Energy Spilling Research Articles

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates

BackgroundLactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth.ResultsUsing glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10 - 0.60 h-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control.ConclusionsThe current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00449-010-0502-y) contains supplementary material, which is available to authorized users.

Substrate consumption and excess sludge reduction of activated sludge in the presence of uncouplers: a modeling approach

A mathematical model with a consideration of energy spilling is developed to describe the activated sludge in the presence of different levels of metabolic uncouplers. The consumption of substrate and oxygen via energy spilling process is modeled with a Monod term, which is dependent on substrate and inhibitor. The sensitivity of the developed model is analyzed. Three parameters, maximum specific growth rate (micro(max)), energy spilling coefficient (q(max)), and sludge yield coefficient (Y(H)) are estimated with experimental data of different studies. The values of micro(max), q(max), and Y(H) are found to be 6.72 day(-1), 5.52 day(-1), and 0.60 mg COD mg(-1) COD for 2, 4-dinitrophenol and 7.20 day(-1), 1.58 day(-1), and 0.62 mg COD mg(-1) COD for 2, 4-dichlorophenol. Substrate degradation and sludge yield could be predicted with this model. The activated sludge process in the presence of uncouplers that is described more reasonably by the new model with a consideration of energy spilling. The effects of uncouplers on substrate consumption inhibition and excess sludge reduction in activated sludge are quantified with this model.

Interaction of molasses and monensin in alfalfa hay- or corn silage-based diets on rumen fermentation, total tract digestibility, and milk production by Holstein cows

Sugar supplementation can stimulate rumen microbial growth and possibly fiber digestibility; however, excess ruminal carbohydrate availability relative to rumen-degradable protein (RDP) can promote energy spilling by microbes, decrease rumen pH, or depress fiber digestibility. Both RDP supply and rumen pH might be altered by forage source and monensin. Therefore, the objective of this study was to evaluate interactions of a sugar source (molasses) with monensin and 2 forage sources on rumen fermentation, total tract digestibility, and production and fatty acid composition of milk. Seven ruminally cannulated lactating Holstein cows were used in a 5×7 incomplete Latin square design with five 28-d periods. Four corn silage diets consisted of 1) control (C), 2) 2.6% molasses (M), 3) 2.6% molasses plus 0.45% urea (MU), or 4) 2.6% molasses plus 0.45% urea plus monensin sodium (Rumensin, at the intermediate dosage from the label, 16g/909kg of dry matter; MUR). Three chopped alfalfa hay diets consisted of 1) control (C), 2) 2.6% molasses (M), or 3) 2.6% molasses plus Rumensin (MR). Urea was added to corn silage diets to provide RDP comparable to alfalfa hay diets with no urea. Corn silage C and M diets were balanced to have 16.2% crude protein; and the remaining diets, 17.2% crude protein. Dry matter intake was not affected by treatment, but there was a trend for lower milk production in alfalfa hay diets compared with corn silage diets. Despite increased total volatile fatty acid and acetate concentrations in the rumen, total tract organic matter digestibility was lower for alfalfa hay-fed cows. Rumensin did not affect volatile fatty acid concentrations but decreased milk fat from 3.22 to 2.72% in corn silage diets but less in alfalfa hay diets. Medium-chain milk fatty acids (% of total fat) were lower for alfalfa hay compared with corn silage diets, and short-chain milk fatty acids tended to decrease when Rumensin was added. In whole rumen contents, concentrations of trans-10, cis-12C18:2 were increased when cows were fed corn silage diets. Rumensin had no effect on conjugated linoleic acid isomers in either milk or rumen contents but tended to increase the concentration of trans-10C18:1 in rumen samples. Molasses with urea increased ruminal NH3-N and milk urea N when cows were fed corn silage diets (6.8 vs. 11.3 and 7.6 vs. 12.0mg/dL for M vs. MU, respectively). Based on ruminal fermentation characteristics and fatty acid isomers in milk, molasses did not appear to promote ruminal acidosis or milk fat depression. However, combinations of Rumensin with corn silage-based diets already containing molasses and with a relatively high nonfiber carbohydrate:forage neutral detergent fiber ratio influenced biohydrogenation characteristics that are indicators of increased risk for milk fat depression.

