CN104818346A - Regulation and control method for fermentation process of L-arginine high in acid yield - Google Patents
Regulation and control method for fermentation process of L-arginine high in acid yield Download PDFInfo
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Abstract
The invention relates to a regulation and control method for a fermentation process of L-arginine high in acid yield. According to the method, when brevibacterium flavum is used as a seed for fermentation production of the L-arginine, temperature, stirring speed and tank pressure of the fermentation process and pH values and dissolved oxygen quantity of different fermentation stages are controlled online by an FPC (flexible printed circuit) type distributed control system, bacterium cell concentration, substrate concentration and product yield during fermentation are measured through timing sampling, and carbohydrates are fed and supplemented when sugar concentration is lower than a critical value. The regulation and control method has the advantages that the distributed control system is adopted for biochemical processes, so that the method is simple and reliable and high performance is achieved; through optimization control of the fermentation process, the acid yield of the L-arginine is increased by 15-20%, fermentation period is shortened to 76-86h, the volume of a lower tank of a fermentation tank is increased from 70% to 80% and stability is high.
Description
Technical field
The present invention relates to technical field of biological fermentation, particularly relate to a kind of regulate and control method of high acid amount L-arginine fermenting process.
Background technology
The forties in 20th century, think when nutritionist is divided into indispensable amino acid and non-essential amino amino acid, L-arginine is non-essential amino acid for adult humans and animals, therefore is not put into indispensable amino acid.Found that its self synthesis in baby and young animal was not enough afterwards, be thus now called semi-dispensable amino acid.To the eighties in 20th century, by widespread use and the further investigation of animal husbandry amino acid additives, find that, for the aquatic animal such as chicken and fish, L-arginine is indispensable amino acid.
The production method of current L-arginine mainly contains hydrolysis method and fermentation method.
Pig hair, hoof first, blood meal, gelatin, protamine, raw fish etc. all contain abundant L-arginine, can be used as raw material and prepare L-arginine through acid hydrolysis, separation.In human hair albumen, the content of L-arginine is 8.7%, and in porcine haemoglobin hydrolyzate, the content of L-arginine is 44.42mg/100mL, accounts for 5.81% of total amino acid content.Chinese people hair wastes amount reaches more than 100,000 tons every year, the pig hair quantity of slag also reaches more than 7.5 ten thousand tons, and Swine blood meal quantity has no report, but quantity also can not be lacked, therefore hydrolysis method produces amino acid whose raw material is abundant, but fails to be promoted owing to there is serious environmental issue.
The progress of domestic and international arginine fermentation:
The process of fermentative Production L-arginine is a very complicated chemical transformation and physiological change combined process, improves two approach of fermentation level, and one is the excellent bacterial classification of seed selection, and two is draw the optimal culture condition, the control device that match with object bacterium.The former is based upon the strain improvement technology in metabolic control fermentation research, and the latter is the ferment control technology be based upon on biochemical reaction engineering basis.Both are only had to combine closely and could finally realize high-caliber fermentative production.
For a long time, focus is more placed in the seed selection of superior strain by people, and the research that relevant fermentation processes is optimized is less.In actual production, required some cultivate control condition, technical indicator obtains often by shaking flask or canister test, and this low-level research method objectively constrains the performance of bacterial classification potentiality.
Fermenting process is optimized to the object of control, be obtain the highest production concentration, the highest production intensity and the highest transformation efficiency simultaneously.And when attempting to obtain higher production concentration with batch fermentation, the further decline of transformation efficiency and production intensity must be meaned.Optimized research is added about fermentation parameter optimal control and stream in recent years, for the production level improving batch fermentation provides possible approach in fermentation industry.At certain initial sugar concentration bottom fermentation, under fermentation parameter optimal control condition, improve total sugar concentration by fed-batch mode, and the sugared concentration maintaining fermenting process is in suitable level, just likely obtains high transformation efficiency, high production intensity and high production concentration simultaneously.
Summary of the invention
The object of the present invention is to provide one effectively to improve acid yield, shorten the regulate and control method of a kind of high acid amount L-arginine fermenting process of fermentation period.
Research finds: the factor affecting L-arginine fermentation can be divided into two classes, and a class is the environmental parameter of fermenting process, and as PH, dissolved oxygen, mixing speed, tank pressure etc., another kind of is key variables, as thalli growth amount, base consumption speed, product synthesis speed etc.
