CN104789618A - Method and device for optimizing and amplifying fermentation process of vitamin B12 - Google Patents

Method and device for optimizing and amplifying fermentation process of vitamin B12 Download PDF

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CN104789618A
CN104789618A CN201510176693.4A CN201510176693A CN104789618A CN 104789618 A CN104789618 A CN 104789618A CN 201510176693 A CN201510176693 A CN 201510176693A CN 104789618 A CN104789618 A CN 104789618A
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physiological metabolism
parameter
tank
bioreactor device
oxygen
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庄英萍
张嗣良
李昆太
刘东洪
储炬
王永红
黄明志
杭海峰
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East China University of Science and Technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH

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Abstract

The invention provides a method and device for optimizing and amplifying the fermentation process of vitamin B12, and particularly comprises a method for optimizing and amplifying the fermentation process by using a biological reactor. The method comprises the following steps: (a), measuring the physiological metabolism parameters, as well as the correlate characteristics or the combination of the metabolism parameters in the fermentation process using the biological reactor, wherein the physiological metabolism parameters are selected from dissolved oxygen, the oxygen uptake rate, pH, the carbon dioxide evolution rate, the respiratory quotient, the quantity of live cells or the cell morphology, the measured metabolic products or substrate consumption, or the combination of the measured metabolic products and substrate consumption; (b), comparing the physiological metabolism parameters and the correlate characteristics of the metabolism parameters measured in step (a) with the physiological metabolism parameters and the correlate characteristics or the combination of the metabolism parameters of the predetermined value; picking out the biological reactor mostly proximate to the predetermined value; determining the biological reactor to be optimized and amplified, wherein the physiological metabolism parameters are as described in step (a).

Description

Vitamins B 12the method and apparatus of optimizing fermentation and amplification
The application number that this divisional application applicant submitted on September 11st, 2008 is 200810042761.8, denomination of invention is " vitamins B 12the method and apparatus of optimizing fermentation and amplification " the divisional application of application for a patent for invention.
Technical field
The present invention relates to method and the device thereof of a kind of optimizing fermentation and amplification, especially relate to a kind of vitamins B 12the method and apparatus of optimizing fermentation and amplification.
Background technology
Test the rule obtained and data to reproduce in large production to make small-sized fermentation, with regard to GPRS with use amplifying technique.In prior art, the general similarity principle that adopts carries out analogy amplification.Particularly, the basic skills of amplifying of comparing in prior art is: first must find out the various parameters characterizing this system, by the several non-dimensional number with certain physical meaning of they compositions, and the functional expression set up between them, then in testing installation, experimentally try to achieve the constant comprised in this functional expression and index, then this relational expression just can be used as the design of the main equipment of testing installation geometricsimilarity therewith.
The object of all scale up principle is all hold from a large amount of test materialss and find out principal contradiction affect production process, thus makes the rule of main equipment and data test the rule that obtains and data can reproduction in large production at small-sized fermentation.Amplified criterion general at present comprises:
(1) geometricsimilarity: its functional expression is as shown in the formula (I)
D 2/ D 1=D i2/ D i1=(V 2/ V 1) 1/3formula (I)
D-------------reactor diameter
D i-------------stirrer diameter
The charging volume of V--------------reactor
(2) constant equal-volume power magnification is constant and determine mixing speed by Pg/V.
(3) constant oxygen transfer coefficient kLa amplifies
(4) constant shear power constant end of blade speed amplification shearing force is directly proportional to stirring rake end of blade speed, generally maintains n when constant volume power magnification 3d 2constant (n be rotating speed of agitator, d be diameter of stirring paddle)
(5) constant mixing time tM amplifies.
In the amplification process of the above-mentioned scale up principle of application (or criterion), those skilled in the art can set up the functional expression of similar formula (I) by limited test, these functional expressions belong to prior art, are not described further at this.
But the weak point of above-mentioned existing amplified criterion is, its be all based on extracellular parameter detecting based on optimised process reference mark be the static state operation method of foundation, be not conceived to the Related Phenomena characteristic parameter based on the change of cellular metabolism material flow point cloth.Can not accomplish that geometricsimilarity, hydrokinematics phase Sihe hydrokinetics are similar when amplifying simultaneously, when important factor to have an impact some when lab scale is studied is not observed, and this factor exactly amplify time become key factor time, quantitative change can be caused to cause qualitative change, thus cause the failure of whole fermenting process.Therefore, this area lacks based on the scale up principle of physiological metabolism characteristic parameter and method thereof.
At present, adopt existing scale up principle to vitamins B 12optimizing fermentation and amplification, encounter problem when being particularly amplified to technical scale level, be embodied in vitamins B 12fermentation unit not higher than 125 μ g/ml, lower than small-sized fermentation test fermentation unit.
In prior art, vitamins B 12typically refer to the cobalami compounds containing cobalt ion.It is a kind of important biologically active substance, is mammiferous Hemopoietic factor, can be used for treating surra, and it is also the somatomedin of many microorganisms and animal simultaneously.The biosynthesizing of vitamin B12 is strictly limited in microorganism, occurring in nature VB 12route of synthesis there are two kinds of different routes: (1) aerobic route of synthesis, in the microorganisms such as such as denitrogenation Pseudomonas (P.denitrificans); (2) anaerobism route of synthesis, in the microorganisms such as such as Bacillus megaterium, P.shermanii, Salmonella typhimurium.Long-term next, VB 12study on the synthesis with production mostly rest on anaerobically fermenting approach; Through the effort of at least 25 years, by means of genetics and molecular biology method and combine with zymetology, chemosynthesis, isotopic labeling, NMR (Nuclear Magnetic Resonance) spectroscopy etc., VB in P.denitrificans 12aerobic route of synthesis just intactly set forth in 1993.Various microorganism vitamin B is shown such as formula the reaction formula shown in II 12the signal of biosynthetic pathway, and aerobic route of synthesis and anaerobism synthesize between difference.
