KR840000014B1 - Automation of glutamic acid fermentation process using exit gas analysis - Google Patents
Automation of glutamic acid fermentation process using exit gas analysis Download PDFInfo
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제1도는 글르타민산발효공정 자동화계통도.1 is a glutamic acid fermentation process automation system.
제2도는 CO2생성속도와 균체농도와의 관계.2 is the relationship between the CO 2 production rate and cell concentration.
본 발명은 공업적인 글루타민산발효공정에 필수적인 페니실린(또는 계면활성제)투여공정을 자동화하는 방법에 관한 것이다. 구체적으로 설명하면, 본 발명은 발효배기가스 분석으로 부터 얻어진 미생물의 성장 및 증식의 간접측정방법에 의하여 글루타민산발효공정에 중요한 페니실린(또는 계면활성제)투여를 자동화하는 방법에 관한 것이다.The present invention relates to a method for automating a penicillin (or surfactant) administration process which is essential for an industrial glutamic acid fermentation process. Specifically, the present invention relates to a method for automating penicillin (or surfactant) administration that is important for glutamic acid fermentation by indirect measurement of growth and proliferation of microorganisms obtained from fermentation exhaust gas analysis.
글루타민산은 공업적으로 코리네박테리움(Corynebacterium), 브레비박테리움(Brevibacterium), 마이크로박테리움(Microbacterium), 마이크로코커스(Micrococcus)등의 미생물을 적정균체량이 될때가지 증식시킨후에 페니실린(또는 계면활성제)을 발효조에 투여함으로서 미생물의 세포막투과성(cellular bermeabilit)을 조절, 미생물내부에 축적되는 글루타민산을 미생물체외로 배출시킴으로서 생산되어 진다.Glutamic acid is industrially used to propagate microorganisms such as Corynebacterium, Brevibacterium, Microbacterium, and Micrococcus until the optimal cell weight is reached before penicillin (or surfactant ) Is produced by regulating cellular bermeabilit of microorganisms and discharging glutamic acid accumulated inside microorganisms out of microorganisms.
이때 공업적으로 사용되는 당밀배지중에는 생육인자인 비오틴(Biotin)과 미네랄(mineral)이 다량함유되어 페니실린(또는 계면활성제)의 적시·적량투여의 정밀도에 따라 미생물의 성장 및 증식의 정도는 크게 달라지며 따라서 글루타민산 생산수율 또한 크게 영향을 받는다.At this time, the molasses medium used industrially contains a large amount of growth factors, biotin and minerals, and the degree of growth and proliferation of microorganisms varies greatly according to the precision of timely and proper dose of penicillin (or surfactant). Therefore, glutamic acid production yield is also greatly affected.
이와같은 페니실린(또는 계면활성제)의 적시·적량투여 공정에 있어서, 종래에는 발효조의 샘플을 직접 채취하여 분광광도계(Spectrophotometer)와 같은 분석기기를 이용, 미생물의 성장 및 증식의 정도를 측정한후 숙련된 기술자의 판단에 의해 수동적으로 이루어져 왔다.In such a timely and proper dose administration process of penicillin (or surfactant), a sample of a fermentation tank is directly collected, and after measuring the extent of growth and proliferation of microorganisms using an analyzer such as a spectrophotometer, It has been done manually at the discretion of the technician.
그러나 이와같은 방법에 의한 주요공정의 관리는 공정상의 많은 불편과 작업상실수에 의한 손실등에 의해 공정상의 불안정요소로서 작용되어 왔으며, 실제로 페니실린(또는 계면활성제)의 투여시기에 있어서의 급격한 균체성장으로 인하여 글루타민산발효의 전공정을 자동화하는데 있어서 정확하고 연속적인 균체측정의 난점은 피할 수 없었다.However, the management of major processes by this method has been acting as a process instability due to many inconveniences in process and loss of work loss, and in fact, due to rapid cell growth at the time of penicillin (or surfactant) administration. Therefore, the difficulty of accurate and continuous cell determination in automating the entire process of glutamic acid fermentation was inevitable.
