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Title:
MICROORGANISM CAPABLE OF OXIDIZING PETROLEUM AND ITS PRODUCTS AND THE TECHNOLOGY FOR ITS USE
Document Type and Number:
WIPO Patent Application WO/2019/073308
Kind Code:
A1
Abstract:
This invention relates to biological degradation of oil hydrocarbons in soil. To achieve this goal, a biopreparation containing non-pathogenic Acinetobacter calcoaceticus BT 8 bacteria, capable of digesting hydrocarbons and, in addition, absorbing nitrogen from the air and using it as a mineral, is used. The amount and mineral composition of the biopreparation are chosen based on the concentration of oil pollutants in the contaminated soil.

Inventors:
BALION STANISLAV (LT)
JURYS ARNOLDAS (LT)
PASKEVICIUS MARTYNAS (LT)
Application Number:
PCT/IB2018/050306
Publication Date:
April 18, 2019
Filing Date:
January 18, 2018
Export Citation:
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Assignee:
UAB BIOVERSIJA (LT)
International Classes:
B09C1/10; C12R1/01
Foreign References:
JP2012228191A2012-11-22
CN102899381A2013-01-30
Other References:
MARYAM BELLO-AKINOSHO ET AL: "Potential of Polycyclic Aromatic Hydrocarbon-Degrading Bacterial Isolates to Contribute to Soil Fertility", BIOMED RESEARCH INTERNATIONAL, vol. 2016, 5798593, 2016, XP055481853, Retrieved from the Internet [retrieved on 20180607]
MERCEDES MARÍN ET AL: "Study of factors influencing the degradation of heating oil by Acinetobacter calcoaceticus MM5", INTERNATIONAL BIODETERIORATION & BIODEGRADATION, 1996, pages 69 - 75, XP055482136, Retrieved from the Internet [retrieved on 20180607], DOI: 10.1016/S0964-8305(96)00027-3
ABDELHALEEM, DESOUKY, ACINETOBACTER: ENVIRONMENTAL AND BIOTECHNOLOGICAL APPLICATIONS., 2003
BARATHI, S.; N. VASUDEVAN: "utilization of Petroleum Hydrocarbons by Pseudomonas Fluorescens Isolated from a Petroleum-Contaminated Soil", ENVIRONMENT INTERNATIONAL, vol. 26, no. 5-6, 2001, pages 413 - 16
ERIKSSON, M.; G. DALHAMMAR; A. K. BORG-KARLSON: "Aerobic Degradation of a Hydrocarbon Mixture in Natural Uncontaminated Potting Soil by Indigenous Microorganisms at 20 Degrees C and 6 Degrees C", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 51, no. 4, 1999, pages 532 - 35
GUTNICK, DL; EA BAYER; C RUBINOVITZ; O PINES; Y SHABTAI; E ROSENBERG: "Emulsan production in Acinetobacter RAG-1", ADV. BIOTECHNOL, vol. 11, 1980, pages 455 - 59
HANSON, K. G.; A. NIGAM; M. KAPADIA; A. J. DESAI: "Bioremediation of Crude Oil Contamination with Acinetobacter Sp. A3", CURRENT MICROBIOLOGY, vol. 35, no. 3, 1997, pages 191 - 93
JAIN, PK; VK GUPTA; RK GAUR; M LOWRY; DP JAROLI; UK CHAUHAN: "Bioremediation of petroleum oil contaminated soil and water", RESEARCH JOURNAL OF ENVIRONMENTAL TOXICOLOGY, vol. 5, no. 1, 2011, pages 1
MARGESIN, R.; F. SCHINNER: "Biodegradation and Bioremediation of Hydrocarbons in Extreme Environments", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 56, no. 5-6, 2001, pages 650 - 63
MISHRA, S.; J. JYOT; R. C. KUHAD; B. LAL: "Evaluation of Inoculum Addition to Stimulate in Situ Bioremediation of Oily-Sludge-Contaminated Soil", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 67, no. 4, 2001, pages 1675 - 81, XP002679275, DOI: doi:10.1128/AEM.67.4.1675-1681.2001
MOHSENZADEH, FARIBA; ABDOLKARIM CHEHREGANI RAD; MEHRANGIZ AKBARI: "Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils", IRANIAN JOURNAL OF ENVIRONMENTAL HEALTH SCIENCE & ENGINEERING, vol. 9, no. 1, 2012, pages 26, XP021136896, DOI: doi:10.1186/1735-2746-9-26
TOREN, AMIR; ELISHA ORR; YOSSI PAITAN; ELIORA Z. RON; EUGENE ROSENBERG: "The Active Component of the Bioemulsifier Alasan from Acinetobacter Radioresistens KA53 Is an OmpA-like Protein", JOURNAL OF BACTERIOLOGY, vol. 184, no. 1, 2002, pages 165 - 70
WALZER, GIL; EUGENE ROSENBERG; ELIORA Z. RON: "The Acinetobacter Outer Membrane Protein A (OmpA) Is a Secreted Emulsifier", ENVIRONMENTAL MICROBIOLOGY, vol. 8, no. 6, 2006, pages 1026 - 32
WANG, Z.; M. FINGAS; S. BLENKINSOPP; G. SERGY; M. LANDRIAULT; L. SIGOUIN; J. FOGHT; K. SEMPLE; D. W. WESTLAKE: "Comparison of Oil Composition Changes due to Biodegradation and Physical Weathering in Different Oils", JOURNAL OF CHROMATOGRAPHY, vol. A 809, no. 1-2, 1998, pages 89 - 107, XP004124745, DOI: doi:10.1016/S0021-9673(98)00166-6
Attorney, Agent or Firm:
PETNIUNAITE, Jurga (LT)
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Claims:
Claims

