Anoxic Culture Research Articles

Effect of sludge fasting/feasting on growth of activated sludge cultures

Reduction of excess sludge in an oxic-settling-anoxic (OSA) activated sludge process might be attributed to a “sludge fasting (insufficient food under an anoxic condition)/feasting (sufficient food under an oxic condition)” treatment. This paper was to examine this explanation by investigating both the sludge fasting/feasting phenomenon and the effect of a fasting/feasting treatment on sludge growth. In this study, five different activated sludge cultures cultivated using synthetic wastewater composed of mainly glucose and other necessary nutrients: (1) an aerobic batch culture, (2) an intermittently aerated batch culture, (3) an anoxic batch culture, (4) a continuous aerobic culture, and (5) an OSA culture, were employed. It was found that only the aerobic batch culture and the aerobic continuous culture are fastable when the oxidation reduction potential (ORP) level is below 100 mV under no-food condition during a 2-h fasting treatment, showing that both the biomass and carbohydrate storage of these two cultures were reduced after the treatment. When the fasted cultures were treated in a feasting environment, an accumulation of carbohydrate storage did not occur, while specific oxygen uptake rates (SOUR) showed a sharp increase. Both the substrate utilization and biomass growth rates were also accelerated. It was therefore confirmed that a sludge feasting did occur after a fasting treatment for the fastable cultures. However, an increase in sludge ATP content was not brought about by the feasting treatment. The sludge fasting/feasting treatment in this paper could not induce a reduction of the observed growth yield ( Y obs) in all the cultures cultivated with glucose-based synthetic wastewater.

Bacterially induced dolomite precipitation in anoxic culture experiments

Bacterially induced dolomite precipitation in anoxic culture experiments

Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli.

Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (-100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (-320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.

Renal tubular epithelial cells tolerate prolonged warm ischaemia in vitro

AS INCREASING numbers of expanded criteria and non–heart-beating donor kidneys are procured, the need for a reliable, accurate test of viability prior to transplantation becomes more acute. An in vitro model of renal cellular ischaemia would facilitate the study and assessment of viability tests and renoprotective compounds. An experimental system for anoxic culture and manipulation of cultured cells has been established and used to study renal cell viability after an ischaemic insult.

Bacterially induced dolomite precipitation in anoxic culture experiments

To study the process of microbial-mediated dolomite formation, growth experiments were carried out with selected bacterial cultures under anoxic environmental conditions simulating those found in Lagoa Vermelha, a hypersaline lagoon in Brazil where dolomite precipitation occurs. Specifically, we report the isolation of a particular strain of sulfate-reducing bacteria, LVform6, from Lagoa Vermelha sediment, which apparently promotes the formation of nonstoichiometric dolomite. Sulfate-reducing bacteria grown in a synthetic liquid medium produced dolomite during 30 days incubation at 30 °C. The precipitates have morphologies similar to those observed in Lagoa Vermelha sediment. Our results demonstrate that sulfate-reducing bacteria can influence dolomite precipitation under controlled lowtemperature, anoxic conditions, and imply that anaerobic microorganisms can play an important role in carbonate sedimentation. They may have been particularly significant in Earth's earliest history when a more reducing atmosphere existed.

Kinetic Behavior of Some Polyphosphate-Accumulating Bacteria Isolates in the Presence of Nitrate and Oxygen

This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen. It is shown that species were able to respire both electron acceptors for phosphate accumulation. A. tumefaciens and A. dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A. hydrophila and K. oxytoca eliminated overall phosphate only in oxic conditions. A. dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas. The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A. dispar have a denitrification rate superior when the cells were adapted to nitrate.

Manganese oxide reduction in laboratory microcosms

Manganese biogeochemistry holds special interest for the characterization of passive treatment systems designed to treat acidic mine waters while meeting enforceable effluent discharge limits set for manganese. In the present study, an initial anoxic enrichment culture was developed for use as an inoculum in experimental systems. Standard anoxic microcosms capable of reducing manganese from Mn4+ to Mn2+ were established from the initial enrichment and altered to study the effects of electron acceptor availability and inhibitors on manganese reduction. Manganese reduction was not significantly inhibited in aerobic and nitrate amended microcosms; however, systems amended with metabolic inhibitors (sodium azide or sodium molybdate) exhibited significant inhibition of manganese reduction relative to standard microcosms. The presence of iron was found to influence the partitioning of reduced manganese with adsorption becoming more important with increasing iron to manganese ratios.

Distinct arginase isoforms expressed in primary and transformed macrophages: regulation by oxygen tension.

Experiments were performed to identify arginase isoforms expressed in primary and transformed rodent macrophages and to determine the molecular mechanisms for the previously observed increase in arginase activity in macrophages cultured in hypoxia or anoxia. Results demonstrate the following: 1) mRNA and protein for hepatic-type AI arginase are expressed in primary cultures of rat and mouse peritoneal macrophages and are enhanced seven- and nine-fold, respectively, by lipopolysaccharide (LPS). 2) mRNA for extrahepatic-type AII arginase is constitutively expressed in mouse, but not rat, peritoneal macrophages and is detected in RAW264.7 cells after LPS treatment; neither J774A.1 nor P388D1 cells contain arginase mRNA. 3) AI arginase mRNA, arginase activity in cell lysates, and L-arginine flux through arginase in intact cells are all increased in rat wound-derived and mouse peritoneal macrophages by hypoxic or anoxic culture; AII arginase mRNA is, in contrast, suppressed > 50% by O2 deprivation. 4) Expression of the L-arginine transporter mCAT-2 is increased greater than twofold by reduced O2 culture. These results demonstrate substantial variability in arginase isoform expression among primary and transformed rodent macrophages. They also identify AI and AII arginase and the mCAT-2 L-arginine transporter as O2-regulated genes.

