CN102643873A

CN102643873A - Method for producing succinic acid by utilizing fermentation of actinobacillus succinogenes

Info

Publication number: CN102643873A
Application number: CN2012101350658A
Authority: CN
Inventors: 张斌
Original assignee: SUZHOU BAIQU FOOD CO Ltd
Current assignee: SUZHOU BAIQU FOOD CO Ltd
Priority date: 2012-05-04
Filing date: 2012-05-04
Publication date: 2012-08-22

Abstract

The invention provides a method for producing succinic acid by utilizing fermentation of actinobacillus succinogenes. The method includes taking canna edulis ker syrup as a main material, producing the succinic acid by using the fermentation of actinobacillus succinogenes CICC11014, determining the optical fermentation medium for bacterial strains through research for medium of bacterial strains; and fermenting the medium after optimization, wherein the medium comprises 89.66g/L of canna edulis ker syrup (by total suger), 38.55g/L of CSL, 2.23g/L of Na2HPO4*12(H2O), 2.23g/L of KH2PO4*3(H2O),60g/L of MgCO3, and 6.5 of the pH; and performing moist heat sterilization at a temperature of 115 DEG C for 20 minutes. The conversion rate of sugar can be improved through fermentation under the optical medium condition, so that the yield of the succinic acid can reach 70g/L.

Description

A kind of method of utilizing succsinic acid pleuropneumoniae fermentation succinic acid-producing

Technical field

The invention belongs to fermentation technical field, be specifically related to a kind of method of utilizing succsinic acid pleuropneumoniae fermentation succinic acid-producing.

Background technology

Succinic Acid (succinic acid) claim succsinic acid again; Be widely used in industries such as medicine, agricultural chemicals, dyestuff, spices, paint, food and plastics; As C4 hardware and software platform compound; Can be used for synthesizing 1, organic chemicals and poly butylene succinate (PBS) type Biodegradable materials such as 4-butyleneglycol, THF, gamma-butyrolactone are thought one of biorefinery product of following 12 kinds of most worthies by USDOE.

The working method of Succinic Acid mainly comprises chemical synthesis and microbe fermentation method; Utilize microbe fermentation method to transform renewable resources (glucose, wood sugar etc.),, pollute little because raw material sources are extensive and cheap; Environmental friendliness, and can absorb fixation of C O during the fermentation ₂, can effectively alleviate Greenhouse effect, opened up the new way that the greenhouse gases carbonic acid gas utilizes, become the focus of research this year.The production bacterial strain of Succinic Acid mainly concentrates on Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, reorganization Corynebacterium glutamicum and recombination bacillus coli.

The bajiao banana taro ( Canna edulisKer.), claim any of several broadleaf plants lotus root, achira etc. again, a kind of unifacial leaf root crop in Lowiaceae, the Canna generalis Bailey genus mainly is distributed in provinces and regions such as the southern Yunnan of China, Guizhou, Sichuan, Guangxi, Hainan.The staple of bajiao banana taro is a starch, and its content is up to 17%-18%, and per mu yield is 2 500-5,500 kg.The dry plate of banana dasheen chemical constitution is: starch 66.0%, ash content 2.80 %, robust fibre 2.65%, fat 0.28%, protein 3.63 %, tannin 0.19 %, moisture 17.10%.Because bajiao banana taro palatability is relatively poor, only as feed, well developed as yet traditionally.

Fermentation is a kind of biological process of complicacy, receives the influence of several factors, all with the output of purpose product confidential relation is arranged like quality, fermentation condition and the fermenting process control etc. of the proportioning of the production performance of thalline self, substratum, seed.In industrial production,, optimum production substratum and culture condition should be provided in order to bring into play the throughput of thalline to greatest extent.Therefore, explore basis that optimal culture conditions that the bacterial strain high yield characteristics gives full expression to is a fermentative prodn and crucial.

Summary of the invention

Having the object of the present invention is to provide a kind of method of utilizing succsinic acid pleuropneumoniae fermentation succinic acid-producing, is main raw material with bajiao banana taro syrup, utilize the succsinic acid pleuropneumoniae ( Actinobacillus succinogenes) the CICC11014 fermentation production of succinic acid, through research, confirmed the righttest fermention medium of bacterial strain: optimize the post-fermentation and culture base: bajiao banana taro syrup (in total reducing sugar) 89. 66 g/L, CSL 38. 55 g/L, Na to strain cultures ₂HPO ₄12H ₂O 2. 23 g/L, KH ₂PO ₄3H ₂O 2. 23 g/L, MgCO ₃60 g/L, pH 6. 5,115 ℃ of moist heat sterilization 20 min.This best medium condition bottom fermentation can improve the transformation efficiency of sugar, makes the output of Succinic Acid reach 70 g/L.

