Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

Considerations on the energy metabolism of Clostridium kluyveri

Summary On the basis of the known fermentation balance and of the enzyme activities reported in Clostridium kluyveri the ethanol-acetate fermentation of Clostridium kluyveri has been analyzed with respect to possible ATP-yielding reactions and to the significance of the evolution of hydrogen gas during the fermentation. The fermentation pathway presented allows the following conclusions: hydrogen gas is an essential end product of the ethanol-acetate fermentation. For each two moles of hydrogen gas evolved one mole of acetyl coenzyme A becomes available to the cells for ATP synthesis, and it is not necessary to assume that ATP is synthesized by Clostridium kluyveri by electron transport phosphorylation. Hydrogen gas must be formed in the dehydrogenation of acetaldehyde. Since Clostridium kluyveri contains a NAD reductase, less than one mole of hydrogen gas is formed per mole of acetaldehyde oxidized, thus explaining that acetate is required for the fermentation of ethanol.It could be demonstrated that growth of Clostridium kluyveri is slow in a hydrogen atmosphere as compared with growth in an argon atmosphere. The general fermentation equation constructed is in accordance with the experimental data of Bornstein and Barker and of Thauer et al.

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Zusammenfassung Auf der Grundlage bekannter Gärungsanalysen und der in zellfreien Extrakten nachgewiesenen Enzymaktivitäten wurde die Äthanol-Acetat-Gärung von Clostridium kluyveri auf mögliche Energie liefernde Reaktionen hin untersucht. Das entworfene Schema der Äthanol-Acetat-Gärung erlaubt folgende Schlußfolgerungen: Die Bildung von molekularem Wasserstoff während der Gärung its von elementarer Bedeutung für den Energiestoffwechsel von C. kluyveri. Je Mol freigesetzten Wasserstoffs stehen C. kluyveri 0,5 Mole Acetyl-Coenzym A für die Energiegewinnung zur Verfügung, und es ist überflüssig, eine ATP-Synthese durch Elektronentransport-Phosphorylierung anzunehmen. Der molekulare Wasserstoff muß bei der Dehydrogenierung des Acetaldehyds gebildet werden. Da hierbei weniger als ein Mol molekularer Wasserstoff je Mol Acetaldehyd entsteht und ein Teil des Wasserstoffs auf NAD übertragen wird, ist Acetat als Wasserstoffacceptor für die Vergärung des Äthanols notwendig.Die abgeleitete Gärungsgleichung stimmt mit den von Bornstein und Barker und von Thauer et al. ermittelten Gärungsbilanzen überein. Es konnte nachgewiesen werden, daß C. kluyveri in einer Wasserstoffatmosphäre sehr viel langsamer wächst als in einer Argonatmosphäre.

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Abbreviations ATP adenosine triphosphate - CoA coenzyme A - Fd_red reduced ferredoxin - NAD(P) nicotinamide-adenine-dinucleotide(phosphate)     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

克氏梭菌硫解酶的鉴定及其功能研究

【目的】硫解酶是梭菌属微生物合成短中链脂肪酸的关键酶。克氏梭菌(Clostridium kluyveri)具有3个高度同源的硫解酶编码基因,对这3个基因的功能鉴定是解析克氏梭菌高己酸合成能力的关键。【方法】通过发酵动力学分析确定克氏梭菌的己酸和丁酸生成动力学特征;转录组测序结合反转录-荧光定量RCR分析克氏梭菌3个硫解酶编码基因的表达水平和时序表达特征;在大肠杆菌中异源表达这3个硫解酶,并对其硫解酶动力学参数进行测定。【结果】克氏梭菌生成丁酸、己酸、辛酸,其中己酸为主要代谢产物;转录组数据显示,在乙酸消耗完全之前,thlA1基因维持恒定表达,thlA2基因表达时序上调,thlA3基因表达时序下调,转录组测序表明3个硫解酶编码基因均具有较高水平的转录活性,thlA2和thlA3的最高表达量分别约为thlA1的29%和43%;硫解酶动力学参数测定结果表明,克氏梭菌3个硫解酶对于四碳底物均显示出相似的底物亲和力(K_m),但ThlA1对四碳底物的催化效率(k_(cat)/K_m)略低于ThlA2和ThlA3。【结论】克氏梭菌的3个硫解酶均具有催化活性,在克氏梭菌体内均呈活跃表达,表明克氏梭菌拥有3个具有催化活性的硫解酶,这为后续深入研究克氏梭菌己酸合成机理奠定了基础。     

