CN103695481A - Method for producing succinic acid through fermentation of sugarcane juice - Google Patents
Method for producing succinic acid through fermentation of sugarcane juice Download PDFInfo
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- CN103695481A CN103695481A CN201310733205.6A CN201310733205A CN103695481A CN 103695481 A CN103695481 A CN 103695481A CN 201310733205 A CN201310733205 A CN 201310733205A CN 103695481 A CN103695481 A CN 103695481A
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Abstract
The invention relates to a method for producing succinic acid through fermentation of sugarcane juice. The method comprises the steps of using actinobacillus succinogenes CCTCC (China Center for Type Culture Collection) M2011399, adding water to adjust the total sugar concentration of the sugarcane juice to 50-100g/L under a CO2 or N2-bearing anaerobic condition, and producing succinic acid through batch fermentation or fed-batch fermentation. The concentration of succinic acid can reach 30-75g/L through fermentation for 30-60h, the utilization ratio of sugar is 85-92%, and the production intensity reaches 0.73-1.46g/(L*h). The method has the prominent advantages that a sugarcane juice raw material replaces expensive glucose to serve as a carbon source for fermentation to produce succinic acid, and since a renewable biomass resource is employed, the method is environment-friendly, and can alleviate the petrifaction resource tension for chemical synthesis of succinic acid.
Description
Technical field
The invention belongs to microbial fermentation technology field, be specifically related to a kind of method of utilizing sugar cane juice fermentation production of succinic acid, relate to Actinobacillus succinogenes (Actinobacillus succinogenes) simultaneously and prepare the application of the method for succinic acid as raw material take sugar cane juice.
Background technology
Succinic acid, claims again succsinic acid (succsinic acid), is a kind of common natural organic acids, is extensively present in animal and plant and microbe, is one of biology intermediate product of carrying out TCA circulation.Succinic acid is a kind of important organic synthesis intermediate, by U.S. FDA, is regarded as GRAS, it is generally acknowledged safety, is mainly used in the fields such as food, medicine, biodegradable plastic, tensio-active agent, washing composition and green solvent.In addition, succinic acid can also be as aspects such as animal feedstuff additive and plant growth stimulators.
At present, it is mainly the method for utilizing chemosynthesis that succinic acid is produced, and produces succsinic acid, mainly by methods such as paraffin oxidation style, light oil oxidation style, butane oxidation method, succinonitrile hydrolysis method, shortenings from butane by Maleic anhydride.Chemically produce succinic acid, need to consume a large amount of non-renewable petrochemical materials, production cost is high, and environmental pollution is serious, thereby has limited the widespread use of succsinic acid.Therefore, people start target diversion to produce succsinic acid by biological fermentation process.Compare with traditional chemical synthesis, fermentation method has the advantages such as cost is low, environmental benefit is good, and has opened up the new way of greenhouse gases Carbon Dioxide Application.At present, the succsinic acid obtaining by fermentative Production, its price is approximately 0.55～1.1 ﹩/kg.In addition, according to Bio-amber company, claim, succsinic acid has the market of 2,500,000,000 Euros, and biological process produces succsinic acid and can compete mutually with the method for petrochemical industry, and plans to utilize plant to carry out industrial production of succinic acid as raw material before 2011.
Succinic acid is the important intermediate of many strictly anaerobic bacteriums and facultative anaerobe metabolism.It has been found that at present multiple-microorganism can produce succsinic acid by fermentation, wherein research mainly concentrates on intestinal bacteria (E.coli), succsinic acid bacterium (Mannheimia succiniciproducens), Anaerobiospirillum succinoproducens (Anaerobiospirillum succiniciproducens) and Actinobacillus succinogenes (Actinobacillus succinogenes) are produced in mannheim.In addition, some milk-acid bacterias, propionic acid produce bacterium and fungi etc. and also can produce a small amount of succinic acid.In numerous product succsinic acid microorganisms, rumen microorganism Actinobacillus succinogenes can utilize various saccharides to ferment, and the concentration that can tolerate glucose and succsinic acid is respectively up to 158g/L and 104g/L; Meanwhile, this bacterium, with advantages such as its high yield, height endurabilities, becomes one of Screening of Succinic-producing Strain most with prospects.
