Effects of oxygen exposure on respiratory activities of Desulfovibrio desulfuricans strain DvO1 isolated from activated sludge

The present study addresses the effects of oxygen exposure on the aerobic and anaerobic respiratory activity of Desulfovibrio desulfuricans strain DvO1. This strain was isolated from the highest sulfate-reduction positive most-probable-number dilution (10(6)) of an activated sludge sample, which had been subjected to 120 h of continuous aeration. Washed cell suspensions of strain DvO1 were aerated at 50% atmospheric oxygen saturation in sulfide-free media for a period of 33 h in the presence or absence of an external electron donor (10 mM lactate). During the aeration periods, samples were removed at intervals for determination of anaerobic INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride]-reducing activity, anaerobic sulfate-reducing activity, and oxygen-reducing activity. The cell suspension aerated in the absence of lactate showed negligible endogenous oxygen reduction rates and therefore did not consume oxygen during the aeration period. In contrast, the cell suspension aerated in the presence of lactate sustained significant rates of oxygen reduction during the entire 33 h aeration period. Despite this, no explicit differences in the potential INT-, oxygen-, or sulfate-reducing activities were evident between the two cell suspensions during the aeration periods. Strain DvO1 remained viable throughout the 33 h aeration periods irrespective of the presence or absence of lactate, however, the oxygen exposure resulted in a dose-dependent reversible metabolic inactivation. Notably, lactate-dependent anaerobic sulfate-reducing activity recovered quickly upon anaerobiosis, and was more oxygen tolerant than lactate-dependent oxygen-reducing activity.     

Effects of perfluorinated oxygen carrier application in yeast, fungi and plant cell suspension cultures

The growth of the yeast Saccharomyces cerevisiae, the fungus Rhizopus nigricans and Nicotiana tabacum cells with perfluorodecalin as an oxygen carrier has been studied. The volumetric mass transfer coefficient (k_La) measured by the dynamic method was higher for the perfluorodecalin oxygenation system than for the conventional aeration system. The results show that perfluorocarbon can be successfully used as an efficient gas carrier, especially for the culture of delicate plant cells. The increase in yeast biomass in the suspension culture aerated by perfluorodecalin was as much as 110% higher than in the culture aerated by air. The fungus R. nigricans grew better when the conventional aeration system was used due to the fact that growth of the mycelium is limited by the transport of oxygen by diffusion in the pellets rather than by interfacial oxygen transport. In the case of isolated tobacco cells, an increase of over 350% in biomass growth was observed for the PFC aeration system.     

Effect of microorganisms on rate of liquid extraction of ethanol from fermentation broths

Liquid extraction is one means of removing metabolic products continuously during a fermentation and so reducing product inhibition. It is known that microbial organisms are attracted to liquid-liquid interfaces, and it is important for the design of extraction systems to establish if this has a detrimental effect on the rate of extraction. The extraction of ethanol from aqueous suspensions of yeast (Saccharomyces cerevisiae) using n- decanol is described in this paper. It was found that the presence of the yeast cells severely reduced the rate of ethanol extraction. The overall mass transfer coefficient was reduced from 5.0 x 10(-6) to 0.7 x 10(-6) m/s. This reduced overall mass transfer coefficient was unaffected by yeast concentration in the range 0.1-20 kg/m(3). The results are consistent with the yeast cells adsorbing to the interface in closely packed layers and preventing mass transfer by simply reducing the available interfacial area. Optical microscope observations confirmed that a yeast layer several cell diameters thick rapidly built up at the interface when a small decanol droplet was added to a yeast suspension.     

A new method of optical detection of yeast acidification power

We describe here a newly developed method for a contact-free optical pH measurement in yeast suspensions supplemented with glucose, and containing the pH sensitive triphenylmethane dye bromocresol green. It is suitable for performing the acidification power test (based on measuring the rate of pH drop of yeast suspension caused by active extrusion of acidity from cells after glucose addition) used for assessing yeast vitality in fermentation industries. Using this methodology we monitored the pH in yeast suspensions in the course of acidification in the pH range of 3.5–5.3. Optical pH measurement allows simultaneous testing of several samples, minimizes the sample volume, simplifies sample handling and reduces the hands-on time in sample processing.     

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells"). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.     

