KR100464107B1 - Microorganism consortia for the degradation of diesel and process for preparation thereof - Google Patents

Abstract

The present invention relates to an oil-degrading microbial agent and a method for manufacturing the same, and after separating microorganisms capable of decomposing oil in soil contaminated with oil, among them, microorganisms for oil-degrading microorganisms are selected and combined. to provide. Oil decomposition microbial agent according to the present invention has the effect of improving the oil degradation rate and ecosystem adaptability.

Description

Microorganism consortia for the degradation of diesel and process for preparation

The present invention relates to an oil-degrading microbial agent, and more particularly, to a method for producing an oil-degrading microbial agent and a method for producing the oil-degrading microbial agent by selecting and combining strains having excellent oil-degrading ability from soil.

Diesel includes various petroleum hydrocarbons and related compounds. Petroleum hydrocarbons and products containing them have contributed greatly to the industrial development of mankind, but on the other hand, they are a major cause of environmental pollution. Petroleum hydrocarbons cause serious environmental problems due to their degradation resistance, bioaccumulation and toxicity to various biological systems.

Oil pollution is caused by leaks, breakage of storage tanks and transport accidents. Diesel is a major source of contamination in soil and groundwater near storage. Contaminants are naturally removed by vaporization, photochemical and soil chemical reactions, and biodegradation, but take considerable time. The physicochemical soil treatment method has been mainly used, but it shows limitations in terms of treatment efficiency and cost. Therefore, in recent years, biological soil purification methods using microorganisms, which play a pivotal role in the natural decomposition of oil, have been studied.

The present inventors have applied for a patent application (Patent Application No. 1999-10776) for separating microorganisms having excellent oil resolution from soil. However, the present inventors have found that by combining the isolates rather than the single strain, the oil degradation rate and the adaptability to the ecosystem can be improved, thereby completing the present invention.

Accordingly, it is an object of the present invention to isolate microorganisms that degrade oil from soil contaminated with oil, and to select microorganisms having excellent oil-degrading ability among them, to provide an oil-decomposing microbial agent combining them.

Another object of the present invention is to provide a method for preparing the oil-degrading microbial agent.

In order to achieve the above object, in the present invention, by separating and screening the hydrolysis strain from the contaminated soil, by combining the selected strains to investigate the oil degradation ability to prepare a hydrolysis microbial formulation.

Hereinafter, the configuration and operation of the present invention.

1 is a graph showing the diesel decomposition rate of the hydrolysis strain selected according to the present invention (cultivated in a minimal nutrient medium containing diesel as the only carbon source for 6 days at 25 ℃),

Figure 2 shows the results of analyzing the residual diesel after gas culture by culturing the oil-degrading microbial agent (incubated for 6 days in a medium containing 4% (v / v) diesel) according to the present invention by gas chromatography Graph shown (large peak is 4% (v / v) diesel as control),

FIG. 3 shows cells according to the time of lyolytic microbial formulation consisting of Pseudomonas Y2G1, Pseudomonas Y2K4 and Sphingmonas D3K1 (incubated at 25 ° C. for 6 days in a minimal medium containing 4% (v / v) diesel as the only carbon source). Graph showing changes in growth, diesel concentration and pH,

4 is a graph showing the change in colony number (CFU) when Pseudomonas Y2G1, Pseudomonas Y2K4, and Sphingomonas D3K1 were cultured alone or in combination with each other (the strain is 4% (v / v) diesel Incubate for 6 days at a minimum medium containing and 25 ℃, the initial inoculum is 1 × 10 ⁶ CFU / mL, diluting the cultured cells and plated in nutrient agar medium and incubated for 3 days at 25 ℃ to measure CFU) ,

FIG. 5 is a graph showing the hydrophobicity (expressed as a percentage of cell turbidity distributed over hexadecane) of selected isolates grown in minimal medium containing 4% (v / v) diesel,

6 is a graph showing the emulsification activity of selected isolate strains grown in a minimal medium containing 4% (v / v) diesel.

The present invention comprises the steps of separating and screening the oil decomposition strain by collecting the contaminated soil; Identifying the isolated strain; Combining the selected strains to investigate the oil resolution of each strain combination; And examining the hydrophobicity and emulsifying activity of the isolate.

