Abstract:
The present invention relates to the area of environmental biotechnology. It describes environment object cleaning from oil pollutants (OPs), when they are treated with oil hydrocarbon emulsifying and oxidizing bacterial preparations and plants suitable for phytoremediation. This method is used for cleaning of soil, briny and fresh water. This invention presents a novel complex OP cleaning method, which fully or partially solves the present shortcomings with environment cleaning from OPs. Invention is different from other known oil pollutant cleaning methods, because OP cleaning is managed with a help from an expert system which comprises the evaluation of primary OP composition and environment parameters, selection of OP cleaning method and OP biodegrading microorganism blends, selection of optimal concentrations for these blends, selection of optimal OP separation and biodegradation parameters and selection of the most suitable plants for phytoremediation.

Description:
TECHNICAL FIELD 
       [0001]    This invention is attributed to the field of environment protection biotechnology. It describes the cleaning of environment objects from oil pollutants (OPs), i.e. their treatment with hydrocarbons emulsifying and oxidizing bacterial preparations, and phytoremediating plants. This method is used for the cleaning of soil, briny and fresh water. 
       BACKGROUND OF INVENTION 
       [0002]    One pollution type that is frequently encountered is pollution with oil and its products. Obtained oil is transported increasingly larger distances, thus heightening the chance of accidents; its use generates the exhaust of gasses responsible for “greenhouse effect”, which affects the ecological state of various regions. It has been determined that tanker accidents alone are responsible for the loss of approximately 1 million tones of oil products a year; ⅓ of those are light fractions that evaporate into the environment and the rest sink or are thrown onto the shore. Mud is created during oil purification, as the by-products are eliminated. They are mostly heavy oil hydrocarbon fractions absorbed into peat or soil. Additionally, large amounts of oil polluted and hard to clean water are generated. Immediately upon their entry into the environment, oil pollutants (OPs) are toxic to the biological sphere. There are lots of different oil products with various properties; their noxiousness to the environment is also not uniform. The most dangerous are volatile products able to quickly disperse in the surroundings; it may be gasoline, kerosene, diesel and other liquid products. Solid state oil products, e.g. bitumen, are only slightly or not at all dangerous to the environment, thus from now on only liquid state OPs will be discussed. Soil and water polluted with oil hydrocarbons are cleaned using physical, chemical and biological methods. However, sometimes a desirable result cannot be achieved by cleaning soil using a biological method, as seasonal temperature fluctuations and overly large OP concentrations in soil have an effect on oil pollutant oxidizing microorganisms. Phytoremediation method is applied increasingly widely, because this cleaning method requires less expenditure than other biological treatments. Polluted soil has to be additionally cleaned before applying phytoremediation, in order to lower OP concentrations to optimal for plant vegetation. A need for the optimization of cleaning treatments arises, as the work scale increases. Only the creation of new complex technologies and their optimal management in addition to the development and application of new biopreparations allows to solve the emerging problems. 
         [0003]    Patent literature describes various microorganisms with oil oxidizing and surface active substance synthesizing properties. Singular oil oxidizing microorganisms (OOM) and their associations are used to clean soil and waters. Bacterial surface active substances (BSAS) and synthetic surface active substances (SSAS) are used for flushing out organic pollutants from the environment (water, soil) and for better biodegradation. Patents that describe OP removal using plants also exist. 
         [0004]    Known OOMs used to clean environment from oil pollutants:  Azotobacter vinelandii  21 strain, described in LT patent No. 3111 B,  Pseudomonas fluorescens  IGN 57, described in LT patent No. 4792 B,  Candida lipolytica  C. 6.1-5, described in LT patent No. 4793 B. The main shortcoming of using those microorganisms is that enzymes synthesized by singular strains are not enough to fully degrade compounds in OPs. 
         [0005]    U.S. Pat. No. 6,652,752 B2 describes a cleaning method when OOMs are isolated from the environment and multiplied, then their mixed culture is used for cleaning of OP infused water and oily mud in a reactor. This method is suitable for use on oily mud when it is polluted with saturated and aromatic hydrocarbons, asphaltenes and resins. Better biodegradation is achieved by using nutrient additives, surface active substances, aeration and keeping an optimal pH. The drawbacks of this method are: it&#39;s hard to control an OP biodegradation process using an unidentified OOM culture; OP biodegradation can only be done ex situ. 
         [0006]    There are known environment cleaning from OPs methods, where pure microorganism culture blends are used for biodegradation. For example, patent LT 5057 B describes a biopreparation composed of a mix of hydrophilic and lipophilic OOM, designed to clean soil and water polluted with oil and its products. The drawback of this biopreparation is that it is effective in a narrow temperature range and only in a presence of small concentrations of oil hydrocarbons. 
         [0007]    Patent RU 2266958 describes OOM strains  Zoogloea  sp. 14H,  Arthrobacter  sp. 13H,  Arthrobacter  sp. 15H,  Bacillus  sp. 3H,  Bacillus  sp. 12 and an association using them as a basis, which are used to clean soil and waters polluted with oil hydrocarbons. Patent shows that the growth of these strains is uninhibited, when the concentration of oil and fuel oil is respectively 15 and 10%. However, these OOMs only fully degrade oil hydrocarbons, when their concentrations are low: 0.5-0.7% for oil and 0.4-0.5% for fuel oil. 
         [0008]    U.S. Pat. No. 6,649,400 describes OOMs belonging to genera  Acinetobacter, Pseudomonas, Alcaligenes, Flavobacterium  and  Moraxella . These OOM strains are used single and in combinations to clean the environment from heavy oil hydrocarbons. 
         [0009]    U.S. Pat. No. 5,494,580 describes a method of cleaning hydrocarbon polluted environment using microorganisms and their blends that are chosen according to the OP composition and quantity and environmental characteristics. Microorganisms  Azotobacter vinelandii  21,  Pseudomonas  sp 0.9,  Pseudomonas  sp. 19,  Pseudomonas  sp. 31 and  Acinetobacter calcoaceticus  23 are used for the degradation of hydrocarbons. The drawback of this patent relates to the long duration of degradation for heavy oil hydrocarbons. 
         [0010]    Patent US 2009/0325271 describes a method of cleaning soil polluted with oil and its products, when the first stage uses oil emulsifying microorganism (OEM) strains  Pseudomonas aeruginosa  IOCX and  Pseudomonas aeruginosa  IOCX DHT, which separate OPs from the soil particles. OOM strains  Pseudomonas putida IOC 5a1,  Pseudomonas putida  IOCR1 and  Baccilus subtilis  were applied at least a fortnight later than OEM. The drawback of this patent is the absence of clarification for the application of OEMs and OOMs in various OP concentrations in the soil, and it is not known what OPs are being removed. 
         [0011]    The method of removing oil hydrocarbons from the soil using higher plants and OOMs is also described. For example, patent US 2004/0101945 describes a method of removing poly-aromatic compounds from the environment using a system made of at least one suitable host-plant, which emits enzymes degrading organic pollutants into the environment, and one microorganism able to degrade organic compounds, improve host-plant viability, growth and survivability. Recommended microorganisms are  Burkholderia  ATCC No. PTA-4755,  Burkholderia  ATCC No. PTA-4756,  Sphingomonas  ATCC No. PTA-4757. The drawback of this patent is the limited application for the soil cleaning from OPs, since there are not much poly-aromatic compounds in oil and its products. 
         [0012]    Patent LT 4593 describes a method for cleaning soil from OPs that is suitable to use in the finishing stage of the biological treatment, when the soil is treated with organic and mineral fertilizers and seeded with less demanding agricultural plant cultures resistant to oil products, whose rhizosphere immobilizes oil oxidizing microorganisms. Cultures are grown until soil pollution drops to the allowed level, and then the soil with the plant biomass is ploughed. The drawback of this patent is that the described method is only used at a low (6000-7000 mg/kg) concentration of oil products in soil. 
         [0013]    Aforementioned OP treatment methods do not fully solve all the problems pertaining to the cleaning of environment from the pollutants generated during industrial processes:
       advanced environment cleaning from OPs methods require human resources of high qualification;   there is no universal technology designed for the cleaning of different objects and territories from OPs;   there is no effective technology for cleaning of the environment from OPs in different climate conditions;   there are no solutions for cleaning the environment from old OPs;   there is no complex method based on the biotechnological processes that could solve the aforementioned problems;   there is no special systematic and effective environment cleaning from OPs management based on process control.       
 
