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In large-scale syngas fermentation, strong gradients in dissolved gas (CO, H2) concentrations are very likely to occur due to locally varying mass transfer and convection rates. Using Euler-Lagrangian CFD simulations, we analyzed these gradients in an industrial-scale external-loop gas-lift reactor (EL-GLR) for a wide range of biomass concentrations, considering CO inhibition for both CO and H2 uptake. Lifeline analyses showed that micro-organisms are likely to experience frequent (5 to 30 s) oscillations in dissolved gas concentrations with one order of magnitude. From the lifeline analyses, we developed a conceptual scale-down simulator (stirred-tank reactor with varying stirrer speed) to replicate industrial-scale environmental fluctuations at bench scale. The configuration of the scale-down simulator can be adjusted to match a broad range of environmental fluctuations. Our results suggest a preference for industrial operation at high biomass concentrations, as this would strongly reduce inhibitory effects, provide operational flexibility and enhance the product yield. The peaks in dissolved gas concentration were hypothesized to increase the syngas-to-ethanol yield due to the fast uptake mechanisms in C. autoethanogenum. The proposed scale-down simulator can be used to validate such results and to obtain data for parametrizing lumped kinetic metabolic models that describe such short-term responses.
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A novel fermentation process was developed in which renewable electricity is indirectly used as an energy source in fermentation, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO2 . As an illustration, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO2 from fermentation offgas followed by electrochemical reduction with renewable electricity. This "closed carbon loop" concept is demonstrated by a case study in which cofeeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica. First, the optimal feed ratio of formic acid to glucose is established using chemostat cultivations. Subsequently, guided by a dynamic fermentation process model, a fed-batch protocol is developed and demonstrated on laboratory scale. Finally, the developed fed-batch process is tested and proven to be scalable at pilot scale. Extensions of the concept are discussed to apply the concept to anaerobic fermentations, and to recycle the O2 that is co-generated with the formic acid to aerobic fermentation processes for intensification purposes.
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Yarrowia , Carbono , Dióxido de Carbono , Fermentación , Formiatos , GlucosaRESUMEN
The compartment model (CM) is a well-known approach for computationally affordable, spatially resolved hydrodynamic modeling of unit operations. Recent implementations use flow profiles based on Computational Fluid Dynamics (CFD) simulations, and several authors included microbial kinetics to simulate gradients in bioreactors. However, these studies relied on black-box kinetics that do not account for intracellular changes and cell population dynamics in response to heterogeneous environments. In this paper, we report the implementation of a Lagrangian reaction model, where the microbial phase is tracked as a set of biomass-parcels, each linked with an intracellular composition vector and a structured reaction model describing their intracellular response to extracellular variations. A stochastic parcel tracking approach is adopted, in contrast to the resolved trajectories used in CFD implementations. A penicillin production process is used as a case study. We show good performance of the model compared with full CFD simulations, both regarding the extracellular gradients and intracellular pool response, using the mixing time as a matching criterion and taking into account that the mixing time is sensitive to the number of compartments. The sensitivity of the model output towards some of the inputs is explored. The coarsest representative CM requires a few minutes to solve 80 h of flow time, compared with approximately 2 weeks for a full Euler-Lagrange CFD simulation of the same case. This alleviates one of the major bottlenecks for the application of such CFD simulations towards the analysis and optimization of industrial fermentation processes.
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Reactores Biológicos , Hidrodinámica , Simulación por Computador , Fermentación , CinéticaRESUMEN
During glucose-limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of ß-lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates (D) of both 0.03 h-1 and 0.05 h-1 are determined to evaluate the quantitative impact on the physiology of a high-yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h-1 , while this is not the case at D = 0.05 h-1 . In addition, a higher cytosolic redox status (NADH/NAD+ ), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h-1 , which are virtually absent at D = 0.03 h-1 . In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario.
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Reactores Biológicos/microbiología , Metabolómica/métodos , Penicillium chrysogenum/crecimiento & desarrollo , Formiato Deshidrogenasas/metabolismo , Formiatos/metabolismo , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Hidrogenasas/metabolismo , Complejos Multienzimáticos/metabolismo , Penicilinas/metabolismo , Penicillium chrysogenum/química , Estrés FisiológicoRESUMEN
Future manufacturing will focus on new, improved products as well as on new and enhanced production methods. Recent biotechnological and scientific advances, such as CRISPR/Cas and various omic technologies, pave the way to exciting novel biotechnological research, development, and commercialization of new sustainable products. Rigorous mathematical descriptions of microbial cells and consortia thereof will enable deeper biological understanding and lead to powerful in silico cellular models. Biological engineering, namely model-based design together with synthetic biology, will accelerate the construction of robust and high-performing microorganisms. Using these organisms, and ambitions towards zero-concepts with respect to emissions and excess resources in bioprocess engineering, industrial biotechnology is expected to become highly integrated into sustainable generations of technology systems.
