JPS6041479A - New mutant species and removal of organic sulfur compound from fossil fuel by using the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention: Pearl, Pseudomonas sp. CB+ and a method using the same to remove organic sulfur compounds from fossil fuels, such as carbonaceous materials such as coal, petroleum and petroleum products. It is.

Many grades of coal and petroleum contain large amounts of sulfur compounds that form corrosive air and water polluting products during combustion.
A number of chemical and physical methods have been developed for the removal of sawmill sulfur compounds, such as S. rites).

Also, some organic sulfur compounds, such as mercaptans, aliphatic sulfides and disulfides, are relatively easy to remove by chemical methods. However, aromatic sulfur compounds such as diphenyl silicide, benzothiophene, dibenzothiophene, etc. have significant B? Exists at a degree.

These aromatic sulfur compounds are resistant to chemical and biological attacks and are often poorly soluble or difficult to remove (ref
) It is characterized as an organic sulfur compound.

A number of chemical methods have been investigated for the removal of organic sulfur. Methods that have been evaluated on a laboratory or industrial scale include oxidative desulfurization, chlorination decomposition, oxidation, hydrodesulfurization, and gravity melting. According to Berry, a significant total product cost in the range of $41 to $58/ton (in 1979) is required for the removal of 10 to 50 T of organic sulfur at bench and one-bar test scales. ( Berr3',
R, 1, (1981ゝ・-Guide to Coal
-Cleaning Methods-.

Chemical F:ngineering, Ja
nuary 26 f3).

Removal of difficult-to-remove organic sulfur compounds by microbiological treatment would have many important advantages. Microbiological methods do not require high-demand, high-pressure or corrosion-resistant equipment and can use inexpensive tB ablation equipment, keeping capital costs low.Also, waste materials can be used to grow microorganisms. Cultivation costs can also be kept low if cultivation is carried out. Additionally, in many sites, biological treatment can be carried out under similar conditions; furthermore, microbiological treatment appreciably alters the structure and composition of the coal or substantially reduces the BTU value of the coal. I won't let you.

Microbiological treatment of petroleum and coal has been studied at the experimental stage using various microorganisms.
bacterium), Beijerinchia (Boi
jerinckia), Rhizobium
Some of these microorganisms have shown the potential to degrade organic sulfur compounds that are difficult to remove, as reported by Edrick et al.
Hcdrick et al. (1982>, Desulfur
Ization of Coal by Biology
cal Pratreatmant”!St+tc
-of the-Art). This article provides a comprehensive summary of research into actual viscosity.However, neither this article nor any other available technical literature describes the mutant strain, Shootmoboos sp.C.
There is no mention of 'B l (ATCC 393g+) or the possibility that it is effective in removing impurities such as organic and sulfur compounds found in carbonaceous materials that are difficult to remove, such as thiophenes. unacceptable.

The present inventors have recently discovered a mutant strain of Pseudomonas sp., which is negative for the urease reaction, grows on Pine Cone base agar medium, but does not grow on 4.1'C, and exhibits an ilu% characteristic. Pgeudmon
have isolated a biologically pure culture of CB I and found it to be extremely useful for the removal of difficult-to-remove organic sulfur compounds from fossil fuels such as coal and petroleum. 39381) Id, a non-motile nogram-negative bacillus of approximately (L 5 Catalase wipe and oxidase test K 144, does not utilize citrate as the sole carbon source, does not grow at 41oC and grows on Pine Conkey agar.Colonies that form on Sakae agar are round, It is regular and white in color. This fungus uses naphthalene, phenol or halesorcinol as the only carbon source for growth and activity, and uses butyrate as the only carbon source. 〉25 Mega Gluton) has 3 plasmids.

A culture of this strain has been deposited with the A 'I' CC and its deposit number is ATCC 11-39381.
select to obtain a strain which obtains benzoate as the sole carbon source,
A strain capable of oxidizing DBT in an aqueous medium was then selected to obtain, and the strain capable of oxidizing DBT in an aqueous medium was further created by treating the strain with a mutagenic factor that increases its ability to oxidize DBT. .

The results of the biochemical properties of the mutant strain CB+ of the present invention (see the table below) show that CB+ was almost certainly acclimatized.
Treatment with mutagenic agents and selection resulted in a pure culture of strain i'3, designated CDI.

Carbon source CBt PIl, Sutsutsue'I Pg,
Ammonium putidabenzoate - Sodium acetate - Cetyl salicylic acid - Citrus salt - CBI (ATCCA393g+)
> All of the refractory aromatic and aliphatic organic sulfur compounds that are difficult to remove, especially coal.

