Anaerobic growth,ergosterol content and sensitivity to a polyene antibiotic,of the yeastSchizosaccharomyces japonicus

WhereasSaccharomyces cerevisiae when grown in continuous culture under anaerobic conditions requires ergosterol,Schizosaccharomyces japonicus (syn.Sch. versatilis) can grow without this substance; sporulation is under anaerobic conditions hardly less prolific than under aerobic conditions.The ergosterol content of anaerobically grown cells ofSch. japonicus was only 0.01 %, that of aerobically grown cells 0.24%. Under anaerobic conditions, cell growth was hardly affected by the polyene antibiotic pimaricin; under aerobic conditions,Sch. japonicus is about as sensitive asS. cerevisiae. Qualitatively, the antagonistic effect of ergosterol on pimaricin action is the same inSch. japonicus and inS. cerevisiae.The relation between ergosterol content and the polyene sensitivity strongly confirms existing views (see Kinsky, 1967) on the mechanism of action of polyene antibiotics.The skilful technical assistance of Miss Marry Reinink is gratefully acknowledged.     

Anaerobic respiration of fumarate as a differential test betweenCampylobacter fetus andCampylobacter jejuni

The effect of formate and of various electron acceptors—fumarate, aspartate, or nitrate—on the growth of 36 catalase-producingCampylobacter strains was quantitatively investigated in a semisynthetic medium, under aerobic (5% oxygen, 10% carbon dioxide, 85% nitrogen) or anaerobic (10% carbon dioxide, 90% nitrogen) conditions. Under anaerobic conditions, 24 strains ofC. jejuni failed to grow, or grew poorly, in the presence of fumarate, whereas ten strains ofC. fetus subsp.fetus and two strains ofC. fetus subsp.venerealis grew abundantly, rather better than under aerobic conditions. The quantitative differences of growth yields were very significant between the two species with fumarate, but less pronounced with aspartate or nitrate. The activity of fumarate-reductase inC. fetus was substantiated by identification of relevant metabolites by gas liquid chromatography and by mass spectrometry. The anaerobic fumarate respiration in the genusCampylobacter has taxonomic implications.     

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration

Cells of Escherichia coli O157:H7 were heat-treated at 59 °C and enumerated in (i) anaerobic medium with a low redox potential, (ii) anaerobic media with the oxidizing agents potassium ferricyanide or 2,6-dichloroindophenol (DPIP) added to raise the redox potential, (iii) aerobic medium with a high redox potential and (iv) aerobic medium with the reducing agent dithiothreitol added to lower the redox potential. The measured heat-resistance was greatest when the enumeration medium was highly anaerobic due to the absence of oxygen and the presence of hydrogen and cysteine HCl. Measured heat resistance was influenced by the redox potential of the enumeration medium independently of the chemical used to adjust it and therefore, independently of the presence of oxygen. Sub-lethally heat-damaged cells regained their ability to grow in media of high redox potential at a similar rate whether the redox potential was increased by the addition of potassium ferricyanide, DPIP or oxygen.     

Specific factor inducing cell division of the algaChlorella pyrenoidosa and of the yeastCandida utilis

From cells of the green algaChlorella pyrenoidosa and of the yeastCandida utilis a substance was isolated which stimulated cell division in these microorganisms. The synthesis of this division-inducing factor (DIF) takes place inChlorella pyrenoidosa during the first half of the development cycle. During the second half of the ontogenetic development it is not possible to restore the inhibited division processes by its application. DIF increases aerobic respiration and aerobic and anaerobic glycolysis. Under aerobic conditions, in the presence of a source of energy and of DIF, cell division takes place without simultaneous synthesis of cell mass. Consequently, the substance can separate the processes of growth and division in that division can take place without growth.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

Aerobacter aerogenes No. 505 isolated from soil by Uyeda produced l-valine extracellularly by an aerobic shaking culture. Under anaerobic conditions the production of this amino acid was inhibited while lactic acid as well as a small amount of alanine were produced. The changes in ORP during the incubation under both conditions were investigated. When l-valine was the main product under aerobic conditions the ORP showed a constant value (rH 8.0) from 16 to 40 hr after inoculation. But when lactic acid was the main product and alanine was produced as the only amino acid under anaerobic conditions, the ORP drifted to rH 0 (zero). The phenomenon of the conversion of fermentation was shown clearly by the ORP of the culture broth.

The endpotentiai of lactic acid fermentation by Rhizopus G-36 was rH 13 to 14 when measured in the presence of trace amounts of redox dye mixtures. Without dyes, the rH was 18 to 22 and this fungal culture was slower in reaching endpotentials than bacterial cultures. It was postulated that the amount of redox substances exhibiting electromotive activity was not sufficient in this culture.

rH value 13 to 14 was not obtained under such conditions that lactic acid was not produced; that is in a medium with higher concentration of the nitrogen source in the presence of Fe²⁺ and Zn²⁺, or in a medium containing acetate in place of glucose as the carbon source.