The present invention adopts advanced Distributed Control System for Fermentation Process-FPC type distributed control system (DCS), on-line Control and monitoring are carried out to first kind environmental parameter, and the mode of fermenting process Equations of The Second Kind key variables is measured in conjunction with timing sampling, systematic study is carried out to the control of arginine fermenting process, the optimum control condition of definite kernel sugar-fermenting different steps parameters, the control condition that convection current adds benefit sugared is on this basis explored, determine a kind of with dissolved oxygen, pH value controls to serve as theme, various parameter in Comprehensive Control fermenting process, and the Effective Regulation method of continuous flow feeding.
More particularly, the regulate and control method of L-arginine fermenting process of the present invention has following characteristics: in the process using brevibacterium flavum for seed fermentation production L-arginine, adopt pH value, the dissolved oxygen amount of temperature, stirring velocity, tank pressure and the fermentation different steps in FPC type distributed control system on-line Control fermenting process, and timing sampling measures cell concentration, concentration of substrate and product formation in fermenting process, benefit sugar is added lower than stream during threshold value in sugared concentration, maintain sugared concentration in certain limit, keep fermentation to be in optimum regime all the time.
The method, by using other parameter in fermenting process distributing system-FPC type distributed control system (DCS) online Comprehensive Control dissolved oxygen, pH value and fermenting process, is determined and keeps optimal conditions of fermentation; By measuring cell concentration, concentration of substrate and product formation, obtain the Changing Pattern of the key variables such as biomass growth rate in fermenting process, base consumption speed, and in conjunction with different initial sugar concentration fermentation test results, determine optimal conditions of fermentation, the composition of best fermentation initial sugar concentration, flow feeding time, feed profile and feed supplement.On this basis, with optimal conditions of fermentation, best fermentation just sugar time ferment, and feed supplement in good time during the fermentation, completes the fermenting process of optimal control thus.
In the above-mentioned methods, the culture temperature in fermenting process is at 30-32 DEG C, and tank pressure is at 0.05-0.06MPa, and mixing speed is at 200-250rpm.
PH in process controls: earlier fermentation (within OD 0.8) PH6.8-6.9; Middle and later periods (OD more than 0.8) PH6.5-6.6.
Dissolved oxygen in process controls: earlier fermentation (within OD 0.8) dissolved oxygen is at 20-30%; Middle and later periods (OD more than 0.8) dissolved oxygen controls at 10-20%.
The best moment of feed supplement is that sugared concentration is down to less than 2%, and adopt Continuous Flow to add, control sugared concentration at 1-2%, feed component is glucose mainly, and suitably add molasses and vitamin H, sugared concentration is at 40-50%, and feeding volume is at 25-30%.
Fermentation processes comprises steady operation conditions and optimizes fermentation parameter two aspects, the former realizes by traditional regulation loop, and the optimal control of system need according to the Changing Pattern of the key variables of fermenting process, optimizing is carried out to PH, dissolved oxygen, feed component, feed profile, to determine the optimal control policy of these variablees.Conventional instrument is adopted to realize to this.And adopt distributed control system to carry out biological process automatically to control that there is simple, reliable, high performance feature.
By the optimal control of fermenting process, the acid yield of L-arginine improves 15-20%, and fermentation period foreshortens to 76-86h, and the lower tank of fermentor tank is long-pending is increased to more than 80% by 70%, and stability is very high.
Embodiment
The present invention is further illustrated below with non-limiting example.The glucose used in following example contains a part crystal water, and pure content is 90%.
Embodiment 1
Use brevibacterium flavum.By glucose 4.0%, corn steep liquor 1.5%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 300L seed culture medium (PH6.7-7.0) that magnesium sulfate 0.05% forms, controls tank pressure 0.05-0.06MPa, rotating speed 200-250rpm, culture temperature 30-32 DEG C, cultivate and access glucose 15% after 18 hours, corn steep liquor 2%, molasses 1%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 2200L fermented liquid that magnesium sulfate 0.05% forms, mixing speed 220rpm, culture temperature 30-32 DEG C.Whole fermenting process PH controls at 6.8-6.9, and dissolved oxygen controls at 20-30%.
