Effect of pH on metabolic pathway shift in fermentation and electro-fermentation of xylose by Clostridium autoethanogenum.

Clostridium autoethanogenum can to convert waste gases (CO2, CO, H2) and xylose from hydrolyzed biomass into acetate, lactate, formate, ethanol and 2,3-butanediol, being a candidate for the transformation of waste streams of lignocellulosic biorefineries. Electro-fermentation (EF) modify the pattern of traditional fermentations resulting in improved product yields as has been shown when using Clostridium strains. The aim of this work was to evaluate the influence of pH on microbial growth and product distribution during fermentation and EF of xylose by C. autoethanogenum DSM10061. Fermentation and EF were carried out in a H-type reactor at three controlled pH: 5.0, 5.5 and 5.8, and at a fixed potential of -600 mV (versus Ag/AgCl) in the EF. The experiments showed that maximum biomass concentration increased as the pH increased in fermentation and EF. In accordance with maximum biomass reached, the highest substrate conversion was observed at pH 5.8 for both systems, with 76.80 % in fermentation and 96.18 % in EF. Moreover, the highest concentrations of acetic acid (1.41 ± 0.07 g L-1) and ethanol (1.45 ± 0.15 g L-1) were obtained at the end of cultures in the EF at pH 5.8. The production of lactic and formic acid decreased by the application of the external potential regardless of the pH value, reaching the lowest productivity at pH 5.8. In contrast, the specific productivity of acetic acid and ethanol was lower in both fermentation and EF at the lowest pH. Furthermore, the presence of 0.06 g L-1 of 2,3-butanediol was only detected in EF at pH 5.8. The results revealed that EF modulated microbial metabolism, which can be explained by a possible increased generation of NADP+/NADPH cofactors, which would redirect the metabolic pathway to more reduced products.

Phenotype-specific estimation of metabolic fluxes using gene expression data.

A cell's genome influences its metabolism via the expression of enzyme-related genes, but transcriptome and fluxome are not perfectly correlated as post-transcriptional mechanisms also regulate reaction's kinetics. Here, we addressed the question: given a transcriptome, how unobserved mechanisms of reaction kinetics should be systematically accounted for when inferring the fluxome? To infer the most likely and least biased fluxome, we present Pheflux, a constraint-based model maximizing Shannon's entropy of fluxes per mRNA. Benchmarked against 13C fluxes of yeast and bacteria, Pheflux accurately estimates the carbon core metabolism. We applied Pheflux to thousands of normal and tumor cell transcriptomes obtained from The Cancer Genome Atlas. Pheflux showed statistically significantly higher glucose yields on lactate in breast, kidney, and bronchus-lung tumoral cells than their normal counterparts. Results are consistent with the Warburg effect, a hallmark of cancer metabolism, suggesting that Pheflux can be efficiently used to study the metabolism of eukaryotic cells.

Kinetic model of Clostridium beijerinckii's Acetone-Butanol-Ethanol fermentation considering metabolically diverse cell types.

Clostridium beijerinckii population branches into metabolically diverse cell types in batch cultures. Here, we present a new kinetic model of C. beijerinckii's Acetone-Butanol-Ethanol fermentation that considers three cell types: producers of acids (acidogenic), consumer of acids and producers of solvents (solventogenic), and spores cells. The model accurately recapitulates batch culture data. Also, the model estimates cell type-specific kinetic parameters, which can be helpful to improve the operation of the ABE fermentation and give a framework to study acidogenic and solventogenic metabolic pathways. To exemplify the latter, we used a constraint-based model to study how the ABE pathways are used among acidogenic and solventogenic cell types. We found that among both cell types, glycolytic production of ATP and consumption of NAD+ varies widely during the fermentation, with their maximum production/consumption rates happening when acidogenic and solventogenic growth rates were at their highest. However, acidogenic cells use the ABE pathway to contribute with an extra 12.5% of the total production of ATP, whereas solventogenic cell types use the ABE pathway to supply more than 75% of the demand for NAD+, alternating between the production of lactate and butyrate, being both coupled to the production of NAD+.

Metabolic flux configuration determination using information entropy.

