CN102226159B - Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol - Google Patents
Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol Download PDFInfo
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Abstract
The invention discloses a strain of Enterobacter cloacae subsp. dissolvens SDM and its application in the preparation of 2,3-butylene glycol. The preservation number of the Enterobacter cloacae subsp. dissolvens SDM is CGMCC No.4230. Pentose, hexose, molasses, starch raw material hydrolysate, lignocellulose raw material hydrolysate are utilized in the strain provided by the invention with high transformation rate, high product concentration and wide substrate spectrum. According to the invention, the application of the strain of Enterobacter cloacae in the preparation of 2,3-butylene glycol has advantages of simple operation method, low cost and high efficiency; the output of 2,3-butylene glycol by the fermentation cylinder fed batch fermentation can reach 125-162 g/L; the transformation rate accounts for 90-95% of the theoretical transformation rate; the maximum productivity of 2,3-butylene glycol is 4.1 g/(L<.>h). Thus, the invention has a great prospect for industrial popularization and application.
Description
Technical field
The present invention relates to an Enterobacter cloacae and dissolve subspecies and application thereof, relate in particular to a plant height and produce the enterobacter cloacae dissolving subspecies of 2,3-butanediol and in the application of preparing in 2,3-butanediol, belong to technical field of bioengineering.
Background technology
2,3-butanediol (2,3-butanediol) is a kind of potential very valuable hardware and software platform compound, be widely used in multiple fields such as medicine, chemical industry, food, fuel and aerospace (
e., Grajek, W., 2009.Biotechnol.Adv.27,715-725.).The 2,3-butanediol of levorotatory form can be used as antifreezing agent because it has lower zero pour.2,3-butanediol can be used as precursor substance for the production of have extremely methylethylketone and the 1,3-butadiene of extensive use at chemical field.The fuel value of 2,3-butanediol is 27200kJ/kg, with quite (29055kJ/kg) of ethanol, is the fuel dope that a kind of potential value is very high.2,3-butanediol also can be used to chiral support of preparing polymkeric substance, ink, perfume, fumigant, moistening agent, tenderizer, softening agent, explosive and medicine etc.2,3-butanediol so widely purposes causes its demand in the international market to rise steadily, and has also caused the extensive concern in the world as the research of liquid fuel additive.
The production of 2,3-butanediol is mainly microbe fermentation method, compared with chemical synthesis, and the method environmental friendliness, technique is simple, technical comparatively ripe, meets the requirement of green chemical industry.Bacterium is the microorganism that fermentative production 2,3-butanediol is had to industrial value.Bacteria genus for fermentative production 2,3-butanediol mainly contains Klebsiella (Klebsiella), serratia (Serratia), bacillus (Bacillus) and Aeromonas (Aeromonas) etc.The report of producing 2,3-butanediol about enterobacter (Enterobacter) is less, and the output of its 2,3-butanediol is lower (Chinese patent CN101709281A, CN101709280A all; Saha, B.C., Bothast, R.J., 1999.Appl.Microbiol.Biotechnol.52,321-326, Zhao Shimin etc., 2008. food and fermentation industries .34 (3), 25-28).
Through retrieval, at present there are no using enterobacter cloacae to dissolve the patent report of subspecies fermentative production 2,3-butanediol.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is: provide a plant height to produce 2, the enterobacter cloacae of 3-butyleneglycol is dissolved subspecies (Enterobacter cloacae subsp.dissolvens), and described bacterium is in the application of preparing in 2,3-butanediol.
Product 2 of the present invention, the enterobacter cloacae of 3-butyleneglycol is dissolved subspecies, it is characterized in that: this bacterial strain enterobacter cloacae by name is dissolved subspecies (Enterobacter cloacae subsp.dissolvens) SDM, bacterial strain on October 19th, 2010 be deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (and be called for short CGMCC; address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), preserving number is CGMCC No.4230.
Above-mentioned enterobacter cloacae is dissolved subspecies (Enterobacter cloacae subsp.dissolvens) bacterial strain SDM and screens and obtain from the agricultural land soil in Jinan, Shandong Province suburb.Through German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (being called for short DSMZ) qualification, its biological property is:
Gram-negative, amphimicrobian, has the excellent bacillus of mobility, long 1.2～2.5 μ m, wide 0.7～0.8 μ m.VP reaction, ONPG reaction, catalase, urine enzyme, arginine dihydrolase, ornithine decarboxylase is positive.Clark and Lubsreaction, oxydase reaction, gelatin hydrolysis reaction and indoles produce reaction and are negative.Its colony colour is yellow-white, circular, protruding, glossy, smooth surface, diameter 2～3mm.It can utilize glucose, fructose, seminose, maltose, D-wood sugar, sucrose, trehalose, L-arabinose, rhamnosyl, semi-lactosi, Citrate trianion and malonate, and 37 DEG C of glucose fermentations produce sour aerogenesis.Can on KCN, grow.Its 16S rDNA sequence is as shown in SEQ ID No.1, and sequence length is 1504bp, and the 16S rDNA sequence alignment similarity that described sequence and many Enterobacter cloacaes dissolve subspecies reaches 99.9%.
