CN1236053C - Bacterium for degrding petroleum and its use - Google Patents
Bacterium for degrding petroleum and its use Download PDFInfo
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- CN1236053C CN1236053C CN 200410038053 CN200410038053A CN1236053C CN 1236053 C CN1236053 C CN 1236053C CN 200410038053 CN200410038053 CN 200410038053 CN 200410038053 A CN200410038053 A CN 200410038053A CN 1236053 C CN1236053 C CN 1236053C
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a bacterium for degrading petroleum and an application thereof. The bacterium for degrading petroleum provided by the present invention is brevibacillus brevis HT CGMCC No. 1142. Experiments show that the bacillus cereus HP CGMCC No. 1141 can effectively improve the crude oil property, the crude oil acid value is enhanced by more than 14 times, the light components are increased, the wax and glue containing reduction rate is from 30 to 50%, the crude oil rheologic property is good, the interfacial tension between oil and water is reduced, the content of organic acid, etc. in a fermentation liquor is increased, the metabolism crude oil path is penultimate oxidation, and a new lipid surfactant is generated. The bacterium can be widely applied to the oil production engineering field, particularly the microbial enhanced oil recovery field.
Description
Technical field
The present invention relates to the microorganism and the application thereof of a strain degraded oil in the bioengineering field, particularly the bacterium of a strain degraded oil and application thereof.
Background technology
The world today is carrying out one with biotechnology and be applied as the new technology revolution of sign, and biological develop energy technology becomes economic potential maximum in the biotechnology, is hopeful one of field with future most, analysis of modernization instrument and means also make the microorganism science enter a brand-new developing period, and these factors have promoted microbial enhanced oil recovery Study on Technology and application.
Oil is a kind of deregenerative energy, and after primary oil recovery and secondary oil recovery, the crude oil of the 60%-70% that still has an appointment in the stratum can't be exploited out.How to improve recovery ratio, from the more crude oil of underground extraction, be the constantly problems of research of many countries for many years always.What generally adopt at present is use the chemical process crude oil extraction, and the ASP ternary composite oil-displacing system of forming as alkali, tensio-active agent, polymkeric substance (Mu Jianhai, prune is helped .[J] chemical science and technology market .2000,23 (7): 17).Since 1980, a lot of countries have carried out the technology that improves oil recovery factor with microbial process, and through the effort of two more than ten years, this technology has obtained very big progress.The technology that this use chemistry or biotechnological formulation improve recovery ratio is called as tertiary oil recovery technology, also claims intensified oil reduction (EOR, Enhanced OilRecovery) technology abroad.For they are made a distinction, improve recovery efficiency technique with microorganism and also can be called four oil recovery techniques or microbial enhanced oil recovery (MEOR, Mcrobial Enhanced Oil Recovery) technology, it is meant and utilizes the useful metabolism product of microorganisms producing or utilize microorganism can decompose the performance of hydrocarbon polymer, thus the technology of raising oil recovery factor.
The microbial enhanced oil recovery technology is a new technology high in technological content, that development is swift and violent, is modern biotechnology ground-breaking application in the petroleum production engineering field, more demonstrates its great vitality for the moisture and approaching exhausted maturing field of height.The microbial enhanced oil recovery technology mainly comprise two classes (Li Yugeng. petroleum microbiology .[M]. Shanghai: the .1996 of press of Shanghai Communications University): a class is to utilize microniological proudcts such as biological polymer and bio-surfactant to carry out the displacement of reservoir oil as the oil field with chemical agent, be called microorganism ground top fermentation and improve recovery ratio technology, be the bioprocess technology method, this technology has become ripe at home and abroad at present; Another kind of is to utilize microorganism and meta-bolites thereof to improve recovery ratio, mainly is the vigor that utilizes microorganism ground bottom fermentation and utilize autochthonous microorganism in the oil reservoir, is called microorganism ground bottom fermentation and improves the recovery ratio method.
The separation of degraded oil microorganism is the basis of realizing the microbial enhanced oil recovery technology, and the quality of bacterial classification is the key of microbe oil production.At present, separated the microorganism that obtains some degraded oils, Beck as Pennsylvania crude oil association has reported the experiment that discharges crude oil with sulphate reducing bacteria, studies show that the good result (nineteen forty-seven) that sulphate reducing bacteria occurs in the bacterium oil decomposes, this experiment was drawn striking by the general Dege of strategic point afterwards and reyn further confirms (1954).Larry dimension thunder (nineteen fifty-five) has observed Pseudomonas aeruginosa, mycobacterium, subtilis, lipolytic enzyme read the surface tension in cultures such as glycerine such as coccus, and surface tension descends obviously, but utilizes hydro carbons and bad.
The innovation and creation content
The purpose of this invention is to provide the bacterium that a strain can degraded oil.
Bacterial strain that can degraded oil is short genus bacillus (Brevibacillus brevis) HT, oneself was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 04 08th, 2004, preserving number is CGMCC № 1142.The CGMCC address is No. 13 Institute of Microorganism, Academia Sinica in north, Zhong Guan-cun, Haidian District, BeiJing, China city, postcode 100080.
