Biotechnol. Prog. 1994, 10, 32-38
32
Fermentation Process Monitoring through Measurement of Aerosol Release Y u-Li Huang, Klaus Willeke,* Arvydas Juozaitis,t and Jean Donnelly Aerosol Research Laboratory, Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267-0056
Andrea Leeson and Robert Wyza Battelle Memorial Institute, Columbus, Ohio 43201-2693
Fermentation involves many complex biological processes, some of which can be difficult to monitor. In this study, aerosol measurement was explored as an additional technique for monitoring a batch aerobic fermentation process using Escherichia coli strain W3110. Using this technique, a small aerosol extraction flow from the fermentor was continuously dried in-line and analyzed with an aerosol size spectrometer and an aerosol photometer, used to measure the size-integrated aerosol concentration. The results of the measurements demonstrated that the bacterial growth rate and the aerosol number Concentration changed in a similar fashion: the effluent aerosol number concentration increased during the exponential growth phase and subsequently decreased after the bacterial cell concentration had reached a stable level. This aerosol concentration increased sharply approximately 1 h after initiation of growth. Thus, this increase in aerosol concentration suggests that the release of aerosols changes as a function of the microbial product formation activity. The products may change the rheological properties of the liquid, especially if surface-active compounds are produced. The increase in aerosol concentration corresponds to a decrease in the values of the measured surface tension during the same time frame. Furthermore, the aerosol size spectrometer and the photometer showed similar time traces of the effluent aerosols. The size distributions of the solid residues from the fermentation broth remained relatively constant, while the concentrations changed with the phase of fermentation. As the photometer is inexpensive, it appears promising as a convenient instrument for monitoring fermentation processes.
Introduction The success of a fermentation depends upon maintaining optimal environmental conditions for biomass and product formation. The provision of such conditions requires careful monitoring of the fermentation. Physical monitoring of the bioprocess as well as physicochemical measurements such as pH, dissolved oxygen, and gas analyses are satisfactory, if care is taken (Stanbury and Whitaker, 1984;Carleysmith, 1987;Ward, 1989). However, despite the fact that many approaches have been studied and applied to the evaluation of fermentation conditions, microbial growth, and product formation (Nyeste et al., 1981;Veres et al., 1981;Crueger, 1982;Cooney, 1983;Wang, 1986;Misev, 1991;O’Connor et al., 1992;Slater et al., 1992), no suitable instrumentation appears to be available for routine on-line analysis of the biomass and broth components. However, some recent investigations have been made in this area. Conductivity, dielectric measurements, and spectrophotometry have long been used to measure cell concentrations in real time, but each has certain limitations [see Zhong et al. (1993)l. These authors recently reported the successful use of an on-line laser turbidimeter to measure the growth of the red-pigmented Perilla frutescens, but also noted limitations with this
* Author to whom correspondence should be addressed. t On leave from the Lithuanian Academy of Sciences, Vilnius,
Lithuania.
instrument. Light reflectance has been evaluated to monitor biomass in solid-state fermentations (Ramana Murthy et al., 1993). Biosensors currently are being developed to monitor metabolic parameters, but are not currently in use in commercial applications (Owen, 1990). Recent progress has been made in the on-line monitoring of intracellular states of fermentation organisms using nuclear magnetic resonance (Chen and Bailey, 1993).Flow cytometry may also have application in areas such as monitoring of cell culture (Al-Rubeai and Emery, 1993). Off-line analysis, however, is commonplace for the measurement of biomass, many substrates, metabolites, enzymes, and cell constituents. Such monitoring methods involve time-consuming procedures that are not suitable for addressing changes in the process conditions. On-line analysis has been studied with gas and liquid chromatographs, continuous-flow analyzers, and enzyme electrodes (Kuhlman et al., 1984; McLaughlin et al., 1985; Picque and Corrieu, 1992). When such devices are used, the fermentation medium is filtered first to remove all the cells. Such sampling systems generally have several disadvantages, among them interference with the aseptic conditions or the normal processes inside the fermentor (Goodhue et al., 1986; Wang, 1986), the mechanical complexity of the system, and limits to use over long periods of sampling time. Also, they are difficult to use in media with complex rheological behavior characteristics. These monitoring systems often involve expensive in-
8756-7938/94/3010-0032$04.50/0 0 1994 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1994,Vol. 10, No. 1
