CN101805757A - Method for producing optical pure L-lactic acid by open type whole-cell recovery cyclic fermentation - Google Patents
Method for producing optical pure L-lactic acid by open type whole-cell recovery cyclic fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing optical pure L-lactic acid by open type whole-cell recovery cyclic fermentation. In the method, bacillus coagulans CASH CGMCC No.2184 is used as a stain for open type fermentation, culture liquid obtained after the fermentation is finished is centrifuged, the bacterium culture liquid is removed to obtain fermentation liquor which contains high optical purity L-lactic acid, the bacteria obtained by centrifugation are used as a seedculture to be inoculated in a culture medium for use in the next time of fermentation for ensuring that the fermentation culture is performed under the same fermentation conditions as the previous fermentation, and the optical pure L-lactic acid is obtained by repeating the previous operation. The method for producing the optical pure L-lactic acid is obviously advantages over the traditional method for producing the optical pure L-lactic acid in terms of high purity, light pollution, high production efficiency, low instrument and equipment requirements, low cost and the like. The method is suitable to be promoted and used widely.
Description
Technical field
The invention belongs to the production method of L-lactic acid in the bio-fermentation engineering field, especially relate to a kind of open type whole-cell and reclaim the method that circulating fermentation is produced optical pure L-lactic acid.
Background technology
Lactic acid (C
2H
5OCOOH), have another name called alpha-hydroxypropionic acid.Lactic acid can be divided into L-lactic acid (levorotation), D-lactic acid (dextrorotatory) and DL-lactic acid (racemism).Lactic acid can be used for fields such as food, printing and dyeing, pharmacy traditionally.In recent years, along with white pollution receives publicity and mineral substance resource non-renewable day by day, be that the biodegradable polymers of raw material production has caused people's extensive concern with the renewable resources.Wherein, poly(lactic acid) is as one of main biodegradable plastic, and its physicals and polystyrene are closely similar, promise to be one of surrogate of petroleum-based plastics.The production of biodegradable polylactic acid needs the L-lactic acid of high-optical-purity, and at present the production cost of the L-lactic acid of high-optical-purity also than higher, this be cause biodegradable polylactic acid can't with one of important factor of petroleum-based plastics competition.
Microbe fermentation method is the main method of producing L-lactic acid.The acquisition of can fermenting from renewable resource such as starch, cellulosic, organic waste even in the waste of L-lactic acid, the wide material sources of raw material.Simultaneously, can be by bacterial strain screening and transformation, obtain producing the fermentative production bacterial strain of the L-lactic acid of high-optical-purity, the high optical pure L-lactic acid of generation is easy to be used for the synthetic of poly(lactic acid).But in extensive batch fermentation process, each batch fermentation all need be in culture medium after sterilization the multistage seed of enlarged culturing, to guarantee having enough production strain cells to support the carrying out of fermentative production in the fermentation culture process.Multistage seed culture has not only prolonged fermentation period, has also increased the input of fermentation equipment such as seeding tank.In addition, the energy expenditure in the multistage seed culture process still can't be avoided.And in modern large scale fermentation process, the single batch fermentation volume is increasing, the also corresponding increase of seed culture volume, and the energy expenditure in the seed culture process can not be ignored.For fear of the multistage seed culture that each batch all will carry out, the operating method of report proposition semicontinuous fermentation is arranged, promptly the seed of the nutrient solution after the part fermentation ends as the next batch fermentation.But this operating method has been brought the metabolic waste in the fermented liquid into new batch fermentation, causes that easily fermentation efficiency descends and bacterial strain is degenerated.At this problem, there is report to propose cell and reclaims fermentation operation, be about to and thalline after fermented liquid separates seed as new batch fermentation.At present, cell reclaims fermentation and adopts filter membrane system separating thallus from fermented liquid.Filter membrane in the filter membrane system stops up easily, must periodic replacement.Conventional cellular segregation operation if can be used for cell as rotating centrifugal and reclaim fermentation, can simplify the operation that full cell reclaims semicontinuous fermentation.But easy pollution microbes when the cell of the mesophilic lactic acid that adopts usually at present production bacterial strain under non-sterile condition reclaims can't use open cell reclaimer operation.
Summary of the invention
The invention provides a kind of open type whole-cell easy and simple to handle, that purity is higher and reclaim the method that circulating fermentation is produced optical pure L-lactic acid.
