Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h^-1 L^-1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)^-1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.     

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg²⁺). I₅₀ values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min^?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg²⁺ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg²⁺ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg²⁺. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg²⁺ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg²⁺ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

The relation between membrane ionic current and ATP synthesis in chromatophores from Rhodopseudomonas capsulata

(1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (J_DIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, J_DIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, J_DIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, J_DIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H⁺ flux through the F₀ channel of the ATPase synthase. The high-power dependence of the F₀ conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F₀ dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from J_DIS. This led to an estimate of 0.77 ± 0.22 for the $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio and of 3.5 ± 1.3 for the $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.     

Different mechanisms of hydroxyl radical production susceptible to purine P2 receptor antagonists between carbon monoxide poisoning and exogenous ATP in rat striatum

Abstract

Previous studies have suggested that carbon monoxide (CO) poisoning stimulates cAMP production via purine P2Y11-like receptors in the rat striatum, activating cAMP signaling pathways, resulting in hydroxyl radical (^?OH) production. Extracellular ATP was thought likely to trigger the cascade, but the present study has failed to demonstrate a clear increase in the extracellular ATP due to CO poisoning. The CO-induced ^?OH production was attenuated by the P2Y11 receptor antagonist NF157, in parallel with its abilities to suppress the CO-induced cAMP production. The ^?OH production was more strongly suppressed by a non-selective P2 receptor antagonist, PPADS, which had no effect on cAMP production. More selective antagonists toward the respective P2 receptors susceptible to PPADS, including NF279, had little or no effect on the CO-induced ^?OH production. The intrastriatal administration of exogenous ATP dose-dependently stimulated ^?OH production, which was dose-dependently antagonized by PPADS and NF279 but not by NF157. Exogenous GTP and CTP dose-dependently stimulated ^?OH production, though less potently. The GTP-induced ^?OH production was susceptible to both of NF279 and PPADS, but the CTP-induced ^?OH production was resistant to PPADS. The mechanism of ^?OH production may differ between CO poisoning and exogenous ATP, while multiple P2 receptors could participate in ^?OH production. The CO-induced ^?OH production was susceptible to the inhibition of NADPH oxidase, but not xanthine oxidase. Also, the NADPH oxidase inhibition suppressed ^?OH production induced by forskolin, a stimulator of intracellular cAMP formation. It is likely that ^?OH is produced by NADPH oxidase activation via cAMP signaling pathways during CO poisoning.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and P_i of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of the pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H⁺ consumption during the synthesis of ATP. The ratio of H⁺ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 $\text{H}{}^{\mathrm{+}}\text{ATP}$.2. The stimulation of the electrical potential decay by ADP and P_i was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No ‘critical’ or ‘threshold’ potential was observed.3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle.4. The stimulation of the electrical potential decay by ADP and P_i is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H⁺-release on the inside of the chromatophore vesicles following a flash.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

ATP production by sorbitol-treated cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201

The phosphorylation system of AMP by sorbitol-treated cells of a methanolutilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201, was investigated for the production of ATP. Firstly, reaction conditions for the ATP production were optimized. Under the optimal conditions, 20–30 g 1^?1 of ATP were produced in the conversion rate of 60–70% from AMP. Activities of reactions involved in the ATP-producing system were compared with cells from different cultures to prepare the cells having the higher activity and to know the essential reaction limiting the rate of the system. The energy efficiency of this system was also discussed.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

Synthesis and possible character of a high-energy intermediate in bacterial photophosphorylation

1. In photophosphorylation with chromatophores from Rhodospirillum rubrum, evidence is presented for the synthesis of activated precursors of ATP in the energy-conversion system coupled to photosynthetic electron transport. 2. A significant amount of ATP is synthesized when a reaction mixture containing chromatophores and ADP is illuminated and then incubated with P_i in the dark. ATP is not synthesized to an appreciable extent, either when a reaction mixture containing chromatophores and P_i is illuminated and then incubated with ADP in the dark, or when one containing chromatophores alone is illuminated and then incubated with ADP and P_i in the dark. The amount of ATP thus synthesized is influenced markedly by concentrations of ADP. 3. The chromatophores illuminated with ADP, if allowed to stand in the dark at 30°, gradually lose the ability to form ATP with P_i in the dark. No loss of the ability occurs when the chromatophores illuminated with ADP are allowed to stand in the dark at 13° or in a frozen state. 4. Mg²⁺ is absolutely required for chromatophores to form ATP in the dark after illumination in the presence of ADP, and for the chromatophores to achieve ATP formation with P_i in the dark. 5. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide and o-phenanthroline strongly inhibit the light-dependent acquisition of the ability to form ATP with P_i in the dark, but not the consequent ATP formation with P_i in the dark. Arsenate, 2,4-dinitrophenol, quinacrine hydrochloride, quinine hydrochloride and pyrophosphate inhibit the former or the latter, or both. Oligomycin inhibits the former somewhat more than the latter. 6. From these findings it is suggested that a high-energy intermediate is formed in photosynthetic ATP formation, and that its formation is dependent on ADP but not P_i.     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Membrane-bound phosphate as driving force for ATP synthesis in chromatophores ofRhodospirillum rubrum

