Biotechnol. Rog. 1994, 10, 284-290
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On-Line Exhaust Gas Analysis of Volatiles in Fermentation Using Mass Spectrometry A. Oeggerlit and E. Heinzle' Chemical Engineering Department, Swiss Federal Institute of Technology (ETH), CH-8092 Ziuich, Switzerland
Liquid-phase concentrations of volatile compounds were determined on-line via gasphase analysis using mass spectrometry. It has been shown theoretically as well as experimentally that almost every volatile compound commonly produced by microorganisms can be measured by the method presented, if its partial pressure in the fermentor off-gas exceeds 1 pbar. This has been shown in a baker's yeast as well as a Bacillus subtilis culture. The experimental results also indicated that these measurements are not controlled by mass-transfer kinetics. A dimensionless parameter, E, allows us to check whether the analysis is determined by thermodynamics (Henry coefficient) or by mass-transfer rates.
Introduction As the market for biologically manufactured products keeps growing, production factors are becoming very important. One major problem in controlling these factors is the lack of suitable, long-term reliable and universal on-line sensors, as all state estimation and control algorithms rely on the quality of on-line measurements. Flavors and fragrances are extremely important for the food, feed, cosmetic, chemical, and pharmaceutical industries (Janssens et al., 1992). With the increasing preference of consumers for natural food additivies, microbial production routes are becoming very popular for the production of these high-value compounds. Probably the oldest microbial processes dealing with flavors are alcoholic fermentations, where volatile compounds strongly influence the strength and taste of the end product (Pons and Wild, 1991; Ueda et al., 1991). Consequently, information about their concentration and their rate of change with time during fermentation is strongly needed to control final product quality. However, the production kinetics of these volatiles are often difficult to obtain online because most of them are present as traces. Some researchers are using membrane sensors to keep track of the trajectories. The membrane probes are usually immersed in the liquid phase and flushed by a carrier gas on the permeate side. The gas mixture is analyzed by gas afterward either by a semiconductor device (SD), chromatography (GC),or by mass spectrometry (MS). Lee et al. (1981) and Axelsson et al. (1988) used an SD quite successfully when only one major volatile was present (ethanol). However, since SDs are nonspecific sensors, they fail in measuring individual compounds from a mixture. Pons et al. (1992) reported mixture analysis results using both a GC and an MS detector. They were able to follow different volatiles (e.g., acetaldehyde, ethyl acetate, up to hexanol). They finally selected GC as the detector of choice, mainly because of its superiority in complex mixture analysis. The analysis interval of up to 20 min was sufficient for monitoring beer and wine production. In contrast, mass spectrometers are able to
* Author to whom all correspondence should be addressed (
[email protected]). + Present address: Department of Chemical Engineering, The University of Queensland, Brisbane 4072, Australia. 8756-7938/94/3010-0284$04.50/0
analyze gas mixtures in a couple of seconds (typically 0.5 s per mass fragment). Mass spectrometric analysis of complex mixtures using electron impact ionization is difficult because of the fragmentation of ionized molecules. A possible solution is the application of soft ionization techniques (Heinzle, 1992) or MS/MS techniques (Hayward et al., 1990). Otherwise, such mixtures have to be separated by gas chromatography prior to the MS analysis (e.g., aroma compounds in wine). Membrane probes give much different results if they are connected directly to the high vacuum of a mass spectrometer (Griot et al., 1987; Heinzle, 1992). The selectivity of the membrane then can cause an enrichment of the desired compounds, increasing its detection limits. In this case, the sensitivity and speed of analysis are determined by the diffusion rates of the individual compounds. This method is, however, less acceptable in industry because of the apprehended risk of infection, multiplexing problems, and the limited distances allowed between MS and reactors (Bohatka, 1985). The sensitivity of the membrane probes, especially when operated in a flow injection mode, was shown to be very high (Lister et al., 1989). In this article, only methods allowing remote gas analysis are discussed further.
Carrier Gas Membrane Probe or Direct Gas Analysis? In gassed reactors, the fermentor off-gas can be analyzed directly, whereas in the rare case of nongassed reactors only a membrane probe can be applied. From a thermodynamic point of view, both methods are equal. With both techniques, the highest concentration reached is the one at the equilibrium between the liquid phase on one hand and the gas phase on the other hand (equal chemical potential). With both methods, a compound must first migrate from the liquid phase to the gas phase-only the different resistances influence the rate at which the equilibrium state is attained. Using the membrane, a compound must first diffuse through the stationary layer in front of the membrane, subsequently permeate through the membrane itself, and diffuse through the stationary layer behind the membrane. Usually the second step will be rate-limiting.
0 1994 American Chemical Society and American Institute of Chemical Engineers
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Biotechnol. Rog., 1994, Vol. 10, No. 3
Table 1. Comparison of Major Properties of Carrier Gas Membrane Sensor and Direct Exhaust Gas Analysis direct off-gas analysis carrier gas membrane probe
increased infection risk probe needs to withstand sterilization process surface properties can be strongly affected during fermentation, leading to changed diffusion kinetics permeation of one compound can be influenced severely by all other species calibration procedure difficult to do because of nonlinearities introduced by the membrane recalibration during run not possible time delay depends only on carrier gas flow rate in transfer line inhomogeneities in liquid phase important because of local sampling foam and aerosol formation do not disturb the measurement
Direct gas analysis could also be mass-transfer-controlled, if diffusion through the stationary boundary layers is slow enough. Additionally, there may be delays caused by mixing in the reactor head space and transport to the analyzer through the transfer lines. Depending on the membrane properties (material, thickness, and pore size) and inert gas flow rate, the partial pressure achieved in the inert gas stream under membrane-transfer limitation will always be equal or less than the one achieved in the exhaust gas, Table 1summarizes characteristic properties of both methods, including practical considerations.
