Analysis of bioreactor parameters online and offline
- 2. ONLINE SENSORS FOR CELL PROPERTIES 1-spectrophotometry 2-flurometry 3-Culture Fluorescence Intensity OFF-LINE ANALYTICAL METHODS 1-measurement of medium properties 2-Analysis of cell population composition 3-Analysis of protein and RNA 4-Particular protein analysis 5- Flow cytometry
- 3. Unfortunately, there are few instruments for continuous monitoring of cell properties in a bioreactor. The most basic measurement needed is total biomass content or concentration or, better still, active biomass concentration. Although a number of possible methods exist ,no approach has yet been invented which provides such data reliably ,consistently, and for a broad class of organisms and media.
- 4. Optical methods based upon light absorbance or scattering have been investigated widely. A sample stream from the reactor may be circulated through a spectrophotometer. A potential difficulty here is the non-linearity between optical density and bio-mass concentration above O.D.=0.5 or 0.5g biomass /l. consequently, sample stream dilution or a shorter light path may be used for measurement of dense cultures.fig.1 shows a flow-through cuvette design developed by Lim and colleagues which has proved very convenient in a number of laboratories. Alternatively ,probes which can be inserted into the process fluid for optical cell density measurements have been developed.
- 6. The only continuous monitoring strategy so far developed that provides information on the biochemical or metabolic state of the cell population is in situ fluorometry. Ultraviolet light(366 nm wavelength) is directed into the culture. Excited by this incident UV radiation, reduced pyridine nucleotides(NADH and NADPH)fluorescence with a maximum intensity at approximately 460nm.the fluorescence emitted from the culture is measured with a suitable detector such as a photodiode or photomultiplier.
- 8. Culture fluorescence intensity depends on cell density, average cell metabolic state and fluorescence emissions, and light absorption by the medium. Experiments in particular- free media have shown that culture fluorescence measurements provide useful information on biomass concentration, oxygen transfer and reactor mixing times, substrate exhaustion, and metabolic transients
- 9. For example, Einsele and coworkers compared the dynamics for liquid mixing in a 40-liter working volume fermentor agitated mechanically at 200 rpm with the dynamics of mixing plus glucose uptake by yeast cells. For the first measurement, fluoroscence of quinine pulsed into 0.05M sulphuric acid in the reactor was monitored (this solute has approximately the same fluorescent properties as NADH). The results, shown in part (a) of fig.2,exhibit oscillations representative of a periodic circulation pattern in the vessel and provide clear evidence of significant dynamic delays in achieving new steady- state conditions in the reactor.
- 10. Part (b) of fig.2 is the reduced pyridine nucleotide fluorescence from a yeast culture following a pulse of glucose added to the reactor at time zero. Here response time is longer, indicating significant dynamic delay in glucose uptake by the cells.
- 12. In this section we consider some of the measurement principles and methods applied to determine the properties of- ◦ Process fluids ◦ Biocatalysts ◦ Biosorbents • Possible methods span the entire spectrum of analytical chemistry , spectroscopy , and biochemistry, making anything approaching a complete presentation impossible in this context.
- 13. After withdrawing a sample from a bioreactor or separation unit, a solid-liquid separation is accomplished by centrifugation or filtration in order to remove cells and any other particulate matter from the fluid phase sample. The desired measurements in a bioreactor are the concentration of substrate and component influencing rates and the concentration of reaction products and inhibitors. For fermentation, analyses of the carbon and nitrogen sources are often desirable .also it may be necessary or useful to determine the levels of certain ions such as magnesium or phosphorus in the medium.
- 14. Liquid-phase quantitative analysis is based usually upon light refraction (measured with a refractive index detector), absorption of light at a particular wavelength (measured with spectrophotometer) or fluorescence due to excitation at one wavelength and subsequent emission at a longer wavelength(measured with a spectrofluorometer) sugars, for example do not absorb light strongly and do not fluoresce but do alter solution refractive index.
- 15. Finer-scale separation among related compounds by chromatographic methods is also commonly in medium chemical analysis. For example, in analyzing mixture of sugars such as maltose and glucose, the different affinities for these two sugars for the primary amino groups on the surface of the support material in a commercially prepared carbohydrate column is used to separate the sugar in an HPLC(high performance liquid chromatography) apparatus. The different sugars emerge from the column at different times, and they may be then detected and quantified separately using a refractive index detector .
- 16. Analytical methods for cell population can be categorized in much the same ways were mathematical models for cell population kinetics. Measurements of :- Total cell mass Protein RNA DNA Plasmid Cellular ATP- by NMR Total cell mass measurement is done by analysis of the elemental composition of cell including carbon, hydrogen and nitrogen. Automatic analyzers are used to determine ion,N,C,Fe,Mg,P,Ca etc.
- 18. Population-average cell content of particular proteins can be determine in several ways. 1-For enzyme ,activity assays are used to monitor the change in enzyme level during process operation. 2-Individual protein :- (a)By protein chromatography (b)By gel electrophoresis (c)By binding of antibodies –ELISA
- 20. Shown in above fig. are the time courses of activity of several key enzymes in this organism during batch cultivation. Here it is seen that the activity level of enzyme associated with acids production decline late in fermentation, while there is increase an enzyme activity associated with solvent production late in batch. Based upon information of this type ,alterations in metabolism may be more directly correlated with strain and bioreactor operating parameter in order to optimize the organism and process condition.
- 22. Enzyme Linked Immunosorbant Assay. Used to quantify individual protein ,in which radioactive labeled antibodies bind with protein at particular region.
- 23. flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers "high- throughput" (for a large number of cells) automated quantification of set parameters