1 . A bacterial preparation for the oxidation of petroleum products, characterized in that it uses Acinetobacter calcoaceticus BT 8 microorganisms and additional trace elements necessary for the growth and development of microorganisms, wherein A. calcoaceticus BT 8 microorganisms carry out gaseous nitrogen fixation.

2. A bacterial preparation according to claim 1 , characterized in that the trace elements are selected from the group consisting of phosphorus, potassium and nitrogen.

3. A bacterial preparation according to claims 1 to 2, characterized in that the amount of nitrogen in the biopreparation is up to 50 mg/kg of contaminated soil.

4. A bacterial preparation according to claims 1 to 3, characterized in that the A. calcoaceticus 16S rRNA sequence is at least 99% identical to SEQ ID NO: 1

5. A bacterial preparation according to claims 1 to 4, characterized in that the resulting preparation is frozen prior to use.

6. Use of a bacterial preparation according to claims 1 to 5 for the degradation of petroleum pollutants in soil.

7. The use of a bacterial preparation according to claim 6, characterized in that soil aeration and irrigation are used to accelerate the degradation of contaminants.

8. The use of a bacterial preparation according to claims 6 to 7, characterized in that the bacterial preparation is sprayed onto the soil 2 times a week.

Description:
MICROORGANISM CAPABLE OF OXIDIZING PETROLEUM AND ITS PRODUCTS AND THE TECHNOLOGY FOR ITS USE

Field of the invention

The present invention relates to the composition of an oil-degrading microorganism and its use for the utilization of petroleum and its derivatives from contaminated soil.

Background of the invention

Petroleum is a complex mixture of hydrocarbons and other organic compounds. It contains hundreds or thousands of aliphatic, branched and aromatic hydrocarbons (Wang et al. 1998), most of which are toxic to living organisms.

The most commonly used methods for the cleaning of contaminated soil are physico- chemical and biological. Physico-chemical treatments include burning, thermal desorption, coking, solvent extraction, landfilling, etc. However, these methods either make the soil unusable (burning) or do not eliminate the real problem (landfills). In addition, in Europe, legislation requires that the amount of landfills is reduced and that waste is recycled as much as possible.