This novel mutant strain CBI has been found to be effective in oxidizing and removing these contaminating impurities from carbonaceous substrates such as petroleum and petroleum products without the use of carbonaceous substances. This microorganism can also be used to remove organic sulfur compounds from various grades of coal with moderate particle sizes ranging from fine to coarse particles. It can also be used to remove sulfur compounds.

Soil samples were obtained in Maryland, USA, and from a Pennsylvania coal mine.
'4), CaC/2 (0,0026'fy), I
The microbial population was isolated from each sample by extraction with a sterile salt solution containing (L/(0,035 g)) and distilled water. Each of the salt extracts containing the microbial population was cultured on nutrient agar. All of the above-mentioned culture cultures were inoculated into tributicase soybean broth medium for culture propagation. Then, minimal culture medium (MgSO40.02%, Queshu-T (.00
,02%, K2HPO41,0%, Na(N[
14) The concentration of 6-DOT, which was converted to HPO40.35 in pi(7) in the presence of DBT, was 100-100
Microbial populations from soil samples of all four animals acclimated to the upper six fish in the presence of selected DBT (Maryland, Maryland, Purge Two and Hypen '7) were in the range of 0/L. and pure cultures of Pseudomonas putida) by inoculation into minimal medium (citrate-free) containing benzoate (benzoate r/sodium 0.251) as a carbon source for k nilg-. An individual was selected to use benzoate as a carbon carrier.

Using benzoate as the only carbon source, all cultures were grown in a minimum base containing 0.25% benzoate in a chamber 1. In order to evaluate the oxidation of DOT in the culture medium, DB'l' was grown overnight at 100 ml of these cultures. It was inoculated to a final concentration of 200 or 400 η/L. The medium for pair j;α was also inoculated with I) BT at the same concentration of BT. After DBT treatment, the cultures were returned to the shaking table and the entire contents of each flask was extracted with methylene chloride to determine the amount of D117 remaining in the aqueous medium that had been cultured for several days. did. Three test cultures (Maryland and Virginia, (soil J33 year old individual g'6 and pure culture of Pseudomonas putida) were prepared with a solvent extractable D B T g3 degree of some 1 t.
It showed 9 less than 1-. These cultures were maintained separately and in mixtures of the three cultures. This combination of crystallization and cultivation is called "Combo".

These three separate cultures and "components" are further I)
11 in the presence of BT and benzoic acid salt can be utilized as the sole carbon source (II', l 14c was selected).

Each of these four cultures and the Pennsylvania coal mine culture were subjected to mutagenesis using diethyl sulfate (DIi:s)b as described by Roth (11) [J, tt, Roth (1971), - Ge
netic Techniquesin 5tudie
s of [1acterial hietaboli
nm-.

Methods in Enzymology, XS1
[, A, gfi pages 3-35]. In each of these five cultures some (so
me ) Ia organisms survived DES treatment and were grown in fresh nutrient broth medium at 11Uelti'[YPs (1/
Loo) L, 35Dc Chi was grown in a single culture overnight and was rescued.

DgS mutagenesis treatment followed by 'rescue' in broth medium was used to inoculate 10 fC microbial cultures into minimal salts medium containing benzoate as a carbon source and grow on a shaking tray. Grow overnight at room temperature.

Each flask of this culture and medium pair 1fi flask with different concentrations of DBT'i (e.g. 1001η/L,
200 Q/L)i;÷12 poor. Return the flask to the shake table and wait for various cultures, e.g. 18 hours, 4 days, 70))y JI L /L E"
e, l) +1 'r For analysis, extract the entire contents of the flask with 3 mIB meburene to prepare a 0-swamp solution.
Only the "compo" culture showed a decrease in B i' 01) The "compo" culture after rDS treatment was treated with a portion of benzoate. This subculture medium C' was subcultured into a minimal salt medium of 8 minutes.
11i--colonies were streaked onto nutrient agar plates for 1F; the recitation was performed in triplicate and the third isolation of single colonies was inoculated into minimal salt medium supplemented with benzoate. Then, DBT oxidation was carried out.
A single colony isolated three times; 1r body? It was named C.

Plasmid analysis The use of various chemicals such as acridine orange and growth at high
) Plasmid DNA molecules can be removed or cured from bacterial K(l) vesicles.
usually replicating circuit
Plasmids present as rDNAduplexes are removed by these chemicals due to interference with acridine (acridine) or due to a change in their fli17717 attachment site (high temperature).

The removal (cut3ng) method used for CBI is 1 annual of Aiethods for
General Bacteriology”Mic
robiology, Washington, D.
It was carried out according to the method described in [C.]. CBI medium beta products in logarithmic growth phase were inoculated at a concentration of 103-104 cells into a series of test tubes containing nutrient broth medium supplemented with 50 or 250 yy/L acridine orange.

