Patent Document:

the invention will be described in details by combining the following embodiments . ( 1 ) adjustive effects of different nutrient systems and temperatures on the growth and metabolism of microorganism ( s ) system composition : formation water of an oil reservoir + crude oil or sucrose + oil flooding microbial strain + baseline microbial strain ( microorganism ( s ) in the formation water and microorganism ( s ) in active sludge )+ nutrient system , wherein the oil flooding bacteria include hydrocarbon degrading bacteria tf2 and biosurfactant producing bacteria hn1 . tf2 is capable of growing by using glucose and n - hexadecane as a carbon source . when using a hydrocarbon mixture of n - hexadecane to n - docosane as a carbon source in the culture process , tf2 gives priority to n - hexadecane . tf2 grows better in the condition of temperature of 50 ° c .˜ 65 ° c ., ph = 6 ˜ 9 , and salinity smaller than 2 % ( nacl ), and grows best in the condition of temperature of 55 ° c ., ph = 7 , and salinity of 0 . 5 % ( nacl ). it is identified that the hydrocarbon degrading bacteria tf2 is g . subterraneus str . 34t . hh1 bacteria is uniform in size and light in color and has high movement frequency . the hn1 bacterial colony is fine and compact , yellow and opaque , its surface is flat , rough and beruffled , the edge of the colony is irregular , single colony is small and is easy to be picked up . it is identified that hn1 is b . subtilis . the carbon source in the nutrient system is sucrose ( 1 %) or crude oil ( 1 %), or their mixture ( 0 . 5 % + 0 . 5 %), the nitrogen source is peptone ( 0 . 25 %) or ammonium chloride ( 0 . 2 %), additionally yeast extract ( 0 . 2 %) and k 2 hpo 4 ( 0 . 08 %), nah 2 po 4 ( 0 . 04 %), mgso 4 . 7h 2 o ( 0 . 02 %), cacl 2 . 2h 2 o ( 0 . 01 %), and nacl ( 0 . 02 %). the cultures are carried out at 37 ° c ., 55 ° c . and 65 ° c ., respectively , and transferred four times to study the adjusting and controlling effects of different carbon sources and nitrogen sources on the metabolic performances and community structures of microorganism ( s ). results of adjusting and controlling cultures are : even starting from the same system , both community and function of a system change obviously under the adjustment and controlling of different carbon sources , nitrogen sources and temperatures . the concentration of microbiome is the highest at 37 ° c ., the dominant bacteria is hn1 , a favorable emulsifying effect is obtained when taking the mixture of sucrose and crude oil as a carbon source whichever of peptone and ammonium chloride is the nitrogen source . when taking sucrose as the carbon source and peptone as the nitrogen source , the content of lipopeptide in the system reaches to 180mg / l , and the surface tension is reduced by 29 %. the concentration of microbiome is slightly lower at 55 ° c ., the dominant bacteria is tf2 , a favorable emulsifying effect is obtained when taking crude oil as a carbon source and peptone as a nitrogen source , and when taking the mixture of sucrose and crude oil as the carbon source and peptone as the nitrogen source , the content of lipopeptide in the system reaches to 320mg / l , and the surface tension is reduced by 25 %. the concentration of microbiome is the lowest at 65 ° c ., the strains in the baseline reproduce to become the dominant bacteria in the microbiome , a favorable emulsifying effect is obtained when taking the mixture of sucrose and crude oil as a carbon source and ammonium chloride as a nitrogen source . when taking sucrose as the carbon source and peptone as the nitrogen source , the content of lipopeptide in the system reaches to 200 mg / l , and the surface tension is reduced by 30 %. therefore , when the nutrient systems are formed by different carbon sources and nitrogen sources to cultivate the same stain at different temperatures , the dominant bacteria cultivated in the system is different , the yield of surfactant obtained in the nutrient system is different , and the reduction of surface tension is different . therefore , the performance of a target microorganism can be maximized by pertinently selecting a nutrient system according to different temperature of an oil reservoir to improve the effect of microbial on enhanced oil recovery . it &# 39 ; s shown in the orthogonal experiments results that the preferred carbon source and nitrogen source for pseudomonas aeruginosa producing glycolipid in fermentation process are soybean oil and sodium nitrate , respectively . the preferred nutrient substrate formula is as follows : 0 . 