Hereinafter, the specific configuration of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Isolation and Screening of Lactic Acid Strains

Soil samples used in the present invention were collected from contaminated soil near oil storage in Korea. Soil samples were taken from the top 15 cm of the soil, filtered through a 2 mm diameter sieve, and stored at 4 ° C. until use. 10 g of soil samples collected at each site were placed in 10 mL minimal nutrient medium and sonicated at 4 ° C. for 15 min on / off cycle. The supernatant (100 μL) of the soil suspension was inoculated in 5 mL of minimal nutrient medium containing 10% (v / v) diesel and incubated at 4 ° C. for 4 weeks. The enriched samples were passaged by inoculating fresh medium every week. As a result of dilution of the inoculum at the last subculture in Nutrient Broth Agar (Difco), colonies with different morphology and growth rates were formed. The minimum nutritional medium composition is shown in Table 1 below.

Minimum Nutritional Medium ingredient Concentration (mg / L Distilled Water) NaNO ₃ 4 KH ₂ PO ₄ 0.15 Na ₂ HPO ₄ 0.5 MgSO _{4 7} H ₂ O 0.2 FeCl ₃ · 6H ₂ O 0.0005 CaCl ₂ · 2H ₂ O 0.01 pH 7.0

The oil resolution of each isolated colony was measured as follows using the DCPIP test method. Each strain colony was incubated for one week at 25 ° C. by adding 5 μL of DCPIP (3 g / L) to 200 μL of a minimal nutrient medium containing 10% (v / v) diesel. The oil resolution was measured by using the color of the reaction tube containing the oil degradation strain colony changed from blue to colorless (Charlotte Schou et al., 1998).

As a result, 128 strains having the activity to decompose diesel were isolated, and 39 strains were isolated from the DCPIP test. Among the 39 isolated strains, the growth characteristics of the 11 isolated strains rapidly grown in diesel-containing minimal nutrient medium were investigated, and the results are shown in Table 2 below.

Isolation and Growth Characteristics of Hydrolysis Strains No. Isolated strain Generic name Growth characteristics Specific growth rate (10 ^-3 / hr) Absorbance _{660 nm} (70 hours culture) Growth Absorbance _{660 nm} Hour (hr) One Y1K3 Aquaspirum 6.53 0.457 0.521 90 2 Y2G1 Pseudomonas genus 7.40 0.48 0.499 100 3 Y2K4 Pseudomonas genus 6.92 0.556 0.568 80 4 Y2C3 Pseudomonas genus 3.67 0.424 0.64 120 5 Y5K2 Pseudomonas genus 1.03 0.3 0.343 140 6 D3K1 Sphingomonas 3.33 0.427 0.474 140 7 D3K3 Stenotropomonas genus 2.97 0.349 0.454 140 8 I2K3 Pseudomonas genus 8.10 0.394 0.441 110 9 I2D2 Flavobacterium genus 5.07 0.491 0.587 140 10 J1D1 Flavobacterium genus 3.47 0.379 0.554 140 11 J1C2 Pseudomonas genus 7.43 0.485 0.596 140

Among the 11 strains, Pseudomonas I2K3 showed the highest growth rate and Pseudomonas Y2K4 showed the fastest initial growth rate.

Example 2. Identification of Strains

The isolates were incubated at 28 ° C. for 48 hours in tryptic soy agar medium. Plated cells were collected by scraping with a sterile loop and used for fatty acid methyl ester (FAME) analysis (Chung et al., 1997). Ester saponification, methylation and extraction were performed using the procedure described in the MIDI Manual (Microbial Identification, Inc.) (Sasser, M, 1990).

Fast growing strains in diesel-containing minimal nutrient medium were identified by FAME analysis. As shown in Table 2, Y1K3 is aquaspirillum sp. , And D3K3 is a Stenotrophomonas sp. ), I2D2 and J1D1 were identified as Flavobacterium sp. , D3K1 was identified as Sphingomonas sp. , And the remaining strains except for the five strains were Pseudomonas sp. It was confirmed.

Among the lyolytic microorganisms isolated from contaminated soil according to the present invention, sphingomonas D3K1 was assigned to accession number KCTC 8935P on March 11, 1999, and Pseudomonas Y2G1 and Pseudomonas Y2K4 on October 30, 2000, respectively. KCTC 18049P and KCTC 18050P were deposited in the Gene Bank of the Institute of Biotechnology.

Example 3. Oil resolution investigation

Experimental Example 1. Diesel decomposition

Strain growth and biodegradation of diesel were monitored by culturing isolated strains in 50 mL of minimal nutrient medium containing 4% (v / v) diesel as the only carbon and energy source, and the culture conditions were 25 ° C. and 120 rpm. Residual diesel concentration and strain growth were measured after 6 days of culture. Initial cell concentration was adjusted to 1 × 10 ⁶ CFU / mL and inoculation amount was 2% (v / v).

