JPH07103379B2 - Biodesulfurization - Google Patents

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a biodesulfurization method using microorganisms. More specifically, by contacting a specific strain with a substance containing an organic sulfur compound, it acts on petroleum (product) such as crude oil or a substance containing an organic sulfur compound such as coal. Regarding the method. The present invention is a method that can be used in the desulfurization step before and after the petroleum refining of the crude oil or the like, or the desulfurization step of the coal or the like.

2. Description of the Related Art Global demand for petroleum is still solid due to the need for global industrial revitalization, the increase in consumption in developing countries, the peak of development of alternative petroleum energy, the limit of energy saving, etc. In particular, demand for gasoline, kerosene, and light oil is firm, and the structure of oil demand is rapidly becoming lighter. With the increase in demand for light petroleum, further progress and development of petroleum refining technology are desired.
For example, the establishment of petroleum refining technology that addresses recent environmental problems and requires less environmental impact is desired, and the administrative side can reduce the sulfur content in diesel oil and kerosene from the current 0.5%. It is being considered to reduce the amount to 0.05% after 5 years. Although the current purification technology centered on distillation and chemical reactions has been completed on one side, it is an energy-intensive process that requires setting operating conditions of high temperature and high pressure. , There are problems with energy load and safety. Therefore, establishment of means capable of refining petroleum at room temperature and pressure, which does not require the energy-intensive process as described above, is now awaited. By the way, the sulfur content in petroleum is 0.0 depending on the source.
There is a range of 5 to 5%, and the forms of the sulfur are sulfate, sulfite, thiosulfate, benzothiophene having a thiophene substituent, dibenzothiophene, in addition to elemental sulfur.
A wide variety of products such as alkylbenzothiophene. In particular, dibenzothiophene and alkylbenzothiophene having a thiophene skeleton, which is difficult to remove in the current desulfurization process, poses a problem in the petroleum refining process, and is not a reaction under aerobic conditions where there is a high risk of accidents such as explosions. It is desired to establish a desulfurization method under anaerobic conditions with less risk.

The present invention is to establish a method utilizing biotechnology as one of the means capable of refining petroleum at room temperature and atmospheric pressure, and more specifically to include petroleum and coal. Isolates microorganisms capable of specifically decomposing sulfur of organic / inorganic compounds that can be converted into compounds that can easily remove the compound from nature,
The purpose is to establish a desulfurization means for petroleum using such microorganisms.

Means for Solving the Problems As a result of extensive studies to solve the above problems, the present inventor has isolated a microorganism having the ability to decompose a specific organic sulfur compound from the natural world and bio-describes the microorganism. By applying to the desulfurization method,
The inventors have completed the present invention by finding that the problem can be solved. That is, the present invention is a microorganism belonging to the genus Pseudomonas, the genus Flavobacterium, or the genus Bacillus, and having at least one microorganism selected from the group having the ability to decompose organic sulfur compounds, under microaerobic or anaerobic conditions. The present invention provides a desulfurization method using a microorganism characterized by decomposing and desulfurizing a substance containing an organic sulfur compound by contacting the substance. Also,
The present invention involves contacting at least one or more microorganisms belonging to the genus Enterobacter, Aeromonas, or Corynebacterium and having the ability to decompose organic sulfur compounds with a substance containing an organic sulfur compound. Desulfurization method using a microorganism characterized by decomposing and desulfurizing the substance by, in particular, the conditions when contacting the microorganism with an organic sulfur compound, microaerobic or anaerobic conditions, characterized The present invention provides a desulfurization method using the microorganism. Hereinafter, the present invention will be described in detail. A, the desulfurization method of the present invention is a desulfurization method using a microorganism belonging to the genus Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebacterium and having the ability to decompose organic sulfur compounds. Is. The mycological properties of the above microorganisms are listed in Table 1.

