TWI384076B - Proteus mirabilis tmr isolate having the abilities to decompose polystyrene and/or styrofoam and uses of the same - Google Patents

Description

Proteus mirabilis TMR isolate having the ability to decompose polystyrene and/or polystyrene foamed plastic and use thereof

The present invention relates to a Proteus mirabilis TMR isolate having the ability to decompose polystyrene and/or polystyrene foamed plastics, which is deposited with the Food Industry Development Institute (FIRDI) under the registration number BCRC 910439. Biological Resource Conservation and Research Center (BCRC). The Proteus mirabilis TMR isolate and its sub-cultured offspring can be used to prepare microbial agents for decomposing polystyrene and/or polystyrene foamed plastics.

Plastic is a product extracted from petroleum. Common plastic types include polystyrene (PS), styrofoam, and polyethylene terephthalate (PET). Polyethylene (PE), polyvinyl chloride (PVC), polyurethane (PU) and polypropylene (PP). Polystyrene {IUPAC designation: poly(1-phenylethane-1,2-diyl) [Poly(1-phenylethane-1,2-diyl)]} is a kind of styrene [it is a kind A polymer of a liquid aromatic hydrocarbon having a chemical formula of (C ₈ H ₈ ) _n .

Polystyrene is a colorless and transparent thermoplastic substance which can be subjected to injection molding, compression molding, extrusion molding, and thermoforming. ) is processed into a variety of different building materials, toys, stationery and tableware.

Polystyrene foamed plastic [also known as polylone] is prepared by adding polystyrene as a blowing agent. In general, polystyrene foamed plastics include: (1) expanded polystyrene foam (EPS), which is commonly used as a cushioning packaging material for packaging articles or has a container for heat insulation; (2) expanded polystyrene (PSP), which is usually used to make disposable dishes, etc.; and (3) extruded polystyrene foam (extruded) Polystyrene foam (XPS), which is commonly used as a thermal insulation material.

Polystyrene and polystyrene foamed plastics have the advantages of low water absorption, thermal insulation, light weight, ease of use, and low cost. Therefore, polystyrene and polystyrene Ethylene foamed plastics have been commonly used in everyday life. However, the excessive use of a large amount of waste generated by polystyrene and polystyrene foamed plastics has caused serious environmental pollution and ecological damage. Currently, known methods for treating wastes of polystyrene and polystyrene foamed plastics include: recycling, burial, and incineration.

In Taiwan, for polystyrene foam used in industrial packaging and building materials, the annual waste can be as high as 12,000 metric tons. Although the recycling rate of polystyrene foamed plastics can reach 67%, polystyrene foamed plastics are easily contaminated during the recycling process, resulting in poor quality. Therefore, more than 4,000 metric tons of polystyrene foamed plastic waste is disposed of in a buried or incineration manner every year. However, polystyrene and polystyrene foamed plastics cannot be decomposed in the general natural environment, so when they are buried in landfills, they will continue to accumulate and remain in the natural environment forever. Furthermore, polystyrene foamed plastics take up a lot of space because of their large size, which in turn makes the life of the landfills shorter.

In addition, if the polystyrene and/or polystyrene foamed plastics are treated by incineration, the temperature of the incinerator usually needs to be as high as 800 ° C to allow them to be completely burned. However, polystyrene and polystyrene foamed plastics have the characteristics of rapid combustion, which can cause oxygen deficient combustion when a large amount of polystyrene and/or polystyrene foamed plastic is incinerated. A large number of carbon monoxide (CO), formaldehyde (HCHO) and "Dixin" are the poisonous gases such as Dioxin, which can cause serious harm to the natural environment and humans.

At present, researchers in the field are working to develop a method for treating polystyrene and/or polystyrene foamed plastics more efficiently and environmentally, wherein the biodegradation using microorganisms is particularly valued.

In PG Ward et al . (2006), Environ. Sci. Technol ., 40:2433-2437, PG Ward et al. disclose a method for converting polystyrene into a biodegradable thermoplastics. The method comprises the following two steps: (1) first, pyrrolysis of polystyrene to styrene oil at 520 ° C; and (2) by Pseudomonas putida CA- 3 ( Pseudomonas putida CA-3) (NCIMB 41162) and bacterial oil is bacterially converse into a biodegradable plastic [also known as polyhydroxyalkanoate (PHA)].

In R. Mor and A. Sivan (2008), Biodegradation , 19: 851-858, R. Mor and A. Sivan mainly monitor biofilm formation of polymorphism of Rhodococcus ruber strain C208 (biofilm). The kinetics of formation and their ability to decompose polystyrene. It was found through experimental results that the Rhodococcus erythropolis strain C208 has high affinity for polystyrene, and thus can adhere to the surface of polystyrene and form a biofilm within a few hours, thereby inducing partial biodegradation of polystyrene.

In O. EISAKU et al . (2003), Proceedings of Environmental Engineering Research , 40: 373-379, O. EISAKU et al . mainly investigated the ability of microorganisms isolated by 16S rDNA analysis to decompose polystyrene. The results showed that Xanthomonas sp. , Sphingobacterium sp. , and Bacillus sp. strain STR-YO can decompose polystyrene.

