Two-tank suspended growth process for accelerating the detoxification kinetics of hydrocarbons requiring initial monooxygenation reactions

An experimental evaluation demonstrated that suspended growth systems operated in a two-tank accelerator/aerator configuration significantly increased the overall removal rates for phenol and 2,4-dichlorophenol (2,4-DCP), aromatic hydrocarbons that require initial monooxygenations. The accelerator tank is a small volume that receives the influent and recycled biomass. It has a high ratio of electrondonor (BOD) to electron acceptor (O₂). Biomass in the accelerator should be enriched in reduced nicotinamide adenine dinucleotide (NADH + H⁺) and have a very high specific growth rate, conditions that should accelerate the kinetics of monooxygenation reactions. For the more slowly degraded 2,4-DCP, the average percentage removal increased from 74% to 93%, even though the volume of the two-tank system was smaller than that of the one-tank system in most experiments. The average volumetric and biomass-specific removal rates increased by 50% and 100%, respectively, in the two-tank system, compared to a one-tank system. The greatest enhancement in 2,4-DCP removal occurred when the accelerator tank comprised approximately 20% of the system volume. Biomass in the accelerator tank was significantly enriched in NADH + H⁺ when its dissolved oxygen (DO) concentration was below 0.25 mg/L, a situation having a high ratio of donor to acceptor. The accelerator biomass had its highest NADH + H⁺ content for the experiments that had the highest rate of 2,4-DCP removal. Biomass in the accelerator also had a much higher specific growth rate than in the aerator or the system overall, and the specific growth rate in the accelerator was inverselycorrelated to the accelerator volume.     

The ACCEL Model for Accelerating the Detoxification Kinetics of Hydrocarbons Requiring Initial Monooxygenation Reactions

The two-tank accelerator/aerator modification of activated sludge significantly increases the biodegradation of hydrocarbons requiring initial monooxygenation reactions, such as phenol and 2,4-dichlorophenol (DCP). The small accelerator tank has a controlled low dissolved oxygen (DO) concentration that can enrich the biomass in NADH + H⁺. It also has a very high specific growth rate (μ_acc) that up-regulates the biomass’s content of the monooxygenase enzyme. Here, we develop and test the ACCEL model, which quantifies all key phenomena taking place when the accelerator/aerator system is used to enhance biodegradation of hydrocarbons requiring initial monooxygenations. Monooxygenation kinetics follow a multiplicative relationship in which the organic substrates (phenol or DCP) and DO have separate Monod terms, while the biomass’s content of NADH + H⁺ has a first-order term. The monooxygenase enzyme has different affinities (K values) for phenol and DCP. The biomass’s NADH + H⁺ content is based on a proportioning of NAD(H) according to the relative rates of NADH + H⁺ sources and sinks. Biomass synthesis occurs simultaneously through utilization of acetate, phenol, and DCP, but each has its own true yield. The ACCEL model accurately simulates all trends for one-tank and two-tank experiments in which acetate, phenol, and DCP are biodegraded together. In particular, DCP removal is affected most by DO_acc and the retention-time ratio, Θ_acc/Θ_total. Adding an accelerator tank dramatically increases DCP removal, and the best DCP removal occurs for 0.2 < DO_acc < 0.5 mg/l and 0.08 < Θ_acc/Θ_total < 0.2. The rates of phenol and DCP utilization follow the multiplicative relationship with a maximum specific rate coefficient proportional to μ_acc. Finally, μ_acc increases rapidly for Θ_acc/Θ_total < 0.25, acetate removal in the accelerator fuels the high μ_acc, and the biomass’s NADH + H⁺ content increases very dramatically for DO_acc < 0.25 mg/l.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

Effects of 2,4-Dichlorophenol, a Metabolite of a Genetically Engineered Bacterium, and 2,4-Dichlorophenoxyacetate on Some Microorganism-Mediated Ecological Processes in Soil

A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10⁷ CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 μg/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to <100 ppm. Coincident with the accumulation of 2,4-DCP was a >400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO₂ evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Experimental determination of control by the H+-ATPase inEscherichia coli

Strains carrying deletions in theatp genes, encoding the H⁺-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of anatp deletion mutant was surprisingly high (some 75–80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H⁺-ATPase at its physiological level, the cellular concentration of H⁺-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H⁺-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H⁺-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H⁺-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H⁺-ATPase level. Moreover, the turnover number of the individual H⁺-ATPase enzymes increased as the H⁺-ATPase concentration was lowered. The negative control by the H⁺-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H⁺-ATPase onE. coli growth rate.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphatase and the effects of enzyme inhibitors

The involvement of membrane (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na⁺ + K⁺)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K⁺ required the simultaneous presence of Na⁺. Ouabain, a specific inhibitor of (Na⁺ + K⁺)-ATPase, significantly inhibited the (Na⁺ + K⁺)-stimulation of respiration. These observations suggest that the (Na⁺ + K⁺)-stimulation of brain slice respiration is related to ADP production as a result of (Na⁺ + K⁺)-ATPase activity. However, ouabain also inhibited non-K⁺-stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na⁺ + K⁺)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na⁺ + K⁺)-ATPase inhibition and acts additionally by increasing the intracellular Na⁺ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na⁺ + K⁺)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na⁺ and K⁺ media and that in choline, K⁺ media. Studies were performed with two (Na⁺ + K⁺)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na⁺ + K⁺)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na⁺ + K⁺)-ATPase and related respiration, but chlorpromazine failed to alter either (Na⁺ + K⁺)-ATPase activity or related respiration.     