       SUMMARY OF THE INVENTION 
       [0020]    Goal of invention is to remove the pollution with oil hydrocarbons from various environment objects and restore their original state by natural means, i.e. OEM and OOM based bioproducts, and plants for phytoremediation, without inducing the secondary pollution. 
         [0021]    Essence of invention is a complex environment cleaning from OPs, based on biotechnological processes, and managed by a special expert system (ES) that chooses optimal cleaning technological parameters: blends of OEMs and OOMs, cleaning conditions and phytoremediating plants. 
         [0022]    This invention offers a novel complex OP cleaning method, which fully or mostly solves shortcomings in the present environment cleaning from OPs. The invention is different from other known oil pollutant cleaning methods, as OP cleaning is controlled by ES, whose operation encompasses the evaluation of primary OP composition and environmental parameters, the selection of OP cleaning method and OP biodegrading microorganism blends, the selection of optimal concentrations for microorganisms composing those blends, the selection of OP separation and biodegradation parameters and the selection of suitable plants for phytoremediation. 
         [0023]    The second difference is that environment objects polluted with oil hydrocarbons are cleaned with microorganism blends selected from OEM group consisting of  Pseudomonas  sp. NJ13,  Acinetobacter  sp. PR82,  Acinetobacter  sp. N3 and OOM group consisting of  Acinetobacter  sp. N3,  Acinetobacter  sp. NJ9,  Acinetobacter  NJ5; it encompasses the following stages:
       a) evaluation of polluted environment and determination of quantity and composition of OPs;   b) OEM selection in order to increase bioaccessibility;   c) OOM selection in such a way that obtained biopreparations would function in wide ranges of oil hydrocarbon concentrations with various oil hydrocarbons at different environmental parameters: relief, temperature, humidity and atmospheric pressure;   d) contact of oil hydrocarbon polluted environment with OP biodegrading microorganism blends;   e) simultaneous OP separation and degradation, employing OEMs and OOMs;   f) application of phytoremediation for the removal of remaining OPs and restoration of soil properties.       
 