JEP in JEP Dayao and MBV Egloso, Department of Biology, University of Manila, Philippines [name: "A preliminary study on the potential biodegrading ability of Xylaria sp. on natural rubber, chicken feather, and polystyrene through scanning electron microscopy"], JEP Dayao and MBV Egloso cultured the genus Xylaria sp. in mineral medium [containing 0.5% glucose for carbon source and polystyrene for co-carbon source] After 50 days, polystyrene was taken out and observed by a scanning electron microscope (SEM). The experimental results show that the mycelia of the genus Trichoderma can adhere to and penetrate the polystyrene to form colonization, thereby achieving the effect of decomposing polystyrene.

Although the above literature has been reported, there is still a need in the art to screen for microorganisms that can decompose polystyrene and/or polystyrene foamed plastics for environmental protection.

Rhubarb mealworm (Latin name: Tenebrio molitor ; Chinese common name: bread bug; English name: meatworm) belongs to Arthropoda, Insecta, Coleoptera, Phytophaga , Tenebrionoidea, Tenebrionidae, Tenebrio . Rhubarb mealworm is a completely metamorphosed insect. Its life history has four periods, including: ovum, larva, pupa and imag. The total life cycle of Rhubarb mealworms is about 80 days, and the process of hatching eggs into larvae lasts about 8 to 10 days; then, the larvae peel off once every 10 days, lasting for 45 to 55 days. During this period, the larvae will become cockroaches; finally, it takes about 7 days to become adult worms (Gonguyu, Entomology, National ZTE University Agricultural College Publishing Committee, pp. 541-604, 1992 revised version).

Previously, it was unexpectedly discovered that the commercially available Rhubarb mealworms have the characteristics of eating styrofoam, and many researchers have been working on the reasons why Rhubarb mealworms can use styrofoam as food and complete the life cycle (Chen Hongru et al. (1995), “Breadworm Waste Treatment-Environmental Protection? Pet Feeding?”, the 35th Primary and Secondary School Science Exhibition of the Republic of China, Taipei County Lixi Kunzhong, pp. 181-188; Zhang Yaqing et al (2002), “The Venus of the Styrofoam-Breadworm”, the winning collection of the 42nd Primary and Secondary School Science Exhibition of the Republic of China, Bo'ai National Primary School, Sanmin District, Kaohsiung; and Liu Jiahui et al. (2006), “Live Garbage Truck” , the 46th Primary and Secondary School Science Exhibition of the Republic of China, National Chaozhou High School). However, these studies only focused on the ecological observation of the yellow worm, the detection of the pH value of the digestive tract, and the analysis of the components contained in the feces. How does the yellow worm have decomposed styrofoam and possible decomposition mechanisms? clear.

Proteus mirabilis is a group of Gram-negative facultative anaerobe belonging to the family Enterobacteriaceae and Proteus . The cells are rod-shaped ( Rod-shape), urea (urea) can be used. They are ubiquitous in a variety of animals as well as in the human intestinal tract.

Upon investigation, the applicant unexpectedly discovered a bacterial isolate isolated from the digestive tract of Rhubarb mealworm (which was later named as Proteus mirabilis TMR), which is different in phylogenetically A strain that has been disclosed in the species and has the ability to decompose polystyrene and/or polystyrene foamed plastic. Therefore, this strain is expected to have great potential in the application of polystyrene and/or polystyrene foamed plastics.

Summary of invention

Thus, in a first aspect, the present invention provides a Proteus mirabilis TMR isolate having the ability to decompose polystyrene and/or polystyrene foamed plastics, which is deposited in the food industry under the accession number BCRC 910439. Development Research Institute (FIRDI) Biological Resource Conservation and Research Center (BCRC).

In a second aspect, the present invention provides a microbial agent for decomposing polystyrene and/or polystyrene foamed plastic comprising a Proteus mirabilis TMR isolate as described above or a subcultured progeny thereof.

In a third aspect, the present invention provides a method for decomposing polystyrene and/or polystyrene foamed plastic, comprising using a Proteus mirabilis TMR isolate as described above or a subcultured progeny thereof Decompose polystyrene and / or polystyrene foam plastic.

The above and other objects, features and advantages of the present invention will become apparent from

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.

In order to effectively treat polystyrene and/or polystyrene foam plastics, and to avoid serious damage to the environment and ecology, countries all over the world have invested a lot of manpower and financial resources to find solutions. Separation, due to the large amount of microorganisms that can be propagated, does not produce toxic by-products during the decomposition of polystyrene and/or polystyrene foamed plastics, causes secondary pollution, and can be operated in situ . Screening out microorganisms suitable for use in decomposing polystyrene and/or polystyrene foamed plastics has become an extremely important research and development topic.

In order to screen out microorganisms suitable for supplying polystyrene and/or polystyrene foamed plastics, the applicant separates the bacterial isolates from the digestive tract of the yellow worm, which eats the styrofoam, and obtains the obtained The isolates were subjected to a polystyrene culture test, and a red strain capable of decomposing polystyrene was selected. The red strain was attributed to Proteus mirabilis , which was named " Proteus mirabilis TMR" by the applicant and was deposited on August 3, 2009, under the registration number BCRC 910439. Center for the Conservation and Research of Biological Resources of the Food Industry Development Institute of Taiwan (BCRC of FIRDI).

Applicants have discovered that Proteus mirabilis TMR has the ability to decompose polystyrene and polystyrene foamed plastics (eg, styrofoam). Further, the bacterial isolate can be grown in an aerobic or anaerobic environment and can be widely used.

