least one polyhydroxyalkanoate (PHA); and/or allowing microorganisms, present in the contaminated waters and capable of metabolizing the hydrocarbons, to develop and degrade the hydrocarbons under an aerobic condition.

Claims

1. A method for bioremediation of waters contaminated with hydrocarbons, the method comprising: putting the contaminated waters in contact with at least one polyhydroxyalkanoate (PHA); and allowing microorganisms, present in the contaminated waters and capable of metabolizing the hydrocarbons, to develop and degrade the hydrocarbons under an aerobic condition; wherein the at least one PHA is dispersed in the contaminated waters in a form of particles, wherein the particles have an average size greater than or equal to 0.1 micron (μm) and less than or equal to 1,000 μm, and wherein the at least one PHA in the form of the particles is dispersed in the contaminated waters without the addition of other substances that stimulate metabolic activity of the microorganisms.

2. The method of claim 1, wherein the at least one PHA is selected from: poly-3-hydroxybutyrate (PHB), poly-3-hydroxyvalerate (PHV), poly-3-hydroxyhexanoate (PHH), poly-3-hydroxyoctanoate (PHO), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyexanoate) (PHBH), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(3-hydroxyoctanoate-co-3-hydroxyundecen-10-enoate) (PHOU), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate (PHBVV), or mixtures thereof.

3. The method of claim 1, wherein the at least one PHA is added to the contaminated waters in an amount so as to obtain a concentration greater than or equal to 0.01 gram (g) per liter of the contaminated waters and less than or equal to 1,000 g per liter of the contaminated waters.

4. The method of claim 1, wherein the particles are in a form of powder or microgranules.

5. The method of claim 1, wherein the particles have an average size greater than or equal to 1 micron (μm) and less than or equal to 500 μm.

6. A method for bioremediation of waters contaminated with hydrocarbons, the method comprising: preparing an aqueous suspension of at least one polyhydroxyalkanoate (PHA); putting the contaminated waters in contact with the aqueous suspension; and allowing at least one first microorganism, present in the contaminated waters and capable of metabolizing the hydrocarbons, to develop and degrade the hydrocarbons under an aerobic condition; wherein the at least one PHA is dispersed in the aqueous suspension in a form of particles, and wherein the particles have an average size greater than or equal to 0.1 micron (μm) and less than or equal to 1,000 μm.

7. The method of claim 6, wherein the at least one PHA comprises at least one nutritive substance suitable for promoting development of the at least one first microorganism.

8. The method of claim 7, wherein the at least one nutritive substance is selected from: ammonium chloride (NH.sub.4E.sub.1), sodium nitrate (NaNO.sub.3), potassium phosphate (K.sub.3PO.sub.4), potassium dihydrogen phosphate (KH.sub.2PO.sub.4), sodium dihydrogen phosphate (NaH.sub.2PO.sub.4), ferrous chloride tetrahydrate (FeCl.sub.2.Math.4H.sub.2O), urea (CH.sub.4N.sub.2O), or mixtures thereof.

9. The method of claim 7, wherein the at least one nutritive substance is added to the contaminated waters in an amount so as to obtain a concentration greater than or equal to 0.01 grams (g) per liter of the contaminated waters and less than or equal to 100 g per liter of the contaminated waters.

10. The method of claim 6, wherein the at least one PHA comprises at least one second microorganism capable of metabolizing the hydrocarbons.

11. The method of claim 6, wherein the at least one PHA comprises at least one surfactant.

12. A method for bioremediation of waters contaminated with hydrocarbons, the method comprising: preparing at least one polyhydroxyalkanoate (PHA); putting the contaminated waters in contact with the at least one PHA; and allowing at least one first microorganism, present in the contaminated waters and capable of metabolizing the hydrocarbons, to develop and degrade the hydrocarbons under an aerobic condition; wherein the at least one PHA comprises at least one second microorganism, capable of metabolizing the hydrocarbons, englobed in the at least one PHA, wherein the at least one PHA is dispersed in the contaminated waters in a form of particles, and wherein the particles have an average size greater than or equal to 0.1 micron (μm) and less than or equal to 1,000 μm.

13. The method of claim 12, wherein the at least one second microorganism comprises one or more bacteria, one or more fungi, or one or more yeasts.

14. The method of claim 12, wherein the at least one second microorganism comprises one or more oil-eating bacteria or one or more hydrocarbon-degrading bacteria.

15. The method of claim 12, wherein the at least one second microorganism is included in the at least one PHA in an amount so as to obtain a concentration of vital cellular units (Unit Forming Colony, UCF) greater than or equal to 10.sup.3 per gram of the at least one PHA and less than or equal to 10.sup.10 per grain of the at least one PHA.

16. The method of claim 12, wherein the at least one PHA further comprises at least one nutritive substance suitable for promoting development of the at least one first microorganism.

17. The method of claim 16, wherein the at least one nutritive substance is selected from: ammonium chloride (NH.sub.4Cl)sodium nitrate (NaNO.sub.3), potassium phosphate (K.sub.3PO.sub.4), potassium dihydrogen phosphate (KH.sub.2PO.sub.4), sodium dihydrogen phosphate (NaH.sub.2PO.sub.4), ferrous chloride tetrahydrate (FeCl.sub.2.Math.4H.sub.2O), urea (CH.sub.4N.sub.2O), or mixtures thereof.

