CN101250561B - Method for producing butanol and butanedioic acid by fermentation - Google Patents
Method for producing butanol and butanedioic acid by fermentation Download PDFInfo
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Abstract
The present invention discloses a method for producing butyl alcohol and succinic acid through fermentation. The method which is the method for producing the butyl alcohol and the succinic acid produces the butyl alcohol and the succinic acid simultaneously through fermenting microbe in different systems and gas which is produced in producing the butyl alcohol through the fermentation is inputted in a system for producing the succinic acid through the fermentation. The method connects a butyl alcohol fermentation system and a succinic acid fermentation system in series, CO2 and H2 which are produced through fermenting the butyl alcohol are utilized to promote succinic acid synthesis, which not only realizes the comprehensive use of the CO2, reduces the discharge of greenhouse gas, but also reduces the using amount of carbonate in the succinic acid producing process. The method does not need reforming complex devices and only needs adding a filtrating and removing bacteria device and then connecting with an air intake duct of a succinic acid fermentor. Experiments show that the method of the invention can keep or even increase the synthesis concentration of the succinic acid under the condition that the carbonate is not additionally added and simultaneously butyl alcohol synthesis is not affected.
Description
Technical field
The present invention relates to the method for a kind of fermentative production butanols and Succinic Acid.
Background technology
Through 4,000,000,000 years very long evolutionary processes, just form on the earth today accurately, efficient, science, the stable ecosystem.Yet the fast development of industrial system not only makes the whole world face serious resource and energy dilemma in the period of short 150, and causes many serious social concerns such as global warming, and China has become greenhouse gases CO
2Main contributor, quantity discharged has occupied the second place of the world, and will continue to increase.
CO
2Being the gas that atmosphere " Greenhouse effect " is had the greatest impact, is again carbon resource the abundantest on the earth.People can pass through CO
2Reduce discharging and two kinds of methods of carbon sequestration, reach in conjunction with means such as production that improves Energy production and service efficiency and increase low-carbon (LC) or non-carbon fuel and utilizations simultaneously and slow down Atmospheric CO
2The target that concentration increases.Utilize microorganism fixation of C O
2It is a very promising development field that organic acid, alcohol etc. are produced in metabolism simultaneously.
Butanols (Butanol) is a kind of important chemical material, is mainly used in to make softening agent, solvent, extraction agent etc., and global annual requirement is above 1,400,000 tons.Butanols is again a kind of novel biological fuel that has potentiality, and its calorific value, octane value and gasoline are suitable; Methyl tertiary butyl ether commonly used in oxygen level and the gasoline is close; Can corrosion pipeline, be convenient to pipe-line transportation; Steam forces down, and is safe, and can with gasoline with any than mixing.
Fermentative Production acetone-butanol (claiming the ABE fermentation again) once was to be only second to alcoholic acid world's second largest fermentation industry.After the eighties in 20th century and since the continuous development of chemical synthesising technology and International Crude Oil continue drop the butylic fermentation market of fading out gradually.Since 2003, along with rising steadily of oil price, countries in the world increase day by day to the worry of energy security and resource security, and butylic fermentation industry has welcome the opportune moment of industry recovery.
Pasteur in 1861 finds that first bacterium can produce butanols, has found that a kind of clostridium Clostridium acetobutylicum can be converted into starch acetone, butanols and ethanol Wei in 1912 graceful now (Weizmann).So far, being used for the wild bacterium of topmost two strains of acetone butanol fermentation is acetone-butanol clostridium (Clostridiumacetobutylicum, be called for short the third fourth bacterium) and Bai Shi clostridium (Clostridium beijerinckii), these two kinds of bacterium are strictly anaerobic bacterium.This two strains bacterium can both utilize starch, but apart from each other (George in phylogeny, H.and e.al, Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn.Clostridium butylicum) and Clostridium aurantibutyricum.Appl.Environ.Microbiol., 1983.45:p.1160-1163).
In C.acetobutylicum, butylic fermentation can be divided into two stages: at the fermentation initial stage, carbon source mainly is converted into acetate and butyric acid; Descend along with fermentation time prolongs fermented liquid pH, when, pH＜4.5 excessive when substrate, acetate and/or butyric acid surpassed threshold concentration and phosphoric acid salt or the growth of vitriol restrictive cell, cell began synthetic in a large number butanols, acetone and ethanol (being collectively referred to as total solvent).According to document, majority without the strain fermentation process of genetic modification in solvent ratio (butanols: acetone: ethanol, mass ratio) is 6: 3: 1 (Nolling substantially, J.and e.al, Genome sequenceand comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum.J.Bacteriol., 2001.183:p.4823-4838).
