KR20170031958A

KR20170031958A - Method of Degradation of Low Rank Coal by Arthrobacter sp., and Method of Producing Energy Source Using the Same

Info

Publication number: KR20170031958A
Application number: KR1020150129583A
Authority: KR
Inventors: 박시재; 요키미코 데이비드; 케이 앤 바리투고; 메리 그레이스 베일런; 채철기; 김유진; 김해정
Original assignee: 명지대학교 산학협력단
Priority date: 2015-09-14
Filing date: 2015-09-14
Publication date: 2017-03-22

Abstract

The present invention relates to a method for decomposing low-grade coal using Arthrobacter sp. , And a method for producing an energy source using the same, and more particularly, to a method for producing Arthrobacter sp. And converting the coal to a dissolved and degraded product. The present invention also relates to a method for decomposing coal using an Arthrobacter sp . The present invention also relates to a method for producing a fermented soybean which comprises culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product; And a method for producing an energy source by decomposition of coal using an Arthrobacter sp. , Comprising obtaining the converted product as an energy source.
The decomposition method of coal using the Arthrobacter sp. According to the present invention can perform the decomposition process of coal under milder conditions than the chemical treatment method and can be carried out by using low-grade coal as a raw material and using alcohol, ammonia, hydrogen, methane Can be safely obtained through a decomposition reaction and the emission of harmful gases such as carbon dioxide and nitrous oxide and sulfoxide can be reduced.

Description

TECHNICAL FIELD The present invention relates to a method of decomposing a low-grade coal using an Asobacter strain, and a method of producing the same,

The present invention relates to a method of decomposing low-grade coal using Arthrobacter sp. And a method of producing an energy source using the same, and more particularly, to a method of producing Arthrobacter sp. And converting the coal to a dissolved and degraded product. The present invention also relates to a method for decomposing coal using an Arthrobacter sp . The present invention also relates to a method for producing a fermented soybean which comprises culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product; And a method for producing an energy source by decomposition of coal using an Arthrobacter sp. , Comprising obtaining the converted product as an energy source.

Coal is a lignin-derived polymer, and plant debris is condensed and accumulated at high temperature and high pressure. The rank of coal is divided according to the degree of polymerization, peat is the lowest level coal, followed by lignite, bituminous coal, bituminous coal and anthracite. According to this classification, lignite is a low grade coal which has lower aromatics and low carbon content, but has a relatively high hydrophilicity and porosity, which is suitable for any pretreatment compared to other types of coal.

Since the coal polymer is not well reacted and is hydrophobic, the chemical process of the coal is limited to severe and extreme conditions, releasing toxic substances to the outside. Higher grade coal has energy inside and it is possible to produce coke. However, non-pure lignite or low-grade coal is not suitable for use as a fuel. Particularly, low-grade coal has high moisture content and therefore low calorific value. In addition, as the cost of handling and storage increases, other processes are needed for the proper utilization of low grade coal.

The complex carbonaceous structure of low grade coal is a good candidate for feedstocks for the production of aromatic, aliphatic such as methanol, hydrogen, corrosive acid, and ammonia, which are industrially important compounds. Pyrolysis of coal and coal tar for the production of aromatic chemical feedstocks and gasification for syngas production are currently used coal processing technologies, which usually involve the release of harmful substances. Current techniques for gasifying or liquefying coal often cause equipment corrosion, contamination, and boiler deposits. In addition, harmful metal oxides can occur during the combustion process of coal. Therefore, it is necessary to develop a technology that can clean the process more efficiently and utilize the low-grade coal efficiently.

As an example of a bioprocess that can replace a chemical process, there is a microbial process. That is, the components contained in the extracellular matrix of the microorganism can be used to dissolve and decompose coal, and to obtain a useful energy source therefrom.

The components contained in the extracellular matrix involved in the solubilization and liquefaction of the coal include hydrolytic enzymes such as esterase or lipase and manganese peroxidase, lignin peroxidase, lacase or tyrosine (Fakoussa RM et al., Appl. Microbiol Biotechnol., 52 (1): 25-40, 1999) , and the like, as well as chelating agents, surfactants and alkaline substrates.