A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates. At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed. The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism. At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased. The specific ethanol flux was not affected. At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased. The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction. Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol. Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production. The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes. Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate. At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased. Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products. Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted. However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance.

Macrophage activation by culture in an anoxic environment.

Abstract The extracellular amino acid composition of experimental wounds in rats during peak macrophage infiltration bears the imprint of the elevated arginase activity present in wound fluid: L-arginine is found in this space in concentrations markedly lower, and L-ornithine in concentrations markedly higher, than those that are detectable in plasma. No evidence, in the form of L-citrulline or NO2- accumulation, can be found at this time for nitric oxide synthase (NOS) activity. Wound-derived macrophages, however, metabolize L-arginine through both arginase and NOS in culture. Given the requirements of NOS for O2 and the reduced O2 tension in wounds, experiments were performed to determine the role of O2 availability on the metabolism of L-arginine by wound-derived macrophages. Results demonstrated that, beyond inhibiting NOS, culture of wound-derived macrophages in an anoxic environment provided an activation signal, markedly increasing total L-arginine metabolism, arginase activity, NOS protein content, and the release of TNF-alpha and IL-6. Neither resident nor Corynebacterium parvum-elicited peritoneal macrophages responded to anoxic culture with increases in L-arginine utilization, arginase activity or, in the case of resident macrophages, in NOS protein content. The enhanced TNF-alpha and IL-6 release induced by anoxia in wound-derived macrophages was also found in resident peritoneal macrophages. Anoxia appears to act, then, as an inducer of activation-associated traits in macrophages obtained from different sites.

Anoxia induces human immunodeficiency virus expression in infected T cell lines

The effects of oxygen deprivation, or anoxia, on human immunodeficiency virus (HIV-1) expression in chronically (ACH.2) and acutely (H9/HIV-1-IIIB) infected cell lines was investigated. Temporary cellular anoxia has previously been shown to activate transcription of endogenous type C leukemia virus sequences, resulting in a significant increase in retroviral RNA within the cell (1). Here we report a 15-fold increase in HIV-1-specific RNA in unstimulated ACH.2 T cells within 24 h of anoxia. This induction of RNA is accompanied by an accumulation of intracellular p24 gag protein as well as an increase in envelope protein. Anoxia induces a further increase in total HIV-1 RNA in ACH.2 cells prestimulated to produce virus by phorbol 12-myristate 13-acetate and in H9 T cells acutely infected with HIV-1-IIIB. The induction of RNA in ACH.2 cells appears to be reversible. Anoxic culture for 24 h followed by a 24-h re-oxygenation period results in a return to "resting state" levels of HIV-1 RNA. These data indicate that oxygen tension within the cellular environment modulates HIV-1 expression, providing a model system in which to study the reversible regulation of HIV-1 RNA and viral gene products within the cell.

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different NADH/NADPH ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulaiton under unaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.

Relation between enzyme release and metabolic changes in reversible anoxic injury of myocardial cells

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 μmol. ATP/g WW, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations.

Absence of reoxygenation damage in isolated heart cells after anoxic injury.

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions and then reoxygenated. When comparing the oxygen deficient organ to the anoxic cell culture, we see that metabolic changes in the latter system proceed in a similar, yet prolonged manner, as in arrested hearts. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds closely correlated to the actual ATP level. Below 2 mumol ATP/gww, an increasing number of cells becomes irreversibly damaged, above this level, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells and is related to reversible membrane alterations. Reoxygenation does not induce changes considered typical of the 'oxygen paradox'. Since mechanical cell-cell interactions are absent in this model, it is suggested that aggravation of tissue damage in heart tissue reoxygenated late is mainly caused by mechanical forces.

Anaerobic Phosphate Uptake by Barley Plants

Considerable uptake of phosphate by both the shoot and root has been demon strated for young barley plants with their roots in anoxic culture solution at con centrations of i to io pM orthophosphate. Consideration of the free space and passive transpirational uptake indicates an accumulatory process, and the immediate efflux caused by respiratory inhibitors supports this. Shoot uptake is much less at higher external concentrations of phosphate and at o-i mM was only 14 per cent of the control. The root accumulation process was unimpaired at an external concentration of 1 pM phosphate when the whole plant was subjected to anaerobic conditions (shoot illuminated) but under similar conditions at a concentration of 100 pM a considerable efflux of phosphate occurred. Analysis of the fate of phosphate taken up from anoxic solution of phosphate (10 pM) indicated that there was a reduction in the level of inorganic phosphate after 4-5 h and steady rise in sugar phosphates up to 6 h with a marked increase in the levels of glucose-6-phosphate, fructose-6-phosphate, and the phosphoglycerate fraction.