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On-Line Assessment of Metabolic Activities Based on Culture Redox Potential and Dissolved Oxygen Profiles During Aerobic Fermentation Simon C. W. Kwong, Lisa Randers, and Govind Rao* Chemical and Biochemical Engineering Program and Medical Biotechnology Center of the Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21228
We report here on the utility of on-line culture redox potential and dissolved oxygen measurements t o identify metabolic changes in fermentation by Corynebacterium glutamicum under aerobic conditions. Metabolic changes were identified by observing discrepancies in the profile of culture redox potential and dissolved oxygen. On the basis of these measurements, we can identify the end of the lag phase, threonine exhaustion, and glucose exhaustion during fermentation.
Introduction In the past two decades, culture redox potential (CRP) measurements in aerobic and microaerobic fermentation have been primarily based on the assumption that extracellular redox potential readings are a function of the intracellular redox environment. Another motivation for these measurements has been that CRP can be used to estimate extremely low levels of dissolved oxygen (DO) (Wimpenny, 1969; Daniels et al., 1970; Kjaergaard, 1976). Many studies have been conducted to investigate the effects of CRP on cell metabolism in microaerobic environments, and it has been observed that different values of CRP during fermentation can lead to changes in endproduct formation (Wimpenny, 1969; Wimpenny and Necklen, 1971; Shibai et al., 1974). These results indicate that optimization of product formation can be achieved by controlling CRP. There are several ways to control CRP. A mechanical method was introduced by Radjai et al. (1984) which varied the agitation rate to control the CRP. Recently, Kwong and Rao (1992) demonstrated that by using reducing agents such as dithiothreitol one can effectively decrease the CRP under aerobic conditions without affecting the dissolved oxygen levels. Although early studies have shown that CRP is related to cellular metabolism, the main focus of these studies has been on the effects of CRP on fermentation performance and intracellular metabolite levels. Few reports exist on the converse, that is, the use of the CRP signal as a source of information about the metabolic state of the fermentation. Among these, the focus has been on CRP changes which are not related to pH and DO in different fermentation systems (Hongo and Uyeda, 1972; Ishizaki et al., 1974;Jacob, 1974;Oktyabr'skii et al., 19Ma). The rationale in these studies has been based on the fact that CRP is normally influenced by both pH and DO, so that any change in CRP under conditions of controlled pH and DO must be caused by cellular metabolism. In the present study, we try to use the same rationale in a quantitative manner. The goal is to interpret the CRP signal in order to assess the metabolic activities under highly aerobic fermentation conditions. It has been shown that there is no thermodynamic equilibrium state in an open biological system (such as fermentation), and the CRP value is considered to be a stationary potential reflecting the redox conditions in the medium (Balakireva et al., 1974). Due to the continuously changing fermentation conditions, it is quite difficult to obtain consistent
CRP values under specific culture conditions. In addition, DO, pH, and microorganisms, as well as the products excreted from them, can also affect the measured CRP values to varying degrees. This indicates that the use of the CRP profile alone is not likely to be of much value. In a highly aerobic environment,oxygenlwateris the major redox couple which dominates the measured CRP. According to Wimpenny (19761, a major redox couple can swamp all other minor redox couples and therefore dictate the measured value of CRP. Consequently, the DO value should correspond to the value of CRP during an aerobic fermentation. Any discrepancy observed between the DO value and CRP may be due to certain metabolic state changes (at constant pH and temperature). On the basis of this assumption, we should be able to assess the metabolic activities during fermentation on the basis of not only CRP itself but also the DO level.
Materials and Methods Briefly, all fermentations were carried out with Corynebacterium glutamicum ATCC 14296 at pH 7.2 and 30 "C in 5-L BioFlo I11 fermentors as previously described (Kwong and Rao, 1991). Data acquisition was on a Macintosh I1 computer. Threonine and glucose were analyzedoff-linewith an HPLC and an enzymaticanalyzer, respectively. Dissolved oxygen, pH, and CRP were measured with appropriate electrodes from Ingold Electrodes, Inc. All CRP values reported here are with respect to the standard hydrogen electrode.