Conventional studies have shown that fungi such as Phanerochaetes chrysosporium and Trichoderma sp. Produce extracellular matrix and secrete it out of the cell, thereby reducing the possibility of biodegrading the coal (European patent registration EP0278973). It has also been found that some bacteria also act as molds to decompose coal (US Patent Application 4914024; Valero N et al. , Braz J Microbiol., 45 (3): 911-8, 2014). Considering industrial applications, bacteria that are easier to handle than molds and have shorter incubation periods are more advantageous (US Patent 6143534; US Patent 5670345). Therefore, for the development of an ideal biological process that can replace chemical processes, it is necessary to find bacteria that have the potential to decompose coal.

Arthrobacter sp. Is a gram-positive aerobic strain that lives in soil or sludge. Coal is used as a carbon source. In particular, the strain is reported to be capable of bioremediation of aromatic compounds . The strain was cultivated in soil (Dussan, Jenny et al., J Bioproces Biotechniq., USA) in ammonium carbonate fuel oil in an open-cast coal mine at Dara Adam Khel coal mine, 02.04, 2012) and weathered low-grade coal.

Under these technical backgrounds, the present inventors have made intensive efforts to develop a method of safely decomposing coal and a method of producing an energy source through the decomposition method. As a result, Arthrobacter sp. And the thus-converted product can be obtained as an energy source, thereby completing the present invention.

It is an object of the present invention to provide a method for decomposing low-grade coal using Arthrobacter sp. And a method for producing an energy source using the same.

In order to achieve the above object, the present invention as comprising the steps of culturing in containing the the bakteo strain (Arthrobacter sp.) Of coal as Aspergillus medium conversion of coal to the melting and the decomposition products, asbestos bakteo strain (Arthrobacter sp The present invention also provides a method of decomposing coal using the above method.

The present invention also relates to a method for producing a fermented soybean which comprises culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product; And obtaining the converted product as an energy source . The present invention also provides a method for producing an energy source by decomposition of coal using an Arthrobacter sp .

The decomposition method of coal using the Arthrobacter sp. According to the present invention can perform the decomposition process of coal under milder conditions than the chemical treatment method and can be carried out by using low-grade coal as a raw material and using alcohol, ammonia, hydrogen, methane Can be safely obtained through a decomposition reaction and the emission of harmful gases such as carbon dioxide and nitrous oxide and sulfoxide can be reduced.

Figure 1 shows the dissolution activity for low grade coal by Arthrobacter sp. At 450 nm wavelength.
Fig. 2 shows the dissolution activity for low-grade coal by Arthrobacter sp. At a wavelength of 513 nm.

The present invention relates to a method for decomposing low-grade coal by Arthrobacter sp. And a method for producing an energy source using the same. Particularly, the coal melting product derived from Arthrobacter sp. Can be used as a substrate for culturing industrially versatile microorganisms.

Arthrobacter sp. Can be isolated from soil or sludge and can degrade aromatic pollutants, especially chlorinated aromatics.

It has been confirmed in the present invention that Arthrobacter sp. Can be cultured in a medium containing low-grade coal to convert the coal to a dissolved and degraded product, and the converted product can be obtained as an energy source.

In one embodiment of the present invention, Arthrobacter sp. Can be cultured in a synthetic medium containing low-grade coal.

Another embodiment of the present invention is directed to a method of inhibiting the growth of a plant which comprises an extracellular matrix involved in solubilization and degradation of coal contained in a synthetic medium inoculated with Arthrobacter sp. The activity of the hydrolytic enzyme esterase or lipase and manganese peroxidase, lignin peroxidase, lacase or tyrosinase, which is an oxidase enzyme, was verified by the chelator, and the drop-plate The presence of biosurfactants according to the test was confirmed.