The Stoichiometry of Respiration-driven Potassium Transport in Corn Mitochondria

Determinations were made for corn (Zea mays L., WF9-Tms × M14) mitochondria of the stoichiometric relationship between K⁺ transport and bond energy produced in respiration (K⁺/~ ratio). With inward pumping of potassium acetate activated by NADH oxidation, the initial rate of K⁺ transported into the sucrose inaccessible space varied between 0.58 and 0.97 K⁺/~, assuming 2 high energy bond equivalents per NADH oxidized. Only small amounts of H⁺ were ejected. Valinomycin did not alter the ratio.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Degradation of chlorophenols catalyzed by laccase

The degradations of 2,4-dichlorophenol (2,4-DCP), 4-chlorophenol (4-CP) and 2-chlorophenol (2-CP) catalyzed by laccase were carried out. The optimal condition regarding degradation efficiency was also discussed, which included reaction time, pH value, temperature, concentration series of chlorophenols and laccase. Results showed that the capability of laccase was the best, while to oxidize 2,4-DCP among the above-mentioned chlorophenols. Within 10 h, the removal efficiency of 2,4-DCP, 2-CP and 4-CP could reach 94%, 75% and 69%, respectively. The optimal pH for laccase to degrade chlorophenols was around 5.5. The increase of laccase concentration or temperature might result in the degradation promotion. The trends of degradation percentage were various among these three chlorophenols with the concentration increase of chlorophenols. Degradation of 2,4-DCP is a first-order reaction and the reaction activation energy is about 44.8 kJ mol⁻¹. When laccase was immobilized on chitosan, crosslinked with glutaraldehyde, the activity of immobilized laccase was lower than that of free laccase, but the stability improved significantly. The removal efficiency of immobilized laccase to 2,4-DCP still remained over 65% after six cycles of operation.     

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis¹. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate², the same is not true for extracellular acid production and its relationship to glycolytic rate ^3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO₂, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate^- + H⁺ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO₂, hydration to H₂CO₃ and dissociation to HCO₃^- + H⁺ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification ⁶. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.     

Effects of 2,4-dichlorophenol on activated sludge

The effects of 2,4-dichlorophenol (2,4-DCP) on both acclimated and unacclimated activated sludge were investigated in batch reactors. The IC(50) values on the basis of maximum specific growth rate ( micro(m)), percent chemical oxygen demand (COD) removal efficiency and sludge activity were found to be 72, 60 and 47 mg l(-1), respectively, for unacclimated culture. The percent COD removal efficiencies of unacclimated culture were affected adversely, even at low concentrations, whereas culture acclimated to 75 mg 2,4-DCP l(-1) could tolerate about 200 mg 2,4-DCP l(-1)on the basis of COD removal efficiency. Although yield coefficient values of unacclimated culture increased surprisingly to very high values with the addition of 2,4-DCP, a linear decrease with respect to 2,4-DCP concentrations was observed for acclimated culture. Although no removal was observed with unacclimated culture, almost complete removal of 2,4-DCP up to a concentration of 148.7 mg l(-1) was observed with acclimated culture. It was showed that the culture could use 2,4-DCP as sole organic carbon source, although higher removal efficiencies in the presence of a readily degradable substrate were observed. Culture acclimated to 4-chlorophenol used 2,4-DCP as sole organic carbon source better than those acclimated to 2,4-DCP.     

Mechanisms of H2O2 formation by leukocytes. Properties of the NAD(P)H oxidase activity of intact leukocytes.

It is postulated that the burst of oxygen consumption and H₂O₂ formation following phagocytosis by polymorphonuclear leukocytes is due to the action of an oxidase located in the plasma membrane. The cyanide-resistant oxygen consumption of resting polymorphonuclear leukocytes was also found to be stimulated by 2,4-dichlorophenol with H₂O₂ being the sole product formed. NADH and NADPH added to the leukocytes greatly enhanced the oxygen consumption and were oxidized in the process without penetrating the leukocytes. Mn²⁺ stimulated this oxidase activity. The apparent K_m values for added NADH and NADPH were 50 and 40 μm, respectively, with a V of 300 nmol/mg protein/min. A stoichiometry of 1 mol H₂O₂ formed per mol of NAD(P)H was found. Whilst the oxidase is similar to the oxidase properties of a peroxidase, myeloperoxidase is not responsible for the activity.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.     

