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REVIEW Techniques for the Estimation of Cell Concentration in the Presence of Suspended Solids M. J. Kennedy,*$+ M. S. Thakur,+JD. I. C. Wang,§ and Gregory N. Stephanopoulod Chemical Engineering Department and Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Measuring cell concentration is of fundamental importance in many biochemical processes. However, this measurement is very difficult t o make when solid particles are present along with the cells. This review examines strategies t h a t have been used t o estimate cell concentration in the presence of solid particles.
Contents Introduction Direct Microscopic Observation Colony Counting on a Plate Prior Separation of the Cells and Solids Preferential Dissolution of the Solid Preferential Dissolution of the Cells Dissolution of the Cells and Solid followed by a Separation Technique Measurement of a Selective Component That Is Not Present in the Solid Metabolic Measurements Measurement of a Physical Property That Is Different in the Cell and the Solid Deconvolution of Light-Based Data Conclusions
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Introduction Many situations involve the growth of cells in the presence of solid particles. For example, solid-state fermentations involvethe growth of organisms on a variety of moist solids. Solid substrates are also used in many industrial submerged culture fermentation media. Lastly, the utilization of cellulosic renewable resources again requires solid substrates to be transformed through biological means. Knowing the cell concentration in such systems is of utmost importance. Agar (1985) states “knowledge of the amount of biomass in a system and its increase with time, the growth rate, is fundamental to any work in biotechnology”. In industrial submerged culture using suspended solids, estimation of the cell concentration is of considerable practical significance. Cell concentration estimates are needed to calculate the specific productivity, to apply control strategies based on substrate consumption, to analyze and diagnose the state of the fermentation, to
* Address correspondence to this author at the following present address: New Zealand Institute of Industrial Research and Development, Box 31-310, Lower Hutt, New Zealand. t Biotechnology Process Engineering Center. Present address: FermentationTechnologyand Bioengineering, Central FoodTechnologicalResearchInstitute,Mysore 570013, India. 8 Chemical Engineering Department.
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develop models, to time inoculum addition, to detect the occurrence of deleterious effects, to determine the phase of the fermentation, and to decide when to harvest. Further uses for biomass estimation measurements are detailed in Chattaway et al. (1992). However, most of the currently available methods of estimating cell concentration do not perform well in the presence of other solid particles. For example, the most commonly used cell mass estimation technique employs dry weight measurements, which include both the cells and the solid particles, and thus fails to correctly estimate cell concentration. There are many reviews on the numerous techniques that have been devised to measure cell concentration in media with no solid particles present (Phillips, 1990; Omstead et al., 1990; Baserga, 1989; Agar, 1985; Harris and Kell, 1985; Karube, 1985; Onken et al., 1985; Carleysmith and Fox, 1984;Joglekar et al., 1983;Cooney, 1981; Wang et al., 1979; Pirt, 1975). In contrast, there are only a small number of papers dealing with the measurement of cell concentration of the presence of solid particles. This review will be limited to those techniques which have been shown to estimate cell concentration in the presence of other solid particles. The methods used to estimate cell concentration in the presence of solid particles have been classifiedin this review according to strategy or concept, as opposed to individual method, to aid the reader in selecting an approach to a new problem or situation. The strategies for measuring cell concentration in the presence of other solids are classified as follows: direct microscopic observation colony counting on a plate prior separation of the cells and solids preferential dissolution of the solid preferential dissolution of the cells dissolution of cells and solids followed by a separation technique measurement of a selective component that is not present in the solid metabolic measurements measurement of a physical property that is different in the cell and the solid deconvolution of light-based data
8758-7938/92/3008-0375$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Englneers
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Max J. Kennedy obtained a Bachelor of Engineering (Chemical and Materials) honors degree from the University of Auckland, New Zealand in 1983. He began work with the New Zealand Government Department of Scientific and Industrial Research (DSIR) in the area of Biotechnology. In 1985,he commenced studying for a Ph.D. at MIT Cambridge, MA. After graduating in 1990 with a Ph.D. in Biochemical Engineering, he returned to New Zealand to the DSIR. He is a member of a DSIR marketing task force representing the Bioprocess Development Group. Currently, he is the Editor of the New Zealand Biotechnology Association Newsletter and the New Zealand Editor of the journal Australasian Biotechnology. He has a special interest in fermentation processes, biotechnology history, and the application of neural networks to biotechnology. He lives with his wife and two children in Wellington, New Zealand.
