746
Anal. Chem. 1989, 61,746-748
Piezoelectric Crystal Monitor for Carbon Dioxide in Fermentation Processes Orlando Fatibello-Filho,' Jose F. d e Andrade? Ahmad A. Suleiman, a n d George G. Guilbault* Department of Chemistry, University of N e w Orleans, N e w Orleans, Louisiana 70148, and Universal Sensors, Inc., P.O. Box 736, N e w Orleans, Louisiana 70148
A plezoelectrlc quartz crystal coated with tetrakls( hydroxyethyl)ethylenedlamlne (THEED) was found to be a good monltor for CO,, whlch can be used In fermentation processes. The response curve is linear over the concentration range 1.8-16% (v/v) COP. The monitor can be used for more than 15 days without signlflcant loss in sensltlvlty and presented good reverslbllity and reproduclbllity In COPcontlnuous monltorlng.
INTRODUCTION Fermentation is a very complex process, whose success depends upon several conditions, including the complete characteristics of microbial culture, the enrichment process, and other factors that affect biomass and product formation. Direct monitoring is required to provide useful information about the state of the process within a short time period. To maximize the efficiency of the control system, fermentation sensors are used, which are either physical or chemical ( I , 2 ) . The measurement of O2and C 0 2 in the entry and exit gases leads to the determination of oxygen uptake, C 0 2 evolution rate, and the respiration rate of microbial culture. Several techniques are available for the detection and determination of carbon dioxide including infrared spectroscopy ( I ) , mass spectrometry ( I $ ) , gas chromatography ( 7 , 8 ) ,colorimetry (1, 2), electrochemical methods ( I ) , and others (2). However, many of these devices are bulky, time-consuming, not practical, and expensive for routine use. We present here a piezoelectric sensor for the detection and determination of C 0 2 (gas) in the fermentation process, which can be portable, simple, low cost, and fast responsing. The principles and applications of these sensors have been previously reviewed (+lI) and sensors for several atmospheric pollutants were reported. Basically, the frequency of vibration of an oscillating crystal is decreased by the sorption process. The linear relationship between the mass added to the crystal surface and the change in frequency can be derived from the Sauerbrey (12) equation
AF = -(2.3
X
10s)F(AM/A)
where A F is the frequency change (hertz), F is the basic frequency of the quartz crystal (megahertz), AM is the mass of the deposited material (grams), and A is the area of the quartz crystal (centimeters squared). With the surface of the crystal coated with a suitable substance that will selectively adsorb a particular gas, the concentration of that gas can be determined quantitatively. Several compounds were evaluated as coating substrates for the detection of carbon dioxide. Among these tetrakis(hydroxyethy1)ethylenediamine (THEED) showed good sensiOn leave from Universidade Federal de S l o Carlos, Departamento de Quimica, Caixa Postal 676, 13560 S l o Carlos, S.P., Brazil. *On leave from Department of Chemistry, FFCLRP-USP, 14049 Ribeir3o Preto, S.P., Brazil. 0003-2700/89/0361-0746$01 SO/O
tivity, response time, recovery time, as well as good stability. In this paper, we describe a continuous C 0 2sensor utilizing a THEED coating that can be used in the fermentation process. EXPERIMENTAL SECTION Apparatus and Reagents. A schematic diagram of the experimental setup is shown in Figure 1. The piezoelectric crystals used were a 10-MHzAT-cut quartz crystal mounted in a HCG/U holder (Bliley, Erie, PA) with a gold-coated metal electrode on both sides. The instrumentation utilized a PZ 105 gas phase detector (UniversalSensors, New Orleans, LA) which is equipped with oscillator circuitry, frequency counter, and a dual-crystal chamber and can be interfaced to a computer or recorder. The response was determined by monitoring the difference in frequency changes between reference (uncoated) and sensing (coated) crystals and was read from either the counter or recorder. Temperature monitoring was made with a thermistor (Thermometrics, Edison, NJ) coupled