Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions

Chlorella pyrenoidosa was cultivated under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. The influence of light on the carbon and energy metabolism of microalgae was investigated by the use of metabolic flux analysis. The respiratory activity of microalgae in the light was assessed from the autotrophic flux distribution. Results showed that the glycolytic pathway, tricarboxylic acid cycle and mitochondrial oxidative phosphorylation maintained high activities during illumination, indicating little effect of light on these pathways, while the flux through the pentose phosphate pathway during illumination was very small due to the light-mediated regulation. The theoretical yields of biomass on ATP decreased in the following order: heterotrophic culture>mixotrophic culture>autotrophic culture, and a significant amount of the available ATP was required for maintenance processes in microalgal cells. The energy conversion efficiency between the supplied energy to culture, the absorbed energy by cells and the free energy conserved in ATP were analyzed for the different cultures. Analysis showed that the heterotrophic culture generated more ATP from the supplied energy than the autotrophic and mixotrophic cultures. The maximum thermodynamic efficiency of ATP production from the absorbed energy, which was calculated from the metabolic fluxes at zero growth rate, was the highest in the heterotrophic culture and as low as 16% in the autotrophic culture. By evaluating the energy economy through the energy utilization efficiency, it was found that the biomass yield on the supplied energy was the lowest in the autotrophic cultivation, and the cyclic culture gave the most efficient utilization of energy for biomass production.     

Integrating flux balance analysis into kinetic models to decipher the dynamic metabolism of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 sequentially utilizes lactate and its waste products (pyruvate and acetate) during batch culture. To decipher MR-1 metabolism, we integrated genome-scale flux balance analysis (FBA) into a multiple-substrate Monod model to perform the dynamic flux balance analysis (dFBA). The dFBA employed a static optimization approach (SOA) by dividing the batch time into small intervals (i.e., ～400 mini-FBAs), then the Monod model provided time-dependent inflow/outflow fluxes to constrain the mini-FBAs to profile the pseudo-steady-state fluxes in each time interval. The mini-FBAs used a dual-objective function (a weighted combination of "maximizing growth rate" and "minimizing overall flux") to capture trade-offs between optimal growth and minimal enzyme usage. By fitting the experimental data, a bi-level optimization of dFBA revealed that the optimal weight in the dual-objective function was time-dependent: the objective function was constant in the early growth stage, while the functional weight of minimal enzyme usage increased significantly when lactate became scarce. The dFBA profiled biologically meaningful dynamic MR-1 metabolisms: 1. the oxidative TCA cycle fluxes increased initially and then decreased in the late growth stage; 2. fluxes in the pentose phosphate pathway and gluconeogenesis were stable in the exponential growth period; and 3. the glyoxylate shunt was up-regulated when acetate became the main carbon source for MR-1 growth.     

A state of the art of metabolic networks of unicellular microalgae and cyanobacteria for biofuel production

The most promising and yet challenging application of microalgae and cyanobacteria is the production of renewable energy: biodiesel from microalgae triacylglycerols and bioethanol from cyanobacteria carbohydrates. A thorough understanding of microalgal and cyanobacterial metabolism is necessary to master and optimize biofuel production yields. To this end, systems biology and metabolic modeling have proven to be very efficient tools if supported by an accurate knowledge of the metabolic network. However, unlike heterotrophic microorganisms that utilize the same substrate for energy and as carbon source, microalgae and cyanobacteria require light for energy and inorganic carbon (CO₂ or bicarbonate) as carbon source. This double specificity, together with the complex mechanisms of light capture, makes the representation of metabolic network nonstandard. Here, we review the existing metabolic networks of photoautotrophic microalgae and cyanobacteria. We highlight how these networks have been useful for gaining insight on photoautotrophic metabolism.     

Insights into metabolic efficiency from flux analysis

The efficiency of carbon and energy flows throughout metabolism defines the potential for growth and reproductive success of plants. Understanding the basis for metabolic efficiency requires relevant definitions of efficiency as well as measurements of biochemical functions through metabolism. Here insights into the basis of efficiency provided by (13)C-based metabolic flux analysis (MFA) as well as the uses and limitations of efficiency in predictive flux balance analysis (FBA) are highlighted. (13)C-MFA studies have revealed unusual features of central metabolism in developing green seeds for the efficient use of light to conserve carbon and identified metabolic inefficiencies in plant metabolism due to dissipation of ATP by substrate cycling. Constraints-based FBA has used efficiency to guide the prediction of the growth and actual internal flux distribution of plant systems. Comparisons in a few cases have been made between flux maps measured by (13)C-based MFA and those predicted by FBA assuming one or more maximal efficiency parameters. These studies suggest that developing plant seeds and photoautotrophic microorganisms may indeed have patterns of metabolic flux that maximize efficiency. MFA and FBA are synergistic toolsets for uncovering and explaining the metabolic basis of efficiencies and inefficiencies in plant systems.     