Daniel I. C. Wang is the Chevron Professor of Chemical Engineering and the Director of the Biotechnology Process Engineering Center a t MIT. He received his B.S. in Chemical Engineering and M.S. in Biochemical Engineering from MIT. He received his Ph.D. in Chemical Engineering from the University of Pennsylvania. He has been a faculty member a t MIT since 1965. Professor Wang is an editorial member for a number of professional journals as well as a member of a number of committees a t the National Research Council, National Academy of Engineering, and National Institutes of Health. His research interests include bioreactor design, biosensor development, protein purification and refolding, and animal cell culture.
Gregory N. Stephanopoulos is Professor of Chemical Engineering in the Department of Chemical Center a t MIT. He received his B.S. from the National Technical University of Athens, his M.S. from the University of Florida, and his Ph.D. from the University of Minnesota, all in the field of Chemical Engineering. Professor Stephanopoulos was a faculty member at the California Institute of Technology from 1978 to 1984. Since then, he has been on the faculty of the Chemical Engineering Department of MIT. His research interests include mixed cultures, parameter identification, process control, and mammalian cell culture.
M. S. T h a k u r is a scientist in the fermentation technology and bioengineering discipline a t Central Food Technological Research Institute, Mysore, India. He received his Ph.D. in Industrial Microbiologyfrom the University of Saugar, India. He has published about 25 research papers and has developed microbial processes based on solid-state and submersed fermentationsspecially for microbial rennet, oils,and glycerol. He is also involved in teaching Industrial Microbiology courses for Food Technology students a t Mysore University. He was awarded a visiting associateship (1988-1990) by the Government of India to work at the Massachusetts Institute of Technology, with Professor D. I. C. Wang. He was also a visiting scientist at the University of Maryland, Baltimore, MD. His current interest is the development of a biosensor for monitoring the food and fermentation processes, terpanoid biotransformations, and mammalian cell culture.
Direct Microscopic Observation In this technique, the cells and solids are viewed under a microscope and various methods are used to aid in counting the cells. An example of a bacterial system is the cell counting of Zymomonas mobilis in the presence of soy flour by the use of a Petroff-Hausser bacterium counter (Ju et al., 1983). Examples involving yeast include the cell counting of Saccharomyces cereuisiae in the
presence of whole soy flour using a Petroff-Hausser bacterium counter (Damiano and Wang, 1985) and the counting of Saccharomyces carlsbergensis in the presence of unrefined soybean flakes using a Levy hemocytometer (Kleyn and Vacano, 1966). The ease of differentiating the cells from the solid can be enhanced by using a staining technique. The stain is usually a fluorescent dye or a fluorescently labeled antibody. Examples include the use of epifluorescence microscopy to detect Thiobacillus ferroonidans in the presence of ore particles with the aid of an acridine orange stain (Yeh et al., 1987)and the use of fluorescent antibody staining to detect T . ferroonidans attached to the surface of coal refuse (Ape1 et al., 1976). Cell distribution on solid particles can also be determined using a scanningelectron microscope. For example, the colonization of T . ferrooxidans on support matrix materials was studied using a scanningelectron microscope (Grishin and Tuovinen, 1989). Determination of cell concentration on a routine basis using a scanning electron microscope is not practical. The disadvantage of direct microscopic observation is that in some cases the cells attach to the solid surface and this makes counting difficult. Also, the technique is very labor intensive and operator fatigue can decrease accuracy. Cell enumeration techniques are usually a "last resort" approach to the problem.
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Colony Counting on a Plate
Preferential Dissolution of the Cells
Colony counting on an agar plate takes advantage of the fact that the cells will grow and produce colonies, whereas the solid particles will not. A dilute suspension is used to inoculate an agar plate, and the colonies are counted after incubation. For example, the estimation of T. ferrooxidans in the presence of ore particles has been reported (Espejo and Ruiz, 1987). Colony counting suffers from the disadvantages of all cell enumeration techniques as listed above. Also, this technique requires considerable time to replicate the cells on a solid growth medium and thus cannot be used for real time estimations.
The principle of these techniques is that the cells are preferentially lysed but the solid remains intact. The mixture is then centrifuged to remove the solid and cell debris. The supernatant,which contains the intracellular cell substance, is then analyzed for any compound which is present in a fixed quantity per cell. Sonication or chemical rupture of the cell followed by the measurement of DNA content or total organic carbon are popular choices for these assays (Solomon et al., 1983;Hashimoto et al., 1982). ATP has been extracted and related to cell concentration in the composting of grape pulp and sheep manure (Thierry and Chicheportiche, 1988). Protein has been solubilized from cells by treatment with sodium hydroxide and related to cell concentration in the fermentation of Aspergillus niger on cassava meal (Raimbault and Alazard, 1980). Dehydrogenase activity was used to estimate the growth rate of a heterogeneous microbial population growing in swine manure (Ghaly et al., 1980; Ghosh et al., 1972).