In another embodiment of the present invention, when an E. coli WL3110 strain is inoculated into a supernatant of a culture derived from an Arthrobacter sp. And cultured, the strain derived from the above-mentioned Arthrobacter sp. The carbon degradation product contained in the cultures was used as a carbon source (carbon substrate), indicating a slight increase in the growth of E. coli WL3110 strain

Thus, in one aspect, the present invention relates to a method for producing Arthrobacter sp. , Which comprises culturing Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product . The present invention also relates to a method for decomposing coal using the above method.

In another aspect, the present invention relates to a method for producing a fermented soybean which comprises culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product; And a method for producing an energy source by decomposition of coal using an Arthrobacter sp. , Comprising obtaining the converted product as an energy source.

In the present invention, the coal may be a low rank coal having a low carbon source content, and the average particle size of the coal may be 50 to 75 μm, Semi-solid, and the coal may be contained in the medium in a weight ratio of 0.1% to 10%.

In the present invention, the strain may be characterized by releasing at least one or more of the extracellular matrix from the group consisting of a hydrolytic enzyme, an oxidase, a chelator, a surfactant and an alkaline substrate, wherein the hydrolytic enzyme is an esterase Characterized in that the oxidizing enzyme is manganese peroxidase, lignin peroxidase, lacase or tyrosinase, and the dissolution and degradation product is hydrogen, nitrogen, carbon dioxide, methane and / or an organic solvent .

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

Example 1: Asperfectant strain ( Arthrobacter sp. ) Culture

In this example, the strains of Arthrobacter ( Arthrobacter ) in a limited / complex medium containing various concentrations of coal sp . ) (Three strains: SLB08, SLB16 and S2A06) were cultured in order to examine the action of the strain on the coal and the decomposition effect of the coal.

material

The low grade coal (Korea Institute of Energy Research, KIER) was physically ground and then filtered using a steel sieve to obtain coal of uniform size. The sieves were composed of wire having an average diameter of 50 mu m and the average pore size of the sieve was 75 mu m. The above-mentioned low-grade coal was sterilized at 121 ° C for 15 minutes and added to the culture medium for culture, which was described later, at a weight ratio of 0.1% to 10%.

Other materials required for culture and analysis were purchased from Sigma Aldrich and Daejeong Chemical.

Culture conditions of strain

Arthrobacter sp. Was cultured in a synthetic medium supplemented with LB (Luria-Bertani) medium (control group) or coal at a weight ratio of 0.1% to 10% described below . If not, it was cultured at 30 ° C and 180 rpm for a designated period.

(1) LB medium (control group): When the strain was shake cultured under aerobic conditions, 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl per 1 L of distilled water were added and cultured in sterilized LB medium Respectively.

(2) Synthetic Medium: When the strain is shake cultured under aerobic conditions, a synthesis comprising 0.1 g KH ₂ PO ₄ , 0.2 g Na ₂ SO ₄ , 0.2 g NaCl, 1 g NH ₄ Cl and 1 g MgSO ₄ per liter of distilled water (N-1) medium (ingredient) medium was sterilized at 121 ° C for 15 minutes, then added with 0.1% to 10% by weight of coal, and the strain was inoculated and cultured. The degree of decomposition of coal was measured.

In addition, if necessary, an additional MR culture medium (composition) medium was prepared and used. The MR-salt medium was prepared by mixing 6.67 g KH ₂ PO ₄ , 4 g (NH ₄ ) ₂ HPO ₄ , 0.8 g MgSO ₄ .7H ₂ O, 0.8 g citric acid, and 5 mL trace metal solution per liter of distilled water The pH of the medium was adjusted to 6.8 with 1M KOH and sterilized at 121 占 폚 for 15 minutes. Next, coal was added to the sterilized medium at a weight ratio of 0.1% to 10%, and the strain was inoculated and cultured. Then, the degree of degradation of the coal by the strain was measured through the following examples.

The rare earth metal solution was prepared by dissolving 10 g FeSO ₄ .7H ₂ O, 2 g CaCl ₂ , 2.2 g ZnSO ₄ .7H ₂ O, 0.5 g MnSO ₄ .4H ₂ O, 1 g CuSO ₄ .5H ₂ O, 0.1 g (NH ₄ ) ₆ Mo ₇ O _{24 揃} 4H ₂ O, and 0.02 g Na ₂ B ₄ O ₇揃 10H ₂ O. 0.8 g MgSO ₄揃 7H ₂ O was used.

