TWI518180B - Pseudomonas taoyuanensis s03 isolate having the emulsifying activity and the scavenging ability for benzene and/or naphthalene and uses of the same - Google Patents

Description

Pseudomonas syriata S03 isolate having emulsifying ability and scavenging ability for benzene and/or naphthalene and use thereof

The present invention relates to a strain of Pseudomonas taoyuanensis S03 having an emulsifying ability and a scavenging ability for benzene and/or naphthalene, which is deposited in the Food Industry Development Research Institute (FIRDI) under the registration number BCRC 910562. Biological Resource Conservation and Research Center (BCRC). The Pseudomonas putida S03 isolate and its subcultured progeny can be used to prepare microbial agents for the removal of crude oil, petroleum refining products, benzene and/or naphthalene present in a contaminated medium.

An aromatic hydrocarbon [also referred to as an arene] refers to a hydrocarbon compound having one or more benzene rings and can be distinguished as a single according to the number of benzene rings. Monocyclic aromatic hydrocarbons (MAHs) and polycyclic aromatic hydrocarbons (PAHs). Aromatic hydrocarbons are commonly found in crude petroleum and petroleum. Refined refined products [eg, diesel, heavy oil, and gasoline) and industrial solvents, and in the preparation of pharmaceuticals, agrochemicals , polymers, explosives, and many daily necessities are often used as starting materials in the process. However, these aromatic hydrocarbons have a very stable molecular structure and have high melting point, high boiling point, and poor decomposition properties, so they are continuously accumulated and exist in nature and living organisms when they are released into the environment, thereby contributing to the entire ecology. The environment or the human body causes serious harm.

Benzene, toluene, ethylbenzene, and xylene are all MAHs and are collectively referred to as BTEX compounds. They are the most common groundwater contaminants and soil contaminants. ), in which benzene is found to be carcinogenic, and thus is highly valued. Benzene (chemical formula C ₆ H ₆ ) is one of the simplest MAHs, which is often used as an organic solvent in the chemical industry or used to prepare benzene derivatives. When benzene enters the human body through skin or eye contact, or through inhalation and intake, it inhibits the central nervous system (CNS) and produces sleepy and dizziness. Symptoms such as (dizzy), headache (headache) and nausea (nausea) can cause red blood cells, white blood cells, and platelets to form, and cause leukemia.

Naphthalene (chemical formula C ₁₀ H ₈ ) is the simplest PAHs composed of two fused benzene rings, which have been widely used in the production of dyes (dyestuffs, resins). Resin, solvent, disinfectant, insecticide, preservative, and mothproofing agent. When naphthalene enters the body, it may cause symptoms such as haemolytic anaemia, nausea, vomiting, diarrhea, jaundice, and liver or kidney damage.

Since benzene and/or naphthalene present in the environment have seriously threatened human health and caused damage to the ecological environment, how to effectively treat these environmental pollutants has become the focus of attention and research in all countries of the world. Currently known treatment methods include: solidification, removal method, incineration, activated carbon adsorption, catalytic reduction, photolysis (photolysis) and bioremediation (bioremediation) and so on.

Bioremediation is the use of microbial biodegradative activities to remove environmental pollutants and recalcitrant xenobiotics. The biological re-cultivation method has been widely used in low cost, does not produce toxic by-products in the process of degrading environmental pollutants, causes secondary pollution, and can be operated in situ . Remediation of contaminated sites. Among all bioremediation techniques, bioaugmentation is particularly valued. It mainly adds microbes with biodegradable activity to a contaminated environment to degrade pollutants. However, aromatic hydrocarbons have low water solubility and strong sorption characteristics to the soil, which causes microorganisms having aromatic hydrocarbon degrading activity to be affected by the bioavailability of aromatic hydrocarbons, thereby limiting the degradation of aromatic hydrocarbons. rate. Therefore, the addition of microorganisms having emulsifying activity in an environment contaminated with aromatic hydrocarbons to increase the desorption rate of aromatic hydrocarbons and the apparent solubility in the aqueous phase can enhance aromatic hydrocarbons. Availability, which in turn promotes the removal of aromatic hydrocarbons.

In the case where indigenous microorganisms are unable to effectively remove aromatic hydrocarbons, biological addition may be the only way to achieve the goal of biological reproduction. Therefore, the separation and screening of microorganisms suitable for use in biological re-cultivation methods has become a goal of researchers in the field.

At present, many strains with emulsifying ability and/or scavenging ability for benzene and/or naphthalene have been isolated from contaminated environments, most of which belong to Pseudomonas spp., pseudo-yellow Pseudoxanthomonas spp., Alicycliphilus spp., Burkholderia spp., Ralstonia spp., Achromobacter 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; RCJohn Et al. (2012), Bulletin of Environmental Contamination and Toxicology , 88: 1014-1019). For example, CN 103045502 A discloses a strain of Rhodococcus erythoropolis T7-3 (CGMCC No. 6104) isolated from a petroleum-contaminated submarine region, which has been shown to emulsify and/or degrade crude oil and petroleum hydroxy (Petroleum hydrocarbons [including alkane and aromatic hydrocarbons (such as benzene and xylene)] are thus expected to be useful for the re-education of oil-contaminated marine organisms. CN 1519312 A discloses a Rhodococcus ruber Em CGMCC No. 0868 isolated from soil contaminated with crude oil, which has been shown to emulsify and/or degrade kerosene and petroleum hydroxy [such as benzene, naphthalene, onion (anthracene) , phenanthrene and pyrene, which are expected to be used in the treatment of oily wastewater and in the biological remediation of oil-contaminated soil.