Prior Separation of the Cells and Solids This technique first separates the cells from the solid and then uses any of the standard cell concentration estimation techniques to quantify the amount of cells. This method is not applicable in most cases because it is very difficult to separate the cells from the solid. However, there are three notable exceptions. The first occurs when there is a large difference in density between the cell and the solid. In this case, the cells and the solid can be separated by centrifugation and then the cells can be recovered for quantification. A case where the solid is much more dense than the cells is the separation of solid ore from T. ferrooxidans (Espejo and Ruiz, 1987). An alternate technique used when the densities of the cell and the solid are similar is to use centrifugation followed by freeze drying of the solid pellet. After freeze drying, the cell layer can be cut from the solid layer more easily and then a dry weight analysis can be performed on the cells (Suzuki, 1988). A problem arises, however, in obtaining a distinct cell/solid interface, and complete separation of the cells from the solid is difficult. The second exception is by the use of filtration to separate the cells and solids. For example, the filtration of blended food samples containing Salmonellae using a 35-pm nylon cloth filter has been reported (Tsen et al., 1989). The disadvantage of this method is that all solid particles smaller than the filter cutoff size will be counted as cells; also, cells adhere to the filter. The third exception occurs when the solid particles are very large and can be easily removed from the medium. Such a case occurs in anchorage-dependent mammalian cell culture. The cells are detached from their solid support matrix by trypsinization (Baserga, 1989). After removal of the solid support, the cells remain in the liquid medium where they are counted by use of a variety of techniques.
Preferential Dissolution of the Solid In cases where the solid particles can be preferentially dissolved, the remaining cell concentration can be determined using standard techniques. For example, the dissolution of ferric iron precipitates with EDTA enables the concentration of T. ferrooxidans to be determined using a Coulter counter (Schuler and Tsuchiya, 1975).In the growth of cells in a semi-solid gel, the liquefication of the gel a t high temperatures allows the cell mass concentration to be determined using traditional techniques, (Marin-Iniesta, 1989; Wei et al., 1983). Solid calcium carbonate particles can be removed from microbial cultures by washing with mineral acid (Agar, 1985). Solid hydrocarbons can be removed from cell samples by extraction with solvents (Amin et al., 1973;Yamada and Yogo, 1970). Sodium hydroxide and heat are used to dissolve cellulosic residues in cell samples prior to cell mass determination (Huang et al., 1971).
Dissolution of the Cells and Solid followed by a Separation Technique In this technique, both the cells and the solids are solubilized. This yields a soluble solution to which a wide range of analytical techniques can be applied. However, some unique property must be found in which the solubilized products of the cells and solids differ. This technique finds most application in measuring the concentration of cells which have been immobilized in various gels. For example, cell growth in polymer resins was measured by hydrolyzing both the cells and the resin in sodium hydroxide. The cell protein fraction and the hydrolyzed resin fractions were then separated using hydrophobic interaction column chromatography. The cell protein estimate was used as a monitor of cell growth (Cheong et al., 1990).