         [0030]    The third difference is that biopreparation used in stage (b) can have properties of both OEM and OOM. 
         [0031]    The fourth difference is that a complex OP cleaning method is used for the biodegradation of oil hydrocarbons characterized with different physical and chemical properties and structure. 
         [0032]    The fifth difference is that using OEM and OOM blends on various environment objects with OP concentrations in range from maximal (˜100%) to minimal (˜0%), the best cleaning results were achieved at concentrations ranging from 35 to 0%. 
         [0033]    As a sixth difference is that OP oxidizing microorganisms can be used in combination with SSAS. 
         [0034]    As a seventh difference is that OP oxidizing microorganisms can be used in combination with BSAS. 
         [0035]    The eighth difference is that water employed for washing OPs from soil can be used for the watering of the same soil, as remaining OPs are removed from it. 
         [0036]    The ninth difference is that BSAS and SSAS can be used multiple times, constantly removing OPs before every use. 
         [0037]    The tenth difference is displayed by observing live OEM cells in a bacterial SAS solution. 
         [0038]    The eleventh difference is that OP emulsification is performed in a pH range of 6-11 and the temperature range of 20-90° C. 
         [0039]    The twelfth difference is that OP degradation by OOM is performed in a pH range of 2-8.5 and the temperature range of 4-40° C., the most preferred pH is 7 and temperature is 30° C. 
         [0040]    The thirteenth difference is that complex OP cleaning can be performed both in situ and ex situ. 
         [0041]    The fourteenth difference is that complex OP cleaning can be started ex situ and continued in situ after the removal of a migrating OP fraction. 
         [0042]    The fifteenth difference is that phytoremediation is employed after the environmental cleaning using OEM and OOM blends. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0043]      FIG. 1  Principal scheme of the OP removal process control 
           [0044]      FIG. 2  Principal scheme of the preparation of OP emulsifying biopreparations 
           [0045]      FIG. 3  Principal scheme of the preparation of OP oxidizing biopreparations 
           [0046]      FIG. 4  Principal schemes of complex OP removal from water (a) and soil (b) 
           [0047]      FIG. 5  Technological scheme of washing OPs from polluted soil 
           [0048]      FIG. 6  Technological scheme of cleaning of water polluted with OPs 
           [0049]      FIG. 7  Technological scheme of open type OP removal from soil 
           [0050]      FIG. 8  Technological scheme of complex OP removal from soil by biodegradation 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0051]    The process of OP removal from various environment objects is coordinated by ES ( FIG. 1 ). The working of such a system is based on the collection and use of information from an OP spill and its application to control OP cleaning processes. Optimal territory cleaning from OP technological parameters are picked with the help of this system and OP removal scenario based on environment protection biotechnological methods is selected (prepared) using them as the foundation. Complex OP removal is performed by employing bioproducts, created using OEMs and OOMs as the basis, and phytoremediation by plants. 
         [0052]    Microorganisms with the most prominent features of oil hydrocarbon emulsification and oxidation were chosen in order to create bioproducts with oil degrading properties. 
         [0053]    Oil pollutants are best emulsified by  Pseudomonas  sp. NJ13,  Acinetobacter  sp. Pr82 and N3 microorganism strains. These strains are preserved in JSC “Biocentras” microorganism collection. 
         [0054]    Their characteristics are as follows: 
         [0055]      Pseudomonas  sp. NJ13 strain (JSC “Biocentras” accession No. B-96-8N) was isolated from oil polluted water body near Nefteyugansk city in Tyumen Oblast (Russia). 
         [0056]    Cells. Cells are in the form of rods with blunt ends, their size is 0.5-0.6Δ1.0-2.3 μm. Cells are mobile, rods can be seen either single or in pairs, Gram negative, do not form endospores. 
         [0057]    Colonies. Glossy, cream-coloured, entire-margined, raised colonies with smooth surface and mucous consistence grow on solid medium after 24 hours. 
         [0058]    Physiological-biochemical properties. It&#39;s an aerobe. Catalase and oxidase reactions are positive, it hydrolyses gelatine. Optimal conditions for strain growth are: temperature range is 25-30° C. and pH is 7.0. Uses glucose, oleic acid, diesel, oil, octadecane, starch, olive and sunflower oil, sodium acetate as a source of carbon and energy. 
         [0059]    Based on 16S rDNA gene analysis, this microorganism is closest to genus  Pseudomonas  sp., as shown in SEQ ID No. 1. 
         [0060]      Acinetobacter  sp. PR82 strain (JSC “Biocentras” accession No. B-94-6N) was isolated from black-earth polluted with heavy oil products in Kaliningrad Oblast (Russia). 
         [0061]    Cells. Cell form and size is dependent on culture age and growth conditions; can range from cocci (0.5-0.7 μm in diameter) to rods (0.6-0.8×1.2-1.6 μm size). Cells are not of even size in culture. Cells are mobile, Gram reaction is variable. 
         [0062]    Colonies. 1-2 mm in diameter, glossy, opaque, raised colonies with smooth surface and whitish entire margin grow on solid medium after 24 hours. 
         [0063]    Physiological-biochemical properties. It&#39;s an aerobe. Catalase reaction is positive, oksidase and urease reactions are negative. Culture is not resistant to acid. Optimal conditions for strain growth: temperature is 30-40° C. and pH is 4.5-9.0. It doesn&#39;t hydrolyse starch and gelatine. Uses glucose, fructose, galactose, saccharose, xylose, ethanol, acetate, citrate, L-alanine, L-phenylanine, D/L-arginine, some hydrocarbons, oil and its products, fats as a source of carbon and energy. 
