CN100451101C - Process for deeply removing organosulfur from fossil fuel by mycobacteria - Google Patents
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- CN100451101C CN100451101C CNB021103755A CN02110375A CN100451101C CN 100451101 C CN100451101 C CN 100451101C CN B021103755 A CNB021103755 A CN B021103755A CN 02110375 A CN02110375 A CN 02110375A CN 100451101 C CN100451101 C CN 100451101C
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Abstract
The present invention discloses a method for deeply eliminating organic sulfur in fossil fuel by using mycobacteria. The method comprises the steps of strain selection, slope culture, a first level of seed culture, culture enlargement, the culture of a fermenting tank, thallus collection, sample treatment, sample separation, sample detection, etc. The present invention has the characteristics of convenient method, wide temperature requirement ((25 to 50 DEG C), low energy consumption, high rate of sulfur elimination, etc. and has large application prospect to treat atmospheric pollution.
Description
(1) technical field
The present invention relates to utilize thermoduric bacteria to remove the method for organosulfur in the fossil oil, relate in particular to the method for utilizing organosulfur in the mycobacterium deep removal fossil oil.
(2) background technology
Along with the development and the application of Vehicle Emission Control new technology, and to the further investigation of fuel oil quality, the sulphur content to oil fuel requires more and more stricter in the world.The burning of fossil oil produces a large amount of toxic gas SO
2Entering atmosphere, cause serious atmospheric pollution, also is simultaneously the major cause that produces acid rain.In order to protect environment, require to use the fossil oil of low sulfur content.The fossil oil deposit of low sulfur content sharply reduces in the world at present, need carry out desulfurization to the high fossil oil of sulfur-bearing and handle.(Hydrodesulfurization HDS) by catalytic process, changes into H with organosulfur in chemical desulfurization method-hydrogenating desulfurization
2S gas, reaction are to carry out under the temperature of the pressure of 1～20Mpa and 290～450 ℃.Because there are strict requirements to the sulphur content in the fossil oil, adding the HDS method expends than higher, and be difficult to the degree of depth and slough organosulfur in the fossil oil, and biocatalytic Desulfurization (Biodesufurization, BDS) cheap, can carry out at normal temperatures, and have high specificity, the biological desulphurization method that therefore develops a kind of fossil oil has been very necessary.This makes the BDS method become a kind of alternative method and the HDS method replaces this method to carry out the deep desulfuration of oil together or directly.
Organosulfur in the fossil oil mainly be dibenzothiophene (Dibenzothiophene, DBT), so the research of biological removal of organic sulfur from is also just carried out for the medelling compound with DBT.Have been found that many microorganisms now,, when sloughing organosulfur, often also destroy C-C, thereby cause fuel value to reduce and inadvisable along alkane degradation approach (Hydrocarbon degradative pathway) degraded DBT.Simultaneously, also find some microorganisms such as Rhod (Rhodococcus), bacillus (Bacillus), Corynebacterium (Corynebacterium) and genus arthrobacter (Arthrobacter) etc., can be along the single-minded approach of sulphur (Sulfur-specific pathway) degraded DBT, they do not open ring, thereby the calorific value that has kept fuel becomes the focus of Recent study.
Is 70% according to relevant report: Ge Dengshi (Gordona) bacterium for the desulfurization degree of middle cut, is 50% for light gas oil; Rhodococcus (Rhodococcus sp.) ECRD-1 can remove 30% with the sulphur of the middle component of virgin oil, simultaneously other 35% sulphur is become the form of oxidation.Energy Biosys Corp. of the U.S. (EnergyBiosystems Corp.) uses a strain through genetic engineering modified bacterial strain rhodococcus erythropolis (Rhodococcuserythropolis) I-19, a kind of sulphur content of oil of the middle cut through hydrotreatment is dropped to 615ppm from 1850ppm, reach 67% decreasing ratio.The investigator of Japan once screened a strain can be under 50 ℃ of conditions be the bacterial strain of unique sulphur source growth with DBT, but the efficiency ratio that removes sulphur is relatively poor, and does not almost have vigor at normal temperatures.Above-mentioned all bacterium and in the practical application of desulfurization, exist as: relatively stricter to temperature requirement, complex process, desulfurization degree is relatively lower wait not enough.
