KR102079536B1 - Microbial community with excellent soil crude oil resolution - Google Patents

Abstract

The present invention relates to a microbial community (KWEN-DT) capable of decomposing contaminated soil, obtained from soil in crude oil contaminated area in Kuwait, including microorganisms such as Achromobacter sp., Bacillus sp., Pseudoxanthomonas sp., and Stenotrophomonas sp. According to the present invention, it is possible to overcome shortcomings of the actual process application of single species of microbial agents or complex microbial agents to the actual crude oil contaminants, and also to contribute to biological environment purification for contaminated environment using the microbial groups in Korea and abroad.

Description

Microbial Community with High Soil Crude Resolution {MICROBIAL COMMUNITY WITH EXCELLENT SOIL CRUDE OIL RESOLUTION}

The present invention relates to a microbial community having a crude oil decomposition capability, and more particularly to the decomposition of soil crude oil using a microbial community having excellent soil crude oil decomposition capability.

Decades of crude oil or refined petroleum spilled on the planet for decades have caused soil or marine pollution, and the environment contaminated with crude oil or petroleum increases the concentration of Petroleum hydrocarbons and changes in ecosystems. Crude oil composition varies depending on the production region, but is generally composed of 15-30% n-alkanes and iso-alkanes, 30-50% cyclic alkanes, and 5-20% aromatic compounds (Arima, Kakinuma, & Tamura). (1968), as well as containing carcinogenic substances, contains toxicities, which can quickly destroy existing environmental ecosystems.

Crude oil contamination with particulate media such as soils and sediments can be treated using physicochemical techniques such as soil washing, heat treatment and excavation. Soil washing facilitates the removal of crude oil from particles by cleaning the soil with chemical cleaners. The problem with soil washing is that it can be subjected to secondary damage from the toxicity of chemicals left behind after washing. The solidification method of the heat treatment method is a method of lowering and fixing the solubility of the pollutants, and it is difficult to predict the solidification retention period of the pollutants, and there is a lower point that the storage cost of the treated soil is significant even if it is fixed indefinitely. In the case of small areas of contaminated area, small amounts of contaminants, and contaminants that directly harm the human body, methods such as soil incineration or excavation have been used to expedite contaminants. However, since both methods target a larger amount of soil than chemical treatment methods, they are not only considered to be expensive and impractical, but are also increasingly being used due to the possibility of secondary pollution to the atmosphere. Recently, many researchers and developers have been trying to develop a low-cost, practical and environmentally friendly treatment technology, and biological treatment has been proposed as an alternative.

Biological treatment refers to the method of breaking down pollutants by biological metabolism, and many studies have used microorganisms as the main biological agent. Biodegradation of organic compounds using microorganisms can be thought of as a series of biological degradation pathways where all compounds are used as substrates and cause oxidation. Methods of biological treatment include in situ methods such as biosparging and bioaugmentation and ex situ methods such as land farming and gioreactor. Biosparging is mainly used in areas where oxygen utilization is limited. It is a treatment method that maximizes the air circulation capacity of the soil with a vacuum pump, increases the oxygen concentration by supplying air below the groundwater layer, and has a mixing effect in the contaminated area. Bioaugmentation is a biological method that improves the efficiency of the degradation process by adding microorganisms necessary to speed up the decomposition of contaminants or microorganisms that help the growth of degraded bacteria. Land farming process is to excavate the soil of contaminated site and spread it to 10 ~ 35 cm to cultivate the soil periodically to aeration and agitation. It is mainly used when the concentration of pollutants is not high or there is a fear of contaminants to settle. Bioreactor is a method of effectively decomposing contaminants or removing elements that limit the decomposition by setting various conditions according to contaminants. Key factors such as environmental conditions, the availability of pollutants and nutrients, and the presence of degraded microorganisms are important for the successful application of these biological purifications.

In areas contaminated with petroleum hydrocarbons, pollutants are a mixture of various substances, so the water solubility, toxicity and bioavailability vary widely. Therefore, there are also several strains of degrading bacteria, and even if the bacteria are not responsible for degradation, there are strains that facilitate the metabolic activity of the colony by interacting in the same community. In this respect, when a single strain or complex microorganism is used for bioaugmentation, the limiting factor is vast compared to the microbial community. Accordingly, the present invention proposes a microbial preparation of bioaugmentation that can be used with various biological treatments as a microbial community.

Korean Laid-Open Patent Publication No. 10-2011-0009403 (2011.01.28.)

Arima, K., Kakinuma, A., & Tamura, G. (1968). Surfactin, a crystalline peptidelipid surfactant produced by Bacillussubtilis: Isolation, characterization and its inhibition of fibrin clot formation. Biochemical and biophysical research communications, 31 (3), 488-494. Deng, M. C., Li, J., Hong, Y. H., Xu, X. M., Chen, W. X., Yuan, J. P.,. . . Wang, J. H. (2016). Characterization of a novel biosurfactant produced by marine hydrocarbon? Degrading bacterium Achromobacter sp. HZ01. Journal of applied microbiology, 120 (4), 889-899. Mangwani, N., Shukla, S., Kumari, S., Rao, T., & Das, S. (2014). Characterization of Stenotrophomonas acidaminiphila NCW? 702 biofilm for implication in the degradation of polycyclic aromatic hydrocarbons. Journal of applied microbiology, 117 (4), 1012-1024. Varma, S. S., Lakshmi, M. B., RAJAGOPAL, P., & Velan, M. (2017). Degradation of Total Petroleum Hydrocarbon (TPH) in Contaminated Soil Using Bacillus pumilus MVSV3. Biocontrol science, 22 (1), 17-23. Youssef, N. H., Duncan, K. E., Nagle, D. P., Savage, K. N., Knapp, R. M., & McInerney, M. J. (2004). Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods, 56 (3), 339-347. Quantitative oil analysis method (Canada K1A 0H3), (1994), Vancouver (Colombie-britannique)

It is an object of the present invention that microorganisms exist in a clustered form in a real environment, and the interaction between microbial species in the clusters is an important power for metabolic activity of the clusters. By enriching and cultivating microbial communities that are capable of degrading crude oil pollution and suggesting the entire microbial community as a single microbial agent, it is possible to limit the practical application of a single microbial agent or a complex microbial agent to actual crude oil contaminants. To overcome.

In order to achieve the above object, the present invention provides a microbial community KWEC-DT [KCTC18648P] excellent in soil crude oil resolution.

The microbial community (KWEC-DT) is a genus of Achromobacter sp., Bacillus sp., Pseudoxanthomonas sp., And Stenotrophomonas sp. Include.

