Jianye Xia

Integration of microbial kinetics and fluid dynamics toward model‐driven scale‐up of industrial bioprocesses

Scale‐up of bioprocesses is hampered by open questions, mostly related to poor mixing and mass transfer limitations. Concentration gradients of substrate, carbon dioxide, and oxygen in time and space, especially in large‐scale high‐cell density fed‐batch processes, are likely induced as the mixing time of the fermentor is usually longer than the relevant cellular reaction time. Cells in the fermentor are therefore repeatedly exposed to dynamic environments or perturbations. As a consequence, the heterogeneity in industrial practices often decreases either yield, titer, or productivity, or combinations thereof and increases by‐product formation as compared to well‐mixed small‐scale bioreactors, which is summarized as scale‐up effects. Identification of response mechanisms of the microorganism to various external perturbations is of great importance for pinpointing metabolic bottlenecks and targets for metabolic engineering. In this review, pulse response experimentation is proposed as an ideal way of obtaining kinetic information in combination with scale‐down approaches for in‐depth understanding of dynamic response mechanisms. As an emerging tool, computational fluid dynamics is able to draw a holistic picture of the fluid flow and concentration fields in the fermentor and finds its use in the optimization of fermentor design and process strategy. In the future, directed strain improvement and fermentor redesign are expected to largely depend on models, in which both microbial kinetics and fluid dynamics are thoroughly integrated.

Euler-Lagrange computational fluid dynamics for (bio)reactor scale down : An analysis of organism lifelines

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.

A novel impeller configuration to improve fungal physiology performance and energy conservation for cephalosporin C production

Effects of impeller configuration on fungal physiology and cephalosporin C production were investigated by an industrial strain Acremonium chrysogenum in a 12 m(3) bioreactor equipped with conventional and novel impeller configuration, respectively. The cell growth and oxygen uptake rate (OUR) profiles were little affected by the impeller configurations. However, differing impeller combinations significantly affected the morphology, which in turn influenced cephalosporin C production. Under the novel impeller configuration, the production of cephalosporin C was 10% higher and an excessive amount of dispersed arthrospores was also observed. Computational fluid dynamics (CFD) simulation further revealed that poor mass and energy exchange as well as inhomogeneous environment existed in the bioreactor equipped with conventional impeller configuration. For equivalent power dissipation, the volume oxygen transfer coefficient (K(L)a) could be enhanced by 15% compared with that of conventional impeller configuration. Power consumption was dramatically decreased by 25% by using novel impeller configuration.

Prelude to rational scale-up of penicillin production: a scale-down study

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.

Integrated isotope-assisted metabolomics and 13 C metabolic flux analysis reveals metabolic flux redistribution for high glucoamylase production by Aspergillus niger

BackgroundAspergillus niger is widely used for enzyme production and achievement of high enzyme production depends on the comprehensive understanding of cell’s metabolic regulation mechanisms.ResultsIn this paper, we investigate the metabolic differences and regulation mechanisms between a high glucoamylase-producing strain A. niger DS03043 and its wild-type parent strain A. niger CBS513.88 via an integrated isotope-assisted metabolomics and 13C metabolic flux analysis approach. We found that A. niger DS03043 had higher cell growth, glucose uptake, and glucoamylase production rates but lower oxalic acid and citric acid secretion rates. In response to above phenotype changes, A. niger DS03043 was characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway in contrast to reduced flux through TCA cycle, which were confirmed by consistent changes in pool sizes of metabolites. A higher ratio of ATP over AMP in the high producing strain might contribute to the increase in the PP pathway flux as glucosephosphate isomerase was inhibited at higher ATP concentrations. A. niger CBS513.88, however, was in a higher redox state due to the imbalance of NADH regeneration and consumption, resulting in the secretion of oxalic acid and citric acid, as well as the accumulation of intracellular OAA and PEP, which may in turn result in the decrease in the glucose uptake rate.ConclusionsThe application of integrated metabolomics and 13C metabolic flux analysis highlights the regulation mechanisms of energy and redox metabolism on flux redistribution in A. niger. Graphical abstractAn integrated isotope-assisted metabolomics and 13C metabolic flux analysis was was firstly systematically performed in A. niger. In response to enzyme production, the metabolic flux in A. niger DS03043 (high-producing) was redistributed, characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway as well as an increased pool size of pentose. The consistency in 13C metabolic flux analysis and metabolites quantification indicated that an imbalance of NADH formation and consumption led to the accumulation and secretion of organic acids in A. niger CBS513.88 (wild-type)

Comprehensive reconstruction and in silico analysis of Aspergillus niger genome‐scale metabolic network model that accounts for 1210 ORFs