In E. Deziel et al. (1996), Appl. Environ. Microbiol. , 62: 1908-1912, E. Deziel et al. isolated 23 strains of PAH-degrading bacteria from bunkers receiving refinery waste. The isolate, in which Pseudomonas aeruginosa 19SJ was found to be able to utilize naphthalene or phenanthrene as the sole substrate, produced a large amount of glycolipid. In further experiments, the strain was found to enhance the apparent solubility of naphthalene by exhibiting the emulsification ability by producing the glycolipid, thereby enhancing the degradation and utilization of naphthalene by the strain itself, and further promoting the formation of the glycolipid. Thus, E. Deziel et al. believe that the production of compounds having interface-activity is a strategy for strains to grow on substrates with low availability. Therefore, this strain is expected to be applicable to bioremediation in an environment contaminated with naphthalene and/or phenanthrene.

In Eun Young Lee et al. (2011), International Proceedings of Chemical, Biological & Environmental Engineering , 20: 37-41, Eun Young Lee et al. used rhizosphere soil from reed for wastewater treatment. Pseudomonas putida AY-10 was isolated from the strain, which was found to be grown in a medium containing benzene, toluene, ethylbenzene or xylene as the sole carbon source, and completely degraded benzene and toluene. , ethylbenzene and xylene. Therefore, Eun Young Lee et al. believe that Pseudomonas putida AY-10 is available for bioremediation in BTEX-contaminated environments.

Although the above literature has been reported, the applicant is actively working to screen microorganisms having emulsifying ability and capable of removing benzene and/or naphthalene for environmental protection. After research, the applicant accidentally isolated a novel bacterial isolate from the soil contaminated with benzene or naphthalene [it was later identified as Pseudomonas taoyuanensis S03]. It is phylogenetically different from the species already disclosed in the genus, and has superior emulsification capacity for heavy oil and diesel oil and scavenging ability for benzene and naphthalene. Therefore, this strain is expected to have great potential in remediating an environment contaminated with crude oil, petroleum refining products, benzene and/or naphthalene.

Summary of invention

Thus, in a first aspect, the present invention provides a Pseudomonas taoyuanensis S03 isolate having emulsifying ability and scavenging ability for benzene and/or naphthalene, which is deposited in the food industry under the registration number BCRC 910562. Development Research Institute (FIRDI) Biological Resource Conservation and Research Center (BCRC).

In a second aspect, the present invention provides a microbial agent for removing benzene and/or naphthalene present in a contaminated medium comprising a Pseudomonas syringae S03 isolate as described above or a subculture thereof Breeding offspring.

In a third aspect, the present invention provides a microbial agent for removing crude oil and/or petroleum refining products present in a contaminated medium, comprising a Pseudomonas sp. S03 isolate as described above or Subculture the offspring.

In a fourth aspect, the present invention provides a method for removing benzene and/or naphthalene present in a contaminated medium comprising: using a Pseudomonas syriata S03 isolate as described above or a subsequent thereof Substituting the progeny to treat the contaminated medium such that the benzene and/or naphthalene present in the contaminated medium is degraded and/or desorbed by the Pseudomonas syringae S03 isolate or its subcultured progeny .

In a fifth aspect, the present invention provides a method for removing crude oil and/or petroleum refining products present in a contaminated medium, comprising: using a Pseudomonas syringae S03 isolate as described above or The subculture is subcultured to treat the contaminated medium such that the crude oil and/or petroleum refining product present in the contaminated medium is emulsified by the Pseudomonas syringae S03 isolate or its subcultured progeny.

The above and other objects, features and advantages of the present invention are The detailed description, the preferred embodiment, and the accompanying drawings will be apparent.

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to effectively deal with environmental pollutants and avoid serious damage to the environment and ecology, countries all over the world have invested a lot of manpower and financial resources to find a solution. Nowadays, among various methods for treating environmental pollutants, the biological re-cultivation method has low cost, does not produce toxic by-products in the process of degrading environmental pollutants, causes secondary pollution, and can be in situ ( in situ It has been widely used in the remediation of contaminated sites.

In order to screen out microorganisms suitable for supply to the biological re-cultivation method, the applicant separated 35 bacterial isolates from the diesel-contaminated plant soil, and then inoculated the obtained isolates to heavy oil containing heavy oil. The emulsification index E ₂₄ (emulsification index E ₂₄ ) was analyzed in a test tube, and a bacterial isolate S03 having an excellent ability to emulsify heavy oil was selected therefrom. Applicants have characterized this bacterial isolate S03 and referred 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 and other microbiological related literature, and judged that the bacterial isolate S03 belongs to a novel Pseudomonas spp., which was named by the applicant as Pseudomonas syria ( Pseudomonas taoyuanensis )S03" was deposited with the Bioresource Conservation and Research Center (BCRC of FIRDI) of the Food Industry Development Research Institute of Taiwan on August 29, 2012 under the registration number BCRC 910562.

Applicants have further found through experiments that the Pseudomonas syriata S03 of the present invention can also effectively emulsify super diesel.

Based on the above, the Applicant believes that Pseudomonas syriata S03 of the present invention or its subcultured progeny can be used for bioremediation in an environment contaminated with crude oil and/or petroleum refining products. Accordingly, the present invention provides a microbial agent for removing crude oil and/or petroleum refining products present in a contaminated medium comprising Pseudomonas syriata S03 as described above or a subcultured progeny thereof.