Measurement of a Selective Component That Is Not Present in the Solid The selectivecomponent to be measured must be present in a fixed quantity per unit cell mass and not present in the solid. This often is the exception rather than the norm. Protein content is often correlated with cell concentration when cells are grown in the presence of nonproteinaceous solids. For example, the growth of cells on cellulose can be monitored using a protein assay (Greene and Gordon, 1989;Moreira et al., 1978). Chitin (poly(N-acetylglucosamine)) present in fungal cell walls can be used to calculate cell concentration. For example, the growth of Aspergillus oryzae in koji fermentations is followed by measuring glucosamine (Ito et al., 1989;Aidoo et al., 1981). Lipid bound phosphates have been used to determine cell concentration in sediments (Findlay et al., 1989). Acid and alkaline phosphatase and phosphoamidase have been used to detect microbial contamination of dust (Kniest et al., 1990). Mixed culture provides the unique challenge of identifying two populations that are very similar in most properties. In this situation, a unique compound in one cell population must not be present in the other. For example, 8-anilino-l-naphthalenesulfonic acid can be used to distinguish between Gram-negative and Gram-positive bacteria (Ramsey et al., 1980). Another example is the differentiation of methanogens from nonmethanogenic organisms using coenzyme F420 (Mink and Dugan, 1977). The cell concentrations of Streptococcus cremoris and
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Table I. Summary of Strategies Used To Estimate Cell Concentration in the Presence of Suspended Solids
strategy direct microscopicexamination colony counting on a plate prior separation of cells and solid preferential dissolution of the solid preferential dissolution of the cells dissolution of cells and solids followed by a separation technique measurement of a selective component that is not present in the solid metabolic measurements measurement of a physical property deconvolution of light-based data
comments labor intensive, cells adhering to solid surface causes problems requires considerable time, numerous cells adhering to one solid particle can lead to one colony not possible in most cases complete separation is very difficult difficult to find reagent that will only dissolve solids difficult to find reagent that will only dissolve cells difficult to find a property in which the solubilized cells and solids differ difficult to find a substance present in cells but not in the solid most metabolites are not a constant proportion of the cell mass during all stages of development metabolite production proportionality to cell concentration may vary with time during the fermentation very few physical properties effectively distinguish between cells and solids shows potential for further development effect of gas bubbles and cell morphology on the technique has not been investigated shows potential for further development
Leuconostoc lactis can be determined in mixed culture by measuring two different enzymes, each specific to only one strain (Boquien e t al., 1989). Gene probes (Steffan et al., 1989) and fluorescent antibodies (Kurane et al., 1979) have been used to estimate cell concentration of specific organisms in mixed culture. The problem in these techniques is finding a substance that is uniquely present in the cells and not in the solid, as well as ensuring that the substance remains a constant proportion of the cell mass during all phases of cellular metabolism. In practice, for most systems, these are very difficult constraints to meet.
Metabolic Measurements The cell mass concentration can be calculated from a substrate consumption rate or a product formation rate. In these cases, the substrate consumption or product formation is assumed to be proportional to the cell accumulation. For example, the ammonium sulfate consumption rate is used to calculate the cell mass concentration during the growth of Trichoderma reesei on leached beet cosette (Schaffeld and Illanes, 1982). a-Amylase and amyloglucosidase concentrations parallel cell mass concentration in A. oryzae in koji fermentations (Aidoo et al., 1981). Lactate production and glucose consumption are used to monitor the growth of mammalian cell cultures. Another method commonly used to monitor cell concentration in fermentations which utilize solid substrate is based on the measurement of the inlet and outlet gas compositions and the gas flow rate. The cell concentration can be estimated from oxygen uptake rate or carbon dioxide evolution rate data. For example, the oxygen uptake rate was used to estimate cell concentration in the fermentation of A. oryzae on steamed rice (Sato et al., 1983). The carbon dioxide evolution rate was used to estimate the growth rates during the fermentation of A. niger on cassava flour (Carrizalez et al., 1981) and of Candida utilis on ryegrass straw (Han, 1987). Another method involves measuring the carbon dioxide evolution rate of soil after fumigation. The carbon dioxide evolution was due to the decomposition of killed microbial biomass by recolonizing populations (Tateishi et al., 1989). Heat is another product of microbial metabolism; heat evolved can be used to estimate cell concentration (Morrison and von Stokar, 1986; Bayer and Fuehrer, 1982). All of the methods in this section suffer from the fact that substrate consumption and product formation rates will not always vary linearly with cell concentration,
especially during the different stages of the fermentation. For example, during the stationary phase the substrate consumption and product formation can occur a t a rate that is different to that during exponential growth and therefore the measurement is not an accurate estimate of cell concentration. An extension of these metabolic measurements is a technique based on mass balancing and stoichiometry which was developed by Cooney et al. (1977). The major disadvantage is that the stoichiometry and cell composition may vary during the fermentation. This technique has yet to be applied when solid substrates are present in the fermentation medium. When solid substrates are present in the fermentation medium, the exact stoichiometry of substrate utilization may be difficult to determine.
Measurement of a Physical Property That Is Different in the Cell and the Solid Methods used in this measurement rely on a physical property which is different (usuallyunder given conditions) in the cells from that of the solid. Since the cells and solid are usually of similar composition, and the fermentation medium often interferes, this property is very difficult to find. However, methods of this type offer promise for overcoming the current difficulties in estimating cell concentration in the presence of solid substrate. One promising method is the measurement of radio-frequency dielectric properties to determine cell concentration, illustrated by the determination of S. cerevisiae concentration in the presence of calcium carbonate (Harris et d.,1987). This method may be used in on-line measurement and has recently been commercialized as the "bugmeter viable cell monitor" (Aber Instruments Ltd.,Aberystwyth Science Park, Aberystwyth, SY23 3 AH, U.K.; 1989).