Claude E. ZoBell had, as far back as 1946, recognized that many microorganisms have the ability to utilize hydrocarbons as the sole source of carbon and energy. He further recognized that the microbial utilization of hydrocarbons were highly dependent on the chemical nature of the components in the petroleum mixture and environmental determinants (Jain et al., 201 1 ). Microbial remediation of a hydrocarbon-contaminated site is accomplished with the help of oil-degrading microorganisms, particularly the indigenous bacteria and fungi present in soil. These microorganisms can degrade a wide range of target constituents present in oily sludge (Eriksson, Dalhammar, and Borg-Karlson 1999; Barathi and Vasudevan 2001 ; Mishra et al., 2001 ). Although fungi also have oil-oxidizing properties, their growth is relatively slow, making them difficult to adapt to outdoor conditions and high levels of pollution (Mohsenzadeh, Chehregani Rad, and Akbari 2012). In order to accelerate the biodegradation of oil products, contaminated soil is treated with artificially produced oil- oxidizing microorganisms that use petroleum hydrocarbons as a source of food and degrade them to harmless substances: C0 2 and H 2 0.

Acinetobacter species are widespread and can be obtained from water, soil, living organisms and even from human skins. They are non-motile, oxidase-negative, strictly aerobic, gram negative coccobacilli. They can use different sources of carbon for growth and can be grown in relatively simple media, including nutrient agar or trypticase soya agar (Abdelhaleem 2003). Due to their diversity, bacteria from genus Acinetobacter produce many materials used in biotechnology. Some types of Acinetobacter produce high levels of polysaccharides, polyesters and lipases. However, emulsifiers, such as emulsan (Gutnick et al. 1980), OmpA (Walzer, Rosenberg, and Ron 2006) or alasan (Toren et al., 2002), are the most important from an industrial point of view. Emulsifiers are particularly useful in hydrocarbon degradation, because they help to emulsify oil and its derivatives, thus accelerating degradation processes. A couple of decades ago it has been observed that some species of Acinetobacter have the ability to degrade oil and its derivatives, but good results have been achieved only under laboratory conditions due to the inhibitory properties of oil and lack of aeration in soil and in liquid media (Hanson et al., 1997). Not only nutrients, but also trace elements such as nitrogen, potassium, phosphorus, sulphur, magnesium, calcium, etc., are essential for the growth and reproduction of all microorganisms. Some of them are vital because they are incorporated in the DNA (nitrogen, phosphorus), amino acids (nitrogen, sulphur) and cell walls (phosphorus, calcium).

Moreover, trace elements can also affect bioremediation of various oil components. The aromatic hydrocarbon biodegradation is particularly sensitive to pH changes. In 1999 Foght et al. investigated the role of nitrogen source in the degradation of oil components under cold seawater conditions (10 °C). Nitrates did not influence the pH, but changes in ammonia led to acidification, which weakened the degradation of aromatic compounds (Margesin and Schinner 2001 ). These results show that it is very important to adapt the conditions in which the microorganisms degrade pollutants and constantly monitor changes in these conditions. Due to the variable weather conditions and lack of observation, many bacterial preparations do not work as expected from the laboratory experiments. Therefore, many inventions related to oil biodegradation can't be used in practice or they work only under enclosed and controlled conditions and, therefore, can't be applied to large areas of contaminated soil.

Summary of the invention

The technology used for the decomposition of oil contaminants utilizing Acinetobacter calcoaceticus BT 8 microorganisms is based on the cultivation of bacteria that were selected from soil and their use for oil degradation. This invention accelerates the process of soil remediation and is superior to other industrially produced preparations, since it involves the ability of the microorganism to absorb nitrogen from the air, thus reducing the need for additional fertilizers.

Detailed description of the invention

The present invention describes a new bacterial preparation capable of efficiently, safely and cost-effectively oxidizing oil contaminants in soil and turning them into harmless chemicals. The bacterial preparation is characterized by a lower mineral nitrogen demand because it employs the bacterium A. calcoaceticus BT 8 which is able to capture gaseous nitrogen from the air and transform it into a nutrient for the maintenance of various vital functions.

