Cell yield (YM) of Thauera selenatis grown anaerobically with acetate plus selenate or nitrate

Thauera selenatis was grown anaerobically in minimal medium with either selenate or nitrate as the terminal electron acceptor and acetate as the carbon source and electron donor. The molar cell protein yields, Y_M-protein (selenate) and Y_M-protein (nitrate), were found to be 7.8 g cell protein/mol selenite formed and 7.5 g cell protein/mol nitrite formed, respectively. These values represent Y_M values of 57 and 55 g (dry weight)/mol acetate when selenate or nitrate was the electron acceptor, respectively. Based upon a calculated Y_ATP value of 10.0 g (dry weight) cells/mol ATP, for growth on acetate in inorganic salts, growth with selenate as the terminal electron acceptor theoretically yielded 5.7 ATP/acetate oxidized, and 5.5 ATP when nitrate was the terminal electron acceptor. The results support the conclusion that energy is conserved via electron transport phosphorylation when selenate or nitrate reduction are the terminal electron acceptors during anaerobic growth with acetate.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Rhizobium ORS 571, isolated from stem nodules of the tropical legumeSesbania rostrata is able to grow in the chemostat with molecular nitrogen as sole nitrogen source at a specific growth rate of 0.1 h^-1. Samples from nitrogenfixing cultures showed high acetylene reduction activities: 1,500 nmol ethylene formed per milligram dry weight per hour. Under nitrogen-fixing conditions an uptake hydrogenase is induced. Ammonia-assimilating cultures, without additional hydrogen, did not induce hydrogenase. The addition of hydrogen to succinate-limited nitrogen-fixing cultures resulted in an increase in the molar growth yield on succinate (Y _succinate) from 27 to 35 and a slight decrease in the molar growth yield on oxygen ( ), showing that hydrogen oxidation is less energy-yielding than the oxidation of endogenous substrates. Respiration-driven proton translocation measured with starved cells indicated the functioning of site 1 and 2 of oxidative phosphorylation. Cytochrome spectra showed that cytochromea ₆₀₀, present at high dissolved oxygen tension (d.o.t.) almost completely disappeared at low d.o.t. In flash-photolysis spectra only thea-type cytochrome could be detected as an oxidase in cells both grown at high and low d.o.t. Growth yields in ammonia-assimilating cultures were higher than those measured in nitrogen-fixing cultures. Assuming two sites of oxidative phosphorylation, a molar growth yield on ATP (Y _ATP) of about 3 and 6 was calculated for respecticely nitrogen-fixing and ammonia-assimilating cultures. TheY _ATP under nitrogen-fixing conditions is dependent on the amount of H₂ formed per mol N₂ fixed (H₂/N₂ ratio). A method has been described to calculate the total amount of ATP use by nitrogenase during the fixation of 1 mol N₂ (ATP/N₂ ratio) and H₂/N₂ ratios in aerobic nitrogen fixing organisms. This calculation yielded that nitrogen fixation inRhizobium ORS 571 is a high ATP-consuming process. The calculated ATP/N₂ and H₂/N₂ ratios were respectively 42 and 7.5.Abbreviations d.o.t. dissolved oxygen tension A preliminary account of this work was presented at the 5th International Symposium on Nitrogen Fixation, September 1983, Noordwijkerhout, The Netherlands     

Hydrogen oxidation and nitrogen fixation in rhizobia,with special attention focused on strain ORS 571

In this survey we describe the influence of hydrogen oxidation on the physiology ofRhizobium ORS 571. The presence of hydrogen is required for the synthesis of hydrogenase. Carbon substrates do not repress the synthesis of hydrogenase. The respiratory system contains cytrochromes of theb- andc-type. Cytochromea ₆₀₀ is present after growth at high oxygen tensions. The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet. → H⁺/O values with endogenous substrates are between 6 and 7. The results show the presence of two phosphorylation sites: site 1 (ATP/2e=1.0) and site 2(ATP/2e=1.33). By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation. A method is described to calculate ATP/N₂ values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N₂) and H₂/N₂ ratios (mol hydrogen formed per mol N₂ fixed) in aerobic organisms. ForRhizobium ORS 571 the ATP/N₂ value is about 40 and the H₂/N₂ ratio is between 5 and 7.5. Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30–40% poly-β-hydroxybutyrate, which explains the high molar growth yields found. Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation. Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed × mol⁻¹ substrate converted) is not very large and that the actual H₂/N₂ ratio is of much more importance. After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Y_succinate value (g dry wt × mol⁻¹ succinate) is followed by a gradual decrease. This is accompanied by a large decrease of the value, and an increased permeability of the cytoplasmic membrane to protons. The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