The microbial community KWEC-DT [KCTC18648P] was deposited with the Korea Biotechnology Research Institute Gene Bank on Dec. 15, 2017 with accession number KCTC18648P.

The microbial community KWEC-DT [KCTC18648P] has been obtained from soils that have long been contaminated with petroleum hydrocarbons, many of which are reported to perform biodegradation of hydrocarbon components in crude oil or produce biosurfactants.

Kuwait soil was used as the soil for a long time contamination by the petroleum hydrocarbon.

According to the present invention as described above, the present invention provides a microbial community KWEC-DT [KCTC18648P] excellent in soil crude oil resolution, thereby limiting the practical process of applying a single species of microbial agent or complex microbial agent to actual crude oil contaminants. Overcoming, there is an effect that contributes to the biological environmental purification to the polluted environment using microbial groups as well as domestic.

1 is a GC-FID chromatograph.
2 is TPH concentration for 4 weeks.
3 is UCM concentration for 4 weeks.
4 is N-alkane concentration for 4 weeks.

Hereinafter, the present invention will be described in detail.

Provided is a microbial community KWEC-DT [KCTC18648P] excellent in soil crude oil resolution according to one embodiment of the present invention.

The microbial community KWEC-DT [KCTC18648P] is a genus of Achromobacter sp., Bacillus sp., Pseudoxanthomonas sp., And Stenotrophomonas sp. It includes.

The microbial community KWEC-DT [KCTC18648P] was deposited with the Korea Biotechnology Research Institute Gene Bank on Dec. 15, 2017 with accession number KCTC18648P.

The microbial community KWEC-DT [KCTC18648P] has been obtained from soils with a long duration of contamination by petroleum hydrocarbons, many of which are reported to carry out biodegradation of hydrocarbon components in crude oil or to produce biosurfactants.

Kuwait soil was used as the soil for a long time contamination by the petroleum hydrocarbon.

The Kuwait soils are characterized by continued oil pollution for about 26 years.

The microbial community KWEC-DT [KCTC18648P] analyzes the actual crude oil degradation activity of crude oil enrichment cultures obtained from the soil of crude oil contaminated area in Kuwait, and simultaneously isolates the diversity of microbial species in the enrichment cultures and isolates the lines of 16S rRNA gene. Based on the findings from the analysis, it includes Surfactant producing strains and TPH degrading strains.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1.

The purpose of this study was to investigate the species diversity of the concentrated culture broth by pure separation. 0.1 ml of the culture was inoculated in nutrient agar (DifcoTM, 212300, beef extract 3.0g / L, peptone 5.0g / L, agar 15.0g / L) and purely separated to isolate a single microorganism. In order to identify single microorganism, 16S rRNA gene was PCR method (1. 94 ℃ denaturation step is to separate double-stranded DNA into single strand, 2. 55 ℃ annealing step is to start and end point of amplification part. Attach the primer to the single-stranded DNA, 3. 72 ℃ extension was amplified by 30 iterations (except for the initial denaturation and final extension) in the DNA taq polymerase DNA replication and amplification step. The amplified 16S rRNA gene was verified by agarose gel electrophoresis (experimental conditions: 100V, 0.05A, 50min), and then queried by Macrogen to obtain a nucleotide sequence. The obtained nucleotide sequence was identified using a classifier of RDP (rdp.cme.msu.edu) and a blast of NCBI (ncbi.nlm.nih.gov).

Genus Achromobacter sp., Arthrobacter sp., Bacillus sp., Georgenia sp., Lysinibacillus sp., Nitratireductor sp., Ochrobactrum sp., Paenibacillus sp., Pseudomonas sp., Pseudoxanthomonas sp., Pusillimonas sp., Staphylococcus sp., Stenotrophomonas sp. Were identified, and Bacillus sp., Pseudoxanthomonas sp., And Stenotrophomonas sp. Were strains that produced TPH degradation and biosurfactants.

The identification results of Example 1 and the production capacity of TPH degradability and biosurfactant are summarized in Table 1 below.

Share
(%) TPH
Resolution PAHs
Resolution n-alkane
Resolution biosurfactant
Productivity Achromobacter sp. 19.5 O O O Atrhrobacter sp. 5.2 O Bacillus sp. 19.5 O O O Georgenia sp. 1.3 Lysinibacillus sp. 3.9 Nitratireductor sp. 3.9 Ochrobactrum sp. 1.3 Paenibacillus sp. 14.3 O Pseudomonas sp. 1.3 Pseudoxanthomonas sp. 14.3 O O O Pusillimonas sp. 1.3 Staphylococcus sp. 7.8 O Stenotrophomonas sp. 6.5 O O O

Example 2.

Some of the bacteria producing biosurfactants produced clear zones in the blood agar due to their hemolytic properties (Youssef et al., 2004). Therefore, in the present invention, the purely isolated microorganisms are blood agar (Comede Life Science, Inc., 500101, digest of casein 15.0g / L, pancreatic digest of soybean meal 5.0g / L, NaCl 5.0g / L, agar 15.0g / L, Sheep blood cultured in 50ml / L) was confirmed whether the clear zone generation. Microorganisms of Bacillus sp., Stenotrophomonas sp., And Pseudoxanthomonas sp. Formed a clear zone in the blood agar and were evaluated for the ability to degrade crude oil of single strains. Crude oil decomposition capacity evaluation method was carried out in the same manner as in the microbial community of Example 3, the measurement was carried out in the same manner as in Measurement Example 1, based on this concentration was calculated by the same formula. The results are shown in Table 2 below.

genus TPH decomposition rate (%) Bacillus sp. 24.1% Stenotrophomonas sp. 14.1% Pseudoxanthomonas sp. 25.8%

Example 3.

Kuwait crude oil contaminated soil was collected in sterile glass bottles, sealed and transported to the laboratory. Thickening culture was inoculated with 10 g of Kuwait crude oil contaminated soil in 300 ml of basal medium I (Table 3 (a)), and the culture conditions were agitated at a temperature of 28 ° C. and 100 RPM. During the enrichment culture, 1 ml of crude oil was supplied to the carbon source every 60 days, and the volume of the culture solution was maintained at 300 ml in basal medium II (Table 3 (b)).

Basal medium I and basal medium II components are shown in Table 3 below.

