Development and Application of Fluxomics Tools for Analyzing Metabolisms in Non-Model Microorganisms

Decoding microbial metabolism is of great importance in revealing the mechanisms governing the physiology of microbes and rewiring the cellular functions in metabolic engineering. Complementing the genomics, transcriptomics, proteinomics and metabolomics analysis of microbial metabolism, fluxomics tools can measure and simulate the in vivo enzymatic reactions as direct readouts of microbial metabolism. This dissertation develops and applies broad-scope tools in metabolic flux analysis to investigate metabolic insights of non-model environmental microorganisms. 13C-based pathway analysis has been applied to analyze specific carbon metabolic routes by tracing and analyzing isotopomer labeling patterns of different metabolites after growing cells with 13C-labeled substrates. Novel pathways, including Re-type citrate synthase in tricarboxylic acid cycle and citramalate pathways as an alternate route for isoleucine biosynthesis, have been identified in Thermoanaerobacter X514 and other environmental microorganisms. Via the same approach, the utilizations of diverse carbon/nitrogen substrates and productions of hydrogen during mixotrophic metabolism in Cyanothece 51142 have been characterized, and the medium for a slow-growing bacterium, Dehalococcoides ethenogenes 195, has been optimized. In addition, 13C-based metabolic flux analysis has been developed to quantitatively profile flux distributions in central metabolisms in a green sulfur bacterium, Chlorobaculum tepidum, and thermophilic ethanol-producing Thermoanaerobacter X514. The impact of isotope discrimination on 13C-based metabolic flux analysis has also been estimated. A constraint-based flux analysis approach was newly developed to integrate the bioprocess model into genome-scale flux balance analysis to decipher the dynamic metabolisms of Shewanella oneidensis MR-1. The sub-optimal metabolism and the time-dependent metabolic fluxes were profiled in a genome-scale metabolic network. A web-based platform was constructed for high-throughput metabolic model drafting to bridge the gap between fast-paced genome-sequencing and slow-paced metabolic model reconstruction. The platform provides over 1,000 sequenced genomes for model drafting and diverse customized tools for model reconstruction. The in silico simulation of flux distributions in both metabolic steady state and dynamic state can be achieved via flux balance analysis and dynamic flux balance analysis embedded in this platform. Cutting-edge fluxomics tools for functional characterization and metabolic prediction continue to be developed in the future. Broad-scope systems biology tools with integration of transcriptomics, proteinomics and fluxomics can reveal cell-wide regulations and speed up the metabolic engineering of non-model microorganisms for diverse bioenergy and environmental applications

Dissecting flux balances to measure energetic costs in cell biology: techniques and challenges

Life is a nonequilibrium phenomenon: metabolism provides a continuous supply of energy that drives nearly all cellular processes. However, very little is known about how much energy different cellular processes use, i.e. their energetic costs. The most direct experimental measurements of these costs involve modulating the activity of cellular processes and determining the resulting changes in energetic fluxes. In this review, we present a flux balance framework to aid in the design and interpretation of such experiments, and discuss the challenges associated with measuring the relevant metabolic fluxes. We then describe selected techniques that enable measurement of these fluxes. Finally, we review prior experimental and theoretical work that has employed techniques from biochemistry and nonequilibrium physics to determine the energetic costs of cellular processes.Comment: 27 pages, 3 figure

Biogas energy development in Slovenia

This paper analyses the development of the biogas energy production and use in Slovenia. The major concern is given to ecological, economic and technical determinants in decision making process for selection among alternative biogas energy production plants and use of energy for heating and electricity. The investments into the biogas plants have reduced ecological problems from large environmental polluters in urban and rural areas. Among them are sewage systems, food wastes, wastes from large-scale animal farms, and some other wastes from food-processing and food consumption places. The measures of economic policy, particularly guaranteed prices for purchased electricity from biogas production plants are presented that have contributed to the development of green energy production

The Physiological Effects of Phycobilisome Antenna Modification on the Cyanobacterium Synechocystis sp. PCC 6803

