Hydrogen production by nitrogen-starved cultures of Anabaena cylindrica.

Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen continuously for 7 to 19 days. Hydrogen production attained a maximum level after 1 to 2 days of starvation and was followed by a slow decline. The maximum rates were 30 ml of H2 evolved per liter of culture per h or 32 mul of H2 per mg of dry weight per h. In 5 to 7 days the rate of H2 evolution by the more productive cultures fell to one-half its maximum value. The addition of 10(-4) to 5 X 10(-4) M ammonium increased the rate of oxygen evolution and the total hydrogen production of the cultures. H2-O2 ratios were 4:1 under conditions of complete nitrogen starvation and about 1.7:1 after the addition of ammonium. Thus, oxygen evolution was affected by the extent of the nitrogen starvation. Thermodynamic efficiencies of converting incident light energy to free energy of hydrogen via algal photosynthesis were 0.4%. Possible factors limiting hydrogen production were decline of reductant supply and filament breakage. Hydrogen production by filamentous, heterocystous blue-green algae could be used for development of a biophotolysis system.     

Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.     

Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

Derepression of nitrogenase gene expression was studied at the mRNA and enzyme activity levels in anaerobic cultures of Anabaena variabilis 29413. Cells, previously grown with ammonium chloride, were incubated in the absence of fixed nitrogen compounds under an Ar atmosphere with dichlorophenyldimethyl-urea present to inhibit oxygen evolution. The appearance of nitrogenase mRNA (measured by dot blot hybridization analysis) and nitrogenase activity (measured as acetylene-reducing activity) was followed, and the cells were also observed by phase-contrast microscopy. Nitrogenase mRNA could be detected after 1.5 to 2.0 h of nitrogen starvation; enzyme activity appeared about 1 h later. Although enzyme activity increased for many hours, mRNA levels reached a steady state rapidly. Neither heterocysts nor proheterocysts formed under these conditions; however, the cells were observed to shrink and become chlorotic. When anaerobic, derepressed cultures were exposed to oxygen, nitrogenase mRNA levels decreased very rapidly.     

H2, N2, and O2 metabolism by isolated heterocysts from Anabaena sp. strain CA.

Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution. These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp. strain CA-V possessing fragile vegetative cell walls. Hydrogen production was observed with isolated heterocysts. The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production. This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients. The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA. H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished. A phenotypic Hup- mutant (N9AR) of Anabaena sp. strain CA was isolated which did not respond to nickel. In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+. Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

Hydrogen production by Anabaena cylindrica: effects of varying ammonium and ferric ions, pH, and light.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.     

Nitrogen deprivation results in photosynthetic hydrogen production in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii is able to use photosynthetically provided electrons for the production of molecular hydrogen by an [FeFe]-hydrogenase HYD1 accepting electrons from ferredoxin PetF. Despite the severe sensitivity of HYD1 towards oxygen, a sustained and relatively high photosynthetic hydrogen evolution capacity is established in C. reinhardtii cultures when deprived of sulfur. One of the major electron sources for proton reduction under this condition is the oxidation of starch and subsequent non-photochemical transfer of electrons to the plastoquinone pool. Here we report on the induction of photosynthetic hydrogen production by Chlamydomonas upon nitrogen starvation, a nutritional condition known to trigger the accumulation of large deposits of starch and lipids in the green alga. Photochemistry of photosystem II initially remained on a higher level in nitrogen-starved cells, resulting in a 2-day delay of the onset of hydrogen production compared with sulfur-deprived cells. Furthermore, though nitrogen-depleted cells accumulated large amounts of starch, both hydrogen yields and the extent of starch degradation were significantly lower than upon sulfur deficiency. Starch breakdown rates in nitrogen or sulfur-starved cultures transferred to darkness were comparable in both nutritional conditions. Methyl viologen treatment of illuminated cells significantly enhanced the efficiency of photosystem II photochemistry in sulfur-depleted cells, but had a minor effect on nitrogen-starved algae. Both the degradation of the cytochrome b ₆ f complex which occurs in C. reinhardtii upon nitrogen starvation and lower ferredoxin amounts might create a bottleneck impeding the conversion of carbohydrate reserves into hydrogen evolution.     

