Phosphate-limited Chemostat Cultures Research Articles

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2741-x) contains supplementary material, which is available to authorized users.

Divergence Involving Global Regulatory Gene Mutations in an Escherichia coli Population Evolving under Phosphate Limitation

Many of the important changes in evolution are regulatory in nature. Sequenced bacterial genomes point to flexibility in regulatory circuits but we do not know how regulation is remodeled in evolving bacteria. Here, we study the regulatory changes that emerge in populations evolving under controlled conditions during experimental evolution of Escherichia coli in a phosphate-limited chemostat culture. Genomes were sequenced from five clones with different combinations of phenotypic properties that coexisted in a population after 37 days. Each of the distinct isolates contained a different mutation in 1 of 3 highly pleiotropic regulatory genes (hfq, spoT, or rpoS). The mutations resulted in dissimilar proteomic changes, consistent with the documented effects of hfq, spoT, and rpoS mutations. The different mutations do share a common benefit, however, in that the mutations each redirect cellular resources away from stress responses that are redundant in a constant selection environment. The hfq mutation lowers several individual stress responses as well the small RNA–dependent activation of rpoS translation and hence general stress resistance. The spoT mutation reduces ppGpp levels, decreasing the stringent response as well as rpoS expression. The mutations in and upstream of rpoS resulted in partial or complete loss of general stress resistance. Our observations suggest that the degeneracy at the core of bacterial stress regulation provides alternative solutions to a common evolutionary challenge. These results can explain phenotypic divergence in a constant environment and also how evolutionary jumps and adaptive radiations involve altered gene regulation.

Ammonium and phosphate limitation in 1,3-propanediol production by Klebsiella pneumoniae

Excretion of 1,3-propanediol (1,3-PD) by K. pneumoniae was compared in ammonium- and phosphate-limited chemostat cultures running with an excess of glycerol. 59 and 43% catabolic flux were directed to 1,3-PD in ammonia-limited cultures and phosphate-limited cultures at dilution rate of 0.1 h(-1), respectively. Ammonia-limited fed-batch cultures produced 61 g 1,3-PD l(-1) and a total of 15 g l(-1) organic acid in 36 h. However, phosphate-limited fed-batch cultures excreted 61 g lactate l(-1) and 44 g 1,3-PD l(-1).

Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-(13)C(6)]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucose- or ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATP-dependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.

Regulation of Carbon and Electron Flow in Clostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

Vancomycin production is enhanced in chemostat culture with biomass-recycle.

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae.

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.

The physiological function of periplasmic glucose oxidation in phosphate-limited chemostat cultures of Klebsiella pneumoniae NCTC 418

Periplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen- and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of butanol by Clostridium puniceum in batch and continuous culture

The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.

Relation between growth rate and erythromycin production inStreptomyces erythraeus

The relation between specific growth rate and specific rate of product formation was studied in phosphate-limited chemostat cultures ofStreptomyces erythraeus. Specific rates of formation were measured for both the final product, erythromycin A, and several of its biosynthetic precursors. In all cases rates of formation increased with inereasing growth rate, indicating that antibiotic production was strongly growth-linked.

Abnormal cellular morphologies ofEscherichia coliK12 mutants defective in plasmid segregation

A collection of mutants of Escherichia coli K12 which had been isolated during phosphate-limited chemostat culture on the basis of their inability to maintain certain plasmids were studied by scanning electron microscopy and found to exhibit a variety of abnormal cellular morphologies including branching, filamentation, and minicell-like sphere formation.

The effect of procaine on the partitioning of plasmid pSC101

The effect of procaine at a sub-lethal concentration (20 mM) on the segregational stability of pSC101 and other plasmids in Escherichia coli was examined in phosphate-limited chemostat culture. Procaine caused segregational instability of pSC101 and a derivative of pBR322 carrying the par function of pSC101. The segregational stability of 2 other unrelated plasmids, RP4 and pDS1109, was unaffected by procaine. It is concluded that the synthesis of a chromosomally encoded membrane protein, under the control of the envZ gene product, is required for pSC101 par function or that the functioning of this protein is impaired by the presence of procaine.

Influence of External pH and Fermentation Products on Clostridium acetobutylicum Intracellular pH and Cellular Distribution of Fermentation Products

Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations up to 200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production, thereby circumventing a decrease in the intracellular pH great enough to be deleterious to the cell.

Intracellular K + and cyclic diphosphoglycerate pools and transients in Methanobacterium thermoautotrophicum

Cultivation of M. thermoautotrophicum under K + - and phosphate-limiting conditions in chemostat cultures provided evidence that this organism can grow rapidly without the need to maintain high internal concentrations of K + and cyclic diphosphoglycerate (cyclic DPG). The internal K + concentration during K + limitation was about 320 mM. For comparison, the K + concentration was near 800 mM in batch-grown cells containing about 150 mM cyclic DPG. Phosphate limitation resulted in a decline in the K + and cyclic DPG pools to 200 mM and 9 mM, respectively. Pulse addition of K + to a K + -limited chemostat culture resulted in a transient increase in cellular K + and cyclic DPG before the new growth rate was reached. A large increase in extracellular K + (from 0.02 to 1 mM) resulted in a transition to a phosphate-limited culture and a concomitant decrease in cell K + and cyclic DPG. These two components declined by approximately 100 mM and 30 mM, respectively, during the transition from K + to phosphate limitation. Pulse addition of phosphate to phosphate-limited chemostat cultures resulted in a transient expansion of the K + and cyclic DPG pools before the shiftup in growth rate occurred. These adjustments of K + to cyclic DPG levels established K + as the major cation balancing the −3 charge of cyclic DPG.

Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli.

Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon- and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinone-sufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH(3), NO(3) (-), or N(2). Expressed as a function of relative growth rate, steady state cellular P contents and PO(4) assimilation rates did not vary with N-source. However, N-source did alter the maximal PO(4)-limited growth rate achieved by the cultures: the NO(3) (-) and N(2) cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH(3) grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO(3) (-)-grown cells were the smallest (627 +/- 34 micromoles C . 10(-9) cells), while NH(3)-grown cells were largest (900 +/- 44 micromoles C . 10(-9) cells) and N(2)-fixing cells were intermediate (726 +/- 48 micromoles C . 10(-9) cells) in size. In the NO(3) (-)-and N(2)-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH(3)-grown cells. Heterocysts were absent in NH(3)-grown cultures but were present in both the N(2) and NO(3) (-) cultures. In the NO(3) (-)-grown cultures C(2)H(2) reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N(2)-fixing cultures the acetylene:N(2) ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.Compared with NH(3), the assimilation of NO(3) (-) and N(2) resulted either in a decrease in cellular C (NO(3) (-) and N(2) cultures) or in a lower maximal growth rate (N(2) culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO(3) (-) or N(2) compared with NH(3).

Cyclic-2,3-diphosphoglycerate levels in methanobacterium thermoautotrophicum reflect inorganic phosphate availability

Methanobacterium thermoautotrophicum was grown in phosphate-limited chemostat cultures at a dilution rate corresponding to a doubling time of 13.2 h. The cyclic-2,3-diphospho-D-glycerate content of these cells was 8 to 10-fold lower than that of cells grown in batch cultures having a doubling time of 11.5 h. This metabolite accounted for 5% of cell dry weight during batch growth on 2 mM phosphate. In the chemostat the steady-state concentration of phosphate was 4 microM, showing that this methanogen is adapted to highly efficient growth at low phosphate concentrations. Since growth rates were similar in both cultures, the growth rate clearly does not depend on intracellular levels of cyclic-2,3-diphosphoglycerate.

Glucose repression of luminescence and luciferase in vibrio fischeri

The autoinduction and glucose repression of luciferase synthesis in batch cultures and continuous cultures of Vibrio fischeri were investigate. As previously reported, a lag in luciferase synthesis occurred in glycerol-grown batch cultures and addition of d-glucose to the medium extended the lag period. A phosphate-limited chemostat culture with d-glucose as energy source (specific growth rate, μ=0.45 h-1) contained uninduced levels of luciferase. Luciferase activity increased to an induced level upon addition of c-AMP or autoinducer to such a chemostat culture while cell mass remained constant. Furthermore, when μ of a phosphate-limited chemostat culture containing d-glucose as energy source was decreased from 0.45 to 0.30 h-1, luciferase activity increased from an uniduced to induced level. After exogenously added c-AMP or autoinducer was diluted out of a phosphate-limited continous culture or after μ was increased to 0.45 h-1, luciferase activity remained at an induced level. Apparently, luciferase in V. fischeri was subject to a catabolite repression by d-glucose that could be overridden by autoinduction or by an autogenous control element.

Cell wall turnover in batch and chemostat cultures of Bacillus subtilis

Wall turnover was studied in Bacillus subtilis. The loss of radioactively labeled wall polymers was followed during exponential growth in batch and chemostat cultures. Turnover kinetics were identical under all growth conditions; pulse-labeled wall material was lost with first-order kinetics, but only after exponential growth for 1 generation time after its incorporation. Similarly, continuously labeled cells showed an accelerating decrease in wall-bound radioactivity starting immediately after removal of the labeled precursor and also reached first-order kinetics after 1 generation time. A mathematical description was derived for these turnover kinetics, which embraced the concept of "spreading" of old wall chains (H. M. Pooley, J. Bacteriol. 125:1127-1138, 1976). Using this description, we were able to calculate from our experimental data the rate of loss of wall polymers from cells and the fraction of the wall which was sensitive to turnover. We found that about 20% of the wall was lost per generation time and that this loss was affected by turnover activity located in the outer 20 to 45% of the wall; rather large variations were found with both quantities and also between duplicate cultures. These parameters were quite independent of the growth rate (the specific growth rate varied from 1.3 h-1 in broth cultures to 0.2 to 0.3 h-1 in chemostat cultures) and of the nature of the anionic polymer in the wall (which was teichoic acid in cultures with an excess of phosphate and teichuronic acid in phosphate-limited chemostat cultures). Some implications of the observed wall turnover kinetics for models of wall growth in B. subtilis are discussed.

Cell wall assembly in Bacillus subtilis: location of wall material incorporated during pulsed release of phosphate limitation, its accessibility to bacteriophages and concanavalin A, and its susceptibility to turnover.

Addition of a pulse of phosphate to a phosphate-limited chemostat culture of Bacillus subtilis W23 led to the synthesis of teichoic acid and the consequent development by the bacteria of the ability to bind phage SP50. In cultures growing at different rates, phage-binding properties became maximal approximately one generation time after addition of the pulse. Removal of the incorporated teichoic acid by turnover also reached its maximum rate after a similar interval. After pulsed release of phosphate limitation in B. subtilis NCTC 3610, the alpha-glucosyl residues of the incorporated teichoic acid, detected by their interaction with concanavalin A, became maximally exposed at the same time that phage binding was maximum. At that time the bacteria bound phage all over the cylindrical part of the surface and at about one-third of the polar caps. That fraction of the receptor material that is exposed soon after its incorporation was distributed along the cylindrical length of most of the bacteria, but few phages bound to the polar caps, except in the case of short bacteria; these bound phages in a markedly asymmetric manner at one pole and along their length. The significance of these results is discussed in relation to the mode of assembly of the cell wall.