Kinetic Analysis of Growth Rate, ATP, and Pigmentation Suggests an Energy-Spilling Function for the Pigment Prodigiosin ofSerratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.

Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint‐based modeling and experimental verification

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.

Continuous biodegradation of single and mixed chlorophenols by a mixed microbial culture constituted by Burkholderia sp., Microbacterium phyllosphaerae, and Candida tropicalis

In this work, the biodegradation of mixed chlorophenols by a microbial consortium was studied in steady state continuous culture. Amplified ribosomal DNA restriction analysis (ARDRA) was used to evaluate the consortium's predominating bacterial species when chemostat was fed with pure or mixed chlorophenols. Removal efficiencies, cell growth yields, volumetric and specific biodegradation rates of phenol and chlorophenols were compared to those obtained with pure strains in the same culture conditions. Using phenol as the primary carbon and energy source, the microbial consortium cometabolically degraded mono-, di- and trichlorophenols, with overall removal efficiencies ranging from 95 to 99.8% and COD removal efficiencies from 85 to 97.8%. In all cultures, the microbial consortium showed better overall removal efficiencies than single strains. Binary mixtures of chlorophenols produced a remarkably increase in specific biodegradation rates of substrates q c, beyond those obtained when the consortium grew on single compounds. The results suggest that chlorophenols collapsed cell's proton motive force; thus, energy spilling, reflected by the q c increase, could be the cell's response to compensate the short-circuit in membrane electrochemical proton gradient.

Comparison between inhibitor and uncoupler for minimizing excess sludge production of an activated sludge process

In order to study the minimization of excess sludge production, the reduction in the excess sludge production in the presence of an inhibitor and uncoupler was studied in this work. The experimental results show that such an addition could effectively reduce the production of excess sludge. With the energy uncoupling model established in this work, energy uncoupling coefficient (E u) was used to evaluate the reduction in excess sludge production. The energy uncoupling coefficients in the presence of dinitrophenol (dNP), Zn2+, and Cu2+ was 0.75, 0.46, and 0.18, respectively. The analysis demonstrated that energy spilling occurred in the presence of dNP, and that dNP was an effective uncoupler for reducing the production of excess activated sludge without affecting the microbial respiration activity.

The ability of acidic pH, growth inhibitors, and glucose to increase the proton motive force and energy spilling of amino acid-fermenting Clostridium sporogenes MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (Deltap) was only -25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZDeltapH) and the electrical membrane potential (DeltaPsi) increased. The Deltap was -125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but DeltaPsi increased to -70 mV. Protein synthesis inhibitors also significantly increased DeltaPsi. Non-growing, arginine-energized cells had a DeltaPsi of -80 mV at pH 6.7 or pH 4.7, but DeltaPsi was not detected if the F1F0 ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and DeltaPsi and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither DeltaPsi nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large Deltap to grow, and Deltap appears to serve as a mechanism of ATP dissipation or energy spilling.

Effects of Diet Fermentability on Efficiency of Microbial Nitrogen Production in Lactating Dairy Cows