From top pathways metabolism as can be seen, during anaerobically fermenting C.acetobutylicum, all can produce a large amount of CO no matter produce acid phase or produce the solvent stage
2: according to document, most is 6: 3: 1 without solvent ratio in the strain fermentation process of genetic modification (butanols: acetone: ethanol, mass ratio) substantially, can produce 1.49 tons of CO at least in the process in 1 ton of total solvent of this ratio calculating fermentative production
2, a certain amount of H of while by-product
2
Bulk fermentation waste gas directly discharges, and not only can increase environmental pressure, and can reduce the utilization ratio of carbon source.Also at the early-stage to the research and the application of fermentation waste gas comprehensive utilization aspect at present, CO in the fermentation waste gas
2When purity is higher, as liquor fermentation (Zhang Guangran, the CO in the liquor fermentation
2Reclaim and utilization wine brewing, 2003.30 (4): p.66-67), can use low pressure, medium-pressure or high pressure method directly with CO
2Liquefaction is reclaimed; Exhaust gas constituents is complicated more, and the cost of Separation and Recovery is high more.
Succinic Acid claims that again (Succinic acid succinate), is a kind of common natural organic acids to succsinic acid.After 1546 separated from amber first, Succinic Acid was in agricultural, food, medicine and other fields widespread use.Succinic Acid is a kind of important chemical, can be used for fields such as tensio-active agent, sanitising agent, solvent, can be used as pH modifying agent, flavour substances or antiseptic-germicide in food service industry, also can be as animal feedstuff additive and plant growth substances etc.Succinic Acid industrial also be a kind of important C4 compound, can be converted into a lot of important chemical, as lipid acid, 1, the 4-butyleneglycol, tetrahydrofuran (THF), (Song such as gamma-butyrolactone, H.and S.Lee, Productionof succinic acid by bacterial fermentation.Enzyme and Microbial Technology., 2006.39 (3): p.352-361.), can also be used for synthesising biological degradation material PBS (poly-butyric acid succsinic acid) and urethane (Willke as monomer, T.and K.Vorlop, Industrial bioconversion of renewable resources asan alternative to conventional chemistry.Appl.Microbiol.Biotechnol., 2004.66:p.131-142).At present existing to surpass 250 kinds be that the Chemicals of raw material can transform by Succinic Acid and obtain with benzene, so the market requirement of Succinic Acid constantly increases.
The Succinic Acid of Xiao Shouing is chemically production with liquefied petroleum gas (LPG) or oil all in the market.Because chemical method is too high by the cost that butane is converted into maleic anhydride production Succinic Acid, the output of Succinic Acid and sales volume are not high, and Application Areas is very limited; In recent years because the progress of biotechnology, the cost of fermentative Production Succinic Acid constantly descends, be lower than chemical synthesis (Willke, T.and K.Vorlop, Industrial bioconversion ofrenewable resources as an alternative to conventional chemistry.Appl.Microbiol.Biotechnol., 2004.66:p.131-142).And the fermentative Production Succinic Acid is a raw material with renewable resourcess such as glucose, and fixing greenhouse gases CO in the biotransformation
2, therefore all obviously be better than chemical synthesis for resource security and environmental safety.
Succinic Acid is biological tricarboxylic acid (TCA) round-robin intermediate product, also is the end product of a lot of anaerobic metabolism processes, and therefore, most microorganisms, animal and plant can both synthesizing succinic acid.But the system of the high succinic acid-producing of energy mainly is fungi and bacterium, have been found that the bacterial strain that can produce Succinic Acid mainly contains Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducensMBEL55E, Aspergillus niger, Aspergillus fumigatus, Byssochlamys nivea, Lentinusdegener, Penicillium viniferum and yeast Saccharomyces cerevisiae etc.The former two's research is more, and the Succinic Acid synthesis capability is also higher.Also there is Many researchers to utilize engineered method to make up recombination bacillus coli in recent years and produces Succinic Acid.
The approach of these bacterial strain synthesizing succinic acids is not quite similar, some mainly is by phosphoenolpyruvic acid carboxylation reaction (Lee, P., et al., Succinic acid production with reduced by-product formtation in thefermentation of Anaerobiospirillum succiniciproducens using glycerol as
According to stoichiometric equation as can be known, every production 1mol Succinic Acid is the 1mol carbonic acid gas fixedly, and 1 ton of Succinic Acid of promptly every production needs to add 0.37 ton of carbonic acid gas at least.The supply of carbonic acid gas mainly contains two kinds of methods in the Succinic Acid fermenting process at present: the one, directly feed carbonic acid gas, and the 2nd, utilize carbonate (as yellow soda ash, lime carbonate or magnesiumcarbonate etc.).These two kinds of methods all need to increase extra fermentation costs.