Based on the above advantageous biological activities, the Proteus mirabilis TMR of the present invention or its subcultured progeny are expected to have potential for decomposing polystyrene and polystyrene foamed plastics. Accordingly, the present invention provides a microbial agent for decomposing polystyrene and/or polystyrene foamed plastic comprising Proteus mirabilis TMR or a subcultured progeny thereof.

As used herein, the term "decomposition" or "decompose" means the metabolically converse transformation of a polymer into a less complex molecule. The degree of decomposition is not particularly limited herein, and both partial decomposition and complete decomposition of the polymer are desirable.

As used herein, the term "polystyrene" means a polymer obtained by polymerizing styrene, or a component containing polystyrene as a main component. The material is copolymerized with a copolymer obtained by another monomer.

As used herein, the term "styrofoam" means a foamed polystyrene product prepared by adding polystyrene as a blowing agent. (foamed polystyrene products). In general, polystyrene foamed plastics include, but are not limited to: (1) expanded polystyrene foam (EPS); (2) expanded polystyrene paper (PSP) And; (3) extruded polystyrene foam (XPS).

The microbial agent according to the present invention may further comprise at least one microorganism capable of decomposing synthetic plastics and/or natural plastics.

As used herein, the term "plastic" means a variety of different complex polymers that are typically produced by polymerization, which can be molded, extruded ( Extruded or cast into a variety of different shapes. According to the present invention, the plastic may be synthetic plastic or natural plastic, and the synthetic plastic includes, but not limited to, polystyrene (PS), polystyrene foam (styrofoam), polyethylene terephthalate ( Polyethylene terephthalate (PET), polyethylene (PE), polyvinyl chloride (PVC), polyurethane (PU) and polypropylene (PP). Natural plastics include, but are not limited to, polylactic acid, polycaprolactone, poly(3-hydroxybutyrate), poly(ethylenedicarboxylate). [poly(ethylene adipate)] and poly(ethylene succinate) [poly(ethylene succinate)].

According to the present invention, the microbial agent may further comprise at least one microorganism selected from the group consisting of decomposable synthetic plastics of the group consisting of: Brevibacillus borstelensis , Rhodococcus ruber , Jane Penicillium simplicissimum YK, Comamonas acidovorans TB-35, Curvularia senegalensis , Fusarium solani , Aureobasidium pullulans , Cladosporium sp. , Pseudomonas chlororaphis , Pseudomonas putida AJ, Ochrobactrum TD, Pseudomonas fluorescens Pseudomonas fluorescens ) B-22, Aspergillus niger , Thermomonospora fusca , Xanthomonas sp. , Sphingobacterium sp. , Spore Bacillus sp. strain STR-YO and woody species ( Xylaria sp .). Preferably, the microorganism capable of decomposing synthetic plastic is selected from the group consisting of Xanthomonas, Sphingomonas, Bacillus sp. STR-YO, and Trichoderma species , Rhodococcus, and combinations thereof.

According to the present invention, the microbial agent may further comprise at least one microorganism capable of decomposing natural plastics selected from the group consisting of: Schlegelella thermodepolymerans , Lehni 's pseudomons Pseudomonas lemoignei , Pseudomonas indica K2, Streptomyces sp. strain SNG9, Rqlstonia pikettii T1, Acidophyte species Sp. ) strain TP4, Alcaligenes faecalis , Pseudomonas stutzeri , Comamonas acidovorans , Caenibacterium thermophilum , Clostridium botulinum , acetone Clostridium acetobutylicum , Fusarium moniliforme , Penicillium roquefort , Amycolatopsis sp ., Bacillus brevis , Dessert Rhizopus (Rhizopus delemer), controversy lust for bacteria (Variovorax paradoxus) LMG 16137 and oats Acidovorax McAlester species (Acidovorax avenae subsp. Avenae) LMG 17238.

The microbial agent according to the present invention can be manufactured into a form suitable for use by techniques well known to those skilled in the art, including, but not limited to, culture solutions, suspensions, granules, powders. , tablets, pills, 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, the Proteus mirabilis TMR 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, alginic acid (alginic). Acid), polyacrylamide, carrageenan, agarose, gelatin, cellulose, cellulose acetate, dextran, and collagen ).

In another preferred embodiment of the invention, the Proteus mirabilis TMR 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 (zeolite). ), aluminum, anthracite, glutaraldehyde, polyacrylic acid, polyurethane, polyvinyl chloride, ion exchange resin ), epoxy resin, photosetting resin, and polyester.

The microbial agent according to the present invention can also be fabricated into a bioreactor or apparatus for decomposing polystyrene and/or polystyrene foamed plastics 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 present invention also provides a method for decomposing polystyrene and/or polystyrene foamed plastic, comprising: decomposing polystyrene and/or using a Proteus mirabilis TMR as described above or a subcultured progeny thereof Or polystyrene foam plastic.

In the method according to the invention, the Proteus mirabilis TMR or its subcultured progeny can be used in combination with at least one microorganism which decomposes synthetic plastic and/or natural plastic.

