CN101531979B - Klebsiella pneumoniae and method of preparing hydrogen and 2, 3-butanediol thereby - Google Patents
Klebsiella pneumoniae and method of preparing hydrogen and 2, 3-butanediol thereby Download PDFInfo
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- CN101531979B CN101531979B CN2009100467268A CN200910046726A CN101531979B CN 101531979 B CN101531979 B CN 101531979B CN 2009100467268 A CN2009100467268 A CN 2009100467268A CN 200910046726 A CN200910046726 A CN 200910046726A CN 101531979 B CN101531979 B CN 101531979B
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Abstract
The invention relates to Klebsiella pneumoniae and a method of preparing hydrogen and 2, 3-butanediol thereby. The Klebsiella pneumoniae is Klebsiella pneumoniae ECU-21 with the preservation number of CGMCC No. 2850. In the preparation method, a ring colony taken from a panel is inoculated to a seed culture medium, the inoculated seed culture medium is arranged in an incubator of 37 DEG C for thestatic culture, and the seed culture medium which is cultured for 12 hours is switched to a fermentation medium for the fermentation culture; after the inoculation, a rubber stopper is used for sealing a fermentation shake flask, the anaerobic culture environment is formed by introduced N2 sweeping, superfluous gas is discharged via an exhaust port, a gas bag is used to be connected with the exhaust port for collecting hydrogen produced during the fermentation after the gas discharge is finished, and the 2, 3-butanediol is obtained in liquid product; the culturing temperature is 37 DEG C, a magnetic stirrer is adopted for stirring, the rotation speed is 150rpm, and the culturing time is 12-15h. The invention has the advantages that the hydrogen and the 2, 3-butanediol are produced by the fermentation under the anaerobic condition, the additional value of the production is increased, the reaction conditions are easy and wide industrial application development prospect is provided.
Description
[technical field]
The present invention relates to the Klebsiella pneumonia technical field, specifically, is a kind of Klebsiella pneumonia and preparation hydrogen and 2 thereof, the method for 3-butyleneglycol, and the bacterial classification that relates to is Klebsiella pneumoniaeECU-21, deposit number is CGMCC No.2850.
[background technology]
The energy is one of mankind's basic substance of depending on for existence, is the power that geoevolution and all things on earth are evolved, and it and expanding economy and human progress are closely bound up.Along with The development in society and economy, human also increasing to the demand of the energy and chemical industrial product raw material.For a long time, caused serious environmental to be polluted and the energy scarcity problem to depending on unduly of Nonrenewable resources such as oil, the task of therefore seeking substitute energy is very urgent.As everyone knows, Hydrogen Energy has cleaning, advantage such as efficient, pollution-free, meet the requirement of Sustainable development, its research has related to a plurality of fields such as the energy, automobile, environment, biology, economy, law, policy, be acknowledged as the most promising energy form, from world wide, Hydrogen Energy economy is just ready to appear.2, the 3-butyleneglycol is widely used in multiple industrial circles such as chemical industry, food, fuel and aviation as a kind of important chemical material, has huge market potential.
The method majority of industrial production hydrogen is the fossil oil cracking process at present, and the chemical industry of comparing high energy consumption, high pollution is produced, adopt Production by Microorganism Fermentation energy product and bio-based chemical to have broad application prospects, it can utilize abandoned biomass to be substrate, adopting fermentation using bacteria is that means are carried out product production, having reduced environmental pollution, do not had energy consumption problem, is the industry of a very meaningful and development prospect.Therefore, if can utilize the method for microbial fermentation, realize hydrogen and 2, the joint process of 3-butyleneglycol will play good promotion promoter action to the whole economy social development.Generation hydrogen and 2 can be used for fermenting, the bacterial classification of 3-butyleneglycol has a lot, comprise Klebsiella, Bacillus, Serratia and Pseudomonas etc., wherein facultative anaerobic bacteria Klebsiella pneumoniae can grow under anaerobism or little oxygen condition, compare other bacterial classification, have substrate utilization scope (comprising glucides such as monose, disaccharide, organic acid, cellulosic hydrolysate etc.) and simple growth conditions widely, therefore be widely used in the industrial fermentation production.
The major cause of restriction biological hydrogen production industrial development is that substrate conversion efficiency is lower at present, fermentation costs is high, for addressing this problem, Chinese scholars has also been done big quantity research, screening and separating highly effective hydrogen yield bacterium, the basic problem that does not still address this problem, also the research for hydrogen-producing bacteria genetic improvement and Physiology and biochemistry provides important Microbial resources; And except improveing, realize that the coproduction fermenting process of high value bio-based chemical also can improve the economy of fermenting process from the bacterial classification aspect, promote the development of bioenergy process of industrialization.