Experimental Example 2. Combination of Isolated Strains

Five strains having excellent oil resolution among the 11 isolated strains were selected, and each strain during the exponential growth phase was combined 1: 1 and inoculated into a minimal nutrient medium. The inoculated cell concentration was 1 × 10 ⁶ CFU / mL and 2% (v / v) of the culture was inoculated. After the combination of the isolates was incubated at 25 ° C. for 6 days, the degradation rate and cell growth (OD 660 nm) of diesel were measured according to the method described below.

The number of colonies formed (CFUs) was measured according to conventional methods (Venkateswaran, K. et al., 1991). In order to monitor the changes of the strains in single strain culture and mixed strain culture, the cells were diluted in 0.85% brine and cultured in nutrient agar medium after 2 and 6 days of culture. Through the nutrient agar plate culture it was possible to distinguish the colonies of all the mixed strains.

Experimental Example 3. Analysis of Diesel Samples

The diesel present in the culture was extracted with hexane according to a conventional method (EPA 3510C), followed by a capillary column (HP-5, 30 m in total length, 0.53 mm in diameter; Hewlette Packard Co., USA) and FID (flame ionization). detector Analysis was performed using gas chromatography (GC-HP 6890, Hewlette Packard Co., USA) equipped with a detector. The operating temperatures of the detector and the injector were 250 ° C and 200 ° C, respectively.

The column (oven) temperature was fixed at 45 ° C. for the first 3 minutes and then increased to 275 ° C. at a rate of 12 ° C./min. The carrier gas was helium, and the gas flow rate was 5 mL / min. The amount of diesel was determined by calculating the sum of each peak area according to the quantitative analysis of total petroleum hydrocarbons (TPH) (EPA i8015B).

Figure 1 shows the diesel decomposition rate of the selected 11 oil-degrading microorganisms. Among them, sphingomonas D3K1 had the highest diesel decomposition rate of 83.77%, and two strains of aquaspirilum Y1K3 and Pseudomonas Y5K2 showed high diesel decomposition rate of 50% or more. Aquaspiryllum Y1K3, Pseudomonas Y2G1 and Pseudomonas Y2K4 showed relatively fast diesel biodegradation rate and cell growth rate, while Pseudomonas Y2C3 showed very low diesel degradation rate. Among the 11 strains, strains having a relatively high oil degradation rate, namely, aquaspirilum Y1K3, Pseudomonas Y2G1, Y2K4, Y5K2, and Sphingomonas D3K1 were used in the diesel decomposition test by culture of the mixed strain, and the results are shown in Table 3 below. Same as

Diesel Degradation of Oil Strain Combination Strain combination Diesel decomposition rate (%) Strain combination Diesel decomposition rate (%) 1 & 2 76.47 1 & 2 & 6 89.46 1 & 3 16.83 2 & 3 & 5 94.39 1 & 5 75.76 2 & 3 & 6 93.53 1 & 6 73.57 2 & 5 & 6 80.06 2 & 3 75.16 3 & 5 & 6 40.41 2 & 5 75.27 1 & 2 & 3 & 5 70.80 2 & 6 82.07 1 & 2 & 3 & 6 78.83 3 & 5 69.01 1 & 2 & 5 & 6 89.86 3 & 6 86.07 1 & 3 & 5 & 6 90.69 5 & 6 93.43 2 & 3 & 5 & 6 70.17 1 & 2 & 3 33.81 1 & 2 & 3 & 5 & 6 72.14 1 & 2 & 5 72.09

Most strain combinations showed higher diesel decomposition rates than single strains. Except for three strain combinations (1 & 3, 1 & 2 & 3, 3 & 5 & 6), all strain combinations showed more than 50% diesel resolution.

Strain combinations 2 & 3 & 5 and 2 & 3 & 6 showed the highest diesel decomposition rates of 94.59% and 93.53%, respectively. 2 is a result of analyzing the diesel decomposition by the combination 2 & 3 & 6 as the peak of the residual diesel using gas chromatography, it was confirmed that most peaks disappeared or decreased after 6 days of culture.

Figure 3 shows the change in cell growth, residual diesel and pH with time when the strain combination was cultured in a medium containing diesel. As the cells grew rapidly, the amount of diesel remaining remained drastically reduced and the pH did not change significantly.

