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.     

Tandem mass spectrometry for on-line fermentation monitoring

Summary Membrane introduction mass spectrometry has been employed for on-line determination of the major products and volatile metabolites ofBacillus polymyxa fermentation. Samples were introduced into the mass spectrometer via a direct insertion membrane probe in which the aqueous solution flowed past a membrane located in the ion source of the mass spectrometer. Concentrations of the products 2,3-butanediol, acetoin, ethanol and acetic acid in fermentation broth were measured by tandem mass spectrometry after permeation through the membrane and ionization by chemical ionization. External standards were employed for quantification and a large linear response range was available for each of the major products observed. Dissolved CO₂ and O₂, as well as CO₂ in the off gases, were also monitored on-line by mass spectrometry. The use of tandem mass spectrometry has allowed the identification of products that were not previously known to be present in measurable amounts.     

On-line monitoring and control of acetone-butanol fermentation by membrane-sensor mass spectrometry

A mass spectrometry (MS) membrane sensor was developed and applied to on-line product measurement in acetone-butanol fermentation. The sensor facilitated the monitoring of acetone, butanol, ethanol, H₂ and CO₂, and single-compound calibration curves for both acetone and butanol showed a linear relationship between the product concentration and the MS response. However, when an actual fermentation was monitored, the product concentration calculated from the MS response was smaller than the concentration determined by gas chromatography, and the relationship between the response and the product concentration was nonlinear. It was found that large amounts of gases (H₂, CO₂) entering the MS analyzation chamber were causing a ‘space charge effect’, which resulted in an MS response ceiling. The problem could be resolved by reducing the surface area of the sensor membrane. Under some fermentation conditions, a by-product, n-butyl butyrate, was produced, and this interfered with the measurement of butanol due to a peak overlapping effect. However, it was found that this could be compensated for by using an empirical equation. Application of the MS membrane sensor in a fed batch culture of acetone-butanol fermentation resulted in successful control of the butanol concentration.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

On-line monitoring of bioreductions via membrane introduction mass spectrometry

Real-time and on-line continuous monitoring of reactants, intermediates, and final products for dicarbonyl compound bioreduction in a continuous plug flow reactor packed with baker's yeast (Saccharomyces cerevisiae) whole cells immobilized on calcium alginate beads was performed by membrane introduction mass spectrometry (MIMS) via selective ion monitoring.     

Construction and performance of plug-in membrane inlet mass spectrometer for fermentor monitoring

An in situ sterilizable plug-in membrane inlet mass spectrometer for monitoring dissolved gases and volatiles in fermentors was constructed and tested. The design ensured a minimal distance to be traveled by analyte molecules from the bulk of the fermentation broth to the ionization chamber of the mass spectrometer. Apart from the specific cross talk due to overlapping mass peaks from different compounds, we found that carbon dioxide interfered unspecifically with all the mass peaks of other substances, changing them by the same factor. The interference changed slowly with time and could be positive or negative depending on the history of the mass spectrometer. Also, the general sensitivity of the instrument changed slowly with time. These effects can be neglected or corrected for empirically in short-term measurements. When the fermentor was aerated with a three-component gas mixture including carbon dioxide, a rapid change in the partial pressure of carbon dioxide in the gas mixture gave rise to a transient in the signal of a gas whose partial pressure was kept constant. This effect revealed a transient change in the composition of the gas mixture in the bubbles caused by net import or export of carbon dioxide during equilibration with the new gas mixture. An experimental method to determine the effective partial pressures of gases in the bubbles during steady-state transport of carbon dioxide was designed. The plug-in membrane inlet mass spectrometer was tried as a probe for oxygen and ethanol in an oxystatic culture of the yeast Pichia stipitis. We found that it was possible to keep a steady-state concentration of as little as 0.5 muM throughout the lifetime of the culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 535-542, 1997.     