[summary of the invention]
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of Klebsiella pneumonia and preparation hydrogen and 2 thereof, the method for 3-butyleneglycol.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of Klebsiella pneumonia, its screening method is:
Klebsiella pneumonia (Klebsiella pneumoniae ECU-21) is a kind of bacterial classification that belongs to Klebsiella, it is Shanghai mayor's bridge purification of water quality factory anaerobic sludge that the present invention screens matrix, mud is diluted coat in the culture plate anaerobism after the different gradients and cultivate; The anaerobism culturing process is carried out in vacuum drier, and culture plate is put into vacuum drier, by vacuumizing/lead to N repeatedly
2Process, make its inside be in anaerobic environment, obtain the anaerobism bacterial strain thereby then vacuum drier is placed 37 ℃ of incubators to cultivate, obtain through the separating for several times purifying at last; Registered preservation on 01 06th, 2009 in Chinese common micro-organisms culture presevation administrative center (CGMCC), deposit number is CGMCC No.2850.
Klebsiella pneumonia of the present invention has following microbial characteristic:
One, morphological specificity:
(1) form of cell and size: tyrothricin, 0.5～1 * 1～3 μ m;
(2) formation of spore: do not have;
Two, the growth conditions on various substratum:
(1) the beef extract agar plate is cultivated (37 ℃, 24 hours)
Colony shape: circle;
Bacterium colony surface elevation: level and smooth, glossy, bacterium colony thickness;
Size: 1～2mm;
Tone: oyster white;
(2) beef extract gelatin stab culture (20 ℃, 6 days)
Liquefy gelatin not;
Three, physiological and biochemical property:
(1) nitrate utilization (28 ℃, 5 days): can utilize;
(2) starch hydrolysis: the positive;
(3) indoles experiment: feminine gender;
(4) hydrolysis of urea: the positive;
(5) methyl red test: feminine gender;
(6) produce H
2S experiment: feminine gender;
(7) to the demand of oxygen: facultative;
(8) growth pH scope: 4.0～9.0;
(9) growth optimal temperature: 34～37 ℃.
Klebsiella pneumonia Klebsiella pneumoniae ECU-21 of the present invention can be with under anaerobic preparing hydrogen and 2, the 3-butyleneglycol, and preparation method's concrete steps are:
(1) Klebsiella pneumoniae ECU-21 seed culture:
Get a ring bacterium colony from flat board and insert seed culture medium, substratum consists of:
Glucose 4.5～5.5g/L, yeast powder 4.5～5.5g/L, peptone 9.5～10.5g/L, extractum carnis 5.5～6.5g/L, NaCl 4.5～5.5g/L, KH
2PO
40.05g/L, K
2HPO
40.05g/L sterilization back pH is 6.0～6.2, shakes the long-pending 250ml of being of bottle, liquid amount is 30ml, leaves standstill to cultivate 12 hours in incubator, and culture temperature is 37 ℃;
(2) Klebsiella pneumoniae ECU-21 fermentation culture:
The seed culture medium of cultivating is inserted in the 600ml fermention medium, and inoculum size is 5%, and fermentation reaction shakes in the bottle at 1L and carries out, and fermention medium consists of:
Glucose 29～31g/L, yeast powder 4.5～5.5g/L, peptone 9.5～10.5g/L, extractum carnis 5.5～6.5g/L, NaCl 4.5～5.5g/L, phosphate buffered saline buffer 200mM, pH are 6.0～6.2; The rubber stopper seal fermentation shake flask is used in the inoculation back, feeds N then
2Purging makes it to form anaerobic culture environment, and unnecessary gas is discharged by venting port, adopts airbag to connect venting port after the gas emptying and collects the hydrogen that fermentation produces, and obtains 2 from liquid product, the 3-butyleneglycol; Culture temperature is 37 ℃, adopts magnetic stirring apparatus to stir, and rotating speed is 150rpm, and incubation time is 12～15h;
In addition, in culturing process, regularly measure the volume of gas in the airbag, and with the content of hydrogen in the gas Chromatographic Determination gas; In culturing process,, and adopt high performance liquid chromatography (HPLC) to measure wherein 2 regularly by the thief hole sampling, the content of 3-butyleneglycol, after the fermentation ends by calculating hydrogen and 2, the output of 3-butyleneglycol and yield etc.