On the other hand, when the LB medium (control group) or the synthetic medium is prepared in a semi-solid state (for example, a plate containing agar medium), 20 g agar is added to the 1 L unsterilized liquid medium And then sterilized at 121 DEG C for 15 minutes. The sterilized agar medium was mixed with coal at a weight ratio of 0.1% to 10%, and then poured into a dish of 8.45 cm in diameter to be hardened. The strain was coated and cultured, and then the degree of degradation of the coal by the strain was measured Respectively.

Asperfectant strains in a medium containing coal ( Arthrobacter sp. ) Growth

In order to confirm the growth ability of Arthrobacter sp. In a semi-solid medium containing coal, 50 μL of the strain cultured overnight in a medium containing no coal was added to a semi-solid synthetic medium (100 mm dish) containing coal And then cultured.

In order to confirm the growth ability of Arthrobacter sp. In the liquid medium, 100 μL of the culture broth cultured overnight was transferred into 10 mL of synthetic medium containing coal and shake cultured.

The extent of growth (growth) of the cultures grown in the semi-solid medium or liquid medium was measured using the Bradford method as the total protein concentration extracted from the strain.

Example 2: Asperfectant strains in a medium containing coal ( Arthrobacter sp. ) And extracellular matrix and its mechanism

The presence of an alkaline substance during shake culture was confirmed by observing the change in pH. Biocatalysts and extracellular enzymes were measured using qualitative plate analysis and spectrophotometer. After addition of ammonium oxalate as a chelator, it was confirmed whether the decomposition ability of coal was increased in the strain without supernatant. Biological surfactants were identified by drop-plate testing.

2-1: Coal dissolution analysis

In order to evaluate the solubilization of coal by Arthrobacter sp. , Coal was added to a shake flask containing 10 mL of a confluent / complex medium at a weight ratio of 0.1% to 10%, then the strain was inoculated, And cultured at 30 ° C for 24 hours. The strains cultivated in the synthetic medium were then washed. The washed strain was prepared in an amount of 100 μL under the condition that the OD600 was 1 bs, and then inoculated on 300 mL synthetic medium (including coal) contained in the 1 L flask.

Cultures cultured in shaking under aerobic conditions were extracted at regular intervals. That is, the strain contained in the culture was centrifuged at 13,000 rpm for 5 minutes and then sieved using a BIOFIL syringe filter (PES membrane; pore size: 0.22 μm). The filtrate was analyzed at 450 nm and 513 nm using a UV spectrometer (SHIMADZU UV-1800 spectrophotometer) to determine the degree of solubilization (activity) of the coal.

Further, the supernatant was filtered with a filter, and the coal residue was dried at 95 DEG C for 24 hours to quantify, and then quantified in Example 5.

As a result, as shown in Fig. 1 and Fig. 2, Arthrobacter sp. SLB08 showed higher coal solubility at 450 and 513 nm than SLB16 strain or S2A06 strain.

2-2: Enzymatic analysis

For enzymatic analysis, 100 μL of Arthrobacter sp. Was inoculated on a nutrient agar medium and then cultured at 30 ° C. for 48 hours. Then, a predetermined amount was taken from the culture, and the various detection reagents described below were added, and the degree of discoloration of the culture was measured to confirm whether or not various enzymes were detected.

(1) Extracellular oxidase detection

The reagent for extracellular oxidase detection is composed of 30 mL of 96% ethanol containing 0.5 g of gum guaiac. When 2 to 3 drops of the above reagent is added to the strain colony, ) Means that the enzyme is present.

As a result, it was confirmed that the extracellular oxidase was present in a dark color around the colony of the plate containing the oxidase hyperacid.

(2) La Case Detection

The laccase detection reagent is composed of 100 mL of 96% ethanol containing 1.24 g of guaiacol. When the reagent is added to 2 to 3 drops of the strain colony, the enzyme turns to purple. It means to exist. The activity of the enzyme was measured by measuring the degree of oxidation of ABTS at 420 nm (Shi et al. , 2009).