(a) Basic Badge I (b) Basic Badge II Total volume 1 L Total volume 1 L Eptone 10.0 g NaCl 5.0 g NaCl 5.0 g CaCl2 · 2H2O 0.1 g CaCl2 · 2H2O 0.1 g

Crude oil decomposition capacity was determined by measuring the concentration of residual petroleum-based total hydrocarbons after mixing the crude oil and the concentrated culture medium. 2.2 g of crude oil was diluted in 100 ml of hexane, subdivided into 1 ml into 20 test tubes, and dried at room temperature for 24 hours on a clean bench. 5 ml of dried crude oil test tubes were put in 5ml of distilled water as a control, and 5ml of concentrated culture medium (OD600nm = 0.550) was added to 15 test tubes. Biodegradation proceeded for 4 weeks. Crude test tube containing the concentrated culture solution was placed at room temperature, and the test tubes were vortex mixed and supplied with air at intervals of 24 hours.

Measurement Example 1.

Residual petroleum total hydrocarbons from one control group and three experimental groups were analyzed at weekly intervals. In order to measure the concentration of petroleum-based total hydrocarbons in the experimental and control groups, the crude oil-rich culture liquid sample was extracted with petroleum-based hydrocarbons by Liquid-Liquid Extraction method. Purification according to ”. The concentration of petroleum-based total hydrocarbons in the purified samples was measured by gas chromatograph (GC-FID, Aglient 6890N, Agilent, DE, USA) equipped with a hydrogen flame ionization detector.

The measured values of Measurement Example 1 for the calculation of TPH, UCM and n-alkane are shown in Tables 4 to 11, and the measurement results are shown graphically in FIG. At this time, Figure 1 (a) is the measurement result of the control group, Figure 1 (b) is the measurement result of the experimental group.

Referring to FIG. 1, the chromatograph of the experimental group after 4 weeks compared to the control chromatograph can be confirmed that there is no significant peak other than surrogate and ISTD (Internal standard). The significant peak means that the peak, which means n-alkanes, is not clearly seen, and the peak is visible in the control group but not visible in the experimental group, indicating that most of the n-alkanes are degraded by the microorganism.

Concentration calculation 1. (Calculation of concentration of TPH and UCM)

The concentrations of petroleum-based total hydrocarbons (TPH) and UCM (Unresolved Complex mixture) were calculated based on Formula 1 based on the measured values of Measurement Example 1.

[Calculation 1]

TPH (UCM) Amount (μg) = (A × W _IS × D × F) / (A _IS × average RRF)

RRF = (A × C _IS ) / (A _IS × C)

average RRF = 0.91 (average calculated based on Table 7)

A = target area,

A _IS = internal standard area,

C _IS = internal standard concentration

C = target concentration

W _IS = Spiked amount in sample (4 ug)

D = Dilution factor (no dilution = 1),

F = Fractionation (volume factor = 2)

Table 4 shows the calculated values of W _IS .

Spiked amount in sample (W _IS ) Stock (ug / ml) ml Amount (ug) OTP (Surrogate std.) 4 ug 80 0.05 4 5a-Andro. (GC-IS.) 4 ug 80 0.05 4

Concentration output 2. (concentration of n-alkane)

Based on the measured value of the said measurement example 1, the concentration of n-alkane was computed based on Formula 2.

[Calculation 2]

Amount (μg) = (A × W _IS × D × F) / (A _IS × each compound RRF)

The target area, internal standard concentration, internal standard area, and target concentration were measured by using a standard solution of each hydrocarbon for calculating RRF (relative response factor) values, which are shown in Tables 5 to 6 below. The calculated RRF (Relative Response Factor) value is shown in Table 7 below. (CAL 1 = TPH standard 1 ppm, CAL 2 = 5 ppm, CAL 3 = 10 ppm, CAL 4 = 50 ppm)

Analytes Area RT CAL1 CAL2 CAL3 CAL4 RT. (min) One 5 10 50 5α-Androstane 5a-Andro. (GC-IS.) 20.782 16.900 16.800 16.400 16.400 o-Terphenyl OTP (Surrogate std.) 19.768 16.700 16.600 16.100 15.100 n-Octane C ₈ 5.222 3.70 19.20 40.30 192.40 n-Decane C ₁₀ 9.607 4.10 20.40 41.10 198.60 n-Dodecane C ₁₂ 12.579 4.20 20.70 41.00 197.10 n-Tetradecane C ₁₄ 14.898 4.30 20.70 40.30 194.20 n-Hexadecane C ₁₆ 16.896 4.10 20.20 38.60 188.30 n-Octadecane C ₁₈ 18.683 4.00 19.60 36.90 184.10 o-Terphenyl OTP 19.768 16.70 16.60 16.10 15.10 n-Eicosane C ₂₀ 20.302 4.10 19.80 36.70 183.60 5α-Androstane 5a-Andro. 20.782 16.90 16.80 16.40 16.40 n-Docosane C ₂₂ 21.782 4.10 19.50 36.80 183.30 n-Tetracosane C ₂₄ 23.143 4.30 19.60 36.80 180.70 n-Hexacosane C ₂₆ 24.402 4.30 19.50 37.00 180.80 n-Octacosane C ₂₈ 25.572 4.30 19.60 37.50 183.00 n-Triacontane C ₃₀ 26.665 4.20 19.60 37.80 184.20 n-Dotriacontane C ₃₂ 27.693 3.90 19.20 37.20 181.00 n-Tetratriacontane C ₃₄ 28.822 4.00 18.80 36.80 178.50 n-Hexatriacontane C ₃₆ 30.231 3.80 17.80 35.20 168.30 n-Octatriacontane C ₃₈ 32.084 3.40 16.10 32.80 152.10 n-Tetracontane C ₄₀ 34.599 2.60 13.10 27.60 126.00

Analytes Concentrations (ug / mL) RT CAL1 CAL2 CAL3 CAL4 RT. (min) One 5 10 50 5α-Androstane 5a-Andro. (GC-IS.) 20.782 4.0 4.0 4.0 4.0 o-Terphenyl OTP (Surrogate std.) 19.768 4.0 4.0 4.0 4.0 n-Octane C ₈ 5.222 1.0 5.0 10.0 50.0 n-Decane C ₁₀ 9.607 1.0 5.0 10.0 50.0 n-Dodecane C ₁₂ 12.579 1.0 5.0 10.0 50.0 n-Tetradecane C ₁₄ 14.898 1.0 5.0 10.0 50.0 n-Hexadecane C ₁₆ 16.896 1.0 5.0 10.0 50.0 n-Octadecane C ₁₈ 18.683 1.0 5.0 10.0 50.0 o-Terphenyl OTP 19.768 4.0 4.0 4.0 4.0 n-Eicosane C ₂₀ 20.302 1.0 5.0 10.0 50.0 5α-Androstane 5a-Andro. 20.782 4.0 4.0 4.0 4.0 n-Docosane C ₂₂ 21.782 1.0 5.0 10.0 50.0 n-Tetracosane C ₂₄ 23.143 1.0 5.0 10.0 50.0 n-Hexacosane C ₂₆ 24.402 1.0 5.0 10.0 50.0 n-Octacosane C ₂₈ 25.572 1.0 5.0 10.0 50.0 n-Triacontane C ₃₀ 26.665 1.0 5.0 10.0 50.0 n-Dotriacontane C ₃₂ 27.693 1.0 5.0 10.0 50.0 n-Tetratriacontane C ₃₄ 28.822 1.0 5.0 10.0 50.0 n-Hexatriacontane C ₃₆ 30.231 1.0 5.0 10.0 50.0 n-Octatriacontane C ₃₈ 32.084 1.0 5.0 10.0 50.0 n-Tetracontane C ₄₀ 34.599 1.0 5.0 10.0 50.0