Summary of the invention
The method that the purpose of this invention is to provide a kind of fermentative production butanols and Succinic Acid.
The method of fermentative production butanols provided by the invention and Succinic Acid is that the while microbial fermentation is produced butanols and Succinic Acid in different systems, and the gas that the fermentative production butanols produces is fed in the system of fermentation production of succinic acid.This technical scheme comprises two microbial fermentation systems---butylic fermentation and Succinic Acid fermentation discharge a large amount of CO in the former fermenting process
2, the latter utilize conversion of glucose for the process of Succinic Acid in fixation of C O simultaneously
2, these two process integration are got up the CO that the butylic fermentation process produces
2And H
2Be used for the Succinic Acid fermentation, the exhaust gas emission when both reducing butylic fermentation provides CO for the Succinic Acid fermenting process again
2And H
2, to realize the recycle and the energy-saving and emission-reduction of carbon source.
In the described method, the microorganism of described fermentative production butanols can be clostridium acetobutylicum (Clostridiumacetobutylicum) or Bai Shi clostridium (Clostridium beijerinckii), the microorganism of described fermentation production of succinic acid can be Anaerobiospirillum succinoproducens (Anaerobiospirillum succiniciproducens), succsinic acid actinobacillus (Actinobacillus succinogenes), produce succsinic acid pasteurella (Mannheimiasucciniciproducens) MBEL55E, aspergillus niger (Aspergillus niger), Aspergillus fumigatus (Aspergillusfumigatus), or yeast (Saccharomyces cerevisiae).
In the described method, the substrate of described fermentative production butanols is Semen Maydis powder or glucose, and the substrate of described fermentation production of succinic acid is a glucose.
In the described method, the microorganism of described fermentative production butanols is preferably clostridium acetobutylicum (Clostridiumacetobutylicum) SB-1 CGMCC No.2287; The microorganism of described fermentation production of succinic acid is preferably succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618.
In the described method, the gas that described fermentative production butanols produces degerming after filtration directly feeds the fermentor tank of described fermentation production of succinic acid or controls the amount of the fermentor tank of feeding fermentation production of succinic acid through surge tank, gas meter.
In the described method, the substratum of described fermentative production butanols is preferably Semen Maydis powder gelatinization liquid; The concentration of Semen Maydis powder is 60-90g/L in the described Semen Maydis powder gelatinization liquid; The substratum of described fermentation production of succinic acid is preferably every liter and contains glucose 30.0-50.0g, yeast powder 10.0g, (NH
4)
2SO
45.5g, K
2HPO
4.3H
2O 8.6g, NaH
2PO
45.0g, MgSO
4.7H
2O 6.0g, FeSO
4.7H
2O 0.005g.
In the described method, the pH of described fermentation production of succinic acid is 5.5-7.0.
In the described method, described fermentation production of succinic acid process flow adds the glucose supplementary carbon source, and glucose concn maintains 5-50g/L and (adds glucose 80-120g l altogether
-1).
In the described method, the described CO that produces
2The microbial fermentation starting point concentration of producing butanols be 10
6-10
7Cfu/L; The described CO that utilizes
2The starting point concentration of producing succinic acid by microbial fermentation be 10
6-10
7Cfu/L.
In the described method, the culture volume of described fermentative production butanols is more than or equal to the culture volume of described fermentation production of succinic acid.
Analyze with experimental verification by following technical conceive and to obtain technique scheme of the present invention: 1) two thinkings that the bulk chemical fermenting process is integrated; 2) butylic fermentation process optimization; 3) Succinic Acid fermentation technology optimization; 4) the butylic fermentation aerogenesis consumes combining of gas process with the Succinic Acid fermentation; 5) the compatibility analysis of butylic fermentation gas production rate and Succinic Acid fermentation air consumption does not need to increase storage unit; 6) H in the butylic fermentation tail gas
2Favourable to keeping Succinic Acid yeasting raising Succinic Acid output.
Method of the present invention is made up of the industrial fermentation system of two important bulk chemical, and is together in series by tail gas/intake ducting: utilize anaerobically fermenting W-Gum or glucose production butanols, acetone etc., the tail gas that produces in this process (mainly is H
2And CO
2) after filtration degerming directly be passed in the Succinic Acid fermentation system CO
2In the Succinic Acid building-up reactions, be utilized, and H
2Then can keep the anaerobic environment of Succinic Acid fermentation system, improve the Succinic Acid productive rate.The application of this technology can make no longer adds carbonate in the Succinic Acid fermenting process, and butanols and Succinic Acid fermenting process efficient are also unaffected.
