# Patent

## Title
Method for scavenging aromatic hydrocarbons, crude petroleum and/or a petroleum refined product

## Abstract

A method for scavenging aromatic hydrocarbons present in a contaminated substrate includes treating the contaminated substrate with Pseudomonas taoyuanensis S03 deposited in DSMZ under an accession number DSM 32185, the aromatic hydrocarbons are benzene, naphthalene, toluene, ethylbezene or xylene. A method for scavenging crude petroleum or a refined product thereof present in a contaminated substrate includes treating the contaminated substrate with Pseudomonas taoyuanensis S03 deposited in DSMZ under an accession number DSM 32185, such that the crude petroleum or the refined product thereof is emulsified by Pseudomonas taoyuanensis S03.

## Background
FIELD
The disclosure relates to a method for scavenging aromatic hydrocarbons, crude petroleum or a petroleum refined product using a Pseudomonas taoyuanensis S03 isolate that was deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) under an accession number DSM 32185.
BACKGROUND
Aromatic hydrocarbons, arenes, are types of hydrocarbon compounds having one or more benzene rings, and may be classified into monocyclic aromatic hydrocarbons (MAHs) and polycyclic aromatic hydrocarbons (PAHs) based on the number of benzene rings. Aromatic hydrocarbons are generally present in crude petroleum, petroleum refined products (e.g., diesel, heavy oil, and gasoline), and industrial solvents, and often used as starting materials for the manufacture of pharmaceuticals, agrochemicals, explosives, and many daily use products. However, aromatic hydrocarbons have stable and hardly degradable structures with high melting and boiling points. When released, aromatic hydrocarbons easily accumulate within and can be harmful to humans and the ecological environment.
The MAHs compounds benzene, toluene, ethylbenzene, and xylene (BTEX) are some of the most common groundwater contaminants and soil contaminants. Among the BTEX compounds, benzene often receives more attention due to its reported carcinogenicity. Benzene (C6H6), the simplest MAH, is often used as a chemical solvent or for the preparation of benzene derivatives in the chemical industry. Upon entering the human body, through skin or eye contact, inhalation, or oral routes, benzene could inhibit the central nervous system (CNS) and cause symptoms such as sleepiness, dizziness, headache, nausea, etc. Long-term contact with benzene could adversely influence the formation of red blood cells, white blood cells and blood platelets, and likely cause leukemia.
Naphthalene (C10H8) is the simplest PAH, composed of two fused benzene rings, and widely used for the production of dyestuffs, resins, solvents, disinfectants, insecticides, preservatives, mothproofing agents, etc. After entering the human body, naphthalene could cause symptoms such as haemolytic anaemia, nausea, vomiting, diarrhea, jaundice, liver or kidney damage, etc.
Because the presence of benzene and/or naphthalene in the environment poses a threat to human health and causes damage to ecological environment, much effort has been made to provide effective methods for the treatment of environmental pollutants. Current methods for the treatment of environmental pollutants include solidification, removal, incineration, activated carbon adsorption, catalytic reduction, photolysis, bioremediation, etc.
Bioremediation is a technique that utilizes the biodegradative activities of microorganisms to remove environmental pollutants and recalcitrant xenobiotics. Advantages of bioremediation include its low cost, lack of toxic by-products and secondary pollution generated during the degradation of the environmental pollutants, in situ operation, etc. Therefore, bioremediation has been widely applied to the remediation of contaminated sites. Bioaugmentation, a process of adding microorganisms with biodegradative activities to contaminated environments for the purpose of degrading pollutants, is particularly well-received among bioremediation techniques. However, certain characteristics of aromatic hydrocarbons, e.g., the low water solubility and strong sorption to soil, could adversely influence their bioavailability to microorganisms capable of aromatic hydrocarbon degradation, thereby limiting the degradation rate of aromatic hydrocarbons. Accordingly, the addition of microorganisms with emulsifying activity to the aromatic hydrocarbon-contaminated environments increases desorption rates and apparent solubility of aromatic hydrocarbons in an aqueous phase, resulting in increased bioavailability and degradation rate of aromatic hydrocarbons.
In the event that indigenous microorganisms are unable to effectively scavenge aromatic hydrocarbons, bioaugmentation may be the only way to achieve bioremediation. Therefore, it has become a goal for researchers in this field to isolate and screen microorganisms suitable for bioremediation.
Many bacterial strains with emulsifying activity and/or benzene- and/or naphthalene-scavenging ability for benzene and/or naphthalene have been isolated from contaminated environments, and mostly belong to Pseudomonas spp., Pseudoxanthomonas spp., Alicycliphilus spp., Burkholderia spp., Ralstonia spp., Achromobacter spp., Hydrogenophaga spp., Rhodococcus spp., Arthrobacter spp., Alcaligenes spp., Micrococcus spp., etc. (Jeong Myeong Kim et al. (2008), Applied and Environmental Microbiology, 74:7313-7320; Shuguang Xie et al. (2011), Biodegradation, 22:71-81; R. C. John et al. (2012), Bulletin of Environmental Contamination and Toxicology, 88:1014-1019). For example, CN 103045502 A discloses a Rhodococcus erythoropolis strain T7-3 (CGMCC No. 6104) isolated from petroleum-contaminated seabed soil samples, which was found to be effective in the emulsification and/or degradation of crude petroleum and/or petroleum hydrocarbons (including benzene and xylene). The Rhodococcus erythoropolis strain T7-3 is expected to be useful in bioremediation of petroleum-contaminated area of the sea. CN 1519312 A discloses a Rhodococcus ruber strain Em CGMCC No. 0868 isolated from crude petroleum-contaminated soils, which was found to be effective in the emulsification and/or degradation of kerosene and petroleum hydrocarbons such as benzene, naphthalene, anthracene, phenanthrene, and pyrene. The Rhodococcus ruber strain Em CGMCC No. 0868 is expected to be useful in oily wastewater treatment and bioremediation of petroleum-contaminated soils.
In E. Deziel et al. (1996), Appl. Environ. Microbiol., 62:1908-1912, E. Deziel et al. isolated twenty-three PAH-degrading bacterial isolates from a sandpit that had previosuly received oil refinery wastes, wherein Pseudomonas aeruginosa 19SJ was found to be able to produce large amounts of glycolipid using naphthalene or phenanthrene as the sole substrate. Furthermore, via glycolipid production, Pseudomonas aeruginosa 19SJ exhibits emulsifying activity that could increase apparent solubility of naphthalene, resulting in enhanced naphthalene degradation and utilization, and in turn, further promotion of glycolipid production. Therefore, production of surface-active compounds by bacteria is likely a part of their strategy for growing on poorly available substrates. For these reasons, Pseudomonas aeruginosa 19SJ is expected to be useful in bioremediation of environments contaminated with benzene and/or naphthalene.
In Eun Young Lee et al. (2011), International Proceedings of Chemical, Biological & Environmental Engineering, 20:37-41, Eun Young Lee et al. isolated Pseudomonas putida AY-10 from the rhizosphere of wastewater treatment reed. Pseudomonas putida AY-10 is able to grow on mediums with benzene, toluene, ethylbenzene, or xylene as the sole carbon source, and also completely degrade benzene, toluene, ethylbenzene, and xylene. Therefore, Pseudomonas putida AY-10 is expected to be useful in bioremediation of BTEX-contaminated environments.
Despite the microorganisms described in the above prior art, the Inventors remain committed in their efforts to screen microorganisms having emulsifying activity as well as benzene- and/or naphthalene-scavenging ability for use in environmental protection. During their research efforts, the Inventors unexpectedly isolated a new bacterial isolate, Pseudomonas taoyuanensis S03, form soils contaminated with benzene and/or naphthalene. The bacterial isolate is phylogenetically different from other bacterial species of Pseudomonas that have been published, and possesses great emulsifying activity against heavy oil and diesel, as well as benzene- and/or naphthalene-scavenging ability. Therefore, the isolate is expected to be useful in the remediation of environments contaminated with crude petroleum, petroleum refined products, benzene, and/or naphthalene.
SUMMARY
Therefore, according to a first aspect of the disclosure, a method for scavenging aromatic hydrocarbons present in a contaminated substrate includes treating the contaminated substrate with Pseudomonas taoyuanensis S03 deposited in DSMZ under an accession number DSM 32185, such that the aromatic hydrocarbons present in the contaminated substrate is degraded or desorbed from the contaminated substrate by the Pseudomonas taoyuanensis S03, wherein the aromatic hydrocarbons are selected from the group consisting of benzene, naphthalene, toluene, ethylbezene, xylene, and combinations thereof.
According to a second aspect of the disclosure, a method for scavenging crude petroleum or a refined product thereof present in a contaminated substrate, includes treating the contaminated substrate with the Pseudomonas taoyuanensis S03 deposited in DSMZ under an accession number DSM 32185, such that the crude petroleum or the refined product thereof is emulsified by the Pseudomonas taoyuanensis S03.