Aspergillus niger is one of the most important cell factories for industrial enzymes and organic acids production. A comprehensive genome‐scale metabolic network model (GSMM) with high quality is crucial for efficient strain improvement and process optimization. The lack of accurate reaction equations and gene‐protein‐reaction associations (GPRs) in the current best model of A. niger named GSMM iMA871, however, limits its application scope. To overcome these limitations, we updated the A. niger GSMM by combining the latest genome annotation and literature mining technology. Compared with iMA871, the number of reactions in iHL1210 was increased from 1,380 to 1,764, and the number of unique ORFs from 871 to 1,210. With the aid of our transcriptomics analysis, the existence of 63% ORFs and 68% reactions in iHL1210 can be verified when glucose was used as the only carbon source. Physiological data from chemostat cultivations, 13C‐labeled and molecular experiments from the published literature were further used to check the performance of iHL1210. The average correlation coefficients between the predicted fluxes and estimated fluxes from 13C‐labeling data were sufficiently high (above 0.89) and the prediction of cell growth on most of the reported carbon and nitrogen sources was consistent. Using the updated genome‐scale model, we evaluated gene essentiality on synthetic and yeast extract medium, as well as the effects of NADPH supply on glucoamylase production in A. niger. In summary, the new A. niger GSMM iHL1210 contains significant improvements with respect to the metabolic coverage and prediction performance, which paves the way for systematic metabolic engineering of A. niger. Biotechnol. Bioeng. 2017;114: 685–695.

Dependence of fungal characteristics on seed morphology and shear stress in bioreactors

The fungal morphology during submerged cultivations has a profound influence on the overall performance of bioreactors. In this research, glucoamylase production by Aspergillus niger has been taken as a model to improve more insights. The morphology engineering could be conducted effectively by changing the seed morphology, as well as specific power input. During the fed-batch cultivations, pellet formation under milder shear stress field helped to reduce the broth viscosity, thus relieving oxygen limitation and promoting the enzyme production. Furthermore, we found that the relation between the shear stress field, which was characterized by energy dissipation rate/circulation function (EDCF), and enzyme activity was consistent with quadratic parabola, which threw light on the process optimization and scale-up for industrial enzyme production.

Advances and Practices of Bioprocess Scale-up

: This chapter addresses the update progress in bioprocess engineering. In addition to an overview of the theory of multi-scale analysis for fermentation process, examples of scale-up practice combining microbial physiological parameters with bioreactor fluid dynamics are also described. Furthermore, the methodology for process optimization and bioreactor scale-up by integrating fluid dynamics with biokinetics is highlighted. In addition to a short review of the heterogeneous environment in large-scale bioreactor and its effect, a scale-down strategy for investigating this issue is addressed. Mathematical models and simulation methodology for integrating flow field in the reactor and microbial kinetics response are described. Finally, a comprehensive discussion on the advantages and challenges of the model-driven scale-up method is given at the end of this chapter.

Multi-omics integrative analysis with genome-scale metabolic model simulation reveals global cellular adaptation of Aspergillus niger under industrial enzyme production condition

Oxygen limitation is regarded as a useful strategy to improve enzyme production by mycelial fungus like Aspergillus niger. However, the intracellular metabolic response of A. niger to oxygen limitation is still obscure. To address this, the metabolism of A. niger was studied using multi-omics integrated analysis based on the latest GEMs (genome-scale metabolic model), including metabolomics, fluxomics and transcriptomics. Upon sharp reduction of the oxygen supply, A. niger metabolism shifted to higher redox level status, as well as lower energy supply, down-regulation of genes for fatty acid synthesis and a rapid decrease of the specific growth rate. The gene expression of the glyoxylate bypass was activated, which was consistent with flux analysis using the A. niger GEMs iHL1210. The increasing flux of the glyoxylate bypass was assumed to reduce the NADH formation from TCA cycle and benefit maintenance of the cellular redox balance under hypoxic conditions. In addition, the relative fluxes of the EMP pathway were increased, which possibly relieved the energy demand for cell metabolism. The above multi-omics integrative analysis provided new insights on metabolic regulatory mechanisms of A. niger associated with enzyme production under oxygen-limited condition, which will benefit systematic design and optimization of the A. niger microbial cell factory.

Gas-liquid mass transfer studies: The influence of single- and double-impeller configurations in stirred tanks

The influence of impeller structure on the mass transfer characteristics was studied with the steady-state method for gas-liquid volumetric mass transfer coefficient (kLa). The single-impeller configurations included eight impeller types (three radial flow impellers, four axial flow impellers and one mixed flow impeller), and the doubleimpeller included three configurations (RT+RT, RT+WHD, WHD+WHD). For single-impeller, the gas-liquid mass transfer rates of radial flow impellers were better than those of axial flow impellers under the same rotation speed and gas flow rate. The mass transfer performance (defined as the volumetric mass transfer coefficient per unit power input) of radial flow impellers were also better than that of axial flow impellers. With the same kLa value under a certain gas flow rate, the local bubble size distribution between radial flow impeller and axial flow impeller was similar. As for double impellers, RT+RT provided the highest mass transfer rate under certain rotation speed and gas flow rate, while WHD+WHD gave the highest values of gas-liquid mass transfer coefficient with the same power consumption.