As used herein, the terms "crude petroleum" and "crude oil" are used interchangeably and refer to the surface of the earth. An untreated or unrefined oil found in the following, which is a hydrocarbon having various molecular weights [including aliphatic hydrocarbons, cyclic hydrocarbons, and A complex mixture of aromatic hydrocarbons and other organic compounds.

As used herein, the terms "petroleum refined product", "refined petroleum" and "petroleum product" are used interchangeably.

According to the present invention, the petroleum refining products include, but are not limited to, diesel, heavy oil, kerosene, fuel oil, gasoline, hydraulic oil, and lubricating oil. (lubricating oil). In a preferred embodiment of the invention, the petroleum refining product is a heavy oil. In another preferred embodiment of the invention, the petroleum refining product is diesel.

According to the invention, the contaminated medium is a solid or liquid environmental medium, including but not limited to: soil, sludge, sediment, aquifer, water body (water body) and waste water. Preferably, the contaminated medium is selected from the group consisting of agricultural land (for example, fields and orchards, etc.), grazing grassland, woodland, gas station land, industrial land, artificial water body (for example, well water) , fish culture ponds, ponds, reservoirs, etc.), natural water bodies [such as groundwater, river water, lake water, seawater, etc.], factory wastewater, domestic sewage, and sludge from sewage treatment plants.

The microbial agent according to the present invention may further comprise at least one microorganism capable of scavenging monocyclic and/or polycyclic aromatic hydrocarbons.

Microorganisms capable of scavenging monocyclic aromatic hydrocarbons suitable for use in the present invention include, but are not limited to, Pseudomonas spp., Pseudoxanthomonas spp., and Alicyclobacillus species ( Alicycliphilus spp.), Burkholderia spp., Ralstonia spp., Achromobacter spp., Hydrogenophaga spp .), Rhodococcus spp. and Arthrobacter spp. Preferably, the microorganism capable of scavenging monocyclic aromatic hydrocarbons is selected from the group consisting of Rhodococcus erythoropolis T7-3, Rhodococcus ruber Em CGMCC No. 0868, love Pseudomonas putida AY-10, and combinations thereof.

Microorganisms capable of scavenging polycyclic aromatic hydrocarbons suitable for use in the present invention include, but are not limited to, Pseudomonas species, Pyrococcus species, Arthrobacter species, Acinetobacter spp., Flavobacterium species ( Flavobacterium spp.), Alcaligenes spp., Micrococcus spp., and Corynebacterium spp. Preferably, the microorganism capable of scavenging polycyclic aromatic hydrocarbons is selected from the group consisting of Rhodococcus erythropolis Em CGMCC No. 0868, Pseudomonas aeruginosa 19SJ, and combinations thereof.

The microbial reagent according to the invention may optionally comprise a pair Useful nutrients for microbial growth, including but not limited to: glycerol, riboflavin, casein, polypeptone, meat extract, soy cake Soybean cake), yeast extract, cellulose, glucose, corn extract, whey powder, starch, vitamins [such as thiamine, biotin, Nicotinic acid amide and calcium panthotenate] and enzymes [such as amylase, protease, and lipase].

The microbial agent according to the present invention can be manufactured into a form suitable for use using techniques well known to those skilled in the art, including, but not limited to, culture solutions, suspensions, granules. ), powder, tablet, pill, capsules, slurries, and the like. In addition, the microbial agent can also be used by immobilizing it on an insoluble support.

The microbial agent according to the present invention may further comprise a biocompatible carrier.

In a preferred embodiment of the invention, Pseudomonas sinensis S03 in the microbial agent 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 (polyacrylamide), carrageenan, agarose, gelatin, cellulose, cellulose acetate, dextran, and collagen.

In another preferred embodiment of the invention, Pseudomonas syriata S03 in the microbial agent 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 Ion exchange resin), epoxy resin, photosetting resin, polyester, and polystyrene.

The microbial agent according to the present invention can also be fabricated into a bioreactor or apparatus for removing crude oil and/or petroleum refining products present in a contaminated medium, using techniques well known to those skilled in the art. For the manufacture of bioreactors, reference is made to, for example, US Pat. No. 5,279,963, US Pat. No. 5,258,303, US Pat. No. 5,552,051, US Pat. No. 5,494, 574, US Pat. No. 6,030, 533, US PCT PCT PCT PCT PCT PCT PCT T. Furuichi (2007), Journal of Hazardous Materials , 148(3): 693-700.

The invention also provides a method for removing the presence of a contaminated A method of crude oil and/or petroleum refining products in a medium, comprising: treating the contaminated medium with a Pseudomonas syriata S03 as described above or a subcultured progeny thereof, such that the contaminated medium is present The crude oil and/or petroleum refining product in the medium is emulsified by the Pseudomonas syriata S03 or its subcultured progeny.

As used herein, the term "emulsified" means that a substance having low water solubility is dispersed into the aqueous phase in the form of fine droplets or particles by changing the interfacial tension. Medium to form a heterogeneous dispersion system.

In the method according to the invention, the Pseudomonas syriata S03 or its subcultured progeny can be used in combination with at least one microorganism capable of scavenging monocyclic and/or polycyclic aromatic hydrocarbons, with respect to the purgable single ring and The microorganism of the polycyclic aromatic hydrocarbon is as described above.

In addition, the applicant confirmed by a test simulating the soil remediation environment that the Pseudomonas sp. S03 can remove benzene and naphthalene present in the soil by desorption and/or degradation.