2 g / l of yeast extract , 120 g / l of soybean oil , 6 . 5 g / l of nano 3 , 1 . 0 g / l of kh 2 po 4 , 1 . 0 l of na 2 hpo 4 . 12h 2 o , 0 . 1 g / l of mgso 4 . 7h 2 o , and 0 . 2 g / l of feso 4 . 7h 2 o . through single factor experiments and orthogonal experiments , the nutrient systems for pseudomonas putida producing glycolipid in fermentation process are studied and the results show that the preferred composition is as follows : the carbon source of 0 . 5 % sucrose + 0 . 5 % crude oil , the nitrogen source of 0 . 2 % ammonium chloride , 0 . 2 % yeast extract , 0 . 08 % kh 2 po 4 , and 0 . 04 % nah 2 po 4 . the bacterial diversity in a water sample produced from an oil reservoir is evaluated via a rflp fingerprint pattern analysis method , the results show that in 74 operational taxonomical units ( otu ), four most abundant otus account for 73 . 6 % of total clones , and the abundances of remaining 70 otus are at low levels , of which 57 otus are represented by a single clone . the dominant microflora is obvious in the oil reservoir environment , the quantity of the primary bacterial accounts for over half of the total quantity , of which the most abundant bacterial accounts for 47 . 7 % of the total , indicating that the bacterial is possibly suitable for high - temperature and high - pressure environmental conditions of the oil reservoir . the 16s rrna gene library analysis method is combined with the rflp fingerprint pattern analysis method to analyze diversity of bacteria and archaea community in a high - temperature water flooding onshore reservoir environment in china to obtain the following bacterial species and amount : gamme - proteobacterial ( 85 . 7 %), thermotogales ( 6 . 8 %), epsilon - proteobacteria ( 2 . 4 %), low - g + c gram - positive ( 2 . 1 %), high - g + c gram - positive , beta - proteobacteria and nitrospira ( all less than 1 . 0 %). among them , thermophilic bacterial is more in species , while mesophilic bacteria , such as pseudomonas is more in amount . archaea obtained mainly belongs to methanogenic archaea , including methanobacteriales , methanococcales , methanomicrobiales and methanosarcinales , among which methanomicrobiales is the dominant archaea . a total of 28 sequence types are divided into three categories : ( 1 ) mesophilic methanogens , mainly including methanosarcina , methanohalophdus , methanocalculus and methanosaeta , etc . ; ( 2 ) thermophilic methanogens , mainly including methanothermobacter , methanococcus and methanoculleus ; ( 3 ) uncultivated archaea . among them , most bacteria are discovered before , but the minority show little similarity to bacteria reported before and may be new types of bacteria . several detected thermophilic methanogens were discovered before in other oil reservoir environments , indicating that they might be widely distributed in high - temperature oil reservoir environments . researches show that hydrogenotrophic methanogenic bacteria co - exists with aceticlastic methanogens in the oil reservoir . diversity of microorganism community in a selected typical offshore high - temperature water flooding oil reservoir in china is studied via the 16s rrna sequence analysis method . the results show that bacterial types mainly belong to firmicutes , thermotogae , nitrospirae and proteobacteria , whereas archaea types mainly belong to methanothermobacter , methanobacter , methanobrevibacter and methanococcus , etc ., and only one clone belongs to thermoprotei . the diversity of bacteria is higher than that of archaea , the dominant bacteria microflora includes a few of types of methanogens , zymocytes and sulfate - reducing bacteria , indicating that the microbial diversity of the oil reservoir environment is relatively lower than other environments . a bacterium having close relationship with hydrocarboniphaga effusa is first discovered in an oil reservoir environment , such bacteria is capable of degrading hydrocarbon and aromatic hydrocarbon , and probably is suitable for growing in oil reservoir environment . in addition , there are still a part of bacteria with sequence type incapable of being found in database for over 97 % of genetic relationship . ( 5 ) simultaneously injecting nutrient system and microbial fermentation broth to improve the application effect of microbial on enhanced oil recovery a microbial flooding experimental area is injection 8 production well in no . 