At present, the substratum of relevant fermentation method succinic acid-producing carbon source used, except glucose, is mainly grain raw material, as U.S. Argonne laboratory carried out the research of Maize Production succinic acid (
www.ipd.anl.gov/biotech/index.html, 2002), Japanese aginomoto and Mitsubishi Chemical Ind jointly combine carry out take W-Gum be fermenting raw materials produce the research of succinic acid (
http:// www.bio168.com, 2005).
But flourish due to the biological industry of grain raw material in recent years, there is shortage in grain, price increase trend is obvious, in addition depending on grain unduly also can affect national grain security, so use cheap non-grain raw material to carry out succinic acid fermentation, is the key that reduces succinic acid production cost and industrialization.
Relevant non-grain raw material is produced succinic acid research some reports both at home and abroad, Korea S Lee PC has reported with Manheimia succiniciprducens MBEL55E fermented whey raw material succinic acid-producing, Japan scientist Inui etc. utilizes cellulosic hydrolysate to produce succinic acid, the Sun Zhi of Southern Yangtze University is great waits people to carry out the research of fermentation succinic acid with raw materials such as molasses, bajiao banana taros, the logical seminar of the Jiang Min of Nanjing University of Technology and the Jiang Shao of HeFei University of Technology reports cellulosic material succinic acid-producing, and Guangxi Academy Of Sciences Huang ripple seminar take cassava and is studied as raw material succinic acid-producing.At present, take sugar cane juice have not been reported as raw material production succinic acid research.
Sugarcane is the general name of saccharum (Saccharum), belongs to C4 plant, and it has very high photosynthetic rate, is that traditional non-grain crop, sugary height, output are high.Sugar cane juice is the juice producing after cane milling, the general 15-20% of sugar degree, contains the nutritive substances such as aspartic acid, aspartic acid, L-Ala, citric acid, vitamin A, vitamins C, nicotinic acid, riboflavin and iron, calcium, phosphorus, manganese, zinc in addition.
Summary of the invention
Object of the present invention is exactly the problem existing for prior art, a kind of method of utilizing sugar cane juice fermenting raw materials to produce succinic acid is provided, provides the application of sugar cane juice in preparing succinic acid and Actinobacillus succinogenes (Actinobacillus succingenes) as raw material, to prepare the application in the method for succinic acid take sugar cane juice simultaneously.
The present invention achieves the above object by the following technical programs: a kind of method of utilizing sugar cane juice fermentation production of succinic acid, comprises the following steps:
1, a method of utilizing sugar cane juice fermentation production of succinic acid, is characterized in that, comprises the following steps:
1) sugar cane stem is squeezed the juice and obtained sugar cane juice, fresh sugar cane juice is directly used in to configuration substratum or is placed in-20 ℃ of refrigerators standby;
2) by fresh or thaw after sugar cane juice add water and adjust sugar to 50-100g/L, add nitrogenous source, inorganic salt, pH buffer reagent to be configured to succinic acid fermention medium;
3) by flat board product amber actinobacillus (Actinobacillus succinogenes) GXAS137 access seed culture medium in, at anaerobic culture box, cultivate 16-20h.
Described product amber actinobacillus (Actinobacillus succinogenes) GXAS137, for acquisition is screened in this experiment from beef cattle cud, on November 18th, 2011, be preserved in the Chinese Typical Representative culture collection center of Wuhan, China university, preservation is numbered CCTCC M2011399, and Chinese patent CN102851224A is open.
The composition of described seed culture medium and concentration thereof are: carbohydrate 10-20g/L, yeast powder 5-10g/L, peptone 5-10g/L, NaHCO
35-10g/L, NaH
2pO
49.6g/L, K
2hPO
41.55g/L.
4) cultured Actinobacillus succinogenes is seeded in fermention medium, inoculum size is 5-10%(V/V), be full of N
2or CO
2environment in ferment, pH buffer reagent regulates pH at 6.0-7.0, and at 30-38 ℃ of fermentation 30-60h, can obtain succinic acid.
The composition of described fermention medium and concentration thereof are: total reducing sugar 40-100g/L, nitrogenous source 10-40g/L, K
2hPO
41-5g/L, CaCl
20.5-1g/L, NaCl0.5-2g/L, (NH
4)
2sO
40.5-2g/L, MgCl
20.5-1g/L.
