On-line monitoring of IPTG induction for recombinant protein production using an automatic pH control signal

The response of IPTG induction was investigated through the monitoring of the alkali consumption rate and buffer capacity during the cultivation of recombinantE. coli BL21(DE3) harboring the plasmid pRSET-LacZ under the control oflac promoter. The rate of alkali consumption increased along with cell growth, but declined suddenly after approximately 0.2 h of IPTG induction. The buffer capacity also declined after 0.9 h of IPTG induction. The profile of buffer capacity seems to correlate with the level of acetate production. The IPTG response was monitored only when introduced into the mid-exponential phase of bacterial cell growth. The minimum concentration of IPTG for induction, which was found out to be 0.1 mM, can also be monitored on-line andin-situ. Therefore, the on-line monitoring of alkali consumption rate and buffer capacity can be an indicator of the metabolic shift initiated by IPTG supplement, as well as for the physiological state of cell growth.     

An automatic,on-line glucose analyzer for feed-back control of fed-batch growth of Escherichia coli

Summary A computer-assisted on-line glucose analyzer was developed for feed-back control of cell growth. Using this system the glucose consumption rate for Escherichia coli was determined to be linear during batch culture at 0.37 g/hr. On-line feed-back control of glucose concentration at 1.5±0.5 g/L was used with fed-batch cultures to produce 31.2 g dry weight of E. coli cells/L in 12 h.     

On-line control of light intensity in a microalgal bioreactor using a novel automatic system

The influence of light intensity upon biomass and fatty acid productivity by the microalga Pavlova lutheri was experimentally studied using a novel device. This device was designed to automatically adjust light intensity in a photobioreactor: it takes on-line measurements of biomass concentration, and was successfully tested to implement a feedback control of light based on the growth rate variation. Using said device, batch and semicontinuous cultures of P. lutheri were maintained at maximum growth rates and biomass productivities – hence avoiding photoinhibition, and consequent waste of radiant energy. Several cultures were run with said device, and their performances were compared with those of control cultures submitted to constant light intensity; the biomass levels attained, as well as the yields of eicosapentaenoic and docosahexaenoic acids were calculated – and were consistently higher than those of their uncontrolled counterpart.     

On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter

Summary On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter was carried out. Effects on the turbidity measurement of operating parameters such as agitation speed, aeration rate, and the concentration of antifoam agent were investigated. Correlations between the on-line-measured turbidity and the off-line dry cell weight concentration were made at various operating conditions. Using the correlations the cell concentration was successfully estimated in a range of up to 8 g/L in batch cultures. The on-line monitoring and regulation of the cell concentration in a range of up to 35 g/L were also satisfactory in continuous and turbidostat cultures with cell recycle.     

On-line monitoring of a two-stage anaerobic digestion process using a BOD analyzer

A computer-controlled biochemical oxygen demand (BOD) analyzer has been developed for fast estimation of biochemical oxygen demand (BODst) automatically with the purpose of on-line monitoring of a process for conversion of biomass under field conditions. The instrument was tested by on-line monitoring of the connecting stream between two stages of a two-stage anaerobic process in laboratory scale. In the first stage, hydrolysis of sugar beet leaves and its conversion into volatile fatty acids and other low molecular weight substrates took place. The effluent from the first reactor was used as a feed stream to the second stage, i.e. an anaerobic contact reactor. The feed stream was sampled intermittently, diluted and analyzed by the BOD analyzer automatically in order to estimate the organic loading rate to the reactor. The results from this study demonstrated that the BOD analyzer could be a stand-alone and promising sensor device for rapid on-line monitoring of easily biodegradable organic substances in biological treatment processes.     

The control of glucose concentration during yeast fed-batch cultivation using a fast measurement complemented by an extended Kalman filter

In this contribution results are presented from the control of glucose during a yeast fed-batch cultivation. For glucose measurements a special flow injection analysis (FIA) system was employed, which uses a glucose oxidase solution instead of immobilized enzymes. To avoid the large delay time caused by probing systems samples containing cells, i.e., samples containing the ordinary culture broth, are injected into the FIA system. Based on a special evaluation method the glucose concentration can be measured with a delay time of about 60 s. Employing an extended Kalman filter, the biomass, the glucose concentration as well as the w_max (Monod model) are estimated. Based on the estimation a feed forward and a PI-control with a set point of 0.5 g/l was carried out. The mean deviation of the set point and the estimated value as well as the set point and the measured value were 0.05 and 0.11 g/l respectively for a control period of 8 h producing a cell dry mass of more than 6 g/l.     

Analysis of pH change and an automatic pH control with A new function: On-line estimation of acetic acid

The pH of microbial culture medium was calculated from equations of equilibrium, material balances for ionic components and electro-neutrality theory. Ammonium ion consumption and Acetic acid production are found out to be the major contributors the alteration of the pH as well as the buffer capacity of the medium. By measuring the buffer capacity on-line, levels of acetic acid were estimated by a software sensor using pH signal in a fermentation process of E.coli growing in a minimal medium. The measured values of acetic acid showed good correlation to those of estimated by the software sensor.     