Effect of diet fermentability on efficiency of microbial N production was evaluated. Eight ruminally and duodenally cannulated Holstein cows (55±15.9 days in milk; mean±SD) were used in a duplicated 4×4 Latin square design with a 2×2 factorial arrangement of treatments. Experimental diets contained either ground high moisture corn (HM) or dry ground corn (DG) at two dietary starch concentrations (32 vs. 21%). All diets were formulated for 18% CP, and the sources of dietary protein were alfalfa silage (50% of forage at DM basis), soybean meal, distillers grain, and blood meal. The amount of OM truly fermented in the rumen varied from 7.7 (DG at 21% dietary starch) to 11.3 kg/d (HM at 32% dietary starch) among treatments, and was greater for high starch diets and HM treatments compared with low starch diets and DG treatments, respectively. Microbial N flow was greater for high starch diets compared with low starch diets, but was not affected by corn grain treatment. Microbial efficiency was lower for HM compared with DG treatment (39.7 vs. 48.4g of microbial N/kg of true ruminally degraded OM), but was not affected by dietary starch concentration. Microbial efficiency was positively correlated with rate of passage for OM and starch (r = 0.77 and 0.75, respectively). Rapid passage rate may have decreased microbial turnover in the rumen, enhancing microbial efficiency. Microbial efficiency was negatively correlated with rate of starch digestion (r = −0.55), consistent with the energy spilling theory. However, energy spilling did not appear to be from lack of ammonia or low ruminal pH. Microbial efficiency was not related to ruminal ammonia concentration, daily mean ruminal pH, or minimum ruminal pH. Rate of starch availability and rates of passage for starch and OM from the rumen are important determinants of efficiency of microbial protein synthesis in vivo.

Metabolic response of biofilm to shear stress in fixed-film culture.

In a biofilm reactor, detachment force resulting from hydraulic shear is a major factor that determines the formation and structure of steady state biofilm. The metabolic response of biofilm to change in shear stress was therefore investigated. A conventional annular reactor made of PVC was used, in which shearing over the rotating disc surface was strictly defined. Results from the steady state aerobic biofilm reactor showed that the biofilm structure (density and thickness) and metabolic behaviour (growth yield and dehydrogenase activity) were closely related to the shear stress exerted on the biofilm. Smooth, dense and stable biofilm formed at relatively high shear stress. Higher dehydrogenase activity and lower growth yield were obtained when the shear stress was raised. Growth yield was inversely correlated with the catabolic activity of biofilm. The reduced growth yield, together with the enhanced catabolic activity, suggests that a dissociation of catabolism from anabolism would occur at high shear stress. Biofilms may respond to shear stress by regulating metabolic pathways associated with the substrate flux flowing between catabolism and anabolism. A biological phenomenon, besides a simple physical effect, is underlying the observed relation between the shear stress and resulting biofilm structure. A hypothesis is proposed that the shear-induced energy spilling would be associated with a stimulated proton translocation across the cell membrane, which favours formation of a stronger biofilm. This research may provide a basis for experimental data on biofilm obtained at different shear stresses to be interpreted in relation to energy.

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus.

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

Minimization of activated sludge production by chemically stimulated energy spilling

Minimization of excess sludge production in activated sludge processes has been pursued around the world in order to meet stringent environmental regulations on sludge treatment and disposal. To achieve this goal, physical, chemical, and biological approaches have been proposed. In this paper, a chemical compound, 3,3′,4′,5-tetrachlorosalicylanilide (TCS) was tested for enhancing microbial energy spilling of the sludgeso as to minimize its growth. In order to examine this, an exploratory study was conducted using both batch and continuous activated sludge cultures. Batch experiments with these two cultures were carried out at different initial concentrations of TCS. It has been confirmed that an addition of TCS is effective in reducing the production of both the sludge cultures, particularly the continuous culture where the observed growth yield was reduced by around 70%, when the initial TCS concentration was 0.8 ppm. Meanwhile, the substrate removal activity of this culture was found not to be affected at this TCS concentration. To further evaluate the TCS effect, a pure microbial culture of E. coli was employed. Batch experiment results with this culture implied that TCS might be able to reduce the cell density of E. coli drastically when an initial TCS concentration was greater than 0.12 ppm. It was also found that TCS was not toxic to this type of bacteria. Microscopic examinations with a 4′, 6-diamidino-2-phenylindole (DAPI) staining technique revealed that TCS neither affected the cell division nor altered the cell size of E. coli. However, both the cell ATP content and the cell dry weight were reduced significantly with the addition of TCS.

