Software development to fermentation gas analysis using mass spectrometry

Summary Fermentation applications of mass spectrometer (Spectramass PC2000) hampered by interface and software difficulties have been successfully solved. A multiplexed exhaust gas analysis, data acquisition and estimation system was designed and implemented. An specific software was developed in C+ + language programming. This system was tested for lipase production by Candida rugosa in batch fermentation.     

Novel calibration method for mass spectrometers for on-line gas analysis. Set-up for the monitoring of a bacterial fermentation

A set-up procedure for the minimisation of signal noise of a capillary inlet mass spectrometer system, enabling direct use of data without noise filtering or drift correction, is described. A reliable calibration method was developed, involving standard calibration mixtures determined by the extreme vertices design. This novel method was shown to be the most accurate in comparison with a number of commonly used methods. These procedures enabled reliable on-line fermenter headspace gas analysis. With a relatively inexpensive mass spectrometer, monitoring of a fermentation and accurate estimation of oxygen uptake rate, carbon dioxide evolution rate, respiratory quotient and biomass concentration was possible.     

A novel method for the on-line analysis of fermentation broth using a sampling device,microcentrifuge and HPLC

Summary This paper describes a novel system for on-line sampling and analysis of whole broth from a fermenter. The system comprises a steam sterilisable sampling device, high speed microcentrifuge and HPLC. We present results characterising the separation efficiency of the microcentrifuge under different conditions, and illustrate the system with glucose and acetate data from an Escherichia coli fermentation using the system. The on-line measurements have been confirmed by off-line analysis.     

An on-line approach to monitor ethanol fermentation using FTIR spectroscopy

Fermentation process control is currently limited by its inability to measure parameters such as substrate, product, and biomass concentrations rapidly for consistent on-line feedback. Physical and chemical parameters, such as temperature and pH, currently can be obtained on-line using appropriate sensors. However, to obtain information on the concentration of the substrate, product, and biomass, samples must be taken off-line for measurement. With the use of spectroscopic techniques, real-time monitoring of process constituents such as product and substrate is possible. Spectroscopic techniques are rapid and nondestructive, require minimal or no sample preparation, and can be used to simultaneously assess several constituents in complex matrices. The production of ethanol is the largest fermentation process in terms of production volume and economic value as a result of its prominence in the food, agricultural, and fuel industries. This study attempts to develop an on-line ethanol fermentation monitoring technique using Fourier transform infrared (FTIR) spectroscopy with a flow-through ATR capability. Models developed using multivariate statistics, employed to obtain on-line FTIR measurements, were successfully validated by off-line HPLC analysis and spectrophotometry data. Standard errors of prediction (SEP) values of 0.985 g/L (R2 = 0.996), 1.386 g/L (R2 = 0.998), and 0.546 (R2 = 0.972) were obtained for ethanol, glucose, and OD, respectively. This work demonstrates that FTIR spectroscopy could be used for rapid on-line monitoring of fermentation.     

In situ determination of nitrogen fixation in Antarctica using a high sensitivity portable gas chromatograph

A portable gas chromatograph was employed in the Vestfold Hills, Antarctica, during the austral summer of 1979-80 for determining nitrogenase activity of the blue-green alga Nostoc commune Vaucher by the acetylene reduction assay. Acetylene reduction was measured in samples taken along a transect where the vegetation changed with respect to differing topography and water availability. Submerged colonies of Nostoc recorded the highest fixation rates (6.89 nmol C₂H₄. cm^-2 h^-1). Damp mosscyanophyte associations growing on shallow slopes showed moderate rates of acetylene reduction (1.99 nmol C₂H₄. cm^-2 h^-1) whilst the drier vegetation of the steeper terrain was the least active (0.19 nmol C₂H₄. cm^-2 h^-1. The employment of a high sensitivity portable gas chromatograph provided an accurate and reliable method of measuring acetylene reduction.     

Application of the headspace gas chromatographic technique for continuous monitoring of the acetone-butanol-ethanol fermentation

An automatic, electropneumatic gas sampling system has been coupled to a gas chromatograph in order to monitor the acetone-butanol-ethanol fermentation. During calibration, signal responses for individual aqueous solutions of acetone, butanol and ethanol were linear up to 20 g l^?1, and negligible deviations from linearity were observed in aqueous solvent mixtures. Although dissolved salts in the medium increased individual solvent activity coefficients, these remained constant throughout the fermentation. After calibration, this quantitative gas chromatographic method proved suitable for rapid, continuous, accurate and precise monitoring of the fermentation.     

Metabolism analysis and on-line physiological state diagnosis of acetone-butanol fermentation

Fermentation equations for acetone-butanol (AB) were applied in a metabolic analysis of the reaction network under various conditions; that is, at different pHs and a high NADH2 turnover rate using methyl viologen, in a Clostridium acetobutylicum culture. The results disclosed variations in the pattern of rate changes that reflected changes in the physiological state. A linear relationship was found to exist between NADH2 generation and butanol production rate. By coupling an automated measurement system with the fermentation model, on-line estimation of the culture state was accomplished. Based on the AB fermentation model, new parameters were defined for on-line diagnosis of the physiological state and determination of the best timing for amplifying NADH2 generation by the addition of methyl viologen to obtain a high level of butanol productivity. A potential means of achieving optimal control for a high level of solvent production, involving the correlation of certain rates, is proposed.     

Local tumor hyperthermia using a computer-controlled microwave system

A minicomputer-based system was designed to control the microwave (2.45-GHz) power to four local hyperthermia applicators. Errors in temperature measurement, due to electromagnetic field interactions with small thermocouple probes, are minimized by sampling the temperature only when the microwave power is off. The programmable controller can regulate the temperature in tumors in 0.1 °C increments from 30 to 60 °C. This technique reduces temperature differences throughout the tumor at steady state to less than 0.4 °C and prevents skin burns.     

