Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).     

Continuous transformation of benzaldehyde to benzyl alcohol by Rhodotorula mucilaginosa immobilized in an ultrafiltration cell

Microbiological transformation of benzaldehyde accomplished by the fungus Rhodotorula mucilaginosa immobilized in the ultrafiltration cell was studied. A polysulfone membrane formed on a sintered PVC support was used for the separation of the transformation product from the cellular material. Kinetic investigations have led to results which are typical of continuously fed stirred tank reactors (CFSTR)-the value of the maximum reaction rate (V(max)) and apparent Michaelis constant (K'(m)) are practically independent of the substrate retention time (calculated in terms of the flow intensity value). A strong relationship was found to occur between V(max) and biomass concentration in the reactor. Study of the apparent enzyme stability shows that the decrease in the biocatalyst activity is chiefly caused by penetration of the cells through the membrane. The experimental results were approximated in terms of the adopted mathematical model. Based on this model, the half-lives (t(1/2)) of enzyme activities were determined. The t(1/2) value varies from 35 to 82 days and depends both on the permeate flux through the membrane and on the separation properties of the membrane.     

Electron Transport-Dependent Chlorophyll-a Fluorescence Quenching by O(2) in Various Algae and Higher Plants

A comparison of chlorophyll-a fluorescence in brown algae (Macrocystis integrifolia, Fucus vesiculosis), green algae (Scenedesmus obliquus, Ulva sp.) and higher plants (bean, corn) show differences in the relative fluorescence intensities and induction time courses which characterize each type of plant. These differences are not reflected in either the maximum fluorescence emission in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (F_max) or the nonvariable fluorescence (F_o). Constancy of F_o and F_max suggests functional similarities of photosystem II and associated antennae pigments in the various classes of plants. The time course differences are observed only in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and appear, therefore, to be electron transport dependent. During induction, the peak in fluorescence (F_p) is much lower in all of the algae studied than in the higher plants. Exogenous O₂ strongly quenches F_p in all plants studied and our data indicate that the low F_p in the algae can be partially accounted for by endogenous O₂ quenching.     

Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes

The galactosylation steps in the biosynthesis of galactolipids involve two different enzymes; a UDP-Gal:diacylglycerol galactosyltransferase and a galactolipid:galactolipid galactosyltransferase. Previous localization studies have shown that in spinach these enzymes are located in the chloroplast envelope. Our results with peas (Pisum sativum var Laxton's Progress No. 9) confirm these results and extend the localization by providing evidence that the galactosyltransferases are in the outer membrane of the envelope. The specific activity of UDP-Gal:diacylglycerol galactosyltransferase in outer membrane preparations was 6 to 10 times greater than that exhibited by inner membrane preparations. In addition, using quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was possible to show that the UDP-Gal:diacylglycerol galactosyltransferase activity associated with inner membrane preparations could be accounted for by outer membrane contamination. It is concluded from these results that this enzyme is located predominantly, if not exclusively, in the outer membrane of the envelope. An analysis of the galactolipid products synthesized by the highly purified outer membrane showed that the galactolipid:galactolipid galactosyltransferase is also present, suggesting that this enzyme is also an outer membrane enzyme. The implication of these results is that the final assembly of galactolipids is carried out on the outer membrane of the chloroplast envelope.     

Expression of zein in long term endosperm cultures of maize

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

A Comparison of Pigment-Protein Complexes among Normal, Chlorophyll-Deficient and Senescent Soybean Genotypes

Pigment-protein complexes were isolated from chloroplasts of normal green and several types of chlorophyll-deficient soybeans. The complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparisons were made between normal and chlorophyll-deficient genotypes of the relative amounts of chlorophyll associated with Photosystem I (PSI), Photosystem II (PSII), light-harvesting, and free pigment complexes.

Chlorophyll-deficient genotypes, compared to normal green genotypes, have fewer light-harvesting complexes and a higher ratio of PSII to PSI complexes. Chlorophyll associated with PSII in yellow genotypes is in relatively higher amounts in spite of the fact that these genotypes have much less grana stacking than normal green genotypes. Although PSII activity has been associated with appressed regions of grana in normal plants, our work shows that the association does not always hold true.