         [0064]    Based on 16S rDNA gene analysis, this microorganism is closest to genus  Acinetobacter  sp., as shown in SEQ ID No. 2. 
         [0065]      Acinetobacter  sp. N3 strain (JSC “Biocentras” accession No. B-92-11AA) was isolated in Norway from OP. 
         [0066]    Cells. Cell form and size is dependent on culture age and growth conditions; can vary from cocci to straight and irregularly-shaped rods (0.6×2.0 μm size). Cells are mobile, mildly positive reaction with Gram dye, however aging culture cells become Gram-negative. 
         [0067]    Colonies. 1-3 mm in diameter, glossy, whitish, smooth-surfaced, circular colonies grow on solid medium after 48 hours. 
         [0068]    Physiological-biochemical properties. It&#39;s an aerobe. Optimal growth conditions: temperature range is 20-30° C., pH is 6.4-7.0. Oxidase reaction is negative, catalase reaction is positive. Uses xylose, galactose, fructose, acetate, L-alanine, D/L-arginine, Tween-80, some aromatic and aliphatic hydrocarbons, oil and oil products as a source of carbon and energy. It weakly assimilates glucose, doesn&#39;t hydrolyse gelatine, denitrification is negative, urease reaction is positive. 
         [0069]    Based on 16S rDNA gene analysis, this microorganism is closest to genus  Acinetobacter  sp., as shown in SEQ ID No. 3. 
         [0070]    OPs are best degraded by OOM  Acinetobacter  sp. NJ9,  Acinetobacter  sp. NJ5 strains. OP emulsifying  Acinetobacter  sp. N3 also displays such properties. These microorganism strains are deposited in JSC “Biocentras” microorganism collection. Their characteristics are: 
         [0071]      Acinetobacter  sp. NJ9 strain (JSC “Biocentras” accession No. B-96-2N) was isolated from oil polluted water body near Nefteyugansk city in Tyumen Oblast (Russia). 
         [0072]    Cells. Single or paired cocci (0.5 μm) or rods (0.5×2.0 μm); rods can form a fake mycelium or be spread in a V or W formation. Gram dyeing is variable—culture is composed of Gram-positive and Gram-negative cells. Very clear cycle cocci-rods-cocci. Cells are mobile. 
         [0073]    Colonies. 1-3 mm in diameter, glossy, raised, smooth-surfaced, translucent and fluorescent grey whitish colonies of paste consistence grow on solid medium after 48 hours. 
         [0074]    Physiological-biochemical properties. It&#39;s an aerobe. Catalase reaction is positive, oxidase reaction is negative. Optimal growth conditions: temperature is 25-30° C., pH is 5.5-7.0. NJ9 strain hydrolyses starch, but doesn&#39;t hydrolyse cellulose and gelatine. Uses glucose, xylose, galactose, maltose, glycerin, ethanol, Tween-80, sodium acetate, L-alanine, some aliphatic and aromatic hydrocarbons, oil and its products as a source of carbon and energy. 
         [0075]    Based on 16S rDNA gene analysis, this microorganism is closest to genus  Acinetobacter sp., as shown in SEQ ID No. 4. 
         [0076]      Acinetobacter  sp. NJ5 strain (JSC “Biocentras” accession No. B-96-1N) was isolated from oil polluted clay near Nefteyugansk city in Tyumen Oblast (Russia). 
         [0077]    Cells. Culture is pleomorphic, evolution cycle (cocci-rods-cocci) depends on the medium composition, growth temperature and aeration. Diameter of cocci is 0.7-0.9 μm, rod size is 0.7-1.1×1.1-1.7 μm. Rods are mobile. Gram dyeing is variable—culture is composed of Gram-positive and Gram-negative cells. 
         [0078]    Colonies. 2-4 mm in diameter, mildly glossy, raised, smooth-surfaced, whitish, entire-margined colonies of a paste consistence grow on solid medium after 48 hours. 
         [0079]    Physiological-biochemical properties. It&#39;s an aerobe. Catalase reaction is positive, oxidaze, methyl red reactions and Voges-Proskauer test are negative. Not resistant to acid. Optimal growth conditions: temperature is 20-30° C. and pH is 7.0-7.5. Doesn&#39;t degrade cellulose, doesn&#39;t hydrolyse starch and gelatine. Uses glucose, xylose, galactose, lactose, L-alanine, some hydrocarbons, oil and its products, fats as a source of carbon and energy. 
         [0080]    Based on 16S rDNA gene analysis, this microorganism is closest to genus  Acinetobacter sp., as shown in SEQ ID No. 5. 
         [0081]    Evaluation of Polluted Environment Parameters, Determination of OP Chemical Origin and Quantity 
         [0082]    After the introduction of OPs into the environment, firstly, according to the standard procedures, their chemical origin, quantity and polluted environment parameters are analyzed. Obtained data is transferred to the ES, whose activities encompass evaluation of primary OP composition and environment parameters, selection of OP removal method, selection of OEM and OOM blends, selection of optimal concentrations for the microorganisms in those blends, selection of optimal OP separation and biodegradation parameters and selection of the most suitable plants for the phytoremediation. With the help of the decision making process, main geographic, geologic, OP origin and quantity, climate, polluted environment characteristics and etc. data is processed and linked within ES module (Table 1). 
         [0083]    ES also processes database information about material, logistic, and human resources needed for OP cleaning and evaluates financial expenditure and losses. 
         [0084]    After primary evaluation of OP cleaning parameters, ES chooses biopreparation compositions and OP cleaning technological and biodegradation parameters. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Principal example of environment evaluation ES module 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Pollutant amount 
                 Pollutants 
                   