Mainly long bacterium period in early stage, consumption sugar is slower comparatively speaking, transition is started when thalline grows to certain phase, at this moment microscopy significantly can find that the bacterium that " eight " word divides has been lacked, majority is single bacterium, more sturdy when form comparatively divides, and at this moment arginine starts accumulation, along with the quickening of the sugared speed of consumption, fermentation also enters produces the acid phase fast.When occurring that dissolved oxygen, PH rebound, starting to flow sugaring (concentration is at 40-50%), adding in batches, control additional amount once, more than 3%, maintains above-mentioned control condition, and cultivating the acid of 96 hours products does not increase, and treats to exhaust lower tank to sugar.Fermentation ends, produce acid 5.4%, lower tank amasss at 3000L.
Comparative example 1-1
All identical with the formula of example 1, constant volume, initial culture conditions, the culture condition in fermenting process slightly does and changes, and PH during below OD0.8 controls at 6.8-6.9, and reduce PH to 6.5-6.6 after dissolved oxygen 20-30%, more than OD0.8, dissolved oxygen controls at 10-20%.Other control is same as Example 1, can promote that thalline makes the transition after reducing PH, dissolved oxygen, and then improves the product acid of thalline.Final fermentation time was at 94 hours, and produce acid 5.8%, lower tank amasss 3000L.
From the fermentation results of this two tank, be all present in long bacterium speed in earlier stage slow, the cycle is long, and thalline is aging, and later transformation rate is low, and product acid is not high.For these situations, adopt and reduce initial sugar concentration, to increasing long bacterium speed in early stage, to shorten the production cycle.
Embodiment 2
Use brevibacterium flavum.By glucose 4.0%, corn steep liquor 1.5%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 300L seed culture medium (PH6.7-7.0) that magnesium sulfate 0.05% forms, control tank pressure 0.05-0.06MPa, rotating speed 200-250rpm, culture temperature 30-32 DEG C, cultivate and access glucose 15% after 18 hours, corn steep liquor 2%, molasses 1%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 2200L fermented liquid that magnesium sulfate 0.05% forms, mixing speed 220rpm, culture temperature 30 DEG C, process control, PH during below OD0.8 controls at 6.8-6.9, dissolved oxygen 20-30%, PH to 6.5-6.6 is reduced after more than OD0.8, dissolved oxygen controls at 10-20%, sugar concentration is down to less than 1%, start to flow sugaring, control sugared concentration at 0.5-1%.Whole process, the long bacterium speed in early stage is obviously accelerated, and the benefit sugar time also obviously shifts to an earlier date, and within 50 hours, namely starts to flow sugaring, fermentation period 85 hours, and produce acid 5.8%, lower tank amasss 3000L.
Comparative example 2-1
Later stage is down to less than 2% when sugared concentration and starts to mend sugar, and maintain sugared concentration at 1-2%, other condition is with example 2.Result shows long bacterium speed can be faster, and within 46 hours, namely start to flow sugaring, fermentation period 81 hours, produce acid 6.2%, lower tank amasss 3000L.
Embodiment 3
Use brevibacterium flavum.By glucose 4.0%, corn steep liquor 1.5%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 300L seed culture medium (PH6.7-7.0) that magnesium sulfate 0.05% forms, control tank pressure 0.05-0.06MPa, rotating speed 200-250rpm, culture temperature 30-32 DEG C, cultivate and access glucose 13% after 18 hours, corn steep liquor 2%, molasses 1%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 2200L fermented liquid that magnesium sulfate 0.05% forms, mixing speed 220rpm, culture temperature 30-32 DEG C, process control, PH during below OD0.8 controls at 6.8-6.9, dissolved oxygen 20-30%, PH to 6.5-6.6 is reduced after more than OD0.8, dissolved oxygen controls at 10-20%, sugar concentration is down to less than 2%, start to flow sugaring, control sugared concentration at 1-2%.Whole process, the long bacterium speed in early stage is obviously accelerated, and the benefit sugar time also obviously shifts to an earlier date, and within 44 hours, namely starts to flow sugaring, fermentation period 84 hours, and produce acid 6.7%, lower tank amasss 3500L.