ATP formation associated with fumarate and nitrate reduction in growing cultures of Veillonella alcalescens

Molar growth yields, fermentation balances and enzyme activities were measured in Veillonella alcalescens grown anaerobically with different substrates in the absence or presence of fumarate or nitrate. The molar growth yields on malate (14.3 g dry wt bacteria/mole substrate) and citrate (19.3) were higher than that on lactate (8.6). The molar growth yield on lactate was increased to 15.5 or 19.8 by the addition of fumarate or nitrate, respectively, to the growth medium, and the molar growth yield on citrate was increased to 25.3 by addition of nitrate. Active growth yield was 25.5. From fermentation balances and fermentation systems similar Y_ATP values (g dry wt bacteria/mole ATP) were calculated for all substrates or mixtures of substrates assuming that one mole of ATP is generated at the electron transport from pyruvate, NADH and NADPH to nitrate or fumarate whereas ATP is not produced in the electron transport from lactate to fumarate or nitrate, and, therefore, this assumption was considered to reflect the actual situation. The mean Y_ATP value at a doubling time of 1 h was 16.5 g dry wt bacteria/mole ATP for growth without an added hydrogen acceptor, 14.4 for growth with fumarate, and 14.2 for growth with nitrate.     

Influence of hydrogen acceptors on growth and energy production ofProteus mirabilis

The generation time ofP. mirabilis in defined and in complex medium is shorter in the presence of hydrogen acceptors than in their absence. In the presence of hydrogen acceptors the molar growth yield for glucose and the acetate production are strongly increased. From the molar growth yield and the acetate production Y_ATP in defined medium was calculated as 5.5 g/mole, whereas in complex medium a value of 12.6 g/mole was obtained. The molar growth yield, the acetate production, the amount of hydrogen acceptor reduced and Y_ATP were used to calculate P/2e⁻ratios for phosphorylation coupled to electron transfer to oxygen, nitrate and tetrathionate as respectively 2.80; 1.48 and 1.23 in defined medium. Under anaerobic conditions in the presence of nitrate or tetrathionate as hydrogen acceptor in complex medium a bend in the growth curve is observed. In the period of rapid growth the P/2e⁻ratio for nitrate reduction is of the same magnitude as that in defined medium, however much lower P/2e⁻ratios are found during the subsequent period of slow growth. The P/2e⁻ratios for tetrathionate reduction in complex medium for both growth periods are lower than those in defined medium. Most probably these results indicate that during this period growth and energy production are uncoupled. Under aerobic conditions in complex medium a constant Y_O value of 32.2 g/atom O is found during a short period of the growth curve. Afterwards when the cell density increases a steady decrease of Y_O is observed.     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Energy production and growth of Pseudomonas oxalaticus OX1 on oxalate and formate

The efficiency of oxidative phosphorylation in Pseudomonas oxalaticus during growth on oxalate and formate was estimated by two methods. In the first method the amount of ATP required to synthesize cell material of standard composition was calculated during growth of the organism on either of the two substrates. The [Y _ATP ^max ] theor. values thus obtained were 12.5 and 6.5 for oxalate and formate respectively, if the assumption were made that no energy is required for transport of oxalate or carbon dioxide. When active transport of oxalate requiring an energy input equivalent to 1 mole of ATP per mole of oxalate was taken into account, [Y _ATP ^max ]theor. for oxalate was 9.4. True Y _ATP ^max values were derived from these data on the assumption that the energy produced in the catabolism of Pseudomonas oxalaticus is used with approximately the same efficiency as in a range of other chemoorganotrophs. P/O ratios were calculated using the equation P/O=Y _O/Y _ATP. The data for Y _O and m _e required for these calculations were obtained from cultures of Pseudomonas oxalaticus growing on oxalate or formate in carbon-limited continuous cultures. The P/O ratios calculated by this method were, for oxalate, 1.3 (or 1.0 if active transport were ignored), and for formate, 1.7.In the second method the stoicheiometries of the respiration-linked proton translocations with oxalate and formate were measured in washed suspensions of cells grown on the two substrates. The H⁺/O ratios obtained were 4.3 with oxalate and 3.9 with formate. These data indicate the presence of two functional phosphorylation sites in the electron transport chain of Pseudomonas oxalaticus during growth on both substrates. A comparison of the P/O ratio on oxalate obtained with the two methods indicated that the energy requirement for active transport of oxalate has a major effect on the energy budget of the cell; about 50% of the potentially available energy in oxalate is required for its active transport across the cell membrane. Translocation of formate requires approximately 25% of the energy potentially available in the substrate. These results offer an explanation for the fact that molar growth yields of Pseudomonas oxalaticus on oxalate and formate are not very different.Abbreviations PMS phenazinemethosulphate - DCPIP 2,6-dichlorophenolindophenol - TMPD N,N,N,N-tetramethyl-1,4-phenylene-diamine dihydrochloride - SD standard deviation - PEP Phosphoenol-pyruvate     

