Cells Of Clostridium Pasteurianum Research Articles

Alcoholic Biofuels Production from Biodiesel Derived Glycerol by Clostridium pasteurianum Whole Cells Immobilized on Silica

Clostridium pasteurianum cells immobilized on silica were used as biocatalysts for bioconversion of both pure and biodiesel-derived crude glycerol to butanol, 1,3-propanediol (PDO) and ethanol. Among five concentrations (1, 2, 3, 5, 10 % w/v) of cell immobilized support studied, 3 % w/v cell immobilized support performed the best and was chosen for further studies. The cross-linked cells produced higher amount of solvents than non-cross linked cells. The flow cytometric results revealed that presence of pure as well as crude glycerol had no adverse effect on cellular morphology of C. pasteurianum. The maximum yields of butanol, ethanol and 1,3-PDO obtained with immobilized cells were 0.36 (25 g/L pure glycerol), 0.11 (10 g/L pure glycerol) and 0.54 g/g (10 g/L pure glycerol), respectively. Among the three crude glycerol concentrations studied, 25 g/L crude glycerol gave maximum yield of n-butanol (0.23 g/g), and 5 g/L crude glycerol formed maximum 1,3-PDO (0.61 g/g). Only traces of ethanol were detected at all concentrations of crude glycerol studied. A comparison of free and immobilized cells revealed that immobilized cells performed better than free cells for mixed alcohols production at higher concentrations of both pure and crude glycerol. Thus, the use of immobilized cells is a viable option for converting high concentrations of biodiesel derived crude glycerol into alternate energy sources.

Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support

Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This article attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3-propanediol (1,3-PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3-PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters, we attribute these results to increased substrate-enzyme affinity and decreased substrate inhibition for 1,3-PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3-PDO.

Production of n-butanol from biodiesel derived crude glycerol using Clostridium pasteurianum immobilized on Amberlite

This paper presents accounts of research on conversion of biodiesel derived crude glycerol to n-butanol via anaerobic fermentation pathway, using immobilized Clostridium pasteurianum cells. Experiments were done to optimize the growth conditions and growth medium, viz. Reinforced Clostridial Media (RCM) and Cooked Meat Media (CMM). Out of these two media, growth was found to be faster in RCM, which was used for experiments. The cells were immobilized on a hydrophilic support Amberlite, an ion exchange resin. Varying concentration of crude glycerol as a substrate was found to have significant effect on product profile with 25gL−1 glycerol giving maximum yield of n-butanol after 120h. High concentration of 150gL−1 glycerol exerted an inhibitory effect on n-butanol production with decreased 1,3-propanediol production, and absolutely no formation of ethanol. Maximum yield of ethanol and 1,3-propanediol was obtained with 5gL−1 and 10gL−1 crude glycerol respectively. To the best of our knowledge, this is the first report of use of the immobilized cells (immobilization of C. pasteurianum cells on Amberlite) for bioconversion of glycerol to butanol.

Physiolgical effects of metronidazole on Clostridium posteurianum

The physiological effects of metronidazole on the growth, viability, fermentation end-product production and cellular morphology of Clostridium pasteurianum cells growing logarithmically were studied. Metronidazole (a 5-nitroimidazole) was found to be the most potent of the nitroimidazole compounds tested against C. pasteurianum. It inhibited the growth rate of C. pasteurianum cultures by varying degrees over a range of drug concentrations (2.5-10 mg/L). Metronidazole had an immediate bactericidal effect at a concentration of 10 mg/L, killing 99.9% of cells within 5 min of drug addition. The same concentration caused an immediate cessation of fermentation end-product (acetate and butyrate) production in these cultures. These observations may be relevant to a proposed cell lysing mechanism which may form an additional mode of action of this important antibiotic.

Evidence for proton motive force dependent transport of selenite by Clostridium pasteurianum.

The proton motive force mediated the transport of selenite (SeO3(2-)) in Clostridium pasteurianum cells by proton symport. The proton conductor, carbonyl cyanide m-chlorophenylhydrazone, inhibited SeO3(2-) uptake while N,N'-dicyclohexylcarbodiimide prevented SeO3(2-) uptake by presumably inhibiting the unidirectional ATPase. Acid pulse studies and antibiotic experiments with valinomycin suggest that the transmembrane delta pH component of the proton motive force mediated the transport of SeO3(2-) into the cells. The SeO3(2-) porter system in C. pasteurianum was found to be dependent upon energy source, temperature, and medium pH.