3 oil field , in which the average porosity of a reservoir is 28 %, the average air permeability is 0 . 7 um 2 , the oil reservoir temperature is 53 ° c ., the formation oil viscosity is 21 mpa . s , the ground degassed oil density is 0 . 92 g / cm 3 , the wax content is 8 . 8 %, the colloidal bitumen content is 14 . 6 %, the freezing point is − 8 ° c ., the geological reserve is 75 × 10 4 t , the type of formation water is nahco 3 , and the salinity is 5528 mg / l . 7426 m 3 of bacteria solution and nutrient solution is first injected into the microbial flooding experimental area , in which the bacteria solution injected is 120 m 3 ( gx - 043 : pseudomonas putida , 60 m 3 ; gx - 104 : geobacillus subterraneus , 40 m 3 ; gx - 118 : bacillus stearothermophilus , 20 m 3 ). the yield of the oil well approximates to the level before the injection of the bacteria after 26 months . the abundances of the injected bacteria are detected by rt - pcr technique : the report fluorophore group marked on the end of probe gene sequence 5 ′ is fam , and the quenching fluorophore group marked on the end of probe gene sequence 3 ′ is tamra . the result shows that compared with that in peak period of response , the concentration of gx - 043 is reduced from 10 2 cell / ml to 10 5 cell / ml , and community structure has changed obviously , the ratio of the injected functional microorganism ( s ) of “ gx - 043 : gx - 104 : gx - 118 ” changes from 7 : 4 : 2 to 5 : 5 : 3 ; the glycolipid content detected in produced fluid of the oil well is only 0 . 06 % of that under laboratory culture condition . therefore , 54 m 3 of gx - 043 fermentation broth and 540 m 3 of corresponding nutrient solution are simultaneously injected into the experimental oil reservoir . according to laboratory study , the bacteria grows vigorously when taking 0 . 5 % sucrose + 0 . 5 % crude oil as a carbon source and 0 . 2 % ammonium chloride as a nitrogen source , and in view of the existence of crude oil in the oil reservoir , actually , the injected nutrient solution is composed of 0 . 5 % sucrose , 0 . 2 % ammonium chloride , 0 . 2 % yeast extract , 0 . 08 % k 2 hpo 4 , and 0 . 04 % nah 2 po 4 . the fermentation broth and the bacterial solution are mixed evenly and then injected into the oil reservoir through water injection well . after the injection of the nutrient solution and the fermentation broth of the main bacteria , the abundance of gx - 043 gradually increases , and the ratio of the three bacteria approximates to the initial level . the average daily oil production of single well of a beneficial oil well increases from 2 . 2 t to 4 . 7 t , the composite water cut decreases from 93 . 7 % to 90 . 2 %, and accumulated oil increase of the area with microbial enhance oil recovery reaches to 3400 t . ( 6 ) single injection of a nutrient system for improving the application effect of microbial on enhanced oil recovery technique a microbial flooding experimental area is injection 5 production well in oil field no . 2 , in which the average porosity of a reservoir is 22 %, the average air permeability is 0 . 83 um 2 , the oil reservoir temperature is 38 ° c ., the formation oil viscosity is 19 mpa . s , the ground degassed oil density is 0 . 90 g / cm 3 , the wax content is 20 . 2 %, the colloidal bitumen content is 10 . 6 %, the geological reserve is 60 × 10 4 t , the type of formation water is nahco 3 , and the salinity is 7137 mg / l . 4320 m 3 of bacteria solution and nutrient solution is first injected into the microbial flooding experimental area , in which the bacteria solution injected is 320 m 3 . the abundances of the injected bacteria are detected by rt - pcr technique after 18 months : the report fluorophore group marked on the end of probe gene sequence 5 ′ is fam , and the quenching fluorophore group marked on the end of probe gene sequence 3 ′ is tamra . analysis shows that compared with peak period of response , community structure has changed , but the abundance of the injected functional microorganism dq - 003 ( b . subtilis ) changes little ( decreases from the highest of 9 . 