Non-invasive online detection of nitric oxide from plants and some other organisms by mass spectrometry

As nitric oxide (NO) is a key messenger in many organisms, reliable techniques for the detection of NO are essential. Here, it is shown that a combination of membrane inlet mass spectrometry (MIMS) and restriction capillary inlet mass spectrometry (RIMS) allows for the fast, specific, and non-invasive online detection of NO that has been emitted from tissue cultures of diverse organisms, or from whole plants. As an advantage over other NO assays, MIMS/RIMS discriminates nitrogen isotopes and simultaneously measures NO and O(2) (and other gases) from the same sample. MIMS/RIMS technology may thus help to identify the source of gaseous NO in cells, and elucidate the relationship between primary gas metabolism and NO formation. Using RIMS, it is demonstrated that the novel fungicide F 500((R)) triggers NO production in plants.     

Applications of thin layer chromatography,high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin

Summary Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.     

Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases

Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO₂ and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO₂ from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO₂ using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O₂ using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate (¹³C₂-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product ¹³CO₂ (m/z 45), avoiding inaccuracies due to endogenous ¹²CO₂.     

On-line mass spectrometry: membrane inlet sampling

Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O₂ evolution versus O₂ uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H₂ generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.

     

On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration

Monitoring CO2 production in systems, where pH is changing with time is hampered by the chemical behavior and pH-dependent volatility of this compound. In this article, we present the first method where the concentration and production rate of dissolved CO2 can be monitored directly, continuously, and quantitatively under conditions where pH changes rapidly ( approximately 2 units in 15 min). The method corrects membrane inlet mass spectrometry (MIMS) measurements of CO2 for pH dependency using on-line pH analysis and an experimentally established calibration model. It is valid within the pH range of 3.5 to 7, despite pH-dependent calibration constants that vary in a non-linear fashion with more than a factor of 3 in this interval. The method made it possible to determine the carbon dioxide production during Lactococcus lactis fermentations, where pH drops up to 3 units during the fermentation. The accuracy was approximately 5%. We used the method to investigate the effect of initial extracellular pH on carbon dioxide production during anarobic glucose fermentation by non-growing Lactocoocus lactis and demonstrated that the carbon dioxide production rate increases considerably, when the initial pH was increased from 6 to 6.8.     

Neural network predictive control by MIMS monitored 2,3-butanediol fermentation by Klebsiella oxytoca

With the aid of a membrane introduction mass spectrometer (MIMS), the major product 2,3-butanediol (2,3-BDL) as well as the other metabolites from the fermentation carried by Klebsiella oxytoca can be measured on-line simultaneously. A backpropagation neural network (BPN) being recognized with superior mapping ability was applied to this control study. This neural network adaptive control differs from those conventional controls for fermentation systems in which the measurements of cell mass and glucose are not included in the network model. It is only the measured product concentrations from the MIMS that are involved. Oxygen composition was chosen to be the control variable for this fermentation system. Oxygen composition was directly correlated to the measured product concentrations in the controller model. A two-dimensional (number of input nodes by number of data sets) moving window for on-line, dynamic learning of this fermentation system was applied. The input nodes of the network were also properly selected. Number of the training data sets for obtaining better control results was also determined empirically. Two control structures for this 2,3-BDL fermentation are discussed and compared in this work. The effect from adding time delay element to the network controller was also investigated.     

Fast atom bombardment mass spectrometry and tandem mass spectrometry in antibiotics: identification of nucleoside antitumor antibiotic toyocamycin in fermentation broth

The presence of the nucleoside antitumor antibiotic toyocamycin in the fermentation broth was determined by a combination of negative and positive ion fast atom bombardment (FAB) mass spectrometry, high resolution FAB mass spectrometry and mass-analysed ion kinetic energy spectrometry (MIKES). A reasonable limit of detection for toyocamycin in the whole broth was obtained by combining the specificity of mass spectrometry/mass spectrometry (also called tandem mass spectrometry) to FAB. The role played by the fermentation matrix upon the production and the observation of characteristic ions by FAB using xenon atoms was examined. High-performance liquid chromatography (HPLC) and FAB mass spectrometry were used to monitor toyocamycin at all stages of strain development, fermentation and recovery.     