Analytes Calibration, RRF = (A * C _IS ) / (A _IS * C) RRF RT CAL1 CAL2 CAL3 CAL4 RT. (min) 5α-Androstane 5a-Andro. (GC-IS.) 20.782 - - - - - o-Terphenyl OTP (Surrogate std.) 19.768 0.99 0.99 0.98 0.92 0.97 n-Octane C ₈ 5.222 0.88 0.91 0.98 0.94 0.93 n-Decane C ₁₀ 9.607 0.97 0.97 1.00 0.97 0.98 n-Dodecane C ₁₂ 12.579 0.99 0.99 1.00 0.96 0.99 n-Tetradecane C ₁₄ 14.898 1.02 0.99 0.98 0.95 0.98 n-Hexadecane C ₁₆ 16.896 0.97 0.96 0.94 0.92 0.95 n-Octadecane C ₁₈ 18.683 0.95 0.93 0.90 0.90 0.92 o-Terphenyl OTP 19.768 1.00 1.00 1.00 1.00 1.00 n-Eicosane C ₂₀ 20.302 0.97 0.94 0.90 0.90 0.93 5α-Androstane 5a-Andro. 20.782 1.00 1.00 1.00 1.00 1.00 n-Docosane C ₂₂ 21.782 0.97 0.93 0.90 0.89 0.92 n-Tetracosane C ₂₄ 23.143 1.02 0.93 0.90 0.88 0.93 n-Hexacosane C ₂₆ 24.402 1.02 0.93 0.90 0.88 0.93 n-Octacosane C ₂₈ 25.572 1.02 0.93 0.91 0.89 0.94 n-Triacontane C ₃₀ 26.665 0.99 0.93 0.92 0.90 0.94 n-Dotriacontane C ₃₂ 27.693 0.92 0.91 0.91 0.88 0.91 n-Tetratriacontane C ₃₄ 28.822 0.95 0.90 0.90 0.87 0.90 n-Hexatriacontane C ₃₆ 30.231 0.90 0.85 0.86 0.82 0.86 n-Octatriacontane C ₃₈ 32.084 0.80 0.77 0.80 0.74 0.78 n-Tetracontane C ₄₀ 34.599 0.62 0.62 0.67 0.61 0.63

Area values of the control group and Examples 1 to 3, and TPH and UCM area values measured according to Measurement Example 1 are shown in Tables 8 to 12.

0 parking Analytes RT Control Experimental Group 1 Experimental Group 2 Experimental Group 3 5α-Androstane 5a-Andro. (GC-IS.) 20.782 16.5 16.6 16.6 16.4 o-Terphenyl OTP
(Surrogate std.) 19.768 3.8 3.9 3.9 3.7 n-Octane C ₈ 6.425 n-Decane C ₁₀ 10.504 n-Dodecane C ₁₂ 13.386 n-Tetradecane C ₁₄ 15.673 3.80 3.80 3.90 3.40 n-Hexadecane C ₁₆ 17.656 7.20 6.70 6.20 5.60 n-Octadecane C ₁₈ 19.430 6.90 6.70 6.80 6.10 o-Terphenyl OTP 20.558 3.80 3.90 3.90 3.70 n-Eicosane C ₂₀ 21.039 5.40 5.30 5.60 4.90 5α-Androstane 5a-Andro. 21.596 16.50 16.60 16.60 16.40 n-Docosane C ₂₂ 22.510 4.10 4.00 4.10 3.70 n-Tetracosane C ₂₄ 23.862 3.90 4.30 4.50 4.00 n-Hexacosane C ₂₆ 25.114 3.70 3.80 3.90 3.50 n-Octacosane C ₂₈ 26.276 2.50 2.10 2.50 2.00 n-Triacontane C ₃₀ 27.361 n-Dotriacontane C ₃₂ 28.462 n-Tetratriacontane C ₃₄ 29.780 n-Hexatriacontane C ₃₆ 31.464 n-Octatriacontane C ₃₈ 33.716 n-Tetracontane C ₄₀ 36.792 TotalPetroleum Hydrocarbons TPH 721.06 730.52 748.29 636.20 Sum of Resolved Peaks GCRTPH 281.40 335.80 331.48 321.74 Unresolved Complex Mixture UCM 439.66 394.72 416.81 314.46

1 parking Analytes RT Control Experimental Group 1 Experimental Group 2 Experimental Group 3 5α-Androstane 5a-Andro. (GC-IS.) 20.782 16.5 18.6 17.8 17.9 o-Terphenyl OTP
(Surrogate std.) 19.768 3.8 5.3 4.5 2.2 n-Octane C ₈ 6.425 n-Decane C ₁₀ 10.504 n-Dodecane C ₁₂ 13.386 1.90 n-Tetradecane C ₁₄ 15.673 3.80 5.00 2.70 6.70 n-Hexadecane C ₁₆ 17.656 7.30 5.80 3.40 7.30 n-Octadecane C ₁₈ 19.430 7.50 4.90 3.90 5.30 o-Terphenyl OTP 20.558 3.80 5.30 4.50 2.20 n-Eicosane C ₂₀ 21.039 5.50 3.10 2.90 3.60 5α-Androstane 5a-Andro. 21.596 16.50 18.60 17.80 17.90 n-Docosane C ₂₂ 22.510 4.40 2.10 2.30 2.50 n-Tetracosane C ₂₄ 23.862 3.70 2.10 2.50 2.30 n-Hexacosane C ₂₆ 25.114 3.50 n-Octacosane C ₂₈ 26.276 2.20 n-Triacontane C ₃₀ 27.361 n-Dotriacontane C ₃₂ 28.462 n-Tetratriacontane C ₃₄ 29.780 n-Hexatriacontane C ₃₆ 31.464 n-Octatriacontane C ₃₈ 33.716 n-Tetracontane C ₄₀ 36.792 TotalPetroleum Hydrocarbons TPH 720.68 679.85 620.36 679.15 Sum of Resolved Peaks GCRTPH 297.37 354.51 249.34 313.05 Unresolved Complex Mixture UCM 423.31 325.34 371.02 366.10