to a multimeter (Honeywell Digitest Model 33R). Air was passed through a water bubbler in the humidity study and the relative humidity was monitored with a digital hygrometer (Model HI8064,Cole-Palmer Ind. Co., Chicago, IL). The various concentrations of carbon dioxide were prepared by dilution of high-purity C 0 2 gas (Matheson Co., East Rutherford, NJ) with air and were verified by a modified photometric method (13). All the reagents used were of analytical grade. Preparation of Coating. The best coating material found was tetrakis(hydroxyethy1)ethylenediamine (THEED) (Alltech Associates, Deerfield, IL) dissolved in acetone. The crystals were coated by addition of known volumes of THEED solutions,onto both electrodesof the piezoelectric crystal using a microsyringe and spreading the solution with a glass rod. The coated crystal was placed in an oven, at 60 "C, for 2 h to allow the solvent to evaporate, leaving a film of coating on the surface. The amount of coating applied to the crystal was determined by monitoring the frequency change of the crystal and calculated from the Sauerbrey equation. RESULTS AND DISCUSSION Investigation of Coating Materials. Several substances were screened as possible substrates for C02including amines, anilines, substituted amines, and different gas chromatography stationary phases containing amino groups. Among those tested, tetrakis(hydroxyethy1)ethylenediamine (THEED) and 7,10-dioxa-3,4-diaza-1,5,12,16-hexadecatetrol (14)showed the highest sensitivities. These compounds were evaluated with respect to sensitivity, stability, selectivity, and reproducibility. Table I shows a partial list of these compounds studied and their responses. The THEED-coated crystal exhibited the best analytical characteristics and subsequent studies were conducted on this diamine coating. Effect of the Amount of Coating. A set of experiments was performed to establish the optimum amount of THEED to be applied to the crystal. It is evident from the plots shown in Figure 2 that A F increases as the amount of coating increases, a t two different sampling times, because of an increased probability of interaction between the coating material and the GO, molecules. However, at high amounts of coating, it was difficult to obtain a uniform dry film and thus poor 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989 4
Table I. Initial Evaluation of Possible Coatings'
n
coating
AIR
AF,b Hz
comments
430
good stability and reproducibility poor reproducibility
THEED
7,10-dioxa-3,4-diaza-1,5,12,16-hex-210 ;~~
adecatetrol 1,8-diamino-p-menthane
@
NflN'-bis(2-hydroxyethy1)ethylene-
diamine tetraethylenepentamine
c02 Flgure 1. Schematic representation of the experimental setup: 1,
control valve; 2, pressure gauge; 3, silica gel column; 4, humidity source: 5, flow meter; 6, four-way valve; 7, hygrometer; 8, molecular sieve column; 9, test cell; 10, temperature control; 11, US PZ 105 gas phase detector; 12, recorder.
300 a h
N
100
I Y 0
triethanolamine GP 22A Carbowax 1000 monostearate tri-n-octylamine diphenylacetylene 1-naphthylamine aniline benzylamine dipropylamine N-(1-naphthy1)ethylenediamine dihydrochloride 1,l-diphenylethylene
900
P
cn
poor stability poor stability
35 30
0 0 0 0
ramine
a 700
a OC.
500
40
56
64
N
72
I
;1000
Y
Amount of Coating(pg1
Ccq, 10% (vlv); temperature, 25 "C;flow rate, 100 mL-min-'; (a) sampling time 60 s, (b) sampling time 30 s.
I
N
I Y
0
P M Q
a
I
15001 CI
Cco2= 10% (v/v); flow rate = 100 mL-mid; temperature = 25 bAverarrefor n = 5.
n
48
Flgrrre 2. Effect of coating amount on the response:
K 0,
10 10
40
1,4,7,10-tetramethyltriethylenetit-
Q
20 20 15 13 13
poor reproducibility good stability and reproducibility good stability and reproducibility poor stability good stability and reproducibility poor sensitivity poor stability poor stability poor stability poor stability poor stability poor stability
60
1,l-dimethylhydrazine monoethanolamine Gp-111 Ucon
v)
140 85 80
Nfl-diethyl-p-phenylenediamine
Q C
747
500
I ~
10
20
30
40
50
Temperature("C) Figure 4. Effect of temperature on the response: Cco,, 50% (v/v); flow rate, 100 mL-min-'; amount of coating, 65 pg; sampling time, 30
5001
S.