Comparative example 3-1
Fermentor tank initial sugar concentration is down to 10%, and other condition is with example 2.Result shows long bacterium speed can be faster, and within 40 hours, namely start to flow sugaring, fermentation period 82 hours, produce acid 6.5%, lower tank amasss 3600L.
Embodiment 4
Use brevibacterium flavum.By glucose 4.0%, corn steep liquor 1.5%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 300L seed culture medium (PH6.7-7.0) that magnesium sulfate 0.05% forms, control tank pressure 0.05-0.06MPa, rotating speed 200-250rpm, culture temperature 30 DEG C, cultivate and access glucose 10% after 18 hours, corn steep liquor 2%, molasses 1%, sulphur ammonium 0.3%, potassium primary phosphate 0.05%, in the 2200L fermented liquid that magnesium sulfate 0.05% forms, mixing speed 220rpm, culture temperature 30-32 DEG C, process control, PH during below OD0.8 controls at 6.8-6.9, dissolved oxygen 20-30%, PH to 6.5-6.6 is reduced after more than OD0.8, dissolved oxygen controls at 10-20%.Add a small amount of corn steep liquor in later stage stream sugaring, when concentration sugared in tank is down to 2%, start to flow sugaring, control sugared concentration at 1-2%.Final fermentation period 84 hours, produce acid 7.1%, lower tank amasss 3900L.
Comparative example 4-1
Embodiment is same as Example 3, and change the corn steep liquor in stream sugaring into molasses, other control condition is the same.Final fermentation period 86 hours, produce acid 7.2, lower tank amasss 4000L.
Claims (6)
1. the regulate and control method of a high acid amount L-arginine fermenting process, it is characterized in that: in the process using brevibacterium flavum for seed fermentation production L-arginine, adopt pH value, the dissolved oxygen amount of temperature, stirring velocity, tank pressure and the fermentation different steps in FPC type distributed control system on-line Control fermenting process, and timing sampling measures cell concentration, concentration of substrate and product formation in fermenting process, add benefit sugar in sugared concentration lower than stream during threshold value.
2. according to the regulate and control method of high acid amount L-arginine fermenting process according to claim 1, it is characterized in that: during the fermentation, control culture temperature at 30-32 DEG C, tank pressure is at 0.05-0.06MPa, and mixing speed is at 200-250rpm.
3., according to the regulate and control method of high acid amount L-arginine fermenting process according to claim 1, it is characterized in that: during the fermentation, earlier fermentation, when OD is within 0.8, control PH6.8-6.9; The fermentation middle and later periods, when OD is more than 0.8, control PH6.5-6.6.
4. according to the regulate and control method of high acid amount L-arginine fermenting process according to claim 1, it is characterized in that: during the fermentation, earlier fermentation, when OD is within 0.8, control dissolved oxygen at 20-30%; The fermentation middle and later periods, when OD is more than 0.8, control dissolved oxygen at 10-20%.
5. according to the regulate and control method of high acid amount L-arginine fermenting process according to claim 1, it is characterized in that: described sugared concentration lower than stream during threshold value add mend sugar refer to when sugared concentration is down to less than 2%, the method adopting Continuous Flow to add mends sugar, controls sugared concentration at 1-2%.
6. according to the regulate and control method of high acid amount L-arginine fermenting process according to claim 1, it is characterized in that: mend sugar and mainly supplement glucose, suitably add molasses and vitamin H, sugared concentration is at 40-50%, and feeding volume is at 25-30%.
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Patent Citations (4)
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| CN1508256A (en) * | 2002-12-18 | 2004-06-30 | 诚志生命科技有限公司 | D-ribose fermentation process regulating method |
| CN101235401A (en) * | 2007-02-02 | 2008-08-06 | 上海祥韦思化学品有限公司 | Fermentation method for preparing L-amino acid |
| CN103476922A (en) * | 2011-04-01 | 2013-12-25 | Cj第一制糖株式会社 | Corynebacterium sp. transformed with fructokinase gene derived from Escherichia sp. and method for producing L-amino acid using the same |
| CN103224964A (en) * | 2011-12-01 | 2013-07-31 | Cj第一制糖株式会社 | Microorganism for simultaneously producing l-amino acid and riboflavin, and method for producing l-amino acid and riboflavin using same |
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Application publication date: 20150805 |