1 % to 7 . 8 %); the concentration still remains at 7 * 10 6 cell / ml , in this case the lipopeptide content detected in produced fluid of the oil well is only 0 . 2 % of that under laboratory culture condition . therefore , 650 m 3 of nutrient solution corresponding to dq - 003 is injected into the experimental oil reservoir . according to laboratory study , the nutrient solution consist of 1 % sucrose and 0 . 25 % peptone , additionally adding yeast extract ( 0 . 2 %), k 2 hpo 4 ( 0 . 08 %) and nah 2 po 4 ( 0 . 04 %). the bacterium grows vigorously in the nutrient system . after the injection of the nutrient solution , the concentration of functional microorganism dq - 003 ( b . subtilis ) returns to 2 * 10 7 cells / ml , the average daily oil production of single well of a beneficial oil well increases from 1 . 2 t to 1 . 9 t , the composite water cut decreases from 95 . 7 % to 95 . 0 %, and accumulated oil increase of the area with microbial enhance oil recovery reaches to 605 t . method for adjusting and controlling microbial oil recovery comprises the following steps : ( 1 ) using molecular biological methods , including 16s rdna library , pcr - dgge , and rt - pcr to analyze microbial community structure in produced fluid of the oil reservoir : firstly , collecting a water sample from an experimental oil reservoir , then according to the method disclosed by the molecular analysis of microbial community diversity of oil reservoir ( li hui , ph . d . dissertation . east china university of science and technology , 2007 ), extracting microbial community genome dna , amplifying 16s rrna gene , establishing a genomic library after sequencing , using a rflp method to analyze microbial community diversity of the oil reservoir to obtain a microbial composition structure in the oil reservoir environment ; and according the method disclosed by the detection of abundance of pseudomonas sp in environmental samples by real - time quantitative pcr ( zhao chuanpeng et al ., journal of southeastern university , 2006 , 36 ( 1 )), analyzing microbial abundance through rt - pcr . the microbial composition structure information of the oil reservoir is obtained by above methods . ( 2 ) adjusting the composition of microorganism ( s ) to be injected into the oil reservoir the concentration of a functional microorganism is 2 × 10 8 cell / ml under laboratory culture condition . based on the analysis results of the step ( 1 ), if the concentration of a functional microorganism is higher than 1 % of the concentration achieved under laboratory culture condition , there is no need to inject the microorganism ; and if the concentration of a functional microorganism is lower that 1 % of the concentration achieved in laboratory culture , then injecting the microorganism into the oil reservoir . the microorganism ( s ) commonly used in oil recovery include bacillus subtilis ( such as b . subtilis , cgmcc1 . 400 ), clostridium acetobutylicum ( such as c . acetobutylicum , cgmcc 1 . 244 ), bacillus stearothermophilus ( such as b . stearothermophilus , cgmcc 1 . 1923 ), g . uzenensis ( such as cgmcc 1 . 2674 ), geobacillus subterraneus ( such as g . subterraneus cgmcc 1 . 2673 ), bacillus lentus ( such as b . lentus , cgmcc1 . 2013 ), pseudomonas aeruginosa ( such as p . aeruginosa , cgmcc1 . 1785 ), enterobacter cloacae ( such as e . cloacae , cgmcc1 . 2022 ), halobacterium halobium ( such as h . salinarium , cgmcc1 . 1952 ), pseudomonas fluorescens ( such as p . fluorescens , cgmcc1 . 1802 ), pseudomonas putida ( such as p . putida , cgmcc1 . 1820 ), etc ., but are not limited to above microorganism ( s ). bacillus subtilis , pseudomonas putida and bacillus stearothermophilus are preferred . ( 3 ) injecting the adjusted microorganism ( s ) or the nutrient system into the oil reservoir through the water injection well the injection mode is that the microbial fermentation solution is injected according to 0 . 