Constraint-based models use steady-state mass balances to define a solution space of flux configurations, which can be narrowed down by measuring as many fluxes as possible. Due to loops and redundant pathways, this process typically yields multiple alternative solutions. To address this ambiguity, flux sampling can estimate the probability distribution of each flux, or a flux configuration can be singled out by further minimizing the sum of fluxes according to the assumption that cellular metabolism favors states where enzyme-related costs are economized. However, flux sampling is susceptible to artifacts introduced by thermodynamically infeasible cycles and is it not clear if the economy of fluxes assumption (EFA) is universally valid. Here, we formulated a constraint-based approach, MaxEnt, based on the principle of maximum entropy, which in this context states that if more than one flux configuration is consistent with a set of experimentally measured fluxes, then the one with the minimum amount of unwarranted assumptions corresponds to the best estimation of the non-observed fluxes. We compared MaxEnt predictions to Escherichia coli and Saccharomyces cerevisiae publicly available flux data. We found that the mean square error (MSE) between experimental and predicted fluxes by MaxEnt and EFA-based methods are three orders of magnitude lower than the median of 1,350,000 MSE values obtained using flux sampling. However, only MaxEnt and flux sampling correctly predicted flux through E. coli's glyoxylate cycle, whereas EFA-based methods, in general, predict no flux cycles. We also tested MaxEnt predictions at increasing levels of overflow metabolism. We found that MaxEnt accuracy is not affected by overflow metabolism levels, whereas the EFA-based methods show a decreasing performance. These results suggest that MaxEnt is less sensitive than flux sampling to artifacts introduced by thermodynamically infeasible cycles and that its predictions are less susceptible to overfitting than EFA-based methods.

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion.

Polyhydroxyalkanoates (PHAs) are ubiquitous prokaryotic storage compounds of carbon and energy, acting as sinks for reducing power during periods of surplus of carbon source relative to other nutrients. With close to 150 different hydroxyalkanoate monomers identified, the structure and properties of these polyesters can be adjusted to serve applications ranging from food packaging to biomedical uses. Despite its versatility and the intensive research in the area over the last three decades, the market share of PHAs is still low. While considerable rich literature has accumulated concerning biochemical, physiological, and genetic aspects of PHAs intracellular accumulation, the costs of substrates and processing costs, including the extraction of the polymer accumulated in intracellular granules, still hampers a more widespread use of this family of polymers. This review presents a comprehensive survey and critical analysis of the process engineering and metabolic engineering strategies reported in literature aimed at the production of chiral (R)-hydroxycarboxylic acids (RHAs), either from the accumulated polymer or by bypassing the accumulation of PHAs using metabolically engineered bacteria, and the strategies developed to recover the accumulated polymer without using conventional downstream separations processes. Each of these topics, that have received less attention compared to PHAs accumulation, could potentially improve the economy of PHAs production and use. (R)-hydroxycarboxylic acids can be used as chiral precursors, thanks to its easily modifiable functional groups, and can be either produced de-novo or be obtained from recycled PHA products. On the other hand, efficient mechanisms of PHAs release from bacterial cells, including controlled cell lysis and PHA excretion, could reduce downstream costs and simplify the polymer recovery process.

Ethanol production improvement driven by genome-scale metabolic modeling and sensitivity analysis in Scheffersomyces stipitis.

The yeast Scheffersomyces stipitis naturally produces ethanol from xylose, however reaching high ethanol yields is strongly dependent on aeration conditions. It has been reported that changes in the availability of NAD(H/+) cofactors can improve fermentation in some microorganisms. In this work genome-scale metabolic modeling and phenotypic phase plane analysis were used to characterize metabolic response on a range of uptake rates. Sensitivity analysis was used to assess the effect of ARC on ethanol production indicating that modifying ARC by inhibiting the respiratory chain ethanol production can be improved. It was shown experimentally in batch culture using Rotenone as an inhibitor of the mitochondrial NADH dehydrogenase complex I (CINADH), increasing ethanol yield by 18%. Furthermore, trajectories for uptakes rates, specific productivity and specific growth rate were determined by modeling the batch culture, to calculate ARC associated to the addition of CINADH inhibitor. Results showed that the increment in ethanol production via respiratory inhibition is due to excess in ARC, which generates an increase in ethanol production. Thus ethanol production improvement could be predicted by a change in ARC.

A generalized disjunctive programming framework for the optimal synthesis and analysis of processes for ethanol production from corn stover.