[Table 1] As described above, from the mycological properties shown in Table 1, Sys of the Berger Manual (Bergys Manual)
tematic Bacteriology Vol. 1
And Vol. From 2 the strains in Table 1 were identified. a) First, the identification of Gram-negative bacteria will be described. In addition, below, (+) shows that the corresponding reaction is positive, and (-) shows that the corresponding reaction is positive. The KMN-89 strain and the KMN-71-1 strain are gram-negative bacilli, which have polar flagella, produce pyocyanin, and grow at 41 ° C., and are arginine dehydrogenase (+). , PHB (polyhydroxybutyrate) does not accumulate and intracellular fatty acids
Due to the inclusion of OH-12: 0 and other ecological and physiological properties, Psudomonas aer
It is believed that it should be identified as uginosa. However, when the GC content in the DNA of the strain was measured, it was 59.6% for 89 strains and 57.4% for 71-1 strains, which is a typical Psudomonas aerugino.
Compared with the GC content of 67.2% of sa, the strain was significantly different from that of Psudomonas aer.
It has proven difficult to identify as uginosa. Since both strains are within the range of the GC content of Psudomonas, which is 58 to 70%,
It was determined that the genus Psudomonas, KMN-89 strain is P
sudomonas SP. KMN-89, Microbiology Research Institute, Institute of Microbial Science and Technology, Institute of Industrial Science 13267
No. (FERMP-13267). The KMN-71-1 strain is Psudomona.
s SP. Similarly, as KMN-71-1
It has been deposited under the deposit number of No. 13266 (FERM P-13266). The KMN-54 strain is a Gram-negative bacillus, which has polar flagella and produces fluorescein (a fluorescent dye), but does not produce pyocyanin, is arginine dehydrogenase (+), and is PHB. It is a typical Psudom due to the fact that dinitrification is (-), intracellular fatty acid contains 2-OH-12: 0, and other ecological and physicochemical properties.
was identified as onas fluorence and its biotype was classified as biovar I. This strain is Psudomonas fluores
Cence KMN-54 as a microtechnology research institute for microbiological research by the Institute of Industrial Science and Technology No. 13265 (FERM
It is deposited under the deposit number of P-13265). KM
The N-86 strain is a Gram-negative bacillus, which has polar flagella,
Arginine dehydrogenase (+) does not accumulate PHB, dinitrification is (-), intracellular fatty acid contains 2-OH-12: 0, and its GC content is also 62.7%. By being, Psudomona
s fluoresence. This strain is
Psudomonas fluorence K
As MN-86, the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microtechnical Laboratory No. 13264 (FERMP-1326)
It is deposited with the deposit number of 4). KMN-91 strain is
Although similar to the above KMN-86 strain, its GC
The content is approximately 59%. Psudomonas fl
uorescience has a GC content of 59.4-63.
Since it is 6%, this strain is Psudomonasfl
was identified as uoresense. This strain is Ps
udomonasfluorescence KMN-
No. 91, it has been deposited with the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology with a deposit number of Micromachine Research Institute No. 13263 (FERM P-13263). The KMN-69 strain is a Gram-negative bacillus, has polar flagella, does not accumulate PHB with arginine dehydrogenase (+), does not liquefy gelatin and does not oxidize with oxidase (+). 2 in body fatty acid
-OH-12: 0 and its GC content is 6
By 0.7%, Psudomonas P
It was identified as utida. In addition, by assimilating sucrose, the biotype was classified into Biotype B. This strain is Psudomonas p
utida KMN-69 as Micro Institute of Microbiology Research Institute No. 13262 (FERM
It is deposited under the deposit number of P-13262). KMN
The -59 strain does not have motility, shows O type in the OF test, both oxidase and catalase are (+), and due to the other properties shown in Table 1, Flavoba
Cerium. This strain is Flao
vbaderium SP. As KMN-59, Institute of Microbial Science and Technology, Industrial Technology Institute
No. (FERM P-13261). The KMN-76 strain is a bacillus bacillus with motility,
Grows both aerobically and anaerobically, catalase (+) and oxidase (-) assimilate glucose to produce acid, and OF
The test shows type F, which decomposes the sugar fermentatively. Furthermore, VP test is (+), DNase is (-),
Decomposes citric acid and the GC content of DNA is 57.1%.
This strain was identified as the genus Enterobacter. This strain is Enterobacter
SP. As KMN-76, the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology, Micromachine Research Institute No. 13260 (FERM P
-13260). KMN-
The 71-2 strain is a non-motile bacillus, grows both aerobically and anaerobically, produces acid from glucose with catalase (+) and oxidase (+), shows F type in the OF test, and nitrate reduction I do. In addition, since the GC content of DNA is 60.4%, this strain is a non-motile Aeromonas genus, and Aeromonas salmonicida