Because mostly the processing of petroleum compound is to carry out under High Temperature High Pressure, so raw material is cooled down and carry out bioprocess again and handle just not too economical; The heat resistant microbe heat resistance is strong, the enzyme system good stability; The raising temperature helps the carrying out of desulphurization reaction.Therefore, but removing the bacterial strain of organosulfur in DBT and other thiophenes around screening some more valuable specificitys ground, and further be applied to the work in the desulfurization of fossil oil, also is the problem that research and solution are badly in need of in present this area.About the patent and the pertinent literature of the method for utilizing the organosulfur in the facultative thermoduric bacteria mycobacterium deep removal fossil oil, do not appear in the newspapers so far by retrieval.
(3) technical scheme
The objective of the invention is at above-mentioned deficiency, a kind of method of utilizing organosulfur in the facultative thermoduric bacteria mycobacterium deep removal fossil oil is provided.
The method of utilizing organosulfur in the mycobacterium deep removal fossil oil of the present invention, its sequence of steps is as follows:
(1) bacterial classification is selected: select the mycobacterium mycobacterium in the middle ancient times (Mycobacterium goodii) DSM44492 for use, " this bacterial strain be preserved in German representative microbial collecting center (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH; Braunschweig; Germany) ", Mycobacterium phlei (Mycobacterium phlei) ATCC 354 " this bacterial strain be preserved in U.S. representative microbial collecting center (12301Park Lawn Drive; Rockville; Md.20852) ", Mycobacterium phlei (Mycobacterium phlei) ATCC11728 " this bacterial strain be preserved in U.S. representative microbial collecting center (12301 Park Lawn Drive; Rockville; Md.20852) ", mycobacterium (Mycobacterium sp.) ATCC 9823 " this bacterial strain be preserved in U.S. representative microbial collecting center (12301 Park Lawn Drive; Rockville; Md.20852) ", one of mycobacterium (Mycobacterium sp.) ATCC 11761 " this bacterial strain be preserved in U.S. representative microbial collecting center (12301Park Lawn Drive; Rockville, Md.20852) ";
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 1.5～2.0% agarose and be added with 0.1～2.0mmol/L DBT, cultivated thalline 20～30 hours for 25～50 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 20～100mL contains the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT (use 300～500mL triangle shake bottle) with inoculation articulating 1～4, under 25～50 ℃ of conditions, shaking culture is 20～30 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connecing first order seed (uses 1～5L triangle to shake bottle) in 300～1000mL contains the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT, under 25～50 ℃ of conditions, shaking culture is 20～30 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 1.8～8L contains the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT, (use the bright automatic fermenter of 2～10L Germany shellfish), under 25～50 ℃ of conditions, cultivated 20～30 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 5～8 minutes, collect the thalline that step (5) makes, and with physiological saline washing 2～3 times; Again thalline is dissolved in the potassium phosphate buffer of 20～100mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 10～30 gram stem cells/liter, promptly make biological catalyst ,-20 ℃ of storages, standby;
(7) handle sample: add 12～40mL sulfur-bearing fossil fuel sample or 10～30 gram solid sulfur-bearing fossil fuel sample, with 3～10: 1 ratio, biological catalyst in the step (6) is mixed (Water-to-oil ratio with fossil oil, WOR), under 25～50 ℃ of conditions, 180 rev/mins vibrated 24～30 hours;
(8) sample separation: with 10,000 rev/mins centrifugal 5～10 minutes, the sample of handling in the separating step (7) obtains oil phase;
(9) sample detection: after treating that sample separation in the step (8) is intact, get 1～5 μ L sample feeding (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the fossil oil, draw decreasing ratio.