According to the method of the present invention, the Proteus mirabilis TMR or its subcultured progeny can be used in combination with at least one microorganism selected from the group consisting of decomposable synthetic plastics in the group consisting of: native cellulose hydrolyzate, reddish Cocci, Penicillium YK, Trichophyton TB-35, Curvularia senegal, Fusarium solani, Aureobasidium, Mycobacterium spp., Pseudomonas aeruginosa, odor Pseudomonas AJ, C. pallidum TD, Pseudomonas fluorescens B-22, C. sphaeroides, S. thermomonas, Xanthomonas species, Sphingomonas species, Bacillus sp. strain STR -YO and wood species. Preferably, the microorganism capable of decomposing synthetic plastic is selected from the group consisting of Xanthomonas, Sphingomonas, Bacillus sp. STR-YO, and Trichoderma species , Rhodococcus, and combinations thereof.

According to the method of the present invention, the Proteus mirabilis TMR or its subcultured progeny can be used in combination with at least one microorganism capable of decomposing natural plastics selected from the group consisting of: Streptomyces thermophilic polymer decomposition Bacillus, Pseudomonas elegans, Pseudomonas syringae K2, Streptomyces species strain SNG9, R. stellaria T1, Phytophthora species strain TP4, Alcaligenes faecalis, S. pseudomonas Phytophthora , C. faecalis, Caenibacterium thermophilum , Botox, Clostridium acetobutylicum, Fusarium oxysporum, Penicillium cholei , Mycobacterium species, Bacillus brevis, Rhizopus genus Controversy about Helminthosporium LMG 16137 and Oats oleaginous oats subspecies LMG 17238.

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 Experimental Materials:

1. The larvae of Tenebrio molitor used in the following examples were purchased from Taichung City.

Luria used in Example 2. The following examples - Bell Thani (LB) broth medium [Luria-Bertani (LB) broth ] (AthenaES TM) having the formulations shown in Table 1 below.

3. The LB agar medium used in the following examples was prepared by adding agar (Amresco) (15 g/L) to the LB broth.

4. The inorganic synthetic (IS) broth medium [inorganic synthetic (IS) broth] used in the following examples has the formulation shown in Table 2 below.

5. The polystyrene-IS broth medium used in the following examples was prepared by adding polystyrene (1 g/L) to the IS broth medium.

6. The polystyrene used in the following examples is a chemical material purchased from Taichung City; the polylone plate is a stationery store purchased from Taichung City; and the oatmeal is oatmeal. Purchased from Quaker Food Co., Ltd.

7. The styrofoam disc (having a diameter of about 7 mm and a thickness of about 0.5 mm) used in the following examples was produced by cutting the styrofoam plate using a punch and a blade.

8. Escherichia coli used in the following examples was purchased from the Center for Bioresource Conservation and Research (BCRC of FIRDI) of the Food Industry Development Institute of Taiwan.

Example 1. Polyllone feeding test of larvae of Rhubarb mealworms experimental method:

In order to confirm that the larvae of the yellow worm can grow into adult worms only by eating the styrofoam, the larvae of the yellow worm (10 days old, weighing about 0.064 g) were randomly divided into three groups (n=45 per group). ), including 2 experimental groups (ie, oatmeal group and styrofoam group) and 1 control group. The larvae of each group of Rhododendron chinensis were separately dispensed into 3 ventilated hard plastic boxes (n=15 per box) and kept in water under a ventilated environment at a temperature of 25 °C.

For the oatmeal group and the styrofoam group, the larvae of the rhododendron were fed with oatmeal (2 g/box/day) and styrofoam board (0.08 g) in addition to a small amount of deionized water. / box / day) lasted 20 days. As for the larvae of the control group of the yellow worm, they were fed with a small amount of deionized water. During the experiment, the survival number of each group of Rhododendron chinensis was counted every 10 days and weighed, and then the average body weight of each group of Rhododendron chinensis was calculated, wherein when the number of survival was counted, the sputum of Rhubarb mealworm was Pupae) was included in the count, and in the calculation of the average body weight, the cockroaches of the yellow worm and the dead worm were excluded.

result:

Figures 1 and 2 show the number of live and the average body weight of Rhubarb mealworms over time in the oatmeal group, the styrofoam group, and the control group, respectively. As can be seen from Fig. 1, when the day 10 was reared, the yellow worm of the control group had all died, while 38 of the styrofoam group and the oatmeal group survived, respectively, and 35 of the yellow worms. In addition, when feeding on the 20th day, 32 and 25 Rhubarb mealworms survived in the styrofoam group and the oatmeal group respectively. The survival rate of the Rhododendron chinensis in the styrofoam group was higher than that in the oatmeal group. By. This result shows that Rhubarb mealworms cannot be grown only by the nutrients originally stored in the body, but the nutrients needed for growth can be obtained by eating styrofoam plates or oatmeal.

In addition, as can be seen from Fig. 2, when the day 10 was raised, the average body weight of the styrofoam group and the oatmeal group was almost the same as that on the day 0; On the 20th day, the average body weight of the yellow worm in the styrofoam group did not change much, while the average body weight of the yellow worm in the oatmeal group increased significantly.

Example 2. Isolation of bacterial isolates from digestive tracts of the yellow mealworm fed with oatmeal or styrofoam plates

From the oatmeal group of Example 1 above and the styrofoam group, one of the rhubarb mealworms that had completed the feeding test was taken out, and they were immersed in 70% alcohol for 2 minutes to disinfect the surface of the insect body, followed by sterilizing. The sterile water was washed a total of 2 times to remove the residual alcohol on the surface of the worm. Thereafter, in the aseptic table, the digestive tracts of the yellow mealworms were taken out and placed in a sterile micro-plastic centrifuge tube, followed by the addition of 100 μL of sterile water and stirred to form a mixture. Subsequently, 50 μL of such a mixture and the mixture were taken so as to spread plate method (spread plate method) are applied on LB agar medium, and then ^{(GasPak TM, BBL) (37} ℃) within the cylinder in an anaerobic Anaerobic culture lasted for 4 to 5 days. Thereafter, colony growth on each LB agar medium was observed with the naked eye.