The system that the present invention will produce the system of carbonic acid gas and consume carbonic acid gas is integrated in the system carries out, and has improved utilization of carbon source rate and plant factor, has reduced the fermentation industry exhaust gas discharging, has reduced fermentation costs; In two fermentation systems, only need add a breather line and an air filter, just can form integrated technique, characteristics such as it is simple, easy to operate to have equipment, and cost is low.
Compare with traditional independent zymotechnique, the integrated technique that method of the present invention adopts has following feature: 1) two system's series connection, simplified the intermediate treatment link, and reduced facility investment; 2) use for the Succinic Acid fermentation after the gas process simple filtering degerming that butylic fermentation produces, simplified the butylic fermentation tail gas treatment process; 3) CO of butylic fermentation generation
2Be used for the Succinic Acid building-up reactions, improved the utilization of carbon source rate, reduced the raw materials cost of Succinic Acid fermentation; 4) CO of butylic fermentation generation
2And H
2Be passed into the Succinic Acid fermentation system simultaneously, can improve Succinic Acid yield and productive rate, shorten fermentation period, reduce fermentation costs.
Description of drawings
Fig. 1 is the fermentative production butanols integrated zymotechnique schema of connecting with Succinic Acid; Among the figure, 1 is the butylic fermentation jar; 2 is the Succinic Acid fermentor tank; 3 is valve; 4 is gas filter.
Fig. 2 is succsinic acid actinobacillus (Actinobacillus succmogenes) ATCC 55618 fermentation production of succinic acid process product growing amount change curves
Fig. 3 is clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (CGMCC No.2287, a Chinese microorganism strain preservation center) fermentative production butanols process product growing amount change curve
Fig. 4 is the required CO of Succinic Acid building-up process
2Produce CO with the butanols building-up process
2Amount the time varied curve figure
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition described in the following embodiment if not otherwise specified, is the quality percentage composition.
The screening and the compliance test result of embodiment 1, fermentative production butanols of the present invention and Succinic Acid
Fermentative production butanols of the present invention and Succinic Acid are respectively with producing CO
2Microbial fermentation produce butanols, with utilizing CO
2Producing succinic acid by microbial fermentation; Its production unit sketch as shown in Figure 1, among Fig. 1,1 is the butylic fermentation jar; 2 is the Succinic Acid fermentor tank; 3 is valve; 4 is gas filter; The tail gas of butylic fermentation jar 1 feeds Succinic Acid fermentor tank 2 by valve 3 and gas filter 4.
Following embodiment is with clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) carries out butylic fermentation, carry out the Succinic Acid fermentation with succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618.
1, the acquisition of the fermentation seed liquid of clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287)
1) acquisition of clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287)
Near 40 parts of the soil samples of the plant rhizosphere of seven Augusts collection Yunnan Tengchong hot spring.Soil sample added respectively 10mL RCM substratum is housed (substratum is formed: every liter of substratum contains yeast powder 3.0g, peptone 10.0g, extractum carnis 10.0g, glucose 5.0g, starch 10.0g, sodium acetate 3.0g, L-cysteine hydrochloride 0.5g, butanols, pH6.8) in the anaerobism pipe, after the mixing, in 100 ℃ of boiling water, heat 90S, kill the nourishing body cell, in 37 ℃ of incubators, cultivate.Routine observation is chosen the anaerobism pipe that has bubble to produce, and uses the Heng Gaite rolling tube technique, separates single bacterium colony.
With single bacterium colony choose spread cultivation after, shift maize powder medium (the quality percentage composition is 7% Semen Maydis powder solution), the fermentation phenomenon is observed in 37 ℃ of cultivations, chooses the test tube that has the wine with dregs lid to form, bacterial strain 105 strains that obtain producing butanols altogether.To these bacterial strains in maize powder medium (the quality percentage composition is 7% Semen Maydis powder solution), 37 ℃ ferment again, karusen is carried out liquid-phase chromatographic analysis, compare the solvent ultimate production of each bacterial strain and the ratio of butanols, through the fermentation screening, it is fast to select a strain fermenting speed, and the bacterial strain that the ratio of butanol of generation is high is clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 CGMCC No.2287.
The form of clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 CGMCC No.2287 vegetative cell is the circular rod-short in two ends, single or duplex, and amphitrichous, movable, cell has 2.6～4.7 * 0.5～0.7 micron.The physiological and biochemical property of SB-1 is identified according to uncle Jie Shi Bacteria Identification handbook and is carried out, the results are shown in Table 1.