In most of bacterium, the biosynthesizing of tetrapyrrole is all from the C-5 skeleton of L-glutamic acid, through polystep reaction, form 5-Aminolaevulic acid (δ-amino-laevulic acid), uroporphyrinogen III (uroporphyrinogen III), and uroporphyrinogen III is methylated on C-2 and C-7 by methyltransgerase, thus corrin-2 (precorrin-2) before synthesis.Such as formula II display, before formation after corrin-2, the biosynthesizing of cobalami starts to form Liang Ge branch approach: in aerobic route of synthesis, front corrin-2 methylates on C-20, thus corrin-3A before being formed; But in anaerobism route of synthesis, front corrin-2, by chelate cobalt ions, generates cobalt-front corrin-2 (cobalt-precorrin-2).Thus, VB 12one of the aerobic difference with anaerobism route of synthesis be that the chelating of cobalt ion is different for opportunity, in anaerobism route of synthesis, the chelating of cobalt ion originates in front corrin-2, however in aerobic route of synthesis the chelating of cobalt ion occur in before after ensuing nine step of reaction of corrin-2.In addition, at VB 12in aerobic route of synthesis, the C-20 atom of front corrin-3A be oxidized by molecular oxygen, result discharges C-20 with the form of acetate, and at VB 12in anaerobism route of synthesis under anaerobic condition, may be present different prices (from+1 to+3) to reach oxygenizement by the cobalt ion of complexing, result discharges C-20 with the form of acetaldehyde.Although VB 12aerobic and anaerobism synthetic route separate when front corrin-2, but they become again identical in adenosine cobyrinamide (adenosyl-cobyric acid) place's synthetic route again.
Between many decades in the past, for the production of VB 12microorganism mainly contain the anaerobism metabolic pathway of synthesizing such as Propionibacterium shermanii, P.freudenreichii, P.denitrificans, and P.denitrificans be in recent years rise for aerobic synthesis VB 12industrial strain, because its production efficiency is much larger than anaerobism metabolic pathway of synthesizing, so be also the focus that current people pay close attention to its applied research.Due to VB 12aerobic route of synthesis and anaerobism route of synthesis have a lot of step to be identical, P.denitrificans is a kind of facultative anaerobe in addition, therefore, production on to P.denitrificans produce VB 12the dissolved oxygen of fermenting process controls to produce certain query and mistaken ideas.
In sum, this area shortage has vitamins B 12the method of optimizing fermentation and amplification, and vitamins B 12plant-scale bio-reactor of fermentation.
Therefore, this area is in the urgent need to exploitation vitamins B 12the method of optimizing fermentation and amplification, and vitamins B 12plant-scale bio-reactor of fermentation.
Summary of the invention
The first object of the present invention is that obtaining one can be used for vitamins B 12the method of optimizing fermentation and amplification.
The second object of the present invention is that obtaining one can be used for vitamins B 12plant-scale bio-reactor of fermentation.
In a first aspect of the present invention, provide a kind of method to using the fermenting process of bioreactor device to be optimized amplification, described method comprises step:
A () measures physiological metabolism parameter in the described fermenting process of described bioreactor device, physiological metabolism parameter correlation characteristic or its combination;
Wherein said physiological metabolism parameter is selected from: the meta-bolites of oxygen-supplying amount, dissolved oxygen, oxygen uptake rate, pH, carbon dioxide evolution rate, respiratory quotient, amount of viable cell or cellular form, mensuration or substrate consumption or its combination;
The physiological metabolism parameter of b described physiological metabolism parameter, physiological metabolism parameter correlation characteristic and preset value that step (a) measures by (), physiological metabolism parameter correlation characteristic or its combine and compare; Select the bioreactor device the most close with described preset value; Determine the amplification bioreactor device optimized;
Wherein said physiological metabolism parameter is as described in step (a).
In a preference, the meta-bolites of described mensuration or substrate consumption are meta-bolites or the substrate consumption of the manual mensuration in laboratory.
In a specific embodiment of the present invention, described bioreactor device is selected from vitamins B 12fermentation unit.
In a specific embodiment of the present invention, described bioreactor device is capacity is 20L--2000m 3fermentor tank.
In a specific embodiment of the present invention, in described step (a), the method by detecting multiple process control parameters of bio-reactor to obtain physiological metabolism parameter in described fermenting process, physiological metabolism parameter correlation characteristic or its combination,
Wherein said multiple process control parameter is mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
In a specific embodiment of the present invention, in described step (b), the difference of the described physiological metabolism parameter of mensuration, physiological metabolism parameter correlation characteristic and preset value is not higher than between 10 ~ 15%.
In a specific embodiment of the present invention, the preset value described in described step (b) is physiological metabolism parameter under canister fermentation condition, physiological metabolism parameter correlation characteristic or its combination;
Wherein, described bioreactor device is not less than between 20 ~ 2000 times relative to the magnification of little tank volume.
In a preference, described canister is as 5,10,20,30,50,100 liters or 1-10M 3.
In a specific embodiment of the present invention, in the comparison procedure of step (b), also comprise the steps:
The physiological metabolism parameter of i described physiological metabolism parameter, physiological metabolism parameter correlation characteristic and preset value that step (a) measures by (), physiological metabolism parameter correlation characteristic or its combine and compare;
(ii) the physiological metabolism characteristic parameter feature of described bioreactor device, physiological metabolism parameter correlation characteristic or its combination is regulated, make the physiological metabolism characteristic parameter feature of described bioreactor device relative to the difference of described preset value not higher than between 10 ~ 15%, the selected bioreactor device the most close with described preset value;
(iii) the amplification bioreactor device optimized is determined.
A kind of automatic control bioreactor device carrying out process optimization and data amplification is provided in a second aspect of the present invention, described automatic control bioreactor device comprises the bio-reactor tank body with agitator, and to the instrument that the process control parameter of described bio-reactor detects and controls, described tank body is arranged:
-sensing device unit, described sensing device unit comprises the dissolved oxygen sensor be arranged on described tank body, amount of viable cell sensor;
-physiological metabolism calculation of characteristic parameters the device that is connected with described sensing device unit, the process control parameter that described sensing device unit obtains is converted into the physiological metabolism characteristic parameter amplifying basis as data by described physiological metabolism calculation of characteristic parameters device.