(GC content 57-59%) was identified. This strain was purchased from Aeromonas SP. As KMN-71-2, Microorganism Research Institute 1st
Deposited under the deposit number of 3259 (FERM P-13259). b) Next, the identification of Gram-positive bacteria will be described. KM
The N-35 strain is a bacillus having motility, grows in both aerobic and anaerobic conditions, and assimilates glucose to produce an acid, and its DNA had a GC content of 41.3%. Although sporulation of this strain was not observed in normal culture, the strain was found to be non-spore type Bacillu due to the properties shown in Table 1.
It was identified as a s genus. This strain is Bacillus SP.
As KMN-35, Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology, Micromachine Research Institute No. 13258 (FERM P-1325)
It has been deposited with the deposit number of 8). KMN-39 strain is
It is a non-motile bacillus, grows both aerobically and anaerobically, does not form spores, and has a catalase reaction (+). In addition, it is a bacterium that assimilates glucose to produce an acid, showed F type in the OF test, and had a GC content of 63.1% in its DNA. In addition, due to the various properties shown in Table 1, Cory
It was identified as the genus nebacterium. This strain is Cor
ynebacterium SP. As KMN-39, it has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology with a deposit number of Micromachine Research Institute No. 13257 (FERM P-13257). Incidentally, the strain used in the desulfurization method of the present invention is not limited to the strain deposited in the above, Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebacterium. There is no particular limitation as long as it is a microorganism that belongs to the group and has the ability to decompose organic sulfur compounds. When the strain is used in the desulfurization method of the present invention, each strain may be used alone or a plurality of strains may be used in combination. B. The desulfurization method of the present invention is characterized in that the substance is decomposed and desulfurized by being brought into contact with a substance containing an organic sulfur compound. Examples of the substance containing an organic sulfur compound include crude oil and coal. These substances can be used as they are, but from the viewpoint of efficiently decomposing the organic sulfur compound as the substrate, for example, the contact interface between the desulfurizing bacterium and the organic sulfur compound as the substrate is made as large as possible. For the purpose, it is possible and preferable to use a surfactant. Further, it is preferable to appropriately shake the reaction system for the same purpose. At the time of the decomposition reaction, the amount of air present is not particularly limited, but under aerobic conditions, during the reaction, for example, oxygen in the air and volatile components in the crude oil are mixed, and in the mixed gas state, due to sparks, etc. There is a risk of explosion due to ignition. From this viewpoint, it is preferable to carry out the reaction under anaerobic conditions where the reaction atmosphere is completely anoxic, under slightly aerobic conditions where there is slight oxygen, or under anaerobic conditions. Further, in consideration of the state of the storage tank at the time of reaction, it is preferable to carry out the reaction under the slightly aerobic condition. The specific amount of oxygen under such aerobic conditions is preferably controlled to be less than or equal to the above explosion limit, specifically 0.5%.
It is particularly preferable to carry out the reaction under the condition (v / v). The temperature during the decomposition reaction is not particularly limited as long as it is a temperature at which the desulfurizing bacterium can act,
A temperature of 10 ° C to 45 ° C is common and preferred. As the constituents of the decomposition reaction system, it is possible to react with the above strain and a substance containing an organic sulfur compound, as well as suitable water. However, in order to improve the reactivity of the desulfurizing bacteria, it is preferable to appropriately add a nutrient source such as an inorganic or organic nitrogen source or inorganic salts to the reaction system. Further, the reaction can be performed by a column method or a batch method.
When the reaction is carried out by the column method, it is necessary to immobilize the desulfurizing bacterium on the reaction column by an appropriate method. After such biodesulfurization reaction, a known separation / purification process is usually applied. In addition, after being subjected to the desulfurization method of the present invention, it can be directly attached to a petroleum refining apparatus (topper) and a petroleum desulfurization apparatus which are currently generally used. However, it is not essential to perform the above separation / purification step after the reaction.

