Cell and ATP Yields of Citrobacter freundii Growing with Fumarate and H2 or Formate in Continuous Culture (original) (raw)
Related papers
1979
phenomenon which is observed when glucose or another readily metabolized carbon compound decreases the intracellular concentration of 3' : 5'-cyclic adenosine monophosphate and prevents the transcription of catabolite-sensitive genes (Magasanik, 1961 ; Tyler et al., 1967; Jacquet & Kepes, 1969). For example, glucose represses the synthesis of tricarboxylic acid cycle enzymes in C. freundii (Keevil et al., 1977a) or in E. coli when this organism is grown in complex media (Gray et al., 19663). Furthermore, nitrate is a powerful oxidizing agent which can replace oxygen as the terminal electron acceptor for the respiratory chain of many bacteria, so its presence substantially influences the products that are formed during anaerobic growth (Krebs, 1937; Pichinoty, 1963). Nitrite, the product of nitrate reduction, can either be further reduced or accumulate as a substance toxic to yeast (Cole & Wimpenny, 1968; Weiner et al., 1975). We have therefore investigated how glucose metabolism by Citrobacter freundii is modified when anaerobic cultures are supplied with the inorganic electron acceptors oxygen, nitrate and nitrite. Our aims were to determine the major regulatory mechanisms which promote a fermentative or respiratory mode of growth, and to correlate activities of individual enzymes (and therefore their rates of synthesis) with the rate at which they function during growth. M E T H O D S Organism and media. Citrobacter freundii strain NCTC 9750 was maintained and grown in the minima] medium of De Graaf & Stouthamer (1971) supplemented with glutamate, lysine, thiamin and methionine as described previously (Keevil et al., 1977b). Samples of each culture were routinely stained by Gram's method and observed microscopically. Single colony isolates were also stabbed into cyanide agar (Munson, 1974): Citrobacter species are resistant to KCN, but most other Enterobacteriaceae are sensitive. The identity of the pure culture from continuous cultures was confirmed with the 'API 10 Enterobacteriaceae System' of bacterial tests (D. A. Pitman, Weybridge, Surrey). Continuous culture studies. The 5 1 fermentation vessel containing 2.8 to 3.0 1 medium was equipped with pH and temperature control (L. H. Engineering, Stoke Poges, Bucks.). The dilution rate for all experiments was 0.035 h-l, and the flow rate of medium into the fermenter was controlled with a peristaltic pump (Watson-Marlow, Falmouth). Anaerobic cultures were sparged at 50 ml min-l with N2/C02 (95 : 5, V/V) (British Oxygen Co., Wolverhampton) : butyl rubber or silicone rubber tubing was used for all connections and no attempt was made to exclude O2 from the feed medium. Although pure 0, (British Oxygen Co. ; 1 1 min-l) was used for experiments with sulphate-limited cultures to ensure that the dissolved 0, conwntration remained above 200 PM, the same dissolved O2 concentration was subsequently achieved with 4 1 air min-l. Air was therefore used in many of the experiments. The impeller speed was 600 rev. min-1. Steady-state growth was established by allowing 14 1 of medium to flow into the fermenter before a 2 1 sample was taken aseptically directly from the vessel. The purity and AGS0 of the culture were checked with 20 ml samples taken aseptically from a sampling port. Butch culture experiments. Exponential phase cultures in 20 ml of aerated nutrient broth were transferred into 500 ml of supplemented minimal medium and incubated for 16 h at 37°C aerobically, or anaerobically with or without nitrate. These cultures provided 5 % (v/v) inocula for 10 1 of identical pre-warmed medium in a stirred fermenter equipped with pH and temperature control (L. H. Engineering). 'White spot' nitrogen was passed into the fermenter at 50 ml min-l during anaerobic growth. For aerobic growth, pure oxygen was supplied to the fermenter through the impeller at 1 or 5 1 min-', and the culture was stirred at 600 rev. min-1. In initial experiments, flasks were aerated in an orbital shaker operating at 200 cycles min-1 (model G-25, New Brunswick Scientific Co.). Neither the flasks nor the 10 1 fermenter were equipped with an oxygen electrode for these experiments. Growth determinations. Cell densities (determined spectrophotometrically), the yield of bacterial dry weight (calculated from calibration curves) and the carbon content of washed cell suspensions were all determined as described by Keevil et al. (19773). Preparation of bacterial extracts. The 2 1 samples were cooled to 4°C and harvested by centrifuging at 15 OOO g for 10 min. Cell-free extracts of soluble and membranebound proteins were prepared as described by Cole & Rittenberg (1971) after bacteria had been broken with a Hughes' (1951) press: 0.1 M-potassium phosphate pH 7.0 was used throughout.. Enzyme assays. Malate dehydrogenase (EC 1. 1. 1 .37), aconitate hydratase (EC 4.2.1 .3), pyruvate dehydrogenase (EC 1. 2. 4. l), succinate dehydrogenase (EC 1 .3.99.1) and NADH oxidase activities were
A Fumarate Reductase in Escherichia Coli Distinct from Succinate Dehydrogenase
Journal of Biological Chemistry
Ample evidence supports the generalization that enzymes capable of catalyzing succinate oxidation also can catalyze the reverse reaction, fumarate reduction, and vice versa. A variety of organisms have yielded highly purified, apparently single enzymes that have both activities (2-6), and where only one activity could be demonstrated it is likely that a different assay system would reveal the other also .