These cultures were incubated overnight at 25°C or at 40°C. Aliquots of each culture were then inoculated into fresh medium.
:1O4OVc diluted and heated to 25"C or 4 without ventilation
Grown at 0°C. To obtain a single colony, a suitable diluted culture material is cultured on nutrient agar! The resulting single colonies were tested for single 19iC iDBT oxidation.

A stock culture of P. putida and two acridine orange-treated butt single color isolates that showed reduced D B i oxidation were plated on nutrient agar plates and plasmid D
Old rnboim and D61y for NA minutes
Method [8irnboim, lf, c, and J, I)
oly.

-A Rapid A11ca order ne Etracti
on Procedure for Screening
Recombinant Plamid DNA
-r NuclejcAcids Re++search
+7.1513-1523] and mM'l (1ysis in'the
yCll)” technology [Newland, J, νV,
et al. (+980).

' Rapid Screening for Pla
smids in Environ+nenta1we
ll” Lysia Technique Usin
g Horizontal Ge1Electroph
oresis ” Abstracts Proc.
3rd AnnualMid-Atlantic R
ogional Extrachromosomal
Flerrr+ntgMoeting, PlaIIm
id, 3.2383 f.

"C'uncurd" CB l CATCCI by these plasmid analysis methods
&39381) was demonstrated to contain a large plasmid with a size of >25 Metz I Daltons. One of the untreated parent strains of Xu contained no plasmids detectable by these assays. After the acridine orange treatment, the above treated C does not oxidize DBT.
An isolate of Ut was also analyzed and found to contain a large plasmid with a molecular extension comparable to that of plasmid p found in CB+.

In this case, the Ryocridine Orange treatment clearly affected the enemy organism 21 without removing the plasmid. The above result shows that CB l (DBT due to fC
oxidation is not plasmid-mediated (not p
lasmid mediated).

biochemical properties C101, Pseudomonas aeruginosa (Ps.

p, erugLHosa), Pseudomonas puguda (PLL.

API 2 OE system CAPI 20 E
SystemΔanalyticalProfile I
ndex+Δnalab Products, Pla
Inview.

NY]. Solj for Pseudomonas h3 strains that are very similar to CBI and Qc, respectively.
Fill fr, S le 1 table. It will be seen that there are large differences in rW between CB+ and each of the other Pseudomonas species.

1st stain API20g system test 0NPG --
-- ADII-Toto- Ll) C---- Ll) C---- 〇DC---, -.

CIT -) - - - H, S - - - IJRE + Tl) A - IND - - - vp - - - GEL l - - - - GLU - - - rhn - - - Table 1 (3 cries) 171Gl ---- A nl y---- 0x! +−1−1−− NO−−−−toN gas −−− Movement r1mi →−+−−1− MAC→ −4−− oy−o →−1−1−→～ 01” -F - 41C-L, 1- - -1- catalase 4- + 1 + naphthalene - phenol - resorcinol - benzoate 10 - CB-1 using C-DOT and 5-DBT As a result of a radioisotope test conducted on culturing DBT, it was observed that the sulfur and carbon skeletons of DBT were oxidized to water-soluble compounds. However, no CO2 was produced. This indicates that CBI does not utilize the carbon skeleton of DB, T as a carbon source. painting body (biomass)
)'i of the solution 1 Hach, removed by centrifugation and passing through a 0.2 micron filter.
(andbook for Water Anxlysi
g @(Hach Chemical Company
The samples were analyzed for sulfate by analytical methods and ion chromatography as described in J.D., 1979).

These two analytical methods gave comparable results. Sulfate concentrations in DBT medium treated with CBI were 100 and 99
K/L, while the sulfate concentration in the control test was 55
and 67q/L.0 These data indicate that CBI is D.
This reaction mechanism has been shown to act on BT to oxidize its sulfur groups and produce inorganic sulfate, which is the main product in the decomposition of DBT by microorganisms, all of which have been reported in other literature. is 3-human axy-2-formylbenzothiophene [Koda+na
, K, et al. (1'/73) , -Identifica
tion of Microbial Product
from Dibenzothiophene and
Its Proposed Oxida-tion P
athway ”' + Agr, Biol, Ch
em. The organic sulfur is determined by subtracting the total content of pyrite-type sulfur and sulfate-type sulfur from the total sulfur content. The reduction in the total sulfur content of the coal after washing ranged from 8 to 34 ml depending on the coal seed size and grain size. CB+ has no effect on pyrite-type sulfur. In order to prove this, the following experiment was carried out. CB+ was cultured at 25°C on a shaking tray using 0.25t of pyrite (approximately 325 mesh) in 100ml of water. After incubation in minimal media containing only benzoate and pyrite, the contents of each flask were centrifuged and the supernatant was filtered through a 0.2 micron filter and removed. ``H!1ndboolc for IIach'' quoted above.
The samples were analyzed for sulfate using the method described in Water Analysis. The pyrite/fungal cells obtained by centrifugation+qta were dried overnight at 60°C and analyzed for total sulfur content hN' using a Fisbar total sulfur analyzer. The total sulfur content of the pyrite in the control E experiment and the inventive experiment treated with CB1 was the same. These results indicate that CB1 does not oxidize pyrite. Therefore, it can be concluded that the organic sulfur type of sulfur in coal is oxidized by CB1.
Examples of the use of BIi in the oxidation of various organic sulfur compounds are shown.Example 1: Untreated Composite Biological Culture Closure and Mutant Strain CB
Oxidation experiments of DBT from an aqueous medium by cultured bacteria were carried out at DIIT [71 degrees and temperature conditions] during various cultures, and the results are shown in Tables 2 and 283. These results demonstrate that CBI reduced significantly more D[lT to 0 in water than the untreated parent culture in the culture medium. 38
50 4 combo 5 days 50 3 component 7 days too 7 component 7 days 200.