Determination of carbon dioxide evolution rate using on-line gas analysis during dynamic biodegradation experiments

Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (K(L)a) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.     

Bleu cheese flavor production by submerged fermentation with on-line monitoring by gas chromatography

Eight cultures ofPenicillium roqueforti and related species were compared in shake-flask cultures on the basis of aroma.P. decumbens IFO 7091 was chosen as having the aroma closest to real Bleu cheese, and was used for fermentation studies. Gas chromatography was employed to obtain a continuous record of 2-pentanone concentration in the fermentor. Methyl ketones are characteristic of Bleu cheese flavor, and a subjective correlation was observed between the aroma of the fermentor off-gas and the 2-pentanone concentration measured. Cell growth and milk fat utilization increased consistently with increasing agitation and aeration, but 2-pentanone production did not correlate with growth and was maximum at intermediate values of agitation and aeration. The maximum 2-pentanone concentration, 100 ppm, was approximately ten times the concentration in Bleu cheese on a dry solids basis. Production of Bleu cheese flavor by submerged fermentation offers the opportunity for a new commercial value-added product from butterfat.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned     

Computer monitoring, capture and analysis of gas/liquid chromatograph traces

The work described here is effectively the computerization ofa gas/liquid chromatograph of the pen recorder type using aBBC microcomputer. The output from the g. l. c. can be capturedby the computer in accordance with a series of parameters. Thesampling rate can be altered, as can an averaging facility toreduce noise, and a threshold level to eliminate the storageof irrelevent and space-consuming data. The unprocessed readingsare initially stored on the computer's floppy disk, then laterretrieved and cut into smaller sections to allow maximum resolutionfor on-screen analysis. In the main analysis stage of the systemall processing is shown graphically on the computer monitorat all stages. The principal steps in analysis involve the mathematicalmodelling and elimination of the solvent peak, the fixing ofthe retention time for each subsequent peak and its disentanglingfrom any following peaks, and finally the calculation of thearea under each individual peak. Several alternative methodsare made available for disentangling peaks, which can be triedsuccessively on a single peak then each printed out with commentsfor comparison later. All readings and results in table formand screen dumps of all trace images are available via a dotmatrix printer. Received on October 29, 1985; accepted on June 27, 1986     

顶空气相色谱法检测破伤风抗毒素中的甲苯残留量

目的建立检测破伤风抗毒素中甲苯残留量的顶空气相色谱法。方法参照《中国药典》三部(2015版)"残留溶剂测定法",采用Agilent DB-1301毛细管色谱柱(15.0 m×530μm×1.00μm)及氢火焰离子化检测器;顶空进样器各参数分别为:平衡温度70℃,平衡时间30 min,定量环温度80℃,传输线温度90℃,进样量1 m L;气相色谱仪各参数为:进样室温度100℃,柱温箱温度60℃,检测器温度220℃,载气氮气流速5 m L/min,采集时间5min;并对该方法的系统适用性、专属性、线性范围、准确度和精密度、检测限和定量限进行验证及初步应用。结果甲苯保留时间2.429 min,峰面积的RSD为1.9%,以甲苯色谱峰计算得到的理论塔板数N=12 900,甲苯色谱峰与其相邻色谱峰的分离度R=6.03。方法的专属性强,制品中的其他物质不干扰甲苯的出峰;标准曲线的范围为1.0×10-4%~2.0×10-3%,相关系数r大于0.99;加标试验的回收率分别为96.0%~102.9%、100.7%~111.0%,重复性和日间精密度RSD均小于5%。检出限为2.6×10-6%,定量限为1.0×10-5%。测定10批破伤风抗毒素成品和10批破伤风抗毒素原液,其甲苯残留量均低于检出限,远低于规定的限度0.089%。结论该方法简便、快速、准确度、灵敏度高、精密度好,适用于破伤风抗毒素类制品中甲苯残留量的检测。     

Continuous monitoring of fermentation outlet gas using a computer coupled MS

Summary Using a quadrupole mass spectrometer (QMS), we continuously monitored N₂, O₂, CO₂, and ethanol in the outlet gas of a batch fermentor. A computer was used to control the QMS, perform correction calculations and account for interferences between some ion peaks. The ability to continously scan multiple peaks and to use N₂ as an internal standard provided rapid and accurate monitoring of fermentation performance. Over the range of interest, a linear response was obtained for all the compounds measured by the QMS.     

Analysis of a fermentation recycle loop for on-line measurements

Summary Recycle lines have been widely used in on-line analysis of fermentation processes. However, like most tools, many practical considerations can remain overlooked. Upon reflecting on this technique, some of these overlooked considerations have been studied. Because of the importance of a debubbler to a recycle line, an improved design was made and tested which can produce a bubble free high flow rate stream to reduce residence times. The effect of the recycle line on the culture growth, and DO was also investigated and found to be negligible.     

Factors influencing on-line analysis of fermentation alcohols by capillary inlet mass spectrometry

Summary The two most important factors for rapid, precise analysis of fermentation alcohols by on-line mass spectrometry were the real response time of the mass spectrometer and the adsorptive capacity of the tube transporting the off-gas. At this scale (20 L), the bulk liquid mixing time, and the rate of attainment of vapour-liquid equilibrium (VLE) in the fermenter were of lesser importance. No departure from fermenter VLE was observed under extremes of agitation speed, aeration rate, low liquid volume and pH. The total system response time (t_s,90%) to a pulse of ethanol injected directly into the fermenter liquid was about 6 minutes.