Short genus bacillus (Brevibacillus brevis) HT CGMCC № 1142 separates in the profit sample of first oil recovery factory of Daqing, Heilongjiang Province and obtains, its bacterium colony oyster white, and moistening, diameter 0.2mm-0.4mm, cell are shaft-like; Cell dia＞1 μ m; Form gemma, gemma expands, and gemma is non-circular; Physiological and biochemical property is as shown in table 1:
The short genus bacillus of table 1. (Brevibacillus brevis) HT physiological and biochemical property
| Test subject | The result | Test subject | The result |
| Carbohydrate produces acid | Gramstaining | Positive | |
| Glucose | - | The starch hydrolysis | - |
| Wood sugar | - | Gelatine liquefication | + |
| L-arabinose | - | Decompose casein | - |
| N.F,USP MANNITOL | - | Catalase | + |
| Utilize the glucose aerogenesis | - | Oxydase | + |
| Utilize Citrate trianion | - | Anaerobic growth | - |
| Nitrate reduction | + | The VP test | - |
| 50 ℃ of growths | + | VP＜pH6 | - |
| The pH5.7 growth | - | VP＞pH7 | - |
| The 7%NaCl growth | - | Methyl red test | - |
Annotate: "+" expression growth or reacting positive; "-" expression is not grown or reaction negative.
Experimental results show that, short genus bacillus (Brevibacillus brevis) HT CGMCC № 1142 can improve the character of crude oil effectively, acid value for crude oil improves more than 14 times, light component increases, the content of wax contains glue reduced rate 30-50%, rheological characteristic of crude oil improves, and interfacial tension reduces between profit, and substances content such as organic acid increases in the fermented liquid; Its metabolism crude oil approach is time terminal oxidation, has produced a kind of novel lipid tensio-active agent.This bacterial strain can be widely used in particularly microbial enhanced oil recovery field, petroleum production engineering field.
Description of drawings
Fig. 1 is the molten oily photo of short genus bacillus (Brevibacillus brevis) HT
Fig. 2 is the haemolysis photo that short genus bacillus (Brevibacillus brevis) HT produces table agent alive
Fig. 3 is that short genus bacillus (Brevibacillus brevis) HT bacterium number is with the variation of cultivating fate
Fig. 4 is short genus bacillus (Brevibacillus brevis) HT bacterium number and nitrogen concentration curve
When Fig. 5 is carbon source for whiteruss and crude oil to the influence curve of short genus bacillus (Brevibacillus brevis) HT bacterium number
Fig. 6 is the culturing bottle photo before and after short genus bacillus (Brevibacillus brevis) the HT effect crude oil
Fig. 7 is short genus bacillus (Brevibacillus brevis) HT effect back crude oil total hydrocarbon color atlas
Fig. 8 is a rheological characteristic of crude oil change curve before and after short genus bacillus (Brevibacillus brevis) the HT effect
Fig. 9 is blank oil sample aromatic hydrocarbon molecule amount
Figure 10 is short genus bacillus (Brevibacillus brevis) HT effect back oil sample aromatic hydrocarbon molecule amount
Figure 11 a be blank oil sample nonhydrocarbon micro--the infrared analysis collection of illustrative plates
Figure 11 b be short genus bacillus (Brevibacillus brevis) HT effect back oil sample nonhydrocarbon micro--the infrared analysis collection of illustrative plates
Figure 12 is an alkane microbiological oxidation general way
Figure 13 is an alkane time terminal oxidative pathway
Figure 14 a is the color atlas of organic acid mixing standard specimen
Figure 14 b is an organic acid color atlas in short genus bacillus (Brevibacillus brevis) the HT sample
Figure 15 a is the mass spectrum of acetate
Figure 15 b is the mass spectrum of propionic acid
Figure 15 c is butyro-mass spectrum
Figure 15 d is the mass spectrum of isovaleric acid
Figure 16 a is the color atlas of organic pure standard specimen
Figure 16 b is the organic pure color atlas of short genus bacillus (Brevibacillus brevis) HT sample
Figure 17 is the alcoholic acid mass spectrum
Figure 18 a is the infrared spectrogram of the B1 material that extracts in short genus bacillus (Brevibacillus brevis) the HT sample
Figure 18 b is the infrared spectrogram of the B2 material that extracts in short genus bacillus (Brevibacillus brevis) the HT sample
Figure 18 c is the infrared spectrogram of the C material that extracts in short genus bacillus (Brevibacillus brevis) the HT sample
Embodiment
Percentage concentration among the embodiment is mass percent concentration.
The separation of embodiment 1, short genus bacillus (Brevibacillus brevis) HT
The separation of short genus bacillus (Brevibacillus brevis) HT is divided into sampling and enrichment culture, screening and two steps of purifying, and concrete grammar is as follows:
1, sampling and enrichment culture
Get behind the profit sample of first oil recovery factory of Daqing, Heilongjiang Province enrichment culture immediately, required bacterial classification is quantitatively preponderated, suppress unwanted growth.What enrichment medium was used is minimal medium: K
2HPO
40.1-1%, NaH
2PO
40.1-0.5%, (NH
4)
2SO
40.05-0.2%, MgSO
47H
2O 0.01-0.5%, FeCl
20.001-0.01%, CaCl
20.001-0.01%, yeast soak powder 0.02-0.2%, crude oil 0.5-20%; PH 6.8-7.5121 ℃, sterilization 15-20min.45 ℃ of 120rpm shaking tables were cultivated 5-7 days.