Effect of Cell Temperature. Temperature is one of the
i most important parameters that influence the complex series
0
50
100
150
200
250
Flow Rate(ml/min) Figure 3. Effect of flow rate on sensitivity: Cm2, 50% (vlv); temperature, 25
OC;
amount of coating, 65 pg; sampling time, 30 s.
precision was observed. It was determined that, for both good response and reproducibility, 50-55 pg of coating is sufficient. Flow Rate and Reversibility Study. Figure 3 shows the effect of flow rate on the sensitivity of the C02detector. The results indicate that the response is practically constant in the flow rate range of 60-230 mL.min-'. Subsequent studies were carried out at a flow rate of 100 rnL.min-' for convenient dilution. Reversibility is the ability of the sensor to reattain the base line frequency after it has been exposed to a gas sample, and is one of the most important factors in evaluating a piezoelectric sensor. Complete reversibility was obtained in 2-5 minutes at sampling times of 3-30 s for a 10% (v/v) COz sample and varied from 2 to 5 min in the linear portion of the calibration curve at a sampling time of 5 s indicating also that reversibility is dependent on analyte concentration.
of chemical reactions which govern any fermentation process. The temperature of the exit gases is a function of the specific fermentation process; hence the effect of temperature on the response of COz sensor was studied. The carrier gas (air) was passed through a coil placed in a controlled thermostated bath. The response of the COz sensor in the temperature range of 20-40 "C decreased by 6% only (Figure 4). This indicates that the adsorption of C 0 2 on the surface of the coating and its desorption in this temperature interval were not seriously affected by temperature. In practical applications this eliminates the need for temperature control giving this sensor an additional advantage. It is evident also that the sensor can be used at higher temperatures, however with lower sensitivity. Effect of Interferents. Interferences from various substances that would be expected to exist in a fermentation process (1,2,15) as potential interferents were tested. Water, ethanol, acetic acid, acetaldehyde, 1-butyric acid, propionic acid, acetoin (3-hydroxy-2-butanol), methanol, and acetone were studied by continuous process. Other trace gases, including SOz,HzS, CSz, CH,-SH, CHI, and NH3,were studied
748
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
800
I
a
600 n
N
I A . I rn 4001 400
\B
Y
C ',
0 Q
rn
100
o!. 0
i
I
10
20
C02
30
Concentration(%)
Response of the sensor to COPat three different condiins: temperature, 25 O C ; flow rate, 100 mL-min-'; coating amount, 52 pg; (a) without sieve 3A (0),(b) with sieve 3A (V),(c)with sieve 3A and 70% humidity (+). These points represent the average value of six measurements. Flgwe 5.
by the syringe dilution method. Water, ethanol, acetaldehyde, and acetone caused high-frequency changes in comparison with the same concentration of C02. Several approaches were evaluated to eliminate the effects of interferences including the use of hydrophobic membranes, desiccants, and molecular sieve. These interferents were eliminated by passing the sampling stream through a column of molecular sieve 3A. Although about 15% of COZ was absorbed by the sieve, a working calibration curve for the determination of COz could be constructed at relative humidity levels of 2-70%. Figure 5 shows the calibration curve obtained at the 70% humidity level. In addition, it was determined that 50 g of molecular sieve 3A was sufficient to eliminate the effect of moisture at this humidity level during 8 h of continuous work. The molecular sieve 3A was easily reactivated by heating and reused without significant loss of its adsorption efficiency. Calibration Curves, Lifetime, and Continuous Monitoring. The effect of the sampling times (3-30 s) on the response of the COz sensor was initially studied a t the concentration range 0-2370 (v/v) C 0 2 . It was concluded that the linear range of the calibration curve decreased as the sampling time increased due to saturation. Figure 5 shows the response curves for COz obtained at a response time of 5 s. The plots were linear over the concentration range 1.8-16% (v/v) COz and exhibited a standard deviation of about 2.5 Hz throughout. Curve a was obtained in dry gas without molecular sieve traps, while curves b and c were obtained in a dried and humidified gas, respectively, with a molecular sieve trap. The recovery time, as indicated eadier, depends on the C02 Concentration, it varied from 2 to 5 min a t 5 s sampling time. The good linearities (r2 = 0.990, curve a; r2 = 0.992, curve b; and 9= 0.994, curve c) and sensitivities of 0.0021, 0.0025, and 0.0025 Hz-g-' found, respectively, show that the use of molecular sieve 3A trap does not affect the quantitative determination of COP. The useful lifetime of a single coating was about 15 days, provided that the crystal was stored in a desiccator when not in use. A slight decrease of about 10% in the response was observed after the 15th day.