Phycobilisomes are the large, membrane extrinsic light harvesting antenna of cyanobacteria. They function to absorb light energy and deliver it efficiently to the photosystems, thereby increasing photosynthetic light absorption. Wild type phycobilisomes in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803) consist of a tricylindrical core from which six rods radiate. The colored phycobiliproteins are held together by colorless linker polypeptides. Several phycobilisome truncation mutants have been generated in Synechocystis 6803. The first, CB, has truncated phycobilisome rods; the second, CK, has only the phycobilisome core; and the third, PAL, has no phycobilisomes at all. Together, these mutants construct a series of increasingly truncated phycobilisomes which are useful for studying the physiology of antenna truncation in cyanobacteria. In this dissertation, the physiological effects of antenna truncation are examined from three perspectives. First, the effect of partial and complete phycobilisome removal on the expression and activity of photosystem II is examined using a variety of assays that center around fluorescence and oxygen evolution. Second, the overall effects of antenna truncation on thylakoid membrane spacing and structure is explored using electron microscopy and small angle neutron scattering. Finally, the effects of antenna truncation on culture-wide biomass productivity are examined in a variety of setting, including a bench-scale photobioreactor. Together, these studies represent a comprehensive examination of the physiological effects of antenna truncation on Synechocystis 6803

Development and software implementation of modelling tools for rapid fermentation process development using a parallel mini-bioreactor system

In order to establish a generic framework for the rapid development and optimisation of scalable fermentation processes, a novel methodology for simplifying model building was explored. This approach integrates small-scale fermentations with model-based experimental design (DoE) and predictive control strategies. In this study, four 1.4 litre vessels were characterised for power input, volumetric oxygen transfer coefficient (KLa) and mixing, to assess its potential for replicating cell culture rapidly. Engineering characterisation results showed excellent propeller operation over a range of 400-1200 rpm and up to the maximum motor output and under various air flow rates in fluid densities up to 4.21 Cp/mPa s (1.211 g/cm3 ). Limits were reached using glycerol (99%) at fluid viscosities of 500Cp/mPa s (1.253g/cm3 ) at 800 rpm and no air flow, hence experiencing the most resistance. This was the most taxing condition in terms of energy input into the system. Furthermore, we determined the efficient gas dispersion which is considered important for oxygen bubble dispersion in viscous fluids. The potential gas dispersion could be calculated as a function of both impeller speed, airflow rate, and the fluid viscosity. The calculations provided a working impeller speed of >263 rpm for >0.5 vvm air flow rate as preliminary parameters in our advanced modelling section. The key outcome of the KLa study was that the results showed suitable potential for mass transfer for high cell density fermentations, for each of the parallel stirred tank bioreactors. To assess the usability of the parallel bioreactors be used for bioprocess rapid development purposes Escherichia coli W3110 was characterised in the 1L WV vessels. So overall the experiments included testing the performance of the vessels engineering parameters and also the biological fermentations confirming that the system was suitable for parallel operation with high reproducibility. For model building, especially suited for the 4-reactor set up the parallel bioreactors a fractional factorial design was used, in which models could be rapidly built and implemented for further research. The screening and model optimisation helped to reduce the development time by using the parallel equipment. Batches of four reactors could be completed in parallel in which comparable experimental results were obtained rapidly for new fermentation models. Optical density measurements provided a quick off-line analysis of the growth curve of microbial populations, as compared to cell plate counts or dry weights that require more time. For the model development and the establishment of our integrated software modelling tool, a modified logistic model was developed to predict microbial growth kinetics. First-order kinetic models, logistic, and Gompertz models were used and comparatively analysed to assess the model fit to test batch data. The logistic model was favourable for mapping and simulating the later phases of bacterial growth, while the well-established exponential growth model predicted the early lag phase in our stoichiometric growth simulation software tool better. The initialisation of the previous fermentation model allowed us to build a statistical model, which was based on the engineering characteristics for optimisation of biomass. Therefore, batch nutrient supply with the aid of stoichiometric models could be tested and modelled. DoE model data was improved with metabolic flux analysis to develop an advanced feeding strategy by testing various metabolic pathways and the nutrients used in experimentation. Bacterial growth predictions and media optimisation were tested for maximising microbial biomass yields. We then modelled the dissolved oxygen concentration and substrate utilisation. The techniques and principles of dynamic flux balance analysis, mechanistic modelling, and stoichiometric mass balancing were used. The aim was to create and validate our integrated software based on advanced modelling for the parallel bioreactor systems and tested through application for E. coli fermentations. Optimising microbial biomass was the main target in this project, with the data collected from fermentation being the strongest comparator and validator. A new software for the integration of DoE and Dynamic flux balance analysis (DFBA) techniques with the intention of creating a working fermentation platform for the Multifors equipment via simulation and fermentation optimisation was the novel outcome of this research. The tool could provide functions for speeding up development time and control of parallel bioreactors