Method of being dissolved subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 fermentative production 2,3-butanediol by enterobacter cloacae of the present invention, its step relating to is:
(1) bacterial classification is selected.
Select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230.
(2) slant culture activation: enterobacter cloacae is dissolved to subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230 bacterial classification and be inoculated in slant medium, under 30～40 DEG C of conditions, leave standstill and cultivate 10～14 hours, for subsequent use;
(3) shake flask fermentation seed culture: the inclined-plane bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 300mL of 40～60mL liquid seed culture medium triangular flask with inoculation articulating 1～2, be placed on the shaking table that rotating speed is 150～200rpm, cultivate 10～16 hours, obtain seed culture fluid for 30～40 DEG C;
(4) ferment tank seed culture:
First order seed is cultivated: the inclined-plane bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 80～120mL liquid seed culture medium triangular flask with inoculation articulating 1～2, be placed on the shaking table that rotating speed is 150～200rpm, cultivate 10～16 hours, obtain first order seed nutrient solution for 30～40 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 0.8～1.2 liter of liquid seed culture medium with the inoculum size of 5～10% (volume ratios), be placed on the shaking table that rotating speed is 150～200rpm, cultivate 10～16 hours, obtain secondary seed nutrient solution for 30～40 DEG C;
(5) fermentation culture:
Shake flask fermentation: with the inoculum size of 5～10% (volume ratios), seed liquor is inoculated in the 500mL triangular flask bottle that 80～120mL fermention medium is housed, be placed on the shaking table that rotating speed is 150～200rpm, cultivate 16～30 hours for 30～40 DEG C, when in fermented liquid 2, when 3-butyleneglycol concentration no longer rises, fermentation stops.
50 liters of ferment tanks: with the inoculum size of volume ratio 5～10%, secondary seed solution is inoculated in 50 liters of fermentor tanks, fermentor tank liquid amount is 30 liters, then taking primary carbon source concentration as 60～120g/L, 30～40 DEG C of temperature, mixing speed 200～600rpm, air flow 0.5～1.5vvm ventilate and stir fermentation culture, incubation time is 20～60 hours; Fermented liquid initial pH value is adjusted to 5.5～7.0, in fermenting process, adds the KOH of 3～6M and the H of 2～5M by stream
3pO
4regulate pH value, make it to be controlled at pH5.5～6.5; In fermenting process, the residual sugar amount in 3～5 hours sampling and measuring fermented liquids and 2,3-butanediol concentration, and add carbon source according to remaining sugar concentration stream, and in the time that 2,3-butanediol concentration no longer rises, fermentation ends.
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 5.5～7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: carbon source 30～40g/L, potassium primary phosphate 3～10g/L, ammonium chloride 3～12g/L, sodium-chlor 1～4g/L, magnesium sulfate 0.05～0.1g/L, ferrous sulfate 0.05～0.1g/L, pH value is adjusted to 5.5～7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned fermention medium consists of: carbon source 60～120g/L, Dried Corn Steep Liquor Powder 5～20g/L, potassium primary phosphate 3～10g/L, ammonium chloride 5～25g/L, potassium acetate 1～4g/L, sodium-chlor 1～4g/L, calcium chloride 0.05～0.1g/L, magnesium sulfate 0.1～0.5g/L, zinc sulfate 0.2～0.6g/L, ferrous sulfate 0.2～0.6g/L, pH value is adjusted to 5.5～7.0, tap water preparation, 115 DEG C of sterilizings 20 minutes;
In above-mentioned 50 liters of ferment tanks cultivation, fed-batch mode preferred pulse stream adds, and its method is in the time that sugared concentration drops to 10～30g/L, and pulse current adding substrate makes sugared concentration reach 50～70g/L.
Carbon source in above-mentioned seed culture medium at least contains glucose or wood sugar.
Carbon source in above-mentioned fermention medium at least contains the one in glucose, wood sugar, sucrose molasses, xylose mother liquid, starch materials hydrolyzed solution, lignocellulosic material hydrolyzed solution; The independent sterilizing of this carbon source, without regulating pH.Wherein, described starch materials hydrolyzed solution makes with following method: get starch materials, enzyme dosage with 2～8U/g starch materials dry weight adds α-amylase, process 0.5h for 95 DEG C, obtain starch materials liquefier, then adjust pH to 4.2, add saccharifying enzyme, the addition of saccharifying enzyme is 100～1000U/g starch materials dry weight, is placed in 60 DEG C of shaking baths and processes to glucose and be thoroughly hydrolyzed, and obtains sugar-containing concentration and counts 15%～40% starch materials hydrolyzed solution with mass volume ratio; Described lignocellulosic material hydrolyzed solution makes with following method: the lignocellulosic material of getting pulverizing, in mass volume ratio, by lignocellulosic material: massfraction is 1% sulfuric acid=1: 10～1: 3 amount adds sulfuric acid, process 1 hour for 120 DEG C, conventional centrifugal, supernatant liquor is sugar-containing concentration and counts 15%～35% lignocellulosic material hydrolyzed solution with mass volume ratio.