In a preference, described process control parameter refers to temperature, mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
Particularly, described agitator employing rotating speed is the high speed agitator between 0 ~ 1000rpm.
Particularly, described bio-reactor is provided with the oxygen supply equipment of oxygen flow between 0 ~ 100 liter/min.
In a specific embodiment of the present invention, the volume capacity of described tank body is at 20L--2000m 3between.Preferred 120m 3~ 2000m 3, more preferably 150m 3~ 2000m 3.
In a specific embodiment of the present invention, described sensing unit also comprises temperature sensor, pH sensor, full tank scale quantity sensor, tail gas CO 2interface, tail gas O 2interface, tachogenerator, pressure transmitter, froth breaking sensor or its combination.
Accompanying drawing explanation
Fig. 1 is at 9m in embodiment 1 3metabolic chart (the The process paramteters of VB that pilot scale fermentation tank obtains 12fermentation in 9m 3fermentor).
Fig. 2 is parameter variation tendency figure (the The process paramteters of VB in embodiment 1 under low Oxygen supplied level 12fermentation in low dissolved oxygen concentration).
Fig. 3 is parameter variation tendency figure (the The process paramteters of VB in embodiment 1 under high Oxygen supplied level 12fermentation in high dissolved oxygen concentration).
Fig. 4 is that in embodiment 1, two kinds of different dissolved oxygens control tanks criticize lower total reducing sugar, amino nitrogen, dry cell weight and VB 12dynamic changing process (Time profiles of total sugar, NH 2-N, cell growth and VB 12production in two DOC control levels).
Fig. 5 is two kinds of different oxygen delivery capacity situation bottom fermentation process specific growth rates and changing conditions (Time profi les of specific growth rate (μ) the and specific production rate (q than product synthesis speed in embodiment 1 p) of VB 12fermentation in two DOC control levels).
Fig. 6 is the schematic diagram of the automatic control bioreactor device of embodiment 2.
Embodiment
The present inventor is through extensive and deep research, by improving preparation technology, obtain with metabolic flux analysis with control to be on the research method basis that adjusts of the Fermentation Process of Parameter of core, obtain one and laboratory scale optimal conditions can be amplified to plant-scale method.Complete the present invention on this basis.
The present invention proposes based on following principle: the function of the biosystem for the purpose of industrial production depends on that external environment stimulates the acting in conjunction with functional gene in born of the same parents.The impact of this external environment cellular function mainly through following several approach, one, ambient conditions (as temperature, pH) directly has an impact to the effect of somatic cells endoenzyme or metabolic rate; Its two, due to reactor mixing transmit caused by matrix (as oxygen, various carbon source) under-supply or excessive, the change of consequent born of the same parents' intracellular metabolite; Its three, the response of the signal transducting system of cell envrionment conditions to external world, causes the change of cell transcription expression system, thus and then cause the change of cellular metabolism network.This just requires that we will carry out optimization and the amplification of industrial bio process on the basis considering born of the same parents' external environment and thalline physiological property, and in actual industrial production, mostly be liquid cultivation, thus namely the research of the outside atmosphere of thalline be summed up as bio-reactor inner fluid mechanics study, obtains the temperature field under different condition, concentration field, shearing force field etc. thus; The behavior of cell is then expressed by thalline physiological property.By both being integrated its interaction rule of research thus opening up a brand-new Scientific Thinking with amplifying for the optimization of bio-reactor.
According to above-mentioned principle, the present invention devises the following concrete route carrying out flow process amplification and optimization: when fermenting process amplifies research, propose first with metabolic flux analysis with control for the fermenting experiment device of core is studied, can obtain for the state parameter of process study or physiological parameter and the correlation properties between them thus, can realize thus gene in bio-reactor, cell, reactor different scale across yardstick research, namely realize optimizing fermentation by multi-scale parameters correlation analysis.When amplifying, as long as we obtain the physiological data change curves such as complete (or basic) identical reflection metabolic fluxes on the equipment amplified, just can overcome the problem in above-mentioned amplification process preferably, thus propose the amplifying technique of Fermentation Process of Parameter adjustment.
Below describe in detail to various aspects of the present invention:
(1) method of process optimization and data amplification is carried out
The present invention relates to a kind of method to using the fermenting process of bioreactor device to be optimized amplification, described method comprises step:
A () measures physiological metabolism parameter in the described fermenting process of described bioreactor device, physiological metabolism parameter correlation characteristic or its combination;
Wherein said physiological metabolism parameter is selected from: the meta-bolites of oxygen-supplying amount, dissolved oxygen, oxygen uptake rate, pH, carbon dioxide evolution rate, respiratory quotient, amount of viable cell or cellular form, mensuration or substrate consumption or its combination;
The physiological metabolism parameter of b described physiological metabolism parameter, physiological metabolism parameter correlation characteristic and preset value that step (a) measures by (), physiological metabolism parameter correlation characteristic or its combine and compare; Select the bioreactor device the most close with described preset value; Determine the amplification bioreactor device optimized;
Wherein said physiological metabolism parameter is as described in step (a).
In a preference, described method comprises:
Obtain the optimization physiological metabolism characteristic parameter intensity of the first bioreactor device (also namely for obtaining the bioreactor device of preset value) or correlation properties etc., described physiological metabolism characteristic parameter is selected from the manual meta-bolites that measures in dissolved oxygen, oxygen uptake rate, pH, carbon dioxide evolution rate, respiratory quotient, amount of viable cell or cellular form, various laboratory or substrate consumption etc. or other combination; Preferably, oxygen uptake rate, respiratory quotient, pH etc. or other combination is selected from.