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of open type whole-cell reclaims the method that circulating fermentation is produced optical pure L-lactic acid, be to produce bacterial strain with thermophilic lactic, preferred Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184 carries out open type fermented for bacterial classification, carry out centrifugal to fermented liquid after the fermentation ends, the supernatant liquor of removing thalline is and contains the high-optical-purity L-lactic acid fermented liquid, centrifugal back gained thalline is re-used as inoculum and inserts in the next batch fermention medium, to carry out fermentation culture with the identical fermentation condition of fermentation finished thoroughly1 last time, so circulation obtains optical pure L-lactic acid.
The present invention adopts Bacillus coagulans as fermented bacterium, described Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 09 24th, 2007, and open in Chinese patent CN200710176060.9.
Described fermentative medium formula is: glucose 130g/L, and yeast powder 2～20g/L, surplus is a water, pH value 6.0.
Described fermentation condition is: 50 ℃～55 ℃ of leavening temperatures, and fermentation time 40～48 hours, 130～150 rev/mins of mixing speed, the auto-feeding weight ratio is that 30%～40% alkali lye control fermented liquid pH value is in 6.0～6.5 scopes therebetween.
Described fermentation condition is preferably: 50 ℃ of leavening temperatures, and fermentation time 48 hours, 150 rev/mins of mixing speed, 33 millimeters of rotation radius, it is that weight ratio is 40% sodium hydroxide that stream adds alkali lye, fermented liquid pH value 6.0.
Outstanding feature of the present invention is that each fermented liquid all reclaims thalline and thalline is used for circulating fermentation with centrifugation under non-sterile condition.
Concrete, the fermented liquid after the described fermentation ends is with 6,000～8,000 rev/min of rotating speed under non-sterile condition centrifugal 20 ± 2 minutes; Described centrifugal rotational speed is preferably 8,000 rev/mins.
Described circulating fermentation number of times is preferably 2～8 times.
For obtaining better ferment effect, described Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classifications also can carry out slant culture and seed culture earlier before fermentation.
The optical pure L-lactic acid that obtains with aforesaid method also belongs to protection scope of the present invention.
The invention provides a kind of open type whole-cell and reclaim the method that circulating fermentation is produced optical pure L-lactic acid.The present invention adopts under the non-sterile condition and to reclaim thalline and be used for circulating fermentation as seed.This open type whole-cell recovery technology can adopt conventional thalline separation unit operation such as rotating centrifugal to reclaim thalline, has avoided the use of film unit, has reduced the cell recovery cost, has improved the cell organic efficiency, makes the cell reclaimer operation more simple and easy to do.Open centrifugal full cell recovery technology can be carried out under non-sterile condition, has reduced the cell reclaimer operation to equipment and environment requirement, can utilize existing installation to carry out cell and reclaim, and has reduced facility investment, has reduced cell reclaimer operation complexity.The genus bacillus that the present invention adopts belongs to thermophilic lactic and produces bacterium, can under the unsterilised condition of substratum, carry out open type fermented production high-optical-purity L-lactic acid, this is because it can and generate lactic acid in growth more than 50 ℃, and common bacterial strain can not so grown under the hot conditions, even sneak into assorted bacterium when centrifugally operated, because its high growth temperature also can become dominant strain in the follow-up fermentation that with it is seed, suppress varied bacteria growing, guarantee lactic acid fermentedly to carry out smoothly.Thermophilic lactic production bacterial strain not only can be realized open type fermented, and its high growth temperature and contamination resistance can also make conventional thalline lock out operation such as rotating centrifugal can be applied to full cell and reclaim fermentation.And common bacterial strain when non-sterile centrifugally operated easily by assorted bacterium.In addition, present method has realized reclaiming the seed of cell as circulating fermentation with conventional centrifugation under the non-sterile condition, has simplified circulating fermentation cell removal process, helps simplifying the operation, and reduces L-lactic fermentation production unit and drops into.The production method of optical pure L-lactic acid of the present invention obviously is better than existing optical pure L-lactic acid production method, has the high and low pollution of purity, production efficiency height, to advantage such as the plant and instrument requirement is low and with low cost, suits large area to popularize and uses.
Below in conjunction with specific embodiment the present invention is described in further details.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.Its objective is content for a better understanding of the present invention, therefore, the example of being given an example does not limit protection scope of the present invention.
Embodiment 1, open type whole-cell reclaim circulating fermentation and produce optical pure L-lactic acid
Employed each substratum is composed as follows in the present embodiment:
Slant medium: glucose 10g/L, yeast powder 10g/L, agar 15g/L, solvent are water; The pH value of described slant medium is 6.5,121 ℃ of sterilizations 20 minutes.