Gas exchange in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133 and Its hydrogenase-deficient mutant strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H(2)), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O(2), CO(2), N(2), and H(2) was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO(2) and O(2). The amount of H(2) produced per molecule of N(2) fixed was found to vary with light conditions, high light giving a greater increase in H(2) production than N(2) fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H(2) produced per molecule of N(2) fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO(2), caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 micro mol (mg of chlorophyll a)(-1) h(-1) to 9 micro mol (mg of chlorophyll a)(-1) h(-1) after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.     

Postmetamorphic Growth and Metabolism of Long-Spined Black Sea Urchin (Diadema antillarum) Reared in the Laboratory

Postmetamorphic growth and metabolism measurements were obtained on two cohorts of laboratory-reared Diadema antillarum. The cohorts grew linearly from less than 1 mm to over 43 mm. Daily growth averaged 0.097 and 0.11 mm d^-1, respectively, for the two cohorts, and was found to differ significantly. Urchin metabolism was examined by a series of simultaneous measurements of oxygen consumption and ammonium excretion over 16 days on starved juveniles ranging 16.5 to 18.3 mm. Metabolic activity under conditions of starvation was used as a test of the viability of urchins reared in the laboratory with cultured food resources. Catabolic activity differed from the first week of starvation compared to the second. Metabolic response included: (1) a 2.2-fold increase in oxygen consumption rate; (2) 50% decline in ammonium excretion rate; and (3) a 5.1-fold increase in oxygen to nitrogen ratio. These measurements are consistent with a shift from almost pure protein catabolism during the first seven days of starvation to a lipid : protein catabolic ratio of 1 : 1 after the first week. Growth and metabolism experiments of this type are seen as a first step towards optimizing laboratory culture techniques of this species.     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.     

Postmetamorphic Growth and Metabolism of Long-Spined Black Sea Urchin (Diadema antillarum) Reared in the Laboratory

Postmetamorphic growth and metabolism measurements were obtained on two cohorts of laboratory-reared Diadema antillarum. The cohorts grew linearly from less than 1 mm to over 43 mm. Daily growth averaged 0.097 and 0.11 mm d^?1, respectively, for the two cohorts, and was found to differ significantly. Urchin metabolism was examined by a series of simultaneous measurements of oxygen consumption and ammonium excretion over 16 days on starved juveniles ranging 16.5 to 18.3 mm. Metabolic activity under conditions of starvation was used as a test of the viability of urchins reared in the laboratory with cultured food resources. Catabolic activity differed from the first week of starvation compared to the second. Metabolic response included: (1) a 2.2-fold increase in oxygen consumption rate; (2) 50% decline in ammonium excretion rate; and (3) a 5.1-fold increase in oxygen to nitrogen ratio. These measurements are consistent with a shift from almost pure protein catabolism during the first seven days of starvation to a lipid : protein catabolic ratio of 1 : 1 after the first week. Growth and metabolism experiments of this type are seen as a first step towards optimizing laboratory culture techniques of this species.     

沼泽红假单胞菌乙酸光合放氢研究

依据光合细菌生长代谢特性和有机废水降解主要产物类型,11种有机物被用于沼泽红假单胞菌（Rhodopseudomonas palustris）Z菌株的光合产氢研究,其中,乙酸反应体系产氢活性最高。在此基础上,研究了该菌株的生长与产氢动力学行为,探求了影响该菌株光合放氢的主要限制性影响因素。结果表明,该菌株产氢与生长部分相关。种子培养基和菌龄对产氢活性有明显影响。细胞最适产氢和生长所需要的光照强度和温度基本一致。当种子来源于硫酸铵高菌龄预培养物或谷氨酸钠对数期预培养物时,该菌株产氢活性显著增加,产氢延滞期明显缩短。氧浓度和接种量对产氢活性也有显著影响。供氢体和氮源浓度直接决定细胞的生长与光放氢活性。在低于70 mmol/L乙酸钠和15 mmol/L谷氨酸钠时,产氢活性随底物浓度的增加而增强。谷氨酸钠浓度高于15mmol/L时,由于游离NH₄⁺的出现,产氢活性受到抑制,但却明显刺激细胞的生长。在标准状况下,该菌株的最大产氢速率可达19.4 mL·L^-1·h^-1。     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