Obtain the physiological metabolism characteristic parameter intensity of the second bioreactor device (bioreactor device also namely for optimizing or amplifying) or correlation properties etc., described physiological metabolism characteristic parameter is selected from the manual meta-bolites that measures in dissolved oxygen, pH, oxygen uptake rate, carbon dioxide evolution rate, respiratory quotient, amount of viable cell or cellular form, various laboratory or substrate consumption etc. or its combination; Preferably, oxygen uptake rate, respiratory quotient, pH etc. or other combination is selected from.
Regulate the physiological metabolism characteristic parameter feature of described second bioreactor device, make it relative to the difference of the optimization physiological metabolism characteristic parameter of described first bioreactor device not higher than 10 ~ 15%, thus obtain the second bioreactor device of process optimization and data amplification.This regulating step makes the physiological metabolism characteristic parameter feature of described second bioreactor device have identical trend with described first bioreactor device.
Particularly, when regulating the physiological metabolism characteristic parameter feature of described second bioreactor device, by the oxygen supply in described second bioreactor device of adjustment and oxygen-consumption, thus the physiological metabolism characteristic parameter feature of described second bioreactor device can be made relative to the difference of the optimization physiological metabolism characteristic parameter of described first bioreactor device not higher than 10 ~ 15%.
More specifically, the technique that the adjustment of described oxygen-supplying amount and oxygen-consumption, OUR can be traditional according to this area, particularly such as by adjustments such as mixing speed, ventilation flow rate, benefit carbon and nitrogen sources or other matrix flows.
In the prior art, to the optimization of reactor and amplify normally based on single or multiple process control parameter (such as temperature, pH etc.), these single or multiple process control parameters are only based on the research of single physiological regulating control mechanism.But the research based on single physiological regulating control mechanism often disclosed local and the feature of a certain period of physiological regulating control, the research only by hyperbranchedization and concrete dispersion is difficult to play a decisive role to whole optimizing fermentation control and amplification.Contriver, through large quantity research, determines " the physiological metabolism characteristic parameter " of most critical in amplification process, biology is combined with engineering science, and solve local and global deformation, time variation state and net result, bacterial classification transform and the relation of process optimization.
Particularly, described oxygen uptake rate (OUR) is by measuring tail gas oxygen concn, ventilation flow rate, fermentating liquid volume and obtaining; Particularly, such as, by adopting following formula:
mol/L·h
In formula:
F in: charge flow rate, (mol)
C feelings inc o2inc cO2in: the concentration % (V) being respectively rare gas element in air inlet, oxygen and carbonic acid gas
C o2outc cO2out: the concentration % (V) being respectively oxygen and carbonic acid gas in exhaust
V: fermented liq, (L)
f = 273 273 + t in · P in · 1 1 + h × 10 - 5
In formula
P in: the pounds per square inch absolute (psia) of air inlet, Pa
T in: the temperature of air inlet, DEG C
H: the relative humidity of air inlet, %
Above-mentioned computation process can also be undertaken by the analysis software package of computer; Such as calculated by fermenting process real-time data analysis software package BIOSTAR.
As described herein, described " bio-reactor " includes but not limited to: fermentor tank, zooblast reactor or vegetable cell reactor.
" the first bioreactor device " in described method typically refers to the bioreactor device of laboratory scale or the bench scale commonly used this area.Particularly, the capacity of described " the first bioreactor device " is between 20 ~ 500 liters.
" preset value " physiological metabolism characteristic parameter of described first bioreactor device can be obtained by the optimization method that this area is traditional, only otherwise to goal of the invention of the present invention produces and limits.Such as.Orthogonal Method is adopted to obtain optimized one or more physiological metabolism characteristic parameter.
" the second bioreactor device " in described method refers to the bioreactor device of industrially scalable.Particularly, the capacity of described " the second bioreactor device " is at 1m 3above.
In a specific embodiment of the present invention, described second bioreactor device is not less than between 20 ~ 2000 times relative to the magnification of described first bioreactor device.
Particularly, the volume of described second bioreactor device is 20 ~ 2000m 3.
(2) the automatic control bioreactor device of process optimization and data amplification can be carried out
The present invention also provides a kind of automatic control bioreactor device carrying out process optimization and data amplification, described automatic control bioreactor device comprises the bio-reactor tank body (100) with agitator (104), and to the instrument that the process control parameter of described bio-reactor detects and controls, described tank body (100) is upper to be arranged:
-sensing device unit (101), described sensing device unit (10) comprises the dissolved oxygen sensor (3) be arranged on described tank body (100), amount of viable cell sensor (10);
-physiological metabolism calculation of characteristic parameters the device (20) that is connected with described sensing device unit (101), the process control parameter that described sensing device unit (101) obtains is converted into the physiological metabolism characteristic parameter amplifying basis as data by described physiological metabolism calculation of characteristic parameters device (20).
Particularly, the volume capacity of described tank body (100) is at 20 ~ 2000M 3between, preferably 100 ~ 2000M 3, more preferably 100 ~ 2000M 3.
Particularly, described sensing unit (101) also comprises temperature sensor (1), pH sensor (2), full tank scale quantity sensor (4), tail gas CO 2interface (5), tail gas O 2interface (6), tachogenerator (7), pressure transmitter (8), froth breaking sensor (9) or its combination.
More specifically, the process control parameter that described instrument measures refers to temperature, mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
Particularly, described amount of viable cell sensor adopts four electrode systems.
Described four electrode systems are particularly such as: according to the viable cell be placed in alternating electric field, because intracellular protoplastis plays ionogen effect, and the electric capacity formed in the polarized action of alternating electric field and biomass are corresponding relation.The frequency changing alternating electric field will produce different polarization effects, and determine best measuring condition thus, the capacitance of measurement is transferred to computer digital animation by unified signal, as the foundation of the biomass of multiparameter correlation analysis.
The various sensors that various sensors in described sensing unit of the present invention can adopt this area traditional, limit as long as it does not produce goal of the invention of the present invention.
The installation site of described instrument of the present invention can be carried out according to the traditional technology of this area, only otherwise to goal of the invention of the present invention produces and limits.
