Unsteady-state operation of continuous fermentor for enhancement of cell mass production

The transient behavior of continuous fermentation is studied concentrating on the time scale intrinsic to the system. The time scale is the time required for the fermentorto reach a stable steady state after the disturbance of cell mass is introduced. When the cell concentration is disturbed from the steady-state value, in particular, at the dilution rate near washout, the transient period becomes extended significantly, and the steady state is resumed sluggishly. This sluggish transient behavior could be turned to an advantage for enhancing the cell mass output rate. The proposed transient operation is a continuous fermentation whereby a positive disturbance in the cell mass is introduced, so that the cell concentration is higher than the steady-state value for an extended transient period. It is shown that a significantly higher cell mass production than that from the steady-state continuous fermentation can be achieved. Simple experiments were performed to demonstrate the improvement of cell (Candida utilis) productivity.     

Continuous production of L-phenylalanine by transamination

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.     

The accountant as triage master: an economist's perspective on voluntary euthanasia and the value of life debate

The author, an economist, rebuts the contention that human life cannot and should not be economically evaluated and argues that such evaluations are made implicitly and inconsistently, resulting in a reduction of human welfare. He presents an economic framework for the analysis of costs and benefits in which the focal point, as in most value systems, is the tradeoff between life and quality of life. Therefore, as the quality of life decreases, society's efforts to preserve life should decrease. If the valuation of life includes self evaluation, then there should be less effort to preserve the life of an individual who wishes to die. Richardson concludes that voluntary euthanasia is a limiting case in which society accepts the individual's valuation of life.     

Low Temperature-Induced Decrease in trans-Delta-Hexadecenoic Acid Content Is Correlated with Freezing Tolerance in Cereals

The effect of growth at 5°C on the trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol was examined in a total of eight cultivars of rye (Secale cereale L.) and what (Triticum aestivum L.) of varying freezing tolerance. In these monocots, low temperature growth caused decreases in the trans-Δ³-hexadecenoic acid content of between 0 and 74% with concomitant increases in the palmitic acid content of phosphatidyl(d)glycerol. These trends were observed for whole leaf extracts as well as isolated thylakoids. The low growth temperature-induced decrease in the trans-Δ³-hexadecenoic acid content was shown to be a linear function (r² = 0.954) of freezing tolerance in these cultivars. Of the six cold tolerant dicotyledonous species examined, only Brassica and Arabidopsis thaliana L. cv Columbia exhibited a 42% and 65% decrease, respectively, in trans-Δ³-hexadecenoic acid content. Thus, the relationship between the change in trans-Δ³-hexadecenoic acid content of phosphatidyl(d)glycerol and freezing tolerance cannot be considered a general one for all cold tolerant plant species. However, species which exhibited a low growth temperature-induced decrease in trans-Δ³-hexadecenoic acid also exhibited a concomitant shift in the in vitro organization of the light harvesting complex II from a predominantly oligomeric form to the monomeric form. We conclude that the proposed role of phosphatidyl(d)glycerol in modulating the organization of light harvesting complex II as a function of growth temperature manifests itself to varying degrees in different plant species. A possible physiological role for this phenomenon with respect to low temperature acclimation and freezing tolerance in cereals is discussed.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Hydroxycinnamoyl-CoA:Putrescine Hydroxycinnamoyltransferase in Tobacco Cell Cultures with High and Low Levels of Caffeoylputrescine

A new hydroxycinnamoyl-CoA:putrescine hydroxycinnamoyltransferase (PHT) was detected in two variant lines of Nicotiana tabacum L. (TX1, TX4) accumulating markedly different levels of caffeoylputrescine. The enzyme accepted only the aliphatic diamines putrescine, cadaverine and 1,3-diaminopropane at a ratio of 100:33:8. Caffeoyl- and feruloyl-CoAs were the best acyl donors. The apparent K_m-values for caffeoyl-CoA and putrescine were near 3 and 10 micromolar, respectively, at the pH-optimum of 10.0. PHT activity was quite similar in low producing TX1 and high producing TX4 cells, while some other biosynthetic enzymes (phenylalanine ammonia-lyase, ornithine decarboxylase) were greatly enhanced in TX4 cells, suggesting that PHT does not catalyze the rate-limiting step in hydroxycinnamoylputrescine formation.     