Hydrogen metabolism and energy costs of nitrogen fixation

Abstract The high energy costs of biological nitrogen fixation are partly caused by hydrogen production during the reduction of dinitrogen to ammonia. Some nitrogen-fixing organisms can recycle the evolved hydrogen via a membrane-bound uptake hydrogenase. The energetic aspects of hydrogen metabolism and nitrogen fixation are discussed.
Studies on both isolated nitrogenase proteins and nitrogen-fixing chemostat cultures show that energy limitation will result in a high hydrogen production by nitrogenase. In plant- Rhizobium symbiosis, the supply of oxygen or photosynthetate is the limiting factor for nitrogen fixation. In both cases, nitrogen fixation is energy-limited, and it is concluded that a large amount of hydrogen is produced during nitrogen fixation in these symbioses.
Hydrogen reoxidation yields less energy than the oxidation of endogenous substrates, and therefore expression of hydrogenase under oxygen-limited conditions is energetically unfavourable. Moreover, hydrogen reoxidation can never completely regain the energy invested during hydrogen production. The controversial reports of the effect of hydrogen reoxidation on the efficiency of nitrogen fixation are being discussed.
The determination of the energy costs of nitrogen fixation (expressed as the amount of ATP needed to fix 1 mol of N₂) using chemostat cultures is described. Calculations show that the nitrogenase-catalysed hydrogen production has more influence on the efficiency of nitrogen fixation than the absence or presence of a hydrogen uptake system.     

Hydrogen Production by the Photosynthetic Bacterium Rhodospirillum rubrum

Continuous photosynthetic production of hydrogen by Rhodospirillum rubrum in batch cultures was observed up to 80 days with the hydrogen donor, pure lactate or lactic acid-containing wastes, supplied periodically. Hydrogen was produced at an average rate of 6 ml/h per g (dry weight) of cells with whey as a hydrogen donor. In continuous cultures with glutamate as a growth-limiting nitrogen source and lactate as a hydrogen donor, hydrogen was evolved at a rate of 20 ml/h per g (dry weight). The composition of the gas evolved remained practically constant (70 to 75% H₂, 25 to 30% CO₂). Photosynthetic bacteria processing specific organic wastes could be an advantage in large-scale production of hydrogen together with food protein of high value, compared to other biological systems.     

Eucaryote thermophily: role of lipids in the growth of Talaromyces thermophilus

Metabolically active heterocysts were isolated from a mutant of Anabaena sp. strain CA with fragile vegetative cells. Heterocysts isolated from cultures grown in 1% CO2 in air reduced C2H2 at 57 and 10 nmol of C2H2 per mg (dry weight) per min under H2 and Ar, respectively. However, if whole filaments were sparged with 1% CO2 in 99% Ar for 12 h before heterocyst isolation, these heterocysts showed C2H2 reduction rates of 83 nmol of C2H4 per mg (dry weight) per min under either H2 or Ar, or 40% the activity of whole filaments grown in 1% CO2 in air. Heterocysts isolated from cultures sparged with 100% Ar or 1% CO2 in 99% N2 had the same C2H2 reduction pattern as heterocysts from cultures grown in 1% CO2 in air, i.e., low activity under Ar and high activity under H2. Labeling of whole filaments incubated with NaH14CO3 for 12 h under 1% CO2 in air or 1% CO2 in 99% Ar resulted in a twofold higher accumulation of 14C-labeled compounds in vegetative cells and heterocysts of Ar-incubated cells. Our results suggest that during incubation under 1% CO2 in 99% Ar, presumably a nitrogen starvation condition, continuing photosynthetic fixation of CO2 leads to accumulation of material(s) in the heterocysts that supports a high, persistent endogenous rate of C2H2 reduction. This material appears to be, in part, glycogen.     

Acetate as a carbon source for hydrogen production by photosynthetic bacteria

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.     

Acetylene reduction (nitrogen fixation) by enterobacteriaceae isolated from paper mill process waters

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.