strumentation. Also, they may not apply to all types of fermentation. Therefore, it is desirable to search for alternative and inexpensive methods of on-line measurement or estimation of the biomass and fermentation products. As we will show, the measurement of the aerosols generated during bacterial fermentation may provide useful process data at a relatively low cost. A large amount of these aerosols is dispersed from the liquid during bacterial growth in aerated and agitated fermentors. The aerosol dispersion is considered to be affected primarily by aeration and agitation (Baron and Willeke, 1986;Ohta et al., 1991). The dispersed aerosol concentration and size distribution depend on the rheological properties of the liquid (Pan et al., 1990;Pilacinski et al., 1990;Szewczyk et al., 1992). During the fermentation process, the metabolic products are generally excreted into the liquid, lowering its pH. These products cause changes in the rheological properties of the liquid, such as the kinematic viscosity and the surface tension, especially if surfaceactive compounds are produced (Georgiouet al., 1992).As liquid dispersion is dependent on the rheological properties of the liquid, the changes in these properties during fermentation should be reflected in the aerosol release from the fermentor. While aerosol dispersion should be dependent on the fermentation process, the dispersed aerosols are present outside the liquid phase, and measuring the aerosol dispersion does not interfere with the process itself. Therefore, measurement of the aerosol release may be a suitable approach for monitoring and controlling bacterial fermentation processes. In earlier studies, Szewczyk et al. (1992) measured the dispersion of aerosols during fermentation over a particle size range of 0.8-20 pm in diameter and found that the amount of aerosol released from the fermentation broth changed with the degree of microbial activity during fermentation. However, microbial parameters were not measured, and the relationship between aerosol characteristics and microbial activities, therefore, was not quantified at that time. The results indicated that the aerosol size distribution peaked at or below the lower limit of the measurement range. Therefore, it was concluded that measurements of the aerosol size distribution in the sub-micrometer range were necessary to evaluate further the change in the aerosol size distribution caused by microbial activities during fermentation. In this study, aerosol release and microbial growth have been monitored simultaneously during fermentation, and the results have been compared with each other. The aerosol concentrations and size distributions were monitored from 0.1 to 3.0 pm with an aerosol size spectrometer. In addition, an aerosol photometer was used to monitor the aerosol concentration over an integrated size range. Materials and Methods Fermentor. A 20-L glass-vessel fermentor (Model CF 2000, Chemap Corp., South Plainfield, NJ) was used in this study. Part of the experimental setup is represented in Figure 1. The fermentor was equipped with a switchable, constant-speed mechanical foam breaker. As the degree of foaming increased during fermentation, the mechanical foam breaker was kept running throughout the aerosol measurements. Three 8-cm impellers, consisting of six blades each, were mounted on a central shaft surrounded by four baffles. The impeller speed was set at 400 rpm during the fermentations. Aeration was provided by the injection of filtered air at the bottom of the fermentor at a typical flow rate, Qaer,of 13.6 L/min.
33
'
PHOTOMETER ;TT
qp;;;;;/
[SIZE SPECTROMETER
AEROSOL
I
-FERMENTOR Figure 1. Schematic diagram of the fermentor headspace and the aerosol sampling train: Qaer = 13.6 L/min, Q, le = Qb = 3.5 L/min, Qphotometer = 2.0 L/min, Qspeetrometer = 0.06 E/rnin, and QpUp = 4.94 L/min. The temperature of the broth, Tbroth, was maintained at 37 OC, and the dissolved oxygen and pH levels in the fermentation broth were monitored to ensure appropriate growth conditions for the microorganisms. Microorganism and Culture Conditions. Escherichia coli strain W3110, the test organism, was maintained on Luria broth agar. The fermentation medium consisted of the following substances (concentration in g/L): glucose, 10.0;tryptone, 22.0; yeast extract, 2.5; KzHPO4,1.74;KH2PO4,0.87; CaC12,O.lO; MgS0~7Hz0,1.135; FeS0~7H20, 0.215; CuC12, 0.105; MnSOcH20, 0.0075; ZnClz, 0.00028; CoCly6H20, 0.00051; NazMoOc2Hz0, 0.00043; H3B03, 0.00011. Ten liters of the broth were added to the fermentor, sterilized, and adjusted to pH 7 with 2 N NaOH. The medium was inoculated with E . coli strain W3110. As the E. coli grew, acids were generated that significantly lowered the pH. The pH of the medium had dropped to approximately 5.8 by the fifth hour after inoculation. Measurement of Aerosol Concentration. The change in aerosol concentration was measured with an aerosol size spectrometer (Model LAS-X, PMS Inc., Boulder, CO) and an aerosol photometer (Model RAM-S, MIE Inc., Billerica, MA). The aerosol size spectrometer is a singleparticle counter which optically measures particle concentrations and size distributions over a size range of 0.13.0 pm in diameter. The aerosol sample is drawn into the small view volume of the device, where light from a laser source is scattered by each particle passing through. The scattered light is collected by a photosensor and converted into an electric pulse. Each signal is classified as to its intensity and is registered in an accumulator with several channels, each for a different size range. Every aerosol size distribution was recorded over a period of 30 s. The data are reported from three measurements for their mean and 95% confidence interval. The photometer has a much larger view volume and measures the light scattered by the aerosol cloud in the view volume. A photometer with an incandescent light source, as used in this study, produces the greatest amount of light scatter per unit particle mass in the size range of about 0.1 pm to several micrometers, with a peak at about 0.5 pm. The integrated size measurement by the photometer, therefore, reflects the aerosol size concentrations of about 0.5 pm diameter particles, particularly if the size distribution (as measured by the LAS-X aerosol size spectrometer) peaks at that value.