Activation and degradation of benzoate, 3-phenylpropionate and crotonate bySyntrophus buswellii strain GA. Evidence for electron-transport phosphorylation during crotonate respiration

A strictly anaerobic, benzoate-degrading bacterium,Syntrophus buswellii strain GA, was able to degrade benzoate or 3-phenylpropionate to acetate, CO₂ and H₂ if the hydrogen partial pressure was sufficiently low. The hydrogen was removed in syntrophic coculture byMethanospirillum hungatei or byDesulfovibrio sp. through interspecies hydrogen transfer or in pure culture by the use of crotonate as reducible cosubstrate. Alternatively,S. buswellii strain GA could grow in pure culture with crotonate. Activities of seven catabolic enzymes were measured in crude cell extracts ofS. buswellii strain GA grown with various substrates and of crotonate-grownS. buswellii strain DSM 2612A. Benzoate, 3-phenylpropionate and crotonate were activated by CoA ligases. Glutaryl-CoA dehydrogenase was found to be involved in the degradation of aromatic compounds and enzymes catalysing -oxidation were involved in the reaction sequence from crotonyl-CoA to acetate. Ac-type cytochrome was present in the cytoplasm, whereasb-type cytochromes were associated with the membranes of bothS. buswelli strains grown on crotonate. These indicated the presence of an electron-transport system. A high growth yield of crotonate-grownS. buswellii strain GA might be explained by electron-transport phosphorylation in addition to substrate-level phosphorylation.     

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H₂ and CO₂: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate

The metabolism of Clostridium kluyveri has been extensively studied, but the range of substrates C. kluyveri can use for growth has not been fully explored. The use of propanol and succinate as growth substrates were established. C. kluyveri grows on acetate with propanol replacing ethanol. The principle carbon containing products were propionate, valerate, butyrate and hexanoate with traces of heptanoate. When grown with ethanol and succinate the principle carbon-containing products were acetate, butyrate and hexanoate. Hexanol was found as a product when incubated with ethanol and succinate 4-hydroxybutyrate or 3-butenoate. 5-Hexenoate was also a product of 3-butenoate and ethanol metabolism. The splitting of succinate into 2 acetates was indicated with ethanol providing the necessary reducing equivalents. Hydrogen was also found as a source of reducing equivalents but could not replace ethanol. A mechanism of succinate metabolism to acetate was proposed which accounts for growth yield, energetics considerations, carbon balances, production of side products and intermediates.Contribution No. 3619     

NaCl-induzierter Crassulaceensäurestoffwechsel bei einer weiteren Aizoacee: Carpobrotus edulis

Summary Carpobrotus edulis grown for 24 days in nutrient solution plus 400 mM of NaCl shows the typical CO₂ gas exchange reactions observed in CAM plants. Control plants grown in nutrient solution alone exhibit CO₂ gas exchange reactions typical for C₃ plants.

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Abkürzungen CAM Crassulaceensäurestoffwechsel - FG Frischgewicht     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Stoffwechselleistung und Enzymaktivität bei Chlamydobotrys (Volvocales)

Zusammenfassung In Versuchen mit Chlamydobotrys, dessen Stoffwechsel vor allem von der Photoassimilation von Acetat abhängt, konnte gezeigt werden, daß Änderungen der Leistung bei der Photoassimilation von Acetat und dessen oxydativem Dunkel-Stoffwechsel von ähnlichen Änderungen der Aktivität gewisser Enzyme begleitet sind. Während des optimalen Wachstums des Organismus auf Acetat ist die Fähigkeit zur Photoassimilation von CO₂ sehr gering, was mit einer niedrigen Aldolaseaktivität einhergeht. Nach Übertragung der Algen auf ein acetatfreies Medium nehmen Photoassimilation von CO₂ und Aldolaseaktivität zu. Eine maximale Aldolaseaktivität konnte nur erzielt werden, wenn eine Stickstoffquelle (NH₄Cl) die Synthese von aktivem Enzymprotein ermöglicht.