Chromatophores ofRhodospirillum rubrum preilluminated in the presence of unlabelled phosphate form labelled ATP in the dark after being separated from the preincubation mixture by gel-filtration and incubated with ADP and³²P_i. The driving force for the synthesis of this labelled ATP was previously shown to be ATP firmly bound to the membrane. The amount of labelled ATP produced is determined by measuring the incorporated³²P_i and extrapolation of the values towards zero time incubation and is shown to correlate with the phosphorylation activity of the chromatophores used. 2.5 nmoles ATP correspond to a phosphorylation activity of 100 moles ATP/mg Bchl. h. The results were compared with those from assays in which chromatophores were preincubated with pyrophosphate in the dark. The results strongly support the hypothesis that the XP formed in the light does not transfer its P to ADP in solution and therefore give further evidence to a double sited coupling factor as proposed previously.     

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (epsilon ADP) to myosin, heavy meromyosin, and subfragment one

The binding of the fluorescent analog of adenosine diphosphate (ADP)¹, 1,N⁶-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (^± .5) × 10^?5 M^?1. This is identical to the values found by Young (J. Biol. Chem., $\text{242}$, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of V_max and K_m were 25 μM phosphate sec^?1 (gm protein)^?1 and 5 × 10^?5 M^?1 for ATP, and 80 μN phosphate sec^?1 (gm protein)^?1 and 45 × 10^?5 M^?1 for εATP. The results indicate that the increased V_max that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in V_max may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex.     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 ± 20 mM^?1 · cm^?1. The molecular weight based on protein and chromophore assays was found to be 1.5 · 10⁵; the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Stability of chromatophores during ATP regeneration in an enzyme reactor

ATP regeneration from ADP and inorganic phosphate catalysed by chromatophores from Rhodo-spirillum rubrum has been coupled to the production of glucose-6-phosphate from glucose catalysed by hexokinase. Different ADP derivatives were tested in respect of their function as substrates for the chromatophores, but only 3′NH₂-ADP was phosphorylated. Several factors causing inactivation of the chromatophores during operational conditions were studied. The addition of radical scavengers resulted in an increased stability of the chromatophores, and it was concluded that free radicals had been formed in the presence of oxygen and caused damage to the membranes. In the presence of catalase, a stable ATP production was obtained for 6 h at an activity of 4.5 mol ATP × (mol bacteriochlorophyll)⁻¹ min⁻¹. In a nitrogen flushed reaction medium, the optimum concentration of the redox buffering ascorbic acid changed during the course of several hours of illumination, while it did not change in an air atmosphere.     

Distribution and change of microbial activity in combined UASB and AFB reactors for wastewater treatment

A thermophilic upflow anaerobic sludge blanket (UASB) reactor was combined with a mesophilic aerobic fluidized bed (AFB) reactor for treatment of a medium strength wastewater with 2,700?mg COD?l^?1. The COD removal efficiency reached 75% with a removal rate of 0.2 g COD?l^?1 h^?1 at an overall hydraulic retention time 14 hours. The distribution of microbial activity and its change with hydraulic retention time in the two reactors were investigated by measuring ATP concentration in the reactors and specific ATP content of the biomass. In the UASB reactor, the difference in specific ATP was significant between the sludge bed and blanket solution (0.02?mg ATP g VS^?1 versus 0.85?mg ATP g VS^?1) even though the ATP concentrations in these two zones were similar. A great pH gradient up to 4 was developed along the UASB reactor. Since a high ATP or biological activity in the blanket solution could only be maintained in a narrow pH range from 6.5 to 7.5, the sludge granules showed a high pH tolerance and buffering capacity up to pH 11. The suspended biomass in AFB reactor had a higher specific ATP than the biomass fixed in polyurethane carriers (1.6?mg ATP g VS^?1 versus 1.1?mg ATP g VS^?1), which implies a starvation status of the immobilized cells due to mass transfer limitation. The aerobes had to work under starvation conditions in this polishing reactor. The anaerobic biomass brought into AFB reactor contributed to an increase in suspended solids, but not the COD removal because of its fast deactivation under aerobic conditions. A second order kinetic model was proposed for ATP decline of the anaerobes. The results on distribution of microbial activity in the two reactors as well as its change with hydraulic retention time lead to further performance improvement of the combined anaerobic/aerobic reactor system.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.