Biotechnol. Prog.. 1994, Vol. 10, No. 1
34
Measurement of Aerosol Release during Fermentation. Figure 1shows a schematic representation of the sampling train used for the aerosol measurements. The sampling train was developed and evaluated in an earlier laboratory study (Wangwongwatanaet al., 1990) and was modified somewhat for the present one. An inoculation needle with an inner diameter of 4 mm was used as the sampling probe. It was inserted through the fermentor head plate near the mechanical foam breaker. As the measured particle size range is from 0.1 to about 3.0 pm in diameter, and the probe, therefore, does not attempt to capture high-inertia particles, the suction flow into the probe does not need to be isokinetic (Brockmann, 1993) and the sampled aerosol does not depend on changes in the aeration conditions, such as gas flow or agitator rpm. The sampling train was connected to the probe through tubing during measurements and was disconnected from the probe when not in use. The connecting tube was clamped when not in use to avoid contamination from the fermentor. Samples were taken at 3.5 L/min (Q-ple) from the sampling port every 0.5-1 h. The sampled air was diluted with dry-filtered air (Qdry, 3.5 L/min), which reduced the relative humidity in the sampled air to less than 50% and, thus, reduced the sampled droplets to the size of their dry residues. A portion of the dried aerosol flow (Qphotometer, 2 L/min) was sampled by the aerosol photometer for the integrated aerosol concentration measurement. The remainder of the dried aerosol flow passed through a diluter (Model 3302, TSI Inc., St. Paul, MN), in which most of the flow was passed through an absolute filter before it was mixed with the remaining aerosols. This further diluted the aerosol flow and, thus, avoided particle coincidence in the small view volume of the aerosol size spectrometer. A small fraction of this flow (Qspectrometer,0.06 L/min) was sampled by the aerosol size spectrometer for the measurement of aerosol concentrations and size distributions in the size range of 0.13.0 pm in diameter. The data from the aerosol size spectrometer were collected and analyzed by a personal computer. Aconstant-flow pump (Qpump,4.94 L/min) drew the remaining diluted aerosol flow through an absolute filter. For all microbial processes, aseptic conditions were used. The fermentation vessel and all fluids were sterilized prior to use. The aerosol sampling probe was sterile, but the sampling train and the aerosol size spectrometer were not. It was assumed that the outward flow of off-gases in the sampling train would reduce the potential for contamination of the culture. The spectrophotometer is a sensitive electronic instrument that cannot be sterilized. To ensure that no measurable bacteria were deposited in the sampling train during periods of sampling, a high-efficiency particulate air (HEPA) filter was inserted several times upstream of the sampling train, which resulted in zero count by the aerosol size spectrometer. Furthermore, the dry-diluted air added to the sampling flow was sterile. After several preliminary experiments, the reported data were from the complete monitoring of one entire fermentation process. For each data point of the process parameters in the figures, dry cell weight measurements were performed in duplicate. The absorbance, surface tension, and viscosity measurements were done once,while the photometer and aerosol size spectrometer readings represent the average of three successive measurements. In the last figure, all measured particle size distributions are normalized and shown as averages with the standard deviations indicated.