Modelling cell metabolism : a review on constraint-based steady-state and kinetic approaches

ABSTRACT: Studying cell metabolism serves a plethora of objectives such as the enhancement of bioprocess performance, and advancement in the understanding of cell biology, of drug target discovery, and in metabolic therapy. Remarkable successes in these fields emerged from heuristics approaches, for instance, with the introduction of effective strategies for genetic modifications, drug developments and optimization of bioprocess management. However, heuristics approaches have showed significant shortcomings, such as to describe regulation of metabolic pathways and to extrapolate experimental conditions. In the specific case of bioprocess management, such shortcomings limit their capacity to increase product quality, while maintaining desirable productivity and reproducibility levels. For instance, since heuristics approaches are not capable of prediction of the cellular functions under varying experimental conditions, they may lead to sub-optimal processes. Also, such approaches used for bioprocess control often fail in regulating a process under unexpected variations of external conditions. Therefore, methodologies inspired by the systematic mathematical formulation of cell metabolism have been used to address such drawbacks and achieve robust reproducible results. Mathematical modelling approaches are effective for both the characterization of the cell physiology, and the estimation of metabolic pathways utilization, thus allowing to characterize a cell population metabolic behavior. In this article, we present a review on methodology used and promising mathematical modelling approaches, focusing primarily to investigate metabolic events and regulation. Proceeding from a topological representation of the metabolic networks, we first present the metabolic modelling approaches that investigate cell metabolism at steady state, complying to the constraints imposed by mass conservation law and thermodynamics of reactions reversibility. Constraint-based models (CBMs) are reviewed highlighting the set of assumed optimality functions for reaction pathways. We explore models simulating cell growth dynamics, by expanding flux balance models developed at steady state. Then, discussing a change of metabolic modelling paradigm, we describe dynamic kinetic models that are based on the mathematical representation of the mechanistic description of nonlinear enzyme activities. In such approaches metabolic pathway regulations are considered explicitly as a function of the activity of other components of metabolic networks and possibly far from the metabolic steady state. We have also assessed the significance of metabolic model parameterization in kinetic models, summarizing a standard parameter estimation procedure frequently employed in kinetic metabolic modelling literature. Finally, some optimization practices used for the parameter estimation are reviewed

Particle production by supercritical antisolvent processing techniques

This thesis discusses particle production by supercritical antisolvent processing (SAS) techniques by looking the fundamentals and applications of the method with some case studies. The final aim of this work is however to consider the SAS particle production process feasibility. In the process studied the solid is dissolved in a conventional solvent and the solution is sprayed continuously through a nozzle into the subcritical or supercritical fluid. The dispersion of solution in the fluid leads to an expansion of the droplets and at the same time an extraction of the liquid into the fluid occurs. The solvent power of the conventional solvent decreases dramatically and supersaturation leads to the precipitation of particles. A static variable volume view cell (VVV-cell) is a useful and fast way to find out the appropriate combinations of the solvent and the gaseous antisolvent for a given solid. Often pharmaceutical materials are expensive and not available in large amounts, which makes it impossible to make phase separation studies with laboratory or production scale SAS equipment. However in VVV-cell experiments it is possible to use small amounts of materials and fast examine the influence of recrystallization temperature, pressure and concentration. But it is not possible to conclude by using only VVV-cell experiments, what kind of particles (size distribution, crystal habit and morphology) there will be produced in SAS process, because of the different formation dynamics and residence times. The present study showed that in the supercritical state the variables, such as density of CO2 and temperature, have a greater effect on the particle size than the model of droplets predicts. The liquid side mass transfer seems to control the studied polymer material particle size. In poly(L-lactic acid) particle formation with dichloromethane solvent and CO2 antisolvent by SAS technique it is advantageous to use low temperature and high pressure, in which conditions the mass transfer effect and volumetric expansion of droplets to produce high supersaturation will be favourable. In SAS process it is not possible to influence the initial droplet size by varying process variables (temperature, pressure and flow rate) in a typical operating range with a similar nozzle. Therefore the mass transfer coefficient of the liquid phase should be maximized to produce a high supersaturation fast when a small particle size is needed. Supercritical fluid technology is considered to be an innovative and promising way to design particles. In this thesis the applicability of two special supercritical precipitation techniques was studied. In the first case the results show, that it is possible to produce completely amorphous particles by spraying a methanol solution of sodium cromoglycate into supercritical carbon dioxide. The most significant parameter affecting the crystallinity was the residual methanol concentration in the particles. In the second case the results show, that cholesterol can be selectively extracted / crystallized at high purity, from a one phase mixture which contains lipids and cholesterol dissolved in pressured CO2. In this simple cholesterol production process no liquid solvents are needed. The egg yolk phospholipid was the most suitable raw material for the tested method. Because the concentration of cholesterol in the fluid stream is low, this method is not economically viable in industrial scale. The work clearly demonstrated that to recrystallize fine particles with SAS techniques in an industrial scale the price of the products must be high. These products would be chemical intermediates, biological and pharmaceutical compounds. On the other hand, if the compound properties are waxy or soft or the products are thermally unstable compounds, it may be feasible to use antisolvent formation techniques also for less expensive products, if the method makes the production possible. The manufacturing cost of SAS process is capital intensive. An estimated manufacturing cost for a new GMP plant is around 50-300 Euro/kg product without a feedstock price. This is for a 4000 to 8000 kg/year production rate and 5-10 wt% feed concentration of the starting material in an organic solvent. An effective way to decrease the manufacturing cost is to increase the raw material concentration in solvent. It is favourable to design a process for production rate over 2000-3000 kg/year and to use over 5 wt% feed concentration. Below those values the manufacturing cost increases dramatically.reviewe