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Biotecnología/métodos , Investigación/tendencias , Biocombustibles , Ingeniería Genética/métodos , Tecnología Química Verde , Microbiología Industrial/métodos , Ingeniería Metabólica/métodos , Consorcios Microbianos/fisiología , ReciclajeRESUMEN
In a 54 m3 large-scale penicillin fermentor, the cells experience substrate gradient cycles at the timescales of global mixing time about 20-40 s. Here, we used an intermittent feeding regime (IFR) and a two-compartment reactor (TCR) to mimic these substrate gradients at laboratory-scale continuous cultures. The IFR was applied to simulate substrate dynamics experienced by the cells at full scale at timescales of tens of seconds to minutes (30 s, 3 min and 6 min), while the TCR was designed to simulate substrate gradients at an applied mean residence time (τc) of 6 min. A biological systems analysis of the response of an industrial high-yielding P. chrysogenum strain has been performed in these continuous cultures. Compared to an undisturbed continuous feeding regime in a single reactor, the penicillin productivity (qPenG ) was reduced in all scale-down simulators. The dynamic metabolomics data indicated that in the IFRs, the cells accumulated high levels of the central metabolites during the feast phase to actively cope with external substrate deprivation during the famine phase. In contrast, in the TCR system, the storage pool (e.g. mannitol and arabitol) constituted a large contribution of carbon supply in the non-feed compartment. Further, transcript analysis revealed that all scale-down simulators gave different expression levels of the glucose/hexose transporter genes and the penicillin gene clusters. The results showed that qPenG did not correlate well with exposure to the substrate regimes (excess, limitation and starvation), but there was a clear inverse relation between qPenG and the intracellular glucose level.
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Reactores Biológicos/microbiología , Medios de Cultivo/química , Penicilinas/biosíntesis , Penicillium chrysogenum/crecimiento & desarrollo , Metabolismo de los Hidratos de Carbono , Carbono/metabolismo , FermentaciónRESUMEN
In the present work, by performing chemostat experiments at 400 and 600 RPM, two typical power inputs representative of industrial penicillin fermentation (P/V, 1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers, respectively) were scaled-down to bench-scale bioreactors. It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures, the previously reported degeneration of penicillin production using an industrial Penicillium chrysogenum strain was virtually absent. To investigate this, the cellular response was studied at flux (stoichiometry), residual glucose, intracellular metabolite and transcript levels. At 600 RPM, 20% more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22% larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration (Cs,out ). At the same time, the biomass specific glucose consumption rate (qs ) did not change but the intracellular glucose concentration was about sixfold higher, which indicates a change to a higher affinity glucose transporter at 600 RPM. In addition, power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae, which was further substantiated by a simple proposed glucose diffusion-uptake model. By analysis of calculated mass action ratios (MARs) and energy consumption, it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs,out appear to trigger a gluconeogenic type of metabolic flux rearrangement, a futile cycle through the pentose phosphate pathway (PPP) and a declining redox state of the cytosol. In support of the change in glucose transport and degeneration of penicillin production at 600 RPM, the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g02880 and Pc06g01340 increased 3.5- and 3.3-fold, respectively, and those of the pcbC gene encoding isopenicillin N-synthetase (IPNS) were more than twofold lower in the time range of 100-200 hr of the chemostat cultures. Summarizing, changes at power input have unexpected effects on degeneration and glucose transport, and result in significant metabolic rearrangements. These findings are relevant for the industrial production of penicillin, and other fermentations with filamentous microorganisms.
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Antibacterianos/biosíntesis , Reactores Biológicos/microbiología , Penicilinas/biosíntesis , Penicillium chrysogenum/crecimiento & desarrollo , Penicillium chrysogenum/metabolismo , Factores Biológicos/metabolismo , Fermentación , Glucosa/metabolismo , Análisis de SistemasRESUMEN
A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.
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Proliferación Celular/fisiología , Glucosa/metabolismo , Análisis de Flujos Metabólicos/métodos , Redes y Vías Metabólicas/fisiología , Modelos Biológicos , Penicillium chrysogenum/fisiología , Simulación por Computador , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Cinética , Tasa de Depuración Metabólica/fisiología , Complejos Multienzimáticos/metabolismo , Penicillium chrysogenum/citología , Factores de TiempoRESUMEN
The trajectories, referred to as lifelines, of individual microorganisms in an industrial scale fermentor under substrate limiting conditions were studied using an Euler-Lagrange computational fluid dynamics approach. The metabolic response to substrate concentration variations along these lifelines provides deep insight in the dynamic environment inside a large-scale fermentor, from the point of view of the microorganisms themselves. We present a novel methodology to evaluate this metabolic response, based on transitions between metabolic "regimes" that can provide a comprehensive statistical insight in the environmental fluctuations experienced by microorganisms inside an industrial bioreactor. These statistics provide the groundwork for the design of representative scale-down simulators, mimicking substrate variations experimentally. To focus on the methodology we use an industrial fermentation of Penicillium chrysogenum in a simplified representation, dealing with only glucose gradients, single-phase hydrodynamics, and assuming no limitation in oxygen supply, but reasonably capturing the relevant timescales. Nevertheless, the methodology provides useful insight in the relation between flow and component fluctuation timescales that are expected to hold in physically more thorough simulations. Microorganisms experience substrate fluctuations at timescales of seconds, in the order of magnitude of the global circulation time. Such rapid fluctuations should be replicated in truly industrially representative scale-down simulators.
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Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.