In one embodiment of the invention, a bacterial preparation consisting of selected A. calcoaceticus BT 8 bacteria and mineral fertilizers that do not comprise nitrogen is used. This preparation has been tested under laboratory conditions with oil contaminant concentration of 181 g/kg.

In another embodiment of the invention, a bacterial preparation consisting of selected A. calcoaceticus BT 8 bacteria and mineral fertilizers comprising nitrogen is used. This preparation has been tested under laboratory conditions with oil concentration of 181 g/kg.

In another embodiment of the invention, a bacterial preparation is used in a specialized manner, after cultivation the bacterial preparation is frozen and defrosted only prior to spraying on the oil contaminated soil; the amount of the preparation and mineral fertilizers is chosen according to the degree of contamination of the soil, and a schedule of additional spraying is set forth.

Description of Figures

Fig. 1 . The amount of oil hydrocarbons after a month-long laboratory experiment with an initial contamination of 181 g/kg is shown. Comparison is made on the effect of the biopreparation with and without a mineral nitrogen source.

Fig. 2. The degradation of oil hydrocarbons under field conditions with an initial oil concentration of 189 g/kg is shown. Experiment duration - 8 weeks.

Materials and methods

Devices: Technical scales KERN PCB 2500, Analytical scales KERN ABJ 220, "Thermo Scientific" laminar air cabinet, "Thermo Scientific" thermosetting shaker MaxQ 4450, "Thermo Scientific" microorganism incubator Heratherm IGS60, fermentor EDF-5.4 1 "Biotehniskais centrs", autoclave AL02-10 "Advantage- Lab", GENESYS 10S UV-Vis spectrophotometer "Thermo Scientific".

Reagents:

Table 1 . Reagents used in the manufacture of the bacterial preparation.

No. Reagent CAS No. Formula Manufacturer

1 . Nutrient medium CM001 B Oxoid

2. Bacteriological agar LP01 1 B - Oxoid

3. Diesel - - Statoil

4. Rectified spirits - - Vilnius Vodka

5. Diammonium 7783-28-0 (NH 4 ) 2 HP0 4 Fisher Scientific hydrophosphate

6. Ammonium chloride 12125-02-9 NH 4 CI Acros Organics

Potassium

7. 7778-77-0 KH 2 P0 4 Fisher Scientific dihydrophosphate

Di-potassium

8. 7758-1 1 -04 K 2 HP0 4 Fisher Scientific hydrophosphate

9. Manganese sulfate 7785-87-7 MnS0 4 Roth

10. Mohr's salt 7783-85-9 FeSC-4 -(NH 4 )S0 4 Fisher Scientific

1 1 . Calcium chloride 10043-52-4 CaCI 2 Fisher Scientific

12. Glucose 492-62-6 - Acros Organics

13. Sodium chloride 7647-13-5 NaCI Fisher Scientific

14. Deionized water 111 ra water quality level - -

15. Tap water - - -

16. Sodium hydroxide 1310-73-2 NaOH Sigma-aldrich

17. Phosphoric acid 13598-36-2 H 3 P0 4 Sigma-aldrich

Potassium

18. 7722-64-7 KMn0 4 Fisher Scientific permanganate

19. Technical oil - - Sanitex

20. Sugar - - Sanitex

21 . Molasses - - Sanitex

Methods:

1. Microorganism selection

In order to create a bacterial preparation for cleaning oil pollutants from soil, a selective sampling method was used to extract microorganisms from hydrocarbon-contaminated soil samples. Samples were taken from an old car repair shop garage, sampling PCS (petroleum contaminated soil). The separation of desired isolates was carried out on an agar medium by growing sampled microorganisms when the only carbon source in the medium was hydrocarbons. The most successful microorganisms were then used in other experiments.