Regulation of Sorbitol Metabolism by Glucose in Clostridium pasteurianum: a Role for Inducer Exclusion

SUMMARY: Cells of Clostridium pasteurianum grown on glucose lacked enzyme systems specific for sorbitol metabolism: the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PTS) and sorbitol-6-phosphate dehydrogenase. These activities were induced by growth on sorbitol, but were partially repressed by glucose. During growth on excess glucose and sorbitol, the sorbitol PTS was absent until the growth rate declined at the onset of stationary phase. Synthesis of at least two PTS proteins, one soluble and one membrane-bound, was repressed in exponentially growing cells. In contrast to the PTS, sorbitol-6-phosphate dehydrogenase was present during the exponential phase. Glucose and methyl α-glucoside inhibited the activity of the sorbitol PTS in permeabilized cells, and this inhibition was competitive at the level of PEP. This implies that in intact cells sorbitol is excluded by glucose, at least in part due to competition between the glucose and sorbitol phosphotransferases for a common pool of phosphate and energy. Exclusion of sorbitol may be an important factor in the repression of sorbitol metabolism by glucose.

Identification of molybdoproteins in Clostridium pasteurianum.

Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N 2-fixing bacterium Clostridium pasteurianum

The H 2 uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H 2 evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N 2-fixing conditions have the highest H 2 uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction ( M r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H 2 oxidation and H 2 evolution at rates of 3000 and 5.9 μmol H 2 consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.

Butyricin 7423 and the membrane H+-ATPase of Clostridium pasteurianum

The bacteriocin butyricin 7423 inhibited the activity of the membrane H+ -ATPase (BFoF1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BFo component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentration of this membrane active bacteriocin.

Transport of molybdate by Clostridium pasteurianum.

The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated. Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate. The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0). The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively. Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki [SO42-] is 3.0 X 10(-5) M; apparent Ki [WO42-] is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect. Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable. Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-.

Characteristics of a reserve alpha-glucan from Clostridium pasteurianum.

An iodophilic intracellular polysaccharide has been isolated from cells of Clostridium pasteurianum. This polysaccharide has been shown to be unbranched and to contain α[1 → 4]-and α[1 → 6]-linked glucopyranose residues in a ratio of 3:2.

Isolation and morphology of native intracellular polyglucose granules from Clostridium pasteurianum.

Intracellular polyglucose granules have been isolated from late log-phase cells of Clostridium pasteurianum. These granules are spherical in shape, heterogeneous in size, and surrounded by a membrane coat which lacks the characteristics of a typical unit membrane. Freeze-etching reveals projections of 12 to 16 nm in diameter in the matrix of the granules, which suggest the presence of polysaccharide fibrils.

Nitrogenase II. Changes in the EPR signal of Component I (iron-molybdenum protein) of Azotobacter vinelandii nitrogenase during repression and derepression

The purified, native iron-molybdenum protein (Component I) of Azotobacter vinelandii nitrogenase shows EPR signals at g values of 4.32, 3.65, and 2.01, when observed below 40° K. The resonances near g = 4.32 and 3.65 are unobscured when whole nitrogen-fixing cells are observed, and we have compared the appearance and disappearance of these resonances with nitrogenase activity as well as Component I protein determined immunochemically, during derepression and repression of nitrogenase. The characteristic resonances are absent in cells grown on ammonium salts. During derepression the activity, EPR signal, and cross-reacting material appear and pass through a maximum in parallel. During repression the immunochemically-detectable protein decays inversely with cell growth, while the activity and signal are depressed considerably more rapidly. This suggests that a short-term control mechanism, including the substantial alteration of the paramagnetic center of Component I without elimination of the antigenic moiety, is responsible for rapid repression of nitrogenase by ammonia. EPR signals similar to those of Component I in Azotobacter were also observed at low temperatures in nitrogen-fixing cells of Clostridium pasteurianum, Klebsiella pneumoniae, and Bacillus polymyxa, suggesting that in vivo studies of Component I of nitrogenase may be feasible generally.

Isolation and Characteristics of Flavodoxin from Nitrogen-fixing Clostridium pasteurianum

The purification, crystallization, and biological activity of flavodoxin is reported. It is a new electron transport protein and has been isolated from extracts of cells of Clostridium pasteurianum grown on nitrogen gas or ammonia and in an iron-deficient medium. Flavodoxin forms yellow crystals from ammonium sulfate solution, has a molecular weight of 14,600, absorption maxima of the oxidized form at 272, 372, 443 mµ with a shoulder at 472 mµ, and contains no iron, molybdenum, cobalt, or labile sulfur. In equimolar amounts, it will substitute for ferredoxin in the metabolism of hydrogen and pyruvate and in the fixation of nitrogen by extracts of C. pasteurianum with an effectiveness of 70, 40, and 30%, respectively. It is suggested that flavodoxin replaces ferredoxin as an electron carrier in cells of C. pasteurianum grown in an iron-deficient medium since these cells contain less than 5% of the ferredoxin found in cells grown in an iron-sufficient medium.