On‐line yeast propagation process monitoring and control using an intelligent automatic control system

The monitoring and control of bioprocesses is a challenging task. This applies particularly if the actions to the process have to be carried out in real‐time. This work presents a system for on‐line monitoring and control of batch yeast propagation under limiting conditions based on a virtual plant operator, which uses the concept of intelligent control algorithms by means of fuzzy logic theory. Process information is provided on‐line using a sensor array comprising the measurement of OD, operating temperature, pressure, density, dissolved oxygen, and pH value. In this context practical problems arising through on‐line sensing and signal processing are addressed. The preprocessed sensor data are fed to a neural network for on‐line biomass estimation. The root mean squared error of prediction is 4 × 10⁶ cells/mL. The proposed system then triggers temperature and aeration by usage of a temperature dependent metabolic growth model and sensor data. The deviation of the predicted biomass from that of the reference trajectory as modeled by the metabolic growth model and its temporal derivative are used as inputs for the fuzzy temperature controller. The inputs used by the fuzzy aeration controller are the deviation of measured extract from that of the reference trajectory, the predicted cell count, and the dissolved oxygen concentration. The fuzzy‐based expert system allows to provide the desired yeast cell concentration of 100–120 × 10⁶ cells/mL at a minimum residual extract limit of 6.0 g/100 g at the required point of time. Thus, a dynamic adjustment of the propagation process to the overall production schedule is possible in order to produce the required amount of biomass at the right time.     

Detection and automatic control of ammonium ion concentration in miceobial culture with an ammonium ion selective electrode

An ammonium ion selective electrode (AISE) had a membrane of polyvinyl chloride in which the antibodies nonactin and monacin were embedded. The detection range was 0.1–200 mM. The step response was 90% in 20 seconds. The output of the AISE increased 6% with a 1°C rise temperature. The output of the AISE was constant between pH 4–7. The selectively coefficient of potassium ion was 0.158 and hence its interferring effect must be considered. The selectivity coeficcients of other cations were small enough to be negligible. Throughout a batch culture of Escherichia coli, values calculated by subtrating (selectivity coefficient) × (potassium ion concentration) from the detected output of the AISE agreed with actual concentrations of ammonium ion. An automatic. constant-value, feebdack control system of ammonium ion concentration was attempted by on-off controlled supply of solution containing both ammonium and potassium ions, the proportion of whose concentration was made equal to the proportion of their average volumetric consumption rates by a microorganism in batch culture. By this control system, ammonium ion concentrations in culture supernatants of fed-batch cultures of Escherichia coli and Saccharomyces cerevisiae could be maintained vitrually at constant levels (5±0.8 mM for the cultivation of E. coli and 50±5 M for the cultivation of S. cerevisiae).     

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 279-286, 1997.     

Elaboration of a semiautomated enzymatic analyzer of glucose for the control of biosynthesis of antibiotics

The results of developing a semiautomatic apparatus with oxygen detection for enzymatic control of glucose concentration are presented. The design of a glucose sensitive electrode is based on an oxygen probe and a membrane with immobilized glucose oxidase. Materials for the probe were chosen and the operating conditions for measuring temperature, pH, linear agitation velocity and other parameters were optimized. The semiautomatic analyzer was constructed and its main characteristics were studied. The results of the apparatus testing during biosynthesis of various antibiotics are presented. It was shown that the required glucose concentration in the cultivation medium was provided for any specific circumstances in relation to the carbohydrate source.     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

On-line membrane inlet mass spectrometry for feed-back control of precursor concentration in penicillin fermentation

Summary A sampling system which enables on-line measurements of the precursor phenoxyacetic acid (POAA) in penicillin fermentation by membrane inlet mass spectrometry is presented and its capacity for feed-back regulation of POAA to a low predefined concentration in a penicillin-V fermentation over 150 hours is demonstrated. The system measures alternately filtered sample and standard solution in a measuring cycle which is shorter than the response time of membrane inlet mass spectrometry (MIMS) but sufficiently long to decide whether the concentration of POAA in the sample is higher or lower than in the standard solution at set point concentration. The decision is used for on-off regulation of the addition of POAA.     

Monitoring and control of methanol concentration during polysaccharide fermentation using an on-line methanol sensor

Combining principles of membrane separation and semiconductor gas sensor technology, we constructed a methanol sensor to follow methanol concentrations on-line. A length of silicone tubing allowed for mass transfer of methanol from the fermentation medium to a carrier gas which then flowed over a semiconductor gas sensor for detection. The sterilizable sensor demonstrated excellent ability in following methanol concentrations during the batch production of a polysaccharide by the organism Methylomonas mucosa, even as the fermentation broth became increasingly viscous. During fed-batch control by feeding methanol to the fermentation to maintain setpoint methanol levels, a drift in the sensor signal was noted and quantified. A drift factor was determined which, after it was incorporated into the calibration calculations, improved methanol concentration control greatly. Methanol concentration was held constant over a range of set point concentrations during fedbatch fermentations.