Assessment of Bacterial Growth. Broth samples were taken aseptically from the fermentor every hour to evaluate the bacterial density by measuring the optical absorbance and dry cell weight. The optical absorbance of the broth sample was measured with a spectrophotometer (Model35, Beckman Instruments, Inc., Fullerton, CA) at a wavelength of 595 nm (A595), with distilled water as the blank. To measure the dry cell weight, each sample was centrifuged a t 3000 rpm for 5 min, washed once with 30 mL of buffered saline solution, and resuspended with 30 mL of distilled water. The solution was separated into 15-mL fractions and put into preweighed tares. The samples were then heated at 80 "C under vacuum for 2448 h to constant weight, and the resulting values were expressed as mg/mL of medium. Rheological Measurements. The effect of bacterial activity on the rheological properties of the fermentation broth was analyzed periodically by measuring the surface tension and kinematic viscosity of the sampled liquid. The surface tension was measured at room temperature (approximately 25 "C) with a DuNouy tensiometer (Model 70535, CSC Scientific Co., Inc., Fairfax, VA). The viscosity of the broth was measured for kinematic viscosity at 37 "C with a Haake viscometer with an M10 measuring head using NV concentric coaxial cylinders. Each 9-mL broth sample was placed into the cylinders, rammed to a final shear rate of 1000 L/s in 1min, and then held a t 1000 L/s for 1 min. The viscosity was measured in centipoise for the time during which the shear rate was held constant. Results and Discussion Figure 2A-C illustrates the measured data of bacterial density and aerosol concentration. Figure 2A presents the time change in bacterial population in the broth recorded by the optical absorbance and dry cell weight measurements. As seen, the exponential growth phase started between 1 and 2 h after inoculation, and a stationary phase was reached at about the fifth hour. Both types of measurements showed the expected pattern of microbial growth. The time-dependent changes of the aerosol concentrations, measured by the aerosol size spectrometer and the aerosol photometer, are shown in Figure 2B,C,respectively. The aerosol size spectrometer recorded relatively few particles above 1 pm. Therefore, the aerosol number concentrations are shown in Figure 2B for the size range of 0.1-1.0 pm in diameter. As seen, the number concentrations of the effluent aerosols began to increase rapidly approximately 3 h after inoculation and reached a maximum level at approximately the fifth hour. After that time, the aerosol concentration started to decrease. This corresponds to the time period during which the culture reached maturation. A similar response was observed from the aerosol photometer, as shown in Figure 2C. Effect of Bacterial Growth on the Change in Rheological Properties and Aerosol Release. The microbial growth rates were estimated from absorbance measurements using exponential growth equations. The instantaneous growth rate, p , was calculated by (In X,- In XI) t where X is the measured absorbance and t is the time interval. A comparison of the growth curve and the aerosol measurement result is shown in Figure 3A. Both the growth rate and the aerosol number concentration changed in a similar fashion, but with an approximately 1-h shift between the curves. This suggests that aerosol release is p
(h-l) =
35
Blotechnol. Prog., 1994, Vol. 10, No. 1
-
-
QAgR 13.8 L / m l n . T,,,,, = 37.C 400 rpm IMPELLER S P E E D WITH YECHANICAL FOAY BREAKER
1.5
= 13.6 L/mln. T,,,,, = 37.C IMPELLER S P E E D = 400 rpm WITH MECHANICAL FOAY BREAKER Q,,,
-] 2.5
30
7
1.0 L
c
1 .o
i 0.s ti c
OPTICAL ABSORBANCE
4
>. K n
m
0.5
X
DRY CELL WEIGHT
" o
.
o
y
4
0.8
c
1 0.0
AEROROL SPECTROYETER RESPONSE
0.4
n W c 4
E
E s t
0.2
4
I
vl
4
W
0.0
1.3
0
- 85
LB
- E - 8 0 &'
.-n0 .-P P)
\ 0
4
E
1.2
cVI
5
0-
v
'
O
1
2
r 3
E
>
i
-
>: 0 V
0
0
vl
t; c
- 55 w
1.1
V -4
c
-
E
3 v)
50
4
5
8
7
TIYE. hr.
0
1
2
3
4
5
8
7
TIME, hr
Figure 2. Comparisons of liquidvs aerosol phase measurements during the growth of Escherichia coli strain W3110: (A) optical absorbance and dry cell weight; (B)O.l-l-Mm particle effluent aerosol concentrationmeasured by the aerosol size spectrometer; (C)light-scatteringresponse from the aerosol photometer.