따라서, 본 발명자는 이와같은 종래의 수동식 투여공정을 자동화하기 위하여 프로세스컴퓨터를 이용, 글루타민산 발효배기가스를 온라인(on-line)으로 연결, 다양한 발효특성변수를 연구한 결과 CO2생성속도(CER)와 균체농도와의 일정한 상관관계를 유도할 수 있었으며 이결과로부터 페니실린(또는 계면활성제)의 적시·적량투여를 자동화함으로써 획기적인 공정의 안정화를 기할수 있었다.Thus, the present inventors this using a process computer to automate the conventional manual administration process, connect the glutamic acid fermentation exhaust gas line (on-line), a result of studying various Fermentation variables CO 2 production rate (CER) The specific correlation between and the cell concentration could be derived, and from this result, the timely and proper dose of penicillin (or surfactant) could be automated to stabilize the process.
본 발명의 내용을 상세히 설명하면 다음과 같다.The content of the present invention will be described in detail as follows.
글루타민산을 생성하는 미생물은 탄소원, 질소원, 산소 그리고 미네랄등이 함유된 발효배지에서 적절한 온도와 pH등을 유지시켜주면 증식됨과 동시에 물과 이산화탄소를 생성하며 이때 이산화탄소는 발효조 외부로 배출시킨다.Microorganisms that produce glutamic acid multiply by maintaining appropriate temperature and pH in fermentation broth containing carbon source, nitrogen source, oxygen, and minerals, and produce water and carbon dioxide.
발효 효배기가스중에 함유된 수분을 제거한후 산소측정기 및 이산화탄소측정기를 사용하여 CO2생성속도(CER)를 다음의 식(Ⅰ)으로부터 계산한다.After removing the water contained in the fermented yeast exhaust gas, the CO 2 production rate (CER) is calculated from the following equation (I) using an oxygen meter and a carbon dioxide meter.
여기서 F는 발효조에 공급되는 공기의 몰수(mole/min), V는 발효배지의 액량(ℓ), PT는 전압, PO2, PCO2는 각각 배기가스중에 함유된 산소와 이산화탄소의 분압을 나타낸다.Where F is the number of moles of air supplied to the fermenter (mole / min), V is the amount of fermentation broth (L), P T is the voltage, P O2 and P CO2 are the partial pressures of oxygen and carbon dioxide contained in the exhaust gas, respectively. .
이와같이 발효초기부터 연속적으로 측정될수 있는 CER은 제2도에서 도시한 바와같이 균체의 농도와는 선형적인 관계가 성립되고, 따라서 연속적으로 CER을 측정함으로서 균체의 농도를 추정할 수 있다.As described above, CERs that can be measured continuously from the beginning of fermentation have a linear relationship with the concentrations of the cells, as shown in FIG. 2. Therefore, the concentration of the cells can be estimated by continuously measuring the CERs.
이러한 CER과 균체농도와의 관계를 이용하여 발효진행과정중 요구하는 균체의 농도에 도달하면 마이크로 프로세서나 프로세스컴퓨터등을 이용하여 용이하게 페니실린(또는 계면활성제)을 적시에 투여할 수 있다.Using the relationship between CER and cell concentration, penicillin (or surfactant) can be easily administered in a timely manner using a microprocessor or a process computer when the required cell concentration is reached during the fermentation process.
또한 CER의 측정을 위하여 상술된 바와같은 산소측정기와 이산화탄소 측정기가 필요하나 배기가스중 산소(또는 이산화탄소)농도만을 측정하더라도 수식(Ⅱ)를 이용하여 나머지 농도(이산화탄소농도)를 추정, 본 발명에 이용할 수 있다.In addition, the oxygen measuring device and the carbon dioxide measuring device as described above are required for the measurement of the CER, but even if only the oxygen (or carbon dioxide) concentration in the exhaust gas is measured, the remaining concentration (carbon dioxide concentration) is estimated using Equation (II) and used in the present invention. Can be.