2 parking Analytes RT Control Experimental Group 1 Experimental Group 2 Experimental Group 3 5α-Androstane 5a-Andro. (GC-IS.) 20.782 18.2 18.2 18.2 18.3 o-Terphenyl OTP (Surrogate std.) 19.768 5.2 4.5 4.3 4.6 n-Octane C ₈ 6.425 n-Decane C ₁₀ 10.504 0.37 n-Dodecane C ₁₂ 13.386 1.40 n-Tetradecane C ₁₄ 15.673 3.30 2.70 2.00 2.70 n-Hexadecane C ₁₆ 17.656 6.40 1.20 1.30 1.60 n-Octadecane C ₁₈ 19.430 7.00 0.94 1.10 1.40 o-Terphenyl OTP 20.558 5.20 4.50 4.30 4.60 n-Eicosane C ₂₀ 21.039 5.40 0.97 1.30 1.50 5α-Androstane 5a-Andro. 21.596 18.20 18.20 18.20 18.30 n-Docosane C ₂₂ 22.510 4.20 0.86 1.40 1.30 n-Tetracosane C ₂₄ 23.862 4.10 0.87 1.20 1.20 n-Hexacosane C ₂₆ 25.114 3.70 1.50 1.60 1.70 n-Octacosane C ₂₈ 26.276 2.30 0.65 0.78 0.88 n-Triacontane C ₃₀ 27.361 0.25 0.36 0.41 n-Dotriacontane C ₃₂ 28.462 0.30 0.46 0.47 n-Tetratriacontane C ₃₄ 29.780 0.73 n-Hexatriacontane C ₃₆ 31.464 n-Octatriacontane C ₃₈ 33.716 n-Tetracontane C ₄₀ 36.792 TotalPetroleum Hydrocarbons TPH 811.72 496.74 435.04 490.23 Sum of Resolved Peaks GCRTPH 283.73 181.46 179.77 199.07 Unresolved Complex Mixture UCM 527.99 315.28 255.27 291.16

3 parking Analytes RT Control Experimental Group 1 Experimental Group 2 Experimental Group 3 5α-Androstane 5a-Andro. (GC-IS.) 20.782 21.3 19.5 20.2 21.5 o-Terphenyl OTP (Surrogate std.) 19.768 3.0 3.3 3.4 3.5 n-Octane C ₈ 6.425 n-Decane C ₁₀ 10.504 n-Dodecane C ₁₂ 13.386 1.10 n-Tetradecane C ₁₄ 15.673 7.80 1.90 1.50 1.50 n-Hexadecane C ₁₆ 17.656 7.80 2.40 1.90 n-Octadecane C ₁₈ 19.430 6.70 2.50 1.40 1.30 o-Terphenyl OTP 20.558 3.00 3.30 3.40 3.50 n-Eicosane C ₂₀ 21.039 5.30 2.00 1.00 5α-Androstane 5a-Andro. 21.596 21.30 19.50 20.20 21.50 n-Docosane C ₂₂ 22.510 4.00 1.50 n-Tetracosane C ₂₄ 23.862 3.60 1.50 n-Hexacosane C ₂₆ 25.114 3.30 1.80 n-Octacosane C ₂₈ 26.276 2.10 0.99 4.10 n-Triacontane C ₃₀ 27.361 2.10 1.50 n-Dotriacontane C ₃₂ 28.462 n-Tetratriacontane C ₃₄ 29.780 n-Hexatriacontane C ₃₆ 31.464 n-Octatriacontane C ₃₈ 33.716 n-Tetracontane C ₄₀ 36.792 TotalPetroleum Hydrocarbons TPH 913.23 500.91 440.50 355.18 Sum of Resolved Peaks GCRTPH 457.26 322.75 306.98 186.20 Unresolved Complex Mixture UCM 455.97 178.16 133.52 168.98

4 parking Analytes RT Control Experimental Group 1 Experimental Group 2 Experimental Group 3 5α-Androstane 5a-Andro. (GC-IS.) 20.782 22.2 20.8 21.1 22.6 o-Terphenyl OTP (Surrogate std.) 19.768 3.2 3.7 3.7 3.6 n-Octane C ₈ 6.425 n-Decane C ₁₀ 10.504 n-Dodecane C ₁₂ 13.386 1.70 0.67 n-Tetradecane C ₁₄ 15.673 7.80 0.93 0.99 0.81 n-Hexadecane C ₁₆ 17.656 7.00 1.40 1.40 1.10 n-Octadecane C ₁₈ 19.430 6.10 0.80 1.00 0.70 o-Terphenyl OTP 20.558 3.20 3.70 3.70 3.60 n-Eicosane C ₂₀ 21.039 4.80 0.33 5α-Androstane 5a-Andro. 21.596 22.20 20.80 21.10 22.60 n-Docosane C ₂₂ 22.510 3.70 n-Tetracosane C ₂₄ 23.862 3.30 n-Hexacosane C ₂₆ 25.114 3.10 n-Octacosane C ₂₈ 26.276 1.80 n-Triacontane C ₃₀ 27.361 2.00 n-Dotriacontane C ₃₂ 28.462 n-Tetratriacontane C ₃₄ 29.780 n-Hexatriacontane C ₃₆ 31.464 n-Octatriacontane C ₃₈ 33.716 n-Tetracontane C ₄₀ 36.792 TotalPetroleum Hydrocarbons TPH 892.02 344.68 313.56 301.37 Sum of Resolved Peaks GCRTPH 330.97 186.00 170.25 163.45 Unresolved Complex Mixture UCM 561.05 158.68 143.31 137.92

Tables 13 to 17 show the amount of each hydrocarbon and the amount of TPH and UCM calculated based on the control values of Tables 8 to 12 and Area values of Examples 1 to 3.