Localization of hydrogenase and nitrate reductase in Campylobacter sputorum subsp. bubulus

Campylobacter sputorum subsp. bubulus contained hydrogenase activity after growth with lactate and nitrate and after growth with hydrogen and nitrate. After growth with hydrogen and nitrate a molar growth yield (g dry cells/mol hydrogen) of 5.6 was measured. Hydrogenase and nitrate reductase were membrane-bound enzymes. In cells with high hydrogenase activity the H⁺/O, H⁺/NO _inf2 ^sup- and H⁺/NO _inf3 ^sup- values with hydrogen as the electron donor were 3.74, 2.61 and 4.36 respectively. In cells with low hydrogenase activity these values were 2.33,-0.86 and 1.31 respectively. These values and the stoichiometry of respiration-driven proton translocation (H⁺/2e=2) led to the conclusion that hydrogenase is located at the periplasmic side of the cytoplasmic membrane. In cells with low lactate dehydrogenase activity or low hydrogenase activity the reduction of nitrate to nitrite could be separated from the reduction of nitrite to ammonia. Positive H⁺/NO _inf3 ^sup- values (between 0.9 and 1.7) with lactate or hydrogen as the electron donor were measured in these cells whereas H⁺/NO _inf2 ^sup- values were negative. From this result it was concluded that nitrate reductase is located at the cytoplasmic face of the cytoplasmic membrane. The results explain the previous observation that molar growth yields with nitrate were somewhat higher than those with nitrite.     

Metabolism of cellobiose and cellulose byBacteroides cellulosolvens

Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y _H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY _H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY _H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY _H.Issued as NRCC No. 27409.     

Lactate metabolism in Propionibacterium pentosaceum growing with nitrate or oxygen as hydrogen acceptor

When anaerobic cultures of Propionibacterium pentosaceum were shifted to low dissolved-oxygen concentration (D.O.C.), acetate production from lactate diminished and propionate production stopped, whereas pyruvate accumulated and oxygen was consumed. Assuming that energy is generated in the electron transfer to oxygen, Y_ATP values (g dry wt bacteria/mole ATP) of between 7.2 and 11.9 were calculated from molar growth yields and product formation. When oxidative phosphorylation in the electron transfer to oxygen was ignored, unreasonably high Y_ATP values were obtained. From these results it is concluded that energy is indeed generated in the electron transfer to oxygen. However, synthesis of cytochrome b was strongly repressed by oxygen. Furthermore, synthesis of all catabolic enzymes studied was impaired in bacteria growing at low D.O.C. Thus, the anaerobic character of P. pentosaceum may be explained by the inhibition of synthesis of both cytochrome b and enzymes in the presence of oxygen.It was demonstrated that nitrate reductase is synthesized constitutively in P. pentosaceum. Synthesis of nitrate reductase was stimulated by nitrate and repressed by oxygen. Synthesis of fumarate reductase was also repressed by oxygen, whereas only a small effect of nitrate on this enzyme was observed.However, propionate formation is inhibited during growth with nitrate. The absence of propionate formation in the presence of oxygen and nitrate is explained by inavailability of NADH needed for the conversion of oxaloacetate into malate in the reductive pathway to succinate, so that succinate and propionate cannot be formed.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.     