Based on the above, the Applicant believes that Pseudomonas syriata S03 of the present invention or its subcultured progeny can be applied to biological re-cultivation in an environment contaminated with benzene and/or naphthalene. Accordingly, the present invention provides a microbial agent for removing benzene and/or naphthalene present in a contaminated medium comprising Pseudomonas syriata S03 as described above or a subcultured progeny thereof.

According to the invention, the contaminated medium, the microbial reagent Substances that may be further included [including: microorganisms, nutrients, and biocompatible carriers that can scavenge monocyclic and/or polycyclic aromatic hydrocarbons] and forms and bioreactors or devices in which the microbial agent is suitable for being manufactured As described above.

The present invention also provides a method for removing benzene and/or naphthalene present in a contaminated medium, comprising: treating the recipient with a Pseudomonas syriata S03 as described above or a subcultured progeny thereof The contaminated medium causes the benzene and/or naphthalene present in the contaminated medium to be degraded and/or desorbed by the Pseudomonas syriata S03 or its subcultured progeny.

As used herein, the term "degradation" means the metabolic decomposition of a compound into a less complex molecule.

As used herein, the term "desorption" means that a compound adsorbed on a solid surface is released from the solid surface by destroying adhesive forces.

In the method according to the invention, the Pseudomonas syriata S03 or its subcultured progeny can be used in combination with at least one microorganism capable of scavenging monocyclic and/or polycyclic aromatic hydrocarbons, with respect to the purgable single ring and The microorganism of the polycyclic aromatic hydrocarbon is as described above.

Other features and effects of the present invention will be apparent from the following description of the drawings, wherein: Figure 1 shows the nucleotide sequence of the 16S rDNA of the Pseudomonas taoyuanensis S03 of the present invention. Figure 2 shows the nucleotide sequence of the gyr B gene of the Pseudomonas syriata S03 of the present invention; Figure 3 shows the Pseudomonas syringae S03 of the present invention in a simulated column device for soil samples containing benzene Benzene clearance rate, wherein the control group represents a soil sample containing benzene that has not been introduced into Pseudomonas syriata S03; FIG. 4 shows the benzene of the soil sample containing benzene in the simulated column device of Pseudomonas sinensis S03 of the present invention. Degradation rate, wherein the control group represents a soil sample containing benzene that has not been introduced into Pseudomonas syriata S03; Figure 5 shows the removal of naphthalene from a soil sample containing naphthalene in a simulated column device of Pseudomonas sinensis S03 of the present invention. Rate, wherein the control group represents a naphthalene-containing soil sample that has not been introduced into Pseudomonas syriata S03; and Figure 6 shows the Pseudomonas sinensis S03 of the present invention in a simulated column device for a soil sample containing naphthalene Naphthalene degradation rate, which represents the control group are not introduced into the S03 Taoyuan Pseudomonas soil samples containing naphthalene.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example General experimental materials: 1. Basic agar medium:

The base agar medium used in the following examples had the formulations shown in Table 1 below.

2. Tryptic soy broth (TSB):

The tryptic soy broth medium used in the following examples had the formulation shown in Table 2 below.

General experimental method: 1. Determination of the concentration of benzene and naphthalene:

In the following examples, volatile organic compounds (VOCs) in each sample to be tested (ie, benzene And naphthalene) was collected using a purge and trap device (Model 4560, OI Analytical), followed by a gas chromatograph (GC) (Model 6890, Agilent Technologies). The concentration of benzene or naphthalene collected is determined. The operating conditions for the air blowing trap and the gas chromatograph are shown in Tables 3 and 4 below, respectively.

Further, for comparison, benzene (5000 μg/mL) and naphthalene (5000 μg/mL) were used as control standards, respectively, and the same analysis was carried out, and these chemicals were purchased from the company.

Example 1. Isolation and screening of bacteria isolates: A. Source and separation of test strains:

The actual soil samples used in this experiment to screen the test strains were obtained from diesel-contaminated soil at the remediation site of Guanyin Township, Taoyuan County (the collection depth was about 1 meter).

First, 10 g of the actual soil sample was added to a flask containing 500 mL of sterile water, and then the soil sample was sufficiently scattered by ultrasonication, and then the resulting suspension was placed in a constant temperature oscillation. The cultivation was carried out in an incubator (28 ° C, 50 rpm) for 7 days. Thereafter, the culture was formed 10-fold serial dilutions (10-fold serial dilution), to obtain a liquid culture having different dilutions ⁽¹⁰⁶ to ¹⁰⁸ fold), then the respective liquid culture containing different dilutions of 0.1 mL of each was taken and uniformly applied to a basic agar medium, followed by standing culture at 30 ° C for 48 to 72 hours. Then, the applicant observed the colony type on each of the basic agar medium and the growth of the strain by naked eye and microscope, respectively, and then selected 35 colonies from the four-quadrant streak method. The methods were separately applied to a basic agar medium, followed by standing culture at 30 ° C for 48 to 72 hours. The above-described strain purification step was repeated several times to obtain 35 strains of purified bacterial isolates including strain numbers S01 to S35. Thereafter, the bacterial isolates were separately inoculated into an appropriate amount of tryptic soy broth medium (TSB), and cultured in a constant temperature shaking incubator (30 ° C, 50 rpm) for 48 to 72 hours, followed by formation. The bacterial culture was added with an appropriate amount of glycerol to a final concentration of 10% (v/v), which was then stored frozen at -80 °C until use.