Deconvolution of Light-Based Data Light-based data (scatter, absorbance, or optical density) are not used frequently in the presence of solid particles because the solid particles interact with the light beam. This means that the contribution to the light behavior from the solids must be separated from that from the cells. Thus, deconvolution techniques are used to obtain a signal that can be related to cell concentration alone. Wei et al. (1983) were one of the first to apply lightbased data to estimate the cell concentration in the presence of solid. Wei et al. (1983) measured the cell
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concentration of S. cereuisiae in a 10%solution of solidified gelatin. A small path length, 0.5 cm, was used so that high cell concentrations could be measured. The authors' assumption that the gelatin and medium components produced no interfering absorbance was successful. However, this assumption is violated in most other solidcontaining media, thus limiting the value of this technique. Hong e t al. (1987) extended the technique further and estimated the concentration of S.cereuisiae in the presence of potato solids. The moisture content of the samples was between 50 and 95 % . After suitable dilution of all the samples to the same water content, the authors measured the absorbance of the cell/solid mixtures relative to a blank of cell-free solid a t the same moisture content. Then authors fitted their variable water content data with an interaction model assuming that the measured absorbance was due to a cell component term, a solid component term, and an interaction term containing the product of the cell concentration and the solid concentration. The solid component term could be discarded because in their case they measured the absorbance relative to a cell-free solid blank. The technique of Hong et al. (1987) suffers in several considerations. It is only applicable if the solids concentration does not change during the fermentation or if the contribution of the solids to the measured absorbance is much less than that of the cells. The technique also requires dilution and a dry weight analysis, and it thus cannot be used as an on-line technique. Since the solid concentration will change significantly in most fermentations and the solids present in most industrial fermentation media will absorb light strongly, this technique is of narrow applicability. A recent development has been the use of light scatter to estimate the cell concentration during a solid substrate fermentation (Kennedy, 1990; Kennedy et al., 1990a,b, 1989, 1988). The principle used to distinguish between cells and solids was that a t certain "invariant regions" of the light scatter spectrum the light scatter intensity is a function of the cell concentration only and is not influenced by the suspended solids. It is hypothesized that the invariant regions are regions of the light scatter spectrum where the solid substrate absorbs as much light as it scatters. Thus, there is no net effect of a changing solid substrate concentration. The light scatter technique has been tested during the growth of Bacillus subtilis var. sakainesis on fishmeal as the sole nitrogen source. Further work is required to test the effect of cellular morphology and gas bubbles on the technique and to establish the cell concentration range over which the technique functions.
Conclusions The main disadvantage of most of the strategies listed above and summarized in Table I is that they are off-line, usually apply under a limited range of conditions, and are labor intensive. Of all the techniques available, the carbon dioxide evolution rate technique is the easiest to operate on-line. However, this technique primarily measures cell metabolic rate and the limitations of using it as a cell concentration measurement technique must be carefully considered. Techniques able to be used on-line that are based on deconvolution of measured signals, or that exploit differences in the physical properties of cells and solids, are the most likely to meet with success in the future.
Acknowledgment We acknowledge and appreciate the funding and support of the Biotechnology Process Engineering Center (with
National Science Foundation ERC initiative under cooperative agreement CDR 88-03014); the New Zealand Government Department of Scientific and Industrial Research, Industrial Development Wellington; the New Zealand National Research Advisory Council; the Department of Biotechnology, Government of India; and the Central Food Technological Research Institute, Mysore, India.
Literature Cited Agar, D. W. Microbial Growth Rate Measurement Techniques. In Comprehensive Biotechnology; Moo-Young, M., Ed.; Pergamon Press: New York, 1985; Chapter 17, Vol. 4. Aidoo, K. E.; Hendry, R.; Wood, B. J. B. Estimation of Fungal Growth in a Solid StateFermentation. Eur. J.Appl. Microbiol. Biotechnol. 1981, 12, 6-9. Amin, P. M.; Nigam, J. N.; Lonsane, B. K.; Baruah, B.; Singh, H. D.; Baruah, J. N.; Iyengar, M. S. Microbial Biomass Production on Solid Hydrocarbon. Fiolia Microbiol. 1973, 18,49-55. Apel, W. A.; Dugan, P. R.; Filppi, J. A.; Rheins, M. S. Detection of Thiobacillus ferrooxidans in Acid Mine Environments by Indirect Fluorescent Antibody Staining. Appl. Environ. Microbiol. 1976 32 (11, 159-165. Baserga, R. Measuring Parameters of Growth. In Cell Growth and Division-A Practical Approach; Baserga, R., Ed.; IRL Press: Oxford, 1989; Chapter 1. Bayer, K.; Fuehrer, F. Computer Coupled Calorimetry in Fermentation. Process Biochem. 1982, July/August. Boquien, C. Y.; Corrieu, G.; Desmazeaud, M. J. Enzymatic Methods for Determining Populations of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in Pure and Mixed Culture. Appl. Microbiol. Biotechnol. 1989, 30,402407.