A mass spectrometry membrane probe and practical problems associated with its application in fermentation processes

A sensor-type, multi-compound monitoring system was constructed to measure several substances dissolved in a culture medium by use of a membrane inlet system coupled with a low-cost quadrupole mass spectrometer. The membrane inlet system was shown to have higher sensitivity with an even faster response than a capillary inlet system. The system was able to monitor on-line gaseous (H₂, CO₂ and O₂) and volatile (ethanol, butanol, acetone, acetic acid and ethyl acetate) compounds at concentrations in a range suitable for fermentation process monitoring applications. The effects of temperature, agitation speed and pressure on measurements using the membrane inlet system were investigated. For volatile compound measurements, temperature and pressure had an effect on the response but the agitation speed did not. A high concentration of compounds caused measurement saturation due to the space charge effect, but this could be overcome by reducing the surface area of the membrane. The system was applied to the monitoring of ethanol in a yeast cultivation. Measurements were affected by CO₂ produced during the fermentation, but this could be compensated for by means of a linear empirical equation. The system demonstrated high potentiality for application to the on-line monitoring of multiple compounds in fermentation processes.     

State analysis of fermentation using a mass spectrometer with membrane probe

A new method of continuous process analysis in fermentation using a mass spectrometer (MS) membrane probe is described. A number of samples from industrial fermentations were analyzed for the occurrence of volatiles detectable with a silicone membrane probe connected to a quadrupole MS. In all fermentations, characteristic spectra were observed which were found to change systematically during the batch process. Factor analysis was used to treat the data. The factor scores were compared with the actual product concentrations (antibiotics, toxins, etc.), which were measured using other analytical methods and were found to correlate with them. On-line analysis was also carried out on a fermentation with an MS and an Apple II microcomputer. Direct monitoring of products, which are not directly measurable with the membrane MS probe requires a new calibration each time conditions such as medium composition are changed.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Degradation of pencillin-V in fermentation media

In Industrial production of penicillin there is a noticeable loss of the product through degradation reactions. It is shown that the degradation of penicillin-V, both in a complex and in a chemically defined medium, can be separated into a phosphate-catalyzed conversion of penicillin-V to penicilloic-V acid, overlaid by at least one other reaction in which penicillin V is degraded to as yet unknown products. Parameter values for the phosphatecatalyzed degradation are found to be independent of the type of fermentation medium. The rate of formation of other degradation products of penicillin-V is found to be significantly higher in a complex fermentation medium with corn-steep liquor in a chemically defined medium. (c) 1994 John Wiley & Sons, Inc.     

Intact cell mass spectrometry as a progress tracking tool for batch and fed-batch fermentation processes

Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth. After the optimization of the ICMS method to penicillin V fermentation broth samples, the obtained ICMS data were evaluated by hierarchical cluster analysis or an in-house software solution written especially for ICMS data comparison. Growth stages of a batch and fed-batch fermentation of Penicillium chrysogenum are differentiated by one of those statistical approaches. The application of two matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) instruments in the linear positive ion mode from different vendors demonstrated the universal applicability of the developed ICMS method. The base for a fast and easy-to-use method for monitoring the fermentation progress of P. chrysogenum is created with this ICMS method developed especially for fermentation broth samples.     

Recovery of purified lactonic sophorolipids by spontaneous crystallization during the fermentation of sugarcane molasses with Candida albicans O-13-1

Numerous studies have focused on how to obtain high yield of sophorolipids using low-cost materials as substrates, and there has been various work on the experimental methods for purifying lactonic sophorolipids. These studies have not yet obtained satisfied results in combining a low-cost fermentation process and the purification of lactonic sophorolipids. This study establishes a fed-batch fermentation process of purifying sophorolipids from Candida albicans O-13-1 using low-cost sugarcane molasses as the substrate. In the optimized conditions of this research, using sugarcane molasses as a substrate and product synthesis based on the temperature stage-controlled fermentation, our result indicates that sophorolipids production could reach 108.7 g/L. More importantly, lactonic sophorolipids can crystallize and precipitate during our established fermentation process. The structures and content of sophorolipids separated from the fermentation broth and sophorolipids crystallized in the fermentation broth were analyzed by a scanning electron microscope (SEM) and liquid chromatography–mass spectrometry (LC–MS). The fermentation process produced 90.5 g/L crystallized lactonic sophorolipids with 90.51% purity. This is an energy-saving and low-cost method to obtain such pure lactonic sophorolipids.     

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016