Culture Fluorescence

NAD(P)H-dependent culture fluorescence has mainly been exploited for metabolic investigations, e.g. [199,227,339-341,410]. The signal is sensitive to variables such as substrate concentration or oxygen supply. Thus, all attempts to exploit this signal as a biomass sensor [478] have been limited to conditions where no metabolic alterations occur [257, 395, 396]. It is well known that a mechanistic or causal-analytical interpretation of the signal trajectory in secondary metabolite cultivations can be very difficult [303]. 

The outstandingly rapid principle of fluorescence measurements served excellently for the controlled suppression of ethanol formation during continuous baker s yeast production [280]. 

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It was known that the intracellular concentrations of the reduced and oxidized forms of the pyridine nucleotides vary in different cell types and under different cell culture conditions.(17) Harrison and Chance applied the NAD(P)H fluorescence technique and found that culture fluorescence can be related to the metabolic state of the cells. 18,19) Since then, more than a hundred papers on NAD(P)H fluorometry have been published. However, they are primarily divided into three major categories ... 

Most of the studies05,20 271 show that a correlation between culture fluorescence and biomass concentration can be obtained mainly in the exponential growth phase. In addition, in order to obtain reproducible correlations, all of the fermentation conditions such as initial substrate concentration, pH, dissolved oxygen level, temperature, and agitation rate have to be the same. However, once the culture is past exponential growth, biomass measurement by following culture fluorescence is no longer accurate. 

Walker and Dhurjati1421 have used culture fluorescence for on-line discrimination of host and plasmid-carrying strains of Escherichia coll In addition, culture fluorescence has also been used in the control of fed-batch fermentation on yeast cell production/29, 431... 

T. Scheper and K. Schugerl, Culture fluorescence studies on aerobic continuous cultures of Saccharomuces cervevisiae, Appl. Microbiol. Biotechnol. 25,440-444 (1986). 

W. Beyeler, A. Einsele, and A. Fiechter, On-line measurements of culture fluorescence Method and application, Eur. J. Appl. Microbiol. Biotechnol. 13, 10-14 (1981). 

J. H. T. Luong and D. J. Carrier, On-line measurement of culture fluorescence during cultivation of Methylomonas mucosa, Appl. Microbiol. Biotechnol. 24, 65-70(1986). 

H.-P. Meyer, W. Beyeler, and A. Fiechter, Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile. 

T. Scheper, T. Lorenz, W. Schmidt, and K. Schugerl, On-line measurement of culture fluorescence for process monitoring and control of biotechnology processes, Ann. NY Acad. Sci. 506, 431—445... 

D. W. Zabriskie, Use of culture fluorescence for monitoring of fermentation systems, Biotechnol. Bioeng, Symp. 9, 117-123(1979). 

D. W. Zabriskie and A.E. Humphrey, Estimation of fermentation biomass concentration by measuring culture fluorescence, Appl. Environ. Microbiol. 35, 337-343 (1978). 

E. L. Winter, G. Rao, and T. W. Cadman, Relationship between culture redox potential and culture fluorescence in Corynebacterium glutamicum. Bioteclmol. Tech. 2, 233-236 (1988). 

G. Rao and R. Mutharasan, NADH levels and solventogenesis in Clostridium acerobutylicum new insights through culture fluorescence, AppL Microbiol Biotechnol. 30, 59-66 (1989). 

K. F. Reardon, T.-H. Scheper, and J. E. Bailey, Metabolic pathway rates and culture fluorescence in batch fermentations of Clostridium Acetobutylicum. Biotechnol Prog. 3, 153-167 (1987). 

C. C. Walker and P. Dhurjati, Use of culture fluorescence as a sensor for on-line discrimination of host and overproducing recombinant Escherichia coli. Biotechnol. Bioeng. 33, 500-505 (1989). 

S. P. Srinivas and R. Mutharasan, Inner filter effects and their interferences in the interpretation of culture fluorescence, Biotech, and Bioeng. 30, 769-774(1987). 

Incubation of Pseudomonas putida with anthracene-labeled carbon-base ferrichrome analog Fe(lll) complex 173 resulted in cellular iron uptake and the appearance of anthracene fluorescence in the culture medium identical to the Fe-ferrichrome uptake. Incubation with the alanyl analog 174 failed to show any significant iron uptake or fiuorescence. This is consistent with the tests described above on the unlabeled analogs. Remarkably, other strains such as Pseudomonas fluorescens S680 or WCS3742 also did not show any iron uptake or culture fluorescence. 

Based on these devices, different biomass estimation experiments were performed based on the culture fluorescence monitoring and feeding strategy studies were developed as well as bioreactor characterizations via mixing time experiments. During the next years smaller fluorescence probes were developed which could be interfaced with bioreactors via standard electrode ports. These open end detector systems measured the fluorescence fight in the backward di-... 

Zabriski and colleagues 145, 46] first used culture fluorescence as an on-line estimate of viable biomass during the batch cultivation of Saccharomyces cere-visiae, a species of Streptomyces, and a species of Thermoactinomyces. They simply linearized the fluorescence to biomass data in order to find a direct function between NADH-dependent culture fluorescence and biomass concentration in the bioreactor. In the following years several other authors reported - on the basis of these results - on the estimation of biomass concentration from culture fluorescence data as shown in Table 1. 

Table 1. On-line estimation of the biomass concentration for different microorganisms by using culture fluorescence ...