In the above-mentioned method of utilizing organosulfur in the mycobacterium degree of depth deep removal fossil oil, the bacterial classification described in the step (1) is selected ancient mycobacterium (Mycobacterium goodii) DSM 44492.
Yeast culture temperature described in step (2), (3), (4), (5) is 37～45 ℃.
The yeast culture time described in step (2), (3), (4), (5) is 24～28 hours.
Dibenzothiophene (DBT) concentration described in step (2), (3), (4), (5) is 0.2～0.5mmol/L.
Biological catalyst described in the step (6) is meant resting cell, contains the cellular component of desulphurase, one of separation and purification desulphurase system or its composition.
The cell concentration of the biological catalyst described in the step (6) be 15～25 the gram stem cells/liter.
Fossil oil described in the step (7) is an oil, coal, brown coal, one of pitch.
The ratio of biological catalyst described in the step (7) and fossil oil is 5～8: 1.
The temperature of handling sample in the step (7) is 37～45 ℃, and the sample duration of oscillation is 25～28 hours.
In the above-mentioned method of utilizing organosulfur in the mycobacterium deep removal fossil oil, the bacterial strain that relates to use improvement A substratum (Modification of A Medium, MAM), it is as follows to fill a prescription:
Glucose 10 grams
KH
2PO
40.5 gram
K
2HPO
44 grams
NH
4Cl 1 gram
1%CaCl
22 milliliters
10%MgCl
26H
22 milliliters of O
1%FeCl
3200 microlitres
5%NaCl 200 microlitres
5 milliliters of 1% yeast extract pastes
Vitamin mixture 200 microlitres
5 milliliters of metal ion mixed solutions
Adding distil water to 1 liter, sterilization is 20 minutes under 115 ℃ of conditions.
The prescription of metal ion mixed solution is as follows in the above-mentioned substratum:
ZnCl
20.5 gram
FeCl
20.5 gram
MnCl
24H
2O 0.5 gram
Na
2MoO
42H
2O 0.1 gram
CuCl
22H
2O 0.05 gram
Na
2WO
42H
2O 0.05 gram
HCl 120mmol/L
Adding distil water to 1 liter.
The prescription of vitamine mixture is as follows in the above-mentioned substratum:
400 milligrams of calcium pantothenate (Calcium pantothenate)
200 milligrams of inositols (Inositol)
400 milligrams in nicotinic acid/nicotinic acid (Niacin)
VB
6(Pyridoxine hydrochloride) 400 milligrams
200 milligrams of para-amino benzoic acid (p-aminobenzoic acid)
VB
12(Cyanocobalamin) 0.5 milligram
Adding distil water to 1 liter.
Add dibenzothiophene (DBT) or dimethyl sulfoxide (DMSO) in above-mentioned substratum, making its concentration is 0.5mmol/L, as the sulphur source of cultivating bacterial strain.
Because mostly the processing of petroleum compound is to carry out under High Temperature High Pressure, so raw material is cooled down and carry out bioprocess again and handle just not too economical.The mycobacterium that the present invention relates to can grow under 25～50 ℃ of wide in range conditions, desulfurization, has reached higher decreasing ratio.The main path reaction formula of its desulfurization is as follows:
Wherein, DszC:DBT monooxygenase; DszA:DBT-sulfone monooxygenase; The DszB:HBPS desulfinase
Diesel oil through the hydrofining processing, wherein most of sulfocompound such as mercaptan, sulfide are all removed by hydrotreatment, sulphur content has only 205ppm, and remaining mainly is the reluctant thiophene type of hydrogenation process organic compounds containing sulfur (experiment is provided by Fushun oil research institute with hydrofining diesel oil).The mycobacterium of the Gu that the present invention adopts can carry out deep desulfuration under more wide in range temperature condition, the sulphur content of hydrofining diesel oil is dropped to 19.8ppm from 205ppm, decreasing ratio has reached 90.3%, can directly use after desulfurization, reaches strict environmental requirement fully.And the following a few strain bacterial strains among the contrast experiment are lower to the result demonstration decreasing ratio of hydrofining diesel oil, and rhodococcus lawq is 80%, and rhodococcus erythropolis IG is 69.1%, and pseudomonas ZCR is 58.5% (the results are shown in Table 1).