Described pH buffer reagent is carbonate, ammoniacal liquor, NaOH, and described carbonate is magnesium basic carbonate or sodium carbonate, and described fermentation condition is aerobic or passes into N
2or CO
2.Preferred version is that magnesium basic carbonate regulates fermentation pH, passes into CO
2; As adopt the alkalescence such as ammoniacal liquor, NaOH to regulate, must pass into CO
2.
Described nitrogenous source comprises organic nitrogen source and inorganic nitrogen-sourced; Described organic nitrogen source comprises yeast powder, peptone, corn steep liquor, soybean cake powder, described inorganic nitrogen-sourced urea, the ammonium sulfate of comprising.
Described fermentation mode can adopt batch fermentation, fed-batch fermentation or continuously ferment.
Analytical procedure:
Sample preparation: fermented liquid is 12000r/min at room temperature, centrifugal 10min, gets supernatant, with aperture, is then the aseptic membrane filtration of 0.22 μ m, with high performance liquid chromatography (HPLC), detects fermented liquid succinic acid and residual concentration of reduced sugar.
Organic acid is measured: HPLC method, wear peace Utimat3000, automatic sampler, chromatographic column: AminexHPX-87H300 * 7.8mm, moving phase 5mmol/L H
2sO
4, pH2.5,45 ℃ of column temperatures, sample size 10uL, flow velocity 0.6mL/min, UV-detector wavelength 210nm.
Residual reducing sugar test: 3,5-dinitrosalicylic acid method (DNS), material with glucose as a standard during mensuration.
Residual total sugar determination: measure reducing sugar content after adopting acid hydrolysis, material with glucose as a standard during mensuration.
Biomass estimation: adopt spectrophotometer (DU800UV/VIS Spectrophotometer, Beckman, USA) to measure at 660nm, sample is first processed with 0.2M HCl, dissolves contained MgCO
3, the centrifugal 10min of 12000r/min, then with distillation washing three times, to remove contained pigment and impurity.
Succinic acid productive rate (%) is defined as the grams of every consumption 1g succinic acid that glucose produces.
Outstanding advantages of the present invention and beneficial effect:
1, take sugar cane juice as fermenting raw materials production succinic acid, succinic acid concentration can reach 30-75g/L, and the utilization ratio of sugar is 85%-92%, and production intensity reaches 0.73-1.46g/(Lh);
2, utilize the glucose of cheap sugar cane juice raw material substitution costliness or food crop to produce succinic acid for carbon source through fermentation, can save a large amount of grain on the one hand, ensure the grain security of China, can reduce nitrogenous source usage quantity in fermentation, reduce succinic acid production cost simultaneously; Can regulate sugared valency on the other hand, ensure peasant's rights and interests.
3, utilize renewable biomass resource to produce succinic acid, also can alleviate the nervous problem of petrochemical industry resource of chemosynthesis succinic acid, and environmentally friendly.
Accompanying drawing explanation
Fig. 1 is the fermentation results figure that the present invention adopts 1.3L fermentor tank batch fermentation sugar cane juice succinic acid-producing.
Embodiment
Below the specific embodiment that utilizes the method for sugar cane juice fermentation production of succinic acid of the present invention, but to not restriction of the present invention.
Embodiment 1
The impact of different initial total sugar concentration on sugar cane juice fermentation succinic acid-producing
To produce in amber actinobacillus bacterial strain access seed culture medium, 30-38 ℃ of static cultivation 18-20h in anaerobic culture box or common incubator, inoculum size access by 8% is equipped with in the 250mL triangular flask of fermention medium, liquid amount is 150mL, magnesium basic carbonate regulates pH at 6.0-7.0, is placed in 37 ℃ of fermentation 48-60h of anaerobic culture box.Anaerobic box culture condition: anaerobic box (Don Whitley Scientific DG250anaerobic workstation), gas mixture N
2: CO
2: H
2=8.5:1.0:0.5,37 ℃ of temperature.Experimental result is as table 1.
The impact of the different sugar cane juice initial total sugar concentration of table 1 on succinic acid output
As can be seen from Table 1: when the sugar cane juice anaerobism batch fermentation of different initial total reducing sugars concentration ferments succinic acid-producing, when initial total reducing sugars concentration is 80g/L, output and the productive rate of succinic acid are the highest, reach respectively 70.08g/L and 87.60%.