BRIEF DESCRIPTION OF THE DRAWINGS
Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which:
FIG. 1 shows the partial 16S rDNA sequence of Pseudomonas taoyuanensis S03 (SEQ ID NO: 1) of the disclosure;
FIG. 2 shows the partial gyrB gene sequence of Pseudomonas taoyuanensis S03 (SEQ ID NO: 2) of the disclosure;
FIG. 3 shows the benzene clearance rate of Pseudomonas taoyuanensis S03 for a benzene-containing soil sample, wherein the control group represents treatment of a benzene-containing soil sample without using Pseudomonas taoyuanensis S03;
FIG. 4 shows the benzene degradation rate of Pseudomonas taoyuanensis S03 for a benzene-containing soil sample, wherein the control group represents treatment of a benzene-containing soil sample without using Pseudomonas taoyuanensis S03;
FIG. 5 shows the naphthalene clearance rate of Pseudomonas taoyuanensis S03 for a naphthalene-containing soil sample, wherein the control group represents treatment of a naphthalene-containing soil sample without using Pseudomonas taoyuanensis S03; and
FIG. 6 shows the naphthalene degradation rate of Pseudomonas taoyuanensis S03 for a naphthalene-containing soil sample, wherein the control group represents treatment of a naphthalene-containing soil sample without using Pseudomonas taoyuanensis S03.