In an embodiment of device of the present invention, by the following technical solutions:
A kind of self-controlled fermentation plant amplified for process optimization and data, this device is by translator and the fermentation tank of agitator, the sensor-based system with multiparameter detection and the instrument controlled and sensing device, band mounting bracket and process pipe system and form with the electrical control cabinet of industrial computer and execution components and parts, it is characterized in that wherein said sensor-based system comprises temperature sensor (1), pH sensor (2), dissolved oxygen sensor (3), full tank scale quantity sensor (4), tail gas CO 2interface (5), tail gas O 2interface (6), tachogenerator (7), pressure transmitter (8), froth breaking sensor (9), ventilation flow rate sensor (26), amount of viable cell sensor (10); Described parameter refers to temperature, mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
The wherein said electrical control cabinet with industrial computer and execution components and parts comprises high precision peristaltic pump (11); Matrix feed supplement electronic scales (12); Precursor or oily electronic scales (13); Soda acid thing electronic scales (14); Recycle pump (25); Magnetic valve (15); D/A converter (18); A/D converter (17); Lower computer (19); Upper computer (20); Rare-earth electric motor (23).
The process pipe system of wherein said band mounting bracket comprises material bottle (22); Full tank scale amount bearing; Special support is used in sampling; Tank body assembly; Rapid-assembling/disassembling support; Electric motor; Pipe support; Water-and-oil separator; Reducing valve; Strainer; Under meter; Air filter; Tensimeter; Water cooler; Pipeline viewing mirror; Water-heater; Without dead volume sampling valve; Froth breaking sensor interface; Tail gas CO 2interface; Tail gas O 2interface; Temperature sensor; PH Sensing interface; DO sensor interface.
The process pipe system of above-mentioned band mounting bracket, each assembly of electrical control cabinet and building form thereof all can carry out according to the technique that this area is traditional, only otherwise limit goal of the invention of the present invention.
The electrical control cabinet of described industrial computer and execution components and parts forms physiological metabolism calculation of characteristic parameters device (20) of the present invention; The wherein various assembly assembly that this area all can be adopted traditional, only otherwise to goal of the invention of the present invention.
Particularly, described is according to on-site data gathering and the demand of operation and the requirement of process optimization with industrial computer and the computer software performed in first device housing, carry out on-line parameter collection, offline parameter calculate, the on-line Control of supplemental characteristic record and partial parameters, synchronously transmitted the data of all parameters to upper computer by local area network, select Configuration Language and C language to carry out Programming respectively to upper computer and lower computer, and employ simple redundancy technology when data logging.
More specifically, described computer software adopts the upper computer software bag BIOSTAR of National Engineering Research Center for Biotechnology of East China University of Science (Shanghai).
Other aspects of the present invention, due to disclosure herein, are apparent to those skilled in the art.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or carry out according to the condition that manufacturer advises.Unless otherwise indicated, otherwise all numbers are weight part, and all per-cent is weight percentage.
Unless otherwise defined or described herein, all specialties used herein and scientific words and those skilled in the art the same meaning be familiar with.In addition any method similar or impartial to described content and material all can be applicable in the inventive method.
The beneficial effect of the invention is:
(1) namely carry out by the fermenting process various parameters characterized in parameter and other metabolic process of cell macroscopic view metabolic fluxes change the optimizing research analyzed based on parameter correlation, P.denitrificans is fermented product VB 12process in the supply of oxygen and consumption etc. conduct in-depth research, found that fermenting process is likely restriction VB for hypoxgia 12one of key influence factor that oxygen-consuming capacity and synthesis capability improve.And on this basis, first at 9m 3by the mutual relationship of regulation and control DO and OUR on pilot scale fermentation tank, make VB 12anabolism normally carried out, finally with the principle that fermenting process oxygen consumption characteristic is consistent, by industrial scale fermentation tank adjustment and improve zymotechnique, achieve technical scale 120m 3vB on fermentor tank 12increasing substantially of production output.
Embodiment 1:B 12optimizing fermentation and amplification
1 materials and methods
1.1 bacterial classifications and substratum
1.1.1 bacterial strain: denitrogenation Pseudomonas (Pseudomonas denitrificans), Huarong pharmaceutical Co. Ltd of pharmacy group provides by Shijiazhuang.
1.1.2 substratum
Primary-seed medium: molasses, corn steep liquor, KH 2pO 4, (NH 4) 2sO 4, (NH 4) 2hPO 4, MnSO 4deng.
Secondary seed medium: molasses, corn steep liquor, KH 2pO 4, (NH 4) 2sO 4, (NH 4) 2hPO 4, MgSO 4deng.
Fermention medium: molasses, sucrose, trimethyl-glycine, (NH 4) 2sO 4, MgSO 4, CoCl 2, DMBI, ZnSO 4deng.
1.2 fermentor tanks: pilot scale fermentation tank volume is 9m 3, agitator is axially; Industrial scale fermentation tank volume is 120m 3, agitator main flow is axial backmixing.
1.3 analytical procedure
1.3.1 biomass measuring: adopt and measure dry cell weight method (Dry cell weight, DCW).Draw 25ml fermented liquid, centrifugal rear distillation washing thalline, after recentrifuge, weighs after being dried to constant weight by thalline 105 DEG C.
1.3.2 the mensuration of total reducing sugar: adopt Fehlings reagent.
1.3.3 the mensuration of amino nitrogen: formol titration.
1.3.4 tails assay: use SIMENS GXH-9022CO 2, O 2analytical system, last data processing and correlation analysis are completed by the upper computer software bag BIOSTAR of National Engineering Research Center for Biotechnology of East China University of Science (Shanghai).
1.3.5 the online dissolved oxygen detection system of dissolved oxygen determination: Mettler Toledo (commercial instruments).
1.3.6pH measure: the online pH detection system of Mettler Toledo (commercial instruments).
1.3.7VB 12measure
By VB multi-form in sample 12be converted into Vitral, moving phase: 250mM phosphate aqueous solution: acetonitrile=30:70, chromatographic column: C8 5FI13513 260*4.6mm; Determined wavelength: 361nm; Sample size: 20ul; Flow velocity: 1.7ml/min.