Mycelia of Rhizopus G-36 after 36 hr-culture produced lactic acid even in the absence of oxygen. But unexpectedly, the ORP under anaerobic secondary culture was exactly the same as that in the aerobic shaking culture (rH 13.2).

A method for homogenization of the culture without secondary oxidation was improved. The ORP of anaerobically homogenized cultures was rH 11, and was thought to be due to the activities of all redox systems in the mycelium.

The respiration system of this strain was switched from cytochrome system to flavin system at the point of change in KGN-sensitivity. The ORP of this strain may be influenced by respiration through the flavin system.     

Transport of glucose intoSaccharomyces cerevisiae RXII

Evidence is submitted that glucose is found in a chemically unaltered form in cells ofSaccharomyces cerevisiae RXII incubated with glucose, if the culture is grown under aerobic conditions at 30°C and is in the logarithmic phase of growth at the moment of harvesting. Under these conditions, the course of formation of a glucose steady state can be studied under aerobic and anaerobic incubation conditions. The steady state glucose concentration in the cells is the linear function of the glucose concentration in the medium.     

Bacterial reduction of hexavalent chromium

Summary Cr(VI)-reducing bacteria are widespread and Cr(VI) reduction occurs under both aerobic and anaerobic conditions. Under aerobic conditions, both NADH and endogenous cell reserves may serve as the electron donor for Cr(VI) reduction. Under anaerobic conditions, electron transport systems containing cytochromes appear to be involved in Cr(VI) reduction. High cell densities are necessary to obtain a significant rate of Cr(VI) reduction. Cr(VI) reduction by bacteria may be inhibited by Cr(VI), oxygen, heavy metals, and phenolic compounds. The optimum pH and temperature observed for Cr(VI) reduction generally coincide with the optimal growth conditions of cells. The optimum redox potential for Cr(VI) reduction has not yet been established.     

Verification of enhanced phosphate removal capability in pure cultures of<Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> under anaerobic/aerobic conditions in an SBR

Laboratory experiments were conducted using pure cultures ofAcinetobacter under anaerobic/aerobic cyclic conditions to explain the release and uptake of soluble phosphate in an activated sludge process showing enhanced biological phosphate removal (EBPR). Under anaerobic/aerobic cyclic conditions in a Sequencing Batch Reactor (SBR), COD uptake concurrent with soluble phosphate release byAcinetobacter was not significant during the anaerobic periods, indicating that EBPR would not be established in pure cultures. However,Acinetobacter cells accumulated higher phosphate content (5.2%) in SBR than that obtained (4.3%) from batch experiments. These results suggest thatAcinetobacter sp. may not follow the proposed pattern of behavior of poly-P bacteria in EBPR activated sludge plants.     

Effect of penicillin on the morphology and reproduction ofAzotobacter chroococcum

The effect of penicillin on the morphology and reproduction of some strains ofA. chroococcum was studied on a number of solid media. When the growth was not entirely suppressed by the penicillin, filamentous cells and spheroplasts were formed. The formation of spheroplasts was stimulated by peptone. Gonidia were sometimes formed inside the spheroplasts and also inside giant cells. They were released from the cell after disruption or after lysis of the cell wall. In some cases they produced dwarf cells. Under certain conditions groups of gonidia present in a cell fused and formed one or more normal-looking cells inside the mother cell. Sometimes one or moreAzotobacter cells developed inside a spheroplast or at the site of a spheroplast with a lysed cell wall. Microcolonies consisting of small cocci representing gonidia and dwarf cells were also observed occasionally at the sites of spheroplasts with lysed cell walls. Occasionally tiny groups of small elements with a less marked structure were found at such sites, probably representing debris of lysed cells. The production of normal-looking cells inside filamentous cells was greatly stimulated on a medium containing 10 percent horse serum, with a drop of sterile water containing 200 or 250 I.U. penicillin added in the centre of the plate. The growth ofA. chroococcum was greatly retarded when the medium contained 10 U/ml penicillin and seemed to be checked entirely at concentrations of 20 U/ml penicillin or higher. Occasionally, however, even at concentrations of 100 and 300 U/ml penicillin, a few filamentous cells were found and also a few microcolonies, visible only through the microscope, consisting of gonidia or regenerative rods. By repeated exposure ofAzotobacter to penicillin populations could be obtained that were adapted to high concentrations of this antibiotic.     

Facile Method for Monitoring Inhibition of Anaerobic Spore Outgrowth

A device is presented for the laboratory monitoring of spore outgrowth under controlled temperature and anaerobic conditions. Alterations in pH, redox potential, headspace composition, and optical density are followed as the activated spores grow out into vegetative cells. An interlock system allows the addition of test solutions or the removal of medium under anaerobic conditions. The device may also be used for rapid (<4 h) chemical inhibition studies or adapted for temperature injury studies of aerobic or anaerobic cells. Data on outgrowth of Clostridium sporogenes and inhibition by nitrite solutions are presented.     