Carleysmith,S. W.; Fox, R. I. Fermentor Instrumentation and Control. In Advances in Biotechnological Processes;Mizrahi, A., van Wezel, A. L., Eds.; Alan R. Lias: New York, 1984; pp 1-51. Carrizalez, V.; Rodriguez, H.; Sardina, I. Determination of the Specific Growth of Molds on Semi-SolidCultures. Biotechnol, Bioeng. 1981,23, 321-333. Chattaway, T.; Demain, A. L.; Stephanopoulos, G. N. Use of Various Measurements for Biomass Estimation. Biotechnol, Prog. 1992,8, 81-84. Cheong, K.-H.; Katayama,Y.; Seto, M.; Kuraishi, H. Estimation of Cellular Protein for Monitoring the Cell Growth of Bacteria in a Photo-CrosslinkedPolymer Resin. J. Ferment. Bioeng. 1990, 70 (2), 136-138. Cooney, C. L. Growth of Microorganisms. In Biotechnology; Rehm, H.-J.,Reed, G., Eds.; VCH: Weinheim, 1981;Chapter 2, Vol. 1, pp 73-112. Cooney, C. L.; Wang, H. Y.; Wang, D. I. C. Computer-Aided Material Balancingfor Prediction of FermentationParameters. Biotechnol. Bioeng. 1977, 19, 55-67. Damiano, D.; Wang,S. S.Improvements in Ethanol Concentration and Fermentor Ethanol Productivity in Yeast Fermentations using Whole Soy Flour in Batch, and Continuous Recycle Systems. Biotechnol. Lett. 1985, 7 (2), 135-140. Espejo, R. T.; Ruiz, P. Growth of Free and Attached Thiobacillus ferrooxidans in Ore Suspension. Biotechnol. Bioeng. 1987, 30, 586-592. Findlay,R. H.; King, G. M.; Watling, L. Efficacy of Phospholipid Analysis of Determining Microbial Biomass in Sediments. Appl. Environ. Microbiol. 1989, 55 (ll),2888-2893. Ghaly, A. E.;Kok, R.; Ingrahm,J. M. Growth Rate Determination of Heterogeneous Microbial Population in Swine Manure. Appl. Biochem. Biotechnol. 1989,22, 59-78. Ghosh, S.; Pohland, F. G.; Gates, W. E. Phasic Utilization of Substratesby AerobicCultures. J. WaterPollut.Control Fed. 1972,44 (3), 376-400. Greene, R. V.; Gordon, S. H. Monitoring Solid-SubstrateFermentations by Fourier Transform Infrared-Photoacoustic Spectroscopy. Presented at PACIFICHEM '89 conference, Hawaii, 1989.