From the observation of the naked eye, it was found that the microorganisms isolated from the digestive tract of the Rhododendron chinensis in the styrofoam group mainly formed red colonies on the LB agar medium, and the white colonies were few. However, the microorganisms isolated from the digestive tract of the rhubarb mealworm of the oatmeal group mainly formed white colonies on the LB agar medium, and the red colonies were few.

In order to confirm that the red and white colonies on the LB agar medium were formed by a single bacterial isolate, one red colony and one white colony separated from the digestive tract of the Phyllostachys pubescens group were four. The four-quadrant streak method was applied to LB agar medium, respectively, and anaerobic culture was carried out at 37 ° C for 4 to 5 days. The experimental results showed that the colonies obtained after subculture of red and white colonies still had the same characteristics as the original colonies on the LB agar medium, indicating the red color separated from the digestive tract of the rhubarb mealworm. Both white colonies are formed from a single bacterial isolate.

This result shows that the type of food affects the microflora of the rhubarb mealworm's digestive tract. When the styrofoam plate is used as the sole source of nutrients, it will cause red colonies on the LB agar medium. The isolate is predominant, so the applicant concludes that the isolate may be associated with the decomposition of styrofoam.

Example 3. Polystyrene culture test of bacterial isolates isolated from the digestive tract of Rhubarb mealworm experimental method: A. Preparation of E. coli inoculum, red strain inoculum and white strain inoculum:

Escherichia coli and the red strain obtained in the above Example 2 and the white strain were separately inoculated into the LB broth medium, and subjected to anaerobic culture overnight in a constant temperature shaking incubator (37 ° C, 160 rpm). . The resulting E. coli culture, red strain culture, and white strain culture were each centrifuged at 8,000 rpm for 10 minutes, and then the supernatant was removed. The remaining precipitates were washed twice with sterile water and then dispersed with an appropriate amount of sterile water, thereby obtaining an E. coli inoculum having a cell density of about 0.06 (OD ₆₀₀ ), respectively. Strain inoculum and white strain inoculum.

B. Polystyrene culture test of Escherichia coli, red strain and white strain:

10 mL of LB broth medium, IS broth medium, and polystyrene-IS broth medium were separately added to an anaerobic culture tube containing a plastic screw cap. Next, each test tube was exposed to nitrogen for 10 minutes to drive off oxygen, and immediately the plastic screw cap was screwed, and then sterilized at a temperature of 121 ° C and a pressure of 15 lb / in ² for 20 minutes. When the temperature of the anaerobic culture tube was lowered to room temperature, 0.1 mL of the red strain inoculum obtained in the above item A was inoculated into each test tube containing different medium, and in a constant temperature shaking incubator (37 ° C, Anaerobic culture was carried out for 7 days in 160 rpm). Thereafter, the growth of the red strain in each test tube was observed with the naked eye. Next, 0.1 mL of the bacterial solution was taken out from each tube using a 1 mL syringe (Becton Dickinson, Singapore) and the bacterial cells were sterilized by small glass beads using a spread plate method. were applied on LB agar medium, and then cultured in an anaerobic tank anaerobically ^{(GasPak TM, BBL) (37} ℃) inside for 7 days. Thereafter, the growth of the red strain on the LB agar medium was observed. In addition, the E. coli inoculum obtained in the above item A and the white strain inoculum were also used for the same experiment, respectively. The experiment was repeated 3 times.

result:

From three experimental results, it was found that Escherichia coli, red strain, and white strain can all grow in the more nutrient LB broth medium, but cannot grow in the IS broth medium lacking the carbon source. In addition, only red strains can be grown in polystyrene-IS broth medium. The results of this experiment show that the red strain obtained in the above Example 2 can be decomposed using polystyrene to obtain nutrients required for growth.

Example 4. Characterization of red strains

In order to confirm the species of the red strain obtained in the above Example 2, the following preliminary test and 16S rDNA sequence analysis were carried out.

experimental method: A. Preliminary test:

Preliminary tests were carried out on red strains, including: gram staining, morphological observation using optical microscopes or scanning electron microscopes (SEM), and mobility (mobility) )Wait.

B, 16S rDNA sequence analysis:

Genomic DNA extraction of the red strain was performed using the Genomic DNA Mini Kit (Geneaid). First, the red strain was inoculated into 10 mL of LB broth medium and subjected to anaerobic incubation overnight at 37 °C. Next, 3 mL of the bacterial solution was taken out and placed in a plastic centrifuge tube, followed by centrifugation at 12,000 rpm for 5 minutes. After removing the supernatant, 200 μL of GT buffer was added and shaken to disperse the cells, and then left to stand at room temperature for 5 minutes. Afterwards, 200 μL of GB buffer was added and shaken for 5 minutes. The resulting mixture was placed in a 70 ° C water bath for 10 minutes and the tube was inverted every 3 minutes during this time. Thereafter, 200 μL of absolute alcohol was added to the mixture and shaken for 10 seconds. If a precipitate formed, the mixture was repeatedly flushed with a micropipette to break up the precipitate. Next, a GD column was placed in a 2 mL collection tube, and all the mixture (including the precipitate) was transferred to the GD column, followed by centrifugation at 12,000 rpm for 5 minutes. Thereafter, the GD column was placed in a new 2 mL collection tube, 400 μL of W1 buffer was added, and then centrifuged at 12,000 rpm for 30 seconds. After removing the effluent, the GD column was placed back into the collection tube, 600 μL of wash buffer was added, and centrifugation was carried out at 12,000 rpm for 30 seconds. After removing the effluent, the GD column was placed back into the collection tube and centrifuged at 12,000 rpm for 3 minutes. Finally, the GD column was placed in a new microcentrifuge tube, and then 100 μL of elution buffer (preheated to 70 ° C) was added to the center of the GD column and allowed to stand. The genomic DNA of the red strain was eluted by centrifugation for 3 to 5 minutes, followed by centrifugation at 12,000 rpm for 30 seconds.

The following table is used by using the obtained genomic DNA as a template and using a set of 16S rRNA genes directed against bacteria to design a universal primer pair F1 and R1 having the nucleotide sequences shown below. Polymerase chain reaction (PCR) of the reaction conditions shown in 3, wherein PCR was performed in GeneAmp PCR System 2400 (Perkin Elmer).

F1 primer

5'-gccacgagccgcggt-3' (sequence identification number: 1)

R1 primer

5'-acgggcggtgtgtac-3' (sequence identification number: 2)

After the completion of the PCR, it was confirmed by agarose gel electrophoresis whether or not a PCR amplification product having a size of about 900 bp was obtained, and the confirmed PCR product was purified from the gel recovery. The purified PCR product was commissioned by the Biotechnology Center of ZTE University (Taiwan, Taichung) for sequencing, and the resulting sequencing result was the use of nucleotide-nucleotide BLAST (nucleotide-nucleotide BLAST) in the NCBI website. The software is used for comparison analysis.

result:

According to the preliminary test results, the red strain obtained in the above Example 2 was a Gram-negative bacterium, and was observed to have a rod shape (a diameter of about 0.4-0.6 μm and a length of about 1.0) under an optical microscope and a scanning electron microscope. -2.0 μm), sporty. In addition, the 16S rDNA sequence analysis result of this red strain is shown in Fig. 3, and after comparison with the gene database in the NCBI website, the 16S rDNA sequence of this strain (SEQ ID NO: 3) and Proteus mirabilis were found. ( Proteus mirabilis ) has up to 98% identity between the 16S rDNA sequences.

Based on the above characterization results, the red strain obtained in the above Example 2 is considered to be a novel isolate of Proteus mirabilis, which was named "Proteus mirabilis TMR" by the applicant and was in 2009. On August 3, the Biosource Collection and Research Center (BCRC) was hosted at the Food Industry Research and Development Institute (FIRDI) in Taiwan under the registration number BCRC 910439 (300 Hsinchu Foods) Road No. 331, Taiwan).

Example 5. Comparative analysis of the characteristics of Proteus mirabilis TMR and conventional strains

In order to prove that the Proteus mirabilis TMR has characteristics different from the conventional Proteus mirabilis strain, in the following examples, it was taken with a conventional strain [i.e., purchased from the American Type Culture Collection Center (American). Characterization of Proteus mirabilis ATCC 29906 by Type Culture Collection, ATCC).

experimental method:

Preliminary experiments were carried out on Proteus mirabilis TMR and Proteus mirabilis ATCC 29906. The experimental items included: LB agar growth test under aerobic and anaerobic conditions, McConnex agar growth test under aerobic environment and Iraq Eosin-methylene blue agar growth test, glucose fermentation test, lactose fermentation test, sucrose fermentation test, citrate utilization test, H ₂ S production test, indole production test , methyl red test, Voges Proskauer test, amylase activity test, protease activity test, lipase activity test, gelatinase activity test And catalase activity test and the like.

result:

Preliminary test results for Proteus mirabilis TMR and Proteus mirabilis ATCC 29906 are shown in Table 4 below. As can be seen from Table 4, the difference between Proteus mirabilis TMR and Proteus mirabilis ATCC 29906 is:

(1) Proteus mirabilis TMR was cultured on LB agar under aerobic conditions to form beige colonies and no swarming motility; and Proteus mirabilis ATCC 29906 was cultured on LB agar under aerobic conditions. Will form white colonies, and there are swimming phenomena;

(2) Proteus mirabilis TMR is cultured on LB agar under anaerobic environment to form red colonies, and there is no migration phenomenon; and Proteus mirabilis ATCC 29906 is cultured on LB agar under anaerobic environment to form white colonies And there is a swimming phenomenon;

(3) Proteus mirabilis TMR could not grow on MacConkey agar under aerobic environment, and the growth on eosin-methylene blue agar was slow;

(4) Proteus mirabilis TMR does not produce gas after using glucose;

(5) Proteus mirabilis TMR cannot utilize lactose and citrate;

(6) Proteus mirabilis TMR cannot utilize organic or inorganic sulfur-containing compounds to produce H ₂ S;

(7) Both the protease activity test and the catalase activity test of Proteus mirabilis TMR were negative.

Based on the above characteristic alignment results, it is known that the Proteus mirabilis TMR of the present invention is different from the conventional strain of the species to which it belongs.