Photosynthetic apparatus of pea thylakoid membranes : response to growth light intensity

We investigated the effect of growth light intensity on the photosynthetic apparatus of pea (Pisum sativum) thylakoid membranes. Plants were grown either in a growth chamber at light intensities that ranged from 8 to 1050 microeinsteins per square meter per second, or outside under natural sunlight. In thylakoid membranes we determined: the amounts of active and inactive photosystem II, photosystem I, cytochrome b/f, and high potential cytochrome b₅₅₉, the rate of uncoupled electron transport, and the ratio of chlorophyll a to b. In leaves we determined: the amounts of the photosynthetic components per leaf area, the fresh weight per leaf area, the rate of electron transport, and the light compensation point. To minimize factors other than growth light intensity that may alter the photosynthetic apparatus, we focused on peas grown above the light compensation point (20-40 microeinsteins per square meter per second), and harvested only the unshaded leaves at the top of the plant. The maximum difference in the concentrations of the photosynthetic components was about 30% in thylakoids isolated from plants grown over a 10-fold range in light intensity, 100 to 1050 microeinsteins per square meter per second. Plants grown under natural sunlight were virtually indistinguishable from plants grown in growth chambers at the higher light intensities. On a leaf area basis, over the same growth light regime, the maximum difference in the concentration of the photosynthetic components was also about 30%. For peas grown at 1050 microeinsteins per square meter per second we found the concentrations of active photosystem II, photosystem I, and cytochrome b/f were about 2.1 millimoles per mol chlorophyll. There were an additional 20 to 33% of photosystem II complexes that were inactive. Over 90% of the heme-containing cytochrome f detected in the thylakoid membranes was active in linear electron transport. Based on these data, we do not find convincing evidence that the stoichiometries of the electron transport components in the thylakoid membrane, the size of the light-harvesting system serving the reaction centers, or the concentration of the photosynthetic components per leaf area, are regulated in response to different growth light intensities. The concept that emerges from this work is of a relatively fixed photosynthetic apparatus in thylakoid membranes of peas grown above the light compensation point.     

Mode of Action Studies on Nitrodiphenyl Ether Herbicides : II. The Role of Photosynthetic Electron Transport in Scenedesmus obliquus

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI) induces light- and O₂-dependent lipid peroxidation and chlorophyll (Chl) bleaching in the green alga Scenedesmus obliquus. Under conditions of O₂-limitation, these effects are diminished by prometyne and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), both inhibitors of photosynthetic electron transport. Mutants in which photosynthetic electron transport is blocked are also resistant to DPEI under conditions of O₂-limitation. Light- and O₂-dependent lipid peroxidation and Chl bleaching are also induced by 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methoxyphthalide (DPEII), a diphenyl ether whose redox properties preclude reduction by photosystem I. However, these effects of DPEII are also inhibited by DCMU. Under conditions of high aeration, DCMU does not protect Scenedesmus cells from Chl bleaching induced by DPEI, but does protect against paraquat. DPEI, but not paraquat, induces tetrapyrrole formation in treated cells in the dark. This is also observed in a mutant lacking photosystem I but is suppressed under conditions likely to lead to O₂ limitation. Our results indicate that, in contrast to paraquat, the role of photosynthetic electron transport in diphenyl ether toxicity in Scenedesmus is not to reduce the herbicide to a radical species which initiates lipid peroxidation. Its role is probably to maintain a sufficiently high O₂ concentration, through water-splitting, in the algal suspension.     

Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.