The result of table 1 bacterial treatment hydrofining diesel oil
# unit is a ppm sulphur; A is 30 ℃, and b is 45 ℃.
(4) embodiment
Embodiment 1: the method for utilizing organosulfur in the mycobacterium deep removal diesel oil
(1) bacterial classification is selected: select the mycobacterium mycobacterium in the middle ancient times (Mycobacterium goodii) DSM44492 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 1.8% agarose and be added with 0.5mmol/L DBT, cultivated thalline 30 hours for 45 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 50mL contains the MAM liquid nutrient medium of 0.5mmol/L DBT (use 300mL triangle shake bottle) with inoculation articulating 2, under 45 ℃ of conditions, shaking culture is 24 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and (use the 1L triangle to shake bottle) in 300mL contains the MAM liquid nutrient medium of 0.5mmol/LDBT, under 45 ℃ of conditions, shaking culture is 24 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 1.8L contains the MAM liquid nutrient medium of 0.5mmol/LDBT, (use the bright automatic fermenter of 2L Germany shellfish), under 45 ℃ of conditions, cultivated 30 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 5 minutes, collect the thalline that step (5) makes, and with physiological saline washing 2 times; Again thalline is dissolved in the potassium phosphate buffer of 20mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 10 gram stem cells/liter, promptly make biological catalyst ,-20 ℃ of storages, standby;
(7) handle sample: add the diesel oil (research institute provides by the Fushun oil) that 26mL contains the sulphur of 205ppm, with 6: 1 ratio, the biological catalyst in the step (6) is mixed with fossil oil (Water-to-oil ratio, WOR), under 43 ℃ of conditions, 180 rev/mins vibrated 25 hours;
(8) sample separation: with 10,000 rev/mins centrifugal 5 minutes, the sample of handling in the separating step (7) obtains oil phase;
(9) sample detection: after treating that sample separation in the step (8) is intact, get 3 μ L sample feedings (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the diesel oil, drawing decreasing ratio is 90.3%.
Embodiment 2: the method for utilizing organosulfur in the mycobacterium deep removal intermediate distillates
(1) bacterial classification is selected: select Mycobacterium phlei in the mycobacterium (Mycobacterium phlei) ATCC11728 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 1.5% agarose and be added with 0.2mmol/L DBT, cultivated thalline 28 hours for 50 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 25mL contains the MAM liquid nutrient medium of 0.2mmol/L DBT (use 300mL triangle shake bottle) with inoculation articulating 1, under 50 ℃ of conditions, shaking culture is 28 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and (use the 2L triangle to shake bottle) in 500mL contains the MAM liquid nutrient medium of 0.2mmol/LDBT, under 50 ℃ of conditions, shaking culture is 28 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 8L contains the MAM liquid nutrient medium of 0.2mmol/LDBT, (use the bright automatic fermenter of 10L Germany shellfish), under 50 ℃ of conditions, cultivated 24 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 5 minutes, collect the thalline that step (5) makes, and with physiological saline washing 2 times; Again thalline is dissolved in the potassium phosphate buffer of 35mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 30 gram stem cells/liter;
(7) with the above-mentioned somatic cells that makes of ultrasonic disruption, 600 watts of power act on 5 seconds, stop 5 seconds, broken 40 minutes, then 10,000 rev/mins centrifugal 10 minutes, collect supernatant liquor make contain desulphurase cellular component as biological catalyst;
(8) handle sample: add the intermediate distillates that 15mL contains 310ppm sulphur, with 9: 1 ratio, the biological catalyst in the step (7) is mixed with fossil oil (Water-to-oil ratio, WOR), under 45 ℃ of conditions, 180 rev/mins vibrated 26 hours;
(9) sample separation: with 10,000 rev/mins centrifugal 5 minutes, the sample of handling in the separating step (8) obtains oil phase;
(10) sample detection: after treating that sample separation in the step (9) is intact, get 3 μ L sample feedings (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the intermediate distillates, drawing decreasing ratio is 86.3%.