DETAILED DESCRIPTION
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Taiwan or any other country.
For the purpose of this specification, it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprise” has a corresponding meaning.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described.
In order to screen microorganisms suitable for bioremediation, the Inventors isolated 35 purified bacterial isolates from diesel-contaminated soil at a remediation site. Each of the bacterial isolates was inoculated in heavy oil to analyze the Emulsification index E24 thereof. Bacterial isolate S03 is found to have excellent emulsifying activity for heavy oil. Based on the results of characteristics identification of the bacterial isolate S03 and referring to Satoshi Yamamoto et al. (1995), Applied and Environmental Microbiology, 61:1104-1109 and Li-Ting Wang et al. (2010), International Journal of Systematic and Evolutionary Microbiology, 60:2094-2098, the bacterial isolate S03 is determined to be a novel Pseudomonas species, and was named as Pseudomonas taoyuanensis S03. Pseudomonas taoyuanensis S03 was deposited under the terms of the Budapest Treaty in the International Strain Depositary Leibniz Institute Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ), Inhoffenstr. 7 B, D-38124 Braunschweig GERMANY, under accession number DSM 32185 on Oct. 14, 2015.
Through further experiments, Pseudomonas taoyuanensis S03 was found to be effective in emulsifying super diesel.
Accordingly, the disclosure provides a method for scavenging crude petroleum or a refined product thereof present in a contaminated substrate, comprising treating the contaminated substrate with the Pseudomonas taoyuanensis S03, such that the crude petroleum or the refined product thereof is emulsified by the Pseudomonas taoyuanensis S03.
As used herein, the term “emulsify” refers to emulsification of a substance having low water solubility that results in a change of interfacial tension between the substance and an aqueous phase so as to allow the substance to be dispersed in the aqueous phase in the form of fine droplets or particles, thereby forming a heterogeneous dispersion system.
As used herein, the term “crude petroleum” refers to untreated or unrefined oil (i.e., crude oil) found beneath the earth's surface, which is a complex mixture containing hydrocarbons of various molecular weights (including aliphatic hydrocarbons, cyclic hydrocarbons, and aromatic hydrocarbons) and other organic compounds.
The term “refined product of the crude petroleum” refers to petroleum refined product, refined petroleum, or petroleum product as well known to a skilled artisan. According to the disclosure, the refined product of the crude petroleum includes, but is not limited to, heavy oil, diesel, kerosene, gasoline, hydraulic oil, and lubricating oil.
In an embodiment of the disclosure, the refined product of the crude petroleum is heavy oil. In another embodiment of the disclosure, the refined product of the crude petroleum is diesel.
Through further experiments, Pseudomonas taoyuanensis S03 was found to be effective in scavenging benzene and naphthalene present in soil by desorption and/or degradation.
Accordingly, the disclosure also provides a method for scavenging aromatic hydrocarbons present in a contaminated substrate, comprising treating the contaminated substrate with the Pseudomonas taoyuanensis S03, such that the aromatic hydrocarbons present in the contaminated substrate is degraded or desorbed from the contaminated substrate by the Pseudomonas taoyuanensis S03, wherein the aromatic hydrocarbons are selected from the group consisting of benzene, naphthalene, toluene, ethylbezene, xylene, and combinations thereof.
As used herein, the term “desorb” refers to release of a compound from a contaminated substrate by breaking the adhesive force therebetween.
According to the disclosure, the contaminated substrate may be a solid or liquid environmental substrate, which includes, but is not limited to, soil, sludge, sediment, aquifer, and water. Preferably, the contaminated substrate is selected from the group consisting of agricultural land (e.g., plowland and orchard land), grazing grassland, woodland, gas station land, industrial land, well water, an aquaculture pond, water reservoir, ground water, river water, lake water, seawater, industrial waste water, domestic sewage, and silt.
According to the disclosure, Pseudomonas taoyuanensis S03 or sub-cultured offspring thereof of the present disclosure may be used to prepare a microbial agent using technology well known to those skilled in the art. The microbial agent may be used for bioremediation of a substrate that is contaminated with crude petroleum or a refined product thereof. The microbial agent may also be used for bioremediation of a substrate that is contaminated with benzene, naphthalene, toluene, ethylbezene, or xylene.
According to the disclosure, the microbial agent may additionally include at least one microorganism capable of scavenging monocyclic and/or polycyclic aromatic hydrocarbons.
The microorganisms capable of scavenging monocyclic aromatic hydrocarbons include, but are not limited to, Pseudomonas spp., Pseudoxanthomonas spp., Alicycliphilus spp., Burkholderia spp., Ralstonia spp., Achromobacter spp., Hydrogenophaga spp., Rhodococcus spp., and Arthrobacter spp. Examples of the microorganisms capable of scavenging monocyclic aromatic hydrocarbons include Rhodococcus erythoropolis T7-3, Rhodococcus ruber Em CGMCC No. 0868, and Pseudomonas putida AY-10.
The microorganisms capable of scavenging polycyclic aromatic hydrocarbons include, but are not limited to, Pseudomonas spp., Rhodococcus spp., Arthrobacter spp., Acinetobacter spp., Flavobacterium spp., Alcaligenes spp., Micrococcus spp., and Corynebacterium spp. Examples of the microorganisms capable of scavenging polycyclic aromatic hydrocarbons include Rhodococcus ruber Em CGMCC No. 0868 and Pseudomonas aeruginosa 19SJ.
According to the disclosure, the microbial agent may optionally include a nutrient that promotes microbial growth, which includes, but is not limited to, glycerol, riboflavin, casein, polypeptone, meat extract, soybean cake, yeast extract, cellulose, glucose, corn extract, whey powder, starch, vitamin (such as thiamine, biotin, nicotinic acid amide, and calcium panthotenate), and enzyme (such as amylase, protease, and lipase).
According to the disclosure, the microbial agent may be manufactured into a suitable form using technology well known to those skilled in the art, which includes, but is not limited to, culture solution, suspension, granule, powder, tablet, pill, capsule, slurry, and the like. In addition, the microbial agent may also be immobilized on an insoluble support for further application.
According to the disclosure, the microbial agent may additionally include a biocompatible carrier.
In an embodiment of the microbial agent of the disclosure, Pseudomonas taoyuanensis S03 is entrapped in the biocompatible carrier. The biocompatible carrier includes, but is not limited to, silica gel, starch, agar, chitin, chitosan, polyvinyl alcohol, polylactic acid, alginic acid, polyacrylamide, carrageenan, agarose, gelatin, cellulose, cellulose acetate, dextran, and collagen.
In another embodiment of the microbial agent of the disclosure, Pseudomonas taoyuanensis S03 is supported on the biocompatible carrier. The biocompatible carrier includes, but is not limited to, glass, ceramic, metal oxide, activated carbon, kaolinite, bentonite, zeolite, alumina, anthracite, glutaraldehyde, polyacrylic acid, polyurethane, polyvinyl chloride, ion exchange resin, epoxy resin, photosetting resin, polyester, and polystyrene.
According to the disclosure, the microbial agent may also be used to manufacture a bioreactor or device for use in scavenging crude petroleum or a refined product thereof using technology well known to those skilled in the art. The bioreactor can be manufactured with reference to, for example, U.S. Pat. No. 5,279,963 A, U.S. Pat. No. 5,258,303 A, U.S. Pat. No. 5,552,051 A, U.S. Pat. No. 5,494,574 A, U.S. Pat. No. 6,030,533 A, US 2003/0008381 A1, US 2006/0270024 A1, EP 0609399 B1, EP 0867238 A3, and K. Ishii and T. Furuichi (2007), Journal of Hazardous Materials, 148(3):693-700.
The present disclosure will be further described by way of the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.
EXAMPLES
Experimental Materials:
1. Minimal Agar Medium:
The recipe of the minimal agar medium used in the examples is shown in Table 1.