2. results and analysis
In 2.1 shaking flasks, different Oxygen supplied level produces VB to fermentation 12impact
VB is synthesized owing to utilizing P.denitrificans 12the metabolic process of an oxygen consumption, at VB 12aerobic route of synthesis in, also relate to adding of O atom.But, different from VB to oxygen supply situation in P.denitrificans fermenting process at present 12synthesize the impact produced and also do not have bibliographical information.Therefore this test is in shake flask fermentation process, and by under the fermented liquid of putting into identical amount in identical shaking flask and same inoculum size situation, the rotation speed change in different fermentations stage to investigate in shaking flask different Oxygen supplied level to VB 12the impact of synthesis.Concrete test design and the results are shown in Table 1:
The change of table 1 shake flask fermentation process medium speed is to VB 12the impact of synthesis
(The effect of different revolutions per minute in shake-flask fermentation on VB12biosynthes is)
As can be seen from Table 1, when the rotating speed of shake flask fermentation process is 200rpm always, VB when to put bottle 12unit is minimum; When the rotating speed of shake flask fermentation process maintains 260rpm always, VB when to put bottle 12unit is the highest in six kinds of process, than contrast raising 24%.The 60-100h of shake flask fermentation process is that P.denitrificans starts to synthesize VB in a large number 12stage, as can be seen from Table 1, the oxygen supply situation of this fermentation stage is the most key on the impact of final Fang Ping unit, this stage rotating speed be three process of 200rpm put a bottle VB 12unit is relatively low, is all three process of 260rpm lower than this stage rotating speed.Can reach a conclusion from above test-results, in shake flask fermentation process, oxygen supply is more conducive to VB in order 12biosynthesizing.
2.2 at 9m 3on pilot scale fermentation tank, different oxygen supply and oxygen consumption situation are to the metabolic effect of denitrogenation Pseudomonas
Know the importance of metabolism during oxygen supply is to denitrified pseudomonas fermenting process in shaking flask research, so first at the pilot scale 9m having installed fermenting process software for data analysis BIOSTAR 3fermentor tank is tested, obtains representative metabolic chart as shown in Figure 1:
Can find from the curve obtained, in denitrified pseudomonas fermenting process, along with the carrying out of fermenting process, the continuous increase of bacterium amount (X), consumption rate (OUR) and carbon dioxide evolution rate (CER) constantly increase, DO also constantly declines, about 20h DO machine has dropped to lower level (about 20%), then OUR, CER all reaches higher level, along with constantly carrying out of fermentation, at about 80h OUR, CER slightly increases again, and DO declines (about 8%) to some extent, and arrived fermentation latter stage, in fact the increase of bacterium amount has not been too large (increase of X is milder), but OUR, CER still maintains higher level, now VB 12growth but maintain higher speed, really VB is described 12building-up process be a stronger oxygen process, VB final under these operation regulation and control 12fermentation unit reaches 195 μ g/ml.
2.3 amplify research based on the technical scale that oxygen consumption feature is consistent
Subsequently at 120m 3industrial scale fermentation tank carries out amplification test, obtains the metabolic condition under following different operating condition.
Regulate 120m 3the physiological metabolism characteristic parameter feature of industrial scale fermentation tank, physiological metabolism parameter correlation characteristic or its combination (regulating Oxygen supplied level particularly).With reference to the physiological metabolism characteristic parameter feature of preset value, the selected bioreactor device the most close with described preset value.
2.3.1 the change of two kinds of different Oxygen supplied level bottom fermentation early stage OUR, CER, DO, pH
Fig. 2 and Fig. 3 is 120m 3on tank under two kinds of different Oxygen supplied levels (air flow F is as figure), the changing trend diagram of OUR, CER, DO, pH of earlier fermentation online acquisition.
Very of short duration as can be seen from the lag phase of Fig. 2 and Fig. 3, P.denitrificans thalline, after thalli growth, pH is in rising trend, after pH rises to climax, starts to decline gradually, then has again a process of ging up, start again afterwards to decline.As Fig. 2 display under low oxygen supply condition, because pH constantly declines, have to maintain the suitable pH of fermented liquid, so there is zigzag pH tendency, and by contrast in the good situation of oxygen supply (Fig. 3) by adding ammoniacal liquor, after 20h, pH is highly stable, the tank do not resembled under low Oxygen supplied level needs Feeding ammonia water to regulate pH criticizing, and may be that carbohydrate metabolism is complete because the supply of oxygen is better, the organic acid of accumulation is less, makes pH more steady.And from the variation tendency of DO, along with the growth DO of thalline declines gradually, CER, OUR also synchronously increase gradually, in Fig. 2 when DO is down to 0 at 17h, OUR and CER still presents ascendant trend, arrives climax, reach 46mol/ (hm respectively to OUR and CER during 24.5h 3) and 44mol/ (hm 3).After 24.5h, OUR and CER obviously starts to decline, and the restriction that this growth metabolism being likely due to thalline is subject to the supply of oxygen causes, and lower VB criticized by this tank 12fang Guan unit be 125 μ g/ml.And in figure 3, the DO tendency in early stage is identical, but reduce degree comparatively Fig. 2 high (about 5%) to DO during 15h, once OUR and CER is made all to a new height by increasing air flow in process, and there is not obvious downtrending in OUR and CER, this illustrates that thalline maintains more vigorous metabolic capacity always, thus is more beneficial to VB 12biosynthesizing, lower VB criticized by this tank 12fang Guan unit be 190 μ g/ml.