Above-mentioned starch materials preferred starch or Tapioca Starch; The preferred wood of lignocellulosic material, corn cob or stalk.
In above-mentioned starch materials hydrolyzed solution, sugared concentration is preferably 25%～35% in mass volume ratio; In above-mentioned lignocellulosic material hydrolyzed solution, sugared concentration is preferably 25%～30% in mass volume ratio.
In above-mentioned application, preferably 37 ± 0.2 DEG C of steps (2), (3), (4), (5) described culture temperature;
In above-mentioned application, the described incubation time of step (2) preferably 12 hours;
In above-mentioned application, the described incubation time of step (3) preferably 12～14 hours;
In above-mentioned application, the described incubation time of step (4) preferably 12～14 hours;
In above-mentioned application, preferably 60～80g/L of step (5) the initial sugared concentration of described 50 liters of ferment tank substratum.
In above-mentioned application, the described 50 liters of ferment tank liquid pH of step (5) preferably 6.0 ± 0.2.
Distinguishing feature of the present invention is:
From the soil layer of farmland screening obtain a strain have fermentation potentiality product 2, the bacterial strain of 3-butyleneglycol, dissolve subspecies through being accredited as enterobacter cloacae, called after enterobacter cloacae is dissolved subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230.
Of the present invention for the preparation 2 of fermenting, the bacterial strain enterobacter cloacae of 3-butyleneglycol is dissolved subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230, there is transformation efficiency high, production concentration is high, the features such as substrate spectrum is wide, can stably carry out 2, the fermentative production of 3-butyleneglycol, fermentor tank fed-batch fermentation 2, the output of 3-butyleneglycol can reach 125～162g/L, and 2,3-butanediol productivity is 4.1g/ (Lh) to the maximum, transformation efficiency reaches 90～95% of theoretical yield, has industrial applications potentiality.
Embodiment
General explanation:
Get starch materials, enzyme dosage with 2～8U/g starch materials dry weight adds α-amylase, process 0.5h for 95 DEG C, obtain starch materials liquefier, then adjust pH to 4.2, add saccharifying enzyme, the addition of saccharifying enzyme is 100～1000U/g starch materials dry weight, be placed in 60 DEG C of shaking baths and process to glucose and be thoroughly hydrolyzed, obtain sugar-containing concentration and count 15%～40% starch materials hydrolyzed solution with mass volume ratio.
Above-mentioned starch materials preferred starch or Tapioca Starch.
The preparation of simultaneous saccharification and fermentation substrate: by 115 DEG C of sterilizings of starch materials liquefier good α-amylaseliquefied 20 minutes, for next step simultaneous saccharification and fermentation.
The preparation of lignocellulosic material hydrolyzed solution: get the diameter of pulverizing at the lignocellulosic material of 0.45～0.9mm, in mass volume ratio, by lignocellulosic material: massfraction is 1% sulfuric acid=1: 10～1: 3 amount adds sulfuric acid, process 1 hour for 120 DEG C, conventional centrifugal, supernatant liquor is sugar-containing concentration and counts 15%～35% lignocellulosic material hydrolyzed solution with mass volume ratio.
The preferred wood of above-mentioned lignocellulosic material, corn cob or stalk.
In above-mentioned starch materials hydrolyzed solution, sugared concentration is preferably 25%～35% in mass volume ratio; In above-mentioned lignocellulosic material hydrolyzed solution, sugared concentration is preferably 25%～30% in mass volume ratio.
Lignocellulosic material hydrolysis solution composition and total reducing sugar analytical procedure are with reference to (Wang AL, et al.Appl Microbiol Biotechnol, 2010,87:965-970).
Glucose is purchased from Licheng District, Jinan City Chang Yingda chemical industry business department, wood sugar is purchased from Ji'nan Healtang Biotechnology Co., Ltd., corn cob, xylose mother liquid are purchased from Shandong Longli Biology Science and Technology Co., Ltd, sucrose molasses are purchased from Guangxi Chinese businessman Guo Tang trade Co., Ltd, and Tapioca Starch is purchased from Shanghai Xiang Heng trade Co., Ltd.
The measuring method of glucose is: centrifugal after fermented liquid dilution, and adopt bio-sensing analyser SBA-40C (Shandong Province academy sciences Biology Research Institute) to measure.Measuring principle is for utilizing fixation glucose dehydrogenation enzyme membrane specificity to measure glucose content.
Wood sugar detection method is: Agilent l100 high performance liquid chromatograph, G1311A quaternary pump, differential refraction analyzing and testing device, sample size 10 μ L.Agilent HPLC 2D chromatographic working station.Chromatographic column is ZORBAX carbohydrate analysis column (4.6 × 150mm), 30～50 DEG C of column temperatures, and moving phase is acetonitrile: water=80: 20 (v/v), flow velocity 1.2mL/min.Qualitative with retention time, external standard method is quantitative.