TABLE 1







Ingredients
Amount




















(NH4)2SO4
1 
g



KH2PO4
5 
g



MgSO4•7H2O
0.1 
g



FeSO4
0.005 
g



Trace metal solution
1 
mL



Agar
23 
g










Deionized water
added to a final




volume of 1 L







The final pH value was adjusted to 7.0 ± 0.2.



Note:



The trace metal solution was prepared by adding 10.75 g MgO, 2 g CaCO3, 4.5 g FeSO4•7H2O, 1.44 g ZnSO4•7H2O, 1.12 g MnSO4•4H2O, 0.25 g CuSO4•5H2O, 0.28 g CoSO4•7H2O, 0.06 g H3BO3, and 51.3 mL HCl in 1 L deionized water.






2. Tryptic Soy Broth (TSB):

The recipe of the tryptic soy broth used in the examples is shown in Table 2.










TABLE 2







Ingredients
Amount




















Tryptone
17
g



Soytone
3
g



Dextrose
2.5
g



NaCl
5
g



K2HPO4
2.5
g










Deionized water
added to a final




volume of 1 L










General Experimental Procedure:

1. Detection of the Concentration of Benzene and Naphthalene:

Benzene and naphthalene were each collected by a purge and trap device (Model 4560, OI Analytical), and concentrations thereof were detected using a gas chromatograph (GC) (Model 6890, Agilent Technologies). The operating conditions of the purge and trap device and the gas chromatograph are shown in Tables 3 and 4 respectively.









TABLE 3





Parameter
Benzene
Naphthalene


















Purge
Temperature (° C.)
25
20



Time (min)
11
11


Desorb
Temperature (° C.)
210
180



Time (min)
2
4


Bake
Temperature (° C.)
260
180



Time (min)
10
20




















TABLE 4







Parameter 
Benzene 
Naphthalene









Column
VOCOL TM capillary
SPB TM-5 capillary 




column having a
column having a 




length of 30 cm and 
length of 30 cm and 




an inner diameter
an inner diameter 




of 0.53 mm and being
of 0.53 mm and being 




equipped with a
equipped with a 




film with a 
film wuth a 




thickness of 3 μm
thickness of 1.5 μm 



Column oven
An initial
An initial



temperature
temperature of 35° C.
temperature of 40° C.



program 
for 5 min, and a
for 2 min, a ramp 




ramp rate of 15° C./
rate of 15° C./min to 




min to 200° C., held
240° C., and a ramp 




at 200° C. for 2 min 
rate of 10° C./min to 





280° C., held at 





280° C. for 2 min 










Carrier gas 
N2 



Flow rate of 
45 mL/min 



the carrier 




gas 




Injector 
200° C. 



temperature




Detector
Flame ionization detector (FID)



Detector 
250° C.



