CZ304792B6 - Halophilic strain of Shewanella haliotis F1 bacteria and use of the strain for biological degradation of petroleum hydrocarbons - Google Patents

Abstract

In the present invention, there is described a halophilic strain of Shewanella haliotis F1 bacteria and the use of the strain for purification of contaminated soil, water and sediments when cleaning up petroleum contaminants in the environment of elevated salinity. The halophilic bacterial strain Shewanella haliotis F1 is deposited in CCM – Czech Collection of Microorganisms of the Masaryk University Faculty of Natural Sciences, Tvrdého 14, 602 00 Brno under the sample deposit number CCM 8486: Shewanella haliotis (F1).

Description

Technical field

The invention relates to a halophilic strain of Shewanella haliotis FI and to the use of this strain for the purification of contaminated soils, waters and sediments in the remediation of petroleum contaminants in a higher salinity environment.

BACKGROUND OF THE INVENTION

The basic principle of bioremediation technology is based on the ability of some microorganisms to use petroleum substances as a source of carbon and energy for their growth. As a result, these substances are decomposed into harmless products. At contaminated sites, the occurrence of microorganisms adapted to the contaminant present can often be observed. However, their concentration in the environment is not high enough to effectively remove the pollutants. This is due to several possible reasons: lack of mineral nutrients, lack of suitable electron acceptor (eg O ₂ in the case of aerobic processes), very low solubility of contaminants in water, increased salinity of the environment.

Studies over the past 30 years have shown a wide diversity of microbial life under extreme conditions such as high temperature, pH, high salinity or high osmotic pressure. Microorganisms that live in these conditions not only hate these extremes, but on the contrary, these extremes are essential for their survival (Madigan and Oren 1999). Some species of bacteria such as Pseudomonas, Micrococcus, Bacillus, Vibrio have been isolated from salinized areas (Triiper et al. 1991). In general, the higher the salinity of the environment, the lower the microbial diversity (Benlloch et al. 2002, Rodriguez - Valera et al. 1985).

Pure cultures of halophilic bacteria, i.e. osmophilic bacteria, which tolerate high osmotic pressure well and require high NaCl concentration in the environment, were further isolated from a number of salt crystallization tanks. Halobacterium sp. was isolated from crystallization tanks in Israel and the United States (Oren and Litchfield 1999). Haloarcula, Haloferax, Halorubrum and Halobacterium sp. are commonly found in crystallization tanks in Spain. (Benlloch et al. 2001, Rodriguez-Valera et al. 1985).

Contaminated sites of high salinity are a significant environmental problem as biodegradation under these extreme conditions is extremely difficult. Some scientific publications show that increased salinity has a negative impact on the biodegradation of petroleum hydrocarbons (Ward and Brock 1978, Mille et al. 1991, Rhykerd et al. 1995). Accordingly, it was proposed that the problem of high salinity and petroleum hydrocarbon contamination should not be eliminated until the second step (Rhykerd et al. 1995). But this approach is not only costly but also considerably environmentally friendly. For this reason, the use of halophilic bacteria in the biodegradation of petroleum substances is a convenient and cheaper solution.

Increased salinity of the environment can pose a significant problem in biodegradation processes, as it significantly affects the metabolic rate of microorganisms and thus the overall rate of biodegradation. The bioremediation method works with strains that are osmotolerant or halophilic and are characterized by their ability to degrade petroleum substances. SUMMARY OF THE INVENTION The object of the invention is to increase the concentration of bacterial strains in the sanitized material to the highest possible level and to maximize their overall metabolic activity. The degradation process then proceeds at a significantly higher rate than in the case of microorganisms that are unable to grow at an increased salt concentration.

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SUMMARY OF THE INVENTION

This task is solved by finding a new halophilic bacterial strain and using it for the biodegradation of petroleum contaminants, especially petroleum hydrocarbons in an environment with increased salinity. The present invention is based on a new halophilic bacterial strain Shewanella haliotis F1, isolated during an expert mission in the Philippines (tanker accident at Guimaras Island), which, on the basis of the tests carried out, exhibits to a significant degree osmotolerance and biodegradability of petroleum substances in increased salinity. It is therefore a bacterial strain suitable for bioremediation applications.

The bacterial strain Shewanella haliotis Fl was isolated from sea water contaminated with pure oil on the coast of Guimaras (Philippines). Isolation was carried out on nutrient agar with 1% pure petroleum and 10% NaCl. Cultivation was carried out aerobically at 30 ° C for 48 h. Isolation was performed in three steps by smearing individual colonies to ensure purity of the isolate.

Sequence analysis of 16S rRNA encoding DNA sequences was used to identify and classify bacteria. Genomic DNA was obtained from pure cultures using classical phenol-chloroform extraction and isolation using a commercially available NucleoSpin Soil kit from Macherey-Nagel. Next, PCR was performed with primers targeting the 16S rRNA region (specifically eub530F / eubl100R) followed by sequencing of the amplification product (ABI PRISM3130x instrument).

Optimal culture conditions are as follows:

° C / 48 hours / nutrient agar with addition of 2.5% NaCl / aerobically, respectively.

° C / 48 hours / BSM medium with addition of 2.5% NaCl, 1% pure C18 hydrocarbon / aerobically, respectively.

° C / 72 hours / BSM medium with addition of 5% NaCl, 1% pure C18 hydrocarbon / aerobically.

The bacterial strain Shewanella haliotis Fl is deposited in the Czech Collection of Microorganisms under accession number CCM 8486.