380
Grishin, S. I.; Tuovinen, 0. H. Scanning Electron Microscope Examination of Thiobacillus ferrooxidans on Different Support Matrix Materials in Packed Bed and Fluidized Bed Bioreactors. Appl. Microbiol. Biotechnol. 1989,31,505-511. Han, Y. W. Oxygen Requirement for Growth of Candida utilis on Semisolid Straw Substrate. Biotechnol. Bioeng. 1987,30, 672-674. Harris, C. M.; Kell, D. B. The Estimation of Microbial Biomass. Biosensors 1985,1, 17-84. Harris, C. M.; Todd, R. W.; Bungard, S. J.; Lovitt, R. W.; Morris, G.; Kell, D. B. DielectricPermittivity of Microbial Suspensions at Radio Frequencies: A Novel Method for the Real-Time Estimation of Microbial Biomass. Enzyme Microb. Technol. 1987,9,181-186. Hashimoto, S.;Fujita, M.; Baccay, R. A. Biomass Determination in the AnaerobicDigestionof Night Soil. J. Ferment. Technol. 1982,60 (l),51-54. Hong, K.; Tanner, R. D.; Malaney, G. W.; Wilson, D. J. A Spectrophotometric Method for Estimating the Yeast Cell Concentration in a Semi-Solid State Fermentation. Process Biochem. 1987,22(5),149-153. Huang, T.-L.; Han, Y. W.; Callihan, C. D. Application of the Lowry Method for Determination of Cell Concentration in Fermentation of Waste Cellulosics. J.Ferment. Technol. 1971, 49 (6),574-576. Ito, K.; Kimizuka, A.; Okazaki, N.; Kobayashi, S. Mycelial Distribution in Rice Koji. J. Ferment. Bioeng. 1989,68 (l), 7-13. Joglekar, R.; Clerman, R.; Ouellette, R.; Cheremisinoff, P. Biotechnology in Industry-Selected Applications and Unit Operations; Ann Arbor Science: Stoneham, MA, 1983. Ju, N.-H.; Damiano, D.; Shin, C.-S.; Kim, N.-K.; Wang, S. S. Continuous Ethanol Fermentation of Zymomonas mobilis using Soy Flour as aProtective Agent. Biotechnol.Lett. 1983, 5 (12),837-842. Karube, I. Biosensors in Fermentation and Environmental Control. In Biotechnology-Applications and Research; Cheremisinoff,P., Ouellette, A., Eds.; Technomic Publishing Co., Inc.: Basel, 1985;Chapter 11. Kennedy, M. J. The Monitoring and Evaluation of a Solid Substrate Submerged Culture Fermentation. Ph.D. Thesis, Massachusetts Institute of Technology,Cambridge,MA, 1990. Kennedy,M. J.; Wang, D. I. C.; Stephanopoulos,G. N. ABiosensor for the Measurement of Cell Concentration in the Presence of Solid Substrate. Microbial and BiochemicalTechnology, 196th American Chemical Society National Meeting, LOBAngeles, 1988; No. 159. Kennedy, M. J.; Wang, D. I. C.; Stephanopoulos, G. N. The Use of Light Scatter Spectra to Estimate Cell Concentration in the Presence of Solid Substrate. American Institute of ChemicalEngineers 1989 Annual Meeting, San Francisco,CA, 1989;No. 148g. Kennedy, M. J.; Wang, D. I. C.; Stephanopoulos, G. N. Measurement of Solid Particle Concentration in Presence of a Second Particle Type. U.S. Patent 4969741,November 13, 1990a. Kennedy, M.J.; Thakur, M. S.; Wang, D. I. C.; Stephanopoulos, G. N. A Light Scatter Biosensor for Cell Concentration Estimation in the Presence of Solid Substrate. Division of Biochemical Technology, 199th American Chemical Society National Meeting, Boston, MA, 1990b;No. 116. Kleyn, J. G.; Vacano, N. L. Some Effects of Soybeans on Yeast Growth and Beer Flavor. Brew. Dig. 1966,41 (ll),95-99. Kniest, F.M.; van Bronswijk, J. E. M. H.; Schober, G.; Bouma, C. Phosphatases as an Index of Biotic Contamination of Dust. Biotechnol. Tech. 1990,4 (4),269-274. Kurane, R.; Suzuki, T.; Takahara, Y.; Kurita, N.; Miyaji, M. Application of Fluorescent Antibody Staining Technique to Trace the MicroorganismInoculated in Biological Treatment Systems. Agric. B i d . Chem. 1979,43 (lo),2093-2098. Marin-Iniesta, F. New Method for the Characterization of the Microbial Growth in Carrageenan Gel. Acta Biotechnol. 1989 9 (l),55-62.