2, screening and the purifying of short genus bacillus (Brevibacillus brevis) HT
Two kinds of selective mediums of screening usefulness are crude oil inorganic salt solid medium and blood agar substratum.
A. the screening on the crude oil inorganic salt solid medium
Do not add any carbon source in this selective medium except crude oil, the bacterial classification that filters out is exactly that department is to be the bacterial classification of sole carbon source growth with crude oil.Preparation process 1 described enrichment inorganic salt solid medium flat board under aseptic condition, crude oil after the dilution is poured into (add-on of crude oil is 1ml crude oil/L substratum) on the inorganic salt solid medium flat board for preparing, after treating that crude oil is tiled in the inorganic salt planar surface equably and solidifies, the enrichment culture liquid in the step 1 is uniformly coated on the flat board.Cultivated 3-5 days for 45 ℃, the result can find out significantly that the crude oil in long bacterium place is utilized as shown in Figure 1, and the former oil reservoir attenuation on the inorganic salt flat board forms transparent circle.Selection can form the bacterial classification of transparent circle, waits to be further purified.
B. the screening of blood agar cultivation
Enrichment culture liquid in the step 1 is uniformly coated on the blood agar.Cultivated 1-2 days for 45 ℃, the result shows that the periphery of bacterial colonies after the cultivation forms tangible haemolysis transparent circle as shown in Figure 2, selects such bacterium colony and waits to be further purified.Because bio-surfactant has can hemolytic characteristic, hemolytic single bacterium colony promptly is the bacterial classification that produces bio-surfactant.The composition of blood agar substratum: extractum carnis 0.3%, peptone 1%, yeast extract paste 0.01%, sodium-chlor 0.5%, agar 2%, sheep blood 5%.
C. will dye down by single colony microscope that above-mentioned selective medium obtains and observe, and, utilize above-mentioned substratum to separate again, up to obtaining pure bacterium colony if impure.In order to obtain anaerobism and facultative bacterial classification, the bacterial classification that obtains by above-mentioned two kinds of selective mediums in the anaerobism workstation 45 ℃ cultivated 5 days, acquisition is a strain bacterium HT of sole carbon source with crude oil, and this bacterial strain has good product tensio-active agent, produce acid, improve characteristic such as crude oil property.
Can select for use following two kinds of methods that this bacterial strain is carried out secular culture presevation: 1. cryopreservation: to get the bacterial classification that is in logarithmic phase, make protective material-70 ℃ of preservations with 15% aseptic glycerine; 2. lyophilize prepares dry powder: centrifugal fermented liquid obtains thalline, makes protective material with skimmed milk, and dry powder is made in lyophilize.
The culture condition of embodiment 2, short genus bacillus (Brevibacillus brevis) HT and process optimization experiment
In order to improve bacterium number in the nutrient solution and favourable meta-bolites, culture condition and the process of short genus bacillus (Brevibacillus brevis) HT of screening is optimized, to obtain comparatively ideal experiment condition.Reach the suitableeest nitrogenous source and the optimization of inorganic salt amount comprising incubation time.Concrete grammar is as follows:
1, the optimization of incubation time
The bacterial classification of short genus bacillus (Brevibacillus brevis) HT inserts liquid nutrient medium: K
2HPO
40.5%, NaH
2PO
40.5%, (NH
4)
2SO
40.1%, MgSO
47H
2O 0.1%, FeCl
20.005%, CaCl
20.001%, yeast soaks powder 0.1%, white oil 10%, and pH6.8-7.5 cultivated 1,3,5,7,9,11,13,15,17 day at 45 ℃ of difference 120rpm, with blood counting chamber meter bacterium number; The result shows that logarithmic growth finishes when fermented liquid is cultivated 30 hours as shown in Figure 3, enters the stage of stable development, and the bacterium number is 6 * 10 to the maximum
9Individual/ml, the stage of stable development can remain to about the 6th day, and back bacterium number slowly descends.
How many Optimum of culture medium is standard with the bacterium number, and when when obtaining thalline and be purpose, incubation time is 30 hours, and the carbon source of substratum is a whiteruss.But to obtain the bacterial metabolism product or when crude oil produced best effects and be purpose, the carbon source of substratum is a crude oil, and was also different when time and whiteruss are carbon source.According to experimental result, make this bacterial strain that crude oil is produced best effect, comprise effects such as emulsification, viscosity reduction, degraded, this bacterial strain meta-bolites accumulation or be 3-15 days to crude oil effect Best Times.