The measuring method of 2,3-butanediol: VARIAN CP-3380 gas chromatograph detects.Butylacetate is extraction agent, and volume ratio extraction in 1: 1, gets upper organic phase and detect.The concrete condition of measuring is: flame ion monitor (FID) temperature is 280 DEG C, and sampler temperature is 220 DEG C, and kapillary column temperature is to be warmed up to 180 DEG C from 50 DEG C, 20 DEG C/min of heat-up rate, and carrier gas is nitrogen.Utilize 2,3-butanediol standard substance (German Sigma-Aldrich company, article No.: 361461) make typical curve, then calculate the content of 2,3-butanediol in fermented liquid according to typical curve.
Embodiment 1: enterobacter cloacae is dissolved separation screening and the qualification of subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230
In this embodiment, substratum used is composed as follows:
Liquid screening substratum: glucose 50g/L, sodium acetate 5g/L, Repone K 0.5g/L, magnesium sulfate 0.15g/L, iron vitriol 0.05g/l, seven water manganous sulfate 0.03g/l, pH is 7.
Base is supported in solid screening: liquid screening substratum, and agar powder 18g/l, pH is 7.
Fermention medium: glucose 100g/L, yeast powder 10g/L, peptone 10g/L, sodium acetate 5g/L, pH is 7.
Nutrient broth medium: glucose 50g/L, yeast powder 10g/L, peptone 10g/L, sodium-chlor 5g/L, pH is 7.
The specific operation process of this embodiment is as follows:
1, separation screening
Obtain soil from farmland, get in the 300mL triangular flask that 1g adds the liquid screening substratum that 50mL sterilizing is housed, under 37 DEG C of conditions, be placed in the rotating speed with 120rpm on shaking table and cultivate 48 hours.Getting 1ml nutrient solution is transferred in identical liquid screening substratum and cultivates 46 hours under the same terms.By nutrient solution with 10
-3, 10
-4two extent of dilution are coated on solid screening and support in the culture dish of base, cultivate 20 hours for 37 DEG C, after growing single bacterium colony, the bacterium colony that choosing colony area is large, be inoculated in fermention medium, be placed in the rotating speed with 180rpm on shaking table, cultivate 20 hours for 37 DEG C, react and carry out primary dcreening operation by VP, filter out positive strain according to reaction times and colour-change.Then measure the output of the 2,3-butanediol of the positive strain of primary dcreening operation, obtain the highest bacterial strain of output.
2, the qualification of bacterial strain
The bacterial strain that above-mentioned screening is obtained is identified at German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (being called for short DSMZ), qualification result shows: this bacterium is Gram-negative, amphimicrobian, there is the excellent bacillus of mobility, long 1.2～2.5 μ l, wide 0.7～0.8 μ l.VP reaction, ONPG reaction, catalase, urine enzyme, arginine dihydrolase, ornithine decarboxylase is positive.Clark and Lubsreaction, oxydase reaction, gelatin hydrolysis reaction and indoles produce reaction and are negative.Its colony colour is yellow-white, circular, protruding, glossy, smooth surface, diameter 2～3mm.It can utilize glucose, fructose, seminose, maltose, D-wood sugar, sucrose, trehalose, L-arabinose, rhamnosyl, semi-lactosi, Citrate trianion and malonate, and 37 DEG C of glucose fermentations produce sour aerogenesis.Can on KCN, grow.Its 16S rDNA sequence is as shown in SEQ ID No.1, and sequence length is 1504bp, and the 16S rDNA sequence alignment similarity that described sequence and many Enterobacter cloacaes dissolve subspecies reaches 99.9%.Comprehensive analysis, identification of strains is that enterobacter cloacae is dissolved subspecies (Enterobacter cloacae subsp.dissolvens), and called after enterobacter cloacae is dissolved subspecies (Enterobacter cloacae subsp.dissolvens) SDM.
Above-mentioned bacterial strains enterobacter cloacae is dissolved subspecies (Enterobacter cloacae ssp.dissolvens) SDM and has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on October 19th, 2010, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, postcode: 100101, its deposit number is CGMCC No.4230.
Embodiment 2, utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 to produce 2,3-butanediol taking glucose as carbon source through fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 37 DEG C of conditions, leaves standstill and cultivate 12 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 37 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain secondary seed nutrient solution for 37 DEG C;
(4) fermentation culture: add 30 liters of fermentation initial mediums in 50 liters of fermentor tanks of German Bei Lang (BIOSTAT B, B.Braun).In fermention medium, access 3 liters of secondary seed nutrient solutions, fermentor tank mixing speed 400rpm, 37 DEG C of condition bottom fermentations 50 hours.Residual sugar amount in 3 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.In fermenting process, glucose is added in pulse, makes the glucose concn in fermented liquid maintain 20～40g/L.
(5) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, glucose concn in suitable Ploidy testing fermented liquid, calculates sugared transformation efficiency, 2,3-butanediol productivity.