Seed culture medium: glucose 50g/L, yeast powder 10g/L, lime carbonate 3g/L, solvent are water; The pH value of described seed culture medium is 6.5,121 ℃ of sterilizations 20 minutes.
Fermention medium: glucose 130g/L, yeast powder 10g/L, surplus is a water; The pH value of described fermention medium is 6.0, and fermention medium is not through sterilization.
Reclaim the method for circulating fermentation with open type whole-cell of the present invention and produce optical pure L-lactic acid, concrete grammar may further comprise the steps:
1. slant culture: Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is inoculated on the slant medium with ordinary method, cultivated 12 hours for 50 ℃.
2. seed culture: the bacterial strain that step 1 is cultivated is encircling in the 100mL triangular flask that the 30mL seed culture medium is housed shaking table shaking culture (150 rev/mins) 12 hours, 50 ℃ of culture temperature with inoculating articulating 2 under the aseptic condition.Make seed culture fluid.
3. fermentation culture (for the first time fermentation): above-mentioned seed culture fluid is inoculated into 3 liters of fermention mediums (fermention medium is through sterilization) that place 5 liters of fermentor tanks with the inoculum size of volume ratio 10%, with 50 ℃ of culture temperature (50 ℃～55 ℃ all can), 150 rev/mins of mixing speed (130～150 rev/mins all can), cultivate 48 hours (40～48 hours all can), finish fermentation.Auto-feeding weight ratio (30%～40% all can) sodium hydroxide that is 40% is regulated fermented liquid pH value and is maintained 6.0 (6.0～6.5 all can) in the culturing process.
4. cell reclaims: fermented liquid in the step 3 under non-sterile condition, is reclaimed thalline with 8,000 rev/mins (6,000～8,000 rev/mins commentaries on classics all can) centrifugal 20 minutes (20 ± 2 minutes all can), and supernatant liquor is used to separate L-lactic acid.
5. repeat fermentation (for the second time fermentation): the thalline that reclaims is not less than 5% inoculum size as inoculum with volume ratio inserts in the next batch fermention medium, to carry out fermentation culture with the identical fermentation condition of the fermentation finished thoroughly1 first time, cultivate and finish the centrifugal recovery thalline in back, supernatant liquor is used to separate L-lactic acid.
6. fermented liquid detects: cell concentration adopts spectrophotometer to detect in the 620nm place.The L-lactic acid concn adopts bio-sensing analyser SBA-40C (Shandong Province academy sciences Biology Research Institute) to measure.L-lactic acid optical purity adopts Agilent1100 liquid chromatograph (Anjelen Sci. ﹠ Tech. Inc) to measure respectively that L-calculates L-lactic acid optical purity as acid and D-lactic acid concn by formula in the fermented liquid.Liquid phase systems adopts chiral separation post (separation of optics allosome is used for Mitsubishi chemical company, MCIGEL-CRS10W (3 μ) 4.6ID * 50mm), 0.002mol/L copper sulfate is moving phase, flow 0.5mL/min, sample size 20 μ L, UV-detector detects wavelength 254nm, 25 ℃ of service temperatures.D-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L0625, and L-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L1750.
L-lactic acid optical purity (%)=L-lactic acid concn/(L-lactic acid concn+D-lactic acid concn) * 100%
After the fermentation ends, get the isolating fermentation broth sample of each batch, detect cell concentration, L-lactic acid content and L-lactic acid optical purity in the fermented liquid according to above-mentioned detection and method of calculation.
The result shows, first batch of fermentation maximal cell concn 8.9 (OD620nm), L-lactic acid concn 63g/L in the supernatant liquor, production intensity 1.4g/L/h, L-lactic acid optical purity 99.4%.Second batch of fermentation maximal cell concn 9.8 (OD620nm), L-lactic acid concn 70g/L in the supernatant liquor, production intensity 1.6g/L/h, L-lactic acid optical purity 99.0%.Hence one can see that, and open type whole-cell reclaims circulating fermentation can improve lactic acid-producing efficient, keeps L-lactic acid optical purity more than 99% simultaneously.