2, nitrogenous source (NH
4)
2SO
4Right bacterium number influence
It is 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.3% (mass percent) (NH that the bacterial classification of short genus bacillus (Brevibacillus brevis) HT adds concentration respectively with identical inoculum size access
4)
2SO
4Liquid nutrient medium: K
2HPO
40.5%, NaH
2PO
40.5%, MgSO
17H
2O 0.1%, FeCl
20.005%, CaCl
20.001%, yeast soaks powder 0.1%, white oil 10%, among the pH6.8-7.5 after, 45 ℃ respectively 120rpm cultivated blood counting chamber meter bacterium number 30 hours.The result shows as shown in Figure 4 as (NH
4)
2SO
4Concentration be 0.2% and 0.225% o'clock bacterium number the highest, reach 2.4 * 10
10Individual/ml.
3, inorganic salt K
2HPO
4And NaH
2PO
4Optimal dose
In the microorganism growth metabolic process, potassium, sodium ion and phosphate anion are not only as the important component of cell but also as important metabolic regulation material, in thalli growth and fermenting process, play an important role, their content in substratum how much, certainly will influence the output of purpose meta-bolites in microbial growth and the fermented liquid.Simultaneously, inorganic salt K
2HPO
4And NaH
2PO
4Be again the pH damping fluid in nutrient solution, crucial effect is played in pair cell adjusting of pH value in fermentation production process.Utilize orthogonal experimental design, the bacterial classification of short genus bacillus (Brevibacillus brevis) HT is added the K of concentration shown in the table 2 with identical inoculum size access respectively
2HPO
4And NaH
2PO
4Liquid nutrient medium: (NH
4)
2SO
40.1%, MgSO
47H
2O 0.1%, FeCl
20.005%, CaCl
20.001%, yeast soaks powder 0.1%, white oil 10%, among the pH 6.8-7.5 after, 45 ℃ respectively 120rpm cultivated blood counting chamber meter bacterium number 30 hours.The result is as shown in table 2:
Table 2. inorganic salt K
2HPO
4And NaH
2PO
4Experimental result
| Experiment numbers | K 2HPO 4 NaH 2PO 4(mass percent concentration %) | The bacterium number (individual/ml) | |
| 1 | 0.06 | 0.15 | 1.7×10 8 |
| 2 | 0.08 | 0.15 | 1.7×10 9 |
| 3 | 0.1 | 0.15 | 3.7×10 9 |
| 4 | 0.12 | 0.15 | 1.0×10 9 |
| 5 | 0.06 | 0.2 | 9.0×10 8 |
| 6 | 0.08 | 0.2 | 1.0×10 10 |
| 7 | 0.1 | 0.2 | 2.4×10 10 |
| 8 | 0.12 | 0.2 | 1×10 10 |
| 9 | 0.06 | 0.25 | 7.33×10 8 |
| 10 | 0.08 | 0.25 | 3.7×10 9 |
| 11 | 0.1 | 0.25 | 9×10 9 |
| 12 | 0.12 | 0.25 | 6.7×10 9 |
Table 2 shows, as two kinds of inorganic salt K
2HPO
4And NaH
2PO
4Amount be respectively 0.1% and 0.2% o'clock bacterial concentration the highest, and the pH value of solution value is about 7 during this concentration, the pH of solution remains unchanged substantially during the fermentation, is easy to the strain fermentation process is controlled.Therefore, these two kinds of inorganic salt both can provide the nutrient of enough growths for this strain growth in this concentration, can play good shock absorption to fermentating liquid PH value again.
When 4, being carbon source, to the influence of bacterium number with whiteruss and crude oil
Verified short genus bacillus (Brevibacillus brevis) HT can utilize crude oil to be carbon source for growth in bacterial screening.With K
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH 7.0-7.2, carbon source is that 2% whiteruss or 4% crude oil are that substratum is at 45 ℃ of fermentation short genus bacillus (Brevibacillus brevis) HT, get the nutrient solution of cultivating 1 day, 3 days, 5 days, 7 days, 10 days, 15 days, blood counting chamber meter bacterium number is surveyed the influence of different carbon sources to growth.The result shows with the whiteruss to be in the substratum of carbon source as shown in Figure 5, and the bacterium number has reached very high value in the time of first day, can remain to the 7th day, and the bacterium number obviously descends after 10 days.Be that the bacterium number reached very high quantity in the culture of carbon source in the time of the 5th day with crude oil, when measuring 15 days, still keep a value stably.When being carbon source with the whiteruss, the bacterium number can reach maximum value very soon.This is because whiteruss is a kind of mixture of saturated alkane, and this bacterial strain is easier to utilize.Can obtain enough cultures in the very short time when therefore, fermenting on ground.After the 7th day, because the minimizing of the accumulation of products of cellular metabolism and nutritive substance and cell wait influence from melting, the bacterium number begins minimizing.And when being carbon source with crude oil 3-5 days the time bacterium number reach higher value, and can keep for a long time, this helps microorganism and utilizes crude oil to breed for carbon source for growth underground under the condition of very a spot of other carbon source of adding, keep very high bacterium number for a long time, to crude oil generation effect, reach the effect of the displacement of reservoir oil.
Determined the prescription of substratum: K according to this experimental result
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH7.0-7.2.Carbon source is 2% whiteruss or 4% crude oil.
