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• W2259782377 abstract "Fermentation systems that utilize insoluble substrates, such as cellulose, are difficult to monitor because few techniques are suitable to analyze solid-state samples. A new technique, photoacoustic spectroscopy (PAS), has been developed and provides information about the UV, visible and JR absorption spectra of solids, gels and other materials not suited for conventional optical analyses (Rosencwaig, 1980a, b; Gerson, Wong and Casper, 1984). PAS has the added advantages of requiring little sample preparation and being non-destructive to the sample. To obtain a PAS spectrum, the sample is placed in a closed cell containing a sensitive microphone. Light is admitted through a window to irradiate the sample. When the sample absorbs light, it becomes heated and warms the surrounding layer of air or gas. The warmed gas expands resulting in increased pressure within the cell. If the light is blocked off, the sample cools and the gas pressure in the cell returns to its original level. Periodically modulating or chopping the light produces gas pressure waves that can be detected as sound. The pitch of the sound corresponds to the modulation frequency, but the intensity depends on the amount of light absorbed by the sample. Light scattering properties of the .sample or optical opacity have virtually no effect in PAS because only absorbed light can produce an acoustic wave. Therefore, the photoacoustic spectrum of a given material is an accurate representation of the optical absorption spectrum of that material. Coincidental with the recent developments in PAS has been the development of Fourier transform (FT) technology, especially for analysis of the mid-JR region (Perkins, 1986). In FTIR, the form of the signal is an interferogram, which is subsequently transformed mathematically into a representative JR spectrum. This allows for rapid data acquisition and processing, which is necessary for application of PAS. Biological materials generally have rather complex chemical structures, which translate into distinct absorption patterns in the mid-IR spectral region. Therefore, FTIR-PAS is ideally suited for analysis of solid-substrate fermentations. Recent experimentation in our laboratory has shown that fungal growth on Solid cellulose discs may be quantitatively determined by monitoring protein absorption bands with FTIR-PAS (Greene, Freer and Gordon, 1988). Cellulose (Whatman #1 filter paper) discs which were impregnated with increasing dry weights of the fungus, Phanerochaete chrysosporikum, exhibited increasing absorbance at 1654 cm(superscript -1) (amide I region )and 1544 cm(superscript -1) (amide II region), These absorbances were attibuted to the peptide backbone of the fungal proteins. By monitoring amide I absorption relative to known quantities of fungal dry weight, it was possible to generate a standard curve. P. chrysosporium, which is cellulolytic, was then grown on filter paper discs. As expected, FTIR-PAS spectra of discs inoculated with a spore suspension exhibited significant increases in amide I and II absorption as a function of culture age. When ‘amide I absorption was converted into fungal dry weight by using the previously described standard curve, a growth curve was generated. The FTIR-PAS growth curve was remarkably similar to one generated by using a Lowry protein assay on the same samples after sonic disruption. FTIR-PAS is subject to instrumental artifacts that can result in data scattering. Lower levels of fungal biomass exhibit such scattering. Although this level of scattering is not of great concern when determining gross fungal biomass, it could introduce significant irreproducibility in attempts to measure lesser constituents, such as secondary metabolites. However, instrumental artifacts may be removed by normalizing to an appropriate internal standard. Ployacrylonitrile (PAN) was selected (Gordon, Greene, Freer and James, 1990).PAN exhibits a sharp absorption peak (2243 cm(superscript -1) far removed from absorptions of biological interest. PAN is easy to apply and uniformly distributes throughout the sample. The reproducibility of FTIR-PAS using PAN for quantitation of microbial biomass on solid surfaces was tested. Four proteins and four microorganisms, selected to cover a range of species and morphologies, were deposited on Millipore filters containing PAN. The use of PAN internal standard greatly enhanced the correlation of the FTIR-PAS assay, routinely enabling biomass measurements to be achieved with less than 10% error. With the degree of accuracy afforded by utilizing the PAN internal standard, it should be possible to measure secondary metabolite production with FTIR-PAS. Peniclillin was selected as a model system. Due to an ester carbonyl and a free carbonyl, the GTIR-PAS spectrum of penicillin exhibited two sharp peaks at 1700 cm(superscript -1) and 1775 cm(superscript -1) A distinct band at 3356 cm(superscript -1) was also observer which was attributed to an NH vibrational stretch. These bands were readily visible when 10-20% penicillin was added to a penicillin-free fungal culture (dry weight basis). In the near future, with the aid of mathematical models for multivariant calibration (a current project in .our laboratory), it should be possible to accurately determine penicillin production at the 1% level from an FTIR spectrum. These results demonstrate the potential ability of FTIR-PAS for analysis of biomass, and constitute a significant advance toward the goal of practical application of the new technique to solid-state assays for microorganisms used in the production of drugs, hormones and other biological agents. With further improvement in technology, FTIR-PAS is potentially a powerful tool for in situ analysis of biological systems." @default.
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• W2259782377 title "Monitoring Solid-Substrate fermentations by Fourier Transform Infrared-Photoacoustic Spectroscopy" @default.
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