               
             
          
           
               
                   
                 OP removal 
                 OP removal 
                 Up to 
                 Up to 
                 Up to 
                 Up to 
                 Up to 
                 Up to 
                 Up to 
                 Up to 
                 burned 
                 Pollutant type 
               
             
          
           
               
                   
                 in situ 
                 ex situ 
                 1 t 
                 5 t 
                 10 t 
                 20 t 
                 50 t 
                 100 t 
                 10 000 t 
                 50 000 t 
                 Yes 
                 No 
                 Gasoline 
                 Diesel 
               
               
                   
               
               
                 City-town 
                 A1 
                 A2 
                 A3 
                 A4 
                 A5 
                 A6 
                 A7 
                 A8 
                 A9 
                 A10 
                 A11 
                 A12 
                 A13 
                 A14 
               
               
                 Technological soil 
                 B1 
                 B2 
                 B3 
                 B4 
                 B5 
                 B6 
                 B7 
                 B8 
                 B9 
                 B10 
                 B11 
                 B12 
                 B13 
                 B14 
               
               
                 Cultivated soil 
                 C1 
                 C2 
                 C3 
                 C4 
                 C5 
                 C6 
                 C7 
                 C8 
                 C9 
                 C10 
                 C11 
                 C12 
                 C13 
                 C14 
               
               
                 Recreational zone 
                 D1 
                 D2 
                 D3 
                 D4 
                 D5 
                 D6 
                 D7 
                 D8 
                 D9 
                 D10 
                 D11 
                 D12 
                 D13 
                 D14 
               
               
                 Preserve 
                 E1 
                 E2 
                 E3 
                 E4 
                 E5 
                 E6 
                 E7 
                 E8 
                 E9 
                 E10 
                 E11 
                 E12 
                 E13 
                 E14 
               
               
                 Ocean 
                 F1 
                 F2 
                 F3 
                 F4 
                 F5 
                 F6 
                 F7 
                 F8 
                 F9 
                 F10 
                 F11 
                 F12 
                 F13 
                 F14 
               
               
                 Sea 
                 G1 
                 G2 
                 G3 
                 G4 
                 G5 
                 G6 
                 G7 
                 G8 
                 G9 
                 G10 
                 G11 
                 G12 
                 G13 
                 G14 
               
               
                 Sea shore 
                 I1 
                 I2 
                 I3 
                 I4 
                 I5 
                 I6 
                 I7 
                 I8 
                 I9 
                 I10 
                 I11 
                 I12 
                 I13 
                 I14 
               
               
                 River 
                 J1 
                 J2 
                 J3 
                 J4 
                 J5 
                 J6 
                 J7 
                 J8 
                 J9 
                 J10 
                 J11 
                 J12 
                 J13 
                 J14 
               
               
                 River shore 
                 K1 
                 K2 
                 K3 
                 K4 
                 K5 
                 K6 
                 K7 
                 K8 
                 K9 
                 K10 
                 K11 
                 K12 
                 K13 
                 K14 
               
               
                 Technological 
                 L1 
                 L2 
                 L3 
                 L4 
                 L5 
                 L6 
                 L7 
                 L8 
                 L9 
                 L10 
                 L11 
                 L12 
                 L13 
                 L14 
               
               
                 waters 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Type of polluted 
               
               
                   
                   
                 Pollutant type 
                   
                   
                   
                 environment 
               
             
          
           
               
                   
                 Crude 
                 Chemical 
                 Climate 
                 Humidity 
                   
                 Briny 
                 Fresh 
               
             
          
           
               
                   
                   
                 oil 
                 Other 
                 composition 
                 Cold 
                 Moderate 
                 Hot 
                 Sufficient 
                 Insufficient 
                 Sand 
                 Clay 
                 Loam 
                 water 
                 water 
               
               
                   
                   
               
               
                   
                 City-town 
                 A15 
                 A16 
                 A17 
                 A18 
                 A19 
                 A20 
                 A21 
                 A22 
                 A22 
                 A24 
                 A25 
                 A26 
                 27 
               
               
                   
                 Technological soil 
                 B15 
                 B16 
                 B17 
                 B18 
                 B19 
                 B20 
                 B21 
                 B22 
                 B23 
                 B24 
                 B25 
                 B26 
                 B27 
               
               
                   
                 Cultivated soil 
                 C15 
                 C16 
                 C17 
                 C18 
                 C19 
                 C20 
                 C21 
                 C22 
                 C23 
                 C24 
                 C25 
                 C26 
                 C27 
               
               
                   
                 Recreational zone 
                 D15 
                 D16 
                 D17 
                 D18 
                 D19 
                 D20 
                 D21 
                 D22 
                 D23 
                 D24 
                 D25 
                 D26 
                 D27 
               
               
                   
                 Preserve 
                 E15 
                 E16 
                 E17 
                 E18 
                 E19 
                 E20 
                 E21 
                 E22 
                 E23 
                 E24 
                 E25 
                 E26 
                 E27 
               
               
                   
                 Ocean 
                 F15 
                 F16 
                 F17 
                 F18 
                 F19 
                 F20 
                 F21 
                 F22 
                 F23 
                 F24 
                 F25 
                 F26 
                 F27 
               
               
                   
                 Sea 
                 G15 
                 G16 
                 G17 
                 G18 
                 G19 
                 G20 
                 G21 
                 G22 
                 G23 
                 G24 
                 G25 
                 G26 
                 G27 
               