Embodiment 2, open type whole-cell reclaim circulating fermentation and produce optical pure L-lactic acid
Do not carry out slant culture and seed culture, directly carry out fermentation culture: will ferment the gained fermented liquid under non-sterile condition for the second time among the embodiment 1, with 8,000 rev/mins of centrifugal 20 minutes recovery thalline.Removal thalline supernatant liquor is and contains the high-optical-purity L-lactic acid fermented liquid.Be no less than 5% inoculum size as inoculum with volume ratio and insert 3 liters of fermention mediums that place 5 liters of fermentor tanks reclaiming thalline, with embodiment 1 in identical fermentation condition carry out circulating fermentation for the third time.
Circulating fermentation finishes for the third time, and aforesaid operations carries out circulating fermentation the 4th time for another example.
Fermented liquid when getting the isolating fermentation ends of each batch detects cell concentration, L-lactic acid content and L-lactic acid optical purity.
Experimental result shows, the 3rd batch of fermentation maximal cell concn 10.8 (OD620nm), L-lactic acid concn 71g/L, production intensity 1.6g/L/h, L-lactic acid optical purity 99.3%.The 4th batch of fermentation maximal cell concn 11.5 (OD620nm), L-lactic acid concn 74g/L, production intensity 1.7g/L/h, L-lactic acid optical purity 99.0%.Hence one can see that, and along with open type whole-cell reclaims the circulation of fermenting, lactic acid-producing efficient has further lifting, and L-lactic acid optical purity remains on more than 99%.
Embodiment 3: reclaim circulating fermentation production with different yeast powder concentration open type whole-cells and contain high optical pure L-lactic acid fermented liquid
Fermention medium: design 4 kinds of substratum, what changed is that yeast powder concentration is respectively 2g/L, 10g/L, 15g/L, 20g/L in the embodiment 1 described fermention medium, glucose 130g/L, and surplus is a water; The pH value of described fermention medium is 6.0, and fermention medium is not through sterilization.
Do not carry out slant culture and seed culture, directly carry out fermentation culture: with the 4th batch of fermented liquid that fermentation obtains among the embodiment 2 with 8, the centrifugal thalline that obtained in 20 minutes is as seed under 000 rev/min of non-sterile condition, is that the fermention medium of nitrogenous source ferments with the 2g/L yeast powder.Again with the fermented liquid that obtains after the fermentation ends with the thalline of centrifugal collection under 8,000 rev/mins of non-sterile conditions as seed, be that the fermention medium of nitrogenous source ferments with the 10g/L yeast powder.Again with the thalline of centrifugal collection after the fermentation ends as seed, be that the fermention medium of nitrogenous source ferments with the 15g/L yeast powder.Again with after the fermentation ends with the thalline of centrifugal collection under 8,000 rev/mins of non-sterile conditions as seed, be that the fermention medium of nitrogenous source ferments with the 20g/L yeast powder.Each fermentation inoculum size is not less than 5%.
After above-mentioned each batch fermentation ends,, detect cell concentration, L-lactic acid concn and L-lactic acid optical purity in the fermented liquid according to the foregoing description 1 described detection and method of calculation.
Test-results sees Table 1, shows that increasing yeast powder concentration helps improving L-lactic acid-producing efficient.The maximum cell photoabsorption can reach 31.8 (OD620nm).The L-lactic acid concn reaches as high as 107g/L, and L-lactic acid optical purity can reach 99.8%, production intensity 2.9g/L/h.
Table 1 yeast powder concentration reclaims the influence of fermentation to open type whole-cell
| Yeast powder concentration (g/L) | Maximal cell concn (OD620nm) | L-lactic acid concn (g/L) | Production intensity (g/L/h) | L-lactic acid optical purity (%) |
| ??2 | ??7.2 | ??26 | ??0.6 | ??99.3 |
| Yeast powder concentration (g/L) | Maximal cell concn (OD620nm) | L-lactic acid concn (g/L) | Production intensity (g/L/h) | L-lactic acid optical purity (%) |
| ??10 | ??13.3 | ??80 | ??1.8 | ??98.9 |
| ??15 | ??22.4 | ??85 | ??1.9 | ??99.3 |
| ??20 | ??31.8 | ??107 | ??2.9 | ??99.8 |
Embodiment 4, open type whole-cell reclaim circulating fermentation and produce optical pure L-lactic acid
Employed each substratum is composed as follows in the present embodiment:
Slant medium is identical with embodiment 1 with the seed culture based formulas.
Fermention medium: glucose 130g/L, yeast powder 20g/L, surplus is a water; The pH value of described fermention medium is 6.0, and fermention medium is not through sterilization.