Growth yields and growth rates of Desulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources

Desulfovibrio vulgaris (Marburg) was grown on H₂ plus sulfate and H₂ plus thiosulfate as the sole energy sources and acetate plus CO₂ as the sole carbon sources. Conditions are described under which the bacteria grew exponentially. Specific growth rates () and molar growth yields (Y) at different pH were determined. and Y were found to be strongly dependent on the pH. Highest growth rates and molar growth yields were observed for growth on H₂ plus sulfate at pH 6.5 (=0.15h^-1; Y _{SO 4} ^2- =8.3g·mol^-1) and for growth on H₂ plus thiosulfate at pH 6.8 (=0.21h^-1; Y _{S 2}O ₃ ² =16.9g·mol^-1).The growth yields were found to increase with increasing growth rates: plots of 1/Y versus 1/ were linear. Via extrapolation to infinite growth rates a Y _SO4 ^2- /max of 12.2g·mol^-1 and a YS₂O ₃ ^2- /max of 33.5g·mol^-1 was obtained.The growth yield data are interpred to indicate that dissimilatory sulfate reduction to sulfide is associated with a net synthesis of 1 mol of ATP and that near to 3 mol of ATP are formed during dissimilatory sulfite reduction to sulfide.     

Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture

The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y _glucose and Y _ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l^-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y _ATP value.These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H₂, is neither coupled to, nor driven by, an energy-yielding reaction.     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

The effect of respiratory chain composition on the growth efficiencies of aerobic bacteria

The relationship between respiratory chain redox carrier composition and the efficiency of aerobic growth in continuous culture under carbon-limited conditions has been investigated for nine species of bacteria.True molar growth yields with respect to molecular oxygen ( ) or dissimilated carbon source (Y _{substrate ox.} ^max ) were not significantly affected by the nature either of the major quinone component (ubiquinone or menaquinone), of the major cytochrome oxidase moiety (cytochrome aa ₃ or o) or of the transhydrogenase segment (energy-dependent transhydrogenase, energy-independent transhydrogenase or no transhydrogenase), but were significantly influenced by the presence or absence of a high potential, membrane-bound cytochrome c. Under glycerol-limited conditions the presence of cytochrome c raised the average from 51.1 to 84.3 g cells · mole O ₂ ^–1 , and the average Y _{glycerol ox.} ^max from 154.6 to 233.2 g cells · mole glycerol oxidised^–1; the presence of this redox carrier also elicited increases in and Y _{substrate ox.} ^max of a similar order during growth under lactate, and glucose-limited conditions.The average efficiencies of aerobic energy conservation calculated from these true molar growth yields were 3.4 mole ATP equivalents · mole O ₂ ^–1 for organisms with respiratory chains which were deficient in cytochrome c and 5.9 mole ATP equivalents · mole O ₂ ^–1 for organisms with respiratory chains which contained cytochrome c.It is concluded from these data, and from parallel measurements of whole cell H⁺/O ratios, that bacterial respiratory systems invariably exhibit energy conservation at sites 1 and 2 but that the presence of a highpotential, membrane-bound cytochrome c is an obligatory prerequisite for energy conservation at site 3.     

Photophosphorylation capacity of stable spheroplast preparations of anabaena

Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H₂O → NADP, H₂O → methyl viologen, and H₂O → ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H₂O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O₂ balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9-aminoacridine fluorescence indicates the formation of a Δ pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH₄⁺. In the presence of NH₄⁺ alone, the membrane potential may act as the driving force for photophosphorylation.     

Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, l-lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H₂. Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO₂ and H₂ with part of the H₂ serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y _Glucose = 20 g/mol, leading to an energy coefficient of Y _ATP = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden–Meyerhof–Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y _Arsenate = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.     

Preparation and characterization of a soluble nitrate reductase from Azotobacter chroococcum

Summary The assimilatory nitrate reductase of the N₂-fixing bacterium Azotobacter chroococcum has been prepared in a soluble form from cells grown with nitrate as the nitrogen source, and some of its properties (electron donors and cofactors, K _mvalues for substrates, molecular weight, inhibitors, activators, etc.) have been studied. The enzyme is of an inducible nature and can exist in two interconvertible forms, either active or inactive.Tungstate very efficiently inhibits growth of the microorganism in media with nitrate. When either nitrite or ammonia are substituted for nitrate as the nitrogen source, growth is unaffected by tungstate concentrations which otherwise completely suppress growth on nitrate. Tungstate interferes by decreasing the cellular level of nitrate reductase activity, preventing, as a consequence, utilization of nitrate.