The physiological and biochemical property of table 1.SB-1
| Clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 | |
| Lonely property | Strictly anaerobic is not grown under the aerobic condition |
| Gram-reaction | Gramstaining is |
| Growth temperature | |
| 20～47 ℃, 37 ℃ of optimums | |
| Protein | Can degrade |
| Gelatin | Can liquefy |
| Starch | Can ferment |
| Fructose | Can ferment |
| Sucrose | Can ferment |
| Maltose | Can ferment |
| Glucose | Can ferment |
| Wood sugar | Can ferment |
| Semi-lactosi | Can ferment |
| Lactose | Can ferment |
| Sorbyl alcohol | Cannot ferment |
| Melibiose | Cannot ferment |
| Glycerine | Cannot ferment |
| Mierocrystalline cellulose | Cannot ferment |
Clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 is preserved in Chinese microorganism strain preservation board of trustee reason person on December 11st, 2007 and understands common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .2287.
Clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 CGMCC No.2287 has been carried out the 16SrDNA sequence amplification, order-checking shows, the nucleotide sequence that the 16S rDNA sequence of SB-1 has sequence 1 in the sequence table, and on the NCBI website, carry out the BLAST comparison.The result shows, the homology of 16S ribosomal RNA (rrn) gene of 16S rDNA sequence C lostridium acetobutylicum ATCC 824 (U16166.1) of SB-1 is 100%, is 99% with the homology of Clostridium acetobutylicum VKPM B-4786 (AM231184.1), Clostridium acetobutylicum 6MSU (AM231182.1), Clostridium acetobutylicum 7MSU (AM231183.1) 16S rRNA.
2) clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) seed culture
1. the activation of clostridium acetobutylicum (Clostridium acetobutylicum) SB-1
Get clostridium acetobutylicum (Clostridium acetobutylicum) frozen storing liquid that 2ml contains spore, boiling water heat shock 60 seconds is taken out, and cold water is inoculated into after the cooling rapidly and contains 10ml RCM substratum (every liter of substratum contains yeast powder 3.0g, peptone 10.0g, extractum carnis 10.0g, glucose 5.0g, starch 10.0g, sodium acetate 3.0g, L-cysteine hydrochloride 0.5g, pH 6.8) the anaerobism pipe in, 37 ℃, 150rpm cultivates 16h and obtains nutrient solution, room temperature is placed, and waits for inoculation culture.
2. the seed culture of clostridium acetobutylicum (Clostridium acetobutylicum) SB-1
Get good clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 of activation (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) nutrient solution, with volumn concentration is that 5% inoculum size is inoculated in the anaerobism bottle that contains 50ml RCM substratum, 37 ℃, 150rpm cultivates 10-12h.And then be inoculated into that (the inoculum size volumn concentration is 5%, 10 in the anaerobism bottle that contains 50ml RCM substratum
7Cfu/L), 37 ℃, 150rpm cultivates 16h, obtains growing to clostridium acetobutylicum (Clostridium acetobutylicum) the SB-1 nutrient solution of logarithmic phase, as seed liquor.
2, the acquisition of the fermentation seed liquid of succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618
1) succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 actication of culture
Getting 100 μ l succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC, 55618 frozen storing liquids by frozen pipe under the oxygen free condition is applied to the LB substratum (every liter of substratum contains glucose 10.0g, yeast powder 5.0g, peptone 10.0g, NaCl 10.0g, pH 6.5.Add agar 15.0g therein and be solid medium) the solid inclined-plane activates, 37 ℃ cultivate 12h after, picking one ring lawn inserts solid LB flat board again, 37 ℃ obtain single bacterium colony after cultivating 12h.
2) succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 seed culture
Single bacterium colony of picking on the LB flat board that obtains from step 1), be inoculated in the anaerobism bottle that contains 50ml LB substratum, 37 ℃, 150rpm, cultivate succsinic acid actinobacillus (Actinobacillussuccinogenes) ATCC 55618 nutrient solutions that 16h obtains growing to logarithmic phase, as seed liquor.