Figure 3. Relationshipbetween the growth of Escherichia coli strain W3110, aerosol release, and rheological properties during fermentation: (A) comparison of estimatedmicrobial growth rate and effluent aerosol concentration during fermentation; (B) changes in surface tension and viscosity in the broth.
related to bacterial activity. As a result of the bacterial activity, different metabolites were released into the liquid that were observed as increased foaming on the liquid surface (Lalchev and Exerowa, 1981;Lalchev et al., 1982; Misev, 1991;Kang et al., 1991;Yasukawam et al., 1991). As the secretion of metabolites and gaseous products usually lags behind the growth (Calam, 1969;van Dijken and Scheffers, 1984;Yasukawam et al., 1991;O'Connor et al., 1992),the observed change in aerosol concentration undoubtedly results from the bacterial metabolism in the liquid. Figure 3B shows the time changes of surface tension and kinematic viscosity in the broth. The viscosity decreased from 1.18 to 1.04 CPduring the peak of cell growth, while the surface tension decreased from 62.5 to 51.3 dyn/cm during the latter part of bacterial growth. It appears that the nutrients were degraded during the growth phase of the Escherichia coli, which reduced the viscosity of the medium. A similar decrease in viscosity was found by Tuffile and Pinho (1970)in their study on the growth of Streptomyces aureofaciens on a starchcontaining medium. The change in surface tension can also be explained. The metabolism of glucose by E. coli produces, in the fermentation medium, products such as ethanol and carbon dioxide, as well as lactic, acetic, formic, and succinic acids IPorter, 1946). In addition, the metabolism of tryptophan, contained in high concentrations in the tryptone, adds more organic acids to the medium (Porter, 1946). Thus, as a result of all these metabolic changes in the composition of the fermentation medium,
the surface tension decreased significantly, as seen in Figure 3B. This decrease in the surface tension increased the foaming tendency of the liquid, which produced higher effluent aerosol concentrations (Figure 3A) by action of the mechanical foam breaker. Thus, the production of metabolites was followed by an increase in aerosol release. The increase in the acidity of the fermentation medium was confirmed by measurements of the pH, which dropped from an initial value of 7 to 5.8 in the fifth hour after inoculation. This reduced the bacterial growth, as seen in Figure 3A. Since the production of organic acids decreases as a result of decreased bacterial growth, and since some of the acids are metabolized further to carbon dioxide, a slight increase in the surface tension of the medium was observed at the end of the experiment (Figure 3B). The foam layer on the liquid surface was observed to have been reduced at the end of the experiment, apparently caused by this rise in surface tension. Thus, a lower effluent aerosol concentration was monitored at the end of the experiment, as shown in Figure 3A. In conclusion, the production of metabolites, therefore, appears to be strongly related to the number concentration of aerosol particles released from the fermentor. The reproducibility of these results was good as the range of variations was only about 2?4 Change i n Aerosol Distribution during Fermentation. Figure 4 presents the number distribution of the dried effluent residues between 0.1 and 3 pm at early vs later stages of growth. The number distribution was calculated by dividing the aerosol number concentration,
.
38
Biotechnol. Prog., 1994, Vol. 10, No. 1
Q,,, = 13.6 L / m i n , T,,, = 37°C IMPELLER S P E E D = 4 0 0 r p m WITH MECHANICAL FOAM BREAKER 105 .
m,
$
01
,
, , ,,
,,,(
,
,-
5 hrs.
0 5
1
1
WITH MECHANICAL FOAM BREAKER
,
,5 h rs. ,
01
os
t.
1
OPTICAL PARTICLE DIAMETER, D , ,urn Figure 4. Effluent aerosol size distributionsduring the growth of Escherichia coli strain W3110: (A) first 5 h of growth; (B) later stages of growth.