B. Preparation of inoculum of bacterial isolates:

35 strains of bacterial isolates obtained according to the above "A, Source and Separation of Test Strains" were inoculated to 5 mL of YM medium (containing 0.4% yeast extract) at a dose of 0.4% (v/v). Yeast extract), 1% malt extract and 0.4% dextrose] (Difco 0711-01, Qixin Biotechnology Co., Ltd.) were cultured at 30 ° C for 18 hours. Thereafter, the obtained bacterial liquid was sequentially subjected to amplification culture of the strain in 200 mL of YM medium and 20 L of YM medium, and the culture thus formed was used as an inoculation source of the bacterial isolate in the following examples.

C. Screening of bacterial isolates having the ability to emulsifie heavy oil:

4 mL of the inoculation source of 35 bacterial isolates obtained according to the above "B, inoculation source for preparing bacterial isolates" were inoculated into 35 test tubes containing 6 mL of heavy oil (purchased from Taiwan National Oil Co., Ltd.) and given The mixture was shaken well for 2 minutes, and then the resulting mixed solution was allowed to stand still at room temperature for 24 hours. Thereafter, the height of the emulsified layer and the oil layer in the test tube was measured. The above experiment was repeated twice.

Emulsifying Index E ₂₄ (emulsification index E ₂₄₎ is by the measured emulsion layer and an oil layer height is substituted into the following equation (1) is calculated by: Equation (1): A = (B / C) × 100

Where: A = emulsification index E ₂₄ (%)

B = height of the emulsion layer (cm)

C = height of the oil layer (cm) If the measured emulsification index E _{24 is} higher, the emulsification ability of the bacterial isolate is stronger.

The experimental results show that among the 35 bacterial isolates purified, the bacterial isolate S03 has an extremely excellent emulsifying ability for heavy oil (data not shown), and the applicant believes that the bacterial isolate S03 is one of the most The potential strain was developed, so it was used for the following characterization.

Example 2. Characterization of bacterial isolate S03:

In order to confirm the bacterial species of the bacterial isolate S03 selected in the above Example 1, the bacterial isolate S03 was subjected to the following preliminary tests, microbial fatty acid identification system analysis, 16S rDNA sequence analysis, DNA rotation enzyme times. Subunit B of DNA gyrase ( gyrB ) gene sequence analysis and DNA-DNA hybridization analysis.

A. Preliminary test:

The preliminary test on the bacterial isolate S03 was carried out on behalf of the Food Industry Research and Development Institute (FIRDI). The test items included: gram staining, morphological observation, Catalase reaction, oxidase reaction, mobility, and growth under aerobic and anaerobic conditions.

According to the preliminary test results, the bacterial isolate S03 is a Gram-negative bacterium, has a catalase, does not have an oxidase, is motility, grows under an aerobic environment, does not grow under an anaerobic environment, and does not produce spore.

B. Analysis of microbial fatty acid identification system:

Related bacterial isolates S03 microbial identification system fatty acid analysis using a MIDI Sherlock ^® Microbial Identification System ^{(MIDI Sherlock ® Microbial Identification System)} (MIDI, Inc., Newark, DE, USA) and commissioned the Institute of Food Industry Development (FIRDI) Come on behalf of it.

The results of the analysis of the microbial fatty acid identification system of the bacterial isolate S03 are shown in Table 5 below, and the analysis results show that the main fatty acids of the bacterial isolate S03 are C _18:1 ω 7c and C _16:1 ω 6c and/or C _16:1 ω 7c, and has C _16:0 , C _10:0 3OH, C _12:0 2OH, and C _12:0 3OH, but lacks C _16:0 2OH and C _16:0 3OH.

C, 16s rDNA sequence analysis:

The 16S rDNA sequence analysis of the bacterial isolate S03 is The Food Industry Development Institute (FIRDI) was carried out on its behalf.

The results of 16S rDNA sequence analysis of the portion of the bacterial isolate S03 are shown in Fig. 1. The 16S rDNA sequence (SEQ ID NO: 1) of the bacterial isolate S03 was compared with the 16S rDNA sequence of the standard strain of the Pseudomonas species in the gene library on the NCBI website. For analysis, the results of the analysis are shown in Table 6 below. As can be seen from Table 6, the 16S rDNA sequence of the bacterial isolate S03 (SEQ ID NO: 1) and the six strains of Pseudomonas spp. (ie, Pseudomonas toyotomiensis , Pseudomonas faecalis ) lubricantis subspecies, basophils Pseudomonas mendocina, having a high degree of sequence among Pseudomonas composti Pseudomonas septicemia and eel) of 16S rDNA sequence similarity (sequence similarity).

According to the experimental results of the above items A to C, the bacterial isolate S03 of the present invention was initially identified as a strain belonging to the genus Pseudomonas, and in order to further confirm whether the bacterial isolate S03 is a novel Pseudomonas spp. The strain of the species, the bacterial isolate S03, was further taken for the analysis of items D and E below.

D, gyrB gene sequence analysis:

The sequence analysis of the gyrB gene of the bacterial isolate S03 was carried out by the Food Industry Development Research Institute (FIRDI).

The results of the gyrB gene sequence analysis of the portion of the bacterial isolate S03 are shown in Fig. 2. Applicants selected six strains of Pseudomonas species (ie Pseudomonas toyotomiensis , Pseudomonas sinensis lubricantis subspecies, hobby ) with higher sequence similarity on the 16S rDNA sequence according to the results of the above analysis. Pseudomonas aeruginosa, Pseudomonas mendocs , Pseudomonas composti, and Pseudomonas septicum as a comparison object, and the gyrB gene sequence of the bacterial isolate S03 (sequence identification number: 2) The gyrB gene sequences of the standard strains of the six Pseudomonas species in the gene database on the NCBI website were compared and analyzed, and the results of the analysis are shown in Table 7 below. Can be seen from Table 7, all the bacterial isolates have a low sequence similarity between portions of the gyrB gene sequence of strain S03 and gyrB gene sequence of the respective standard strains.