Compared with prior art, positively effect of the present invention is:
Current ferment for hydrogen production industry is subjected to the serious restriction of problems such as substrate cost height, transformation efficiency be low, influenced the development of its process of industrialization, adopt Klebsiella pneumonia of the present invention to have significant advantage: at first, this bacterial classification is a facultative anaerobic bacteria, can under the anaerobic condition, produce hydrogen and 2, the 3-butyleneglycol does not need the supply of oxygen, and this has just reduced a series of problems of being brought by the oxygen transfer restriction; Secondly, when utilizing glucose, have higher transformation efficiency, and the gas that reaction generates only contains H for substrate
2With CO
2, there is not other foreign gas, reduced the difficulty of later stage to two gas delivery, also generated 2 in the fermenting process, organic chemicals such as 3-butyleneglycol, ethanol, this makes fermenting process have higher production value added, the hydrogen transformation efficiency can reach 2.1moH
2/ mol glucose, 2,3-butyleneglycol transformation efficiency reaches 0.5g/g glucose; Once more, can use ligno-cellulose hydrolysate through experimental verification the present invention is that substrate carries out the coproduction fermenting process, in ligno-cellulose hydrolysate, there is the wood sugar that generates by hydrolysis of hemicellulose, most microorganisms can not utilize wood sugar, and the present invention can be than the metabolism wood sugar of good utilisation, this has further enlarged substrate utilization scope of the present invention, thereby can use more cheap abandoned biomass ferments, and the reaction conditions that the present invention uses is simple, has wide industrial application DEVELOPMENT PROSPECT.
[embodiment]
Below provide Klebsiella pneumonia of the present invention and preparation hydrogen and 2 thereof, the embodiment of the method for 3-butyleneglycol.
Embodiment 1
Utilize Klebsiella pneumoniae ECU-21 to prepare hydrogen and 2, the method for 3-butyleneglycol
Fermentative medium formula is: glucose 10g/L, yeast powder 5g/L, peptone 10g/L, extractum carnis 6g/L, NaCl 5g/L, KH
2PO
40.05g/L, K
2HPO
40.05g/L pH 6.0～6.2; 121 ℃ of high-temperature sterilization 20min.
Get the slant strains of 4 ℃ of preservations, picking one ring is seeded to the triangle that the 30ml seed culture medium is housed and shakes in the bottle, leaves standstill under 37 ℃ and cultivates 12 hours, then seed culture medium is seeded in the 600ml fermention medium with 5% inoculum size, adopt rubber plug that fermentation shake flask is sealed, the back feeds N
2Purging makes and forms anaerobic environment in the reactor, and unnecessary gas is discharged by venting port, N
2The purging back that finishes adopts the gas collection bag to connect venting port, and the hydrogen that fermentation produces is collected by airbag, and regularly (1 hour) is from the thief hole sampling and measuring and calculate final cell amount, liquid product amount and hydrogen output.And the about 2.15g/L of final cell concentration, hydrogen output is 1.3V/V, average hydrogen conversion is 1.21mol H
2/ mol glucose, 2,3-butyleneglycol output reaches 4.68g/L, and average conversion reaches 0.48g/g glucose.
Embodiment 2
Utilize Klebsiella pneumoniae ECU-21 to prepare hydrogen and 2, the method for 3-butyleneglycol
Fermentative medium formula: glucose 30g/L, yeast powder 5g/L, peptone 10g/L, extractum carnis 6g/L, NaCl 5g/L, phosphate buffered saline buffer 200mM regulates different pH and is respectively 5.0～7.5,121 ℃ of high-temperature sterilization 20min.
Get the slant strains of 4 ℃ of preservations, picking one ring is seeded to the triangle that the 30ml substratum is housed respectively and shakes in the bottle, leaves standstill under 37 ℃ and cultivates 12 hours, inoculum size by 5% is switched to and fills 600ml, in the fermention medium that pH has nothing in common with each other, adopt the rubber stopper seal fermentation shake flask, logical N
2Emptying air wherein forms anaerobic environment, and unnecessary gas is discharged by venting port, N
2Purging finishes back employing gas collection bag connection venting port in order to collect the hydrogen that fermentation produces, and culture temperature is constant to be 37 ℃, the employing magnetic stirrer, and mixing speed is 150rpm.Fermentation culture 16h, regularly (1 hour) from thief hole sampling and measuring cell, hydrogen, liquid product etc., and adopts pH meter to measure fermented liquid pH value, by NaOH adjusting pH value, makes every batch fermentation liquid keep constant pH in the fermenting process.Calculate final cell amount, liquid product amount and hydrogen output after the fermentation ends.Under different pH, record result's (table 1) as shown in the table.
The different pH bottom fermentation of table 1 product result
Embodiment 3
Utilize Klebsiella pneumoniae ECU-21 to prepare hydrogen and 2, the method for 3-butyleneglycol
Fermention medium is the hydrolyzed solution that enzymolysis obtains after the swollen quick-fried pre-treatment of maize straw process steam, and hydrolyzed solution is mainly formed: glucose 60g/L; Wood sugar 12g/L; Cellobiose 4g/L; With 6 times of hydrolyzed solution dilutions, add yeast powder 5g/L, peptone 10g/L, extractum carnis 6g/L, NaCl 5g/L, phosphate buffered saline buffer 200mM, pH 6.0～6.2,115 ℃ of high-temperature sterilization 20min.
