Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

Protoheme,a dispensable growth factor forBacteroides fragilis grown by batch and continuous culture in a basal medium

The growth yields of 10 strains ofBacteroides fragilis isolated from a variety of clinical sites were determined in (a) basal medium, (b) basal medium plus heme, and (c) basal medium plus heme and menadione. The molar growth yield values, expressed as a function of glucose (Y_G) and ATP produced (Y_ATP) for 24 h and 48 h were used for a comparison of different strains. Considerable variation occurred among strains, but in general only the results from 24-h grown cells were reproducible. After this period, the microscopic appearance of cells changed dramatically from well-formed, intact cells to large collections of extracellular vesicles and lysed cells. All strains were stimulated by heme, but marked differences occurred among strains. The addition of heme and menadione to the basal medium increased the Y_G values of some strains, whereas others were unaffected. Heme-cultured cells produced acetate, propionate, and succinate as major metabolic end products and possessed cytochrome b, menaquinone-10, and fumarate reductase activity. Strain NCTC 9343 grown without added heme by continuous culture or batch culture produced cells that were morphologically and biochemically similar. Under both conditions these cells lacked cytochromes, menaquinones, and fumarate reductase activity, but produced high levels of lactate and fumarate together with lower levels of acetate, propionate, and succinate.     

Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.     

The Physiology of Encystment of Hartmannella castellanii*

SYNOPSIS. Some aspects of the physiology of encystment of the soil amoeba Hartmannella castellanii in a replacement encystment medium consisting of 5 × 10^-2 M MgCl₂ have been investigated. It is suggested that measurement of the cellulose produced during encystment in the synthesis of the cyst wall is a more reliable measure of the process than other methods tried. The degree of encystment was dependent on the physiologic state of the amoebae and the composition of the growth medium, but the initial pH of the encystment medium (C. 4.0-8.5) had little effect on the process. The requirement for Mg during encystment was probably not due to its deficiency during growth. Encystment was inhibited to varying extents by inhibitors of protein synthesis, tetracycline and chloramphenicol and also by arsenate, arsenite and iodoacetate; sodium fluoride, malonate and 2, 4-dinitrophenol were without marked effect. Addition of glucose and α-ketoglutarate to the replacement medium led to improvement in the encystment response. The presence of glutamate and histidine during encystment led to cell death. Other carbon and nitrogen sources had no effect. During encystment there was an increase in the metabolic activity of the amoebae, as measured by their oxygen consumption. This was accompanied by a decrease of about 40% in cellular dry weight and protein content. Of the other chemical components, there were marked initial increases in the levels of total carbohydrates and pentose which were followed by their depletion during cellulose synthesis. Encystment was completed after about 64 hr when the synthesis of cellulose was complete and the oxygen uptake of the amoebae fell to an immeasurable level.     

Effects of fumarate,l-malate,and anAspergillus oryzae fermentation extract ond-lactate Utilization by the ruminal bacteriumSelenomonas ruminantium

Growth ofSelenomonas ruminantium HD4 in medium that contained 21mm d-lactate was stimulated to varying degrees by 10mm l-malate, 10mm fumarate, and 2% (v/v)Aspergillus oryzae fermentation extract (Amaferm). Amaferm treatment caused the greatest growth stimulation. Initial uptake rates (30s) and long-term uptake rates (30 min) ofd-lactate by whole cells ofS. ruminantium were increased in the presence of 10mm l-malate. Amaferm (25 l/ml) also stimulated long-term uptake rates ofd-lactate, whereas fumarate had no effect. Initial uptake ofd-lactate was depressed in the presence of fumarate or Amaferm. When eitherl-malate, fumarate, or Amaferm was included in thed-lactate growth medium, a homosuccinate fermentation resulted and an inverse relationship was observed between growth (protein synthesis) and succinate production. Recent research demonstrated that Amaferm containsl-malate, and this dicarboxylic acid may be involved in stimulatingd-lactate utilization byS. ruminantium.     

Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Isolation and characterization of siderophore from cowpeaRhizobium (peanut isolate)

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.     

Effect of NaCl salinity on growth and mineral partitioning in Quercus robur L., a rhythmically growing species

 Sodium salt sensitivity of common oak (Quercus robur L.) was evaluated in hydroponic culture using INRA-Morizet solution. Addition of NaCl to the nutrient solution reduced only length and weight of roots and first flush stems. In contrast, the second flush was properly expanded even in the presence of 40 mM of NaCl in culture medium. Both leaf number and leaf area were not affected by increasing salt concentration in medium culture while this increase induced significant leaf damage especially in first flush leaves. Stem starch storage was reduced only at 40 mM NaCl treatment. Common oak seedlings seemed to be able to better compartmentalize sodium than chloride when the NaCl concentration increased in the medium culture. Chloride presented a lower uptake than sodium. Sodium was preferentially accumulated in roots and this accumulation occurred at the expense of potassium uptake. The decrease of ATP content in leaves of common oak seedlings submitted to NaCl treatments could indicate that it was used for sodium exclusion out of the leaves, especially in second flush leaves. Relationships between growth responses, starch and mineral element distribution in common oak seedlings will be discussed. Received: 20 November 1997 / Accepted: 3 March 1998     

Effect of reduction of culture medium sodium, using different sodium chloride substitutes, on the proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts

We have substituted choline chloride, tetramethylammonium chloride, sucrose, or glucose for culture medium sodium chloride. When culture medium sodium is reduced below physiological levels (143 mM) by replacement of graded concentrations of sodium chloride with equivalent concentrations of choline chloride, normal fibroblasts approach proliferative inactivity in the presence of 90 mM Na, while their Rous sarcoma virus (RSV)-infected counterparts proliferate actively; both normal and neoplastic cells die with further sodium reduction. When culture medium NaC; is replaced with tetramethylammonium chloride, however, both normal and RSV-infected fibroblasts alike approach proliferative inactivity in the presence of 110 mM Na and both die off in the presence of 90 mM Na. When culture medium NaCl is replaced with sucrose or glucose yet another set of results is obtained: both normal and RSV-infected fibroblasts proliferate at reduced, although significant, rates in the presence of 42 mM Na. It is clear from our experimental results that the effects of reduction of culture medium sodium on cell proliferation differ markedly with the use of different sodium chloride substitutes. Caution must be exercised, therefore, in drawing inferences concerning the role of sodium in mitogenesis from experimental studies based on the tactic of reduction of external sodium.     

Energy metabolism of some representatives of the Haemophilus group

The purpose of this investigation was to characterize the carbohydrate catabolism and the constellation of the respiratory chain components of Haemophilus influenzae RAMC 18 Bensted, H. parainfluenzae 1 Fleming, H. parainfluenzae 429 Pittman and H. aegyptius 180a Pittman. These strains represent several physiological types with respect to respiratory quinones and glucose catabolism.On addition of glucose or lactate to the complex growth medium a remarkable increase in cell mass was observed. Depending on the growth rate, carbohydrate degradation varied with the strains examined so that at the end of the exponential growth phase only small amounts of the supplements could be demonstrated.All strains were found to possess functional enzymes of Embden-Meyerhof-Parnas-, Entner-Doudoroff-pathways, hexosemonophosphate shunt, tricarboxylic acid cycle and gluconeogenesis with an extremely high activity of malate dehydrogenase.The concentration of cytochromes varied according to culture conditions. The cytochromes a₁, d, o and b+c were found to occur under aerobic conditions. In cells grown anaerobically in the presence of fumarate cytochromes a₁ and d could not be demonstrated. Under aerobic conditions preparations of H. parainfluenzae 1 Fleming exhibited an -maximum at 558 nm, whereas under anaerobic culture conditions with fumarate as terminal electron acceptor an -maximum at 552 nm occurred, suggesting different roles of b and c type cytochromes in aerobic and anaerobic electron transport to fumarate, respectively.     

不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

Organic acid metabolism in Sclerotinia sclerotiorum

Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Degradation of fenitrothion byBacillus stearothermophilus adhering to silica

B. stearothermophilus strain AG-49, when cultivated in mineral medium in the presence of silica (SA), adhered to SA. Adhesion depended on age of culture, contact time and glucose concentration of the culture medium. Mid-exponential phase culture (5 h) required minimum contact time (30 min) for maximum adhesion. 0.6% glucose concentration was optimum. Quantitative variation in protein and saccharide extractable in sodium chloride and sodium dodecyl sulfate (SDS) was observed. Five % degradation of fenitrothion by adherentB. stearothermophilus could be achieved in 4 d.     

Formation of the exopolysaccharide ethapolan by <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. IMV B-7005 on a fumarate-glucose mixture

Our studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4: 1 resulted in a 1.3–2.2-fold increase of the EPS amount synthesized and in a 1.3–2-fold increase of the EPS yield relative to the biomass compared to cultivation on monosubstrates. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Metabolic characterization of lactic acid bacterium Lactococcus garvieae sk11, capable of reducing ferric iron, nitrate, and fumarate

A lactic acid bacterium capable of anaerobic respiration was isolated from soil with ferric iron-containing glucose basal medium and identified as L. garvieae by using 16S rDNA sequence homology. The isolate reduced ferric iron, nitrate, and fumarate to ferrous iron, nitrite, and succinate, respectively, under anaerobic N2 atmosphere. Growth of the isolate was increased about 30-39% in glucose basal medium containing nitrate and fumarate, but not in the medium containing ferric iron. Specifically, metabolic reduction of nitrate and fumarate is thought to be controlled by the specific genes fnr, encoding FNR-like protein, and nir, regulating fumarate-nitrate reductase. Reduction activity of ferric iron by the isolate was estimated physiologically, enzymologically, and electrochemically. The results obtained led us to propose that the isolate metabolized nitrate and fumarate as an electron acceptor and has specific enzymes capable of reducing ferric iron in coupling with anaerobic respiration.     

Improved Growth Media for Vibrio succinogenes

Five new culture media for Vibrio succinogenes are described. Of these, a medium composed of 0.4% yeast extract, 100 mM ammonium formate, 120 mM sodium fumarate, and 0.05% sodium thioglycolate, pH 7.3, supports the best growth.     

Some Characteristics of Central Metabolism in Acinetobacter sp. Grown on Ethanol

Ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The ratio of these enzymes was different in the exponential and the stationary growth phases. The addition of the C₄-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of microbial exopolysaccharide ethapolan on C₂-substrates.     

Culture conditions for efficient induction of green plants from isolated microspores of barley

Subculture regime and carbohydrate concentration of the medium had a marked effect on the regeneration of green plantlets from mechanically isolated microspores of Hordeum vulgare L. cv. Kymppi. A sevenfold increase in the yield of green plants was obtained by shortening the suspension culture time of the developing proembryo mass from 4 to 3 weeks. A further twofold increase was obtained by increasing the maltose concentration of the microspore isolation medium and of the culture medium. Under optimal conditions, a mean of 169±97 green plants per spike were regenerated.