The Regulation of the Plasma Membrane Redox System and H+-transport in Adaptation of Reed Ecotypes to Their Habitats

The redox system and H⁺-transport activities in the plasma membranes from two ecotypes of reed (Phragmites communis Trin.), named swamp reed (SR) and dune reed (DR) according to their habitats, were investigated. Compared to the SR, the DR possessed the very high rates of NADH oxidation and Fe(CN)₆ ^3– and EDTA-Fe³⁺ reduction when NADH was taken as the electron donor. As NADPH was an electron donor, the rate of NADPH oxidation was also significantly higher in the DR than that in the SR. In addition, the H⁺-transport activity in the plasma membranes was also significantly higher in the DR than in the SR.     

The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential

In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H⁺ or Na⁺. In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na⁺/H⁺ antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na⁺ transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.     

SHIFTS IN UBIQUINONE REDOX STATUS OF RAT BRAIN IN VITRO WITH CATIONIC STIMULATION

–Changes in respiratory rate, ubiquinone (Q) redox status, lactate and pyruvate levels in chopped rat telencephalon were studied after cationic stimulation of respiration. When chopped telencephalon was incubated with glucose as substrate, increasing the K⁺ concentration from 5 mm to 55 mm was associated with a 57% increase in the respiratory rate and a reduction in Q redox status from 76% oxidized to 64% oxidized. Lactate increased by over 200%. Substitution of pyruvate for glucose with 5 mm -K⁺ resulted in a respiratory rate that was 78% of that seen with glucose and 55 mm -K⁺. Increasing K ⁺ from 5 mm to 55 mm increased the respiratory rate by only 6%, with pyruvate as substrate. Q underwent a reduction that was half that seen with glucose. Ouabain largely prevented the K ⁺-induced increase in respiratory rate with glucose as substrate, but Q was still reduced by 5 percentage points while lactate and pyruvate were unchanged. When respiration was uncoupled with 2,4-dinitrophenol, increasing the K⁺ concentration from 5 mM to 55 mm had no further effect on any of the metabolic parameters measured. Deletion of Ca²⁺ from the medium resulted in an increase in respiration of 18%, but neither the Q redox status nor the levels of lactate or pyruvate were significantly changed. The results demonstrate that the K⁺-induced stimulation of respiration results from a coordinated metabolic response whereas the stimulation of respiration associated with Ca²⁺ depletion is probably mediated through ion fluxes at the cell membrane and activation of Na⁺-K⁺-activated ATPases.     

Hormone action on transmembrane electron and h transport

A possible involvement of two different systems in proton translocation was investigated by simultaneous measurement of transmembrane electron flow and proton secretion in a pH-stat combined with a redoxstat. The pH gradient between cytoplasm and apoplast is probably maintained by an H⁺ -pumping ATPase and by a second proton extrusion system, which seems to be linked to a redox chain with NAD(P)H as electron donor. Indole acetic acid inhibits both e⁻ and H⁺ efflux, but only if the `electron draw' from the outside is not too high. The electron draw depends on the hexacyanoferrate level at the plasmalemma surface and on the Ca²⁺ concentration. The inhibiting effect of auxin on e⁻ and H⁺ efflux in the presence of hexacyanoferrate can be only detected at low levels of bivalent cations and of the artificial electron acceptor. The inhibition of e⁻ and H⁺ efflux by auxin requires high oxygen levels. The influence of auxin on both e⁻ and H⁺ transfer disappears below 2 kilopascals O₂, a level which does not influence respiration. Ethanol and fusicoccin do not increase the e⁻ flux, probably because the electron transfer from the plasma membrane to HCF III is the limiting step. If electron transfer is reduced by IAA pretreatment, ethanol increases e⁻ flux. Fusicoccin decreases e⁻ and increases H⁺ efflux if the rates have been lowered previously by indole acetic acid pretreatment. This effect depends on high oxygen levels and is reversible by lowering oxygen pressure. Auxin and Ca²⁺ change e⁻ flow and H⁺ ejection in a 1:1 ratio.     

Modeling chlorophenols degradation in sequencing batch reactors with instantaneous feed-effect of 2,4-DCP presence on 4-CP degradation kinetics

Two instantaneously fed sequencing batch reactors (SBRs), one receiving 4-chlorophenol (4-CP) (SBR4) only and one receiving mixture of 4-CP and 2,4-dichlorophenol (2,4-DCP) (SBRM), were operated with increasing chlorophenols concentrations in the feed. Complete degradation of chlorophenols and high-Chemical oxygen demand (COD) removal efficiencies were observed throughout the reactors operation. Only a fraction of biomass (competent biomass) was thought to be responsible for the degradation of chlorophenols due to required unique metabolic pathways. Haldane model developed based on competent biomass concentration fitted reasonably well to the experimental data at different feed chlorophenols concentrations. The presence of 2,4-DCP competitively inhibited 4-CP degradation and its degradation began only after complete removal of 2,4-DCP. Based on the experimental results, the 4-CP degrader’s fraction in SBRM was estimated to be higher than that in SBR4 since 2,4-DCP degraders were also capable of degrading 4-CP due to similarity in the degradation pathways of both compounds.