0 parking (unit: μg) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 o-Terphenyl OTP (Surrogate std.) 24 24 24 23 n-Octane C ₈ 0.00 0.00 0.00 0.00 n-Decane C ₁₀ 0.00 0.00 0.00 0.00 n-Dodecane C ₁₂ 0.00 0.00 0.00 0.00 n-Tetradecane C ₁₄ 1.87 1.86 1.91 1.69 n-Hexadecane C ₁₆ 3.68 3.41 3.15 2.88 n-Octadecane C ₁₈ 3.64 3.51 3.56 3.24 n-Eicosane C ₂₀ 2.83 2.76 2.91 2.58 5a-Andro. (GC-IS.) 4.00 4.00 4.00 4.00 n-Docosane C ₂₂ 2.15 2.09 2.14 1.96 n-Tetracosane C ₂₄ 2.03 2.22 2.33 2.09 n-Hexacosane C ₂₆ 1.92 1.96 2.02 1.83 n-Octacosane C ₂₈ 1.29 1.08 1.28 1.04 n-Triacontane C ₃₀ 0.00 0.00 0.00 0.00 n-Dotriacontane C ₃₂ 0.00 0.00 0.00 0.00 n-Tetratriacontane C ₃₄ 0.00 0.00 0.00 0.00 n-Hexatriacontane C ₃₆ 0.00 0.00 0.00 0.00 n-Octatriacontane C ₃₈ 0.00 0.00 0.00 0.00 n-Tetracontane C ₄₀ 0.00 0.00 0.00 0.00 Total petroleum hydrocarbons TPH 385.70 388.40 397.85 342.38 Unresolved Complex Mixture UCM 235.18 209.87 221.61 169.23

1 parking unit (μg) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 o-Terphenyl OTP (Surrogate std.) 24 29 26 13 n-Octane C ₈ 0.00 0.00 0.00 0.00 n-Decane C ₁₀ 0.00 0.00 0.00 0.00 n-Dodecane C ₁₂ 0.00 0.00 0.00 0.86 n-Tetradecane C ₁₄ 1.87 2.19 1.23 3.04 n-Hexadecane C ₁₆ 3.73 2.63 1.61 3.44 n-Octadecane C ₁₈ 3.95 2.29 1.91 2.58 n-Eicosane C ₂₀ 2.88 1.44 1.41 1.74 5a-Andro. (GC-IS.) 4.00 4.00 4.00 4.00 n-Docosane C ₂₂ 2.31 0.98 1.12 1.21 n-Tetracosane C ₂₄ 1.92 0.97 1.20 1.10 n-Hexacosane C ₂₆ 1.82 0.00 0.00 0.00 n-Octacosane C ₂₈ 1.14 0.00 0.00 0.00 n-Triacontane C ₃₀ 0.00 0.00 0.00 0.00 n-Dotriacontane C ₃₂ 0.00 0.00 0.00 0.00 n-Tetratriacontane C ₃₄ 0.00 0.00 0.00 0.00 n-Hexatriacontane C ₃₆ 0.00 0.00 0.00 0.00 n-Octatriacontane C ₃₈ 0.00 0.00 0.00 0.00 n-Tetracontane C ₄₀ 0.00 0.00 0.00 0.00 Total petroleum hydrocarbons TPH 385.50 322.60 307.60 334.87 Unresolved Complex Mixture UCM 226.43 154.38 183.97 180.51

2 parking units (μg) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 o-Terphenyl OTP (Surrogate std.) 29 25 24 26 n-Octane C ₈ 0.00 0.00 0.00 0.00 n-Decane C ₁₀ 0.00 0.17 0.00 0.00 n-Dodecane C ₁₂ 0.00 0.00 0.00 0.62 n-Tetradecane C ₁₄ 1.47 1.21 0.89 1.20 n-Hexadecane C ₁₆ 2.97 0.56 0.60 0.74 n-Octadecane C ₁₈ 3.35 0.45 0.53 0.67 n-Eicosane C ₂₀ 2.56 0.46 0.62 0.71 5a-Andro. (GC-IS.) 4.00 4.00 4.00 4.00 n-Docosane C ₂₂ 2.00 0.41 0.67 0.62 n-Tetracosane C ₂₄ 1.93 0.41 0.57 0.56 n-Hexacosane C ₂₆ 1.74 0.71 0.75 0.80 n-Octacosane C ₂₈ 1.08 0.30 0.36 0.41 n-Triacontane C ₃₀ 0.00 0.12 0.17 0.19 n-Dotriacontane C ₃₂ 0.00 0.15 0.22 0.23 n-Tetratriacontane C ₃₄ 0.00 0.36 0.00 0.00 n-Hexatriacontane C ₃₆ 0.00 0.00 0.00 0.00 n-Octatriacontane C ₃₈ 0.00 0.00 0.00 0.00 n-Tetracontane C ₄₀ 0.00 0.00 0.00 0.00 Total petroleum hydrocarbons TPH 393.64 240.89 210.97 236.43 Unresolved Complex Mixture UCM 256.04 152.89 123.79 140.42

3 parking units (μg) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 o-Terphenyl OTP (Surrogate std.) 15 17 17 17 n-Octane C ₈ 0.00 0.00 0.00 0.00 n-Decane C ₁₀ 0.00 0.00 0.00 0.00 n-Dodecane C ₁₂ 0.42 0.00 0.00 0.00 n-Tetradecane C ₁₄ 2.98 0.79 0.60 0.57 n-Hexadecane C ₁₆ 3.09 1.04 0.00 0.75 n-Octadecane C ₁₈ 2.74 1.12 0.60 0.53 n-Eicosane C ₂₀ 2.15 0.89 0.43 0.00 5a-Andro. (GC-IS.) 4.00 4.00 4.00 4.00 n-Docosane C ₂₂ 1.63 0.67 0.00 0.00 n-Tetracosane C ₂₄ 1.45 0.66 0.00 0.00 n-Hexacosane C ₂₆ 1.33 0.79 0.00 0.00 n-Octacosane C ₂₈ 0.84 0.43 1.73 0.00 n-Triacontane C ₃₀ 0.84 0.66 0.00 0.00 n-Dotriacontane C ₃₂ 0.00 0.00 0.00 0.00 n-Tetratriacontane C ₃₄ 0.00 0.00 0.00 0.00 n-Hexatriacontane C ₃₆ 0.00 0.00 0.00 0.00 n-Octatriacontane C ₃₈ 0.00 0.00 0.00 0.00 n-Tetracontane C ₄₀ 0.00 0.00 0.00 0.00 Total petroleum hydrocarbons TPH 378.41 226.72 192.47 145.80 Unresolved Complex Mixture UCM 188.94 80.64 58.34 69.37