As a result, purple appeared around the colony of the plate containing guaiacol, and green appeared around the colony of the plate containing 1 mM ABTS, indicating that the case was active.

(3) Tyrosinase detection

The tyrosinase detection reagent is composed of 100 mL of 96% ethanol containing 1.08 g of p-creasol. When 2 to 3 drops of the above reagent is added to the strain colony, 4 hours The color of the enzyme turns orange or brown to indicate that the enzyme is present.

As a result, a yellowish brown color appeared around the colony of the plate containing p-cresol, and it was confirmed that tyrosinase activity was present.

(4) Detection of peroxidase

The reagent for detecting peroxidase is composed of the same amount of 0.4% (v / v) hydrogen peroxide (H ₂ O ₂ ) and 1% pyrogallol dissolved in distilled water, It means that the enzyme is present when it is discolored to orange or yellowish brown.

As a result, a yellowish brown color appeared around the colony of the plate containing hydrogen peroxide and pyrogallol solution, and it was confirmed that there was peroxidase activity. In particular, it was confirmed that the activity of hydrogen peroxide was increased by an increase in the brown color around the colonies cultured for 5 to 7 days in plates containing LB agar (or nutritional agar).

(5) Detection of lipase and esterase

Lipase detection was performed by spreading or streaking the strain on a plate (Himedia) (Lakshmipathy et al. , 2010) containing tributyrin cold and incubating at 30 ° C for 48 hours Means that lipase is present when a transparent part is formed around the colonies after incubation.

The activity of the esterase was confirmed by the formation of a transparent region on a plate prepared by adding a 10% (v / v) solution in which 0.3 mL of ferulic acid was dissolved in dimethylformamide. In addition, esterase detection was carried out using 0.92 mmol / L p-nitrophenyl (pNP) acetate (Torres et al , 2009).

As a result, when the strain was plated on the TBA medium, a transparent part was formed, confirming the presence of lipase and esterase. The TBS medium was prepared from 5 g of peptone per liter, 3 g of beef extract, 10 mL of tributyrin and 20 g of agar.

In addition, ROA (Rhodamine B-Olive Oil agar) plate analysis for the activity of specific lipase was also performed by streaking the strain on the prepared medium. Lipase production was confirmed by the presence of orange fluorescent color around the colonies when exposed to ultraviolet light.

In addition, the hydrolytic activity of lipase was confirmed by a white precipitate formed around the colonies on a plate containing Tween 20/80 medium. The Tween 20/80 medium was prepared from 10 g of peptone, 5 g of NaCl, 0.1 g of CaCl ₂ .2H ₂ O and 10 mL of Tween 20 (or Tween 80).

Example 3: Biosurfactant analysis

To confirm the presence of biosurfactants, a drop-collapse test was performed. The drop-collapse test shows that when the supernatant derived from 20 μL of the strain is placed on the oil-coated surface, the droplets are disintegrated when the surfactant is included. However, if the non-surfactant is included, Is present.

Two coating oils were used for the drop-collapse test. To each well of a polystyrene microwell plate with an 8 mm inner diameter and a 0.30 mm deep well, 7 μL of mineral oil was applied (or 7 μL of mineral oil to the glass slide) and left at room temperature for 24 hours. Then 20 μL of supernatant and 20 μL of sodium dodecyl sulfate or distilled water were added to each well (or glass slide) using a 10 μL glass syringe (Hamilton microliter syringe 701-N 80300) Lt; / RTI > After one minute, the droplets were visually confirmed. The syringe was used three times with distilled water, three times with alcohol and three times with ether. All experiments were repeated three times.

