Nobuhiro Aoki

Kinetic evaluation and monitoring of methanogen culture based upon fluorometry

SummaryAs the fluorescent coenzyme F420 (factor 420) is present exclusively in methanogenic bacteria, its fluorescence was measured for Methanobacterium thermoautotrophicum ΔH and Methanobrevibacter arboriphilus A2. Cell extracts from acetone-treated methanogenic bacteria showed the highest intensities of fluorescence under excitation (EXW) and emission wavelengths (EMW) of 404 and 470 nm, respectively. No significant fluorescence was observed in the extracts from non-methanogenic microorganisms such as Escherichia coli. On the basis of fluorescence spectra, the fluorescence of the extract was considered to be caused by the coenzyme F420. In both methanogen cultures, the specific fluorescence change rate was nearly equal to the specific growth rate, and the ratio of the intensity to the cell concentration was almost constant during the logarithmic growth phase. It was thus possible to monitor the cultures of the methanogenic bacteria using the intensity of fluorescence as an indicator.

Colominic acid production from Escherichia coli in a fed-batch culture under the control of ammonium ions using an FIA system

Abstract A fed-batch culture was applied for the production of colominic acid from Escherichia coli . It was found that the copper ion, which is a trace element in medium and an essential element in N -acetylneuraminic acid synthase, affected the colominic acid production and the highest production was obtained using 1.6 mg/ l CuCl 2 . Ammonia formed from nitrogen source utilization greatly affected colominic acid production and only slightly affected the cell growth. A flow injection analysis (FIA) system was fabricated and applied to monitor and control the ammonia concentration in the culture broth. It was found that a concentration of 0.3 g/ l of ammonia was optimal for colominic acid production where 1.9 g/ l was obtained.

Estimation of microbial mass concentration based on fluorometric measurement of cell protein

Abstract To develop a convenient method for the estimation of microbial cell mass, the fluorescence intensity of protein ( F 290 ) was measured in cell extracts from Escherichia coli, Ruminococcus albus , and Aspergillus niger . Cell extracts obtained by sodium dodecyl sulfate (SDS)-boiling treatment after sonication had high F 290 intensities under an excitation wavelength (EXW) of 290 nm and an emission wavelength (EMW) of 340 nm. On the basis of the fluorescence spectra, we confirmed that the fluorescence of the extract was caused by protein in the cell, especially by tryptophan residues in the proteins. Carbohydrate, lipids, nucleic acids, and vitamins from the cells did not inerfere significantly with the F 290 intensity. The intensity and cell mass concentration were related linearly below 0.15 g-cell/ l , and the ratio of the intensity to the cell concentration was evaluated as a constant for each microorganism. It was thus possible to monitor the microbial cultures using the F 290 intensity as an indicator, even in culture systems where optical and gravimetric measurements of cell mass were not effective.