Response of nicotinamide adenine dinucleotide and cytochromes to aerobiosis and anaerobiosis on addition of glucose to saccharomyces cerevisiae

When aerated suspensions of whole yeast cells were either made anaerobic or were supplied with substrate (glucose), changes in absorption occurred which indicated cytochrome reduction. No such change was observed on addition of substrate to anaerobic suspension of yeast. These results were confirmed by obsevations of changes in absorbance at a series of fixed wavelengths corresponding to the reduced absorption peaks of cytochromesa, b andc. NAD* on the other hand was reduced on addition of substrate under both aerobic and anaerobic conditions. These results are discussed in relation to the possible role of NAD* in the Pasteur Effect.     

Mass transfer and mixing rates in fermentation vessels

The effects on mass-transfer and overall mixing rates of varying impeller geometry and operating speed have been studied for flat-bladed turbines in laboratory fermentors, in aerated aqueous solutions, and in unaerated and aerated suspensions (1.6% w/v) of paper pulp. In the absence of suspended solid, oxygen absorption rates could be correlated directly with power input. In the pulp suspension, oxygen absorption at a given power input was influenced by impeller geometry and operating speed. The data for the three-phase system can be correlated by a dimensionless equation relating oxygen-transfer rates and mixing times to the geometrical and operating parameters of the impellers.     

Different characteristics between menadione and menadione sodium bisulfite as redox mediator in yeast cell suspension

Menadione promoted the production of active oxygen species (AOS) in both yeast cell suspension and the crude enzymes from the cells, but menadione sodium bisulfite (MSB) had little effect on the production of AOS in the cell suspension. MSB kept the stable increase in the electron transfer from intact yeast cells to anode compared to menadione, but the electron transfer promoted by MSB was inhibited in permeabilized yeast cell suspension. Menadione promoted oxidation of NAD(P)H much faster than MSB in permeabilized yeast cell suspension, suggesting the oxidative stress due to consumption of NAD(P)H. The proliferation of yeast cells was inhibited by menadione under aerobic conditions rather than anaerobic conditions, and the inhibitory effect was reduced by superoxide dismutase and catalase. The effect of MSB on the proliferation was much smaller than that of menadione. The above facts suggest that harmless MSB promotes the electron transfer from plasma membrane of yeast cells to anode. On the other hand, harmful menadione might promote the electron transfer from cytosol and plasma membrane to anode and dissolved oxygen.     

Studies on in vivo inactivation of gramicidin S synthetase and its retardation

The oxygen-dependent in vivo inactivation of gramicidin S synthetase was investigated in Bacillus brevis ATCC 9999. Inhibitors of energy metabolism and of protein synthesis added to aerated cell suspensions did not provide any protection against inactivation, thus indicating that the process does not depend on energy-yielding metabolism nor on de novo protein synthesis. Organic thiols added to anaerobic long-term incubations retarded synthetase inactivation for several hours, whereas in short-term incubations of previously air-exposed cells they resulted in partial restoration of activity. The in vivo inactivation of the enzyme was found to be accompanied by a parallel drop in intracellular thiols. These results implicate enzyme SH oxidation as a mechanism of in vivo inactivation. Retardation of inactivation was achieved upon addition of utilizable carbon sources (glycerol, fructose, inositol) to aerated cell suspensions in buffer, the degree of stabilization being proportional to the ease of uptake and to the concentration of C source. This effect involves actual consumption of the exogenous C source and is accompanied by lower dissolved oxygen levels in the cell suspension. Pulsed additions of C source could retard inactivation but could not restore partly or fully lost activity. The C-source effect was blocked by the uncoupler dinitrophenol, while dissolved oxygen levels in the suspension remained low. C-source-supplemented cell suspensions incubated under air had a decreased intracellular redox state, as revealed by intracellular SH concentration.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10-20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer (F1H*) and ADP are involved.     

Monitoring the rate of oxygen consumption in plant cell suspensions

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of cellular glutathione elevation on the oxygen enhancement ratio

The radiation responses at various oxygen tensions were evaluated in V79 Chinese hamster cells under conditions where their nonprotein thiols, primarily glutathione (GSH), were elevated by 2-oxothiazolidine-4-carboxylate (OTZ). OTZ, when cleaved by intracellular oxoprolinase, provides the cell with cysteine which stimulates GSH synthesis. A 2-hr pretreatment with 10 mM OTZ elevated GSH to 200% of controls. This elevation in GSH offered no protection to aerated cells; however, for O2 tensions less than or equal to 40,000 ppm modest protection was observed as evidenced by an increase in oxygen enhancement ratio. GSH elevation afforded maximal protection between 1000 and 10,000 ppm O2; however, the extent of protection was relatively small (protection factor = 1.3).     