01 % of pore volume controlled by experimental well group , and the microbial solution is injected into the experimental reservoir through the water injection well . according to the normal working system of oil field development , crude oil is directly obtained from the beneficial oil production well without changing any oil recovery process parameter . method for adjusting and controlling nutrient system corresponding to microorganism ( s ) for oil recovery comprises the following steps : analyzing glycolipid content in the produced fluid of the oil well according to the method disclosed by studies on optimum conditions of preparation of rhamnose by microbial fermentation ( li zuyi et al ., chinese journal of biotchnology , 1999 ( 1 )) and analyzing lipopeptide content according to the method disclosed by determination of the lipopeptide biosufactant in cell - free broth ( chen tao et al ., oilfield chemistry , 2004 ( 4 )) to obtain metabolites information of functional microorganism ( s ) in the oil reservoir . the abundance and activity information of the microorganism ( s ) producing the metabolites are obtained according to the analysis of the change of the metabolites . ( 2 ) adjusting composition of a nutrient system to be injected into the oil reservoir : obtaining metabolite information by analyzing glycolipid content or lipopeptide content in the produced fluid , and making a decision on the basis of the analysis results of the step ( 1 ). if the concentration of a functional microorganism is higher than 1 % of the concentration achieved under laboratory culture condition ( the concentration of the functional microorganism is 2 × 10 6 cell / ml under laboratory culture condition ), there is no need to inject the nutrient system corresponding to the microorganism into the oil reservoir ; whereas if the concentration of a functional microorganism is lower that 1 % of the concentration achieved under laboratory culture condition and the concentration of metabolites is higher than 0 . 1 % of the concentration achieved under laboratory culture condition , injecting the nutrient system corresponding to the microorganism into the oil reservoir ; and by taking sucrose as a carbon source and peptone as a nitrogen source , adjusting the mass ratio of “ carbon source to nitrogen source ” to be “ 5 : 1 ” so as to stimulate the functional microorganism ( s ) or metabolites to become the dominant microorganism ( s ) or main metabolites . ( 3 ) injecting the adjusted nutrient system into the oil reservoir through a water injection well : amount of the nutrient system injected into the experimental reservoir through the water injection well is determined according to 0 . 1 % of pore volume controlled by experimental well group . method for adjusting and controlling microorganism and nutrient system corresponding to the microorganism ( s ) for oil recovery comprises the following steps : ( 1 ) microbial community structure in produced fluid of an oil reservoir are analyzed by using molecular biological methods including 16s rdna library , pcr - dgge , and rt - pcr and biological metabolites of biosurfactant , organic acid , etc . in the produced fluid are detected by utilizing instrument analysis methods . collecting a water sample from an experimental oil reservoir , according to the method disclosed by the molecular analysis of microbial community diversity of oil reservoir ( li hui , ph . d . dissertation . east china university of science and technology , 2007 ), extracting microbial community genome dna , amplifying 16s rrna gene , establishing a genomic library after sequencing , analyzing evolution information of a microbial system , building a phylogenetic tree , using a rflp method to analyze microbial community diversity of the oil reservoir to obtain a microbial community structure in the oil reservoir environment ; adopting the method disclosed by the molecular analysis of microbial community diversity of oil reservoir ( li hui , ph . d . dissertation . east china university of science and technology , 2007 ) to analyze the diversity of alkane degradation gene ( aikb ) in the oil reservoir environment via pcr - dgge fingerprint pattern method ; according the method disclosed by the detection of abundance of pseudomonas sp in environmental samples by real - time quantitative pcr ( zhao chuanpeng et al ., journal of southeastern university , 2006 , 36 ( 1 )), analyzing the abundance