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Summary In experiments with Chlamydobotrys, the metabolism of which depends mainly on the photoassimilation of acetate, changes in the metabolic activity for the photoassimilation of acetate and its oxidative dark-metabolism could be shown to be accompanied by similar changes in the activity of certain enzymes. During optimal growth of the organism on acetate the ability for photoassimilation of CO₂ is extremely weak which is corresponded by low aldolase-activity. After transferance of the algae to an acetatefree medium the photoassimilation of CO₂ and the aldolase-activity rise. Maximum aldolase-activity could only be obtained, if a nitrogen-source (NH₄Cl) makes possible a synthesis of active enzyme-protein.

     

Die Biosynthese der Poly-β-hydroxybuttersäure durch Knallgasbakterien

Zysammenfassung Bereits nach 8 sec ¹⁴CO₂-Fixierung ist die aus Hydrogenomonas H 16 isolierte Poly--hydroxybuttersäure (PHBS) gleichmäßig radioaktiv markiert.Es werden Beweise dafür erbracht, daß die PHBS aus Kohlendioxyd über 3-Phosphoglycerinsäure, Brenztraubensäure, Acetyl-CoA und Acetacetyl-CoA synthetisiert wird.Während der PHBS-Synthese geht so eines von drei fixierten CO₂-Molekülen durch oxydative Decarboxylierung der Brenztraubensäure wieder verloren. Damit steht im Einklang, daß die CO₂-Fixierungsleistung wachsender Zellen größer ist als die PHBS-speichernder.Nur ein Zehntel der Ribulose-1,5-diphosphat-Carboxylase, die für die gemessene autotrophe CO₂-Fixierungskapazität erforderlich wäre, konnte im Rohextrakt von Hydrogenomonas H 16 nachgewiesen werden. Das Enzym ließ sich 20 fach anreichern.

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Summary Poly--hydroxybutyric acid (PHBA) isolated from Hydrogenomonas H 16 following an 8 sec ¹⁴CO₂-incorporation is already uniformly labelled.It was shown, that the synthesis of PHBA from carbon dioxide takes place via 3-phosphoglyceric acid, pyruvic acid, acetyl-CoA and acetoacetyl-CoA.During the synthesis of PHBA, one of three CO₂-molecules previously fixed is lost in an oxydative decarboxylation of pyruvic acid. It is therefore evident that the CO₂-fixation of growing cells will be larger than that of cells storing PHBA.Only one tenth of the ribulose-1,5-diphosphate carboxylase, which would be necessary for the measured CO₂-fixation, could be determined in the crude extract of Hydrogenomonas H 16. The carboxylase was purified about 20-fold.

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Auszug aus der gleichlautenden Dissertation der mathematisch-naturwissenschaftlichen Fakultät der Universität Göttingen 1963.     