EXAMPLES Examples will be shown below, but the scope of the present invention is not limited to these examples. [Example 1] Decomposition test of persistent organic sulfur compound The organic sulfur compound dibenzothiophene (DBT) or alkyldibenzothiophene having a thiophene skeleton which is difficult to remove by the current desulfurization process is the only salt medium shown below. As a carbon source and a sole sulfur source, the decomposition test was carried out to produce biodesulfurizing bacteria under slightly aerobic (anaerobic) conditions. It should be noted that the formula (1) and the formula (2) represent the hardly removable organic sulfur compounds in the petroleum fraction. - Bio desulfurization medium composition _{- NH 4 NO 3 1.0g K 2} HPO 4 2.0g KH 2 PO 4 1.0g MgCl 2 · 6H 2 O 0.1g CaCl 3 · 6H 2 O 0.01g FeCl 3 · 6H ₂ O 0.01 g ZnCl ₂ 0.01 g MnCl ₂ .4H ₂ O 0.01 g Meat extract 0.03 g Yeast extract 0.03 g Peptone 0.03 g Substrate 1.0% (v / v) or (w / w) pH 6.8-7.0

[Chemical 1] "Culture of desulfurization bacterium" Culture of desulfurization bacterium is carried out by washing the microorganism cultured in the agar growth medium with the screening liquid medium containing no organic sulfur compound, and then suspending the same in the same solution, and then in a vial bottle (30 ml).
The above-mentioned bacterial suspension was added to 20 ml of the screening medium, and the mixture was filled with nitrogen. Then, the vial was sealed in the same manner as at the time of screening, and shake culture was carried out at 30 ° C. under anaerobic condition for 1 month. The shaking culture was adopted to enhance the contact between the substrate and the desulfurizing microorganism because DBT as a substrate is a solid substance. "Quantification of consumption rate of substrate" Regarding the quantification of DBT, gas chromatography (G
The analysis was performed in C). That is, the culture solution was collected in a screw cap test tube and the pH was made lower than 2.5. Next, add 20 ml of ethyl acetate to the vial that had been cultivated,
Washed well. This washing solution (ethyl acetate) was added to the culture solution collected in a screw cap test tube, and after stirring, the upper layer was taken. As a result, the substrate DBT was efficiently extracted in the ethyl acetate layer. (GC analysis conditions) Separation column OV-17 inlet temperature 290 ° C detector temperature 290 ° C column temperature 120 ° C to 5 ° C / min. -270 ° C Internal Standard Carbazole "Measurement of Desulfurization Bacteria" The growth of microorganisms was measured by measuring turbidity with a spectrophotometer. The measurement wavelength was 660 nm. [Example 2] Screening for a novel microaerobic (anaerobic) desulfurizing bacterium A microorganism that assimilates and decomposes an organic sulfur compound in a petroleum fraction, which is difficult to remove in the current desulfurization process, shown in the above formula (1) Was searched under natural aerobic conditions (anaerobic conditions) close to the actual conditions such as stockpiling tanks. "Development and search of screening method for new microaerobic desulfurization bacterium" of hard-to-remove organic sulfur compounds (dibenzothiophene, thiophene, ethylmethylsulfide) in petroleum sulfur as carbon source and sulfur source Microorganisms that grow under oxygen suppression conditions close to the above conditions were screened from soil and the like. After about 1 month of enrichment culture, the operation of subculturing and enriching 10% of the culture broth was repeated 4 times, and a well-diluted dilution of the culture broth was applied to the agar growth medium to form colonies that appeared. Bacteria were picked up and further cultured twice in an agar screening medium to separate those that grow. In the integration culture, the liquid culture was performed by adding 50 ml of a medium to a vial (100 ml), adding a soil sample and an organic sulfur compound, filling with nitrogen, sealing with a butyl rubber stopper coated with silicon and an aluminum cap, and culturing at 30 ° C. . In the agar medium, thiophene and ethylmethyl sulfide as substrates were placed on a glass Petri dish lid, vaporized and given, and dibenzothiophene was dissolved in ether, inoculated, and sprayed on the medium surface. The composition of the screening medium and growth medium using dibenzothiophene, which is a difficult-to-remove organic sulfur compound, as a carbon source and a sulfur source, is the same as that of the medium composition shown in Example 1 (hard-degrading organic sulfur compound decomposition test). It is the same. "Acquisition of new microaerobic desulfurization bacteria by new screening" Using soils (about 400 points) collected from the Tohoku region etc., difficult-to-remove organic sulfur compounds dibenzothiophene (DBT), thiophene or The accumulation culture of the assimilating and degrading microorganisms was carried out under aerobic conditions using ethylmethyl sulfide as a carbon source and a sulfur source. As a result, 92 strains using DBT as a substrate, 112 strains using thiophene as a substrate,
109 strains were obtained using ethyl methyl sulfide as a substrate. [Example 3] Measurement of assimilation decomposition rate of difficult-to-remove organic sulfur compound dibenzothiophene by newly obtained biodesulfurization bacterium "Method for quantifying decomposition and consumption of organic sulfur compound and measuring method for growth of desulfurization bacterium" New screening result Quantification of DBT consumption of each of the obtained desulfurization microorganisms under slightly aerobic (anaerobic) conditions, and microorganism measurement as described above, (cultivation of desulfurization bacteria), (quantification of consumption rate of substrate), and (desulfurization bacteria) Measurement of growth degree). “Decomposition and consumption of petroleum fraction difficult-to-remove organic sulfur compounds by newly acquired microaerobic desulfurization bacteria” Table 2 shows the degree of DBT decomposition of deposited biodesulfurization bacteria.