Building a cascading multi-product biorefinery process for Ascophyllum nodosum: a green chemistry approach

Brown macroalgae are an attractive third-generation feedstock for biofuel, as well as a source of natural products. A cascading biorefinery approach extracts potentially bioactive compounds, i.e., polyphenols, fucoidan, and commodity products i.e., alginate, proteins in the same process. In order to design a green chemistry-compliant process and reduce the use of organic solvents in bioactive product extraction, aqueous two-phase systems (ATPS) and low-concentration biodegradable acid extractions were applied. The present work aimed to develop a multi-product biorefinery concept using Ascophyllum nodosum as a model feedstock using life cycle assessment (LCA), techno-economic analysis (TEA), and technical feasibility trials (TFT) as early-design tools for its development. After a biochemical characterisation of three potential model species, A. nodosum was selected as model feedstock based on the high accumulation of high-value products with potential breakthrough in the market. A.nodosum exhibited higher contents of polyphenols, lipids, protein, and minerals than the other species analysed, with 4.63% DW, 8.13% DW, 11.33% DW, and 29.54% DW, respectively. Once the biochemical characterisation was completed, three biorefining scenarios using different technology pathways (solvent, physicochemical, and green techniques) were modelled to process 1,000 metric tonnes (MT) biomass/year, in order to evaluate their economic and environmental metrics. From all evaluated scenarios, a green chemistry-compliant cascading sequence showed the lowest capital expenditure (CAPEX) (£30 million), operational expenditure (OPEX) (£11 million), cost of goods per kg of feedstock processed (CoG/kg) (£0.08) and production costs (£0.03/kg), along with the highest internal rate of return (IRR) (75.0%). Additionally, this scenario exhibited the lowest environmental impacts in all categories assessed, around 2 – 10 times lower than the other scenarios. In addition, the cascading sequence performance was evaluated to obtain first-hand data and re-iterate the models. The cascading sequence approach has been proposed to maximise resource efficiency and, in this work, a cascading sequence aimed at the sequential extraction of fucoidan, alginate, polyphenols, and proteins. Bioprocessing hotspots were identified for polyphenol and fucoidan extraction steps and further optimised using automated high-throughput screenings (HTS) and Design of Experiments (DoE), recovering 89% of total polyphenols, and showing a 33% increase in fucoidan recovery. Finally, after completing the bioprocessing hotspots, economic and environmental models were re-iterated to confirm the robustness of the biorefinery concept developed. The re-iterated version of the green chemistry-compliant cascading sequence exhibited better recovery performance in the optimised extraction stages, and thus showed better sales revenues (£91 million) than its previous version, a higher IRR (82.1%), and lower CoG/kg (£0.05) and production costs (£0.01/kg). The re-iterated version of the cascading sequence also exhibited the lowest environmental impacts in every category assessed, of all scenarios analysed in this project. These findings confirm that a holistic approach to early bioprocesses design is a valuable addition for decision-making tool options in the development of green-compliant multi-product third generation biorefineries