Fermentation ends, the concentration that records glucose is 2.7 ± 0.5g/L (mean+SD), 2,3-butyleneglycol concentration 161.3 ± 0.7g/L (mean+SD), sugar transformation efficiency reaches 92.1 ± 0.8% (mean+SD) of theoretical yield, 2,3-butanediol productivity is 3.23 ± 0.01g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 6.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: glucose 40g/L, and potassium primary phosphate 8g/L, ammonium chloride 5g/L, sodium-chlor 1g/L, magnesium sulfate 0.05g/L, ferrous sulfate 0.05g/L, pH value is adjusted to 6.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned fermention medium consists of: glucose 65g/L, Dried Corn Steep Liquor Powder 6g/L, potassium primary phosphate 4g/L, ammonium chloride 20g/L, potassium acetate 4g/L, sodium-chlor 2g/L, calcium chloride 0.08g/L, magnesium sulfate 0.4g/L, zinc sulfate 0.3g/L, ferrous sulfate 0.3g/L, pH value is adjusted to 6.0, tap water preparation, 115 DEG C of sterilizings 20 minutes; The independent sterilizing of glucose.
Embodiment 3, utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 to produce 2,3-butanediol taking wood sugar as carbon source through fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 35 DEG C of conditions, leaves standstill and cultivate 12 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 35 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain secondary seed nutrient solution for 35 DEG C;
(4) fermentation culture: add 30 liters of fermention mediums in 50 liters of fermentor tanks of German Bei Lang (BIOSTAT B, B.Braun).In fermention medium, access 3 liters of secondary seed nutrient solutions, fermentor tank mixing speed 300rpm, 35 DEG C of condition bottom fermentations 24 hours.Residual sugar amount in 3 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.
(5) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, xylose concentration in suitable Ploidy testing fermented liquid, calculates sugared transformation efficiency, 2,3-butanediol productivity.
Fermentation ends, the concentration that records wood sugar is 2.5 ± 0.2g/L (mean+SD), 2,3-butyleneglycol concentration 53.6 ± 0.9g/L (mean+SD), sugar transformation efficiency reaches 91.2 ± 1.5% (mean+SD) of theoretical yield, 2,3-butanediol productivity is 2.23 ± 0.04g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: wood sugar 40g/L, and potassium primary phosphate 6g/L, ammonium chloride 5g/L, sodium-chlor 3g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, pH value is adjusted to 7.0, distilled water preparation, sterilizing 20 minutes under 115 DEG C of conditions;
Above-mentioned fermention medium consists of: wood sugar 120g/L, Dried Corn Steep Liquor Powder 15g/L, potassium primary phosphate 7g/L, ammonium chloride 15g/L, potassium acetate 4g/L, sodium-chlor 2g/L, calcium chloride 0.075g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.4g/L, ferrous sulfate 0.2g/L, pH value is adjusted to 7.0, tap water preparation, 115 DEG C of sterilizings 20 minutes; The independent sterilizing of wood sugar.
Embodiment 4, utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 to produce 2,3-butanediol taking sucrose molasses as carbon source through fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 40 DEG C of conditions, leaves standstill and cultivate 10 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 40 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 13 hours, obtain secondary seed nutrient solution for 40 DEG C;
(4) fermentation culture: add 30 liters of fermentation initial mediums in 50 liters of fermentor tanks of German Bei Lang (BIOSTAT B, B.Braun).In fermention medium, access 3 liters of secondary seed nutrient solutions, fermentor tank mixing speed 600rpm, 40 DEG C of condition bottom fermentations 40 hours.Wherein, fermentation was by 5 hours, and beginning stream of pulses adds total sugar concentration and counts 55% sucrose molasses with mass volume ratio.Residual sugar amount in 4 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.In the time that total sugar concentration in fermented liquid drops to 20g/L left and right, stream of pulses makes total sugar concentration arrive 50g/L left and right with sucrose molasses.
(5) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, total sugar concentration in suitable Ploidy testing fermented liquid, calculates 2,3-butanediol productivity.
Fermentation ends, the concentration that records total reducing sugar is 3.1 ± 0.6g/L (mean+SD), 2,3-butyleneglycol concentration 112.4 ± 2.2g/L (mean+SD), 2,3-butanediol productivity is 2.81 ± 0.06g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: glucose 35g/L, and potassium primary phosphate 6g/L, ammonium chloride 8g/L, sodium-chlor 3g/L, magnesium sulfate 0.06g/L, ferrous sulfate 0.08g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned fermention medium consists of: total sugar concentration is counted 55% sucrose molasses 100g/L, Dried Corn Steep Liquor Powder 8g/L, potassium primary phosphate 12g/L with mass volume ratio, ammonium chloride 12g/L, potassium acetate 4g/L, sodium-chlor 3g/L, calcium chloride 0.05g/L, magnesium sulfate 0.3g/L, zinc sulfate 0.4g/L, ferrous sulfate 0.4g/L, tap water preparation, the initial pH of this fermention medium is 6.5,115 DEG C of sterilizings 20 minutes; The independent sterilizing of sucrose molasses.