Figure 4 compares the change in colony number (CFU) during the single and mixed culture of the 2, 3 and 6 strains. Pseudomonas Y2G1 increased by 40% on the second day of culture compared to when cultured alone, and Pseudomonas Y2K4 increased by 370% on the second day of culture. In the case of Sphingmonas D3K1, the number of colonies in the mixed culture was increased by 220% on the 6th day of culture compared to the single culture. As shown in Figure 3, the amount of residual diesel rapidly decreased during the initial 2 days in the culture of the strain combination. Pseudomonas Y2K4 was mainly involved in the initial decomposition of diesel based on the results, it was confirmed that the sphingomonas D3K1 acted on the diesel decomposition in the second half of the culture.

Example 4 Determination of Hydrophobicity and Emulsification Activity of Separated Strains

Experimental Example 4. Hydrophobicity Measurement

The relative hydrophobicity of the isolate was measured according to the BATH test method (Zhang et al., 1994), and the cells to be analyzed were prepared by the following procedure. The cultured cells were collected by centrifugation at 10,000 × g, washed twice to remove any material that could interfere with the assay, and then the cells were minimally adjusted to adjust the optical density (OD) to 400 at 400 nm. Resuspend in nutrient medium. The cell solution (4.0 mL) and hexadecane (1.0 mL) were mixed in a test tube, the tube was vortexed for 60 seconds, and the hexadecane and aqueous phases were allowed to stand for 30 minutes to separate the two phases. It was. The aqueous phase was carefully removed with a Pasteur pipette and the turbidity of the aqueous phase was measured at 400 nm. The hydrophobicity of the cells was expressed as a percentage of cell turbidity distributed over hexadecane, and was calculated according to the following formula (1).

Hydrophobicity = {(1-OD in aqueous solution) x 100} / OD of cell suspension ---- [1]

Experimental Example 5. Measurement of Emulsification Activity

The emulsification activity of each isolate was measured according to the method described by Ko et al. (1998). Cell cultures were centrifuged to free the cells and filtered through a cellulose acetate filter (0.45 μm diameter, Adventec MFS. Inc., Japan). Cellular material collected in the filter (0.5 mL) was added to a test tube containing 2.5 mL of 50 mM potassium phosphate buffer with 0.1 mL of n-hexadecane added. The emulsion activity of the aqueous phase was measured by optical density at 610 nm.

5 and 6 show hydrophobicity and emulsifying activity of 11 isolates selected, respectively. As shown in FIG. 5, in most isolates except D3K1 and Y2C3, there was a proportional relationship between hydrophobicity and diesel removal rate, that is, the higher hydrophobicity, the higher diesel removal rate. As shown in Fig. 6, except for Pseudomonas Y2G1, 10 isolates showed low emulsification activity.

As a result, strain combinations 2 & 3 & 5 (Pseudomonas Y2G1, Pseudomonas Y2K4 and Pseudomonas Y5K2) and strain combinations 2 & 3 & 6 (Pseudomonas Y2G1, Pseudomonas Y2K4 and Sphingomonas D3K1) were the most suitable strain combinations for diesel decomposition. . Each strain in the strain combination was a strain having excellent diesel resolution, Pseudomonas Y2G1 and Y2K4 showed a fast growth rate in the minimum nutrient medium containing diesel. In strain combinations 2 & 3 & 6, spingomonas D3K1, the fastest diesel degradation strain, acted on diesel degradation in the second half of the culture, and Pseudomonas Y2G1 and Y2K4 were involved in the diesel degradation at the beginning of the culture.

Pseudomonas Y2G1 with high hydrophobicity and emulsifying activity showed high diesel decomposition rate (57.69%). However, it was difficult to establish the proportional relationship between diesel decomposition rate, hydrophobicity and emulsifying activity. In addition, only a combination of strains with good resolution could not be confirmed as having a high diesel decomposition rate. Therefore, in order to manufacture an appropriate microbial agent that decomposes diesel, the separated isolates must be variously combined with each other to have excellent oil resolution in the early and late stages, and also have high adaptability to the ecosystem. The oil-degrading microbial preparations according to the invention fulfill the above requirements.

As described above in detail with reference to the specific configuration of the present invention, the present invention provides an excellent microorganism formulation consisting of mixed strains having excellent oil resolution ability to improve the oil decomposition rate and ecosystem adaptability than when using a single strain Since it is effective, it is a very useful invention for the environmental industry.

Claims (2)

An oil-degrading microbial agent comprising Pseudomonas Y2G1 (Accession No .: KCTC 18049P), Pseudomonas Y2K4 (Accession No .: KCTC 18050P), and Sphingonanas D3K1 (Accession No .: KCTC 8935P), isolated from contaminated soil. delete