MS Data Handling and Calibration Mass spectrometers basically measure ion currents on different masses (mlz). It is a characteristic of this technique that a small background ion current is always measured (even when no gas of interest is fed to the analyzer). Therefore, background calibrations are necessary, particularly at low intensities. Finally, the changes in the ion current intensities are linearly related to the gas concentration. Modern instruments perform background calibration and subtraction automatically and offer either raw data (ion currents) or estimated final concentrations. The measured ion current Imi on mass mi (mlz)represents the sum of all the compounds contributing to a mass fragment. If we assign the concentrations cl-cq to the q different species sj, this can be mathematically expressed as Im,
= fmi,sfl + fmi,s?2 + .*.+ fmi,s:q
(1)
where fmi,sj represents the calibration factor for compound s j on mass mi. By measuring ion currents on several masses, say ml-mp, the following matrix equation is obtained:
no infection risk not an issue mass-transfer coefficienta can vary due to liquid properties, stirring rate, and gassing rate partial pressure (fugacity) of one compound is much less influenced by other species calibration is easier (linear response) recalibration during run possible with calibration gas time delay depends on residence time of exhaust gas in reactor head space and also on gas flow rate in transfer line integral sample of gassed reactor region is obtained foam must not enter the MS; aerosols can introduce errors
measurements than analyzed compounds usually are available (p > q ) , leading to a regression problem. This solution will normally be more accurate since measurement noise will be damped. The simplest least-squares solution is given by
C = (F*F)-'FTI
(4)
Calibration can be performed by independently determining each set of factors [ f m l a j , fm*gj, ..., fmpfjlTfrom a pure solution of compound sj. Another possibility consists of using different calibration mixtures, as described by Babalievski (1987). To understand which influences are lumped into an individual calibration factor f m i d j ,let's recall a general gasphase mass balance at constant temperature, pressure, and volume for compound si under constant pressure P in the gas-phase volume, VG: accumulation = flow in
- flow out - transfer from liquid phase
The meanings of the variables and their dimensions are as follows: P (bar), total pressure; R (bar-L mol-' K-9, general gas constant (value = 0.083 14);T (K),temperature; y, molar fraction in the gas phase; CL (mol L-9, concentration in the liquid phase; G (mol s-l), total molar gas flow rate; kLa (s-l)?mass-transfer coefficient; VGand VL (L), gas and liquid volumes; indices 0 and 1, input and output streams, respectively; G and L, gas and liquid phases, respectively; *, equilibrium value as given in eq 6. Usually the inlet gas does not contain any volatile compounds. Therefore, the concentration yo is 0. Gasphase mixing is generally fast enough that the accumulation term can be neglected (e.g., with an aeration rate of 1w m and VG = 0.3V~,the gas residence time 7 G yields 18 SI. If we assume equilibrium between the liquid and gas phases at the interfacial layer, Henry's law can be applied:
After the gas-phase molar fraction yl,sj is measured on a unique mass, yielding the ion current ImiJj,
I t can be abbreviated
I=FC
(7)
(3)
with f and being column vectors of dimension (p X 1) and (q X l),respectively,and F representing the calibration factor matrix of dimension (p X q). In order to solve eq 3, the number of measured ion currents needs to be equal to or larger than the number of analyzed compounds QI 1 q). In the case where p = q, a unique solution is possible. However, more ion current
eq 5 is simplified to
rearranged to yield the liquid-phase concentration,
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Table 2. Increase in Partial Pressure, p m d o ,of Acetoin in e Typical Culture Medium Compared to a Pure Water Solution.
mass ImiAc (A)/ fmiAc Pm& (m/& CAc (g L-') (g L-l A-1) (%I 43 6.568 X 10-10 1.522 X log 20.5 54 3.113 X 3.212 X loll 22.4 86 3.349 x lo-" 2.986 X 1010 18.9 88 5.607 X 1.783 X 10" 23.1 Acetoin concentration, 4 g L-1; aeration rate, 2.4 L min-l (0.3 wm); stirrer speed, 400 rpm; temperature, 30 "C;SEM voltage, 2000 V.
and lumped into the calibration factor fmipj at constant conditions GI, T, kLhj, Hsj, P, and S m i a j ,
The additional variables and their dimensions are as follows: HsJ(bar-Lmol-'), Henry coefficient of compound S, at temperature T;p , (bar), partial pressure of compound s, at temperature T; SmtaJ (A-l), sensitivity of the MS detector on mass (mlz)m,for speciess,; ImtaJ(A),ion current of compound s, on mass m,.
Most Important Measurement Factors and Checks for Mass-Transfer Limitation. Equations 9 and 10 explain the dependency of the calibration factor on both MS-specific characteristics (Smta>as well as thermodynamic properties (HsJ)and operating conditions (P,T,GI, VL, and kLa, ). This requires controlled (preferably constant) conditions during both calibration and fermentation runs. Most of the variables are easily kept constant (P, T, GI, VL, and Sm,,sJ). In a fed-batch situation, the varying VL has to be considered. The two variables to be examined further are the Henry coefficient HsJand the mass-transfer coefficient kLas . H, is influenced by the totaf salt concentration, as well as otker components in the broth, as has been studied in detail for dissolved oxygen by Schumpe (1985). Table 2 shows an average difference of 21.2 % of theacetoin partial pressure between a pure water solution on one hand and a typical Bacillus subtilis nutrient mixture on the other. This strongly implies that the calibration should be carried out with culture medium. In order to rely on a constant calibration factor, the total salt concentration needs to be constant as well. This requirement is usually fulfilled, at least in low cell density cultures (e.g.,