The aim of this study is to analyze the techno-economic performance of process configurations for ethanol production involving solid-liquid separators and reactors in the saccharification and fermentation stage, a family of process configurations where few alternatives have been proposed. Since including these process alternatives creates a large number of possible process configurations, a framework for process synthesis and optimization is proposed. This approach is supported on kinetic models fed with experimental data and a plant-wide techno-economic model. Among 150 process configurations, 40 show an improved MESP compared to a well-documented base case (BC), almost all include solid separators and some show energy retrieved in products 32% higher compared to the BC. Moreover, 16 of them also show a lower capital investment per unit of ethanol produced per year. Several of the process configurations found in this work have not been reported in the literature.

In silico analysis of Clostridium acetobutylicum ATCC 824 metabolic response to an external electron supply.

The biological production of butanol has become an important research field and thanks to genome sequencing and annotation; genome-scale metabolic reconstructions have been developed for several Clostridium species. This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 to analyze its metabolic capabilities and response to an external electron supply through a constraint-based approach using the Constraint-Based Reconstruction Analysis Toolbox. Several analyses were conducted, which included sensitivity, production envelope, and phenotypic phase planes. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power (electrofermentation), results in an increase in the butanol-specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth, which coincides with results reported in literature. Phenotypic phase planes showed that the reducing capacity becomes more limiting for growth at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimality line. Although the maximum butanol flux does not coincide with the growth optimality line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, being a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824.

Repeated-batch operation for the synthesis of lactulose with ß-galactosidase immobilized by aggregation and crosslinking.

Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae ß-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.5gdeglutaraldehyde/g enzyme at 5h of cross-linking, obtaining a specific activity of 15,000IUg(-1), with 30% immobilization yield. CLAG was more stable than the free enzyme under non-reactive conditions with a half-life of 123h at 50°C and when operated in repeated-batch mode, yield and productivity was 3.8 and 4.3 times higher. Maximum number of batches was determined considering biocatalyst replacement at 50% residual activity. 98 and 27 batches could be performed under such criterion at fructose/lactose molar ratio of 4 and 20 respectively, reflecting that enzyme stability is strongly affected by the sugars distribution in the reaction medium.

Transgalactosylation and hydrolytic activities of commercial preparations of ß-galactosidase for the synthesis of prebiotic carbohydrates.

ß-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: ß-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of ß-galactosidases could be tuned by changing the concentration of galactose donor.

Corn stover semi-mechanistic enzymatic hydrolysis model with tight parameter confidence intervals for model-based process design and optimization.

Uncertainty associated to the estimated values of the parameters in a model is a key piece of information for decision makers and model users. However, this information is typically not reported or the confidence intervals are too large to be useful. A semi-mechanistic model for the enzymatic saccharification of dilute acid pretreated corn stover is proposed in this work, the model is a modification of an existing one providing a statistically significant improved fit towards a set of experimental data that includes varying initial solid loadings (10-25% w/w) and the use of the pretreatment liquor and washed solids with or without supplementation of key inhibitors. A subset of 8 out of 17 parameters was identified, showing sufficiently tight confidence intervals to be used in uncertainty propagation and model analysis, without requiring interval truncation via expert judgment.

Genome-scale NAD(H/(+)) availability patterns as a differentiating feature between Saccharomyces cerevisiae and Scheffersomyces stipitis in relation to fermentative metabolism.

Scheffersomyces stipitis is a yeast able to ferment pentoses to ethanol, unlike Saccharomyces cerevisiae, it does not present the so-called overflow phenomenon. Metabolic features characterizing the presence or not of this phenomenon have not been fully elucidated. This work proposes that genome-scale metabolic response to variations in NAD(H/(+)) availability characterizes fermentative behavior in both yeasts. Thus, differentiating features in S. stipitis and S. cerevisiae were determined analyzing growth sensitivity response to changes in available reducing capacity in relation to ethanol production capacity and overall metabolic flux span. Using genome-scale constraint-based metabolic models, phenotypic phase planes and shadow price analyses, an excess of available reducing capacity for growth was found in S. cerevisiae at every metabolic phenotype where growth is limited by oxygen uptake, while in S. stipitis this was observed only for a subset of those phenotypes. Moreover, by using flux variability analysis, an increased metabolic flux span was found in S. cerevisiae at growth limited by oxygen uptake, while in S. stipitis flux span was invariant. Therefore, each yeast can be characterized by a significantly different metabolic response and flux span when growth is limited by oxygen uptake, both features suggesting a higher metabolic flexibility in S. cerevisiae. By applying an optimization-based approach on the genome-scale models, three single reaction deletions were found to generate in S. stipitis the reducing capacity availability pattern found in S. cerevisiae, two of them correspond to reactions involved in the overflow phenomenon. These results show a close relationship between the growth sensitivity response given by the metabolic network and fermentative behavior.