temperature










Furthermore, benzene (5000 μg/mL) and naphthalene (5000 μg/mL) purchased from SUPELCO were used as control standards.
Example 1. Isolation and Screening of Bacterial Isolates
A. Source and Isolation of Test Strains
A soil sample was collected from diesel-contaminated soil in a remediation site in Guanyin District, Taoyuan City (collected at a depth of about 1 meter below the soil surface).
A 10 g soil sample was sufficiently dispersed in 500 mL steriled water by ultrasonication, and the resultant suspension was incubated in a thermo shaker incubator (28° C., 50 rpm) for 7 days to obtain a bacteria culture. Subsequently, the bacteria culture was subjected to 10-fold serial dilutions so that 106 to 108-fold diluted bacteria culture solutions were formed. 0.1 mL of each of the diluted bacteria culture solutions was evenly applied onto a minimal agar medium, followed by cultivation at 30° C. for 48 to 72 hours. Afterwards, based on colonial morphology and the results of microscopic examination, 35 colonies were selected. Each of the colonies was applied onto a minimal agar medium by virtue of the four-quadrant streak method, followed by cultivation at 30° C. for 48 to 72 hours. After repeating the above four-quadrant streak method several times, 35 purified bacterial isolates (i.e., S01 to S35) were obtained. Each of the purified bacterial isolates was inoculated into a proper amount of TSB, followed by cultivation in a thermo shaker incubator (30° C., 50 rpm) for 48 to 72 hours to obtain a bacterial culture. The bacterial culture of each of the purified bacterial isolates was mixed with glycerol at a volume ratio of 9:1, followed by storage at −80° C. for further use.
B. Preparation of Inoculum of Each of the Purified Bacterial Isolates
Each of the purified bacterial isolates obtained from section A of this example was inoculated into 5 mL YM medium (containing 0.4% yeast extract, 1% malt extract, and 0.4% dextrose) (Difco 0711-01) at a concentration of 0.4% (v/v), followed by cultivation at 30° C. for 18 hours. Afterwards, the bacteria culture was subjected to amplification cultivation in 200 mL YM medium and 20 L YM medium so as to obtain an inoculum of the purified bacterial isolate.
C. Screening of Bacterial Isolates Having Heavy Oil Emulsifying Activity
4 mL of the inoculum of each of the purified bacterial isolates obtained from section B of this example was inoculated into 6 mL heavy oil (purchased from CPC Corporation, Taiwan), followed by shaking for 2 minutes. Then, the resultant mixture stood at room temperature for 24 hours to form an emulsified layer and an oil layer, and the heights of the emulsified layer and oil layer were measured. The experiment was repeated twice.
The Emulsification index E24 was calculated using the following Equation (I):

A=(B/C)×100  (I)

where A=emulsification index E24 (%)

B=height of the emulsified layer (cm)
C=height of the oil layer (cm)
The higher the Emulsification index E24, the higher the emulsifying activity of the bacterial isolate.
The results thus acquired show that among 35 bacterial isolates, the bacterial isolate S03 has excellent emulsifying activity for heavy oil (data not shown). Accordingly, the Inventors believe that the bacterial isolate S03 showed the greatest potential for treatment of crude petroleum, and thus was subjected to characteristics identification below.
Example 2. Characteristics Identification of the Bacterial Isolate S03
In order to identify the bacterial species of the bacterial isolate S03, the following as says were conducted: preliminary tests, fatty acid analysis, 16S rDNA sequence analysis, gyrB (subunit B of DNA gyrase) gene sequence analysis, and DNA-DNA hybridization analysis.
A. Preliminary Tests
The preliminary tests for the bacterial isolate S03 including gram staining, morphological observation, catalase activity, oxidase activity, mobility, and growth phenomium under aerobic and anaerobic environments were conducted by Food Industry Research and Development Institute (FIRDI).
According to the preliminary test results, the bacterial isolate S03 is a gram-negative bacilli, has catalase activity, does not have oxidase activity, has motility, is able to grow under aerobic condition but not under anaerobic condition, and is not able to generate endospores.
B. Fatty Acid Analysis
The fatty acid analysis for the bacterial isolate S03 was conducted using MIDI Sherlock® Microbial Identification System) (MIDI, Inc., Newark, Del., USA) by FIRDI.
The results of the fatty acid analysis are shown in Table 5, and indicates that the bacterial isolate S03 has C18:1 ω7c and C16:1 ω6c and/or C16:1 ω7c as major fatty acids, C16:0, C10:0 3OH, C12:0 2OH, and C12:0 3OH, and is in absence of C16:0 2OH and C16:0 3OH.










TABLE 5







Fatty acid
Content (%)



















C18:1 ω7c
29.91



C16:1 ω7c/C16:1 ω6c
29.01



C16:0
11.14



C12:0
9.71



C10:0 3OH
4.25



C12:0 3OH
3.87



C17:1 ω8c
2.99



C17:1 ω6c
1.44



C14:0
1.37



C17:0 cyclo
0.91



C17:0
0.68



C13:0
0.66



C11:0
0.42



C15:1 ω6c
0.41



C11:0 3OH
0.38



C13:0 iso
0.30



C10:0
0.28



C17:0 iso
0.27



C16:1 ω5c
0.23



C19:1 ω6c
0.23



C15:1 ω8c
0.21



C18:1 ω7c 11-methyl
0.19



C11:0 iso 3OH
0.16



C12:0 2OH
0.15



C18:0
0.15



C9:0 3OH
0.13



C14:1 ω5c
0.11



C15:0 iso
0.08



C13:0 3OH/C15:1 i H
0.07



C19:0 cyclo ω8c
0.07



C18:1 ω5c
0.06



C17:1 iso ω9c
0.05



C11:0 iso
0.04



C12:1 3OH
0.04



C16:0 iso
0.03










C. 16S rDNA Sequence Analysis

The 16S rDNA sequence analysis for the bacterial isolate S03 was conducted by FIRDI.
The partial 16S rDNA sequence of the bacterial isolate S03 (SEQ ID NO:1) is shown in FIG. 1, and was compared to 16S rDNA sequences of type strains of Pseudomonas genus from a gene database on the NCBI website. The results are shown in Table 6. It can be seen from Table 6 that the partial 16S rDNA sequence of the bacterial isolate S03 (SEQ ID NO:1) has high sequence similarity to the 16S rDNA sequence of each type strain of Pseudomonas spp., i.e., Pseudomonas toyotomiensis, Pseudomonas oleovorans subsp. lubricantis, Pseudomonas alcaliphila, Pseudomonas mendocina, Pseudomonas composti, and Pseudomonas anguilliseptica.