3, fermentative production butanols and/or Succinic Acid
Carry out the method for following method one to five respectively and carry out fermentative production butanols and/or Succinic Acid, relatively the effect of traditional zymotic method and the inventive method:
Method one, succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 fermentation production of succinic acid---with Na
2CO
3Regulate pH and the required CO of afterfermentation process
2
(every liter of substratum contains glucose 30.0g, yeast powder 10.0g, (NH with 4 liters of sterilized succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC, 55618 fermention mediums are housed in 7 liters of Succinic Acid fermentor tanks 2 shown in Figure 1
4)
2SO
45.5g, K
2HPO
4.3H
2O 8.6g, NaH
2PO
45.0g, MgSO
4.7H
2O6.0g, FeSO
4.7H
2O 0.005g), be 5% (10 with the cultured succsinic acid actinobacillus of aforesaid method (Actinobacillussuccinogenes) ATCC 55618 seed liquor with volumn concentration
7Cfu/L) inoculum size inserts in the fermentor tank 2, and 37 ℃, 200rpm cultivated 80 hours, in the fermenting process with Na
2CO
3Regulating pH is 6, and stream adds glucose (keeping the concentration of fermented liquid glucose is 5-50g l simultaneously
-1, add glucose 100g l altogether
-1) supplementary carbon source.In the fermenting process, get fermented liquid, change with liquid chromatographic detection Succinic Acid, acetate, lactic acid, concentration of ethanol.
Fermenting process product formation curve respectively as shown in Figure 2; The primary product concentration of fermenting 60 hours is as shown in table 2, and the primary product concentration of fermenting 80 hours is as shown in table 3, and the result shows that 60 hours Succinic Acid concentration of fermentation reaches 61.5g/L, and fermentation consumed Na in 60 hours in the fermenting process
2CO
360g/L; Continue to prolong fermentation time, Succinic Acid concentration is up to 80.0g/L (fermenting 80 hours).The Succinic Acid fermentation byproduct is few, can acetate, ethanol and lactic acid only be arranged detected by product, and concentration is very low.
Butanols and Succinic Acid fermentation system primary product concentration (60 hours) in each method of table 2.
| Method four | 10.5 | 3.4 | 1.7 | 15.4 | 63.0 | 0.5 | 2.0 | 1.2 | 60.0 |
| Method five | 10.5 | 3.3 | 1.7 | 15.3 | 61.8 | 1.1 | 2.3 | 2.3 | 0 |
Butanols and Succinic Acid fermentation system primary product concentration (80 hours) in each method of table 3.
Method two, additionally do not feed CO
2Reach succsinic acid actinobacillus (Actinobacillussuccinogenes) ATCC 55618 fermentation production of succinic acid when adding carbonate
To 4 liters of sterilized succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 fermention mediums (substratum is with method one) be housed in 7 liters of Succinic Acid fermentor tanks 2 shown in Figure 1, cultured succsinic acid actinobacillus ATCC 55618 seed liquor are 5% (10 with volumn concentration according to the method described above
7Cfu/L) insert in this fermentor tank 2,37 ℃, 200rpm cultivates, and regulating pH with NaOH in the fermenting process is 6, and stream adds glucose (keeping the concentration of fermented liquid glucose is 5-50g l simultaneously
-1, add glucose 100g l altogether
-1) supplementary carbon source.In the fermenting process, get fermented liquid, change with liquid chromatographic detection Succinic Acid, acetate, lactic acid, concentration of ethanol.
The primary product concentration of fermenting 60 hours is as shown in table 2, the primary product concentration of fermenting 80 hours is as shown in table 3, the result shows, fermenting, Succinic Acid concentration reaches 53.0g/L after 60 hours, continue to prolong fermentation time, fermenting, Succinic Acid concentration is up to 65.0g/L after 80 hours, well below adding Na
2CO
3Technology.
Method three, clostridium acetobutylicum (Clostridium acetobutylicum) fermentative production butanols
Clostridium acetobutylicum (Clostridium acetobutylicum) fermention medium (corn culture medium): the Semen Maydis powder ultimate density is 75g/L, sterilizes behind the gelatinization 30min.The gelatinizing method of Semen Maydis powder: the Semen Maydis powder of certainweight is put into the normal-temperature water of certain volume, and soak about 5min the back that stirs, and pours in the ebullient water, stirs in order to avoid stick with paste the end, and making the Semen Maydis powder ultimate density is 75g/L.
To 4 liters of RCM substratum of sterilization in advance be housed in 7 liters of butylic fermentation jars 1 shown in Figure 1, will be according to the method described above cultured clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) seed liquor be 5% (10 with volumn concentration
7Cfu/L) insert in this butylic fermentation jar 1,37 ℃ leave standstill cultivation.Fermentation tail gas directly discharges after the degerming after filtration.In the fermenting process, get fermented liquid, with the quantitative changeization of liquid chromatographic detection butanols, acetone, ethanol, total solvent (generating butanols, acetone, alcoholic acid total amount).
Butylic fermentation process product growing amount change curve as shown in Figure 3; The primary product concentration of fermenting 60 hours is as shown in table 2, and the primary product concentration of fermenting 80 hours is as shown in table 3, and the result shows that fermenting, butanol concentration reaches 10.5g/L after 60 hours.The main by product of butylic fermentation jar 1 is acetone and ethanol, and the two is important solvent and chemical.