Combo 10 days 200l.

Combo 10 days too 7 (:Bl 18 hours too 62 C1ll 18 hours +00 65 CB+ 18 hours + 00 60 Table 3 Effects on acid ratio of D B T by Cl31 gum
Between cultivation 'Jj L'jjr degree original [) B 'I''
1i) IIJ D B T reduction rate 18 ・ 25 2
Example 2 CBI in aqueous medium l) Existence other than B T In order to evaluate the oxidizing ability of the sulfur compound, the two tests described above were conducted. The CBI culture was inoculated into a minimum culture medium containing benzoate (0.25%) and incubated on a shaking table for 2 hours.
Grow overnight at 5°C. The selected organic sulfur compound was added at a specified concentration to the flask containing the CB+ overnight culture. After the flask containing the microbial culture and organic sulfur compound was incubated for 24 to 48 hours, the entire contents of the flask were After extraction, the total content of organic sulfur compounds was measured. Briefly, the contents of each flask were extracted three times with 10 methylene chloride and the extracts were analyzed by gas chromatography. Zero medium control test flasks contained minimal medium, benzoate, and the organic sulfur compound to be evaluated. For the second set of culture medium control test flasks containing a specific concentration of 0, extraction was performed immediately after charging to determine the extraction efficiency of each organic sulfur compound 2 fs 'fr fl! The results are shown in Table 4. These results demonstrate that the mutant strain CB+ of the invention removed significant amounts of solvent extractable organic sulfur compounds from the aqueous medium.

Table 4 Other organic sulfur compounds by CBI Initial cultivation n-mectylsulfate 24 too 4424
200 85 24 200 81 Benzyl methyl sulfide 24 too 3124
too 32 Thioanilin 24 100 45 24 10 (151 1-benzothiophene 24 200 g + Example 3 This example shows that the mutant strain CB+ of the present invention removed sulfur from weighed coal samples with various particle sizes. This experiment was carried out to test the ability of CBI to obtain 1000 ml of bacteria, and a culture of a.CBI was inoculated into a minimal medium containing benzoate (0.25%) and grown overnight at 25°C on a shaking table. To each 18-24 hour culture, 5 volumes of coal powder were added.The flask containing the 15 charcoal mixture was returned to the shaking table and incubated for 24-48 hours. After that, the coal powder was removed by centrifugation, it was washed with distilled water, and the total sulfur content of the coal powder was analyzed.

It should be noted that the results of these experiments are given by the total sulfur content t!・CB l'ff Wentz' coal and Mont's coal were selected as the coals for the purpose of frugality.The reason is that the sulfur content of these coals is It has been reported that the total sulfur content constitutes about 90% of the total sulfur content.
Because there is. The results are shown in Table 5. The results in Table 5 show that treatment of coal formula 4F with the mutant strain CB+ of the present invention significantly reduces the total sulfur content of the coal when the grain size of the coal particles is 250 microns (60 mesh) or less. I am truly grateful for what I have brought to you.

/ Example 4

Claims (1)

[Scope of Claims] 1. A mutant strain of the Pseudomonas group, negative for urease reaction, grown on pine conchy agar! A biologically pure culture of Pseudomonas sp. CB+ which has the characteristic of growing at 1-1 but not at 410C. 2. Carbonaceous! A method for removing organic sulfur compounds from carbonaceous materials consisting of treating the materials with a culture of Pseudomonas sp. CBI. 36. The removal force method according to claim group 2, wherein the carbonaceous substrate is coal. 4. The carbonaceous substrate is petroleum or petroleum products (removal force method described in item 2)