TABLE 6






Genbank
sequence



Accession
similarity


Type strain
number
(%)







Pseudomonas toyotomiensis
AB453701
98.78


HT-3T




Pseudomonas oleovorans
DQ842018
98.68


subsp. lubricantis RS1T




Pseudomonas alcaliphila
AB030583
98.64


AL15-21T




Pseudomonas mendocina LMG
Z76664
98.21


1223T




Pseudomonas composti C2T
FN429930
97.82


Pseudomonas
X99540
97.80


anguilliseptica NCIMB




1949T




Pseudomonas indoloxydans
DQ916277
97.77


IPL-1T




Pseudomonas oleovorans DSM
Z76665
97.55


1045T




Pseudomonas stutzeri ATCC
CP002881
97.49


17588T




Pseudomonas putida DSM 291T
Z76667
97.42


Pseudomonas
AB009457
97.29


plecoglossicida FPC951T




Pseudomonas taiwanensis
EU103629
97.24


BCRC 17751T




Pseudomonas benzenivorans
FM208263
97.22


DSM 8628T




Pseudomonas borbori
AM114527
97.22


R-20821T




Pseudomonas flavescens
U01916
97.22


B62T




Pseudomonas monteilii CIP
AF064458
97.22


104883T




Pseudomonas punonensis
JQ344321
97.15


LMT03T




Pseudomonas fuscovaginae
AB021381
97.15


MAFF 301177T




Pseudomonas xanthomarina
AB176954
97.12


KMM 1447T




Pseudomonas argentinensis
AY691188
97.08


CH01T









Based on the identification results of sections A to C of this Example, the bacterial isolate S03 according to this disclosure is determined to be a strain of Pseudomonas spp. In order to further identify whether the bacterial isolate S03 is a novel Pseudomonas species, the following analyses were conducted.
D. gyrB Gene Sequence Analysis
The gyrB gene sequence analysis for the bacterial isolate S03 was conducted by FIRDI.
The partial gyrB gene sequence of the bacterial isolate S03 (SEQ ID NO:2) is shown in FIG. 2. In view of the results of section C of this Example, the six Pseudomonas spp. showing higher 16S rDNA sequence similarity to the bacterial isolate S03 were used for comparison in the gyrB gene sequence. The partial gyrB gene sequence of the bacterial isolate S03 (SEQ ID NO:2) was compared to gyrB gene sequences of type strains of the six Pseudomonas spp. from a gene database on the NCBI website. The results are shown in Table 7. It can be seen from Table 7 that the partial gyrB gene sequence of the bacterial isolate S03 has low sequence similarity to the gyrB gene sequence of each of the type strains.









TABLE 7






Genbank
sequence



Accession
similarity


Type strain
number
(%)







Pseudomonas toyotomiensis
AB494447
86.87


HT-3T




Pseudomonas oleovorans
EF675630
86.87


subsp. lubricantis RS1T




Pseudomonas alcaliphila
AB494446
85.46


JCM 10630T




Pseudomonas mendocina ATCC
AJ633103
85.66


25411T




Pseudomonas composti C2T
HE800474
86.33


Pseudomonas
FN554168
86.91


anguilliseptica LMG 21629T









E. DNA-DNA Hybridization Analysis

The DNA-DNA hybridization analysis for the bacterial isolate S03 was conducted by FIRDI using nine type strains of Pseudomonas spp. purchased from BCRC of FIRDI, i.e., Pseudomonas toyotomiensis, Pseudomonasoleovorans subsp. lubricantis, Pseudomonas alcaliphila, Pseudomonas mendocina, Pseudomonas composti, Pseudomonas anguilliseptica, Pseudomonas benzenivorans, Pseudomonas oryzihabitans, and Pseudomonas japonica to obtain a DNA relatedness value between each of the type strains and the bacterial isolate S03.
The results are shown in Table 8. It can be seen from Table 8 that the DNA relatedness values between the bacterial isolate S03 and each of the nine strains is below 60%. The results indicate that the bacterial isolate S03 shows a distant relatedness to each of the type strains.