Different simple correlations between biomass and fluorescence data showed that on-line estimation is possible under strictly defined culture conditions. Even a strictly finear relation between biomass and culture fluorescence was found for the growth of Zymomonas mobilis, Methylomonas mucosa, and Pseudomonas putida under non-limited conditions [47]. 

While most appfications were performed in suspended cell cultures some authors showed that the application of NADH-dependent fluorescence monitoring is also possible in immobifized cell systems. Here the growth of Clostridium acetobutylicum and the Saccharomyces cerevisiae immobilized in different calcium alginate structures was studied. However, calibration of the culture fluorescence signal with the biomass concentration was not possible but qualitatively an increasing biomass also led to an increase in the fluorescence signals. 

In conclusion it can be stated that accurate biomass estimation based on the culture fluorescence monitoring is possible, when the NAD(P)H-pool per cell is... 

The different applications listed in Table 2 show that culture fluorescence offers the opportunity to have non-invasive insight not only into the fluorophor behavior of the medium but also in the metabolic state. It gives information about the redox status of the cultivation, but still the danger of interference cannot be excluded. Thus, the interpretation of the data measured is often complicated and only successful for a standard cultivation process [51-65]. 

Fig. 4. Courses of culture fluorescence diu-ing a high-cell-density cultivation of . coli...

Fluorescence sensors have been used since 1957 to measure cell internal NAD(P)H at 450 nm. Later on they were applied for in situ determination of the cell concentration. However, the culture fluorescence intensity is not only influenced by the cell concentration, but also by the physiological state of the cells [56] and, in addition to that, there are several other compounds that participate in the fluorescence emission besides NAD(P)H. To identify the fluorophores in the cells and cultivation medium, the excitation and the emission wave lengths are varied in a broad range [57,58]. Two instruments were applied for the 2D-fluorescence spectroscopy Model F-4500 (Hitachi) and the BioView Sensor (Delta light Optics). Each of them uses an excitation range of 250-560 nm,an emission range of 260/300-600 nm and the measuring time of 1 min [59,60]. The application of this technique for CPC production was performed by Lindemann [61]. 

NAD(P)H concentration in biomass and its ratio to NAD(P) gives a measure for the culture reduction status [35]. NAD(P)H can, in principle, be detected directly in vivo by in situ fluorescence measurement (see the chapter by Sonnleitner in this volume - the section on culture fluorescence). This measure serves as a biomass concentration sensor if the specific NAD(P)H concentration stays constant. If not, the ratio of culture fluorescence and biomass concentration, the specific fluorescence, can be a measure for the culture reduction state, or indicate other more complex events like metabolic pathway shifts [35] or even the formation of a variant in a culture [36] (see also Figs. 2 and 3). 

Fig. 2a-c. Growth of Bacillus stearothermophilus PV72 in continuous culture on a synthetic medium containing glucose (8 gH) as the sole carbon and energy source. The dissolved oxygen concentration was controlled at 50 % and the dilution rate was 0.3 h-1. As derived from the measured process variables, variant formation started at about 15-16 h after inoculation. Shown are the measures for a Respiration activity b External and internal reduction state (redox potential and culture fluorescence) c Cell density (Reprinted from J. Biotechnol. 54, K.C. Schuster et al., p. 19,1997, with permission from Elsevier Science)... 

As indicated in Table 6, the biochemical sensor can be used for intracellular activities, which are closely related to the level of key intermediates such as NAD/NADH and ATP/ADP/AMP. Only one sensor for monitoring on-line NADH on the intracellular level is commercially available (BioChemTechnology, Malvern, PA). The Jluorometer sensor can measure continuously the culture fluorescence, which is based on the fluorescence of NADH at an emission wavelength of 460 nm when excited with light at 360 nm. The sensor response corresponds to the number of viable cells in the lower range of the cell concentration. It should be especially noted that the sensor reflects the metabolic state of microorganisms. 

However, since the adenine dinucleotides are important coenzymes for several anabolic and catabolic reactions in the cell, the NAD(P)H pool changes when the metabolism of the microorganisms is affected. These changes as well as some abiotic factors (eg, bubbles or fluorescent components in the medium) could cause problems during biomass estimation with culture fluorescence. 

The more analytical tools that are available and the better the understanding of critical biochemical pathways, the more rapidly fermentation processes can be developed. Besides those previously mentioned, a munber of different parameters have been monitored on-line in fermentation development [7], including exhaust gas analysis and gas fluxes [46], cell density [47], redox potential [48], IR [49], culture fluorescence [50], biological activities [45 ], and viscosity. It is important to iterate that small-scale fermentation studies should aim to develop relatively simple control systems that are easily scaled. As an example, although HPLC systems are routinely set-up on line to measure and control laboratory scale fermentations, the robustness of such a system and its utility in a manufacturing facility remains debatable. 

A recent development has been the use of two-dimensional fluorescence spectroscopy as a new method for on-Hne monitoring of bioprocesses [108]. As ergot alkaloids fluoresce, the formation of the product during cultivation can be observed by two-dimensional fluorescence spectroscopy. Substraction spectra offered on-line real time information about the productivity during the cultivation. It was possible to follow the biomass concentration on-line by monitoring the culture fluorescence intensity in the region of riboflavine and its derivatives. This is a powerful application of this new sensor since the on-Une determination of biomass is extremely complicated for this fimgus. 

Key Words Anaplasma phagocytophilum, granulocytic anaplasmosis cell culture fluorescent labeling neutrophils. 

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