Effects of Hydrocarbons on the Morphology of Candida tropicalis pK 233

Candida tropicalis pK 233 exhibited marked morphological changes depending upon carbon sources for growth. Although the yeast showed a typical yeast-like development when grown on glucose, the cells grown on hydrocarbon or ethanol were composed of a mixture of filamentous-form (F-cells) and yeast-form cells (Y-cells). The carbon chain lengths of n-alkanes tested as growth substrates had a significant influence on the ratio of F-cells to Y-cells. Electronmicroscopic observation revealed that a hypha was divided by septa into several cells.

Separation of Y-cells and F-cells was achieved by using a suitable filter cloth. F-cells gave a high Qo₂ value compared with Y-cells when hydrocarbon was used as oxidation substrate, even though there was little difference between the respiratory activities of these two cells measured with glucose.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Dinitrophenol influences the rate and yield of Acetobacter methanolicus during the growth on glucose

Acetobacter methanolicuswas grown on glucose in the presence of dinitrophenol (DNP) under carbon/energy-limited conditions. DNP affected both the growth yield and the growth rate (D_sh) at which the energy generation was shifted from a complete to an incomplete substrate oxidation by using the PQQ-linked glucose dehydrogenase. The more the growth yield was decreased, the higher both the DNP concentration and the growth rate became. At about 0.53 mM DNP, growth was completely stopped. D_sh decreased from 0.21h^?1in the absence of DNP to 0.175 h^?1and 0.075 h^?1in the presence of 0.2 mM and 0.4 mM DNP, respectively. The experimental data are discussed in terms of the limitations in the generation of energy and some stress situations which are exerted by the presence of the uncoupler.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture

The influence of agitation and aeration on growth and on production of glucose oxidase of Asp. niger has been studied. It was found that both rate of growth and glucose oxidase production was higher at an agitation speed of 700 rpm than at 460 rpm. Further increase in speed of agitation resulted in neither a higher rate of growth nor a higher glucose oxidase activity. Total glucose oxidase activity was highest in a medium containing 5% sugar (at an agitation speed of 700 rpm) and did not get higher when the sugar concentration of the medium was increased to 7%. When pure oxygen was bubbled through the culture the rate of growth of the culture (in the linear phase) was 95 mg. mycelial dry wt./100 ml./hr., and only 61 mg. when air was applied. The glucose oxidase activity of oxygenated culture was double the activity of aerated culture. Viscosity of the homogenized culture became higher with higher concentration of mycelia. The viscosity of oxygenated culture was found to be lower than that of aerated culture.     

Hydrogen evolution by a thermophilic blue-green alga Mastigocladus laminosus

The thermophilic blue-green alga (cyanobacterium), Mastigocladuslaminosus isolated from a hot spring, evolved hydrogen gas undernitrogen-starved conditions in light when algal cells were grownin a nitrate-free medium. Cells grown in a nitrate-medium evolvedno detectable hydrogen gas in light. The optimal temperatureand pH for hydrogen evolution were 44–49?C and 7.0–7.5.High activity of hydrogen evolution. 1.6 ml H₂/mg chl.hr, wasinduced when algal cells grown in the nitrate medium were activelyforming heterocysts in the nitrate-free medium in air. Hydrogenevolution in light was depressed by nitrogen gas and inhibitedby salicylaldoxime or DNP. This hydrogen evolution by M. laminosusis attributed to the action of nitrogenase. (Received June 20, 1979; )     

Sul Controllo Della Glicolisi Nel Lievito

Abstract

On the control of carbohydrate utilization in yeast. — The results of a previous investigation showed that in higher plants the stimulating action of 2,4 dinitrophenol (DNP) on oxygen uptake and glycolysis is accompained by a fall of the level of reducing sugars, due to an increase of their respiratory utilization, and thus — according to every evidence — of the rate of hexose phosphate synthesis.

In the present work, the occurrence of a similar phenomenon in yeast (where the inhibiting effect of DNP on glucose uptake is not so much marked as in higher plant tissue) was investigated.