E, DNA-DNA hybridization analysis:

Standard DNA-DNA hybridization analysis of bacterial isolates used in S03 is stored in the 9 available from the Center for Biological Resources Research and development of food industry (BCRC of FIRDI) of species of the genus Pseudomonas strain [i.e. Pseudomonas toyotomiensis, Pseudomonas sinensis lubricantis subsp., Pseudomonas aeruginosa, Pseudomonas mendoc , Pseudomonas composti , Pseudomonas syringae, Pseudomonas benzenivorans , Pseudomonas oryzihabitans and Pseudomonas Japonica ] is used for comparison and commissioned by the Food Industry Development Institute (FIRDI).

The DNA relatedness values measured between the bacterial isolate S03 and each of the standard strains are shown in Table 8 below. As can be seen from Table 8, the bacterial isolate S03 and each standard strain The DNA parental value between them is less than 60%, which means that the bacterial isolate S03 has a distant relationship with each standard strain.

To summarize the characterization results of the above, refer 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 and other microbiological related literature, the applicant believes that the bacterial isolate S03 of the present invention is a novel strain of Pseudomonas species, which was named by the applicant as " Pseudomonas taoyuanensis". )S03", and was deposited at the Bioresource Conservation and Research Center (BCRC of FIRDI) of the Food Industry Development Research Institute on August 29, 2012 with the registration number BCRC 910562 (300 Food Road, Hsinchu City, Taiwan) .

Example 3. Evaluation of the emulsifying ability of Pseudomonas syriata S03 for diesel:

In the present embodiment, the Pseudomonas sinensis S03 of the present invention is substantially analyzed by the method described in the item C of "Cleaning the bacterial isolate having the ability to emulsify heavy oil" of Example 1, The difference is that 6mL of super diesel (purchased from Taiwanese Oil Company) is used to replace heavy oil.

The experimental results showed that the emulsification index E ₂₄ measured by the super diesel fuel inoculated with Pseudomonas syriata S03 after 24 hours of culture was as high as 105%. Thus, the Pseudomonas sp. S03 of the present invention has an excellent ability to emulsify superdiesel.

Example 4. Pseudomonas syriata S03 in the simulated soil remediation environment for the removal of benzene and naphthalene:

In order to confirm whether the Pseudomonas syriata S03 of the present invention has the ability to remove benzene and naphthalene present in contaminated soil in a simulated soil remediation environment, the following experiment was carried out.

Experimental Materials: 1. de-oxygen water:

9 mg of sodium sulfite (Na ₂ SO ₃ ) and 1 mg of cobalt chloride (CoCl ₂ ) were added to 1 L of sterile water and mixed well, and then allowed to stand at room temperature for 2 minutes, and the obtained deoxygenated water was taken as a Simulate the flow of groundwater through the soil.

2. Simulated column device:

The simulated column device used in this experiment consists of an upright cylindrical column (30 cm long, 5 cm inside diameter) that can be filled with soil, and two Teflon that can be used to seal the top and bottom of the column, respectively. a plug, a Teflon plug connected to the bottom end of the column and a peristaltic pump (Model 323U, WATSON-MARLOW) for introducing deoxidized water into the column, and a connection to the tube A Teflon plug at the top of the column and a conduit available for collecting effluent.

3. Benzene-containing soil sample:

The benzene-contaminated soil (collecting depth of about 5 meters) taken from a remediation site in Miaoli County was crushed with a raft, then air-dried for 7 to 10 days, followed by an aperture. The screen was sieved to a 0.85 mm screen, and the sieved soil sample thus obtained was measured according to the method described in "1. Determination of benzene and naphthalene concentration" in the "General Experimental Method" above. One is a benzene concentration of 1231.1 mg/kg, which is then stored at 4 ° C for use.

4. Naphthalene-containing soil sample:

The soil contaminated with naphthalene (collecting a depth of about 1 meter) from a remediation site in Kaohsiung City was crushed with a raft, then air dried for 7 to 10 days, followed by a sieve with a pore size of 0.85 mm. The sieve was sieved, and the sieved soil sample obtained was measured according to the method described in "1. Determination of benzene and naphthalene concentration" in the "General Experimental Method" above. The amount was adjusted to have a naphthalene concentration of 191.7 mg/kg, which was then stored at 4 ° C for use.

experimental method: A. Test for removing benzene from soil samples:

First, a gauze is laid on the inner side of the bottom end of the column to prevent the soil from leaking out, and then sealed with a Teflon plug, and then the following substances are sequentially filled into the column: the bottom layer is sequentially from bottom to top. Glass beads with a diameter of 0.1 cm, 0.2 cm, and 0.5 cm (filling height is about 2 cm), and the middle layer is 653.85 g of a soil sample containing benzene (having a benzene concentration of 1231.3 mg/kg, and a filling height of about 20 cm) And the topmost layer of glass beads having a diameter of 0.5 cm, 0.2 cm, and 0.1 cm from bottom to top (filling height is about 2 cm). Next, the top end of the column is sealed with a Teflon plug.