[Table 2] As a typical example, Pseudoml of gram-negative bacteria
as putida KMN-69 strain (FERM P-1
3262) and the gram-positive bacterium Corynebact
erium SP. KMN-39 strain (FERM P-1
The decomposition process of DBT according to 3257) is shown in FIGS. As a result, it was confirmed that each strain of desulfurization bacteria certainly decomposes and consumes DBT as an added substrate as compared with the control, although there is a difference in the decomposition rate. In addition, the degree of desulfurization bacteria growth under microaerobic conditions was also confirmed for all strains. It should be noted that, as represented by the genus Pseudomonas in the present invention, not utilizing and decomposing useful components in petroleum such as dodecane and hexadecane is an extremely advantageous point in practical application of the desulfurization method of the present invention. Furthermore, when the DBT was decomposed under aerobic conditions using the above-mentioned microbial species, all strains utilized and decomposed the DBT. Among them, Pseudomonas putida K
The degree of decomposition of MN-69 is high, and about 50 days after 28 days of contact
0 ppm of DBT was assimilated and decomposed. [Example 4] Utilization / decomposition of microorganisms under slightly aerobic (anaerobic) conditions of alkyldibenzothiophene A compound that is not removed by the current chemical desulfurization process and remains as an organic sulfur compound derived from crude oil until the end in petroleum products. Alkyldibenzothiophene which is is subjected to the desulfurization method of the present invention. “Synthesis of alkyldibenzothiophene” Since alkyldibenzothiophene is not currently sold as a reagent, 4,6-dimethyldibenzothiophene, which is a kind of alkyldibenzothiophene, was synthesized according to the synthetic reaction method shown in Formula (3). .

[Chemical 2] "Application of slightly aerobic (anaerobic) biodesulfurizing bacteria to alkyldibenzothiophenes" As a result of screening for application to alkyldibenzothiophenes by microaerobic (anaerobic) biodesulfurizing bacteria that utilize and decompose DBT With respect to the obtained microaerobic desulfurization bacteria (DBT-assimilating / decomposing bacteria), Table 3 shows the degree of decomposition of alkyldibenzothiophene under anaerobic conditions.

[Table 3] In addition, as a typical example, Pseudo which is a gram-negative bacterium
monas putida KMN-69 (FERM
FIG. 3 shows the decomposition process of P-13262), Corynebacterium SP. KMN
Fig. 4 shows the decomposition process of -39 (FERM P-13257).
It was shown to. As a result, even under slightly aerobic (anaerobic)
It was confirmed that assimilation and degradability was achieved by using alkyldibenzothiophene as the sole carbon source and nitrogen source. At present, there is no report of a bacterium capable of assimilating and degrading that grows using such an alkyldibenzothiophene as the sole carbon source and nitrogen source.

Industrial Applicability According to the present invention, there is provided a biodesulfurization method capable of refining petroleum, coal and the like at room temperature and atmospheric pressure.

[Brief description of drawings]

FIG. 1 Pseudomonas putida K
Degradation process of DBT by MN-69 strain.

FIG. 2 Corynebacterium SP. K
Degradation process of DBT by MN-39 strain.

FIG. 3 Pseudomonas putida K
Decomposition process of alkyldibenzothiophene by MN-69 strain.

FIG. 4 Corynebacterium SP. K
Decomposition process of alkyldibenzothiophene by MN-39 strain.

 ─────────────────────────────────────────────────── ─── Continuation of front page FERM P-13265 microbial accession number FERM P-13266 microbial accession number FERM P-13267 Applicant has permission to prepare. (56) References JP 57-195187 (JP, A) JP 60-41479 (JP, A) JP 45-23831 (JP, B1) JP 45-20176 (JP, B1) JP Japanese Patent Publication No. 54-36601 (JP, B1) US Patent 2574070 (US, A) US Patent 2641564 (US, A)

Claims (3)

[Claims] 1. At least one or more kinds of microorganisms belonging to the genus Pseudomonas, the genus Flavobacterium, or the genus Bacillus and having an ability to decompose an organic sulfur compound, are treated under microaerobic or anaerobic conditions. A desulfurization method using a microorganism, characterized in that the substance is decomposed and desulfurized by bringing the substance into contact with a substance containing an organic sulfur compound. 2. The genus Enterobacter, the genus Aeromonas,
Alternatively, at least one or more kinds of microorganisms belonging to the genus Corynebacterium and capable of decomposing organic sulfur compounds are contacted with a substance containing an organic sulfur compound to decompose and desulfurize the substance. A desulfurization method using a microorganism characterized by the following. 3. The desulfurization method according to claim 2, wherein the conditions for contacting the microorganism with the organic sulfur compound are microaerobic or anaerobic conditions.