Effects of low and elevated CO2 on C3 and C4 annuals

Abutilon theophrasti (C₃) and Amaranthus retroflexus (C₄), were grown from seed at four partial pressures of CO₂: 15 Pa (below Pleistocene minimum), 27 Pa (pre-industrial), 35 Pa (current), and 70 Pa (future) in the Duke Phytotron under high light, high nutrient, and wellwatered conditions to evaluate their photosynthetic response to historic and future levels of CO₂. Net photosynthesis at growth CO₂ partial pressures increased with increasing CO₂ for C₃ plants, but not C₄ plants. Net photosynthesis of Abutilon at 15 Pa CO₂ was 70% less than that of plants grown at 35 Pa CO₂, due to greater stomatal and biochemical limitations at 15 Pa CO₂. Relative stomatal limitation (RSL) of Abutilon at 15 Pa CO₂ was nearly 3 times greater than at 35 Pa CO₂. A photosynthesis model was used to estimate ribulose-1,5-bisphosphate carboxylase (rubisco) activity (Vc_max), electron transport mediated RuBP regeneration capacity (J _max), and phosphate regeneration capacity (PiRC) in Abutilon from net photosynthesis versus intercellular CO₂ (A–C _i) curves. All three component processes decreased by approximately 25% in Abutilon grown at 15 Pa compared with 35 Pa CO₂. Abutilon grown at 15 Pa CO₂ had significant reductions in total rubisco activity (25%), rubisco content (30%), activation state (29%), chlorophyll content (39%), N content (32%), and starch content (68%) compared with plants grown at 35 Pa CO₂. Greater allocation to rubisco relative to light reaction components and concomitant decreases in J _max and PiRC suggest co-regulation of biochemical processes occurred in Abutilon grown at 15 Pa CO₂. There were no significant differences in photosynthesis or leaf properties in Abutilon grown at 27 Pa CO₂ compared with 35 Pa CO₂, suggesting that the rise in CO₂ since the beginning of the industrial age has had little effect on the photosynthetic performance of Abutilon. For Amaranthus, limitations of photosynthesis were balanced between stomatal and biochemical factors such that net photosynthesis was similar in all CO₂ treatments. Differences in photosynthetic response to growth over a wide range of CO₂ partial pressures suggest changes in the relative performance of C₃ and C₄ annuals as atmospheric CO₂ has fluctuated over geologic time.     

Two Pathways for Glutamate Biosynthesis in the Syntrophic Bacterium Syntrophus aciditrophicus

The anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate by Syntrophus aciditrophicus grown syntrophically with Methanospirillum hungatei provides a model to study syntrophic cooperation. Recent studies revealed that S. aciditrophicus contains Re-citrate synthase but lacks the common Si-citrate synthase. To establish whether the Re-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-¹³C]acetate and [1-¹⁴C]acetate as well as [¹³C]bicarbonate as additional carbon sources during axenic growth of S. aciditrophicus on crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and ¹³C nuclear magnetic resonance (NMR) spectroscopy of the obtained [¹³C]glutamate, as well as decarboxylation of [¹⁴C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route used Re-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium of S. aciditrophicus when grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. Besides Clostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.     

Leaf temperature of soybean grown under elevated CO2 increases Aphis glycines (Hemiptera: Aphididae) population growth

Abstract Plants grown under elevated carbon dioxide (CO₂) experience physiological changes that influence their suitability as food for insects. To determine the effects of living on soybean (Glycine max Linnaeus) grown under elevated CO₂, population growth of the soybean aphid (Aphis glycines Matsumura) was determined at the SoyFACE research site at the University of Illinois, Urbana‐Champaign, Illinois, USA, grown under elevated (550 μL/L) and ambient (370 μL/L) levels of CO₂. Growth of aphid populations under elevated CO₂ was significantly greater after 1 week, with populations attaining twice the size of those on plants grown under ambient levels of CO₂. Soybean leaves grown under elevated levels of CO₂ were previously demonstrated at SoyFACE to have increased leaf temperature caused by reduced stomatal conductance. To separate the increased leaf temperature from other effects of elevated CO₂, air temperature was lowered while the CO₂ level was increased, which lowered overall leaf temperatures to those measured for leaves grown under ambient levels of CO₂. Aphid population growth on plants grown under elevated CO₂ and reduced air temperature was not significantly greater than on plants grown under ambient levels of CO₂. By increasing Glycine max leaf temperature, elevated CO₂ may increase populations of Aphis glycines and their impact on crop productivity.     