PCO2=O.2lxPT-PO2(Ⅱ)P CO2 = O.2lxP T -P O2 (II)
표 1은 산소측정기를 사용하여 측정한 배기가스중에 함유된 산소의 농도와 이산화탄소 측정기로 측정된 이산화탄소농도로 부터 식(Ⅱ)를 이용 계산된 산소농도와의 비를 나타낸다.Table 1 shows the ratio of the oxygen concentration in the exhaust gas measured using the oxygen meter and the oxygen concentration calculated using the formula (II) from the carbon dioxide concentration measured by the carbon dioxide meter.
[표 1]TABLE 1
산소농도의 실측치와 계산치의 비Ratio of measured value and calculated value of oxygen concentration
표 1에서 알수 있는 바와같이 실제측정치와 계산으로부터 얻어진 계산치와의 상대오차범위는 ±3%이내임을 보여주고 있어 산소측정기(또는 이산화탄소측정기)만으로도 CER을 계산, 간편하게 공업적으로 이용할 수 있다.As can be seen from Table 1, the relative error range between the actual measured value and the calculated value obtained from the calculation is within ± 3%, so the CER can be calculated and used industrially simply by the oxygen measuring device (or carbon dioxide measuring device).
[실시예 1]Example 1
30ℓ 소형발효조를 이용하여 Mg++, Mn++, Fe++, PO4 -3등이 함유된 공업용 당밀배지에 코리네박테리움 속종균을 식균하여 공기유량 1VVM, 온도 31℃, pH7.6, 교반속도 400rpm에서 증식시켰다.Using a 30 ℓ small fermenter, Corynebacterium spp. Was inoculated into industrial molasses containing Mg ++ , Mn ++ , Fe ++ , PO 4 -3 and air flow rate 1VVM, temperature 31 ℃, pH7.6 The mixture was grown at agitation speed of 400 rpm.
이때 사용된 본 배지의 조성은 다음과 같다.The composition of the medium used at this time is as follows.
균체의 증식과 더불어 생성되는 배기가스의 일부는 건조기를 통과하여 제1도에 도시한 바와같이 이산화탄소측정기 및 산소 측정기에 유입되어 각각의 농도가 측정되며 이들 측정기에서 발생되는 전류는 씨그널 콘디셔너에서 증폭되어 컴퓨터입력에 적합한 ±5V이내의 전압으로 바뀌어져 A/D컨버터를 통해 프로세스컴퓨터에 입력되어 이 결과를 분석, 발효가 진행되면서 CER의 값이 연속적으로 계산되어 프로세스컴퓨터에 기억되며, CER의 설정치에 도달하게 되면 D / A컨버터를 통해 나온 출력전압에 의해 솔레노이드밸브를 작동시켜 페니실린(또는 계면활성제)의 자동투여가 이루어지는 것이다.Part of the exhaust gas generated along with the growth of the cells is passed through the dryer to the carbon dioxide meter and the oxygen meter as shown in FIG. 1, and the concentration of each cell is measured. The current generated by the meter is amplified by the signal conditioner. It is changed to voltage within ± 5V suitable for computer input and input to process computer through A / D converter. As the result of analysis and fermentation is progressed, the value of CER is continuously calculated and stored in process computer. When it arrives, the solenoid valve is operated by the output voltage from the D / A converter, so that penicillin (or surfactant) is automatically administered.
표 2는 발효진행중 상술된 식을 이용하여 측정된 CER과 이를 근거로 계산된 균체농도와 측정된 균체농도와의 오차를 보여준다.Table 2 shows the CER measured using the above-described formula during fermentation, and the error between the measured cell concentration and the measured cell concentration.