Embodiment 5, utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 to produce 2,3-butanediol taking xylose mother liquid as carbon source through fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 30 DEG C of conditions, leaves standstill and cultivate 14 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 37 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 13 hours, obtain secondary seed nutrient solution for 37 DEG C;
(4) fermentation culture: add 30 liters of fermentation initial mediums in 50 liters of fermentor tanks of German Bei Lang (BIOSTAT B, B.Braun).In fermention medium, access 3 liters of secondary seed nutrient solutions, fermentor tank mixing speed 400rpm, 37 DEG C of condition bottom fermentations 44 hours.Wherein, fermentation was by 5 hours, and beginning stream of pulses adds total sugar concentration and counts 68% xylose mother liquid with mass volume ratio.Residual sugar amount in 4 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.In the time that total sugar concentration in fermented liquid drops to 20g/L left and right, stream of pulses adds xylose mother liquid makes total sugar concentration arrive 50g/L left and right.
(5) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, total sugar concentration in suitable Ploidy testing fermented liquid, calculates 2,3-butanediol productivity.
Fermentation ends, the concentration that records total reducing sugar is 4.7 ± 0.8g/L (mean+SD), 2,3-butyleneglycol concentration 67.9 ± 1.7g/L (mean+SD), 2,3-butanediol productivity is 1.54 ± 0.04g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: wood sugar 35g/L, and potassium primary phosphate 6g/L, ammonium chloride 8g/L, sodium-chlor 3g/L, magnesium sulfate 0.06g/L, ferrous sulfate 0.08g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned fermention medium consists of: total sugar concentration is counted 68% xylose mother liquid 90g/L, Dried Corn Steep Liquor Powder 10g/L, potassium primary phosphate 10g/L with mass volume ratio, ammonium chloride 5g/L, potassium acetate 4g/L, sodium-chlor 3g/L, calcium chloride 0.05g/L, magnesium sulfate 0.3g/L, zinc sulfate 0.4g/L, ferrous sulfate 0.4g/L, tap water preparation, the initial pH of this fermention medium is 6.5,115 DEG C of sterilizings 20 minutes; The independent sterilizing of xylose mother liquid.
Embodiment 6, utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 to produce 2,3-butanediol taking Tapioca Starch hydrolyzed solution as carbon source through fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 40 DEG C of conditions, leaves standstill and cultivate 12 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 40 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 13 hours, obtain secondary seed nutrient solution for 40 DEG C;
(4) fermentation culture: add 30 liters of fermentation initial mediums in 50 liters of fermentor tanks of German Bei Lang (BIOSTAT B, B.Braun).In fermention medium, access 3 liters of secondary seed nutrient solutions, fermentor tank mixing speed 400rpm, 40 DEG C of condition bottom fermentations 51 hours.Residual sugar amount in 3 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.Carry out stream of pulses according to remaining sugar concentration and add the Tapioca Starch hydrolyzed solution that total sugar concentration is 320g/L, maintain sugared concentration at 40～80g/L.
(5) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, sugared concentration in suitable Ploidy testing fermented liquid, calculates sugared transformation efficiency, 2,3-butanediol productivity.
Fermentation ends, the concentration that records glucose is 4.0 ± 0.4g/L (mean+SD), 2,3-butyleneglycol concentration 58.3 ± 1.5g/L (mean+SD), sugar transformation efficiency reaches 70.6 ± 1.6% (mean+SD) of theoretical yield, 2,3-butanediol productivity is 1.14 ± 0.03g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: glucose 40g/L, and potassium primary phosphate 6g/L, ammonium chloride 5g/L, sodium-chlor 3g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, pH value is adjusted to 7.0, distilled water preparation, sterilizing 20 minutes under 115 DEG C of conditions;
Above-mentioned fermention medium consists of: the Tapioca Starch hydrolyzed solution 100g/L that total sugar concentration is 320g/L, Dried Corn Steep Liquor Powder 15g/L, potassium primary phosphate 3g/L, ammonium chloride 5g/L, potassium acetate 4g/L, sodium-chlor 2g/L, calcium chloride 0.075g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.4g/L, ferrous sulfate 0.2g/L, pH value is adjusted to 7.0, tap water preparation, 115 DEG C of sterilizings 20 minutes.