聚球藻7942光自养培养的碳代谢和能量代谢

代谢通量分析是研究微藻光自养培养过程中CO2和光能利用的一个非常有用的工具。本研究建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。研究结果表明,CO2固定是代谢能量和还原力消耗的主要途径,分别约占总消耗能量的85%和70%。研究还发现在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80g/molATP和2.95g/molATP,但基于总吸收光能的细胞得率和对应的光能转换效率则随着光强的增加而降低。     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Carbon metabolism and energy conversion of Synechococcus sp. PCC 7942 under mixotrophic conditions: comparison with photoautotrophic condition

To investigate the carbon metabolism and energy conversion efficiency of the cyanobacterium Synechococcus sp. PCC 7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and with acetate, respectively, and further discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could enhance the growth of Synechococcus sp. PCC 7942. The metabolic flux through the glycolytic pathway, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation was affected by the two organic substrates. Additionally, the cellular composition was also modulated by glucose and acetate. Under mixotrophic conditions, glucose exerts more significant impact on the diminishment of photochemical efficiency. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher compared with that of photoautotrophic one. On the basis of chlorophyll fluorescence analysis, the actual energy conversion efficiencies based on ATP synthesis in the photoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures were evaluated to be 4.59%, 5.86%, and 6.60%, respectively.     

Characterization of energy conversion of Synechococcus sp. PCC7942 under photoautotrophic conditions based on metabolic flux and chlorophyll fluorescence analysis

Energy conversion efficiency of photoautotrophic microalgae plays an important role in the utilization of light energy for cell growth and production of metabolites. To understand the utilization of light energy, Synechococcus sp. PCC7942 was cultivated at different incident light intensities of 15.8, 47.3, and 94.6 μmol/m²/sec in continuous culture. The influence of light on the carbon and energy metabolism of microalgae was investigated by combining metabolic flux analysis (MFA) and chlorophyll fluorescence analysis (CFA). Results showed that the yields of biomass based on ATP (Y _ATP) and total light energy (Y _E) both declined with increasing light, and the maximal values of Y _ATP and Y _E were estimated to be 4.73 g/mol-ATP, and 17.10 × 10^?3 g/kJ respectively, at the examined conditions. The overall efficiency of energy conversion against total absorbed energy changed with the varying irradiances. However, the actual conversion efficiency of total energy based on CFA was almost constant, regardless of the different irradiances used in the present study.     

A hybrid kinetic and constraint-based model of leaf metabolism allows predictions of metabolic fluxes in different environments

While flux balance analysis (FBA) provides a framework for predicting steady-state leaf metabolic network fluxes, it does not readily capture the response to environmental variables without being coupled to other modelling formulations. To address this, we coupled an FBA model of 903 reactions of soybean (Glycine max) leaf metabolism with e-photosynthesis, a dynamic model that captures the kinetics of 126 reactions of photosynthesis and associated chloroplast carbon metabolism. Successful coupling was achieved in an iterative formulation in which fluxes from e-photosynthesis were used to constrain the FBA model and then, in turn, fluxes computed from the FBA model used to update parameters in e-photosynthesis. This process was repeated until common fluxes in the two models converged. Coupling did not hamper the ability of the kinetic module to accurately predict the carbon assimilation rate, photosystem II electron flux, and starch accumulation of field-grown soybean at two CO₂ concentrations. The coupled model also allowed accurate predictions of additional parameters such as nocturnal respiration, as well as analysis of the effect of light intensity and elevated CO₂ on leaf metabolism. Predictions included an unexpected decrease in the rate of export of sucrose from the leaf at high light, due to altered starch–sucrose partitioning, and altered daytime flux modes in the tricarboxylic acid cycle at elevated CO₂. Mitochondrial fluxes were notably different between growing and mature leaves, with greater anaplerotic, tricarboxylic acid cycle and mitochondrial ATP synthase fluxes predicted in the former, primarily to provide carbon skeletons and energy for protein synthesis.     