Example 4: HPLC analysis of the product

The sample obtained by centrifugation at 13,000 rpm for 5 minutes was filtered using a BIOFIL injection filter of a PES membrane having a pore size of 0.22 탆. The filtered sample was analyzed at 450 nm and 513 nm wavelength of a UV spectrophotometer to measure the solubilized coal material. The organic carbon released from the coal was quantified by COD analysis. The filtered supernatant was added to various solutions of various concentrations of 20 mg / L to 1500 mg / L for COD analysis and the samples were treated at 150 ° C for 2 hours (Dry Block heater, JS Research Inc.). The absorbance at 620 nm was then measured with a COD detector (Hach). For the released organic matter, it was measured using HPLC. The measuring instrument for HPLC analysis was BIORAD Aminex A RID detector with HPX-87H column (300 mm x 7.8 mm) was used. The column conditions were 0.1N sulfuric acid as the mobile phase and the temperature was adjusted to 37 ° C.

Example 5 Analysis of Coal Residues

When the supernatant of the coal sediment was collected, the precipitate was dried at 95 ° C for 24 hours, and then stored for SEM (Scanning Electron Microscope) or TEM (Transmission Electron Microscope) observation. In addition, XRD and FTIR analyzes were performed to determine the functional groups and structural changes of the coal polymer. Elemental analysis of coal was also performed to determine the composition of coal before and after pretreatment.

As a result, a pictogram representing decomposition of coal was obtained.

Example 6: Asperfectant strain ( Arthrobacter sp. ) The resulting product E. coli Effect of Growth on Growth

In order to verify the solubilization ability of coal products, the supernatant of the culture derived from Arthrobacter sp. Was filtered, and E. coli WL3110 was inoculated in the filtered supernatant and incubated at 37 ° C for 24 hours with shaking (Park JH et al., Proc. Natl. Acad. Sci., USA 104: 7797-7802, 2007) were used to determine the absorbance at 600 nm .

As a result, when the E. coli WL3110 strain was inoculated into the supernatant of the culture derived from Arthrobacter sp. , The culture was inoculated into the culture solution derived from the above-mentioned Arthrobacter sp. The carbon degradation product was used as a carbon source (carbon substrate), indicating a slight increase in the growth of E. coli WL3110 strain.

The results indicate that culturing the Arthrobacter sp. In a medium containing low-grade coal converts the coal to a dissolved and degraded product, and confirms that the converted product can be obtained as an energy source Respectively. Particularly, it has been reported that the esterase (hydrolytic enzyme) contained in the extracellular matrix involved in the solubilization and degradation of coal contained in a synthetic medium inoculated with Arthrobacter sp. Or lipase, and manganese peroxidase, lignin peroxidase, lacase or tyrosinase, which is an oxidase enzyme, were confirmed, and the decomposition ability of coal according to the chelator was confirmed, and the bio-surfactant . In addition, when cultured in a culture supernatant derived from water bakteo strain (Arthrobacter sp.) In Aspergillus inoculated with E. coli strain WL3110, the carbon included in the cultures derived from bakteo strain (Arthrobacter sp.) In the Aspergillus The degradation product was used as a carbon source (carbon substrate), indicating a slight increase in the growth of E. coli WL3110 strain.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

A method for degrading coal using an Arthrobacter sp. , Comprising culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product.

Culturing an Arthrobacter sp. In a medium containing coal to convert the coal to a dissolved and degraded product; And a step of obtaining the converted product as an energy source . &Lt; Desc / Clms Page number 20 >

The method according to claim 1 or 2, wherein the coal is low rank coal having a low carbon source content.

The method according to claim 1 or 2, wherein the average particle diameter of the coal is 50 to 75 占 퐉.

The method according to claim 1 or 2, wherein the medium is liquid or semi-solid.

The method according to claim 1 or 2, wherein the coal is contained in the medium in a weight ratio of 0.1% to 10%.

3. The method according to claim 1 or 2, wherein the strain releases at least one or more of an extracellular substrate in the group consisting of a hydrolase, an oxidase, a chelator, a surfactant, and an alkaline substrate.

8. The method according to claim 7, wherein the hydrolytic enzyme is an esterase or a lipase, and the oxidase is a manganese peroxidase, a lignin peroxidase, a lacase or a tyrosinase.

3. The process according to claim 1 or 2, wherein the dissolved and decomposed product is hydrogen, nitrogen, carbon dioxide, methane and / or an organic solvent.