of a functional microorganism through rt - pcr ; analyzing bacteria concentration via a microscope - blood counting chamber counting method ; and obtaining microbial composition structure information of the oil reservoir by above methods . analyzing glycolipid content in the produced fluid of an oil well according to the method disclosed by studies on optimum conditions of preparation of rhamnose by microbial fermentation ( li zuyi et al ., chinese journal of biotchnology , 1999 ( 1 )) and analyzing lipopeptide content according to the method disclosed by determination of the lipopeptide biosufactant in cell - free broth ( chen tao et al ., oilfield chemistry , 2004 ( 4 )) to obtain metabolites information of functional microorganism ( s ) in the oil reservoir . the abundance and activity information of the microorganism ( s ) producing the metabolites are obtained according to the analysis of the change of the metabolites . ( 2 ) adjusting composition of microorganism and a nutrient system to be injected into the oil reservoir : according to the analysis results of the step ( 1 ), if the concentration of a functional microorganism is higher than 1 % of the concentration achieved under laboratory culture condition ( the concentration of the functional microorganism is 2 × 10 6 cell / ml under laboratory culture condition ), there is no need to inject the microorganism and the nutrient system corresponding to the microorganism into the oil reservoir ; if the concentration of a functional microorganism is lower that 1 % of the concentration achieved under laboratory culture condition and the concentration of metabolites is higher than 0 . 1 % of the concentration achieved under laboratory culture condition , injecting the nutrient system corresponding to the microorganism ( s ) into the oil reservoir ; and if the concentration of a functional microorganism is lower that 1 % of the concentration achieved under laboratory culture condition and the concentration of metabolites is lower than 0 . 1 % of the concentration achieved under laboratory culture condition , injecting the microorganism ( s ) and the nutrient system corresponding to the microorganism ( s ) into the oil reservoir . the microorganism ( s ) commonly used in oil recovery include bacillus subtilis ( such as b . subtilis , cgmcc1 . 400 ), clostridium acetobutylicum ( such as c . acetobutylicum , cgmcc 1 . 244 ), bacillus stearothermophilus ( such as b . stearothermophilus , cgmcc 1 . 1923 ), g . uzenensis ( such as cgmcc 1 . 2674 ), geobacillus subterraneus ( such as g . subterraneus cgmcc 1 . 2673 ), bacillus lentus ( such as b . lentus , cgmcc1 . 2013 ), pseudomonas aeruginosa ( such as p . aeruginosa , cgmcc1 . 1785 ), enterobacter cloacae ( such as e . cloacae , cgmcc1 . 2022 ), halobacterium halobium ( such as h . salinarium , cgmcc1 . 1952 ), pseudomonas fluorescens ( such as p . fluorescens , cgmcc1 . 1802 ), pseudomonas putida ( such as p . putida , cgmcc1 . 1820 ), etc ., but are not limited to above microorganism ( s ). bacillus subtilis , pseudomonas putida and bacillus stearothermophilus are preferred . by taking glucose as a carbon source and peptone as a nitrogen source , adjusting the mass ratio of “ carbon source to nitrogen source ” to be “ 25 : 1 ” to stimulate the functional microorganism ( s ) or metabolites to be the dominant microorganism ( s ) or main metabolites . ( 3 ) injecting the adjusted microorganism ( s ) or the nutrient system into the oil reservoir through a water injection well the injection mode is that the microbial fermentation solution is injected according to 0 . 01 % of pore volume controlled by experimental well group , and the nutrient system is injected in the amount according to 0 . 1 % of pore volume controlled by experimental well group ; if the nutrient solution and the microbial solution are simultaneously needed , they should be mixed well before the injection ; and the microbial solution and the nutrient solution are injected into the experimental reservoir through a water injection well . according to the normal working system of oil field development , crude oil is directly obtained from the beneficial oil production well without changing any oil recovery process parameter .

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