Assumptions and Rationale The basic conjecture of using CRP to identify metabolic state changes during fermentation is based on the observed discrepancies between CRP and DO that occur at different stages of a fermentation. According to the Nernst equation, CRP is linear with respect to logarithmic DO at constant temperature and pH. However, Kjaergaard (19771,Jacob (19741, and other authors (HongoandUyeda, 1972; Ishizaki et al., 1974; Oktyabr'skii et al., 1984a) have shown that DO is not the only factor affecting CRP, and any observed discrepancy always involves metabolic state changessuch as product formation and substrate depletion. In order to quantify this assumption, a model of the CRP profile during fermentation was developed. In this CRP model, we adapted our approach from the work of Wimpenny (1976), who found that the CRP is a measure of a few dominant redox couples which swamp all other
8756-7938/92/3008-0576$03.00/00 1992 American Chemlcal Society and American Institute of Chemical Engineers
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minor potentials and thus determine the value of the measured CRP. Several half-cell redox reactions during a microaerobic or aerobic fermentation can be written as follows: [Reli
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O,(dissolved)
[Oxli + ne-
+ 4H+ + 4e-
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2H20
(2)
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[Reli and [Oxli are particular activities of different unknown reduced and oxidized couples which are dominant during fermentation. By applying the Nernst equation, we can break down the measured CRP of a fermentation as follows:
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20
150
0
0
CRP = E h ( 0 2 ) kpH + Eh(Re) (3) where k is a constant based on the electron balance of eqs 1and 2, &(Re) represents the redox potentials of specific redox couples, and Eh(O2) represents the redox potentials of dissolved oxygen/water redox couples. For constant temperature and pH, the rate of change of redox potential is
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ACRP h E h ( 0 , ) + hE,(Re) (4) During a fermentation, only overall redox potential CRP and DO can be measured. Since DO measurement is a measure of Eh(Oz), the discrepancy between CRP and dissolved oxygen is due to other redox couples which are involved in the changes of the metabolic state during the fermentation. In a practical sense, when the trends of ACRP and ADO are the same, the metabolic states of the cells in a fermentation are possibly the same. On the other hand, when the trends of ACRP and ADO are different, we can expect that the cells are undergoing a metabolic state change. These simple arguments serve as the basis for using CRP to identify metabolic state changes during fermentation.
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Results and Discussion Figure l a shows the profiles of dissolved oxygen (DO) and culture redox potential (CRP) of a 350 rpm fermentation. There are two significant discrepancies between the trends of CRP and DO. One occurs in the first 10 h of the fermentation. Another occurs between 20 and 30 h. When we examined this fermentation in detail, we found that several metabolic state changes are involved. During the first 10 h of fermentation, the DO level remained constant at about 100% saturation. However, the CRP trend was strikingly different. It fell to a minimum a t 4 h and then increased again to a maximum at 9 h. Accompanied with this maximum CRP at 9 h, the dry cell weight started to increase (Figure lb). This indicates that the CRP can be used to identify the end of the lag phase. This observation is not new and was one of the first observations made when early investigations were made on CRP measurements in microbialsystems (Hewitt, 1936). However, the cause of the decrease in CRP remains unknown. In addition, some of our experimental results show no CRP drop at the beginning of the experiment. We believe that the initial CRP drop is dependent on the state of the cell innoculum. After cell growth was initiated, both CRP and dissolved oxygen started to decrease. Another discrepancy in CRP and DO trends occurred at 20 h. The DO dropped to a minimum at 20 h while CRP continued to decrease. Upon examining the off-line data, we observed that this discrepancy occurred when threonine was depleted (Figure
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Figure 1. (a)CRP and DO profiles froma duplicate fermentation at 350 rpm. Vertical lines in all figures show the points (from
left to right) at which growth commences, threonine is depleted, and glucose is depleted,respectively. (b) CRP, DO, dry cell weight (DCW),and glucose concentration profiles from a fermentation at 350 rpm. (c) CRP, DO, and threonine concentration profiles from a fermentation at 350 rpm.