Fed-batch synthesis of galacto-oligosaccharides with Aspergillus oryzae ß-galactosidase using optimal control strategy.

Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with ß-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed-batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L(-1) as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g(-1) in fed-batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g(-1) ). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS-5 and GOS-6) was observed in the fed-batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis.

Attainable region analysis for continuous production of second generation bioethanol.

BACKGROUND: Despite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed. RESULTS: We obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate. CONCLUSIONS: In this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

Selection of process alternatives for lignocellulosic bioethanol production using a MILP approach.

This work proposes a decision-making framework for the selection of processes and unit operations for lignocellulosic bioethanol production. Process alternatives are described by its capital and operating expenditures, its contribution to process yield and technological availability information. A case study in second generation ethanol production using Eucalyptus globulus as raw material is presented to test the developed process synthesis tool. Results indicate that production cost does not necessarily decrease when yield increases. Hence, optimal processes can be found at the inflexion point of total costs and yield. The developed process synthesis tool provides results with an affordable computational cost, existing optimization tools and an easy-to-upgrade description of the process alternatives. These features made this tool suitable for process screening when incomplete information regarding process alternatives is available.

Process design and sustainability in the production of bioethanol from lignocellulosic materials

Background: Bioethanol is produced mainly from sugar cane and corn. In the last years it has been subject of debate due to the effects in food prices and land use change. The use of lignocellulosic materials for bioethanol production, such as agroindustry, forestry and municipal residues, wood or dendroenergetic species, has been proposed as a sustainable way for producing this biofuel. The design of a sustainable process for producing bioethanol requires a methodological approach whereby economical, environmental and social criteria are systematically integrated from the early stages of process design. Results: Until now a methodology for guiding the design of a sustainable process for bioethanol production is not available, and there are just a few studies on this subject. Moreover, with the recent global concerns on climate change, developed technologies have been confronted with additional requirements to validate their sustainability. In this sense, the inclusion of sustainability criteria on process design becomes necessary for defining a systematic methodology to select the most appropriate operations in the process stages to achieve a sustainable bioethanol production. Conclusions: A description of the stages for the production of bioethanol from lignocellulosic materials is provided in this review and the main findings in relation to the more important sustainability indicators are presented.

Synthesis of galacto-oligosaccharides by ß-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose.

The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae ß-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55°C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38 g GOSh(-1) mg enzyme(-1) was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 gGOSh(-1) mg enzyme(-1) were obtained, respectively.

A pseudo steady-state model for galacto-oligosaccharides synthesis with ß-galactosidase from Aspergillus oryzae.

A pseudo steady-state model for the kinetically controlled synthesis of galacto-oligosaccharides (GOS) with Aspergillus oryzae ß-galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta-oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl-enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl-enzyme complex is higher than that of the free enzyme.

Towards a long-term model construction for the dynamic simulation of HIV infection.

This study involves the mathematical modelling of long-term HIV dynamics. The proposed model is able to predict the entire trajectory of the disease: initial viremia in the early weeks of the infection, latency, and progression to AIDS; a range spanning approximately ten years. The model outcomes were compared to clinical data and significant agreement was achieved. The formulated model considers all important population compartments including macrophages, latently-infected CD4+ T-cells, and cytotoxic T-lymphocytes (CTLs), an attempt which in many respects is novel in the area of HIV modelling. The ranges of the model parameters and initial conditions were obtained from literature, and their values were determined in this work directly by fitting published clinical data. Furthermore, the simulation results emphasize the importance of macrophages in HIV infection and progression to AIDS and show a clear correlation between the level of CTLs and HIV progression. The ability of the model to correlate analytical data gives credibility to its predictions, a fact that will be exploited in future research in modelling immunological and pharmacological avenues of treatment.