Here again DNP, at a 10^-4M concentration, induced a rapid decrease of the disaccaride trehalose and of glycogen, such as to account for the increased rate of respiration and of fermentation. The ratio between the contributions to CO₂ of Carbons 1 and respectively 6 of glucose was not significantly changed by DNP, which suggests that at least part of the DNP induced increase of glycolysis was mediated by the Embden Meyerhof pathway, and thus that a larger amount of fructose diphosphate was formed in the presence of the uncoupler.

In other experiments the effects of DNP on the dissimilation of C¹⁴ labeled glucose, glycerol and pyruvate to CO₂ and ethanol, and on the incorporation of the radioactive isotope into various fractions, 15 minutes after feeding the labeled substrates, was investigated. It was found that:

1) Glucose and glycerol uptake is not markedly inhibited by DNP at the concentration employed (^10–4M).

2) In the absence of DNP, a considerable portion of the radioactivity fed as glucose or glycerol and taken up by the yeast cells is recovered in the glycogen and trehalose fractions. (35% of the glucose, and 22% of the glycerol taken up). This is also observed for carbons 2 and 3, but not for carbon 1 of pyruvate. This indicates a reversibility of the glycolitic processes comprehended in the region between phospho-enol pyruvate andpolysac-carides; while the pyruvate kinase reaction appears to represent a sharp barrier at the « lower » end of glycolysis.

3) DNP almost completely inhibited the incorporation of C¹⁴ from glucose and glycerol into glycogen and trehalose, although it increased the rate of its dissimilation to CO₂ and ethanol. The total amount of glucose and glycerol transformed in the various metabolites (and thus — according to every evidence — phosphorylated) was somewhat lowered and proteins synthesis severely depressed. These effects are interpreted as due to the uncoupling action of DNP at the mitochondrial level, and to the consequent general decrease of the ATP and UTP levels required for protein and for polysaccharide synthesis.     

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters,F390-A and F390-G,on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum

Intracellular levels of F₃₉₀ (AMP and GMP adducts of the 5-deazaflavin cofactor F₄₂₀) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F₃₉₀ was detected in growing cells, hydrogen starved cells and CO₂ starved cells prior to O₂ contamination. Also, no F₃₉₀ was found in hydrogen depleted cells after O₂ treatment. Exposure of exponentially growing cells and CO₂ starved cells to oxygen lead to the formation of F₃₉₀ species; the increase in the detected amount of F₃₉₀ was coupled to a decrease of the F₄₂₀ level. As soon as anaerobiosis was reestablished F₃₉₀ cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O₂ exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

Effects of the pO2 on swimming activity and food utilization in ophiocephalus striatus

The obligatory air-breathing fish Ophiocephalus striatus, fed on goat-liver, surfaced 906 times, swimming 270 m/day in aerated water (mean pO₂: 151 mm Hg) and 883 times, travelling 325 m/day in non-aerated water (92 mm Hg); surfacing and swimming activities increased below the pO₂ of 74 mm Hg. Hanging duration was more (8.4 hr/day) in the aerated series than that in the non-aerated series (5.9 hr/day). Rates of feeding, absorption and metabolism of either series averaged to 131,126 and 93 (= 0.8 ml O₂/g/hr) g cal/g live fish/day. Conversion rate and efficiency slightly increased from 22 g cal/g/day and 17% in the aerated series. Culturing O. striatus in aerated waters may not be advantageous. Starving groups in the aerated and non-aerated aquaria surfaced 317 times, hung for 14.7 hr, and swam 61 m/day, expending 15 g cal/ g/day (= 0.1 ml O₂/g/hr).     