Embodiment 2
The impact of different nitrogen sources on sugar cane juice fermentation succinic acid-producing
According to the method for producing amber actinobacillus strain fermentation succinic acid-producing in embodiment 1, the flat board of the bacterial strain of deposit number CCTCC M2011399 is cultivated after 48h in anaerobic box, with inoculation articulating one, encircle to 30mL seed culture medium, cultivate 20h for 37 ℃, according to 8% inoculum size, access initial total reducing sugars 80g/L, the 250mL triangular flask of the fermention medium of different nitrogen sources, liquid amount is 150mL, be placed in 37 ℃ of fermentation 60h of anaerobic culture box, adopt HPLC to measure succinic acid and Main By product content, the results are shown in Table 2.
The impact of table 2 different nitrogen sources on sugar cane juice fermentation succinic acid-producing
As can be seen from Table 2: the suitableeest nitrogenous source is yeast powder (70.13g/L), be secondly corn steep liquor (68.42g/L), poor when ammonium sulfate is nitrogenous source (34.77g/L).As known from the above, yeast powder is that nitrogenous source output is slightly better than corn steep liquor, but corn steep liquor price is far below yeast powder, considers the factors such as production cost and industrialization production, selects corn steep liquor as the nitrogenous source of sugar cane juice succinic acid-producing fermentation.
Embodiment 3
The impact of different pH buffer reagents on sugar cane juice fermentation succinic acid-producing
According to the method for producing amber actinobacillus strain fermentation succinic acid-producing in embodiment 1, for the ease of pH monitoring and pH buffer reagent stream, add, adopt 1.3L ferment tank, thin up sugar cane juice, initial total sugar concentration is 80g/L, nitrogenous source is corn steep liquor, liquid amount is 700mL, passes into 100%CO
2, air flow is 0.2mL/min, and leavening temperature is 37 ℃, and fermentation time is 60h, regulates pH at 6.5-7.0, and buffer reagent used and fermentation results are in Table 3.
The different pH buffer reagents of table 3 affect sugar cane juice succinic acid-producing
As shown in Table 3: take magnesium basic carbonate during as pH buffer reagent, succinic acid output is the highest, can reach 70.26g/L.This is because add magnesium basic carbonate solid, pH can be maintained to suitable level and the key enzyme in succinic acid route of synthesis---the activator of phosphoenolpyruvic acid carboxylation kinases (PEPCK) is Mg
2+, therefore, thereby thalline shows higher metabolic activity during the fermentation, produce more meta-bolites.
Embodiment 4
1.3L fermentor tank batch fermentation succinic acid-producing
According to the method for producing amber actinobacillus strain fermentation succinic acid-producing in embodiment 3, adopt 1.3L ferment tank, thin up sugar cane juice, initial total sugar concentration is 80g/L, and nitrogenous source is corn steep liquor, and liquid amount is 700mL, passes into 100%CO
2, air flow is 0.2mL/min, and leavening temperature is 37 ℃, and fermentation time is 60h, adopts magnesiumcarbonate condition fermented liquid pH, and concentration is 70g/L, fermentation results is shown in Fig. 1.
As can be seen from Figure 1: in initial sugar cane juice total sugar concentration, be 80g/L, through 60h fermentation, succinic acid concentration reaches 70.23g/L, and fermentation residual sugar is 3.15g/L, and succinic acid productive rate is 87.78%, and production intensity is 1.17g/(Lh).
Embodiment 5
1.3L fermentor tank fed-batch fermentation sugar cane juice succinic acid-producing
According to embodiment 4 methods, in 1.3L fermentor tank, carry out fed-batch fermentation, initial total sugar concentration is 50g/L, when fermented liquid residual sugar is down to 20g/L, adopt peristaltic pump to take certain speed stream and add the sugar cane juice that total sugar concentration is 160g/L, in fermented liquid, sugared concentration is controlled at 30g/L left and right, fermentation 60h, and experimental result result is as table 4.
Table 41.3L fermentor tank batch feeding test-results
As can be seen from Table 4: in initial sugar cane juice total sugar concentration, be 50g/L, through fed-batch fermentation 60h, succinic acid concentration reaches 75.14g/L, and fermentation residual sugar is 3.15g/L, and succinic acid productive rate is 87.78%, and production intensity is 1.17g/(Lh).