IC).A t this time, amino acid production commences, and in some cases (depending on oxygen availability), lactate formation reaches a maximum (Kwong and Rao, 1991). Similar observations on ammonia and glucose depletions of synchronized cultures of Escherichia coli, Bacillus subtilis, and Serratia marcescens in transition experiments were also reported by Oktyabr’skii (Oktyabr’skii et al., 1984b; Oktyabr’skii and Smirnova, 1986). However, Oktyabr’skii’sexperiments mainly focused on the physical
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characteristics of CRP and do not contain information from a complete fermentation to check the profiles of CRP and DO. Contrary to Oktyabr'skii's reports, we did not observe any discrepancy between CRP and DO during glucose depletion in our fermentation system. However, at the end of the discrepancy between CRP and DO trends (24 h), the CRP value ceased to decrease and started to follow the same trend as the dissolved oxygen and was accompanied by the exhaustion of glucose (Figure lb). In the last fermentation (350 rpm), the DO level ranged between 35 and 100%. The threonine and glucose depletions occurred at a DO level between 35 and 50%. According to the basic assumption stated earlier, at a low DO level, a larger portion of CRP is contributed by the unknown redox couples. This may enhance the discrepancy between DO and CRP (eq 4) and therefore make it more easier to identify metabolic activities during fermentation. In order to confirm the capability of CRPand DO-based identification, we ran a fermentation with a dominant DO level using a higher agitation rate. Figure 2a shows the profiles of DO and CRP from a 500 rpm fermentation. The CRP profile was similar to that from the 350 rpm fermentation. The DO level in this experiment remained between 85 and 100%. Similar to the results of the 350 rpm fermentation, there were two significant discrepancies between the trends of CRP and DO. The first discrepancy signaling the end of the lag phase was easy to identify (Figure 2b). As in the 350 rpm case, the steep CRP drop and subsequent rise at about 10 h coincided with the initiation of cell growth (Figure 2b). As shown in Figure 2a, the DO profile leveled off to about 90% at 18h and then continued to decrease at a slow rate to 88% at 25 h. It then slowly increased again to 90 5% at 30.5 h. From 18to 30.5 h, the DO remained between 88% and 90%. On a plot with a normal scale, this variation is almost undetectable (Figure 2b). On the other hand, while the DO leveled off at 18h, the CRP continued to decrease at a faster rate. This discrepancy between CRP and DO occurred at about 18 h. The discrepancy between CRP and DO at 18 h coincided with the depletion of threonine (Figure 2c). When CRP started to increase at 30.5 h, it corresponded to the depletion of glucose. On the basis of these results, it appears that one can use the combined information in CRP and DO to assess cellular metabolicactivities during fermentation. This is especially the case under highly aerobic conditions where the DO signal does not vary much and the CRP measurement offers greater resolution. The high resolution of this technique is the key for fermentation computer analysis and control, especially for event-driven fed batch fermentations. We are currently conducting a fed batch study using this analysis and will report the results separately. We also note that this technique has the advantage of allowing precise determination of substrate exhaustion on-line. For example, in the 350 rpm experiment, our off-line sampling missed the precise point of threonine depletion (Figure IC),while it happened to coincide with glucose exhaustion (Figure lb). The reverse was true with the 500 rpm experiment where the off-line data shows the precise point of threonine depletion (Figure 2c) but not of glucose (Figure 2b). It is also important to note that threonine analysis is not straightforward, is expensive, and takes a few hours to complete. Glucose analysis is faster, but still requires expensive instrumentation. The approach we have suggested here is simple and inexpensive and is especially valuable for multiple substrate fermentations, such as those using auxotrophs.
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Figure 2. (a) CRP and DO profiles from a fermentation at 500 rpm. (b) CRP, DO, dry cell weight (DCW), and glucose concentrationprofiies from a fermentation at 500 rpm. (c) CRP, DO, and threonine concentration profiles from a fermentation at 500 rpm.