Varied curve during according to the fermenting process meta-bolites of Fig. 2 and Fig. 3 two individual system can calculate the CO that butylic fermentation produces
2The CO that amount and Succinic Acid fermenting process consume
2Amount, result as shown in Figure 4, the result shows the CO that butylic fermentation produces
2The CO that amount and Succinic Acid fermenting process consume
2Varied curve is identical substantially during amount.Illustrate, in the fermentation system of same volume, the CO that butylic fermentation produces
2Substantially it is required to satisfy the Succinic Acid fermentation.
Method four, use integrated technique provided by the invention are realized butanols and Succinic Acid cascade fermentation
Device as shown in Figure 1, the exhaust pipe of fermentor tank 1 connect an air filter 4 backs and link to each other with the ventilation inlet of fermentor tank 2.Wherein, 4 liters of corn culture medium of sterilization in advance are housed in 7 liters of butylic fermentation jars 1 shown in Figure 1, is 5% (10 with cultured clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) seed liquor with volumn concentration
7Cfu/L) insert in the fermentor tank 1,37 ℃ leave standstill cultivation.4 liters of sterilized succsinic acid actinobacillus fermention mediums are housed in 7 liters of fermentor tanks 2 shown in Figure 1, are 5% (10 with cultured succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 seed liquor with volumn concentration
7Cfu/L) insert in the fermentor tank 2,37 ℃, 200rpm cultivates, in the fermenting process with Na
2CO
3Regulating pH is 6, and stream adds glucose (keeping the concentration of fermented liquid glucose is 5-50g l simultaneously
-1, add glucose 100g l altogether
-1) supplementary carbon source.In the fermenting process, get fermented liquid in the fermentor tank 1, detect the quantitative changeization of butanols, acetone, ethanol, total solvent (generating butanols, acetone, alcoholic acid total amount) with liquid phase chromatography; Get fermented liquid detection Succinic Acid, acetate, lactic acid, concentration of ethanol variation in the fermentor tank 2.
The primary product concentration of fermenting 60 hours is as shown in table 2, the primary product concentration of fermenting 80 hours is as shown in table 3, and the result shows that the integrated fermentation of butanols and Succinic Acid is after 60 hours, the yellow soda ash addition is 60g/L, and butanols and Succinic Acid concentration reach 10.5g/L and 63.0g/L respectively.Prolong fermentation time (fermenting 80 hours), butyric acid and Succinic Acid concentration best result do not reach 11.0g/L and 82.0g/L.The synthetic concentration of Succinic Acid is a little more than independently Succinic Acid fermentation under these processing condition.The main by product of butylic fermentation (fermentor tank 1) is acetone and ethanol, and Succinic Acid fermentation (fermentor tank 2) by product is acetate, ethanol and lactic acid.
Method five, use integrated technique provided by the invention realize that butanols and Succinic Acid (do not add carbonate and CO
2) cascade fermentation
As the experimental installation of Fig. 1, the exhaust pipe of fermentor tank 1 connects an air filter 4 backs and links to each other with the ventilation inlet of fermentor tank 2.Wherein, 4 liters of RCM substratum of sterilization in advance are housed in 7 liters of butylic fermentation jars 1 shown in Figure 1, is 5% (10 with cultured clostridium acetobutylicum (Clostridium acetobutylicum) SB-1 (Chinese common micro-organisms culture presevation administrative center, CGMCC No.2287) seed liquor with volumn concentration
7Cfu/L) insert in the fermentor tank 1,37 ℃ leave standstill cultivation.4 liters of sterilized succsinic acid actinobacillus fermention mediums are housed in the fermentor tank 2, are 5% (10 with cultured succsinic acid actinobacillus (Actinobacillus succinogenes) ATCC 55618 seed liquor with volumn concentration
7Cfu/L) insert in the fermentor tank 2,37 ℃, 200rpm cultivates, and regulating pH with NaOH in the fermenting process is 6.2, and stream adds glucose (keeping the concentration of fermented liquid glucose is 5-50g l simultaneously
-1, add glucose 100g l altogether
-1) supplementary carbon source.
The primary product concentration of fermenting 60 hours is as shown in table 2, and the primary product concentration of fermenting 80 hours is as shown in table 3, and the result shows that butanols and Succinic Acid fermentation are after 60 hours, and butanols and Succinic Acid concentration reach 10.5g/L and 61.8g/L respectively; Prolong fermentation time (fermenting 80 hours), butyric acid and Succinic Acid concentration best result do not reach 11.0g/L and 80.8g/L.Independently ferment, add in the Succinic Acid fermentation system basically identical as a result of carbonate with butanols, need not add Na but save under these processing condition
2CO
3The main by product of butylic fermentation (fermentor tank 1) is ethanol and acetone, and the main by product of Succinic Acid fermentation (fermentor tank 2) is acetate, ethanol and lactic acid.