Closure to “Modeling of Energy Spilling in Substrate‐Sufficient Cultures” by Guang‐Hao Chen and Yu Liu

Closure to “Modeling of Energy Spilling in Substrate‐Sufficient Cultures” by Guang‐Hao Chen and Yu Liu

Energy spilling by thermophilic aerobes in potassium-limited continuous culture

Multiple continuous-flow reactors were operated at 55°C under potassium-limited conditions to investigate energy spilling metabolism during thermophilic aerobic biological wastewater treatment. Energy spilling metabolism has been previously shown to reduce the observed cell growth yield at short mean cell residence times, which could potentially be used to promote autothermal operation during the biological treatment of high-strength wastewater. Under the potassium-limited conditions imposed during these experiments, cell yields decreased at lower residence times, commensurate with the characteristic pattern of energy spilling conditions. The minimum growth yield measured was 0.19 mg TSS per mg COD removed, which was believed to be too high to promote autothermal reactor operation for thermophilic treatment. These results suggest that operation of conventional biological treatment processes under energy spilling conditions could be used to reduce the net production of residual biomass.

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture.

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.

Effect of chemical uncoupler on the observed growth yield in batch culture of activated sludge

Microbial metabolism is the sum of biochemical transformation that includes interrelated catabolic and anabolic reactions. It has been demonstrated that coupling between anabolism and catabolism can be seriously dissociated by chemical uncoupler. Based on the knowledge on energy uncoupling resulting from substrate excess, a growth yield model was developed for chemical uncoupler-containing batch culture of activated sludge, and was verified with experimental and literature data. The observed growth yield was found to decrease with increasing the ratio of initial uncoupler concentration to initial biomass concentration ( C u/ X o), which represents the real strength of chemical uncoupler exerted on unit biomass. The presence of chemical uncoupler in the culture medium can cause significant energy spilling. It appears that energy spilling may be an effective pathway for bacteria to protect themselves against abnormal living conditions, but at the cost of consuming nongrowth energy.

Cellular cycling of substrate as a possible cryptic way for energy spilling in suspended cellular continuous cultures

Mass balances analysis of polyphasic dispersed systems showed that cellular cycling of the substrate involves (a) an increase in the specific metabolisation rate and (b) a decrease in the biomass yield coefficient (energy spilling). In biomass calculations, the substrate release rate can replace the maintenance coefficient concept. Thus, decreasing the substrate transfer rates could improve cells or microorganisms strains of industrial importance.

Model of dissolved organic carbon distribution for substrate-sufficient continuous culture.

The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism. It appears that the excess substrate could determine metabolic pathways of microorganisms, which further control dissolved organic carbon (DOC) distribution under substrate-sufficient conditions. However, how to quantitatively describe the DOC distribution remains unclear in substrate-sufficient continuous culture. Based on a balanced DOC reaction, a DOC distribution model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. Results showed that a considerable portion of the DOC consumed was directly oxidized to carbon dioxide through energy spilling under substrate-sufficient conditions. The proposed model for the first time quantified the DOC distribution between nongrowth-associated and growth-associated metabolisms of cells. The proposed model was verified with literature data very well.

A kinetic model incorporating energy spilling for substrate removal in substrate-sufficient batch culture of activated sludge.

Batch assays are currently used to study the kinetic behavior of microbial growth. However, it has been shown that the outcome of batch experiments is greatly influenced by the initial ratio of substrate concentration (S(o)) to biomass concentration (Xo). Substrate-sufficient batch culture is known to have mechanisms of spilling energy that lead to significant nongrowth-associated substrate consumption, and the Monod equation is no longer appropriate. By incorporating substrate consumption associated with energy spilling into the balance of the substrate oxidation reaction, a kinetic model for the observed specific substrate consumption rate was developed for substrate-sufficient batch culture of activated sludge, and was further verified by experimental data. It was demonstrated that the specific substrate consumption rate increased with the increase of the S(o)/Xo ratio, and the majority of substrate was consumed through energy spilling at high S(o)/Xo ratios. It appears that the S(o)/Xo ratio is a key parameter in regulating metabolic pathways of microorganisms.