Embodiment 3: the method for utilizing organosulfur in the mycobacterium deep removal gasoline
(1) bacterial classification is selected: select Mycobacterium phlei in the mycobacterium (Mycobacterium phlei) ATCC 354 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 2.0% agarose and be added with 0.4mmol/L DBT, cultivated thalline 30 hours for 37 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 100mL contains the MAM liquid nutrient medium of 0.4mmol/L DBT (use 500mL triangle shake bottle) with inoculation articulating 4, under 37 ℃ of conditions, shaking culture is 24 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and (use the 2L triangle to shake bottle) in 600mL contains the MAM liquid nutrient medium of 0.4mmol/LDBT, under 37 ℃ of conditions, shaking culture is 24 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 6L contains the MAM liquid nutrient medium of 0.4mmol/LDBT, (use the bright automatic fermenter of 10L Germany shellfish), under 45 ℃ of conditions, cultivated 26 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 5 minutes, collect the thalline that step (5) makes, and with physiological saline washing 3 times; Again thalline is dissolved in the potassium phosphate buffer of 26mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 20 gram stem cells/liter, promptly make biological catalyst;
(7) handle sample: add the gasoline that 40mL contains the sulphur of 260ppm, with 3: 1 ratio, the biological catalyst in the step (6) is mixed with fossil oil (Water-to-oil ratio, WOR), under 35 ℃ of conditions, 180 rev/mins vibrated 28 hours;
(8) sample separation: with 10,000 rev/mins centrifugal 8 minutes, the sample of handling in the separating step (7) obtains oil phase;
(9) sample detection: after treating that sample separation in the step (8) is intact, get 2 μ L sample feedings (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the diesel oil, drawing decreasing ratio is 83.3%.
Embodiment 4: the method for utilizing organosulfur in the mycobacterium deep removal diesel oil
(1) bacterial classification is selected: select mycobacterium in the mycobacterium (Mycobacterium sp.) ATCC 9823 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 1.5% agarose and be added with 1.3mmol/L DBT, cultivated thalline 30 hours for 25 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 25mL contains the MAM liquid nutrient medium of 1.3mmol/L DBT (use 300mL triangle shake bottle) with inoculation articulating 1, under 27 ℃ of conditions, shaking culture is 28 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and (use the 3L triangle to shake bottle) in 700mL contains the MAM liquid nutrient medium of 1.3mmol/LDBT, under 25 ℃ of conditions, shaking culture is 29 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 7L contains the MAM liquid nutrient medium of 1.3mmol/LDBT, (use the bright automatic fermenter of 10L Germany shellfish), under 27 ℃ of conditions, cultivated 30 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 6 minutes, collect the thalline that step (5) makes, and with physiological saline washing 3 times; Again thalline is dissolved in the potassium phosphate buffer of 80mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 30 gram stem cells/liter, promptly make biological catalyst;
(7) handle sample: add the diesel oil (research institute provides by the Fushun oil) that 15mL contains 205ppm sulphur, with 8: 1 ratio, the biological catalyst in the step (6) is mixed with fossil oil (Water-to-oil ratio, WOR), under 27 ℃ of conditions, 180 rev/mins vibrated 29 hours;
(8) sample separation: with 10,000 rev/mins centrifugal 9 minutes, the sample of handling in the separating step (7) obtains the diesel oil phase;
(9) sample detection: after treating that sample separation in the step (8) is intact, get 4 μ L sample feedings (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the diesel oil, drawing decreasing ratio is 88.7%.