               
                   
                 Sea shore 
                 I15 
                 I16 
                 I17 
                 I18 
                 I19 
                 I20 
                 I21 
                 I22 
                 I23 
                 I24 
                 I25 
                 I26 
                 I27 
               
               
                   
                 River 
                 J15 
                 J16 
                 J17 
                 J18 
                 J19 
                 J20 
                 J21 
                 J22 
                 J23 
                 J24 
                 J25 
                 J26 
                 J27 
               
               
                   
                 River shore 
                 K15 
                 K16 
                 K17 
                 K18 
                 K19 
                 K20 
                 K21 
                 K22 
                 K23 
                 K24 
                 K25 
                 K26 
                 K27 
               
               
                   
                 Technological 
                 L15 
                 L16 
                 L17 
                 L18 
                 L19 
                 L20 
                 L21 
                 L22 
                 L23 
                 L24 
                 L25 
                 L26 
                 L27 
               
               
                   
                 waters 
               
               
                   
                   
               
             
          
         
       
     
         [0085]    OEM Evaluation 
         [0086]    Soil, due its structural properties, can absorb OPs that enter it. Sorption capacity depends on the soil type and OP fractional composition. Thus BSAS are used in order to increase OOM bioaccessibility to OPs and in such way increase the degradation speed of oil hydrocarbons. 
         [0087]    One of the most important properties of BSAS is the ability to decrease surface tension within the phase interface. OEM strains were grown separately in liquid nutrient media. Surface tension of an OEM culture liquid was measured with a tensiometer at a temperature of 21° C. after 16 hours of incubation (Table 2). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The evaluation of BSAS producing microorganisms 
               
               
                 according to surface tension 
               
             
          
           
               
                   
                   
                 Surface tension, 
               
               
                 No. 
                 Strain 
                 mN/m 
               
               
                   
               
               
                 1 
                   Acinetobacter  sp. N3 
                 32.7 
               
               
                 2 
                   Acinetobacter  sp. Pr82 
                 34.0 
               
               
                 3 
                   Pseudomonas  sp. NJ13 
                 24.8 
               
               
                   
               
             
          
         
       
     
         [0088]    OOM Evaluation According to the Degradation of Oil Hydrocarbons of Various Composition and Structure 
         [0089]    OP composing hydrocarbons are divided into light (C 6 -C 10 ), medium (C 10 -C 28 ) and heavy (C 28 -C 40 ) depending on the amount of carbon atoms in their molecules (Table 3). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 OP degradation with OOMs 
               
             
          
           
               
                   
                 OP degradation, % (after 24 h) 
               
             
          
           
               
                   
                   
                   
                 Distillation 
                 Blend of heavy and 
                   
                   
               
               
                   
                   
                 Crude 
                 of medium 
                 medium fraction 
                   
                 Fuel 
               
               
                 No. 
                 Microorganisms 
                 oil 
                 fractions 
                 distillations 
                 Diesel 
                 oil 
               
               
                   
               
             
          
           
               
                 1 
                 
                   Acinetobacter 
                 
                 65.3 
                 68.5 
                 38.5 
                 71 
                 30.9 
               
               
                   
                 sp. N3 
               
               
                 2 
                 
                   Acinetobacter 
                 
                 46.8 
                 52.5 
                 40.9 
                 56.2 
                 20.9 
               
               
                   
                 sp. NJ5 
               
               
                 3 
                 
                   Acinetobacter 
                 
                 40.6 
                 45.7 
                 31.5 
                 47.4 
                 15.0 
               
               
                   
                 sp. NJ9 
               
               
                   
               
             
          
         
       
     
         [0090]    Oil hydrocarbons of such structure are usually found in places of “aged pollution”. 
         [0091]    Spatial structure also influences degradation degree of OP hydrocarbons 
         [0092]    (Table 4). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Degradation of heavy OPs with various 
               
               
                 spatial structures using OOMs 
               
             
          
           
               
                   
                 Degradation, % 
               
             
          
           
               
                   
                   
                 Unbranched chain 
                 Branched chain 
                 Aromatic 
               
               
                   
                   
                 hydrocarbons 
                 hydrocarbons 
                 hydrocarbons 
               
               
                   
                 Micro- 
                 after 48 h 
                 after 48 h 
                 after 72 h 
               
               
                 No. 
                 organisms 
                 Hexatriacontane 
                 Squalane 
                 Pyrene 
               
               
                   
               
               
                 1 
                   Acineto - 
                 30.1 
                 30.9 
                 14.3 
               
               
                   
                 
                   bacter 
                 
               
               
                   
                 sp. N3 
               
               
                 2 
                   Acineto - 
                 46.1 
                 54.5 
                 18.9 
               
               
                   
                 
                   bacter 
                 
               
               
                   
                 sp. NJ5 
               
               
                 3 
                   Acineto - 
                 34.0 
                 43.6 
                 10.1 
               
               
                   
                 
                   bacter 
                 
               
               
                   
                 sp. NJ9 
               
               
                   
               
             
          
         
       
     
         [0093]    Degradation of OPs in Soil 
         [0094]    An ability of singular OOMs to degrade OPs in various types of soil was determined (Table 5). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Degradation of oil:fuel oil (1:1) mix by 
               
               
                 singular OOMs in various types of soil 
               
             
          
           
               
                   
                 Degradation, % (after 6 weeks) 
               
             
          
           
               