2.3.2 the total reducing sugar of mensuration-two kinds different Oxygen supplied level bottom fermentation process of amplification effect, amino nitrogen, dry cell weight and VB 12deng Parameters variation situation
Corresponding with above-mentioned test-results, the tank that the different dissolved oxygens of this test determination two kinds control criticizes bottom fermentation process total reducing sugar, amino nitrogen, dry cell weight and VB 12changing conditions, the results are shown in lower Fig. 4:
During the fermentation, when total sugar concentration is reduced to about 4g/100ml, start to carry out adding sugar, in control fermented liquid, total sugar concentration is at 3-4g/100ml.As can be seen from Fig. 4 .A, earlier fermentation sugar consumption basic simlarity, it is slightly better that low oxygen supply tank is criticized, and this is likely that the strain quality of this batch fermentation is better caused, but after 20h, the good tank of oxygen supply criticize its sugar consumption speed obviously lower oxygen supply tank criticize and will get well; Fermenting process amino nitrogen variation tendency be lower oxygen supply tank to criticize before 36h amino nitrogen content comparatively the good tank of oxygen supply criticize low, but afterwards, the tank that the amino nitrogen that lower oxygen supply tank is criticized controls higher than high dissolved oxygen is criticized, and especially after 120h, the amino nitrogen rise amplitude that low dissolved oxygen control tank is criticized is very large.The change of amino nitrogen content describes the bacterial metabolism change that tank in two kinds of oxygen supply situations is criticized equally, and after 36h, low dissolved oxygen controls the bacterial metabolism ability that tank criticizes and declines obviously due to dissolved oxygen restriction, the aggravation of thalline self-dissolving after causing 120h especially.
As can be seen from Fig. 4 .B, before 30h, the dry cell weight that lower oxygen supply tank is criticized is criticized a little more than the good tank of oxygen supply, this and consume at the sugar in this stage, amino nitrogen content etc. all has good dependency.But after 30h, the dry cell weight that the good tank of oxygen supply is criticized then is greater than lower oxygen supply tank to be criticized always, the different tanks under different oxygen supply control strategies criticize under maximum dry cell weight be respectively 35.44g/L and 31.26g/L.This illustrates again, and because seed quality better waits some factors, the thalli growth metabolic capacity that lower oxygen supply tank is criticized is better than the good tank of oxygen supply at earlier fermentation and criticizes.But along with biomass is increasing, this needs increasing oxygen to meet the growth metabolism of thalline, its oxygen delivery capacity when not carrying out technique adjustment criticized by lower oxygen supply tank can not meet the demand of thalline for oxygen, make dissolved oxygen be reduced to 0, make dissolved oxygen become the restrictive factor of thalli growth metabolism especially.In addition, from thalline synthesis VB 12, during 36h, the VB that lower oxygen supply tank is criticized 12unit also criticizes a little more than the good tank of oxygen supply, is respectively 20.13 μ g/ml and 18.65 μ g/ml.But after 60h, the VB that the good tank of oxygen supply is criticized 12unit but criticizes higher than lower oxygen supply tank, especially after 108h, and the VB that the good tank of oxygen supply is criticized 12the increasing degree of unit is criticized apparently higher than lower oxygen supply tank especially, final VB 12the good tank of Fang Guan unit oxygen supply is criticized the fermentation unit that lower oxygen supply tank criticizes and is improve 50%, and final fermentation unit reaches 190 μ g/ml.
Total reducing sugar, amino nitrogen, dry cell weight and VB that comprehensive above two kinds of different Oxygen supplied level tanks are criticized 12deng Parameters variation process, a conclusion clearly can be drawn, P.denitrificans during the fermentation, at the aerobic earlier fermentation the most violent of thalline, if the demand of thalline to oxygen can not be met, the growth metabolism of thalline can be had a strong impact on because of the restriction of the supply of oxygen, thus cause thalline to shift to an earlier date self-dissolving, can VB be affected especially 12synthesis.We do certain adjustment to the mixing speed of fermenting process and air flow quantity accordingly, to improve the oxygen supply of fermenting process, mixing and mass transfer situation, by guaranteeing oxygen supply and the oxygen consumption of fermenting process, especially oxygen supply situation is conducive to growth and the VB of fermenting process thalline after improving 12synthesis.
2.3.3 two kinds of different Oxygen supplied level bottom fermentation process specific growth rates and the changing conditions than product synthesis speed
Under the tank of above-mentioned two kinds of different Oxygen supplied levels is criticized, μ and q in its fermenting process pchange see Fig. 5:
As can be seen from Fig. 5 .A, the tank of lower oxygen supply is criticized the specific growth rate when 12h and is just reached maximum value, is 8.89 × 10 -2h -1, and the better tank of oxygen supply is criticized and is reached maximum value when 16h, is 9.13 × 10 -2h -1.In the stage of thalline vigorous growth, the specific growth rate that the better tank of oxygen supply is criticized is always higher than the specific growth rate that lower oxygen supply tank is criticized.After 48h, the thalline specific growth rate of the two is 1 × 10 -2h -1below, specific growth rate is especially close to 0 and after 96h.As can be seen from Fig. 5 .B, in whole fermenting process, the q that the better tank of oxygen supply is criticized palways higher than the q that lower oxygen supply tank is criticized p.Result illustrates again, produces VB in P.denitrificans fermentation 12process in, the restriction of the supply of oxygen can have a strong impact on growth and the VB of thalline 12synthesis, and the Oxygen supplied level improved in fermenting process is advantageously in growth and the VB of fermenting process thalline 12synthesis.
3. conclusion
Have at present and much synthesize VB about P.denitrifican 12report, but mainly just to concentrate on VB 12the research of route of synthesis, and adopt random mutagenesis and genetic engineering means to improve product VB 12level.And produce VB at P.denitrifican 12the report of relevant overall metabolism process control in real time less, be mostly also all confined to the optimization etc. of substratum.The cell oxygen consumption feature inquired in fermenting process macroscopic view metabolic regulation is concentrated P.denitrifican fermentation to be produced to the impact of VB12 herein.