Sequence table
Claims (3)
1. a method of producing butanols and Succinic Acid is that the while microbial fermentation is produced butanols and Succinic Acid in different systems, and the gas that the fermentative production butanols produces is fed in the system of fermentation production of succinic acid;
The microorganism of described fermentative production butanols is clostridium acetobutylicum (Clostridium acetobutylicum) SB-1CGMCC No.2287; The microorganism of described fermentation production of succinic acid is succsinic acid actinobacillus (Actinobacillussuccinogenes) ATCC 55618;
The substratum of described fermentative production butanols is a Semen Maydis powder gelatinization liquid; The concentration of Semen Maydis powder is 60-90g/L in the described Semen Maydis powder gelatinization liquid; The substratum of described fermentation production of succinic acid is every liter and contains glucose 30.0-50.0g, yeast powder 10.0g, (NH
4)
2SO
45.5g, K
2HPO
4.3H
2O 8.6g, NaH
2PO
45.0g, MgSO
4.7H
2O 6.0g, FeSO
4.7H
2O 0.005g;
The pH of described fermentation production of succinic acid is 5.5-7.0;
Described fermentation production of succinic acid process flow adds the glucose supplementary carbon source, and glucose concn maintains 5-50g/L;
The gas that described fermentative production butanols produces degerming after filtration directly feeds the fermentor tank of described fermentation production of succinic acid or controls the amount of the fermentor tank of feeding fermentation production of succinic acid through surge tank, gas meter.
2. method according to claim 1 is characterized in that: the starting point concentration of the microorganism of described fermentative production butanols is 10
6-10
7Cfu/L; The starting point concentration of the microorganism of described fermentation production of succinic acid is 10
6-10
7Cfu/L.
3. method according to claim 2 is characterized in that: the culture volume of described fermentative production butanols is more than or equal to the culture volume of described fermentation production of succinic acid.
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| CN101613722B (en) * | 2009-07-31 | 2011-09-07 | 安徽丰原发酵技术工程研究有限公司 | Alcohol and succinic acid production method by fermenting cellulosic raw material |
| CN102286547B (en) * | 2011-08-18 | 2013-06-12 | 江南大学 | Method for producing biological butyl alcohol by semicontinuous fermentation |
| CN103131732B (en) * | 2011-12-02 | 2015-10-14 | 中国科学院青岛生物能源与过程研究所 | A kind of method improving butanol concentration in acetone butanol fermentation process |
| CN103205468A (en) * | 2013-04-24 | 2013-07-17 | 南京工业大学 | Method for preparing succinic acid by using sucrose fermentation |
| CN104611385B (en) * | 2015-03-09 | 2017-10-31 | 吕涛 | A kind of method for preparing lactic acid and succinic acid using same bacterial strain CGMCC 1593 fermentation couplings |
| FR3053357B1 (en) * | 2016-06-30 | 2019-07-26 | IFP Energies Nouvelles | PROCESS FOR RECOVERING ALCOHOLS IN A FERMENTER |
| CN106801026B (en) * | 2017-02-13 | 2020-05-12 | 广西科学院 | Bacterial strain for producing succinic acid by fermentation of xylose mother liquor and production method thereof |
| CN111349661A (en) * | 2019-12-30 | 2020-06-30 | 淮阴师范学院 | Method and device for efficiently and biologically fixing butanol fermentation tail gas and industrial ethylene oxide tail gas |
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| CN1143677A (en) * | 1995-08-23 | 1997-02-26 | 中国科学院上海植物生理研究所 | High-butanol ratio clostridium acetobutylicium and culturing method and use thereof |
| CN101153294A (en) * | 2007-08-30 | 2008-04-02 | 合肥工业大学 | Immobilized cell single-tank high-strength continuous fermentation process for succinic acid |
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| CN1143677A (en) * | 1995-08-23 | 1997-02-26 | 中国科学院上海植物生理研究所 | High-butanol ratio clostridium acetobutylicium and culturing method and use thereof |
| CN101153294A (en) * | 2007-08-30 | 2008-04-02 | 合肥工业大学 | Immobilized cell single-tank high-strength continuous fermentation process for succinic acid |
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| Title |
|---|
| 苏潥等.Actinobacillus succinogenes 130Z厌氧发酵产丁二酸条件初步研究.《生物加工过程》.2007,第5卷(第2期),第67-72页，. * |
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