1. a method of utilizing succsinic acid pleuropneumoniae CICC11014 fermentation succinic acid-producing is characterized in that doing carbon source with bajiao banana taro syrup, and substratum is loaded on (long-pending 50 mL of dress liquid) in the 100 mL anaerobism bottles, and inoculum size 5% charges into 100% CO ₂3 parallel appearance are done in each experiment, average and miscalculation.

2. bajiao banana taro stem tuber (China Sichuan Province, the place of production) is got in the syrupy preparation of the described bajiao banana taro of step 1, and smash on kibbler section, oven dry back, soaks 2 h; Transfer pH 6.7, increase temperature glycase (Wuxi Saden zymin factory) 20 U/g dry powder, 105 ℃ of liquefaction 20-40 min; Be cooled to 60 ℃ again, transfer pH 4. 5, add saccharifying enzyme 300 U/g dry powder, saccharification 8-12 h passed through filtering bajiao banana taro syrup again, and syrup contains total reducing sugar 15%-20% (w/w).

3. the described seed culture medium of step 1: glucose 10 g/L, yeast extract paste 15 g/L, Na ₂HPO ₄12H ₂O 1 g/L, KH ₂PO ₄3H ₂O 1 g/L, 121 ℃ of moist heat sterilization 20 min postcooling, and add 1 mol/L NaHCO after 5% membrane filtration ₃, the pH of seed liquor changes when cultivating with buffering.

4. the described initial fermention medium of step 1: bajiao banana taro syrup (in total reducing sugar) 65 g/L, steeping water (CSL, solid content 55%) 20 g/L, Na ₂HPO ₄12H ₂O 1 g/L, KH ₂PO ₄3H ₂O 1 g/L, MgCO ₃60 g/L, 6.5,115 ℃ of moist heat sterilization 20 min of pH value.

5. the described concrete medium optimization method of step 4 is, carries out carbon source when experiment, except that total reducing sugar according to the experimental concentration, other compositions are with initially fermention medium is identical; Nitrogenous source when experiment, except that nitrogenous source according to the experimental concentration, carbon source is according to outside the optimal concentration, other compositions are with initially fermention medium is identical; CSL when experiment, except that carbon source concentration and nitrogen concentration according to the optimal concentration, other compositions are with initially fermention medium is identical; Phosphoric acid salt when experiment, remove phosphoric acid salt concentration (2 kinds of phosphate concn summations) according to the experimental concentration, carbon source, nitrogenous source and CSL concentration are according to optimal concentration, and other compositions are with initially fermention medium is identical.

6. the described method of step 5 is characterized in that optimum carbon source concentration is 80 g/L.

7. the described optimum nitrogen source of step 5 is CSL.

8. the described optimum CSL concentration of step 6 is 30 g/L.

9. the described best phosphate concn of step 5 is 6 g/L.

Description of drawings

The different carbon source concentrations of Fig. 1 are to the influence of fermentation production of succinic acid.

Fig. 2 different nitrogen sources is to the influence of bajiao banana taro syrup fermentation production of succinic acid.

The different CSL concentration of Fig. 3 are to the influence of bajiao banana taro syrup fermentation production of succinic acid.

The different phosphate concns of Fig. 4 are to the influence of bajiao banana taro syrup fermentation production of succinic acid.

Embodiment

Following embodiment elaborates to the present invention, but to not restriction of the present invention.

The used bacterial strain of this patent is succsinic acid pleuropneumoniae CICC11014, available from ATCC, is numbered: 55618.

Embodiment 1

Present embodiment is explained the influence of different carbon source concentrations to fermentation production of succinic acid, is different carbon sources with glucose with bajiao banana taro syrup respectively, according to total sugar concentration 20 g/L, and 40 g/L, 60 g/L, 80 g/L and 100 g/L preparation fermention medium, relatively A. succinogenesCICC11014 is to the situation of utilizing of bajiao banana taro syrup and glucose, and experimental result is shown in Fig. 1.

Fig. 1 result shows, A. succinogenesUtilize bajiao banana taro syrup will obviously be better than glucose as the effect of carbon source.When using glucose to do carbon source, Succinic Acid output reaches 36. 45 g/L for the highest when containing 60 g/L total reducing sugars with initial fermention medium; And when using bajiao banana taro syrup to do carbon source, Succinic Acid output reaches 52. 14 g/L for the highest when containing 80 g/L total reducing sugars with initial fermention medium.

Embodiment 2

Present embodiment explanation different nitrogen sources is to the influence of bajiao banana taro syrup fermentation production of succinic acid, and wherein the addition of each organic or inorganic nitrogenous source is a benchmark with identical nitrogen element content in the fermention medium, is respectively: NH ₄Cl 4.5 g/L, KNO ₃8 g/L, yeast extract paste 12.5 g/L, peptone 10 g/L, steeping water (CSL) 20 g/L (solid content 55%), trypticase soya peptone albumen (TSB) 15 g/L, Carnis Bovis seu Bubali cream 12 g/L, blank (not adding any nitrogenous source).