Embodiment 7: utilize enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDMCGMCC No.4230 and produce 2,3-butanediol taking starch hydrolyzate as carbon source simultaneous saccharification and fermentation
The sequence of steps that application method relates to is as follows:
(1) bacterial classification is selected: select enterobacter cloacae to dissolve subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230;
(2) slant culture: bacterial classification is inoculated on slant medium, under 40 DEG C of conditions, leaves standstill and cultivate 12 hours;
(3) seed culture:
First order seed is cultivated: the bacterial strain that step (2) is cultivated, under aseptic condition, encircle in the 500mL of 100mL liquid seed culture medium triangular flask with inoculation articulating 2, be placed on the shaking table that rotating speed is 150rpm, cultivate 12 hours, obtain first order seed nutrient solution for 40 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 1 liter of liquid seed culture medium with the inoculum size of 10% (volume ratio), be placed on the shaking table that rotating speed is 150rpm, cultivate 13 hours, obtain secondary seed nutrient solution for 40 DEG C;
(4) preparation of starch processing and synchronous saccharification batch fermentation substratum:
350g starch is added water and is modulated into starch slurry and is settled to 1L, regulate pH to 6.0, be that 3U/g starch dry weight adds α-amylase by enzyme concentration, boiling 4 hours under 95 DEG C of conditions, obtain liquefied starch, 115 DEG C of sterilizings 20 minutes, cooling rear as carbon source, for the preparation of the fermention medium that carries out synchronous saccharification batch fermentation;
(5) 50 liters of fermentor tank synchronous saccharification batch fermentations are produced 2,3-butanediol:
The inoculum size that is 5% by volume by cultured enterobacter cloacae dissolving subspecies (Enterobacter cloacae subsp.dissolvens) SDM CGMCC No.4230 seed liquor under aseptic condition accesses the fermention medium of sterilizing, add the saccharifying enzyme of filtration sterilization, saccharifying enzyme addition is 200U/g starch dry weight simultaneously; The total liquid amount of fermentor tank is 30L, and taking leavening temperature as 37 DEG C, mixing speed is 350rpm, and air flow is that 1.0vvm ferments; Fermentation initial pH value is 6.5, and in fermenting process, pH value is controlled at pH 6.0; Fermentation time is 40 hours.Residual sugar amount in 5 hours sampling and measuring fermented liquids and 2,3-butanediol concentration.In fermenting process, add the liquefied starch of sterilizing and the saccharifying enzyme of filtration sterilization, saccharifying enzyme addition is 200U/g starch dry weight, and in maintenance fermented liquid, glucose concn is at 15～40g/L.
(6) determination and analysis: get above-mentioned fermented liquid, centrifugal 5 minutes of 8,000rpm, gets supernatant liquor and dilute 2,3-butanediol concentration, sugared concentration in suitable Ploidy testing fermented liquid, calculates sugared transformation efficiency, 2,3-butanediol productivity.
Fermentation ends, the concentration that records total reducing sugar is 3.6 ± 0.4g/L (mean+SD), 2,3-butyleneglycol concentration 82.5 ± 1.7g/L (mean+SD), 2,3-butanediol productivity is 2.06 ± 0.04g/ (Lh) (mean+SD).
Above-mentioned slant medium consists of: glucose 15g/L, and peptone 4g/L, yeast extract paste 4g/L, sodium-chlor 2g/L, Repone K 2g/L, magnesium sulfate 1.5g/L, agar powder 18g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned seed culture medium consists of: glucose 30g/L, and potassium primary phosphate 3g/L, ammonium chloride 8g/L, sodium-chlor 4g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.05g/L, pH value is adjusted to 6.5, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Above-mentioned fermention medium consists of: liquefied starch, and its consumption is 500mL/L, Dried Corn Steep Liquor Powder 7g/L, potassium primary phosphate 6g/L, ammonium chloride 15g/L, potassium acetate 4g/L, sodium-chlor 3g/L, calcium chloride 0.1g/L, magnesium sulfate 0.4g/L, zinc sulfate 0.2g/L, ferrous sulfate 0.2g/L, tap water preparation, the initial pH of this fermention medium is 6.5,115 DEG C of sterilizings 20 minutes; The independent sterilizing of liquefied starch.
Claims (1)
1. the enterobacter cloacae dissolving subspecies of 2,3-butanediol are produced in a strain, it is characterized in that: this bacterial strain enterobacter cloacae dissolving by name subspecies (
enterobacter cloacae subsp. dissolvens) SDM, bacterial strain has been deposited in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number is CGMCC No. 4230 on October 19th, 2010.
Described in claim 1 enterobacter cloacae dissolve subspecies in the application of preparing in 2,3-butanediol, it is characterized in that: by enterobacter cloacae dissolve subspecies (
enterobacter cloacae subsp. dissolvens) SDM CGMCC No. 4230 fermentative production 2,3-butanediols; The method wherein relating to is:
Slant activation cultivate: by enterobacter cloacae dissolve subspecies (
enterobacter cloacae subsp. dissolvens) SDM CGMCC No. 4230 bacterial classifications are inoculated in slant medium, under 30～40 DEG C of conditions, leave standstill and cultivate 10～14 hours, for subsequent use;
First order seed is cultivated: by the inclined-plane bacterial strain of above-mentioned cultivation, under aseptic condition, encircle in 500 mL triangular flasks of 80～120 mL liquid seed culture mediums with inoculation articulating 1～2, be placed on the shaking table that rotating speed is 100～200 rpm, cultivate 10～16 hours, obtain first order seed nutrient solution for 30～40 DEG C;
