Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H ؉ because of respiration and to ATP hydrolysis by the F o F 1 -ATPase. Such current was inhibited by inhibitors of the respiratory chain or F o F 1 -ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.The patch clamp technique is an excellent method to measure ion movement across cell membranes as current (1). An extremely small glass pipette (about 1 m in diameter) is attached to the membranes, and activity of ion translocating proteins (ion channels, ion pumps, or ion transporters) is directly measured. So far, however, this important method has been mainly utilized for studies on animal or plant cells but scarcely for bacterial cells (2). Bacterial cells are usually too small to be measured by this method.Escherichia coli, a Gram-negative bacterium, is the best characterized organism from both biochemical and genetical points of view. Ion pumps and ion transporters in E. coli are biochemically well characterized. Many mutant E. coli cells are available. Thus, genetical manipulations are very easy with this microorganism. Therefore, development of a patch clamp method applicable to E. coli membranes must be extremely valuable. Cells of E. coli are surrounded by an outer membrane and an inner membrane (cytoplasmic membrane) separated by a peptidoglycan layer and a periplasmic space. All of the major ion pumps and ion transporters such as the respiratory chain, F o F 1 -ATPase, various ion transporters, and ion-coupled solute transporters are located in the cytoplasmic membrane. To measure ion translocation via such ion pumps or transporters of the cytoplasmic membrane, we have to overcome the following three hurdles: 1) we have to prepare giant vesicles, the diameter of which must be at least 10 m (this is important to get high success rate and accuracy of measurement), 2) the pipette must be directly accessible to the cytoplasmic membrane, and 3) the substrates or effectors of the ion pumps or transporters must be easily accessible to the active site of the proteins and easily removable from the system.It would be essential to prepare giant protoplasts to overcome the first two hurdles. Many attempts have been made by many research groups to prepare giant bacterial cells or giant protoplasts. So far, however, no giant protoplasts surrounded by cytoplasmic membranes and suitable for patch clamp analysis have been prepared....
Gluconic acid production was investigated using an enzymatic hydrolysate of waste office automation paper in a culture of Aspergillus niger. In repeated batch cultures using flasks, saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). The average gluconic acid yields were 92% (SM) and 80% (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yields were 60% (SM) and 67% (GM) with 80-100 g/l of gluconic acid. When pure oxygen was supplied the production rate increased to four times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM.
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