Applied and Environmental Microbiology, 2006
Citrate metabolism by Enterococcus faecium FAIR-E 198, an isolate from Greek Feta cheese, was studied in modified MRS (mMRS) medium under different pH conditions and glucose and citrate concentrations. In the absence of glucose, this strain was able to metabolize citrate in a pH range from constant pH 5.0 to 7.0. At a constant pH 8.0, no citrate was metabolized, although growth took place. The main end products of citrate metabolism were acetate, formate, acetoin, and carbon dioxide, whereas ethanol and diacetyl were present in smaller amounts. In the presence of glucose, citrate was cometabolized, but it did not contribute to growth. Also, more acetate and less acetoin were formed compared to growth in mMRS medium and in the absence of glucose. Most of the citrate was consumed during the stationary phase, indicating that energy generated by citrate metabolism was used for maintenance. Experiments with cell-free fermented mMRS medium indicated that E. faecium FAIR-E 198 was able to ...
Journal of General Microbiology, 1979
Glutamate induced the synthesis of 2-oxoglutarate dehydrogenase 50-fold during anaerobic growth of Citrobacter freundii and, in the absence of glutamate, this enzyme was even more active in cultures sparged with N,/CO, (95 : 5, v/v). Enzyme synthesis was partially repressed when the inlet gas was passed through heated copper but totally repressed when the inlet gas was passed through alkaline pyrogallol and reduced benzyl viologen (a treatment which would remove CO, as well as 0,). Fumarate hydratase activity also decreased but alcohol dehydrogenase and the sum of the succinate dehydrogenase and fumarate reductase activities increased when residual 0, was removed from the sparging gas. Soluble cytochromes a, and cs52.5 were detected in rigorously anaerobic cultures. Thus traces of 0, which contaminate commercial compressed N, are sufficient to induce 2-oxoglutarate dehydrogenase synthesis and to affect significantly the synthesis and incorporation of respiratory chain components into the cytoplasmic membrane.
Effects of Acetate on the Growth and Fermentation Performance of Escherichia coli KO11
Applied Biochemistry and Biotechnology, 1999
Escherichia coli KO11, in which the genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) encoding the ethanol pathway from Zymomonas mobilis were inserted into the chromosome, has been shown to metabolize all major sugars that are constituents of hemicellulosic hydrolysates to ethanol, in anaerobic conditions. However, the growth and fermentation performance of this recombinant bacteria may be affected by acetic acid, a potential inhibitor present in hemicellulose hydrolysates in a range of 2.0-15.0 g/L. It was observed that acetate affected the growth of E. coli KO11, prolonging the lag phase and inducing loss of biomass production and reduction of growth rate. At lower pH levels, the sensitivity to acetic acid was enhanced owing to the increased concentration of the protonated species. On the other hand, the recombinant bacteria showed a high tolerance to acetic acid regarding fermentative performance. In Luria broth medium with glucose or xylose as a single sugar source, it was observed that neither yield nor productivity was affected by the addition of acetate in a range of 2.0-12.0 g/L, suggesting some uncoupling of the growth vs ethanol production.
Inhibitory effect of fumarate on growth of Bacteroides fragilis
CHEMICAL & PHARMACEUTICAL BULLETIN, 1988
The growth of the strictly anaerobic bacterium Bacteroides fragilis on glucose was inhibited in the presence of fumarate.The molar growth yields for glucose were 29.6 and 20.0g of dry cells/mol of glucose in the absence and presence of fumarate,respectively.In the culture with fumarate,the ratio of lactate production increased and that of succinate was slightly decreased.Fumarate addition also affected the levels of some enzyme activities involved in fermentation of glucose .
Fermentation of fumarate and L-malate by Clostridium formicoaceticum
Journal of Bacteriology, 1978
The fermentation of fumarate and L-malate by Clostridium formicoaceticum was investigated. Growing and nongrowing cells degraded fumarate by dismutation to succinate, acetate, and CO2; on the other hand, only small amounts of succinate were detected when the organism was grown on L-malate. This dicarboxylic acid was mainly converted to acetate and CO2. The fermentation balances were modified if bicarbonate or formate were present in the medium. When C. formicoaceticum was grown in the presence of both dicarboxylic acids, fumarate was consumed before L-malate. The latter was mainly converted to acetate, whereas fumarate was fermented to acetate and succinate. Molar growth yields were determined to be 6 g of dry weight per mol of fumarate and 8 g of dry weight per mol of L-malate fermented.
Pathway of Succinate and Propionate Formation in Bacteroides fragilis
Journal of Bacteriology, 1978
Cell suspensions of Bacteroides fragilis were allowed to ferment glucose and lactate labeled with 14 C in different positions. The fermentation products, propionate and acetate, were isolated, and the distribution of radioactivity was determined. An analysis of key enzymes of possible pathways was also made. The results of the labeling experiments showed that: (i) B. fragilis ferments glucose via the Embden-Meyerhof pathway; and (ii) there was a randomization of carbons 1, 2, and 6 of glucose during conversion to propionate, which is in accordance with propionate formation via fumarate and succinate. The enzymes 6-phosphofrucktokinase (pyrophosphate-dependent), fructose-1,6-diphosphate aldolase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, and methylmalonyl-coenzyme A mutase could be demonstrated in cell extracts. Their presence supported the labeling results and suggested that propionate is formed from succinate via succinyl-, methylmalonyl-, and pro...
Citrate Metabolism by Enterococcus faecalis FAIR-E 229
Applied and Environmental Microbiology, 2001
Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as wel...