CO2-Fixierung durch Knallgasbakterien

Zusammenfassung Die Gewinnung, Aufbereitung und papierchromatographische Analyse ¹⁴C-markierter Extrakte aus PHBS-speichernden Zellen von Hydrogenomonas H 16 wird beschrieben und diskutiert. Einige chromatographische Trennsysteme wurden verglichen, insbesondere mit dem von Metzner (1960) vorgeschlagenen System. R_f-Werte wichtiger Intermediärverbindungen und Positionskonstanten von Phosphatestern wurden ermittelt.Für die papierchromatographische Trennung von Zuckern, Aminosäuren und Hydroxamsäuren sowie für organische Säuren und Phosphatester wurden zweckmäßige Systeme angegeben, Sprühmittel verglichen und Farbreaktionen beschrieben.Chromatogramme von Extrakten aus Hydrogenomonas H 16 und Chlorella pyrenoidosa, die ¹⁴CO₂ im Kurzzeitversuch unter vergleichbaren Bedingungen eingebaut hatten, zeigten voneinander verschiedene Markierungsmuster. Unter den Phosphatestern sind bei Hydrogenomonas (nur 63% des fixierten ¹⁴C) vorwiegend Hexosemonophosphate, Sedoheptulose-7-phosphat, Phosphoglycerinsäure und AMP markiert, bei Chlorella (95% des fixierten ¹⁴C) hauptsächlich Phosphoglycerinsäure, Triosephosphat und Phosphoenolbrenztraubensäure. Bei Hydrogenomonas waren einige organische Säuren und Aminosäuren (Äpfelsäure, Citronensäure, Bernsteinsäure, Fumarsäure, Glutaminsäure, Alanin u.a.) relativ stark markiert, die bei Chlorella kaum oder nicht radioaktiv waren.

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CO₂-fixation by Knallgas bacteriaII. Chromatographical identification of early labeled fixation products

Summary The production, preparation, and analysis by paperchromatography of ¹⁴C-labeled extracts from cells of Hydrogenomonas H 16 storing poly--hydroxybutyric acid (PHBS) is described and discussed. Some chromatographical solvent systems were compared, especially with one proposed by Metzner (1960). R_f-values of important intermediates and phosphate ester position constants were determined.Separation by paperchromatography of sugars, amino acids, and hydroxamic acids was tested in various systems. Sprays for these compounds, as well as for organic acids and phosphate esters, were compared and their colour reactions described.Chromatograms of extracts from Hydrogenomonas H 16 and Chlorella pyrenoidosa after incorporation of ¹⁴CO₂ in short time experiments under comparable conditions showed labelling patterns different from each other. In the case of Hydrogenomonas, 63% of the activity fixed was found in the phosphate esters, mainly in hexose monophosphates (incl. sedoheptulose-7-phosphate), phosphoglyceric acid, and AMP. The phosphate esters of Chlorella contained 95% of the activity fixed, the bulk of which appeared in phosphoglyceric acid, triose phosphate and phosphoenolpyruvic acid. Much less was found in the hexose monophosphates.Some organic and amino acids such as malic, citric, succinic, fumaric glutamic acid and alanine of the Hydrogenomonas extracts were rather heavily labelled, while this was not the case with Chlorella.

     

Untersuchungen über die Aufnahme von Glykolat bei Chlorogonium

Summary The alga Chlorogonium was cultured either heterotrophically or autotrophically under different partial pressures of CO₂ by aerating with pure air of air enriched with 2% CO₂. Cells were harvested in the logarithmic phase, transferred to phosphate buffer containing 0.01 M 1C¹⁴-glycolate and incubated with shaking in the dark. Under these conditions the rate of glycolate uptake was higher when the cells had been grown in the light. Cells grown in the light at the lower CO₂-concentration took up more glycolate than those grown with 2% CO₂. Approximately 90% of the radioactivity taken up with the glycolate was released as CO₂. The radioactivity remaining in the algae was somewhat higher in those cells which had been cultured heterotrophically or autotrophically under air than in cells grown autotrophically under air enriched with 2% CO₂.Addition of glycolate increased the uptake of oxygen by the cells. The consumption of the oxygen was quantitatively correlated to the uptake of glycolate.     

Study of some enzyme systems by means of the differential microrespirometer

Three enzymic phenomena, inactivation by oxygen, Q_OO2 measurements of a dehydrogenase system, and hydrolysis of acetyl phosphate by a phosphatase of Clostridium kluyveri, were studied by means of the differential microrespirometer. Respiration of yeast cells was also measured with the same instrument. All results obtained in the four types of study agreed closely with earlier results obtained with the Warburg apparatus. The amount of sample needed was of the order of 1/100 to 1/200 of that necessary for comparable study with the Warburg apparatus. The advantages of the instrument in enzyme isolation and purification studies are discussed.