TABLE 8








DNA relatedness




value (%) with 



Type 
the bacterial



strain
isolate S03









Pseudomonas toyotomiensis
51



BCRC 80682T




Pseudomonas oleovorans
59



subsp. lubricantis BCRC




80656T




Pseudomonas alcaliphila BCRC
51



80654T




Pseudomonas mendocina BCRC
35



10458T




Pseudomonas composti BCRC
59



80701T




Pseudomonas anguilliseptica
58



BCRC 80691T




Pseudomonas benzenivorans
47



BCRC 80692T




Pseudomonas oryzihabitans
39



BCRC 16007T




Pseudomonas japonica BCRC
19



80101T










Based on the aforesaid results, and further reference to certain related references such as Satoshi Yamamoto et al. (1995), Applied and Environmental Microbiology, 61:1104-1109 and Li-Ting Wang et al. (2010), International Journal of Systematic and Evolutionary Microbiology, 60:2094-2098, the bacterial isolate S03 of this disclosure was determined by the Inventors to be a novel Pseudomonas species, named as Pseudomonas taoyuanensis S03, and deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) under an accession number DSM 32185 on Oct. 14, 2015.
Example 3. Evaluation for Diesel Emulsifying Activity of Pseudomonas taoyuanensis S03
In this example, the evaluation for diesel emulsifying activity of Pseudomonas taoyuanensis S03 was performed substantially according to the procedures as set forth in section C of Example 1, except that super diesel (purchased from CPC Corporation, Taiwan) was used in place of heavy oil.
The Emulsification index E24 thus determined is 105%. The result shows that Pseudomonas taoyuanensis S03 has excellent emulsifying activity for super diesel.
Example 4. Effect of Pseudomonas taoyuanensis S03 on Scavenging Benzene and Naphthalene
Experimental Materials:
1. De-Oxygenated Water:
9 mg Na2SO3 and 1 mg CoCl2 were added to 1 L sterilized water, followed by mixing homogeneously and reacting at room temperature for 2 minutes to obtain de-oxygenated water, simulating groundwater that flows through the soil.
2. Simulated Column Apparatus:
A simulated column apparatus used in this experiment includes an upright cylindrical column having a length of 30 cm and an inner diameter of 5 cm, two Teflon plugs for respectively sealing top and bottom ends of the column, a peristaltic pump (Model 323U, WATSON-MARLOW) connected to the column through the Teflon plug at the bottom end of the column for pumping de-oxygenated water into the column, and a conduit connected to the column through the Teflon plug at the top end of the column for collecting effluent from the column.
3. Benzene-Containing Soil Sample:
A soil sample collected from benzene-contaminated soil in a remediation site in Miaoli county (collected at a depth of about 5 meters below the soil surface) was crushed with a mallet. The crushed soil sample was air-dried for 7 to 10 days, and was then filtered with a filter having a pore size of 0.85 mm. The filtered soil sample was determined to have 1231.1 mg/kg of benzene based on the method as described in the previous section of “General Experimental Procedure”, followed by storage at −80° C. for further use.
4. Naphthalene-Containing Soil Sample:
A soil sample collected from naphthalene-contaminated soil in a remediation site in Kaohsiung city (collected at a depth of about 1 meter below the soil surface) was crushed with a mallet. The crushed soil sample was air-dried for 7 to 10 days, and was then filtered with a filter having a pore size of 0.85 mm. The filtered soil sample was determined to have 191.7 mg/kg of naphthalene based on the method as described in the previous section of “General Experimental Procedure”, followed by storage at −80° C. for further use.
Experimental Procedures:
A. Scavenging Ability of Pseudomonas taoyuanensis S03 for Benzene in the Soil Sample
The bottom end of the upright cylindrical column was covered with gauze and sealed with one of the Teflon plugs. The upright cylindrical column was packed with glass beads having diameters of 0.1 cm, 0.2 cm, and 0.5 cm from the bottom end to the top end as a bottom layer with a height of 2 cm, 653.85 g of the benzene-containing soil sample having a benzene concentration of 1231.1 mg/kg as an intermediate layer with a height of 20 cm, and glass beads having diameters of 0.5 cm, 0.2 cm, and 0.1 cm from the bottom end to the top end as a top layer with a height of 2 cm. The top end of the upright cylindrical column was then sealed with the other Teflon plug.
The de-oxygenated water was pumped into the column through the bottom end of the column using the peristaltic pump at a flow rate of 0.2 mL/min. As the effluent steadily flowed out of the column through the conduit, a suitable amount of calcium peroxide (1000 mg/L) was pumped into the column using the peristaltic pump to adjust the dissolved oxygen (DO) of the soil sample packed in the column. Subsequently, a suitable amount of Pseudomonas taoyuanensis S03 was pumped into the column using the peristaltic pump, such that the concentration of Pseudomonas taoyuanensis S03 in the soil sample was 1×105 CFU/g. Furthermore, a benzene-containing soil sample without Pseudomonas taoyuanensis S03 was used as a control group and was subjected to the same procedure.
The effluent was collected, and was then subjected to the detection of DO and pH value using a DO meter (Model D0200, CLEAN) and a pH meter respectively. The DO and pH value of soil sample are deemed to be equal to the DO and pH value of the effluent thus detected. The experiment was performed for 15 days. During the experiment, the DO of the effluent was adjusted to be in the range of 6 to 7 mg/L and the pH value of the effluent was controlled to be in a range of 6.0 to 7.5 so as to provide favorable growth conditions in the soil sample for Pseudomonas taoyuanensis S03.
Furthermore, the effluent was subjected to detection of benzene concentration based on methods as described in the previous section of “General Experimental Procedure”, and the detected concentration of benzene was multiplied by the volume of the effluent to obtain the benzene content (g) in the effluent.
At the end of the experiment, the benzene content in the effluent which was measured daily was summed up to obtain the total benzene content in the effluent. Furthermore, a suitable amount of the soil sample in the column was subjected to detection of the concentration of benzene based on methods as described in the previous section of “General Experimental Procedure”, and the detected concentration of benzene was multiplied by the weight of the soil sample to obtain the remaining benzene content in the soil sample. Similarly, the initial benzene content in the soil sample (g) was calculated before the experiment was performed.
Benzene clearance rate (%) and benzene degradation rate (%) were calculated using the following Equations (II) and (III) respectively:

D=(E−F)/E×100  (II)

where D=Benzene clearance rate (%)

    
    
        E=Initial benzene content in the soil sample (g)
        F=Remaining benzene content in the soil sample (g)

G=(E−F−H)/E×100  (III)

where G=Benzene degradation rate (%)

        E=Initial benzene content in the soil sample (g)
        F=Remaining benzene content in the soil sample (g)
        H=Total benzene content in the effluent (g)

B. Scavenging Ability of Pseudomonas taoyuanensis S03 for Naphthalene in the Soil Sample

    
    


