Near-infrared spectroscopy for bioprocess monitoring and control.

This article describes the calibration of a spectroscopic scanning instrument for the measurement of selected contaminants in a complex biological process stream. Its use is for the monitoring of a process in which contaminants are to be removed selectively by flocculation from yeast cell homogenate. The main contaminants are cell debris, protein, and RNA. A low-cost instrument has been developed for sensitivity in the region of the NIR spectrum (from 1900 to 2500 nm) where preliminary work found NIR signatures from cell debris, protein, and RNA. Calibration models have been derived using a multivariate method for concentrations of these contaminants, such as would be found after the flocculation process. Two strategies were compared for calibrating the NIR instrument. In one case, samples were prepared by adding materials representative of the contaminants to clarified yeast homogenate so the contaminant levels were well known but outside the range of interest. In the other case, where samples were like those from the process stream after flocculation and floc removal, there was uncertainty of analysis of contaminant level, but the calibration was in the range of interest. Calibration using process stream samples gave results close to those derived from traditional assays. When the calibration models were used to predict the contaminant concentrations in previously unseen samples, the correlation coefficients between measurements and predictions were above 90% in all cases but one. The prediction errors were similar to the errors in the traditional assays.     

THE EFFECT OF GROWTH ILLUMINATION ON THE PIGMENTATION OF A MARINE DINOFLAGELLATE12

Amphidinium klebsii cultures grown under different light intensities exhibited similar chlorophyll a content per cell. Among the accessory pigments, chlorophyll c concentration decreased slightly in cells exposed to increasing light intensities up to 0.129 ly/min. The concentration of the 2 major xanthophylls present in A. klebsii cells–peridinin and diadinoxanthin–however, varied according to the light background of the cells. Some biochemical pathways in the formation of peridinin in dinoflagellates are discussed.     

2-Deoxy-D-galactose metabolism in ascites hepatoma cells results in phosphate trapping and glycolysis inhibition.

The metabolism of 2-deoxy-D-galactose has been studied in AS-30D rat ascites hepatoma cells in suspension. Using 2-deoxy-D-(1-14C)galactose and an alkaline ethanol deproteinization procedure, the quantitatively identified metabolites included 2-deoxy-D-galactose 1-phosphate comprising 99.3%, and UDP-2-deoxy-D-galactose and UDP-2-deoxy-D-glucose, together amounting to 0.4% of the total metabolites. After incubation for 5 h in the presence of 2-deoxy-D-galactose (1 mmo1/1), the content of 2-deoxy-D-galactose 1-phosphate reached 35 mmo1x(kg cells)-1. The rate of phosphorylation of 2-deoxy-D-galactose was rapid during the first 30 min and decreased to approximately 20% of this rate during the subsequent hours. The rapid trapping of Pi in the form of 2-deoxy-D-galactose 1-phosphate resulted in a depression of free intracellular Pi in spite of a concomitant increase in net 32Pi uptake from the medium and a decrease of ATP and other 5'-nucleotides. The rates of glucose utilization and lactate production were depressed by more than 80% in the presence of 2-deoxy-D-galactose (1 mmo1/1). Interruption of Pi trapping by removal of 2-deoxy-D-galactose from the medium reversed the depressions of Pi and ATP and resulted in a rapid but incomplete relief of glycolysis inhibition. Crossover analysis of glycolytic intermediates indicated an inhibition at the 6-phosphofructokinase step. The depression of glucose utilization may be mediated by the increased level of glucose 6-phosphate, a potent inhibitor of hexokinase. An additional inhibitory effect of a metabolite of 2-deoxy-D-galactose at the 6-phosphofructokinase step was indicated by crossover analysis after reversal of Pi and ATP depressions in the presence of a high intracellular content of 2-deoxy-D-glactose 1-phosphate. The quantitative analysis of the metabolites of 2-deoxy-D-galactose demonstrated the predominance of the monophosphate and the negligible formation of UPD derivatives of this sugar analog in AS-30D hepatoma cells. This provides a system for the investigation of a galactose analog as a phosphate-trapping agent in the virtual absence of uridylate trapping.