19F-nuclear magnetic resonance: measurements of [O2] and pH in biological systems.

19F-nuclear magnetic resonance (NMR) has been used to determine both intracellular pH and oxygen concentrations in cell suspensions. Oxygen concentrations in Paracoccus denitrificans and insulinoma cells, RINm5F, in the NMR probe can be monitored directly by 1/T1 measurements of perfluorotripropylamine (FTPA)/lecithin emulsion added to the suspensions. With FTPA oxygen monitoring, we investigated the relative aeration capabilities of two types of NMR chambers. Both normal and transformed eucaryotic cells can be maintained in either chamber for at least 1-2 h at cytocrits of up to 20-25%, with 30% oxygen saturation and cell viabilities of 90-95%. Similar concentrations of procaryotes were maintained aerobic with high FTPA concentrations in the more efficient of the two NMR chambers. A new precursor molecule for the 19F-NMR pH indicator difluoromethylalanine, the para-chlorophenyl ester, has been tested and used in RINm5F cells and P. denitrificans, neither of which hydrolyzes methyl esters.     

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae)

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.     

A rapid method for enumerating Salmonella in milk powders

A one-day procedure for enumerating Salmonella in milk powder is described. Milk powder suspension was treated with a mixture of trypsin and Tween 80, then centrifuged to give a sediment of microbial cells that was plated on the xylose lysine desoxycholate agar for the direct enumeration of Salmonella. Known populations (1–200 cfu/25 g of sample) of salmonellas inoculated into milk powder suspensions were recovered with almost 100% efficiency in each of 152 trials of the method, covering 17 different serotypes.     

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.     

The possible use of PKZh-type devices (liquid purity control devices) in the analysis of cell suspensions

PKZh means "device for liquid purity control". A possibility is considered to use the native PKZh type device for carrying out quantitative analyses of cellular suspension components, for routine bacterial suspension, agglutinated bacterial suspension and erythrocyte suspension. The flowing photometric principle of particle recording, used in the device, allows to analyse biological suspensions with small amounts of components. The device provides a differential count of some cells and their conglomerates in six dimensional ranges, within the frames of 1-25 micron or higher. The time consumption for one sample analysis is 10-15 seconds.     

Oxygen transfer properties of a bioreactor for use within a nuclear magnetic resonance spectrometer

Nuclear magnetic resonance (NMR) spectroscopic analysis of whole cells is an important emerging technique for noninvasive and nondestructive monitoring of cell physiology. However, this technique requires extremely high cell densities. Attempts to maintain densities above the carrying capacity of a maintenance system result in the demise of the entire culture. To define conditions for maintaining mammalian cells at high densities for NMR studies, we have designed a bioreactor to operate under defined, oxygen-limited conditions within an NMR spectrometer. The bioreactor utilizes hollow fibers to deliver nutrients and remove wastes from an agitated cell suspension. The mass transfer properties of the fibers with respect to oxygen were determined. Ehrlich Ascites Tumor (EAT) cells were supplied with glutamine as the respiratory carbon source. The maximum viable cell density supported by a given oxygen concentration in the fluid flowing through the fiber lumen was predicted and then confirmed experimentally on the bench and in the spectrometer.     

Turbulent flow of red cells in dilute suspensions. Effect on kinetics of O2 uptake.

The turbulent flow properties of dilute (0.06% by volume) suspensions of human red blood cells in 4-mm-bore glass tubing were estimated by laser anemometry. The flow properties of the dilute red cell suspension were similar to those of a dilute suspension of polystyrene spheres (0.5 micron diameter) in isotonic NaCl solution. Flow was found to be laminar when the Reynolds number was below 2,000, transitional in the range of Reynolds numbers from 2,000 to 3,000, and fully turbulent above Reynolds number 3,000. These results differ from previous studies of more concentrated red cell suspensions. The length scales of the turbulence were also estimated: at a Reynolds number near 4,000 the macroscale is about 1.25 mm, the Taylor microscale is about 0.85 mm, and the Kolmogoroff scale is near 0.075 mm. The results are discussed in relation to previous measurements of the rate of oxygen uptake by dilute red cell suspensions in the flow-type rapid reaction apparatus. Our results suggest that under the conditions of most of these oxygen uptake measurements, the turbulent flow is characterized by eddies about 1 mm across, mixing with each other on a time scale of about 45 ms. Since most of the reported oxygen uptake measurements involve a similar time scale, it is possible that an effective "unstirred layer" influenced the reported rate of oxygen uptake.