Influence of Light on Carbon Utilization in Aerobic Anoxygenic Phototrophs

Aerobic anoxygenic phototrophs contain photosynthetic reaction centers composed of bacteriochlorophyll. These organisms are photoheterotrophs, as they require organic carbon substrates for their growth whereas light-derived energy has only an auxiliary function. To establish the contribution of light energy to their metabolism, we grew the phototrophic strain Erythrobacter sp. NAP1 in a carbon-limited chemostat regimen on defined carbon sources (glutamate, pyruvate, acetate, and glucose) under conditions of different light intensities. When grown in a light-dark cycle, these bacteria accumulated 25% to 110% more biomass in terms of carbon than cultures grown in the dark. Cultures grown on glutamate accumulated the most biomass at moderate light intensities of 50 to 150 μmol m⁻² s⁻¹ but were inhibited at higher light intensities. In the case of pyruvate, we did not find any inhibition of growth by high irradiance. The extent of anaplerotic carbon fixation was detemined by radioactive bicarbonate incorporation assays. While the carboxylation activity provided 4% to 11% of the cellular carbon in the pyruvate-grown culture, in the glutamate-grown cells it provided only approximately 1% of the carbon. Additionally, we tested the effect of light on respiration and photosynthetic electron flow. With increasing light intensity, respiration decreased to approximately 25% of its dark value and was replaced by photophosphorylation. The additional energy from light allows the aerobic anoxygenic phototrophs to accumulate the supplied organic carbon which would otherwise be respired. The higher efficiency of organic carbon utilization may provide an important competitive advantage during growth under carbon-limited conditions.     

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic cultures of Euglena gracilis

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic culture of Euglena gracilis were studied. Under a low light supply coefficient, these two metabolic activities seem to proceed independently. The cell growth rate in photoheterotrophic culture was about the sum of the growth rates in pure photoautotrophic and heterotrophic cultures. However under a high light supply coefficient, both photoautotrophic and heterotrophic (glucose assimilation) metabolic activities were inhibited, resulting in a low photoheterotrophic growth rate. The photoheterotrophic culture was more sensitive to photoinhibition compared to the pure photoautotrophic culture. Inhibition of glucose assimilation in the photoheterotrophic culture was due to both direct and indirect (through photosynthesis) effects of high light intensity. Cell growth, glucose assimilation and alpha-tocopherol content of the cells were higher when ambient air was used for aeration than when a mixture of carbon dioxide and air was used. Even when photosynthesis was inhibited by addition of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea to photoheterotrophic culture, light stimulated alpha-tocopherol synthesis by E. gracilis.     

GROWTH,PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM,CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N₂ fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O₂-sensitive N₂ fixation from photosynthetic O₂ evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.     

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ?-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin–Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.     

Production of α-tocopherol by sequential heterotrophic–photoautotrophic cultivation of Euglena gracilis

Photoautotrophic cultivation of Euglena gracilis results in cells with high α-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the α-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of α-tocopherol was therefore investigated for efficient α-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular α-tocopherol content increased from 120 μg g⁻¹ at the end of heterotrophic phase to more than 400 μg g⁻¹ at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the α-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 μg g⁻¹. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for α-tocopherol accumulation. In this way, it was possible to produce 7 g l⁻¹ cells containing about 1100 μg α-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in α-tocopherol productivity of 100 μg l⁻¹ h⁻¹ which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively.     

Flux balance analysis of photoautotrophic metabolism

Photosynthesis is the principal process responsible for fixation of inorganic carbon dioxide into organic molecules with sunlight as the energy source. Potentially, many chemicals could be inexpensively produced by photosynthetic organisms. Mathematical modeling of photoautotrophic metabolism is therefore important to evaluate maximum theoretical product yields and to deeply understand the interactions between biochemical energy, carbon fixation, and assimilation pathways. Flux balance analysis based on linear programming is applied to photoautotrophic metabolism. The stoichiometric network of a model photosynthetic prokaryote, Synechocystis sp. PCC 6803, has been reconstructed from genomic data and biochemical literature and coupled with a model of the photophosphorylation processes. Flux map topologies for the hetero-, auto-, and mixotrophic modes of metabolism under conditions of optimal growth were determined and compared. The roles of important metabolic reactions such as the glyoxylate shunt and the transhydrogenase reaction were analyzed. We also theoretically evaluated the effect of gene deletions or additions on biomass yield and metabolic flux distributions.     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。     

Heterotrophic (Dark) CO2 Fixation by Euglena gracilis. Possible regulation by tricarboxylic acid cycle intermediates

SYNOPSIS. Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture and mode of nutrition. Dark CO₂ fixation increased transiently during the growth of cells under photoautotrophic (CO₂, light) and heterotrophic (glucose, dark) conditions. Cells grown heterotrophically with acetate or ethanol had no transient increase in fixation. The addition of acetate to a heterotrophically growing culture during the period of increasing dark CO₂ fixation resulted in rapid elimination of this fixation. The results suggest that dark CO₂ fixation in Euglena functions in anaplerotic feeding of the tricarboxylic acid cycle, drained by biosyntheses during growth. Induction of the glyoxylate cycle by acetate may provide an alternate source of tricarboxylic cycle intermediates, obviating the requirement for dark CO₂ fixation as a source of the intermediates.