Biotechnol. Prog., 1992, Vol. 8, No. 5
Marison, I. W.; von Stokar, U. The Application of a Novel Heat Flux Calorimeter for Studying Growth of Escherichia coli W in Aerobic Batch Culture. Biotechnol. Bioeng. 1986,28,17801793. Mink, R. W.; Dugan, P. R. Tentative Identification of Methanogenic Bacteria by FluorescenceMicroscopy. Appl. Environ. Microbiol. 1977,33 (3),713-717. Moreira, A. R.; Phillips, J. A.; Humphrey, A. E. Method of Determining the Concentration of Adsorbed Protein and Cell Biomass in Cellulose Fermentations. Biotechnol. Bioeng. 1978, 20,1501-1505. Omstead, D. R.; Phillips, J. A.; Humphrey, A. E. Indirect Parameter Estimation. In Computer Control of Fermentation Processes; Omstead, D. R., Ed.; CRC Press Inc.: Boca Raton, FL, 1990;Chapter 4. Onken, U.; Buchholz, R.; Sittig, W. Measurement and Instrumentation. In Biotechnology; Rehm, H.-J., Reed, G., Eds.; VCH: Weinheim, 1985;Chapter 29. Phillips, J. A. Novel Sensors for the Monitoring of Fermentation Processes. In Computer Control of Fermentation Processes; Omstead, D. R., Ed.; CRC Press, Inc.: Boca Raton, FL, 1990; Chapter 2. Pirt, S. J. InPrinciples ofMicrobe and Cell Cultivation;Blackwell Scientific Publications: Boston, MA, 1975. Raimbault, M.; Alazard, D. Culture Method to Study Fungal Growth in Solid Fermentation. Eur. J . Appl. Microbiol. Biotechnol. 1980,9,199-209. Ramsey, W. S.; Nowlan, E. D.; Simpson, L. B.; Messing, R. A,; Takeguchi, M. M. Applications of Fluorophore-Containing Microbial Growth Media. Appl. Environ. Microbiol. 1980,39 (2),372-375. Sato, K.; Nagatani, M.; Nakamura, K.-I.; Sato, S. Growth Estimation of Candida lipolytica from Oxygen Uptake in a Solid State Culture with Forced Aeration. J . Ferment. Technol. 1983,61 (6),623-629. Schaffeld, G.; Illanes, A. Determination of Cell Mass in an Insoluble Lignocellulosic Substrate in Submerged Fermentation. Biotechnol. Lett. 1982,4 (lo),667-672. Schuler, M. L.; Tsuchiya, H. M. Determination of Cell Numbers and Cell Size for Thiobacillus jerrooxidans by Electrical Resistance Method. Biotechnol. Bioeng. 1975,17, 621-624. Solomon, B. 0.; Erickson,L. E.; Yang, S. S. Estimation of Biomass Concentration in the Presence of Solids for the Purpose of Parameter Estimation. Biotechnol. Bioeng. 1983,25 (lo), 2469-2478. Steffan, R. J.; Breen, A.; Atlas, R. M.; Sayler, G. S. Application of Gene Probe Methods for Monitoring Specific Microbial Populations in Freshwater Ecosystems. Can. J . Microbiol. 1989,35,681-685. Suzuki, T. Government Industrial Research Institute, Nagoya Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Hirate-cho,Kita-ku Nagoya, Japan. Private communications, 1988. Tateishi, T.; Horikoshi,T.; Tsubota, H.; Takahashi,F. Application of the Chloroform Fumigation-Incubation Method to the Estimation of Soli Microbial Biomass in Burned and Unburned Japanese Red Pine Forests. FEMS Microbiol. Ecol. 1989,62, 163-172. Thierry, A.; Chicheportiche, R. Use of ATP Bioluminescence Measurements for the Estimation of Bioprocess during Biological Humidification. Appl. Microbiol. Biotechnol. 1988, 28,199-202. Tsen, H.-Y.; Chen, M.-H.; Shieh, J.-S.;Wang, S.-J.; Hu, N.-T. Possible Use of a 1.8 kb DNA Fragment for the Specific Detection of Salmonella in Foods. J . Ferment. Bioeng. 1989, 68 (l),1-6. Wang, D. 1. C.; Cooney, C. L.; Demain, A. L.; Dunnill, P.; Humphrey, A. E.; Lilly, M. D. Fermentation and Enzyme Technology; John Wiley and Sons: New York, NY, 1979. Wei, C. J.; Tanner, R. D.; Malaney, G. W.; Charles, M. An OnLine Indirect Measurement Technique for Monitoring Yeast Cell Biomass in Semi-Solid Gels. Process Biochem. 1983, March/April.
Biotechnol. Prog., 1992, Vol. 8, No. 5
Yamada,K.; Yogo, M. Studies on the Utilization of Hydrocarbons by Microorganisms: Part 15: Yeast and Bacterial Cell Production from Paraffin Wax. Agric. Biol. Chem. 1970,34 (2),296-301. Yeh, T.Y.; Godshalk J. R.;Olson, G. J.; Kelly, R. M. Use of Epifluorescence Microscopy for Characterizing the Activity of Thiobacillus ferrooxidans on Iron Pyrite. Biotechnol. Bioeng. 1987,30, 138-146.
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Suggested Additional Reading Pons, M.-N., Ed. Bioprocess Monitoring and Control; Hanser Publishers: New York, 1992. Omstead,D. R. Ed. Computer Control of FermentationProcesses; CRC Press, Inc.: Boca Raton, FL, 1990. Accepted June 10,1992.