1 2 3 Time(hours1 Flgure 6. Continuous monitoring of C02: COP concentration, (a) 4.2, (b) 8.1, (c) 10, and (d) 12% (vlv); temperature, 25 O C , flow rate, 100 mL-min-'; coating amount, 52 pg.
0
Figure 6 shows the performance of the sensor in a continuous monitoring mode study for four different C 0 2 concentrations (4.2,8.1, 10, and 12% (v/v)). The stability was tested by exposing the sensor to a known concentration for 25 min after which the concentration of COz was varied before complete reversibility was achieved. It is evident that the proposed sensor is then capable of real time monitoring of C 0 2 concentration with good reproducibility. It is envisioned that an alarm could be incorporated to alert to dangerous levels of C 0 2 present.
CONCLUSIONS The piezoelectric sensor coated with THEED presented here possesses good selectivity, sensitivity, reversibility, and reproducibility for the continuous monitoring of C 0 2 in the fermentation process. The main advantage of this sensor over alternative methods of continuous monitoring is simplicity, low cost, and fast response. Registry No. THEED, 140-07-8;COz, 124-38-9. LITERATURE CITED (1) Wang, D. C.; Cooney, C. L.; Demain, A. L.; Dunhill, P.; Humphrey, A. E.; Lilly, M. D. Fermentation & Enzyme Technology; Wiley: New York, 1979;p 212. (2) Stanbury, P. F.; Whitaker, A. Rinciples of Fermentation Technology; Pergamon Press: New York, 1984; p 145. (3) Lloyd, D.; James, C. J. FEMS Microbial. Lett. 1987, 42(1),27. (4) Szilagyi, J.; Bohatka, S.;Langer, G.; Santha, Gy.; Seres, P. Repr.Eur. Congr. Biofechnoi. 3rd 1984, 3 , 609. (5) Buckland, B.; Brix, T.; Fasted, H.; Gbewonyo, K.; Hunt, G.; Jaln, D. 6io.lTechnology 1985, 3(1 l), 987. (6) Heinzle, E.; Moes, J.; Griot, M.; Kramer, H.; Dunn, 1.; Bourne, J. R. Anal. Chim. Acta 1984, 763,219. (7) Ross, L. F. J. Chromatogr. 1987, 474(2), 405. (8) Meyer, C. L.; McLaughlin, J. K.; Papoutsakis, E. T. Ann. N . Y . Acad. Sci. 1986, 469 (Biochem. Eng. 4),350. (9)Guilbault, G. G.; Jordan, J. M. CRC Crit. Rev. Anal. Chem. 1988, 79,
1.
(IO) Alder, J. F.; McCallum, J. J. Analysf 1983, 708, 1169. (11) Guilbault. G. G. Ion-Sei. Electrode Rev. 1980, 2,3. (12) Sauerbrey, G. 2. 2.Phys. 1959, 755, 206. (13) Maxon, W. D.; Johnson, M. J. Anal. Chem. 1952, 2 4 , 1541. (14) Jordan, J. M. UNO MSc. Thesis, University of New Orleans, New Orleans, LA, 1985;pp 64-66. (15) Gastineau, C. F.; Darby, W. J.: Turner, T. B. Fermented Food Beverages in Nutrition; Academic Press: New York, 1979; pp 507-517.
RECEIVED for review October 4, 1988. Accepted December 7, 1988. The scholarships furnished by CNPq (process # 20.0749/86-QU-2 and 20.0832/86.7-QU-2) and FAPESP (process # 86/2196-0) to Brazilian authors are gratefully acknowledged. The financial assistance of the National Science Foundation (SBIR Grant 151-8861450) is gratefully acknowledged.