First oxygen supply situation has been investigated to VB by the shake flask test of P.denitrifican 12the impact of synthesis, result shows that oxygen supply is more conducive to VB in order 12synthesis; On this basis, the correlation analysis changed by Fermentation Process of Parameter trend curve, first at 9m 3on pilot scale tank, the supply having found oxygen in fermenting process and the trend guaranteeing OUR ensure VB 12the key factor that synthesis capability improves, therefore by adjusting technique and improving, improves the Oxygen supplied level of fermenting process, makes VB 12output had and significantly improved; Last at technical scale 120m 3fermentor tank amplifies according to same principle, and oxygen supply when being operated by adjusting process and OUR correlation parameter (as Fig. 3 regulates), made the fermentation unit of VB12 bring up to 190 μ g/ml by 125 μ g/ml before process modification, improve 50%.
Embodiment 2:
As shown in Figure 6, automatic control bioreactor device of the present invention comprises the bio-reactor tank body (100) with agitator (104), this device is by the fermentation tank of translator and agitator (104), there is the sensor-based system of multiparameter detection and the instrument controlled and sensing device, with mounting bracket and process pipe system and with industrial computer and the electrical control cabinet composition performing components and parts, wherein said sensor-based system comprises temperature sensor (1), pH sensor (2), dissolved oxygen sensor (3), full tank scale quantity sensor (4), tail gas CO 2interface (5), tail gas O 2interface (6), tachogenerator (7), pressure transmitter (8), froth breaking sensor (9), ventilation flow rate sensor (26), amount of viable cell sensor (10), described parameter refers to temperature, mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
Sensing device unit (101) comprises the dissolved oxygen sensor (3) be arranged on described tank body (100), amount of viable cell sensor (10).
The process pipe system of described band mounting bracket comprises material bottle (22) etc.
The described electrical control cabinet with industrial computer and execution components and parts comprises; The wherein said electrical control cabinet with industrial computer and execution components and parts comprises high precision peristaltic pump (11); Matrix feed supplement electronic scales (12); Precursor or oily electronic scales (13); Soda acid thing electronic scales (14); Recycle pump (25); Magnetic valve (15); D/A converter (18); A/D converter (17); Lower computer (19); Upper computer (20); Rare-earth electric motor (23), ventilation flow rate sensor (26).
Described is according to on-site data gathering and the demand of operation and the requirement of process optimization with industrial computer and the computer software performed in first device housing, carry out on-line parameter collection, offline parameter calculate, the on-line Control of supplemental characteristic record and partial parameters, synchronously transmitted the data of all parameters to upper computer by local area network, select Configuration Language and C language to carry out Programming respectively to upper computer and lower computer, and employ simple redundancy technology when data logging.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (8)

1., to the method using the fermenting process of bioreactor device to be optimized amplification, it is characterized in that, described method comprises step:
A () measures physiological metabolism parameter in the described fermenting process of described bioreactor device, physiological metabolism parameter correlation characteristic or its combination;
Wherein said physiological metabolism parameter is selected from: the meta-bolites of oxygen-supplying amount, dissolved oxygen, oxygen uptake rate, pH, carbon dioxide evolution rate, respiratory quotient, amount of viable cell and cellular form, mensuration or substrate consumption or its combination;
The physiological metabolism parameter of b described physiological metabolism parameter, physiological metabolism parameter correlation characteristic and preset value that step (a) measures by (), physiological metabolism parameter correlation characteristic or its combine and compare; Select the bioreactor device the most close with described preset value; Determine the amplification bioreactor device optimized;
Wherein said physiological metabolism parameter is as described in step (a);
Preset value described in described step (b) is physiological metabolism parameter under canister fermentation condition, physiological metabolism parameter correlation characteristic or its combination;
Wherein, described bioreactor device is not less than between 20 ~ 2000 times relative to the magnification of little tank volume.
2. the method for claim 1, is characterized in that, described bioreactor device is selected from vitamins B 12fermentation unit.
3. the method for claim 1, is characterized in that, described bioreactor device is capacity is 20L--2000m 3fermentor tank.
4. the method for claim 1, it is characterized in that, in described step (a), the method is by detecting multiple process control parameters of bio-reactor to obtain physiological metabolism parameter in described fermenting process, physiological metabolism parameter correlation characteristic or its combination
Wherein said multiple process control parameter is mixing speed, ventilation flow rate, tank pressure, froth breaking, pH, dissolved oxygen concentration, fermented liquid true volume and weight, and feed supplement amount comprises matrix, precursor, oil, soda acid thing, tail gas CO 2and O 2.
5. the method for claim 1, is characterized in that, in the comparison procedure of step (b), also comprises the steps:
The physiological metabolism parameter of i described physiological metabolism parameter, physiological metabolism parameter correlation characteristic and preset value that step (a) measures by (), physiological metabolism parameter correlation characteristic or its combine and compare;
(ii) the physiological metabolism characteristic parameter feature of described bioreactor device, physiological metabolism parameter correlation characteristic or its combination is regulated, make the physiological metabolism characteristic parameter feature of described bioreactor device relative to the difference of described preset value not higher than between 10 ~ 15%, the selected bioreactor device the most close with described preset value;
(iii) the amplification bioreactor device optimized is determined.
6. one kind can be carried out the automatic control bioreactor device of process optimization and data amplification, described automatic control bioreactor device comprises the bio-reactor tank body (100) with agitator (104), and to the instrument that the process control parameter of described bio-reactor detects and controls, it is characterized in that, described tank body (100) is upper to be arranged:
-sensing device unit (101), described sensing device unit (101) comprises the dissolved oxygen sensor (3) be arranged on described tank body (100), amount of viable cell sensor (10);
-physiological metabolism calculation of characteristic parameters the device (20) that is connected with described sensing device unit (101), the process control parameter that described sensing device unit (101) obtains is converted into the physiological metabolism characteristic parameter amplifying basis as data by described physiological metabolism calculation of characteristic parameters device (20).
7. device as claimed in claim 6, it is characterized in that, the volume capacity of described tank body (100) is at 20L--2000m 3between.
8. device as claimed in claim 6, it is characterized in that, described sensing unit (101) also comprises temperature sensor (1), pH sensor (2), full tank scale quantity sensor (4), tail gas CO 2interface (5), tail gas O 2interface (6), tachogenerator (7), pressure transmitter (8), froth breaking sensor (9) or its combination.
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