Claims (4)
1. a method of utilizing sugar cane juice fermentation production of succinic acid, is characterized in that, comprises the following steps:
1) sugar cane stem is squeezed the juice and obtained sugar cane juice, fresh sugar cane juice is directly used in to configuration substratum or is placed in-20 ℃ of refrigerators standby;
2) by fresh or thaw after sugar cane juice add water and adjust sugar to 50-100g/L, add nitrogenous source, inorganic salt, pH buffer reagent to be configured to succinic acid fermention medium;
3) by product amber actinobacillus (Actinobacillus succinogenes) the access seed culture medium on flat board, at anaerobic culture box, cultivate 16-20h,
The composition of described seed culture medium and concentration thereof are: carbohydrate 10-20g/L, yeast powder 5-10g/L, peptone 5-10g/L, NaHCO
35-10g/L, NaH
2pO
49.6g/L, K
2hPO
41.55g/L,
4) cultured Actinobacillus succinogenes is seeded in fermention medium, inoculum size is 5-10V/V%, is being full of N
2or CO
2environment in ferment, pH buffer reagent regulates pH at 6.0-7.0, and at 30-38 ℃ of fermentation 30-60h, can obtain succinic acid,
The composition of described fermention medium and concentration thereof are: total reducing sugar 40-100g/L, nitrogenous source 10-40g/L, K
2hPO
41-5g/L, CaCl
20.5-1g/L, NaCl0.5-2g/L, (NH
4)
2sO
40.5-2g/L, MgCl
20.5-1g/L.
2. the method for utilizing sugar cane juice fermentation production of succinic acid according to claim 1, is characterized in that, described pH buffer reagent is carbonate, ammoniacal liquor, NaOH, and described carbonate is magnesium basic carbonate or sodium carbonate.
3. the method for utilizing sugar cane juice fermentation production of succinic acid according to claim 1, is characterized in that, described nitrogenous source comprises organic nitrogen source and inorganic nitrogen-sourced; Described organic nitrogen source comprises yeast powder, peptone, corn steep liquor, soybean cake powder, described inorganic nitrogen-sourced urea, the ammonium sulfate of comprising.
4. the method for utilizing sugar cane juice fermentation production of succinic acid according to claim 1, is characterized in that, described fermentation mode adopts batch fermentation, fed-batch fermentation or continuously ferments.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846021A (en) * | 2015-06-02 | 2015-08-19 | 广西科学院 | Method for producing succinic acid through fermentation by utilizing lemna minor |
| CN105219808A (en) * | 2015-10-20 | 2016-01-06 | 江南大学 | A kind of method dextran-fermentation waste liquid being used for succinic acid fermentative production |
| CN106801026A (en) * | 2017-02-13 | 2017-06-06 | 广西科学院 | Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing |
| CN109517088A (en) * | 2018-11-08 | 2019-03-26 | 东华大学 | A method of curdlan is prepared by raw material of sugar-cane juice |
| CN111676252A (en) * | 2020-07-15 | 2020-09-18 | 广西民族大学 | Method for reducing generation of acetic acid serving as succinic acid fermentation by-product |
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| CN102851224A (en) * | 2012-01-19 | 2013-01-02 | 广西科学院 | Actinobacillus succinogenes strain and method for producing succinic acid by screening and fermentation of same |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102851224A (en) * | 2012-01-19 | 2013-01-02 | 广西科学院 | Actinobacillus succinogenes strain and method for producing succinic acid by screening and fermentation of same |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846021A (en) * | 2015-06-02 | 2015-08-19 | 广西科学院 | Method for producing succinic acid through fermentation by utilizing lemna minor |
| CN105219808A (en) * | 2015-10-20 | 2016-01-06 | 江南大学 | A kind of method dextran-fermentation waste liquid being used for succinic acid fermentative production |
| CN105219808B (en) * | 2015-10-20 | 2018-09-28 | 江南大学 | A method of dextran-fermentation waste liquid is used for succinic acid fermenting and producing |
| CN106801026A (en) * | 2017-02-13 | 2017-06-06 | 广西科学院 | Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing |
| 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 |
| CN109517088A (en) * | 2018-11-08 | 2019-03-26 | 东华大学 | A method of curdlan is prepared by raw material of sugar-cane juice |
| CN111676252A (en) * | 2020-07-15 | 2020-09-18 | 广西民族大学 | Method for reducing generation of acetic acid serving as succinic acid fermentation by-product |
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Application publication date: 20140402 |