Example 6. Polystyrene growth test of Proteus mirabilis TMR in aerobic or anaerobic environment experimental method: A. Preparation of Proteus mirabilis TMR inoculum:

The Proteus mirabilis TMR was inoculated into 10 mL of LB broth medium and subjected to anaerobic incubation overnight in a constant temperature shaking incubator (37 ° C, 160 rpm) to obtain Proteus mirabilis TMR inoculum.

B. Growth test of Proteus mirabilis TMR:

100 mL of LB broth and polystyrene-IS broth were separately added to common flasks. In addition, 100 mL of LB broth and polystyrene-IS broth were separately added to the anaerobic flask, and then each anaerobic flask was exposed to nitrogen for 15 minutes to drive off the oxygen, and then immediately tighten the cap. . Next, the ordinary culture flasks and the anaerobic culture flasks were sterilized at a temperature of 121 ° C and a pressure of 15 lb / in ² for 20 minutes. When the temperature of the flasks was lowered to room temperature, 1 mL of the Proteus mirabilis TMR inoculum obtained in item A above was inoculated into each flask and placed in a constant temperature shaking incubator (37 ° C, 160). The culture was carried out in rpm for 24 hours. During the culture process, 0.1 mL of the bacterial liquid was taken out from each culture flask every 4 hours, and the bacterial liquids were separately subjected to 10-fold serial dilution to prepare tests with different concentrations. Bacteria. 0.1 mL of 10 ⁵ , 10 ⁶ , 10 ⁷ and 10 ⁸ dilutions of the test bacterial solution were taken out and uniformly applied to LB agar medium, and cultured at 37 ° C for 3 days, and the number of colonies was observed. The amount of Proteus mirabilis TMR contained in the original bacterial solution is converted according to the dilution factor. The experiment was repeated twice.

result:

Figure 4 shows the growth curve of Proteus mirabilis TMR in LB broth medium and polystyrene-IS broth medium under aerobic or anaerobic conditions. As can be seen from Fig. 4, when cultured in LB broth medium, the growth of Proteus mirabilis TMR in aerobic or anaerobic environment is not much different, and when cultured in polystyrene-IS broth medium, singularity Proteobacteria TMR grows better in an aerobic environment than in an anaerobic environment. In addition, Proteus mirabilis TMR grows better in LB broth medium than in polystyrene-IS broth medium, whether in an aerobic or anaerobic environment. The results of this experiment show that Proteus mirabilis TMR can grow under aerobic or anaerobic conditions.

Example 7. Evaluation of the ability of Proteus mirabilis TMR to decompose styrofoam experimental method: A. Preparation of Proteus mirabilis TMR inoculum:

Proteus mirabilis TMR was inoculated into 400 mL of LB broth medium and aerobic culture was performed overnight in a constant temperature shaking incubator (37 ° C, 160 rpm). The resulting culture was centrifuged at 8,000 rpm for 10 minutes, and then the supernatant was removed. The remaining precipitate was washed twice with sterile water and then the precipitate was sprinkled with sterile water to form a Proteus mirabilis TMR inoculum having a volume of 40 mL.

B. Evaluation of the decomposition ability of the styrofoam:

Three styrofoam disks sterilized by UV light irradiation were separately added to four sterilized and nitrogen-filled anaerobic culture tubes containing 10 mL of IS broth medium. Next, 1 mL of the Proteus mirabilis TMR inoculum obtained in the above item A was inoculated into each test tube, and subjected to anaerobic culture in a constant temperature shaking incubator (37 ° C, 160 rpm), at 0, At 24, 48, and 72 hours, one tube was taken out, and the trinocular biological microscope (Zoomkop T-KOP, Laid Technology Co., Ltd.) was used to observe the decomposition of the styrofoam disc.

Knot fruit:

Figure 5 shows the situation in which Proteus mirabilis TMR decomposes the styrofoam disc in IS broth medium at different culture times. As can be seen from Fig. 5, the decomposed area gradually becomes larger as the culture time increases. In particular, at the 72nd hour of the culture, the 1/3 to 1/2 area of the styrofoam disc has been break down. The results of this experiment show that Proteus mirabilis TMR can decompose and use styrofoam to obtain the nutrients needed for growth.

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.

<110> National Chung Hsing University

National Tsinghua University

<120> Proteus mirabilis TMR isolate having the ability to decompose polystyrene and/or polystyrene foamed plastic and use thereof

<130> TMR

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Universal forward primer F1 for PCR amplification of bacterial strain 16S rRNA gene

<210> 2

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Universal reverse primer R1 for PCR amplification of the 16S rRNA gene of bacterial strains

<400> 2

<210> 3

<211> 847

<212> DNA

<213> Proteus mirabi1is TMR

<400> 3

Figure 1 shows the survival of Rhubarb mealworms over time in the oatmeal group, the styrofoam group, and the control group;

Figure 2 shows the average body weight of Rhubarb mealworms over time in the oatmeal group, the styrofoam group, and the control group;

Figure 3 shows the 16S rDNA nucleotide sequence of the Proteus mirabilis TMR of the present invention;

Figure 4 shows the growth curve of Proteus mirabilis TMR in LB broth medium and polystyrene-IS broth medium under aerobic or anaerobic conditions;

Figure 5 shows the situation in which Proteus mirabilis TMR decomposes the styrofoam disc in IS broth medium at different culture times.