여기에서 건조균체량은 배지의 고형성분을 제외한 균체만의 농도로서 3000rpm에서 15분간 원심분리하여 침전된 균체를 2회에 걸쳐 수세하여 재원심분리한후 105℃에서 20시간 건조한후 측정된 무게이다.Here, the dry cell weight is the concentration of only cells excluding the solid component of the medium, and centrifuged at 3000 rpm for 15 minutes to wash the precipitated cells twice, followed by recentrifugation and drying at 105 ° C. for 20 hours.
배지의 고형성분은 블랭크(Blank)로서 식균전 채취하여 측정하였다.The solid component of the medium was measured by phagocytosis as a blank.
[표 2]TABLE 2
CER로부터 계산된 균체량 및 측정된 균체량대비Cell mass calculated from CER and cell weight measured
이때 컴퓨터에 의해 계산된 단위 균체당 CER은 0.473mmole/g cell/min이었으며, 이를 근거로 계산된 건조균체량은 측정된 건조균체량과 ±0.15g/ℓ이내의 오차를 보였다.At this time, the CER per unit cell calculated by computer was 0.473mmole / g cell / min, and the dry cell weight calculated based on this showed an error within ± 0.15g / ℓ with the measured dry cell weight.
[실시예 2]Example 2
실시예 1과 동일조건으로 공기유량을 0.5VVM으로 하여 증식시켰다.Under the same conditions as in Example 1, the air flow rate was increased to 0.5 VVM.
표 3은 표2와 같은 방법으로 배기가스중의 산소와 이산화탄소분석에 의하여 간접적으로 계산된 건조균체량과 실측된 균체농도과의 대비 결과이다.Table 3 shows the results of the comparison between the dry cell mass and indirectly calculated cell concentrations indirectly calculated by oxygen and carbon dioxide analysis in the exhaust gas in the same manner as in Table 2.
[표 3]TABLE 3
CER로부터 계산된 균체량 및 측정된 균체량과의 대비Cell mass calculated from CER and contrast with cell mass measured
표3에서 나타난 바와같이 공기의 유량이 바뀌어도 이에 따른 계산된 건조균체량은 측정된 건조균체량과 ±0.1g/ℓ의 오차범위에 있음을 알수 있었다.As shown in Table 3, even though the air flow rate was changed, the calculated dry cell mass was found to be in the error range of ± 0.1 g / l and the dry cell mass measured.
따라서 공기의 유량변화에 따른 CER을 이용한 간접적인 균체농도측정이 가능하며 이때 계산된 단위균체당 CER은 0.564mmole/gcell/min이었다.Therefore, indirect cell concentration measurement using CER according to the flow rate of air was possible, and the calculated CER per unit cell was 0.564mmole / gcell / min.
[실시예 3]Example 3
실시예 1과 동일조건으로 초당농도와 온도, 교반속도등을 각각 변화시켜 증식시켰다. 표 4는 표 1과 표 2에서 보여준 것과 같이 계산된 건조균체량과 측정된 건조균체량의 차이를 보여주고 있으며 각각의 경우에 계산된 단위 균체당 CER을 표시하였다.Under the same conditions as in Example 1, growth was performed by varying the concentration per second, the temperature, and the stirring speed. Table 4 shows the difference between the calculated dry cell mass and the measured dry cell mass as shown in Tables 1 and 2, and shows the calculated CER per unit cell in each case.
좀더 구체적으로 설명하면 발효조건이나 발효조 상태에 따라 단위 균체당 CER의 값은 변할수 있으나 본 발명에 의한 균체농도의 측정은 ±0.1g/ℓ정도의 오차를 보임으로서 완벽하게 페니실린(또는 계면활성제)의 투여를 자동화함으로서 공정의 안정을 획기적으로 기할수 있었다.More specifically, the value of CER per unit cell may vary depending on fermentation conditions or fermenter conditions, but the measurement of cell concentration according to the present invention shows an error of ± 0.1 g / L, which is a perfect penicillin (or surfactant). By automating the administration, it was possible to dramatically stabilize the process.
[표 4]TABLE 4
발효조건에 따른 균체농도 측정결과Cell concentration measurement results according to fermentation conditions
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