The scavenging ability of Pseudomonas taoyuanensis S03 for naphthalene in the soil sample was determined substantially according to the procedures as set forth in section A of this example, except that the naphthalene-containing soil sample was used in place of the benzene-containing soil sample.
Naphthalene clearance rate (%) and naphthalene degradation rate (%) were calculated using the following Equations (IV) and (V) respectively:

I=(J−K)/J×100  (IV)

where I=Naphthalene clearance rate (%)

    
    
        J=Initial naphthalene content in the soil sample (g)
        K=Remaining naphthalene content in the soil sample (g)

L=(J−K−M)/J×100  (V)

where L=Naphthalene degradation rate (%)

        J=Initial naphthalene content in the soil sample (g)
        K=Remaining naphthalene content in the soil sample (g)
        M=Total naphthalene content in the effluent (g)

Results:

A. Scavenging Ability of Pseudomonas taoyuanensis S03 for Benzene in the Soil Sample

    
    


The benzene clearance rate of Pseudomonas taoyuanensis S03 is shown in FIG. 3. It can be seen from FIG. 3 that the benzene clearance rate of Pseudomonas taoyuanensis S03 (about 95%) is significantly higher than that of the control group. The results indicate that Pseudomonas taoyuanensis S03 is effective in scavenging benzene present in soil.
The benzene degradation rate of Pseudomonas taoyuanensis S03 is shown in FIG. 4. It can be seen from FIG. 4 that the benzene degradation rate of Pseudomonas taoyuanensis S03 (about 60%) is significantly higher than that of the control group. The results indicate that Pseudomonas taoyuanensis S03 has excellent benzene degrading ability.
Based on the results of FIGS. 3 and 4, it is revealed that Pseudomonas taoyuanensis S03 can scavenge benzene present in soil by desorption and degradation.
B. Scavenging Ability of Pseudomonas taoyuanensis S03 for Naphthalene in the Soil Sample
The naphthalene clearance rate of Pseudomonas taoyuanensis S03 is shown in FIG. 5. It can be seen from FIG. 5 that the naphthalene clearance rate of Pseudomonas taoyuanensis S03 (between 72% and 80%) is significantly higher than that of the control group. The results indicate that Pseudomonas taoyuanensis S03 is effective in scavenging naphthalene present in soil.
The naphthalene degradation rate of Pseudomonas taoyuanensis S03 is shown in FIG. 6. It can be seen from FIG. 6 that the naphthalene degradation rate of Pseudomonas taoyuanensis S03 is slightly higher than that of the control group.
It can be seen from FIGS. 5 and 6 that the naphthalene clearance rate of Pseudomonas taoyuanensis S03 is significantly higher than the naphthalene degradation rate, indicating that the effect of Pseudomonas taoyuanensis S03 on scavenging naphthalene in soil is reached mainly via desorbing naphthalene from the soil sample into the effluent.
In view of the foregoing, Pseudomonas taoyuanensis S03 has excellent emulsifying activity, and is effective in scavenging benzene, naphthalene, toluene, ethylbezene, or xylene present in soil by desorption and degradation so as to achieve bioremediation. Accordingly, Pseudomonas taoyuanensis S03 of the present disclosure is expected to have potential in developing a microbial agent for use in scavenging crude petroleum, a petroleum refined product (e.g. diesel and heavy oil), benzene, naphthalene, toluene, ethylbezene, or xylene in a contaminated substrate.
All the patents and references cited in this specification are incorporated herein in their entirety as reference. When there is conflict, the detailed descriptions in this case, including the definitions, would prevail.
While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

## Claims

1. A method for scavenging aromatic hydrocarbons present in a contaminated substrate, comprising treating the contaminated substrate with Pseudomonas taoyuanensis S03 deposited in DSMZ under accession number DSM 32185, such that the aromatic hydrocarbons present in the contaminated substrate are degraded or desorbed from the contaminated substrate by the Pseudomonas taoyuanensis S03, the aromatic hydrocarbons being selected from the group consisting of benzene, naphthalene, toluene, ethylbezene, xylene, and combinations thereof.

2. The method of claim 1, wherein the aromatic hydrocarbons are selected from the group consisting of benzene, naphthalene, and the combination thereof.

3. The method of claim 1, wherein the contaminated substrate is selected from the group consisting of soil, sludge, sediment, aquifer, and water.

4. The method of claim 3, wherein the contaminated substrate is selected from the group consisting of plowland, orchard land, grazing grassland, woodland, gas station land, industrial land, well water, an aquaculture pond, ground water, river water, lake water, seawater, industrial waste water, domestic sewage, and silt.

5. A method for scavenging crude petroleum or a refined product thereof present in a contaminated substrate, comprising treating the contaminated substrate with Pseudomonas taoyuanensis S03 deposited in DSMZ under accession number DSM 32185, such that the crude petroleum or the refined product thereof is emulsified by the Pseudomonas taoyuanensis S03.

6. The method of claim 5, wherein the refined product is selected from the group consisting of heavy oil, diesel, kerosene, gasoline, hydraulic oil, and lubricating oil.

7. The method of claim 5, wherein the contaminated substrate is selected from the group consisting of soil, sludge, sediment, aquifer, and water.

8. The method of claim 7, wherein the contaminated substrate is selected from the group consisting of plowland, orchard land, grazing grassland, woodland, gas station land, industrial land, well water, an aquaculture pond, ground water, river water, lake water, seawater, industrial waste water, domestic sewage, and silt.