Claims (17)

A Proteus mirabilis TMR, deposited at the Center for Bioresource Conservation and Research of the Food Industry Development Institute under the accession number BCRC 910439. A microbial agent for decomposing polystyrene and/or polystyrene foamed plastic, comprising a Proteus mirabilis TMR as claimed in claim 1 or a subcultured progeny thereof. The microbial agent of claim 2, further comprising at least one microorganism capable of decomposing synthetic plastic and/or natural plastic. The microbial reagent of claim 3, wherein the microorganism capable of decomposing the synthetic plastic is selected from the group consisting of: Brevibacillus borstelensis , Rhodococcus ruber , simple blue Penicillium simplicissimum YK, Comamonas acidovorans TB-35, Curvularia senegalensis , Fusarium solani , Aureobasidium pullulans , Cladosporium sp. , Pseudomonas chlororaphis , Pseudomonas putida AJ, Ochrobactrum TD, Pseudomonas Fluorescens ) B-22, Aspergillus niger , Thermomonospora fusca , Xanthomonas sp. , Sphingobacterium sp. , Bacillus Bacillus sp. strain STR-YO and Xylaria sp. The microbial agent of claim 4, wherein the microorganism capable of decomposing the synthetic plastic is selected from the group consisting of a strain of Xanthomonas, a species of Sphingomonas, a strain of Bacillus species STR-YO, Trichoderma species, Rhodococcus, and combinations thereof. The microbial reagent of claim 3, wherein the biodegradable microorganism is selected from the group consisting of Schlegelella thermodepolymerans , Pseudomonas elegans ( Pseudomonas lemoignei ), Pseudomonas indica K2, Streptomyces sp. strain SNG9, Ralstonia pikettii T1, Acidophyte sp . strain TP4, Alcaligenes faecalis , Pseudomonas stutzeri , Comamonas acidovorans , Caenibacterium thermophilum , Clostridium botulinum , Acetone Clostridium acetobutylicum , Fusarium moniliforme , Penicillium roquefort , Amycolatopsis sp. , Bacillus brevis , Dezhigen Rhizopus delemer , Variovorax paradoxus LMG 16137, and oats acid oats subspecies ( Ac Idovorax avenae subsp. avenae ) LMG 17238. The microbial agent of claim 2, which is manufactured in a dosage form selected from the group consisting of a culture solution, a suspension, a granule, a powder, a tablet, a pill, a capsule, and a thick slurry. The microbial agent of claim 2, further comprising a biocompatible carrier. The microbial agent of claim 8, wherein the Proteus mirabilis TMR is captured by the biocompatible carrier. The microbial agent of claim 9, wherein the biocompatible carrier is selected from the group consisting of tannin extract, starch, agar, chitin, chitosan, polyvinyl alcohol, Alginic acid, polyacrylamide, carrageenan, agarose, gelatin, cellulose, cellulose acetate, polydextrose, and collagen. The microbial agent of claim 8, wherein the Proteus mirabilis TMR is carried on the biocompatible carrier. The microbial agent of claim 11, wherein the biocompatible carrier is selected from the group consisting of glass, ceramics, metal oxides, activated carbon, kaolinite, bentonite, zeolite, Aluminum, anthracite, glutaraldehyde, polyacrylic acid, polyurethane, polyvinyl chloride, ion exchange resins, epoxy resins, photoplastic resins, and polyesters. A method for decomposing polystyrene and/or polystyrene foamed plastic, comprising: using a Proteus mirabilis TMR as in claim 1 or a subcultured progeny thereof to decompose polystyrene and/or Polystyrene foam plastic. The method of claim 13, wherein the Proteus mirabilis TMR or its subcultured progeny can be used in combination with at least one microorganism that decomposes synthetic plastic and/or natural plastic. The method of claim 14, wherein the Proteus mirabilis TMR or a subcultured progeny thereof can be used in combination with at least one microorganism selected from the group consisting of decomposable synthetic plastics: a native fiber Hydrolysate, Rhodococcus, Penicillium YK, Trichophyton TB-35, Curvularia sinensis, Fusarium solani, Aureobasidium, Mycobacterium spp., Green needle Phytophthora, Pseudomonas aeruginosa AJ, C. pallidum TD, Pseudomonas fluorescens B-22, C. sphaeroides, S. thermophilus, Xanthomonas species, Sphingomonas species, spores Bacillus sp. strain STR-YO and wood fungus species. The method of claim 15, wherein the microorganism capable of decomposing the synthetic plastic is selected from the group consisting of: a Xanthomonas species, a Sphingomonas species, a Bacillus sp. strain STR -YO, Trichoderma species, Rhodococcus, and combinations thereof. The method of claim 14, wherein the Proteus mirabilis TMR or a subcultured progeny thereof can be used in combination with at least one microorganism capable of decomposing natural plastics selected from the group consisting of: Shi Thermophilic polymer-decomposing bacteria, Pseudomonas elegans, Pseudomonas syringae K2, Streptomyces species strain SNG9, R. stellaria T1, Acidophilus species strain TP4, Alcaligenes faecalis , Pseudomonas stutzeri, Pseudomonas acidophilus, Caenibacterium thermophilum , Botox, Clostridium acetobutylicum, Fusarium oxysporum, Penicillium rosenbergii , Mycobacterium species, Bacillus brevis Rhizopus genus, Arguemophilus LMG 16137 and Oats oleaginous oats subspecies LMG 17238.