Conclusions On the basis of the agitationlaeration that the culture is subjected to, the DO profile may not be sufficiently sensitive to identify metabolic events on line. However, combined with CRP measurements, the points at which cell growth begins and substrate exhaustion takes place can be precisely identified. For fermentations that are normally carried out under the same conditions, this technique should be valuable for on-line monitoring and control purposes.
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Acknowledgment G.R. acknowledges funding from the National Science Foundation (BCS-8911957and BCS-9157852). We thank Mr. Rui Costa for his assistance.
Literature Cited Balakireva, L. M.; et al. The Redox Potential in Microbiological Media. Biotechnol. Bioeng. Symp. 1974,4,769-780. Daniels, W. F.; et al. The Relationship of Oxidation-Reduction Potential to the Growth Performance of Tissue Culture Media Poised Prior to Incubation Biotechnol. Bioeng. 1970,12,409417. Hewitt, L. F. Oxidation-Reduction Potentials In Bacteriology and Biochemistry; London County Council: London, 1936. Hongo,M.; Uyeda,M. Studieson Oxidation-ReductionPotentials (ORP) of Microbial Cultures. Part IV. Lactic Acid Fermentation by Rhizopus G-36(2). Agric. Biol. Chem. 1972,36(21, 279-284. Ishizaki, A.; et al. Basic Aspects of Electrode Potential Change in Submerged Fermentation. Agric. B i d . Chem. 1974,38(12), 2399-2406. Jacob, H. E. Reasons for the Redox Potential in Microbial Cultures. Biotechnol. Bioeng. Symp. 1974,4,781-788. Kjaergaard, L. Influence of Redox Potential on Glucose Catabolism of Chemostat Grown Bacillus Iicheniformis. Eur. J . Appl. Microbiol. 1976,2, 215-220. Kjaergaard, L. The Redox Potential: Its Use and Control in Biotechnology. Adv. Biochem. Eng. 1977,131-149. Kwong, S. C. W.; Rao, G. The Utility of Culture Redox Potential for Identifying Metabolic State Changes in the Amino Acid Fermentation. Biotechnol. Bioeng. 1991,38, 1034-1040. Kwong, S.C. W.; Rao, G. Effect of a Reducing Environment in an Aerobic Amino Acid Fermentation. Biotechnol. Bioeng. 1992,40,851-857.
Oktyabr’skii, 0. N.; Smirnova, G. V. Changes in the Redox Potential of the Medium for B. subtilis Cultures and Their Connection with the Electrical Parameters of the Cells. Biophysics 1986,31 (3),503-508. Oktyabr’skii, 0. N.; et al. Dynamics of Redox Potential in an E. coli Culture in a Regime of Periodic Cultivation. Biophysics 1984a,29 (5),908-912. Oktyabr’skii, 0. N.; et al. Changes in the Redox Potential in Transitional Processes in Pure and Mixed Cultures of Escherichia coli and Serratia Marcescencs. Biophysics 1984b, 29 (21,325-328. Radjai, M. K.; et al. Optimization of Amino Acid Production by Automatic Self-Tuning Digital Control of Redox Potentid. Biotechnol. Bioeng. Symp. 1984,14,657-679. Shibai, H.; et al. SimultaneousMeasurement of DissolvedOxygen and Oxidation-Reduction Potentials in the Aerobic Culture. Agric. Biol. Chem. 1974,38 (12),2407-2411. Wimpenny, J. W. T. The Effect of Eh on Regulatory Processes in Facultative Anaerobes. Biotechnol. Bioeng. 1969,11,623629. Wimpenny, J. W. T. Can Culture Redox Potential be a Useful Indicator of Oxidation Metabolism by Microorganism? J. Appl. Chem. Biotechnol. 1976,26,48-49. Wimpenny, J. W. T.; Necklen, D. K. The Redox Environment and Microbial Physiology. I. The Transition from Anaerobiosis to Aerobiosis in Continuous Cultures of Facultative Anaerobes. Biochim. Biophys. Acta 1971,253,352-359. Accepted August 24, 1992. Registry No. Oxygen,7782-44-7; threonine, 72-19-5; glucose, 50-99-7.