Embodiment 5: the method for utilizing mycobacterium deep removal organosulfur in intermediate distillates
(1) bacterial classification is selected: select mycobacterium in the mycobacterium (Mycobacterium sp.) ATCC 11761 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated on the MAM solid slant culture base that contains 1.5% agarose and be added with 1.8mmol/L DBT, cultivated thalline 27 hours for 30 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition down encircles in 25mL contains the MAM liquid nutrient medium of 1.8mmol/L DBT (use 300mL triangle shake bottle) with inoculation articulating 1, under 30 ℃ of conditions, shaking culture is 27 hours on shaking table, makes first order seed;
(4) enlarged culturing: with 5% (volume ratio) inoculum size, connect first order seed and (use the 4L triangle to shake bottle) in 900mL contains the MAM liquid nutrient medium of 1.8mmol/LDBT, under 30 ℃ of conditions, shaking culture is 27 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with 5% (volume ratio) inoculum size, connect secondary seed and in 8L contains the MAM liquid nutrient medium of 1.8mmol/LDBT, (use the bright automatic fermenter of 10L Germany shellfish), under 30 ℃ of conditions, cultivated 28 hours;
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 7 minutes, collect the thalline that step (5) makes, and with physiological saline washing 2 times; Again thalline is dissolved in the potassium phosphate buffer of 35mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 30 gram stem cells/liter;
(7) with the above-mentioned somatic cells that makes of ultrasonic disruption, 600 watts of power act on 5 seconds, stop 5 seconds, broken 40 minutes, then 10,000 rev/mins centrifugal 10 minutes, collect supernatant liquor make contain desulphurase cellular component as biological catalyst;
(8) handle sample: add the intermediate distillates that 30mL contains 310ppm sulphur, with 7: 1 ratio, the biological catalyst in the step (7) is mixed with fossil oil (Water-to-oil ratio, WOR), under 40 ℃ of conditions, 180 rev/mins vibrated 27 hours;
(9) sample separation: with 10,000 rev/mins centrifugal 8 minutes, the sample of handling in the separating step (8) obtains oil phase;
(10) sample detection: after treating that sample separation in the step (9) is intact, get 5 μ L sample feedings (the comprehensive dynamically microcoulomb analyser of WK-2D microcomputer, Jiangsu Electrical Analysis Instrument Factory), measure residual sulphur content in the intermediate distillates, drawing decreasing ratio is 84.5%.
Claims (9)
1. method of utilizing organosulfur in the mycobacterium deep removal fossil oil, its sequence of steps is as follows:
(1) bacterial classification is selected: select the mycobacterium mycobacterium in the middle ancient times (Mycobacterium goodii) DSM 44492 for use;
(2) slant culture: above-mentioned bacterial strains is inoculated in agarose that contains 1.5～2.0% and the MAM that is added with 0.1～2.0mmol/LDBT, on the solid slant culture base, cultivated thalline 20～30 hours for 25～50 ℃;
(3) first order seed is cultivated: with the bacterial strain of step (2) cultivation, aseptic condition encircles in 20～100mL with inoculation articulating 1～4 down and contains in the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT, under 25～50 ℃ of conditions, shaking culture is 20～30 hours on shaking table, makes first order seed;
(4) enlarged culturing: with volume ratio 5% inoculum size, connect first order seed and contain in the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT in 300～1000mL, under 25～50 ℃ of conditions, shaking culture is 20～30 hours on shaking table, makes secondary seed;
(5) fermentor cultivation: with volume ratio 5% inoculum size, connect secondary seed and contain in the MAM liquid nutrient medium of 0.1～2.0mmol/L DBT, under 25～50 ℃ of conditions, cultivated 20～30 hours in 1.8～8L;
In the method for sulphur, the MAM substratum of use is the A substratum of improvement in above-mentioned mycobacterium deep removal fossil oil, and it is as follows to fill a prescription: 10 gram glucose, 0.5 gram KH
2PO
4, 4 gram K
2HPO
4, 1 gram NH