Embodiment 3, short genus bacillus (Brevibacillus brevis) HT are to the degraded of crude oil
1, dispersion and emulsion effect
Short genus bacillus (Brevibacillus brevis) HT bacterial classification is containing K
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH7.-7.2, carbon source is in the substratum of 10% crude oil 45 ℃, the 120rpm shaking table cultivate after 5 days the result as shown in Figure 6, show and contrast (not adding bacterium) and compare, the color of fermented liquid is obviously deepened, and effect back crude oil has the advantages that not hang bottle.Because microorganism is the sole carbon source growth with crude oil, changed the character of crude oil, make the component of crude oil that variation take place.Among Fig. 6, the left side culturing bottle is contrast, and the right side culturing bottle is the fermentation culture of short genus bacillus (Brevibacillusbrevis) HT.Crude oil after the fermentation is examined under a microscope, and finds to have in the crude oil bacterium liquid to exist.Bacterium exists on the water-oil interface, and is carbon source with oil, conforms to produce displacement of reservoir oil material, thereby plays emulsification, wetting, the effect that disperses crude oil.
2, crude oil total hydrocarbon color atlas before and after short genus bacillus (Brevibacillus brevis) the HT effect
Get in the step 1 crude oil of fermentation after 5 days and do the total hydrocarbon analysis of crude oil according to a conventional method.Result such as table 3 and shown in Figure 7, table 3 shows from the variation of total hydrocarbon stratographic analysis parameters to be seen, this bacterial strain is some the high carbon number alkane in the degrading crude oil optionally, and long chain hydrocarbon content reduces relatively, short hydrocarbon or low chain hydrocarbon content increase relatively, and the light component of crude oil increases; In addition, ∑ C
21/ ∑ C
22And C
21+ C
22/ C
28+ G
29Be the parameter of describing oil-gas migration, ∑ C so
21/ ∑ C
22And C
21+ C
22/ C
28+ G
29Ratio increase, the direction of expression oil migration, along with macromolecular compound content reduces relatively, the light constituent compounds content increases relatively.Fig. 7 shows because the thermal destruction of high-molecular weight hydrocarbon and shifting to the low carbon number scope, and color atlas gradually becomes preceding high cutting edge of a knife or a sword type, and normal alkane carbon number distribution curve moves to light component direction.The flowability of crude oil improves.
The stable hydrocarbon chromatogram detected result of the crude oil after the table 3. bacterial strain HT fermentation
| Sample | Carbon number range | Main peak carbon | ∑C21 ∑C22 | C21+C22 C28+C29 |
| Blank | Nc9-nc35 | nc23 | 0.67 | 1.66 |
| HT | Nc8-nc35 | nc23 | 1.62 | 1.89 |
3, rheological characteristic of crude oil changing conditions
Short genus bacillus (Brevibacillus brevis) HT bacterial classification is containing K
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH7.0-7.2, carbon source is in the substratum of 10% crude oil 45 ℃, respectively after the 120rpm shaking table is cultivated 5 days, analyzes rheological characteristic of crude oil before and after the microbial process according to a conventional method, the result shows that effect back rheological characteristic of crude oil obviously improves as shown in Figure 8, and viscosity reduces.When rotor revolution number was 61/s, viscosity of crude was 101mPas before the effect, and viscosity of crude is reduced to 56.9mpas after this bacterial strain effect, and viscosity of crude has reduced by 43.7%.
4, the crude oil content of wax, contain glue and change
After in step 2, having analyzed crude oil alkane distribution situation, in order further to inquire into the changing conditions of the bacterial strain effect front and back crude oil content of wax, glue, do the crude oil content of wax according to a conventional method, contained glue variation experiment, the result is as shown in table 4, show that the wax of crude oil before and after the bacterial classification effect and gelationus content all have variation in various degree, wax content in crude oil drops to 12.6% by barren 20.2% after this bacterial strain effect, and gel content drops to 15.1% by barren 19.8%.
The change list of crude oil wax, glue before and after the effect of table 4. bacterial classification
| The sample title | Content of wax % | Contain glue % |
| Blank crude oil | 20.2 | 19.8 |
| HT | 12.6 | 15.1 |
5, the variation of aromatic hydrocarbons in the crude oil
Get in the step 1 crude oil of fermentation after 5 days and carry out GC-MS according to a conventional method, the relative content of the Sino-Philippines series of analyse crude oil aromatic hydrocarbons, the result is as shown in table 5, shows optionally degraded different components in the luxuriant and rich with fragrance series of this bacterial strain, causes MPI, DPI index variation.Luxuriant and rich with fragrance series in the crude oil is commonly considered as the cracking from steroid terpene compound.Because alkyl position difference on phenanthrene ring, stability is also variant, generally be in the methyl of β position, more stable as 3-methyl and 2-methylphenanthrene, in the 1-of α position methyl, 4-methylphenanthrene and to be in the 9-methylphenanthrene of meta more active, dimethylphenanthrene also has similarity rules, to β β type, stability increases successively from α α type, α β type, after this bacterial strain effect, the MPI of crude oil and DPI index all improve, and illustrate that the comparatively active component of chemical property in the luxuriant and rich with fragrance series is easily by this strains for degrading.Its total ion current figure such as Fig. 9 and shown in Figure 10.