1
0.8
1
N , in each size channel of the aerosol size spectrometer by the logarithmic difference between the upper and lower particle size limits of that channel. The recorded size distributions of the effluent aerosolsshow that the majority of the particles are of a diameter, D, below 1 pm, as predicted by Szewczyk et al. (1992). During the first 3 h of fermentation, no significant change was observed in the aerosol concentrations and size distributions. After 4 h of fermentation, foam had reached the mechanical foam breaker, and the effluent aerosol concentration increased nearly 100 times over the initial concentration and continued to increase during the fifth hour. The effluent aerosol concentration decreased afterward, but by a much smaller amount. Regardless of the changes in concentration, the size distribution curves remained approximately the same between 0.1 and 3.0 pm throughout the process. This may be an effect of the mechanical foam breaker on top of the fermentor, as the centrifugal force broke down the foam bubbles near the top of the fermentor and produced sizespecific droplets. This observation that the sub-micrometer size distribution remained relatively constant throughout the fermentation process suggests that the effluent aerosol concentration may be monitored by a narrow or integrated wide size range below 1 pm. As discussed previously, the aerosol photometer measures the aerosol concentrations of an aerosol cloud over a wide size range, with the peak sensitivity at about 0.5 pm. If the peak of the particle size distribution is at or near 0.5 pm, the concentration measurement primarily reflects the aerosols of that size range. As the scattering intensity recorded by the aerosol photometer depends on the amount of aerosol particle surface available for light scattering, the aerosol surface distribution is the relevant size distribution to examine. We have, therefore, converted all aerosol number distributions measured by the aerosol size spectrometer to surface distributions, and we present the normalized average number and surface distributions in Figure 5. Each normalized number distribution was calculated by dividing each number distribution by the total number concentration in the measured 0.1-3.0-pm size range. By assuming that each aerosol particle is spherical and that the linear midpoint of each size channel in the aerosol size spectrometer represents the average diameter of the particles in that size range, the surface distribution was
SURFACE DISTRIBUTION
/\
0.6
0.4
0.2 0.0 0.1
0.5
1
O P T I C A L P A R T I C L E D I A M E T E R , D , pm
Figure 5. Average normalized distributions of effluent aerosol from fermentor broth (A) normalized number distribution;(B) normalized surface distribution. calculated by multiplying the number of particles in each size range by the particle surface, S, of the mid-sized particle. Each average normalized distribution was calculated from the average of all size distribution measurements taken during the fermentation process. The average normalized number distribution, Figure 5A, indicates two peaks in the effluent aerosol: one between 0.1 and 0.2 pm, and the other a t about 0.5 pm. The modifier “optical” is added to “particle diameter” in Figure 5 to indicate that the measured particle size is based on the optical scattering response (Willeke and Liu, 1976). The average surface distribution of the effluent aerosol shown in Figure 5B indicates a peak at about 0.5 pm. This demonstrates that the conversion from number distribution to surface distribution suppresses the peak of the smaller particles and accentuates the peak of the larger particles. As 0.5 pm is the most sensitive size for the aerosol photometer, the photometric measurement essentially reflects the concentrations of that size. This explains why the photometric response is very similar to the total number concentrations from 0.1 to 1.0 p m , as shown in Figure 2B,C and suggeststhe photometer as an inexpensive measuring device for monitoring fermentation processes. Light-scattering smoke detectors are the least expensive photometers on the market today, optimized to detect smoke in homes and offices. General Applicability of Measuring Bioaerosols for Determining Fermentation Stages. Aerosol measurementa in a fermentation process can indicate the stage of the process in which microorganisms bring about chemical reactions within a short period of time. In many cases, these reactions involve the degradation of proteins, fats, or carbohydrates or a combination of this with the
Bbtechnol. Rog., 1994, Vol. 10, No. 1
utilization, usually, of oxygen to produce degradation products (such as acids) and carbon dioxide. The aerosol measurement method used in this study to detect the microbial growth stage will probably work best for a vat fermentation, such as one yielding alcohol, yeast cells, antibiotics, enzymes, amino acids, vinegar, or citric acid (Rebd, 1982). The applicability of aerosol measurement as an on-line method can be considered for selected fermentations where the fermentation process is well understood and predictable operating conditions can be achieved. Investigators wishing to use this aerosol measurement method for a new process will be concerned about the difference from the present study in temperature, humidity, agitation rate, and air flow rate required to bring about optimal operating conditions. An example would be to increase the oxygen concentration during aerobic fermentation by increasing the air flow rate, which in turn also increases foaming. Changes in these parameters do bring about changes in measured aerosol concentrations. Szewczyk et al. (1992),using a similar fermentation process with Escherichia coli, measured the bioaerosols with an aerosol size spectrometer. They found that, a t a fixed agitation rate of 450 rpm and air flow rates of 1.7-8.5 m3/ h, the aerosol concentration was independent of the air flow rate for particles 2-pm particles, a 5-fold decrease in air flow rate resulted in an increase in aerosol concentration up to 100%, because the number of large aerosol particles generated by mechanical agitation remains approximately constant for a fixed agitation rate. The aerosol particle size range