Enlarged culturing: by the first order seed of above-mentioned cultivation, under aseptic condition, be inoculated in 5 liters of triangular flasks of 0.8～1.2 liter of liquid seed culture medium with the inoculum size of volume ratio 5～10%, be placed on the shaking table that rotating speed is 100～200 rpm, cultivate 10～16 hours, obtain secondary seed nutrient solution for 30～40 DEG C;
50 liters of ferment tanks: with the inoculum size of volume ratio 5～10%, secondary seed solution is inoculated in 50 liters of fermentor tanks, fermentor tank liquid amount is 30 liters, then taking primary carbon source concentration as 60～120 g/L, 30～40 DEG C of temperature, mixing speed 200～600 rpm, air flow 0.5～1.5 vvm ventilate and stir fermentation culture, incubation time is 20～60 hours; Fermented liquid initial pH value is adjusted to 5.5～7.0, in fermenting process, adds the KOH of 3～6 M and the H of 2～5 M by stream
3pO
4regulate pH value, make it to be controlled at pH 5.5～6.5; In fermenting process, the sugared residual in 3～5 hours sampling and measuring fermented liquids and 2,3-butanediol concentration, and according to the concentration current adding substrate of substrate, in the time that in fermented liquid, 2,3-butanediol concentration no longer rises, fermentation ends;
Wherein: the mediums and formulae relating in above-mentioned cultivation is:
Slant medium consists of: glucose 15 g/L, and peptone 4 g/L, yeast extract paste 4 g/L, sodium-chlor 2 g/L, Repone K 2 g/L, magnesium sulfate 1.5 g/L, agar powder 18 g/L, pH value is adjusted to 5.5～7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Seed culture medium consists of: carbon source 30～40 g/L, potassium primary phosphate 3～10 g/L, ammonium chloride 3～12 g/L, sodium-chlor 1～4 g/L, magnesium sulfate 0.05～0.1 g/L, ferrous sulfate 0.05～0.1 g/L, pH value is adjusted to 5.5～7.0, distilled water preparation, 115 DEG C of sterilizings 20 minutes;
Fermention medium consists of: carbon source 60～120 g/L, Dried Corn Steep Liquor Powder 5～20 g/L, potassium primary phosphate 3～10 g/L, ammonium chloride 5～25 g/L, potassium acetate 1～4 g/L, sodium-chlor 1～4 g/L, calcium chloride 0.05～0.1 g/L, magnesium sulfate 0.1～0.5 g/L, zinc sulfate 0.2～0.6 g/L, ferrous sulfate 0.2～0.6 g/L, pH value is adjusted to 5.5～7.0, tap water preparation, 115 DEG C of sterilizings 20 minutes.
3. enterobacter cloacae is dissolved subspecies in the application of preparing in 2,3-butanediol as claimed in claim 2, it is characterized in that: in described 50 liters of ferment tanks cultivation, fed-batch mode is that stream of pulses adds.
4. enterobacter cloacae is dissolved subspecies in the application of preparing in 2,3-butanediol as claimed in claim 3, it is characterized in that: the mode that described stream of pulses adds is in the time that sugared concentration drops to 10～30 g/L, and pulse current adding substrate, makes sugared concentration reach 50～70 g/L.
5. enterobacter cloacae is dissolved subspecies in the application of preparing in 2,3-butanediol as claimed in claim 2, it is characterized in that: the carbon source in described seed culture medium at least contains glucose or wood sugar.
6. enterobacter cloacae is dissolved subspecies in preparation 2 as claimed in claim 2, application in 3-butyleneglycol, is characterized in that: the carbon source in described fermention medium at least contains the one in glucose, wood sugar, sucrose molasses, xylose mother liquid, starch materials hydrolyzed solution, lignocellulosic material hydrolyzed solution; The independent sterilizing of this carbon source, without regulating pH; Wherein, described starch materials hydrolyzed solution makes with following method: get starch materials, enzyme dosage with 2～8 U/g starch materials dry weights adds α-amylase, process 0.5 h for 95 DEG C, obtain starch materials liquefier, then adjust pH to 4.2, add saccharifying enzyme, the addition of saccharifying enzyme is 100～1000 U/g starch materials dry weights, is placed in 60 DEG C of shaking baths and processes to glucose and be thoroughly hydrolyzed, and obtains sugar-containing concentration and counts 15%～40% starch materials hydrolyzed solution with mass volume ratio; Described lignocellulosic material hydrolyzed solution makes with following method: the lignocellulosic material of getting pulverizing, in mass volume ratio, by lignocellulosic material: the amount that massfraction is 1% sulfuric acid=1:10～1:3 adds sulfuric acid, process 1 hour for 120 DEG C, conventional centrifugal, supernatant liquor is sugar-containing concentration and counts 15%～35% lignocellulosic material hydrolyzed solution with mass volume ratio.
7. enterobacter cloacae is dissolved subspecies in the application of preparing in 2,3-butanediol as claimed in claim 6, it is characterized in that: described starch materials selects starch or Tapioca Starch; Described lignocellulosic material selects wood, corn cob or stalk.
8. enterobacter cloacae is dissolved subspecies in the application of preparing in 2,3-butanediol as claimed in claim 6, it is characterized in that: in described starch materials hydrolyzed solution, sugared concentration counts 25%～35% with mass volume ratio; In described lignocellulosic material hydrolyzed solution, sugared concentration counts 25%～30% with mass volume ratio.
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| CN104046586B (en) * | 2014-07-02 | 2016-08-17 | 山东大学 | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof |
| CN104593285A (en) * | 2014-09-30 | 2015-05-06 | 广西科学院 | Enterobacter cloacae gxas-h1 and use thereof |
| KR101745079B1 (en) * | 2015-04-15 | 2017-06-09 | 고려대학교 산학협력단 | Method for producing 2,3-butanediol comprising of adding acetate |
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