Comparative Physiology of Respiration in Aquatic Fungi II. The Saprolegniales, Especially Aphanomyces astaci

The endogenous respiration of six members of Saprolegniaceae (Oomycetes), Saprotegnia sp., Thraustotheca sp., Achlya sp., Dichtyuchus sp., Aphanomyces euteiches, and A. astaci were studied in the presence and in the absence of exogenous substrate using conventional manometric techniques. Glucose stimulated the rate of oxygen uptake of unstarved mycelia to some extent in all the fungi. Attempts to increase the weak stimulation of respiration by glucose in A. astaci were not successful. The respiratory quotients of the fungi tested were usually in the range of 0.7 to 0.9 during endogenous respiration, and addition of glucose increased these values more than expected. L-leucine and L-glutamic acid stimulated respiration of A. astaci only when the fugus was starved, and acetic acid and butyric acid were inhibitory. Fructose and acetic acid increased respiration in starved mycelium of A. euteiches while L-leucine and L-glutamic acid had little effect. Antimycin A, HOQNO, HCN, and fluoroacetate strongly inhibited endogenous oxygen uptake by A. astaci. Amytal and azide were also markedly inhibitory while rotenone and CO had little effect. DNP and diphenylamine inhibited respiration at a high concentration but at a lower concentration DNP was stimulatory. In contrast the respiration of Saprolegnia sp. was resistant to cyanide, antimycin A, and HOQNO. Spectrophotometric observations on homogenized mycelia of Saprolegnia sp. and of A. astaci indicated the presence of cytochrome c (551 nm), two b-type cytochromes (557 and 564 nm) and cytochrome a-a₃ (605 nm) all in approximately equimolar concentrations. In both strains CO combined with cytochrome a₃ and an unidentified pigment. A remarkable similarity in the cytochrome system seems to exist between these two strains and some members of the Leptomitales.     

Respiration and glycolysis in the human diploid cell strain WI-38

Assessment of the respiratory and glycolytic capacity of non-growing WI-38 cells shows that, in the absence and presence of added glucose, the mean rates of oxygen consumption were 247 (QO₂ = 5.61) and 208 (QO₂ = 4.73) mμmoles/mg dry wt/hr., respectively. Mean glucose consumption was 225 mμmoles/mg dry wt/ hr. With uniformly labeled ¹⁴C glucose as substrate, 36 mμg atoms of carbon dioxide were produced, corresponding to 15–20% of the total cellular respiration. Mean values for lactate production in the presence and absence of glucose were 345 (Q_L^O2 = 7.85) and 196 (Q_L^O2 = 4.45) mμmoles/mg dry wt/hr., respectively. Human diploid cells in culture age, in the sense that their ability to proliferate decreases with time during serial subcultivation. Studies of their respiratory and glycolytic capacity as a function of the aging process showed that total respiration, glucose respiration and glycolytic capacity were relatively constant for cells in the middle and late passages and indicate that aging in this sense is not directly related to the respiratory and glycolytic capacity of the cell.     

Production and stabilization of cells of Bacillus popilliae and Bacillus lentimorbus

Bacillus popilliae and B. lentimorbus grew most rapidly and to the greatest extent in aerated cultures at 30 to 32 C with oxygen absorption rates of 1 mmole of O₂ per min per liter, or above. The control of pH also increased the maximal populations attained. Media were developed which consistently produced cell populations of about 10⁹ within 24 to 48 hr in aerated cultures of these two species. The acetic acid produced in highly aerated cultures was shown not to be responsible for the rapid loss of cell viability in stationary phase cultures. However, H₂O₂ was very lethal to cells of B. popilliae, and this species is known to have the capacity to produce it. Stationary-phase cells were partially stabilized by reducing the availability of oxygen after 24 hr of incubation on a shaker, and the addition of low levels of glucose further stabilized the cells. The most stable cells were those produced in a medium in which 4% Trypticase (BBL) and 0.1% barbituric acid were incorporated. A high percentage of these cells contained refractile bodies visible under a phase microscope. Although these bodies were not heat-resistant and lacked other characteristics of endospores, cells in cultures containing them had reasonably high viability for extended periods, as compared with those in control cultures.     