                 No. 
                 Microorganisms 
                 Loam 
                 Clay 
                 Sand 
               
               
                   
               
               
                 1 
                   Acinetobacter  sp. N3 
                 43.1 
                 38.8 
                 35.2 
               
               
                 2 
                   Acinetobacter  sp. NJ5 
                 52.3 
                 32.2 
                 43.6 
               
               
                 3 
                   Acinetobacter  sp. NJ9 
                 37.5 
                 47.9 
                 49.2 
               
               
                   
               
             
          
         
       
     
         [0095]    Degradation of OPs using OOM and OEM blends in various types of soil was also evaluated (Table 6). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Degradation of oil:fuel oil (1:1) mix by OOM 
               
               
                 and OEM blends in various types of soil 
               
             
          
           
               
                   
                 Degradation, % (after 6 weeks) 
               
             
          
           
               
                   
                 Loam 
                 Clay 
                 Sand 
               
               
                   
                 OEM 
                 OEM 
                 OEM 
               
             
          
           
               
                 No. 
                 OOM 
                 N3 
                 Pr82 
                 NJ13 
                 N3 
                 Pr82 
                 NJ13 
                 N3 
                 Pr82 
                 NJ13 
               
               
                   
               
               
                 1 
                 
                   Acinetobacter 
                 
                 56.1 
                 49.5 
                 50.8 
                 51.6 
                 46.0 
                 53.3 
                 55.6 
                 60.0 
                 47.3 
               
               
                   
                 sp. N3 
               
               
                 2 
                 
                   Acinetobacter 
                 
                 77.0 
                 55.4 
                 58.7 
                 60.1 
                 44.4 
                 41.7 
                 64.0 
                 81.4 
                 78.7 
               
               
                   
                 sp. NJ5 
               
               
                 3 
                 
                   Acinetobacter 
                 
                 56.5 
                 44.7 
                 51.9 
                 50.8 
                 55.3 
                 60.8 
                 72.1 
                 64.9 
                 52.4 
               
               
                   
                 sp. NJ9 
               
               
                   
               
             
          
         
       
     
         [0096]    Degradation of OPs in Fresh Water 
         [0097]    Degradation of OP in fresh water was performed using OOM cultures. All the microorganisms were more effective at degrading oil, instead of fuel oil (Table 7). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Degradation of oil and fuel oil (1:1) by OOMs in fresh water 
               
             
          
           
               
                   
                 Degradation, % (after 3 days) 
                   
               
               
                   
                 Fresh water 
               
             
          
           
               
                 No. 
                 OOM 
                 Oil 
                 Fuel oil 
               
               
                   
               
               
                 1 
                   Acinetobacter  sp. N3 
                 71.2 
                 60.4 
               
               
                 2 
                   Acinetobacter  sp. NJ5 
                 55.4 
                 54.3 
               
               
                 3 
                   Acinetobacter  sp. NJ9 
                 59.6 
                 43.4 
               
               
                   
               
             
          
         
       
     
         [0098]    Degradation of OPs in Briny Water 
         [0099]    Cleaning of briny water from OPs was also performed using OOM cultures (Table 8). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Degradation of oil and fuel oil (1:1) 
               
               
                 by OOMs in sea and ocean water 
               
             
          
           
               
                   
                 Degradation, % (after 4 days) 
               
             
          
           
               
                   
                 Microorganisms 
                 Sea 3.5‰ 
                 Ocean 35‰ 
               
             
          
           
               
                 No. 
                 and their blends 
                 Oil 
                 Fuel oil 
                 Oil 
                 Fuel oil 
               
               
                   
               
               
                 1 
                   Acinetobacter  sp. N3 
                 54.8 
                 45.2 
                 31.6 
                 26.7 
               
               
                 2 
                   Acinetobacter  sp. NJ5 
                 39.7 
                 38.4 
                 13.3 
                 21.4 
               
               
                 3 
                   Acinetobacter  sp. NJ9 
                 38.0 
                 25.2 
                 27.6 
                 24.2 
               
               
                   
               
             
          
         
       
     
         [0100]    Selection of Biopreparation Composition 
         [0101]    Aside from the primary cleaning data, the data regarding OEM and OOM abilities to remove OPs from various types of soil and from water of different salinity is also entered into ES. With the help of ES decision making management process, this data is evaluated and the results help to make a choice of the best OEM and OOM blends. 
         [0102]    Selection of Plants for Phytoremediation 
         [0103]    The stage of OP removal using hydrocarbon biodegrading OEM and OOM blends is finished once OP concentration in soil decreases to 25 g/kg. 
         [0104]    Phytoremediation is used for remaining oil pollution. This process can employ singular plants like red clover ( Trifolium pratense  L.), Timothy-grass ( Phleum pratense ), perennial ryegrass ( Lolium perenne ) or their combinations. 
         [0105]    OP cleaning process is finished when the concentration of oil hydrocarbons does not exceed environmental regulations. All the data is entered into ES. 
         [0106]    Management of OP Cleaning-Process 
         [0107]    ES chooses the most optimal OP removal technological scenario for a particular environmental object and controls OP removal progress by processing all the present and newly entered OP removal technological parameters. If OP removal progress does not satisfy a chosen scenario, it is immediately replaced with another, more suitable to reach a maximal degree of OP degradation. 
         [0108]    When OP concentrations satisfy environmental regulations, ES frames a final OP removal report, evaluating not only OP removal process, but also its costs. 
         [0109]    Complex Soil Cleaning from OPs In Situ 
         [0110]    This data is entered into ES:
       polluted area is 10 ha;   soil type is loam;   average soil temperature is 20° C.;   soil humidity is 20%;   soil pH is 7.2;   OP concentration in the soil is about 162 g/kg;   OP chemical composition: saturated compounds—68%, aromatic compounds—14%, resins—8%, asphaltenes—10%.       
 