2. Selection of nitrogen-fixing microorganisms

The selected oil-oxidizing isolates were grown on an agar medium, without a nitrogen source. During the experiment, only those oil-oxidizing isolates, that were able to capture gaseous nitrogen in the air and transform it into a mineral for the maintenance of various vital functions, remained.

3. Determination of microorganism species

In order to confirm the isolated microorganism's classification, it was identified by the BASECLEAR laboratory in the Netherlands. The microorganism was identified using a validated MicroSeq system from Applied Biosystems. This automatic system is based on amplification of the bacterial 16S rRNA gene during PCR and DNA sequencing. An A. calcoaceticus BT8 16S rRNA gene sequence was identified - SEQ ID No. 1 :

GATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGTGATGGTGyTTGC

ACTATCACTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGG G

ACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGA TC

TTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAA GG

CCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACT G

AGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGC

AAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTT TA

AGCGAGGAGGAGGCTACTGAAGTTAATACCTTCAGATAGTGGACGTTACTCGCAGAA TA

AGCACCGGCTAACTCTGT.

The gene sequence was found to coincide by 99.94% with the sequence of A. calcoaceticus 16S rRNA from the database used in the laboratory. This identification of the species confirmed that the bacteria we isolated is non-pathogenic and suitable for use in soil bioremediation.

4. Production of A. calcoaceticus BT 8 biopreparation

In order to ensure that A. calcoaceticus BT 8 is effective under field conditions, the microorganism is cultured in a simple nutrient medium with reduced mineral nitrogen content under aerobic conditions. At the end of cultivation, the biopreparation is prepared for freezing at -18 °C, adding 10% (by volume) of molasses. The culture liquid with molasses is mixed well, bottled and frozen.

Examples

Examples of use of the bacterial preparation are indicated here. The invention is not limited to the following examples.

Example 1

The cultured and frozen A. calcoaceticus BT8 preparation was thawed and diluted with water enriched with mineral fertilizers (P, K), and then 50 ml of the preparation was poured into 1 L containers with oil contaminated soil. The containers were kept at room temperature (-22 °C) for a month. Each week, the soil was mixed and additional biopreparation with water was added. Every week, the amount of hydrocarbons in the soil was measured. The experimental conditions are presented in Table 2.

Table 2. Biopreparation content, ml 0,5 1 2 5

Water, ml 49,5 49 48 45

Final oil content, g/kg 101 91 84 93

Example 2

The produced and frozen A. calcoaceticus BT 8 preparation was thawed under laboratory conditions, diluted with water enriched with mineral fertilizers (N, P, K) and then 50 ml of the preparation was poured into 1 L containers with oil contaminated soil. The containers were kept at room temperature (-22 °C) for a month. Each week, the soil was mixed and additional biopreparation with water was added. Every week, the amount of hydrocarbons in the soil was measured. The experimental conditions are presented in Table 3.

During this experiment, it was found that the addition of an extra nitrogen source to the medium resulted only in a slight change in the degradation of hydrocarbons. A comparison of the results of the first and second experiment is presented in Figure 1 .

Table 3.

Example 3

Cultured and frozen A. calcoaceticus BT 8 preparation was thawed prior to use and 1 L of the preparation was poured on a 4 m 2 soil contaminated with petroleum products (189 g/kg). The preparation was diluted with water enriched with mineral fertilizers (P, K, N) at a ratio of 1 :20. The amount of nitrogen in the mineral fertilizer mixture was reduced to 50 mg/kg of contaminated soil. Half of the diluted biopreparation was sprayed on the -30 cm thick plowed soil layer, after spraying, the soil was plowed again and the second portion of the biopreparation was sprayed. Each week, the soil was mixed and additionally supplemented with biopreparation and water. Every week, the amount of hydrocarbons in the soil was measured. Outdoor experiment lasted for 2 months. The experimental conditions are presented in Table 4. Table 4.

This experiment has shown that while using our invention an effective degradation of hydrocarbons in field conditions requires lower nitrogen fertilizer levels.