Aromatic hydrocarbons GC-MS analytical results in the crude oil before and after the effect of table 5.HT bacterial strain
| Sequence number | The sample title | Luxuriant and rich with fragrance serial content (%) | | DPI | |
| 1 | Blank oil | 7.26 | 0.67 | 0.87 | |
| 2 | HT oil | 7.21 | 0.72 | 0.91 | |
| Remarks: MPI=1.5 * (2-methylphenanthrene+3-methylphenanthrene)/(phenanthrene+1-methylphenanthrene+9-methylphenanthrene) DPI=4 * (dimethylphenanthrene 1.+dimethylphenanthrene 2.+dimethylphenanthrene 3.+ | |||||
6, the variation of nonhydrocarbon in the crude oil
Get in the step 1 crude oil of fermentation after 5 days and adopt micro--infrared method to analyze before and after this bacterial strain effect the variation of non-hydrocarbon constituents and structure in crude oil, the result is shown in Figure 11 a and Figure 11 b, and Figure 11 a shows 2924cm
-1And 2853cm
-1The absorption peak difference represent methylidene at place and the asymmetric and symmetrical stretching vibration of C-H of methylene radical, methylene radical is dominant, because the C-H stretching vibration peak of general methyl is at 2960cm
-1And 2870cm
-1The place is covered by methylene peak, adds 722cm
-1The peak crude oil nonhydrocarbon is described based on long carbon saturated chain, and the side chain degree is little for the absorption peak of methylene radical in the long carbochain (n＞4); 1461cm
-1And 1376cm
-1The C-H flexural vibration peak of place's methyl, methylene radical is the circumstantial evidence of above deduction.1601cm
-1The absorption peak at place may be that the two key stretching vibrations of C=N, C=C cause, but according to fingerprint region 910cm
-1Following not obvious infrared absorption peak (relevant with the position of substitution of phenyl ring) can be inferred and be contained a certain amount of aromatic hydrocarbons composition in the crude oil nonhydrocarbon.1650--1900cm
-1Absorption peak in the scope mainly shows as carbonyl compound, as the C-O stretching vibration of aldehyde, ketone, acid, ester, acid anhydrides and acid amides etc., from infrared spectra, a little less than the absorption intensity at the interior peak of this scope, and becoming broadband shape, may be because contain the mixing cpd of a small amount of band carbonyl in the crude oil nonhydrocarbon.Figure 11 b show after this bacterial strain effect with microbial process before to compare the key distinction be new infrared absorption peak to occur at the 2483cm place, peak shape is wide and blunt, this since hydroxyl produce at intermolecular or intramolecularly formation hydrogen bond, but at 1603cm
-1The place does not have tangible infrared absorption peak, illustrates that hydrogen bond is not to cause because contain water in the sample.According to the skew rule of peak shape feature and chemical shift, inferring after this bacterial strain effect has a certain amount of carboxylic acid to generate in the luxuriant and rich with fragrance hydrocarbon of crude oil, in the carboxylic acid because the strong association of carbonyl and hydroxyl, can be according to the different infrared absorption peaks of carboxylic acid content from 3300cm
-1Be extended to 2500cm
-1Below.
For further confirmation, measured the acid number of this bacterial strain effect front and back crude oil nonhydrocarbon, the result is as shown in table 6, show after this bacterial strain effect, the acid number of crude oil nonhydrocarbon is increased to original 14 times, illustrate that this bacterial strain in the degradation process to the crude oil different components biooxidation reactions has taken place, generated high-carbon organic acid, improved the acid number of crude oil.
Crude oil nonhydrocarbon acid number analytical results before and after the effect of table 6.HT bacterial strain
| Sequence number | The sample title | Nonhydrocarbon acid number (%) | Acid number increases (doubly) |
| 1 | Blank oil | 0.1037 |
| 2 | HT oil | 1.4799 | 14.27 |
7, stable hydrocarbon biological degradation Analysis on Mechanism in the crude oil
From above research as can be seen, this bacterial strain to the degraded of crude oil to be degraded to main path.Most of microbe can be summarized with Figure 12 the main oxidative pathway of alkane, at first is alkane to be carried out terminal oxidation (or β-Yang Hua) generate lipid acid, press β-Yang Hua approach degraded alkane again, and association acyl group-CoA participates in the Metabolic activity of microorganism.Usually, for a specific alkane, microbiological deterioration product lipid acid is one to differ the mixing acid of two carbon each other, and just degradation condition and carbon number is different, and the carbon number distribution of mixing acid is different.This bacterial strain shows that to the composition analysis result of oil degradation the carbon number that mixes fat acid acid concentrates on C
2-C
20In the scope.This illustrates that this bacterial strain may be since a comparatively special approach to the bio-oxidation of crude oil---inferior terminal oxidation (subterminal oxidation) (as Figure 13).Crude oil medium high carbon chain stable hydrocarbon is the lipid acid of carbochain in time terminal oxidation generates two kinds at first, carries out terminal oxidation and β-Yang Hua again.