         [0118]    ES chose this OP removal technological scenario, after processing present and entered data:
       OEM strain  Pseudomonas  sp. NJ13; NOM— Acinetobacter  sp. N3;   OEM and OOM ratio in the blend is 1:2.   primary blend concentration in a work suspension is 2.7×10 7  CFU/mL;   nutrient additives (N and P);   foreseeable cleaning duration is up to 18 months   foreseeable frequency for taking of control samples is 1 time/3 months.       
 
         [0125]    ES chosen scenario foresees that soil phytoremediation with a combination of Timothy-grass ( Phleum pratense ) and ryegrass ( Lolium perenne ) seeds will be performed after OP concentration decreases to 25 g/kg. Once OP concentration in soil decreases to 2 g/kg, OP removal works are terminated and a final report regarding OP cleaning process and its costs is prepared. 
         [0126]    Complex Soil Cleaning from OP Ex Situ 
         [0127]    This data is entered into ES:
       the amount of oily mud is 1400 t;   soil type is loam;   humidity of oily mud is 50%;   pH of oily mud is 6.8;   OP concentration in a mud is about 285 g/kg;   OP chemical composition: C 28 -C 40  OPs—42.5%, other OP fractions—57.5%.       
 
         [0134]    ES chose this OP removal technological scenario, after processing present and entered data: 
         [0135]    1. OP emulsification.
       OP separation in a washing device;   used OEM strain is  Acinetobacter  sp. Pr82;   OP separation temperature is 45-50° C.;   pH of emulsifying suspension is 8.5;   OP emulsification process is terminated when OP concentration decreases to 170 g/kg.       
 
         [0141]    2. OP degradation.
       OP biodegradation is performed in a specially constructed cleaning site;   spreading layer thickness is 0.4 m;   OOM strains:  Acinetobacter  sp. NJ5 and  Acinetobacter  sp. NJ9.   OOM ratio in a blend is 1:1;   primary blend concentration in a work suspension is 5×10 7  CFU/mL;   nutrient additives (N and P);   OP degradation process is terminated when OP concentration decreases to 25 g/kg.       
 
         [0149]    3. Phytoremediation.
       soil restoration is performed in a special phytoremediation field;   soil spreading layer thickness is 0.2-0.3 m;   ploughing and cultivation   plants used for phytoremediation are red clovers ( Trifolium pretense  L.)   phytoremediation process is terminated when OP concentration decreases to 2 g/kg.       
 
         [0155]    4. Final OP removal report.
       data about OP removal process;   data about OP removal costs.       
 
         [0158]    Complex Cleaning of Freshwater Body from OPs 
         [0159]    This data is entered into ES:
       polluted area of freshwater body is 1 km 2 ;   average water temperature is 18° C.;   water pH is 7.1;   OP concentration on the surface of the water is about 0.5 g/L;   OP chemical composition: diesel.       
 
         [0165]    ES chose this OP removal technological scenario, after processing present and entered data:
       used OOM strains are  Acinetobacter  sp. N3,  Acinetobacter  sp. NJ9;   ratio in the blend is 2.5:1;   primary blend concentration in a work suspension is 1.8×10 6  CFU/mL;   cleaning duration is up to 6 months;   foreseeable frequency of taking control samples is every 0.5 months;   treatment frequency is 1 time/month.       
 
         [0172]    ES chosen scenario foresees that OP cleaning process will be terminated once OP concentration drops to 0.4 mg/L. After that a final report about OP cleaning process and its costs will be prepared. 
         [0173]    Complex Cleaning of Briny Water from OPs 
         [0174]    This data is entered into ES:
       accident on an oil platform;   oil amount in the sea is 200 t;   oil amount on the shore is 5 t;   polluted sea area is 20 km 2 ;   polluted shore length is 15 km;   water salinity is 8.5‰;   OP chemical composition: crude oil.       
 
         [0182]    ES chose this OP removal technological scenario, after processing present and entered data: 
         [0183]    1. Water cleaning.
       used OOM strain is  Acinetobacter  sp. NJ9;   primary concentration in a work suspension is 1.1×10 7  CFU/mL;   cleaning duration is 3 months;   foreseeable frequency of taking control samples is 2 times/month;   treatment frequency is 2 times/month.       
 
         [0189]    2. Shore cleaning.
       used OEM strain is  Acinetobacter  sp. N3 and OOM strain is  Acinetobacter  sp. NJ9;   ratio in a blend is 1:1;   primary concentration in the main suspension is 1.3×10 7  CFU/mL;   dosing volume is 1 L/metre of shore length;   cleaning duration is 3 months;   foreseeable frequency of taking control samples is 2 times/month;   treatment frequency is no less than 1 time/month.       
 
         [0197]    ES chosen scenario foresees that OP cleaning process will be terminated once OP concentration drops to 0.1 mg/L in water and 1 g/kg on the shore. After that a final report about OP cleaning process and its costs will be prepared. 
         [0198]    Principal schemes for ES operation, OEM and OOM biosynthesis and technological schemes for main OP removal processes are presented further.