Seed culture medium is with embodiment 1, get the slant strains of 4 ℃ of preservations, picking one ring is seeded to the triangle that the 30ml seed culture medium is housed and shakes in the bottle, under 37 ℃, leave standstill and cultivated 12 hours, inoculum size by 5% is forwarded in the 1L fermentation shake flask that fills the 600ml fermention medium, the employing rubber stopper seal is cultivated, logical N
2Emptying air wherein, unnecessary gas is discharged by venting port, N
2Adopt the gas collection bag to connect venting port after purging finishes and collect the hydrogen that produces in the fermenting process.Culture temperature is constant to be 37 ℃, adopts magnetic stirrer, and mixing speed is 150rpm.Fermentation culture 14～16h, per hour sampling and measuring cell, hydrogen, liquid product etc.And the about 2.68g/L of final cell concentration, hydrogen output is 0.6V/V, average hydrogen conversion is 0.54molH
2/ mol reducing sugar, 2,3-butyleneglycol output reaches 5.05g/L, and average conversion reaches the 0.51g/g reducing sugar.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (2)
1. Klebsiella pneumonia, it is characterized in that, Klebsiella pneumonia is Klebsiellapneumoniae ECU-21, in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) registration preservation, preservation date is on 01 06th, 2009, and deposit number is CGMCCNo.2850.
2. one kind is utilized the described Klebsiella pneumonia of claim 1 to prepare hydrogen and 2, and the method for 3-butyleneglycol is characterized in that, concrete steps are:
(1) Klebsiella pneumoniae ECU-21 seed culture
Get a ring bacterium colony from flat board and insert seed culture medium, substratum consists of:
Glucose 4.5～5.5g/L, yeast powder 4.5～5.5g/L, peptone 9.5～10.5g/L, extractum carnis 5.5～6.5g/L, NaCl4.5～5.5g/L, KH
2PO
40.05g/L, K
2HPO
40.05g/L sterilization back pH is 6.0～6.2, shakes the long-pending 250ml of being of bottle, liquid amount is 30ml, leaves standstill to cultivate 12 hours in incubator, and culture temperature is 37 ℃;
(2) Klebsiella pneumoniae ECU-21 fermentation culture
The seed culture medium of cultivating is inserted in the 600ml fermention medium, and inoculum size is 5%, and fermentation reaction shakes in the bottle at 1L and carries out, and fermention medium consists of:
Glucose 29～31g/L, yeast powder 4.5～5.5g/L, peptone 9.5～10.5g/L, extractum carnis 5.5～6.5g/L, NaCl4.5～5.5g/L, phosphate buffered saline buffer 200mM, pH are 6.0～6.2; The rubber stopper seal fermentation shake flask is used in the inoculation back, feeds N then
2Purging makes it to form anaerobic culture environment, and unnecessary gas is discharged by venting port, adopts airbag to connect venting port after the gas emptying and collects the hydrogen that fermentation produces, and obtains 2 from liquid product, the 3-butyleneglycol; Culture temperature is 37 ℃, adopts magnetic stirring apparatus to stir, and rotating speed is 150rpm, and incubation time is 12～15h.
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| CN101531979B true CN101531979B (en) | 2011-07-13 |
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| CN102031231B (en) * | 2010-11-03 | 2013-04-03 | 甘肃农业大学 | Growth-promoting rhizobacteria and fungicide thereof, preparation method and application of fungicide |
| CN102477448B (en) * | 2010-11-24 | 2013-11-06 | 上海工程技术大学 | Method for bio-hydrogen production |
| CN106434489B (en) * | 2016-11-18 | 2021-04-30 | 中国人民解放军疾病预防控制所 | High-yield wine Klebsiella pneumoniae strain and application thereof |
| CN110256730A (en) * | 2019-06-03 | 2019-09-20 | 华南理工大学 | Light stabilizer and the preparation method and application thereof for weather-proof pvc material |
| CN111548959B (en) * | 2020-05-13 | 2022-03-22 | 内蒙古工业大学 | Klebsiella pneumoniae and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033453A (en) * | 2006-03-07 | 2007-09-12 | 中国科学院过程工程研究所 | Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels |
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|---|---|---|---|---|
| CN101033453A (en) * | 2006-03-07 | 2007-09-12 | 中国科学院过程工程研究所 | Klebsiella and application of the same for eliminating organic nitrogen in fossil fuels |
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|---|
| Niu, K et al..登录号EU360793.《GenBank》.2008,1-2. * |
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