The analysis of embodiment 4, bacterial strain fermentation liquor
(1) organic acid The qualitative analysis
Utilize 6890N/5973N gas chromatograph-mass spectrometer (GC-MS) (U.S. Agilent company), (0.25mm * 30m), carrier gas is high-purity helium to the FFAP quartz capillary column, flow 1ml/min, 250 ℃ of temperature of vaporization chamber, column temperature adopts temperature programming, and the beginning temperature is 160 ℃, speed is 2 ℃/min, outlet temperature is 180 ℃, and sample size 0.5 μ l carries out the organic acid qualitative test according to ordinary method, the color atlas of organic acid standard specimen such as Figure 14 a, the retention time under chromatographic condition is as shown in table 7.
The organic acid gas chromatogram of HT sample such as Figure 14 b.Contrast through mass spectrometric detection with the organic acid standard specimen, containing four kinds of organic acids in the HT sample is acetate, propionic acid, butyric acid, isovaleric acid, organic acid mass spectrometric detection figure such as Figure 15 a, 15b.Shown in 15c and the 15d.
The retention time of table 7. organic acid standard specimen
| Acetate | Propionic acid | Butyric acid | Isovaleric acid | |
| Retention time (min) | 2.16 | 2.50 | 2.99 | 3.26 |
(2) organic acid quantitative analysis results
1. water sample distilling effect test
This extracting method (is measured pretreated sample 100ml, is placed distilling flask, add the acidifying of 7ml phosphoric acid, distillation.When distilling flask is left small volume of solution, cold slightly, respectively add 20ml distilled water more at twice and continue distillation.With in the NaOH solution and distillate, PH is 8～9 with acidometer control.After being concentrated into about 5ml with Rotary Evaporators, sample is changed in the 10ml volumetric flask, with hcl acidifying to PH≤3, constant volume, organic acid to be measured) can extract various organic acids fully, done the distilling effect test.Promptly respectively get five portions of HT fermented liquids of 100ml, handle through aforesaid method and measure, the result is as shown in table 8:
The test of table 8. organic acid distilling effect
| Organic acid | (five times average: mg/L) for measured value | Standard is inclined to one side | The variation coefficient (%) |
| Acetate | 24.35 | 1.02 | 4.2 |
| Propionic acid | 20.41 | 0.73 | 3.6 |
| Butyric acid | 3.082 | 0.16 | 5.3 |
| Isovaleric acid | 1.269 | 0.37 | 1.6 |
Table 8 shows: five measurement result circulation ratios of same sample are better, illustrate to adopt the distillatory method fully the low molecular weight organic acid in the water sample to be steamed.
2. the organic acid quantitative analysis of HT sample
Short genus bacillus (Brevibacillus brevis) HT bacterial classification is containing K
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH 7.0-7.2, carbon source is in the substratum of 10% crude oil, 45 ℃, nutrient solution after the 120rpm shaking table is cultivated 5 days is made three parallel samples respectively respectively, utilizes 6890N/5973N gas chromatograph-mass spectrometer (GC-MS) (U.S. Agilent company), and the FFAP quartz capillary column (0.25mm * 30m), carrier gas is high-purity helium, flow 1ml/min, 250 ℃ of temperature of vaporization chamber, column temperature adopts temperature programming, the beginning temperature is 160 ℃, speed is 2 ℃/min, and outlet temperature is 180 ℃, and sample size 0.5 μ l measures peak value, obtain organic acid concentration mean value, the result is as shown in table 9, shows that acetic acid content is bigger in the HT sample, based on acetate.
Calculation formula:
(ρ is a standard specimen density to standard specimen concentration (mg/L)=20/50 * ρ * 1000=400 ρ, g/ml) treats the concentration of test sample
Organic acid quantitative analysis results in the table 9.HT sample
| Concentration (mg/L) | Standard deviation | The variation coefficient (%) | Volumetric molar concentration (mmol/L) | |
| Acetate | 574.0 | 45.3 | 7.9 | 9.5667 |
| Propionic acid | 8.318 | 0.44 | 5.3 | 0.1124 |
| Butyric acid | 14.68 | 1.15 | 7.8 | 0.1668 |
| Isovaleric acid | 7.131 | 0.83 | 6.1 | 0.0699 |
| Total acid content | 9.9158mmol/L | |||
2, the organic pure qualitative and quantitative analysis of fermented liquid
Utilize 6890N/5973N gas chromatograph-mass spectrometer (GC-MS) (U.S. Agilent company), the FFAP quartz capillary column (0.25mm * 30m), carrier gas is high-purity helium, flow 1ml/min, 200 ℃ of temperature of vaporization chamber, column temperature adopts temperature programming, the beginning temperature is 80 ℃, keeps 1 minute, and speed is 4 ℃/min, 110 ℃ of outlet temperatures, sample size 1.0 μ l carry out the qualitative test of organic alcohol according to ordinary method, and the color atlas of organic pure standard specimen is shown in Figure 16 a, show that under this chromatographic condition four kinds of organic pure separating effects are fine; Retention time under chromatographic condition is as shown in table 10.
The organic pure gas chromatogram of HT sample is shown in Figure 16 b.Through mass spectrometric detection, only contain ethanol (Figure 17) in the HT sample.