Production of fatty acids and lipid by a Candida sp. growing on a fraction of n-alkanes predominating in tridecane

A Candida sp. was grown on a fraction of n-alkanes (dodecane 22%, tridecane 48%, tetradecane 28%) as sole carbon source. The growth rate was increased most markedly by using high concentrations of n-alkanes (16.7% v/v). When grown in a 5 liter fermentor, the yeast reached its highest yield (60 g. of cell dry wt/l) with a concomitant high yield of fatty acids (21 g of fatty acids/l), by using a nitrogen-deficient medium. To achieve good growth, it was essential to use an inoculum (1 part into 10) of rapidly growing cells and beneficial to increase the agitation rate gradually once growth had begun. After 108 hr maximum conversions of substrate to product were: 71.5% (w/w) for alkanes into cells and 24.8% (w/w) for alkanes into fatty acids. Of the, total fatty acids at the end of the fat-accumulating phase of growth 54% were shorter in chain length than palmitic acid (C₁₆H₃₂O₂). When grown on glucose, as sole carbon source, less than 2% of the total fatty acids were shorter than palmitic acid. When n-alkanes were added to cells growing on glucose, short-chain fatty acids (C₁₀ to C₁₄) were synthesized immediately, indicating a derepressed enzyme system for hydrocarbon assimilation and the absence of diauxie. The production of these acids was at the apparent sacrifice of linoleic acid synthesis. In spite of the high conversion ratios, it is concluded that it would be uneconomical to produce fatty acids, even expensive ones such as lauric acid, by microbial transformation of n-alkanes.     

Experimental Studies on the Physiology of the Phycobiont and Mycobiont Ramalina ecklonii By

The lichenized fungus and alga of the fruticose lichen Ramalini ecklonii were isolated into pure cultures. The ascospores of the fungus failed to germinate in less than five weeks incubation in spite of the use of a variety of cultural conditions. The fungus showed a considerable increase in growth on malt extract agar. Both organisms showed a marked tolerance for high concentrations of glucose although growth was quantitatively reduced. The fungus was able to use a variety of carbon and nitrogen sources as well as an extract of algal cells. Cultivation in the absence of biotin and thiamine failed to yield significant amounts of growth. The alga yielded 27 mg of dry weight after three weeks in a synthetic medium under low light intensities. The alga could be grown in satisfactory amounts on CO₂ and inorganic salts with moderate light intensities. Experiments using ¹⁴CO₂ showed the fungus able to incorporate the extra-cellular and intra-cellular products of algal metabolism. The rate of incorporation of extra-cellular products was inhibited by high concentrations of biotin and thiamine. The alga assimilated ^l4CO₂ which was retained by the cells over a period of 14 days, at which time 78 per cent of the activity was insoluble in 80 per cent ethanol. An extract of the fungus labelled with ¹⁴C glucose was partially taken up by the alga and 50 per cent of the label was insoluble in 80 per cent after three days incubation in the light. No lichen acids were found in either the fungal cultures or the algal cultures although large amounts (e.g. 2 liters) of material were extracted and chromatographed. Usnic acid was produced by the intact lichen thallus.     

Removal of inorganic ions from wastewaters by immobilized microalgae

Anabaena doliolum and Chlorella vulgaris immobilized on chitosan were more efficient at removing NO₃ ^–, NO₂ ^p–, PO₄ ^3– and CR₂O₇ ^2– from wastewaters than cells immobilized on agar, alginate, carrageenan or even free cells. Carrageenan-immobilized cells, however, were better at removing NH₄ ⁺ and Ni²⁺. The PO₄ ^3– uptake capacity was significantly increased in cells starved of PO₄ ^3– for 24 h. Agar-immobilized cells, though having good metal and nutrient uptake efficiency, had only a slow growth rate. Chitosan is recommended as an algal support for wastewater detoxification.The authors are with the Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi-221005, India     

The role of sugars in the respiration of differently cultivated green algae

A study of the effect of exogenous sugars (fructose, glucose, arabinose and xylose) on Qo₂ inChioreila pyrenoidosa (A 82) and inScenedesmus obliquus (A 125) showed that the effect of sugars on respiration depends on previous cultivation of the alga. In autotrophically cultivated algae all sugars contribute to the increase of Qo₂, in those cultivated rnixotrophically (with the addition of glucose or together with yeast extract, and with xylose) this occurs only in Scenedesmus when glucose or xylose are applied. The effect of cultivation was apparent in that mixotrophically cultivated algae had larger endogenous reserves of substrates than the autotrophic ones. The exogenous substrate is not preferentially respired and does not affect respiration solely as its substrate. In Chlorella, the effect of different cultivation conditions is more pronounced, the QO₂ being usually higher than inScenedesmes.     