The optimal conditions for our bacterial preparation and explanations are shown in Table 5.

Table 5.

Parameter Range Explanation

The concentration of petroleum products does not

Petroleum product

1 -300 significantly affect the efficiency of the degradation of content, g/kg

petroleum hydrocarbons

In the case of lower titres, the soil is further treated with

Oil-oxidizing the biological preparation

>10°

microorganisms, CFU/g

<35 - additional watering is required

Moistu e, % 35-60

>60 - more frequent soil mixing is required

In the absence of orthophosphates, an estimated

Orthophosphates,

1000-3000 amount of fertilizer is added

mg/kg pH is corrected with 0,1 % NaOH/H 3 P0 4 aqueous

PH 5,7-8,2

solution

References Abdel aleem, Desouky. 2003. Acinetobacter: Environmental and Biotechnological Applications. T. 2.

Barathi, S., and N. Vasudevan. 2001 . utilization of Petroleum Hydrocarbons by Pseudomonas Fluorescens Isolated from a Petroleum-Contaminated Soil." Environment International 26 (5-6): 413-16.

Eriksson, M., G. Dalhammar, and A. K. Borg-Karlson. 1999.„Aerobic Degradation of a Hydrocarbon Mixture in Natural Uncontaminated Potting Soil by Indigenous Microorganisms at 20 Degrees C and 6 Degrees C." Applied Microbiology and Biotechnology 51 (4): 532-35.

Gutnick, DL, EA Bayer, C Rubinovitz, O Pines, Y Shabtai, and E Rosenberg. 1980. „Emulsan production in Acinetobacter RAG-1 ". Adv. Biotechnol 1 1 : 455-59.

Hanson, K. G., A. Nigam, M. Kapadia, and A. J. Desai. 1997. „Bioremediation of Crude Oil Contamination with Acinetobacter Sp. A3." Current Microbiology 35 (3): 191 -93.

Jain, PK, VK Gupta, RK Gaur, M Lowry, DP Jaroli, and UK Chauhan. 201 1 . „Bioremediation of petroleum oil contaminated soil and water". Research journal of environmental toxicology 5 (1 ): 1 .

Margesin, R., and F. Schinner. 2001 . „Biodegradation and Bioremediation of Hydrocarbons in Extreme Environments." Applied Microbiology and Biotechnology 56 (5-6): 650-63. Mishra, S., J. Jyot, R. C. Kuhad, and B. Lai. 2001 . Evaluation of Inoculum Addition to Stimulate in Situ Bioremediation of Oily-Sludge-Contaminated Soil." Applied and Environmental Microbiology 67 (4): 1675-81 . doi:10.1 128/AEM.67.4.1675-1681 .2001 . Mohsenzadeh, Fariba, Abdolkarim Chehregani Rad, and Mehrangiz Akbari. 2012. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils". Iranian Journal of Environmental Health Science & Engineering 9 (1 ): 26. doi:10.1 186/1735-2746-9-26.

Toren, Amir, Elisha Orr, Yossi Paitan, Eliora Z. Ron, and Eugene Rosenberg. 2002. „The Active Component of the Bioemulsifier Alasan from Acinetobacter Radioresistens KA53 Is an OmpA-like Protein." Journal of Bacteriology 184 (1 ): 165— 70. H . Walzer, Gil, Eugene Rosenberg, and Eliora Z. Ron. 2006.„The Acinetobacter Outer Membrane Protein A (OmpA) Is a Secreted Emulsifier." Environmental Microbiology 8 (6): 1026-32. doi:10.1 1 1 1/j.1462-2920.2006.00994.X.

12. Wang, Z., M. Fingas, S. Blenkinsopp, G. Sergy, M. Landriault, L. Sigouin, J. Foght, K.

Semple, and D. W. Westlake. 1998. Comparison of Oil Composition Changes due to Biodegradation and Physical Weathering in Different Oils." Journal of Chromatography. A 809 (1 -2): 89-107.