4 parking (unit: μg) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 o-Terphenyl OTP (Surrogate std.) 15 18 18 16 n-Octane C ₈ 0.00 0.00 0.00 0.00 n-Decane C ₁₀ 0.00 0.00 0.00 0.00 n-Dodecane C ₁₂ 0.62 0.00 0.00 0.24 n-Tetradecane C ₁₄ 2.86 0.36 0.38 0.29 n-Hexadecane C ₁₆ 2.66 0.57 0.56 0.41 n-Octadecane C ₁₈ 2.39 0.33 0.41 0.27 n-Eicosane C ₂₀ 1.87 0.00 0.00 0.13 5a-Andro. (GC-IS.) 4.00 4.00 4.00 4.00 n-Docosane C ₂₂ 1.45 0.00 0.00 0.00 n-Tetracosane C ₂₄ 1.28 0.00 0.00 0.00 n-Hexacosane C ₂₆ 1.20 0.00 0.00 0.00 n-Octacosane C ₂₈ 0.69 0.00 0.00 0.00 n-Triacontane C ₃₀ 0.77 0.00 0.00 0.00 n-Dotriacontane C ₃₂ 0.00 0.00 0.00 0.00 n-Tetratriacontane C ₃₄ 0.00 0.00 0.00 0.00 n-Hexatriacontane C ₃₆ 0.00 0.00 0.00 0.00 n-Octatriacontane C ₃₈ 0.00 0.00 0.00 0.00 n-Tetracontane C ₄₀ 0.00 0.00 0.00 0.00 Total petroleum hydrocarbons TPH 354.64 146.26 131.16 117.69 Unresolved Complex Mixture UCM 223.05 67.33 59.95 53.86

The concentrations of the control groups and Examples 1 to 3 calculated on the basis of Tables 8 to 17, and the concentrations of TPH, UCM and n-alkane calculated using Formula 1 and Formula 2 are shown in Tables 18 to 22.

0 parking (unit: μg / ml) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 n-Octane C ₈ 0.000 0.000 0.000 0.000 n-Decane C ₁₀ 0.000 0.000 0.000 0.000 n-Dodecane C ₁₂ 0.000 0.000 0.000 0.000 n-Tetradecane C ₁₄ 1.873 1.862 1.911 1.686 n-Hexadecane C ₁₆ 3.682 3.406 3.152 2.881 n-Octadecane C ₁₈ 3.638 3.511 3.564 3.236 n-Eicosane C ₂₀ 2.827 2.758 2.914 2.581 n-Docosane C ₂₂ 2.154 2.089 2.141 1.956 n-Tetracosane C ₂₄ 2.028 2.222 2.326 2.092 n-Hexacosane C ₂₆ 1.923 1.964 2.015 1.831 n-Octacosane C ₂₈ 1.290 1.077 1.282 1.038 n-Triacontane C ₃₀ 0.000 0.000 0.000 0.000 n-Dotriacontane C ₃₂ 0.000 0.000 0.000 0.000 n-Tetratriacontane C ₃₄ 0.000 0.000 0.000 0.000 n-Hexatriacontane C ₃₆ 0.000 0.000 0.000 0.000 n-Octatriacontane C ₃₈ 0.000 0.000 0.000 0.000 n-Tetracontane C ₄₀ 0.000 0.000 0.000 0.000 Total petroleum hydrocarbons TPH 385.7 388.4 397.9 342.4 Unresolved Complex Mixture UCM 235.2 209.9 221.6 169.2 Total n-alkane C8 ~ C40 19.42 18.89 19.31 17.30

1 parking (unit: μg / ml) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 n-Octane C ₈ 0.000 0.000 0.000 0.000 n-Decane C ₁₀ 0.000 0.000 0.000 0.000 n-Dodecane C ₁₂ 0.000 0.000 0.000 0.862 n-Tetradecane C ₁₄ 1.873 2.187 1.234 3.045 n-Hexadecane C ₁₆ 3.733 2.631 1.612 3.441 n-Octadecane C ₁₈ 3.955 2.292 1.906 2.576 n-Eicosane C ₂₀ 2.880 1.440 1.408 1.738 n-Docosane C ₂₂ 2.312 0.979 1.120 1.211 n-Tetracosane C ₂₄ 1.924 0.969 1.205 1.102 n-Hexacosane C ₂₆ 1.819 0.000 0.000 0.000 n-Octacosane C ₂₈ 1.135 0.000 0.000 0.000 n-Triacontane C ₃₀ 0.000 0.000 0.000 0.000 n-Dotriacontane C ₃₂ 0.000 0.000 0.000 0.000 n-Tetratriacontane C ₃₄ 0.000 0.000 0.000 0.000 n-Hexatriacontane C ₃₆ 0.000 0.000 0.000 0.000 n-Octatriacontane C ₃₈ 0.000 0.000 0.000 0.000 n-Tetracontane C ₄₀ 0.000 0.000 0.000 0.000 Total petroleum hydrocarbons TPH 385.5 322.6 307.6 334.9 Unresolved Complex Mixture UCM 226.4 154.4 184.0 180.5 Total n-alkane C8 ~ C40 19.63 10.50 8.48 13.97

2 parking (unit: μg / ml) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 n-Octane C ₈ 0.000 0.000 0.000 0.000 n-Decane C ₁₀ 0.000 0.166 0.000 0.000 n-Dodecane C ₁₂ 0.000 0.000 0.000 0.621 n-Tetradecane C ₁₄ 1.475 1.207 0.894 1.200 n-Hexadecane C ₁₆ 2.967 0.556 0.603 0.738 n-Octadecane C ₁₈ 3.346 0.449 0.526 0.666 n-Eicosane C ₂₀ 2.563 0.460 0.617 0.708 n-Docosane C ₂₂ 2.001 0.410 0.667 0.616 n-Tetracosane C ₂₄ 1.933 0.410 0.566 0.563 n-Hexacosane C ₂₆ 1.744 0.707 0.754 0.797 n-Octacosane C ₂₈ 1.076 0.304 0.365 0.409 n-Triacontane C ₃₀ 0.000 0.117 0.169 0.191 n-Dotriacontane C ₃₂ 0.000 0.145 0.223 0.227 n-Tetratriacontane C ₃₄ 0.000 0.356 0.000 0.000 n-Hexatriacontane C ₃₆ 0.000 0.000 0.000 0.000 n-Octatriacontane C ₃₈ 0.000 0.000 0.000 0.000 n-Tetracontane C ₄₀ 0.000 0.000 0.000 0.000 Total petroleum hydrocarbons TPH 393.6 240.9 211.0 236.4 Unresolved Complex Mixture UCM 256.0 152.9 123.8 140.4 Total n-alkane C8 ~ C40 17.10 5.29 5.38 6.74