The photosynthetic apparatus and photoinduced electron transfer in the aerobic phototrophic bacteria <Emphasis Type="Italic">Roseicyclus mahoneyensis</Emphasis> and <Emphasis Type="Italic">Porphyrobacter meromictius</Emphasis>

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q_A) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q_A of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of +463 mV and −25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of +415 mV for P and +94 mV for Q_A. Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.     

Effect of hydrogen peroxide on the biodegradation of PCBs in anaerobically dechlorinated river sediments

The ability to initiate aerobic conditions in dechlorinated anaerobic sediments was tested using hydrogen peroxide as an oxygenation agent. Hydrogen peroxide additions to the sediment induced aerobic polychlorinated biphenyl (PCB) degraders as indicated first, by an increase in bacterial count and second by a decline in PCB concentration from 135 µg/g to 20 µg/g over a 96-day period. Dechlorinated anaerobic sediment seems also to harbor indigenous anaerobic and aerobic microorganisms with high PCB degradation abilities. Those results support the potential ofin situ degradation of PCBs using a sequential anaerobic-aerobic technique.     

Copper ions and the regulation ofSaccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions

We have previously reported that theSaccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression ofCRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, theCUP1 metallothionein gene ofS. cerevisiae was found to be subject to a similar down-regulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the coppertrans-activator ofCUP1 andCRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression ofCRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics ofCUP1 andCRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

Anaerobically grown <Emphasis Type="Italic">Thauera aromatica</Emphasis>, <Emphasis Type="Italic">Desulfococcus multivorans</Emphasis>, <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> are more sensitive towards organic solvents than aerobic bacteria

The effect of seven important pollutants and three representative organic solvents on growth of Thauera aromatica K172, as reference strain for nitrate-reducing anaerobic bacteria, was investigated. Toxicity in form of the effective concentrations (EC50) that led to 50% growth inhibition of potential organic pollutants such as BTEX (benzene, toluene, ethylbenzene, and xylene), chlorinated phenols and aliphatic alcohols on cells was tested under various anaerobic conditions. Similar results were obtained for Geobacter sulfurreducens and Desulfococcus multivorans as representative for Fe³⁺-reducing and sulphate-reducing bacteria, respectively, leading to a conclusion that anaerobic bacteria are far more sensitive to organic pollutants than aerobic ones. Like for previous studies for aerobic bacteria, yeast and animal cell cultures, a correlation between toxicity and hydrophobicity (log P values) of organic compounds for different anaerobic bacteria was ascertained. However, compared to aerobic bacteria, all three tested anaerobic bacteria were shown to be about three times more sensitive to the tested substances.     

Selenium enhances the antimutagenic activity of probiotic bacterium Enterococcus faecium M-74

The antibacterial activity of the probiotic bacterium Enterococcus faecium M-74 was assessed on De Man–Rogosa–Sharpe (MRS), Todd–Hewitt (T–H), M17 (M-17) and brain heart infusion (BHI) media with sodium selenite pentahydrate (+Se) and without sodium selenite pentahydrate (–Se) under aerobic or anaerobic conditions against nine bacterial pathogens. The highest antibacterial activity was found to be in the MRS medium under anaerobic conditions. There were no differences in the antibacterial activity between MRS(+Se) and MRS(–Se) media. The antimutagenic activity of MRS(+Se) and MRS(–Se) extracts after culture with E. faecium M-74 as well as of live and killed cells of E. faecium M-74 grown in the presence or absence of Se against the genotoxicity of ofloxacin (OFL) and acridine orange (AO) was determined in the Euglena gracilis assay. The MRS(+Se) extracts showed a significantly higher activity in reducing the genotoxicity of OFL and AO than MRS(–Se) extracts. The live cells of the probiotic strain M-74 exhibited higher antimutagenic activity than the killed bacterial cells, but differed depending on the mutagen used. However, the live bacterial cells grown in the presence of Se showed significantly higher antimutagenic activity. These results suggest a potential benefit for the future development of new Se-enriched probiotics exhibiting higher antimutagenic properties.     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

Phosphate uptake by immobilizedAcinetobacter calcoaceticus cells in a full scale activated sludge plant

Anin situ study of the P-uptake ability ofAcinetobacter calcoaceticus was carried out using the alginate immobilization technique. ImmobilizedA. calcoaceticus cells displayed a high P-uptake ability (>97% P-accumulating cells) when immersed in the aerobic zone of an activated sludge system for 30–240 min. The overall P-accumulation pattern of the anaerobic zone depicted a typical P-release mechanism. However, limited P-accumulation was also observed at this stage. Growth and anaerobiosis were not prerequisites for P-uptake. The immobilized cell retention time in the anaerobic zone did not affect remarkably the inherent P-uptake ability of immobilizedA. calcoaceticus when exposed to the aerobic stage. P-uptake and release were reversible and depended on the environmental conditions to which immobilized cells were exposed. Immobilization ofA. calcoaceticus using alginate can be regarded as a reliable method of studying pure cultures in the activated sludge process.