18. The method of claim 16, wherein the at least one nutritive substance is added to the contaminated waters in an amount so as to obtain a concentration greater than or equal to 0.01 grams (g) per liter of the contaminated waters and less than or equal to 100 g per liter of the contaminated waters.

19. The method of claim 12, wherein the at least one PHA further comprises at least one nutritive substance suitable for promoting development of the at least one first microorganism and the at least one second microorganism.

20. The method of claim 12, wherein the at least one PHA further comprises at least one surfactant.

Description

EXAMPLE 1

(1) 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 950 kDa. The suspension contained 190 g of PHB per litre of suspension.

(2) The PHA suspension was subjected to a drying process by means of spray-drying at a temperature of 230° C.

(3) The final product was a powder of PHB with an apparent density of 0.35÷0.45 kg/L and an average particle size equal to 20-30 μm. The moisture content was lower than 1%. The product was ready for bagging and direct use.

EXAMPLE 2

(4) 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 800 kDa. The suspension contained 120 g of PHB per litre of suspension.

(5) A mixture of nutritive substances was added to the PHA suspension, consisting of an aqueous solution of mineral salts thus composed:

(6) ammonium chloride (NH.sub.4Cl) 80 g/L, potassium dihydrogenphosphate (KH.sub.2PO.sub.4) 8 g/L, sodium nitrate (NaNO.sub.3) 20 g/L.

(7) The PHA suspension containing the above mixture was subjected to a drying process by means of spray-drying at a temperature of 220° C.

(8) The final product was a powder containing PHB and mineral salts, with an apparent density of 0.25÷0.35 kg/L and an average particle size equal to 20-30 μm. The moisture content was lower than 1%. The product was ready for bagging and direct use.

EXAMPLE 3

(9) A suspension was prepared of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvale-rate (PHBVV) in water starting from the polymer in powder form, having a weight average molecular weight (determined by GPC) of about 500 kDa. The suspension contained 90 g of PHBVV per litre of suspension.

(10) 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 106 cell bodies per gram of PHBVV present in suspension.

(11) The suspension of PHBVV containing the above mixture was subjected to an orthogonal filtration process, obtaining a cake having 35% of moisture. The cake thus obtained was subjected to a drying process using a bed-dryer at a temperature of 60° C.

(12) The product thus obtained, containing PHBVV and the bacterial mixture, was in powder form with an apparent density of 0.55÷0.65 kg/L. The moisture content was lower than 0.8%. The product was ready for bagging and subsequent direct use.

EXAMPLE 4

(13) In order to verify the effectiveness of the materials prepared according to Examples 1 and 2 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.

(14) The following products were introduced into a tank having dimensions of 78 cm×33 cm×42 cm (total volumetric capacity equal to 108 L):

(15) a) 90 L of coastal seawater; in order to favour the elimination of metazoans, particulate and/or debris possibly present, the water, before being introduced into the tank, was filtered on a filter having a porosity equal to 300 μm:

(16) b) 45 mL of oil Dansk Blend Crude Oil (gravity API: 33.50).

(17) The content of the tank was kept in motion by means of an internal pump, with recycling equal to 5 L/hr, which allowed a non-turbulent stirring to be maintained. The system also included an “overflow” system and a continuous charge of seawater (1 L/hr) in order to guarantee continuous replacement and simulate the conditions present in a marine environment.

(18) Treatment with PHB alone (OIL-PHA) After the oil had been introduced, 51 g of PHB, prepared according to Example 1, 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.

(19) A representative sample was collected at regular time intervals, and the following parameters were measured: measurement of the total bacterial abundance (DAPI count): the direct cell count was effected with an epifluorescence microscope after colouring with a specific fluorochrome, according to the standard method described in the publication of APAT and IRSA-CNR “Analytical methods for water” 29/2003, chapter 9040 (pages 1149-1153); the values are expressed as logarithm of the number of cells per mL of sample; measurement of the quantity of residual hydrocarbons with respect to the initial quantity (weight %), measured by means of ionizing flame gas-chromatography (GC-FID).

(20) The results are indicated in the graphs of FIGS. 1 and 2. FIG. 1 also shows the value of the microbial abundance present in seawater as such (NSW, natural seawater).

(21) 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 (30 days), when the total abatement proved to be equal to about 60%, whereas the degradation peak (about 65%) was observed on the 20.sup.Th day of experimentation (FIG. 2).

(22) Treatment with PHB and nutritive substances (OIL-PHA-MIX1).

(23) The experiment was carried out according to the same operative procedures described above, using, instead of PHB alone as in Example 1, a composition consisting of

(24) PHB and nutritive substances prepared according to Example 2, which was added in a quantity of 100 g.

(25) The results are indicated in FIGS. 1 and 2, 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 70% already after 14 days).

(26) For comparative purposes, the same experiment was carried out without the addition of PHB and/or nutritive substances, i.e. pouring only OIL into the tank. The results are also indicated in FIGS. 1 and 2, from which the improvement in terms of abatement of hydrocarbons due to the addition of PHB or PHB and nutritive substances, is evident.