Reclaim the method for circulating fermentation with open type whole-cell of the present invention and produce optical pure L-lactic acid, concrete grammar may further comprise the steps:
1. slant culture: identical with embodiment 1.
2. seed culture: identical with embodiment 1.
3. fermentation culture: above-mentioned seed culture fluid is inoculated into 3 liters of fermention mediums (fermention medium is through sterilization) that place 5 liters of fermentor tanks with the inoculum size of volume ratio 10%,, cultivated 40 hours, finish to ferment with 55 ℃ of culture temperature.The auto-feeding weight ratio is that 30% sodium hydroxide adjusting fermented liquid pH value maintains 6.5 in the culturing process.
4. cell reclaims: under non-sterile condition, with 6,000 rev/mins of centrifugal 20 minutes recovery thalline, supernatant liquor is used to separate L-lactic acid with nutrient solution in the step 3.
5. repeat fermentation: identical with embodiment 1.
After the fermentation ends, get the isolating fermentation broth sample of each batch, detect cell concentration, L-lactic acid content and L-lactic acid optical purity in the fermented liquid.
The result shows, first batch of fermentation maximal cell concn 10.5 (OD620nm), L-lactic acid concn 88g/L, production intensity 2.2g/L/h, L-lactic acid optical purity 99.4%.Second batch of fermentation maximal cell concn 12.1 (OD620nm), L-lactic acid concn 95g/L, production intensity 2.4g/L/h, L-lactic acid optical purity 99.3%.Hence one can see that, and open type whole-cell reclaims circulating fermentation can improve lactic acid-producing efficient, keeps L-lactic acid optical purity more than 99% simultaneously.
Claims (10)
1. an open type whole-cell reclaims the method that circulating fermentation is produced optical pure L-lactic acid, be with thermophilic lactic produce bacterium be bacterial classification in fermention medium, carry out 50 ℃～55 ℃ open type fermented down, after the fermentation ends fermented liquid is carried out centrifugally, the supernatant liquor of removing thalline is and contains the high-optical-purity L-lactic acid fermented liquid; Centrifugal back gained thalline is re-used as inoculum and inserts in the next batch fermention medium, carrying out fermentation culture with the identical fermentation condition of fermentation finished thoroughly1 last time, and then this fermented liquid is carried out centrifugally operated; So circulation repeatedly obtains optical pure L-lactic acid.
2. production method according to claim 1 is characterized in that: it is Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 that described thermophilic lactic is produced bacterium.
3. production method according to claim 1 and 2 is characterized in that: described fermentative medium formula is: glucose 130g/L, and yeast powder 2～20g/L, surplus is a water, pH value 6.0.
4. according to claim 1 or 2 or 3 described production methods, it is characterized in that: described fermentation condition is: 50 ℃～55 ℃ of leavening temperatures, fermentation time 40～48 hours, 130～150 rev/mins of mixing speed, the auto-feeding weight ratio is 30%～40% alkali lye therebetween, and control fermented liquid pH value is in 6.0～6.5 scopes.
5. production method according to claim 4 is characterized in that: the inoculum size of each fermentation is no less than 5%, 50 ℃ of leavening temperatures, fermentation time 48 hours, it is that weight ratio is 40% sodium hydroxide that 150 rev/mins of mixing speed, stream add alkali lye, fermented liquid pH value 6.0.
6. according to the described production method of aforementioned arbitrary claim, it is characterized in that: each fermented liquid all reclaims thalline and thalline is used for circulating fermentation with centrifugation under non-sterile condition; Concrete, the fermented liquid after the described fermentation ends is with 6,000～8,000 rev/min of rotating speed under non-sterile condition centrifugal 20 ± 2 minutes; Described centrifugal rotational speed is preferably 8,000 rev/mins.
7. according to the described production method of aforementioned arbitrary claim, it is characterized in that: described circulating fermentation number of times is 2～8 times.
8. production method according to claim 7 is characterized in that: in the used fermention medium, yeast powder content increases progressively one by one in described each circulating fermentation; For example, when fermenting for the first time in the fermention medium yeast powder content be 2g/L, when fermenting for the second time in the fermention medium yeast powder content be 10g/L, when fermenting for the third time in the fermention medium yeast powder content be 15g/L, during the 4th fermentation in the fermention medium yeast powder content be 20g/L.
9. according to each described production method of claim 1-8, it is characterized in that: described Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classifications also need carry out slant culture and seed culture before fermentation.
10. the optical pure L-lactic acid of using each described method of claim 1-9 to obtain.
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