Can find out by Fig. 2, any when adding nitrogenous source when not adding, utilize the fermentation of bajiao banana taro syrup can produce the Succinic Acid of 13.69 g/L; Then produce Succinic Acid (data are unlisted) when using glucose fermentation under the same conditions hardly.The explanation of this phenomenon, in bajiao banana taro syrup except containing abundant carbon source, also contain some can by A. succinogenesThe nitrogenous source nutritive substance that utilizes, thus promote the growth of thalline and produced acid.This bacterium is relatively poor to inorganic nitrogen-sourced assimilation, almost can not utilize inorganic nitrogen-sourced.When fermention medium adds each organic nitrogen source at home and abroad, be the highest (62.32 g/L) with the succinic acid-producing amount of CSL (20 g/L).Though the product of yeast extract paste acid amount is suitable basically with CSL, because the price of yeast extract paste is higher, considers industrial production cost from now on, all adopts the nitrogenous source additive of CSL as fermention medium in the experiment afterwards.

Embodiment 3

Present embodiment is explained the influence of different CSL concentration to bajiao banana taro syrup fermentation production of succinic acid, and CSL concentration is respectively: 10 g/L, and 20 g/L, 30 g/L, 40 g/L, 50 g/L, Succinic Acid content is measured in the fermentation back.

Can know that by Fig. 2 when CSL concentration was 30 g/L, Succinic Acid output was up to 68.42 g/L.

Embodiment 4

Present embodiment is explained the influence of different phosphate concns to bajiao banana taro syrup fermentation production of succinic acid; Under other optimal conditionss, in initial fermention medium, add phosphate concn respectively and be respectively: 0 g/L, 1.5 g/L in the above; 6 g/L; 10.5 g/L, 15 g/L, Succinic Acid content is measured in the fermentation back.

Fig. 4 can find out that Succinic Acid output was up to 70 g/L when phosphate concn was 6 g/L.

Claims

1. a method of utilizing succsinic acid pleuropneumoniae CICC11014 fermentation succinic acid-producing is characterized in that doing carbon source with bajiao banana taro syrup, and substratum is loaded in the 100 mL anaerobism bottles, and inoculum size 5% charges into 100% CO ₂3 parallel appearance are done in each experiment, average and miscalculation.

2. method according to claim 1 is characterized in that the syrupy preparation of bajiao banana taro gets bajiao banana taro stem tuber, and smash on kibbler section, oven dry back, soaks 2 h; Transfer pH 6.7, increase temperature glycase 20 U/g dry powder, 105 ℃ of liquefaction 20-40 min; Be cooled to 60 ℃ again, transfer pH 4. 5, add saccharifying enzyme 300 U/g dry powder, saccharification 8-12 h passed through filtering bajiao banana taro syrup again, and syrup contains total reducing sugar 15%-20% (w/w).

3. method according to claim 1 is characterized in that seed culture medium: glucose 10 g/L, yeast extract paste 15 g/L, Na ₂HPO ₄12H ₂O 1 g/L, KH ₂PO ₄3H ₂O 1 g/L, 121 ℃ of moist heat sterilization 20 min postcooling, and add 1 mol/L NaHCO after 5% membrane filtration ₃, the pH of seed liquor changes when cultivating with buffering.

4. method according to claim 1 is characterized in that initial fermention medium: bajiao banana taro syrup (in total reducing sugar) 65 g/L, steeping water (CSL, solid content 55%) 20 g/L, Na ₂HPO ₄12H ₂O 1 g/L, KH ₂PO ₄3H ₂O 1 g/L, MgCO ₃60 g/L, 6.5,115 ℃ of moist heat sterilization 20 min of pH value.

5. method according to claim 4 is characterized in that carrying out carbon source when experiment, except that total reducing sugar according to the experimental concentration, other compositions are with initially fermention medium is identical; Nitrogenous source when experiment, except that nitrogenous source according to the experimental concentration, carbon source is according to outside the optimal concentration, other compositions are with initially fermention medium is identical; CSL when experiment, except that carbon source concentration and nitrogen concentration according to the optimal concentration, other compositions are with initially fermention medium is identical; Phosphoric acid salt when experiment, remove phosphoric acid salt concentration (2 kinds of phosphate concn summations) according to the experimental concentration, carbon source, nitrogenous source and CSL concentration are according to optimal concentration, and other compositions are with initially fermention medium is identical.

6. method according to claim 5 is characterized in that carbon source concentration is 80 g/L.

7. method according to claim 5 is characterized in that the experiment for CSL.

8. method according to claim 6 is characterized in that CSL concentration is 30 g/L.

9. method according to claim 5 is characterized in that phosphate concn is 6 g/L.