Thereafter, the peristaltic pump was used and the deoxygenated water was continuously introduced into the column from the bottom end of the column at a rate of 0.2 mL/min. When the effluent flows stably from the conduit at the top end of the column, an appropriate amount of calcium peroxide (CaO ₂ ) (1000 mg/L) is introduced into the column by the peristaltic pump so that The dissolved oxygen content of the soil sample in the column falls within the range of 6 to 7 mg/L. Next, using the peristaltic pumping, an appropriate amount of the inoculum of Pseudomonas syriata S03 was introduced into the column, so that the soil sample in the column had a bacterial concentration of 1 × 10 ⁵ CFU/g. Further, for comparison, a soil sample containing benzene which was not introduced into Pseudomonas syriata S03 was used as a control group, and the same experiment was conducted.

Thereafter, the effluent is collected and measured by using a dissolved oxygen (DO) Dissolved oxygen (DO) meter (model DO200, CLEAN) was used to measure the dissolved oxygen amount and pH value was measured using a pH meter. Further, the benzene concentration of the effluent is measured according to the method described in "1. Determination of benzene and naphthalene concentration" of the "general experimental method" above, and then the obtained benzene concentration is multiplied by the volume of the effluent. The benzene content (g) of the effluent was obtained.

The total duration of the experiment was 15 days, and the effluent was collected every day according to the above method and its dissolved oxygen amount, pH value and benzene content were measured, while the measured dissolved oxygen amount was continuously monitored and the column was adjusted according to the above manner. The amount of dissolved oxygen in the soil sample is maintained while the pH of the effluent is maintained in the range of 6.0 to 7.5 to ensure that the strain is grown in an aerobic environment with a preferred pH. After the end of the experiment, the benzene content (g) of the effluent measured per day was added to obtain the total benzene content (g) of the effluent.

In addition, on the 15th day after the introduction of Pseudomonas syriata S03, an appropriate amount of soil sample was taken out from the column, and it was described in "1. Determination of benzene and naphthalene concentration _" according to the "general experimental method" above. The method is to measure the benzene concentration, and then the measured benzene concentration is multiplied by the weight of the soil sample to obtain the residual benzene content (g) of the soil sample. In addition, the initial benzene content (g) of the soil sample was also calculated in the above manner before the start of the experiment. The above experiment was repeated twice.

The benzene clearance rate (%) is calculated by substituting each benzene content (g) obtained above into the following formula (2): formula (2): D = (EF) / E ×100

Where: D = benzene clearance rate (%)

E = initial benzene content of soil samples containing benzene (g)

F=Residual benzene content of soil samples containing benzene (g)

Further, the benzene degradation rate (%) is calculated by substituting each benzene content (g) obtained above into the following formula (3): Formula (3): G = (EFH) /E×100

Where: G = benzene degradation rate (%)

E = initial benzene content of soil samples containing benzene (g)

F=Residual benzene content of soil samples containing benzene (g)

H = total benzene content of the effluent (g)

B. Test for removing naphthalene from soil samples:

The test for removing naphthalene from soil samples is generally carried out by referring to the methods and procedures described in "A. Test for removing benzene from soil samples" in the "Experimental Methods" above, except that the column is filled. The soil sample containing benzene was replaced by 653.85 g of a naphthalene containing soil sample having a naphthalene concentration of 184.6 mg/kg. Similarly, a total of 15 days were spent throughout the experiment, the effluent was collected daily and its dissolved oxygen, pH and naphthalene content were measured, and the total naphthalene content (g) of the effluent was calculated on the last day of the experiment. In addition, the initial naphthalene content (g) and the residual naphthalene content (g) of the soil sample containing naphthalene are also carried out by referring to the method described in "A. Test for removing benzene in soil samples" of the "Experimental Method" above. Measurement and calculation.

The naphthalene clearance rate (%) is calculated by substituting each naphthalene content (g) obtained above into the following formula (4): Formula (4): I = (JK) / J ×100

Where: I = naphthalene clearance rate (%)

J = initial naphthalene content of soil samples containing naphthalene (g)

K = residual naphthalene content of soil samples containing naphthalene (g)

Further, the naphthalene degradation rate (%) is calculated by substituting each naphthalene content (g) obtained above into the following formula (5): Formula (5): L = (JKM) /J×100

Where: L = naphthalene degradation rate (%)

J = initial naphthalene content of soil samples containing naphthalene (g)

K = residual naphthalene content of soil samples containing naphthalene (g)

M = total naphthalene content of the effluent (g)

result: A. Test for removing benzene from soil samples:

Figure 3 shows the benzene removal rate of P. sphaeroides S03 for soil samples containing benzene in a simulated column apparatus. As can be seen from Fig. 3, the benzene clearance rate measured by the soil sample introduced with Pseudomonas syriata S03 was significantly increased (more than 95% of the results of the experiment). The results of this experiment show that Pseudomonas sinensis S03 can effectively remove benzene present in the soil.

Figure 4 shows Pseudomonas syriata S03 in a simulated column device Benzene degradation rate for soil samples containing benzene. As can be seen from Fig. 4, the benzene degradation rate measured by the soil sample introduced with Pseudomonas syriata S03 was significantly increased compared with the control group (the results of the two experiments were all above 60%). The results of this experiment show that Pseudomonas sinensis S03 has excellent benzene degradation ability.

Based on the results of Figures 3 and 4, the Applicant believes that Pseudomonas sinensis S03 can remove benzene present in the soil by desorption and degradation.