3 parking (unit: μg / ml) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 n-Octane C ₈ 0.000 0.000 0.000 0.000 n-Decane C ₁₀ 0.000 0.000 0.000 0.000 n-Dodecane C ₁₂ 0.419 0.000 0.000 0.000 n-Tetradecane C ₁₄ 2.979 0.793 0.604 0.568 n-Hexadecane C ₁₆ 3.090 1.039 0.000 0.746 n-Octadecane C ₁₈ 2.737 1.115 0.603 0.526 n-Eicosane C ₂₀ 2.150 0.886 0.428 0.000 n-Docosane C ₂₂ 1.628 0.667 0.000 0.000 n-Tetracosane C ₂₄ 1.450 0.660 0.000 0.000 n-Hexacosane C ₂₆ 1.329 0.792 0.000 0.000 n-Octacosane C ₂₈ 0.839 0.432 1.728 0.000 n-Triacontane C ₃₀ 0.842 0.657 0.000 0.000 n-Dotriacontane C ₃₂ 0.000 0.000 0.000 0.000 n-Tetratriacontane C ₃₄ 0.000 0.000 0.000 0.000 n-Hexatriacontane C ₃₆ 0.000 0.000 0.000 0.000 n-Octatriacontane C ₃₈ 0.000 0.000 0.000 0.000 n-Tetracontane C ₄₀ 0.000 0.000 0.000 0.000 Total petroleum hydrocarbons TPH 378.4 226.7 192.5 145.8 Unresolved Complex Mixture UCM 188.9 80.6 58.3 69.4 Total n-alkane C8 ~ C40 17.46 7.04 3.36 1.84

4 parking (unit: μg / ml) Analytes Control Experimental Group 1 Experimental Group 2 Experimental Group 3 n-Octane C ₈ 0.000 0.000 0.000 0.000 n-Decane C ₁₀ 0.000 0.000 0.000 0.000 n-Dodecane C ₁₂ 0.622 0.000 0.000 0.241 n-Tetradecane C ₁₄ 2.858 0.364 0.382 0.292 n-Hexadecane C ₁₆ 2.661 0.568 0.560 0.411 n-Octadecane C ₁₈ 2.391 0.335 0.412 0.269 n-Eicosane C ₂₀ 1.868 0.000 0.000 0.126 n-Docosane C ₂₂ 1.445 0.000 0.000 0.000 n-Tetracosane C ₂₄ 1.275 0.000 0.000 0.000 n-Hexacosane C ₂₆ 1.198 0.000 0.000 0.000 n-Octacosane C ₂₈ 0.690 0.000 0.000 0.000 n-Triacontane C ₃₀ 0.769 0.000 0.000 0.000 n-Dotriacontane C ₃₂ 0.000 0.000 0.000 0.000 n-Tetratriacontane C ₃₄ 0.000 0.000 0.000 0.000 n-Hexatriacontane C ₃₆ 0.000 0.000 0.000 0.000 n-Octatriacontane C ₃₈ 0.000 0.000 0.000 0.000 n-Tetracontane C ₄₀ 0.000 0.000 0.000 0.000 Total petroleum hydrocarbons TPH 354.6 146.3 131.2 117.7 Unresolved Complex Mixture UCM 223.1 67.3 59.9 53.9 Total n-alkane C8 ~ C40 15.78 1.27 1.35 1.34

The principle of the concentration calculation method of TPH and UCM is the same. However, TPH (petroleum-based total hydrocarbons) can be seen as UCM + N-alkanes + a (intermediate or unknown peak), and the longer it is weathered, the more the focus of biological treatment is UCM.

Average Petroleum Total Hydrocarbons (TPH) Concentration by Week and Average Petroleum Total Hydrocarbons (TPH) Degradation Rate by Week

Using the Tables 8 to 22, the average petroleum total hydrocarbon (TPH) concentrations per week and the average petroleum total hydrocarbon (TPH) decomposition rate for each week based on this were calculated by Equation 3 and shown in Table 23. Schematically shown in FIG.

[Calculation 3]

TPH degradation rate = 100-(average of experimental / control values for each parking × 100)

Amount (μg / ml) Week 0 1 week 2 weeks 3 weeks 4 Weeks Control 385.7 385.5 393.6 378.4 354.6 Experimental group 376.2 321.7 229.44 188.3 131.7 TPH decomposition rate (%) 0.9 15.3 39.6 50.4 65.3

Degradation rate of total petroleum-based unresolved complex mixture (UCM) by liquor and mean petroleum-based total unresolved complex mixture (UCM) decomposition by liquor

The average unresolved complex mixture (UCM) concentration and the unresolved complex mixture (UCM) decomposition rate based on the week numbers using Tables 8 to 22 were calculated in Equation 4, and are shown in Table 24. Shown.

[Calculation 4]

UCM degradation rate = 100-(average of experimental / control values for each parking × 100)

Amount (μg / ml) Week 0 1 week 2 weeks 3 weeks 4 Weeks Control 235.2 226.4 256.0 188.9 223.1 Experimental group 200.2 173.0 139.0 69.4 60.4 UCM decomposition rate (%) 11.4 23.4 38.5 39.3 73.3

Average n-alkane Concentration by Week and Average n-alkane Degradation Rate by Week

The average concentration of n-alkane per week and the decomposition rate of n-alkane based on the same using Tables 8 to 22 were calculated in Equation 5 and shown in Table 25, and are shown in FIG. 4.

[Calculation 5]

n-alkanes decomposition rate = 100- (experimental group of each parking / control group of each parking × 100)

Amount (μg / ml) Week 0 1 week 2 weeks 3 weeks 4 Weeks Control 19.4 19.6 17.1 17.5 15.8 Experimental group 18.5 11.0 5.8 4.1 1.3 n-alkanes decomposition rate (%) -3.5 38.6 67.5 77.2 92.6

Referring to FIG. 1 and Table 23, the average TPH concentration of the control group during the four weeks was 379.6 ± 14.9 ㎍ / ml, while in the experimental group it was confirmed that the concentration decreases over time and the final TPH concentration after 4 weeks was 65.3% ( 131.7 ± 14.3 ㎍ / ml) it can be seen that the degradation efficiency.

Referring to Figure 2, Table 24 and Figure 3, Table 25, it can be seen that UCM and N-alkanes also showed degradation efficiency of 73.3% and 92.6%, respectively, compared to the control.

The microbial community obtained through enrichment of indigenous microorganisms from contaminated soil is not involved in pollutant degradation, but the case of increasing efficiency through interaction with decomposing bacteria shows that the microbial community enriched in this experiment directly Although not involved, the efficiency is thought to be increased by interacting with crude microorganisms or microbial surfactant producing microorganisms.

As mentioned above, specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (2)

Microbial community KWEC-DT [KCTC18648P], a microorganism with excellent degradable soil crude oil, including the genus Paenibacillus sp. And Staphylococcus sp .

The method of claim 1,
The microbial community KWEC-DT [KCTC18648P] is a genus of Achromobacter sp. , Bacillus sp ., Pseudoxanthomonas sp. , And Stenotrophomonas sp. The microbial community KWEC-DT [KCTC18648P] with excellent soil crude oil resolution further comprising.

Images