4Cl, the 2mL mass percent is 1% CaCl
2, the 2mL mass percent is 10% MgCl
26H
2O, 200 μ L mass percents are 1% FeCl
3, 200 μ L mass percents are 5% NaCl, the 5mL mass percent is 1% yeast extract paste, the vitamin mixture of 200 μ L, the metal ion mixed solution of 5mL, adding distil water to 1 liter; Sterilization is 20 minutes under 115 ℃ of conditions; The prescription of metal ion mixed solution is that every liter of distilled water contains: ZnCl
20.5g; FeCl
20.5g; MnCl
24H
2O 0.5g; Na
2MoO
42H
2O 0.1g; CuCl
22H
2O 0.05g; Na
2WO
42H
2O 0.05g; HCl120mmol/L; The prescription of vitamine mixture is that every liter of distilled water contains: the 400mg calcium pantothenate; The 200mg inositol; The 400mg nicotinic acid; 400mg VB
6The 200mg para-amino benzoic acid; 0.5mg VB
12
(6) collect thalline: under 5000 rev/mins of conditions centrifugal 5～8 minutes, collect the thalline that step (5) makes, and with physiological saline washing 2～3 times; Again thalline is dissolved in the potassium phosphate buffer of 20～100mmol/L of 120mL, regulates pH to 7.5, make the concentration of thalline reach 10～30 gram stem cells/liter, promptly make biological catalyst ,-20 ℃ of storages, standby;
(7) handle sample: add 12～40mL sulfur-bearing fossil fuel sample or 10～30 gram solid sulfur-bearing fossil fuel sample, with 3～10: 1 ratio, biological catalyst in the step (6) is mixed with fossil oil, and under 25～50 ℃ of conditions, 180 rev/mins vibrated 24～30 hours;
(8) sample separation: with 10,000 rev/mins centrifugal 5～10 minutes, the sample of handling in the separating step (7) obtains oil phase;
(9) sample detection: after treating that sample separation in the step (8) is intact, get 1～5 μ L sample feeding, measure residual sulphur content in the fossil oil, draw decreasing ratio.
2. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that the yeast culture temperature described in step (2), (3), (4), (5) is 37～45 ℃.
3. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that the yeast culture time described in step (2), (3), (4), (5) is 24～28 hours.
4. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that, dibenzothiophene (DBT) concentration described in step (2), (3), (4), (5) is 0.2～0.5mmol/L.
5. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1, it is characterized in that the biological catalyst described in the step (6) is meant resting cell, contains the cellular component of desulphurase, one of separation and purification desulphurase system or its mixture.
6. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that, the cell concentration of the biological catalyst described in the step (6) be 15～25 the gram stem cells/liter.
7. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that the fossil oil described in the step (7) is an oil, coal, brown coal, one of pitch.
8. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that the ratio of biological catalyst described in the step (7) and fossil oil is 5～8: 1.
9. the method for utilizing organosulfur in the mycobacterium deep removal fossil oil as claimed in claim 1 is characterized in that, the temperature of handling sample in the step (7) is 37～45 ℃, and the sample duration of oscillation is 25～28 hours.
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| CN101134944B (en) * | 2007-04-20 | 2012-06-06 | 浙江大学 | Mycobacterium applied for fuel oil thiirane desulfuration and uses thereof |
| CN106994394B (en) * | 2017-04-13 | 2020-01-10 | 中国地质科学院矿产综合利用研究所 | Method for reducing combustibility of high-sulfur coal gangue |
| CN109293448A (en) * | 2018-09-21 | 2019-02-01 | 卫卓然 | A kind of preparation method of humic acid fertilizers |
| CN110510758A (en) * | 2019-09-10 | 2019-11-29 | 中交铁道设计研究总院有限公司 | A method of utilizing lead ion in Mycobacterium graminis removal sewage |
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| JP2001231546A (en) * | 2000-02-18 | 2001-08-28 | Petroleum Energy Center | Method for desulfurizing with microorganism at high temperature |
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| 黄铁矿烧渣微生物脱硫. 冯雅丽等.北京科技大学学报，第24卷第2期. 2002 |
| 黄铁矿烧渣微生物脱硫. 冯雅丽等.北京科技大学学报，第24卷第2期. 2002 * |
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