B. Test for removing naphthalene from soil samples:

Figure 5 shows the naphthalene scavenging rate of Pseudomonas syriata S03 in a simulated column apparatus for soil samples containing naphthalene. As can be seen from Fig. 5, the naphthalene scavenging rate measured by the soil sample introduced with Pseudomonas syriata S03 was significantly increased compared with the control group (both of the experimental results reached 72% or more). The results of this experiment show that Pseudomonas sinensis S03 can effectively remove naphthalene present in the soil.

Figure 6 shows the degradation rate of naphthalene for soil samples containing naphthalene in P. syringae S03 in a simulated column apparatus. As can be seen from Fig. 6, the degradation rate of naphthalene measured by the soil sample introduced with Pseudomonas syriata S03 was slightly higher than that of the control group compared with the control group.

By comparing Fig. 5 with Fig. 6, it can be found that the naphthalene scavenging rate of Pseudomonas sinensis S03 is significantly higher than that of naphthalene, which means Pseudomonas sp. S03 is mainly by desorbing naphthalene in soil to water. In phase to achieve the effectiveness of the cleanup.

Based on the above experimental results, the applicant believes that: the present invention Pseudomonas syriata S03 has an excellent emulsifying ability, and can remove benzene and naphthalene present in the soil by desorption and/or degradation, thereby achieving the purpose of biological re-cultivation. Therefore, the Pseudomonas syriata S03 of the present invention has been developed to be used for the removal of crude oil, petroleum refining products (such as diesel and heavy oil), benzene and/or naphthalene present in a contaminated medium, particularly soil. High potential for microbial agents.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

【Biomaterial Storage】 Domestic registration information [please note according to: registration authority, date, number order]

1. Pseudomonas taoyuanensis S03: Center for Bioresource Conservation and Research, Food Industry Development Institute (BCRC of FIRDI); August 29, 2012; BCRC 910562.

Foreign deposit information [please note: ordering country, organization, date, number order]

(no)

<110> Pang Renjie He Yizheng Zhou Jindong Chai Yulan

<120> Pseudomonas sylvestris ( PSEUDOMONAS TAOYUANENSIS ) S03 isolate having emulsifying ability and scavenging ability for benzene and/or naphthalene and use thereof

<130> Pseudomonas syriata S03

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1489

<212> DNA

<213> Pseudomonas sp. S03

<400> 1

<210> 2

<211> 995

<212> DNA

<213> Pseudomonas sp. S03

<400> 2

Claims (16)

A Pseudomonas taoyuanensis S03, which is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration number BCRC 910562. A microbial agent for removing benzene and/or naphthalene present in a contaminated medium comprising Pseudomonas syriata S03 as claimed in claim 1 or a subcultured progeny thereof. A microbial agent for removing crude oil and/or petroleum refining products present in a contaminated medium, comprising Pseudomonas syriata S03 as claimed in claim 1 or a subcultured progeny thereof. The microbial reagent of claim 3, wherein the petroleum refining product is selected from the group consisting of heavy oil, diesel, kerosene, fuel oil, gasoline, hydraulic oil, and lubricating oil. The microbial agent of claim 2 or 3, wherein the contaminated medium is selected from the group consisting of: soil, sludge, sediment, aquifer, water body, and wastewater. The microbial reagent of claim 5, wherein the contaminated medium is selected from the group consisting of: land, orchard land, grazing grassland, woodland, gas station land, industrial land, well water, fish culture pond, Groundwater, river water, lake water, sea water, factory wastewater, domestic sewage, and sludge from sewage treatment plants. The microbial agent of claim 2 or 3, further comprising at least one microorganism capable of scavenging monocyclic and/or polycyclic aromatic hydrocarbons. A microbial reagent according to claim 2 or 3, which is manufactured as one selected from Dosage forms for the following groups: culture solutions, suspensions, granules, powders, lozenges, pills, capsules, and thick pastes. The microbial agent of claim 2 or 3, further comprising a biocompatible carrier. The microbial agent of claim 9, wherein the Pseudomonas putida S03 is captured by the biocompatible carrier. The microbial agent of claim 10, wherein the biocompatible carrier is selected from the group consisting of tannin extract, starch, agar, chitin, chitosan, polyvinyl alcohol, polylactic acid, Alginic acid, polyacrylamide, carrageenan, agarose, gelatin, cellulose, cellulose acetate, polydextrose, and collagen. The microbial agent of claim 9, wherein the Pseudomonas putida S03 is carried on the biocompatible carrier. The microbial agent of claim 12, wherein the biocompatible carrier is selected from the group consisting of glass, ceramic, metal oxide, activated carbon, kaolinite, bentonite, zeolite, aluminum, anthracite , glutaraldehyde, polyacrylic acid, polyurethane, polyvinyl chloride, ion exchange resin, epoxy resin, photoplastic resin, polyester, and polystyrene. A method for removing benzene and/or naphthalene present in a contaminated medium, comprising: treating the contaminated medium with Pseudomonas syriata S03 as claimed in claim 1 or a subcultured progeny thereof And causing the benzene and/or naphthalene present in the contaminated medium to be degraded and/or desorbed by the Pseudomonas syriata S03 or its subcultured progeny. A method for removing crude oil and/or stone present in a contaminated medium A method of refining an oil product, comprising: treating the contaminated medium with Pseudomonas syriata S03 as claimed in claim 1 or a subcultured progeny thereof, such that the crude oil present in the contaminated medium and/or Or the petroleum refining product is emulsified by the Pseudomonas syriata S03 or its subcultured progeny. The method of claim 14 or 15, wherein the contaminated medium is selected from the group consisting of: soil, sludge, sediment, aquifer, water, and wastewater.