The retention time of the organic pure standard specimen of table 10.
| Ethanol | Propyl alcohol | Butanols | Amylalcohol | |
| Retention time (min) | 1.93 | 2.47 | 3.51 | 5.00 |
Ethanol quantitative result such as table 11 in the HT sample.Wherein 20 μ l ethanol constant volumes are in the 50ml volumetric flask, and concentration is 315.7m g/L, the same organic acid of quantitative analysis method of calculation.
Table 11.HT sample alcoholic acid quantitative analysis results
| Concentration (mg/L) | Standard deviation | The variation coefficient (%) | Volumetric molar concentration (mmol/L) | |
| Ethanol | 759.9 | 28.5 | 3.8 | 16.52 |
3, the evaluation of bio-surfactant
(1) preliminary evaluation of bio-surfactant
1. the evaluation of lipopeptid type biological surfactant
Get short genus bacillus (Brevibacillus brevis) HT bacterial classification and containing K
2HPO
40.1%, NaH
2PO0.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, and PH 7.0-7.2, carbon source is in the substratum of 10% crude oil, 45 ℃, the nutrient solution 50ml after the 120rpm shaking table is cultivated 5 days transfers pH value to 2 with concentrated hydrochloric acid respectively, 4 ℃ of standing over night, whether observe has precipitation to produce.The result shows that this fermented liquid does not have precipitation and produces, and proves thus and does not contain the lipopeptid type biological surfactant in this fermented liquid.
2. uv-absorbing is identified
Bio-surfactant after purifying has according to a conventional method been done determination of uv absorption, and they all do not have absorption in ultraviolet range.Can tentatively judge and not contain aromatics.
(2) ultimate analysis of bio-surfactant
The three kinds of bio-surfactants that obtain of purifying have according to a conventional method been carried out C, H, N Determination on content, and data are as shown in table 12:
The results of elemental analyses of table 12. bio-surfactant
| C(％) | H(％) | N(％) | |
| Extract the B1 material among the HT | 73.06 | 11.29 | 3.50 |
| Extract the B2 material among the HT | 77.30 | 15.53 | 2.93 |
| Extract the C material among the HT | 66.33 | 8.71 | 2.38 |
Table 12 shows the content of these three kinds of bio-surfactant N all below 3.5%, and the content of N is all more than 10% in the amino acid of reporting on the document, and as seen, these three kinds of bio-surfactants are not amino acid and lipopeptid class.And detect with ninhydrin solution, amino acid whose characteristic reaction do not occur, further determined above-mentioned conclusion.
(3) Infrared spectroscopy of bio-surfactant
The B1 that the HT sample obtains after purifying, the infrared spectrum of B2 material shown in Figure 18 a, 18b, show 2924-2853cm respectively
-1, 1456cm
-1Be the C-H vibration peak of methyl, methylene radical, do not have the characteristic peak of other bio-surfactant.
The infrared spectrum of the C material that the HT sample obtains after purifying shows 2922-2872cm shown in Figure 18 c
-1, 1455cm
-1It is the C-H vibration peak of methyl, methylene radical; At 1724cm
-1Near peak is the stretching vibration of C=0; At 1103cm
-1Neighbouring is the symmetrical stretching vibration of C-O-C, shows the existence that ester is arranged.At 3382cm
-1The stretching vibration peak that tangible O-H is arranged, showing has hydroxyl in the compound.
From ultraviolet and infrared spectrum analysis, the C material that the HT sample obtains after purifying is a glycolipid class tensio-active agent.But using the qualitative method to carbohydrate is that the anthrone reagent method is measured, and does not show the characteristic reaction of carbohydrate again.Therefore the C material should be a kind of novel lipid tensio-active agent.
4, the interfacial tension analysis of HT bacterial strain fermentation liquor
The drop volume interfacial tensimeter that utilizes German LAUDA company to produce is containing K to short genus bacillus (Brevibacillus brevis) HT bacterial classification under 45 ℃ of current intelligences
2HPO
40.1%, NaH
2PO
40.2%, (NH
4)
2SO
40.2%, CaCl
22H
2O 0.001%, FeSO
47H
2O 0.001%, yeast 0.1%, PH7.0-7.2, carbon source is in the substratum of 10% crude oil, and 45 ℃, interfacial tension detects between nutrient solution after the 120rpm shaking table is cultivated 5 days and crude oil respectively, detected result is as shown in table 13, shows that the HT bacterial strain can well the metabolism biological tensio-active agent.Bio-surfactant has hydrophilic and hydrophobic grouping in the meta-bolites of bacterium, aligns on water-oil interface, improves interfacial activity, reduces interfacial tension.
The interfacial tension determination data of table 13.HT bacterial strain fermentation liquor
| The sample title | Interfacial tension (mN/m) |
| Local water | 30.24 |
| HT | 13.39 |
Claims (2)
1, short genus bacillus (Brevibacillus brevis) HT CGMCC № 1142.
2, short genus bacillus (Brevibacillus brevis) HT CGMCC № 1142 is in the petroleum production engineering Application for Field.
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