Advantages of the invention are that the bacterial strain Shewanella haliotis F1 exhibits high biodegradation activity, survival in extreme conditions, in particular in a high salinity environment, and high durability. The bacterial strain Shewanella haliotis Fl multiplies rapidly, so that in a contaminated environment it reaches high concentrations and high metabolic activity in a short time, which significantly accelerates the biodegradation process.

Clarification of drawings

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be explained in more detail by means of the accompanying drawings, in which:

Fig. 1 Shewanella haliotis Fl osmotolerance Fig. 2 Shewanella haliotis Fl microcultivation biodegradation test of oil hydrocarbons Fig. 3 Correlation between Shewanella haliotis F1 optical density and number of colonies constituting the unit Fig. 4 Shiftanella haliotis biodegradation biodegradation test.

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DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the specific embodiments of the invention described and illustrated below are presented by way of illustration and not by way of limitation of the examples to the examples. Those skilled in the art will find or will be able to provide, using routine experimentation, more or less equivalents to the specific embodiments of the invention described herein. These equivalents will also be included within the scope of the following claims.

Testing of petroleum hydrocarbon biodegradation capability in Examples 1-3 was performed as described below. Microplate Reader ELx808 was used for microculture. Glucose, pure C18 hydrocarbon and NaCl were used as chemical and microbial agents. The test microorganism was a model net population of Shewanella haliotis F1.

- Microculture was performed in a Microplate Reader EFx808; the device is able to measure turbidity of the microbial culture in the liquid growth medium by means of turbidity filters. The Microplate Reader EEx808 consists of an incubation and measurement unit and a computer that has the appropriate Gen5® software installed to record the growth curves and evaluate them. Incubation, mixing and kinetic measurements are performed automatically according to the set parameters of the experiment.

- the ability of microorganisms to grow in individual model solutions of selected pollutants in three replicates was monitored, control culture was performed without loading with possible growth inhibitor - NaCl.

- growth is expressed as the change in optical density (O. D.), which was measured automatically every half hour at 450 and 630 nm,

- shaking was performed automatically every 30 minutes, prior to measurement, for 10 seconds with variable shaking intensity set,

- the culture space has a capacity of 96 wells (1 culture plate), each of 250 μΕ, the medium (whose composition depends on the nature of the test being performed) was inoculated with 30 µL of cell suspension (culture medium of defined O.D at specified wavelength), sterile,

- the inoculum was prepared by submerged cultivation of microorganisms in a medium chosen according to the nature of the experiment with the addition of a C-source of appropriate concentration

- Inoculum cultivation was carried out in Erlenmeyer flasks on shaker at room temperature.

Model solutions are prepared in laboratory conditions, using input parameters, using the measuring technique, so their composition does not need to be verified by analysis. Microplate Reader ELx808 allows to monitor increase or decrease of optical density in culture medium, this parameter reflects increase or decrease of concentration of present microbial population.

The test was evaluated by increasing or decreasing the optical density of the culture medium, depending on the defined composition of the model solution, which makes it possible to evaluate the stimulatory or inhibitory effect of this solution on the given microbial population.

The influence of the presence of pollutant and NaCl is determined from the increase or decrease of the optical density in the tested solutions. An increase in optical density (alone or as compared to control) is considered to be a stimulating effect of the test solution.

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A decrease in optical density (alone or compared to control) is considered to be an inhibitory effect of the test solution.

The evaluation of the effect of the presence of the pollutant (Cl8) and the inhibitory agent (NaCl) also took into account the results of supplementary submerged cultures, in a volume of 100 ml, in Erlenmeyer flasks on shakers - see Example 4.

Example 1

The osmotolerance of the bacterial strain Shewanella haliotis Fl was tested under laboratory conditions. The only carbon and energy source used was the hydrocarbon Cl8 at concentrations of 200, 500 and 1000 mg / L. Salinity ranged from 0.5 to 10% NaCl.

Example 2

Biodegradation of petroleum hydrocarbons by the bacterial strain Shewanella haliotis Fl was tested under laboratory conditions. The only carbon and energy source used was hydrocarbon 08 with a concentration range of 0 to 2000 mg / L in 5% NaCl solution.

Example 3

In addition to the above tests, a correlation between the optical density of the bacterial strain Shewanella haliotis F1 and the number of colonies per unit per milliliter of medium was found under laboratory conditions.

Example 4

Biodegradation of petroleum hydrocarbons by the bacterial strain Shewanella haliotis Fl was tested under laboratory conditions. Cultivation was carried out batch-shaking at room temperature in BSM medium with the addition of 5% NaCl. Hydrocarbon Cl 8 was used as the sole source of carbon and energy. The loss of oil was monitored by means of the NEL (FTIR spectrometry) parameter.

Industrial applicability

The halophilic bacterial strain Shewanella haliotis F1 according to the invention can be used for the purification of contaminated soils, water and sediments in the remediation of oil contaminants, especially in an environment with increased salinity.

Claims (1)

PATENT CLAIMS 5 1. Halophilic bacterial strain Shewanella haliotis Fl with biodegradable activity against petroleum hydrocarbons deposited in CCM - Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Tvrdého 14, 602 00 Brno under deposit number CCM 8486: Shewanella haliotis (Fl). Use of the halophilic bacterial strain Shewanella haliotis F1 according to claim 1 for the biodegradation of petroleum substances in an environment with increased salinity.