Enantioselective toxicological response of the green alga Scenedesmus obliquus to isocarbophos

Chiral compounds usually behave enantioselectively in phyto‐biochemical processes. Isocarbophos (ICP) is a chiral pesticide that is widely used. To evaluate the toxicological response of ICP and its enantiomers to Scenedesmus obliquus, algal growth, total chlorophyll, total soluble protein, and the superoxide anion radicals (O₂^?‐) were investigated. The microalgae were treated with ICP and its enantiomers at 0.01–10 mg/l for 96 h. The growth of S. obliquus was stimulated at low levels of ICP and its enantiomers (0.01–1 mg/l), but all were inhibited at high concentrations (10 mg/l). The total soluble protein content and total chlorophyll content of the tested green alga S. obliquus gradually increased, depending on the growth of algal cells in the medium. Meanwhile, the content of O₂^?‐ was decreased. Interestingly, the cell number and content of the chlorophylls and protein decreased with increasing levels of concentration, whereas O₂^?‐ increased. Our results indicated that enantioselectivity was observed in the dose–response of ICP and its enantiomers in S. obliquus. The high O₂^?‐ level might lead to the death of S. obliquus. The stimulation of growth suggests a regulatory mechanism that is related to the capability of the algae to adapt to the O₂^?‐. Chirality 24:481–485, 2012. © 2012 Wiley Periodicals, Inc.     

Environmental regulation of enzymes in the microbodies and mitochondria of dark-grown, greening, and light-grown Euglena graclis

Catalase activity is demonstrated histochemically in the microbodies of aerated cultures of Euglena gracilis strain Z grown on inorganic media supplemented with acetate or glucose. Although this enzyme can also be assayed photometrically in cell-free extracts of acetate-supplemented cells, it is below the level of detectability in extracts of glucose-supplemented cells, there being an order of magnitude fewer microbodies in the latter than the former. Even acetate-supplemented cultures (dark-grown, greening, or continuously light-grown) fail to exhibit detectable catalase activity when CO₂ is removed from the air by Ascarite.Negative results were obtained with histochemical techniques considered optimal for the demonstration of cytochrome oxidase; under other conditions, however, a KCN-sensitive enzyme was revealed in the mitochondrial matrix. This (unidentified) enzyme is first observed in mitochondria after 20–24 hr of greening, reaches a maximum intensity at about 48 hr, and becomes undetectable by 72 hr of greening. Poisoning of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in loss of activity of this mitochondrial enzyme.     

Light-dependent hydrogen evolution by Scenedesmus

Summary The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D₃. Cl-CCP at 10^-5M concentration completely inhibited the evolution of H₂ in the dark and increased the apparent rate of H₂ evolution in the light. At 10^-5M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O₂ evolution reappeared after approximately 1 hr of illumination if CO₂ was present. Increasing the Cl-CCP concentration to 5 x 10^-5M led to a maximum rate of photohydrogen production and fully inhibited H₂ evolution, photoreduction and dark H₂ evolution. H₂ evolution was accompanied by a release of varying amounts of CO₂ in the light, as well as in the dark. Dark CO₂ production was stimulated by Cl-CCP. H₂ evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released ¹⁴CO₂ from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H₂ released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O₂ was not inhibited in its capacity to evolve H₂ in the light. These data indicate that the evolution of H₂ in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O₂ by photosystem II.The following abbreviations are used: Cl-CCP = carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DNP = 2,4-dinitrophenol.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission.