Building blocks needed for mechanistic modeling of bioprocesses: A critical review based on protein production by CHO cells.

This paper reviews the key building blocks needed to develop a mechanistic model for use as an operational production tool. The Chinese Hamster Ovary (CHO) cell, one of the most widely used hosts for antibody production in the pharmaceutical industry, is considered as a case study. CHO cell metabolism is characterized by two main phases, exponential growth followed by a stationary phase with strong protein production. This process presents an appropriate degree of complexity to outline the modeling strategy. The paper is organized into four main steps: (1) CHO systems and data collection; (2) metabolic analysis; (3) formulation of the mathematical model; and finally, (4) numerical solution, calibration, and validation. The overall approach can build a predictive model of target variables. According to the literature, one of the main current modeling challenges lies in understanding and predicting the spontaneous metabolic shift. Possible candidates for the trigger of the metabolic shift include the concentration of lactate and carbon dioxide. In our opinion, ammonium, which is also an inhibiting product, should be further investigated. Finally, the expected progress in the emerging field of hybrid modeling, which combines the best of mechanistic modeling and machine learning, is presented as a fascinating breakthrough. Note that the modeling strategy discussed here is a general framework that can be applied to any bioprocess.

The effect of light intensity on microalgae biofilm structures and physiology under continuous illumination.

The interest by biofilm-based microalgae technologies has increased lately due to productivity improvement, energy consumption reduction and easy harvesting. However, the effect of light, one key factor for system's operation, received less attention than for planktonic cultures. This work assessed the impact of Photon Flux Density (PFD) on Chlorella vulgaris biofilm dynamics (structure, physiology, activity). Microalgae biofilms were cultivated in a flow-cell system with PFD from 100 to 500 [Formula: see text]. In the first stage of biofilm development, uniform cell distribution was observed on the substratum exposed to 100 [Formula: see text] while cell clusters were formed under 500 [Formula: see text]. Though similar specific growth rate in exponential phase (ca. 0.3 [Formula: see text]) was obtained under all light intensities, biofilm cells at 500 [Formula: see text] seem to be ultimately photoinhibited (lower final cell density). Data confirm that Chlorella vulgaris showed a remarkable capability to cope with high light. This was marked for sessile cells at 300 [Formula: see text], which reduce very rapidly (in 2 days) their chlorophyll-a content, most probably to reduce photodamage, while maintaining a high final cell density. Besides cellular physiological adjustments, our data demonstrate that cellular spatial organization is light-dependent.

Water vapor transport properties of bio-based multilayer materials determined by original and complementary methods.

To enhance PLA gas barrier properties, multilayer designs with highly polar barrier layers, such as nanocelluloses, have shown promising results. However, the properties of these polar layers change with humidity. As a result, we investigated water transport phenomena in PLA films coated with nanometric layers of chitosan and nanocelluloses, utilizing a combination of techniques including dynamic vapor sorption (DVS) and long-term water vapor adsorption-diffusion experiments (back-face measurements) to understand the influence of each layer on the behavior of multilayer films. Surprisingly, nanometric coatings impacted PLA water vapor transport. Chitosan/nanocelluloses layers, representing less than 1 wt.% of the multilayer film, increased the water vapor uptake of the film by 14.6%. The nanometric chitosan coating appeared to have localized effects on PLA structure. Moreover, nanocelluloses coatings displayed varying impacts on sample properties depending on their interactions (hydrogen, ionic bonds) with chitosan. The negatively charged CNF TEMPO coating formed a dense network that demonstrated higher resistance to water sorption and diffusion compared to CNF and CNC coatings. This work also highlights the limitations of conventional water vapor permeability measurements, especially when dealing with materials containing ultrathin nanocelluloses layers. It shows the necessity of considering the synergistic effects between layers to accurately evaluate the transport properties.

The impact of light/dark regimes on structure and physiology of Chlorella vulgaris biofilms.

Introduction: Biofilm-based microalgae production technologies offer enormous potential for improving sustainability and productivity. However, the light pattern induced by these technologies is a key concern for optimization. Methods: In this work, the effects of light/dark cycles on architecture, growth, and physiology of Chlorella vulgaris biofilms were assessed in a millifluidic flow-cell with different time cycles (15 s to 3 min) keeping the average light constant at 100 µmol·m-2·s-1. Results and discussion: Results showed that photoinhibition can be mitigated by applying a light fraction of 1/3 and a cycle time of 15 s. By contrast, when the cycle time is extended to 90 s and 3 min, photoinhibition is high and photoefficiency dramatically decreases. To cope with light stress, cells acclimate and organize themselves differently in space. A high peak light (500 µmol·m-2·s-1) triggers a stress, reducing cell division and inducing clusters in the biofilm. This work provides guidelines for optimizing rotating microalgae production systems in biofilms and assesses the minimum rotating frequency required to maintain the net growth rate close to that of continuous light of the same average intensity, mitigating photo-inhibition. The overall gain in productivity is then provided by the total surface of the biofilm turning in the illuminated surface area.

Improvement of Sinapine Extraction from Mustard Seed Meal by Application of Emerging Technologies.

Sinapine is a phenolic compound found in mustard (Brassica juncea) seed meal. It has numerous beneficial properties such as antitumor, neuroprotective, antioxidant, and hepatoprotective effects, making its extraction relevant. In this study, the extraction of sinapine was investigated using three methods: (i) from a mustard seed meal defatted by a supercritical CO2 (SC-CO2) pretreatment, (ii) by the implementation of high-voltage electrical discharges (HVEDs), (iii) and by the use of ultrasound. The use of SC-CO2 pretreatment resulted in a dual effect on the valorization of mustard seed meal, acting as a green solvent for oil recovery and increasing the yield of extracted sinapine by 24.4% compared to the control. The combination of ultrasound and SC-CO2 pretreatment further increased the yield of sinapine by 32%. The optimal conditions for ultrasound-assisted extraction, determined through a response surface methodology, are a temperature of 75 °C, 70% ethanol, and 100% ultrasound amplitude, resulting in a sinapine yield of 6.90 ± 0.03 mg/g dry matter. In contrast, the application of HVEDs in the extraction process was not optimized, as it led to the degradation of sinapine even at low-energy inputs.

A Novel Strategy to Enhance Antioxidant Content in Saccharomyces Cerevisiae Based on Oxygen Pressure.

Antioxidant foods represent a potent lever to improve diets while creating value. Yet, their cultivation is often tied to a specific area and climate, limiting availability and increasing market cost. Therefore, microorganism-based antioxidant production emerges as a promising technology to solve these problems. In this view, a novel process was investigated for antioxidant accumulation in yeast culture. S. cerevisiae cells were exposed to various hyperbaric air conditions from 1 to 9 bar (A). Yeast cultures exhibited an increased reactive oxygen species content, which induced oxidative defense expression. After a few hours, reactive oxygen species levels decreased while antioxidant contents remained high, leading to a net increase in antioxidant power. At 6 bar (A), yeast achieved the highest net antioxidant power (phenolics content +48.3 ± 18.6 %, reducing power +120 ± 11.4 %) with an acceptable growth rate (0.27 h-1). Regarding time evolution, a 2 h exposure seems to be the optimum: cells have the lowest reactive oxygen species level while their antioxidant power is increased. From a biotechnological perspective, this finding highlights air pressure as an antioxidant-manipulating stress strategy. Moreover, the proposed process led to a patent that could potentially reduce energy and chemical consumption in such antioxidant accumulation processes.

Principal factors affecting the yield of dilute acid pretreatment of lignocellulosic biomass: A critical review.

This review provides a critical analysis of the state of the art of dilute acid pretreatment applied to lignocellulosic biomass. Data from 63 publications were extracted and analysed. The majority of the papers used residence times of<30 min, temperature ranges from 100 °C to 200 °C, and acid levels between 0 % and 2 %. Yields are quantified directly after pretreatment (xylose content) or after enzymatic hydrolysis (glucose content). Statistical analyses allowed the time-temperature equivalence to be quantified for three types of biomass: they were formulated by non-linear expressions. In further works, investigating less explored areas, for example moderate temperature levels with longer residence times, is recommended. Pretreatment material (time-temperature kinetics, reactor type) and analytical methods should be standardized and better described. It becomes mandatory to promote the development of an open, findable, accessible, interoperable, and reusable data approach for pretreatments research.

Three-dimensional pore characterization of poly(lactic)acid/bamboo biodegradable panels.

In the context of novel environmental and energy regulations in construction (RE2020), biocomposites derived from bamboo fibers, bamboo powders, and biodegradable poly(lactic)acid polymer, all of which are renewable resources, have been investigated to meet the criteria of the novel regulations. In this work, the biocomposites were manufactured by twin-screw internal mixing at 170 °C for 5 min with a rotation speed of 60 rpm. The composites sheets were then shaped on a hydraulic press at 185 °C. Pore characterization including pore volume fraction, 3D-pore structure and morphology, and pore distribution of these materials were investigated using X-ray tomography combined with image processing (Avizo). The results show that when the bamboo fibers content is increased, an augmentation in the pore volume fraction and the number of large-volume pores could be observed. In turn, the bamboo powder-containing sheet had a significant increase in pore volume fraction, while a higher quantity of smaller pores, with uniform size, could be observed. The water absorption capacity of these composite increases with the increase of the amount of pore distribution, pore connection, and pore volume fraction. In addition, the orientation of the fibers in 3D observation, flexural mechanical properties, and thermal stability of the biocomposites are also reported in this study.

A macroscopic Washburn approach of liquid imbibition in wood derived from X-ray tomography observations.

Imbibition of water and silicone oil in poplar and spruce is investigated at the anatomical level by X-ray tomography observations and at the macroscopic level by imbibition kinetics. Imbibition mechanisms depend on both liquid and species. In poplar, oil penetrates vessels with a small contact angle, consistent with the value measured on solid wood (ca. [Formula: see text]). Surprisingly, no direct penetration of water was observed in vessels. The large contact angle for water blocks the capillary rise at the scars between vessel cells. In spruce, oil and water penetrate primarily in latewood, where bordered pits remain open. Subsequently, water slowly invades the rest of the growth ring, while transversal migration is quasi-absent for oil. These 3D observations were quantified to feed a simple imbibition model that satisfactorily simulates macroscopic imbibition kinetics. A 1D approach is sufficient for oil imbibition while a 2D approach is required for water, revealing dual scale effects.

A predictive model of organic acids separation by chromatography with strong anionic resins in sulfate form.

Organic acids commonly have quite symmetrical chromatography profiles at low pH (< 1.5) with strong anionic resins, but a significant tailing can be observed with succinic and citric acids. Classical adsorption models, like the Langmuir model, fail to predict this behavior, which can have a major influence on mean retention times and profile shapes, therefore on chromatography performances. A new retention model was developed to better predict organic acid separation with strong anionic resin. This model combines a refined Langmuir adsorption model and an ion-exchange model. Organic acid adsorption is assumed to be due to hydrogen bonding with sulfate and hydrogen sulfate counter-anions on the resin. The adsorption capacity depends mostly on molecular size: up to sixteen formic acid molecules could be adsorbed per counter-anions, meanwhile only two succinic acid or one citric acid molecules could be adsorbed. This adsorption model was then embedded in a generic and accurate modeling approach (continuous column with mass balance equations solved by the conservation element/solution element (CE/SE) method). All parameters of this column model were identified by fitting the simulation to experimental results (equilibrium curves and pulse tests). Then, the column model was validated with original experimental results from a binary mixture pulse test (formic and succinic acids). Results show that simulations are much more predictive for multi-component pulse tests, both in terms of profile shape and retention time, which cannot be captured without considering ion-exchange.

Morphological growth pattern of Phanerochaete chrysosporium cultivated on different Miscanthus x giganteus biomass fractions.

BACKGROUND: Solid-state fermentation is a fungal culture technique used to produce compounds and products of industrial interest. The growth behaviour of filamentous fungi on solid media is challenging to study due to the intermixity of the substrate and the growing organism. Several strategies are available to measure indirectly the fungal biomass during the fermentation such as following the biochemical production of mycelium-specific components or microscopic observation. The microscopic observation of the development of the mycelium, on lignocellulosic substrate, has not been reported. In this study, we set up an experimental protocol based on microscopy and image processing through which we investigated the growth pattern of Phanerochaete chrysosporium on different Miscanthus x giganteus biomass fractions. RESULTS: Object coalescence, the occupied surface area, and radial expansion of the colony were measured in time. The substrate was sterilized by autoclaving, which could be considered a type of pre-treatment. The fastest growth rate was measured on the unfractionated biomass, followed by the soluble fraction of the biomass, then the residual solid fractions. The growth rate on the different fractions of the substrate was additive, suggesting that both the solid and soluble fractions were used by the fungus. Based on the FTIR analysis, there were differences in composition between the solid and soluble fractions of the substrate, but the main components for growth were always present. We propose using this novel method for measuring the very initial fungal growth by following the variation of the number of objects over time. Once growth is established, the growth can be followed by measurement of the occupied surface by the mycelium. CONCLUSION: Our data showed that the growth was affected from the very beginning by the nature of the substrate. The most extensive colonization of the surface was observed with the unfractionated substrate containing both soluble and solid components. The methodology was practical and may be applied to investigate the growth of other fungi, including the influence of environmental parameters on the fungal growth.

A validated Distributed Activation Energy Model (DAEM) to predict the chemical degradation of biomass as a function of hydrothermal treatment conditions.

This study proposes a DAEM (Distributed Activation Energy Model) approach to predict the chemical alterations of lignocellulosic biomass as a function of hydrothermal treatment conditions. The model is first tuned by an original device allowing the sample shrinkage to be continuously assessed during hydrothermal treatment in saturated water vapor up to 190 °C. The shrinkage dynamic is supplied in the DAEM model as an indicator of the degree of biomass conversion. A set of chemical analyses was performed at selected residence times and treatment temperatures to correlate this degree of conversion with the resulting chemical molecules. A set of functions was then derived from this database to correlate the degree of conversion with the components concentrations. Finally, a validation database was built with different combinations of temperature levels and residence times. The model was proved to be predictive on this new dataset.

CO2 Absorption Using Hollow Fiber Membrane Contactors: Introducing pH Swing Absorption (pHSA) to Overcome Purity Limitation.

Recently, membrane contactors have gained more popularity in the field of CO2 removal; however, achieving high purity and competitive recovery for poor soluble gas (H2, N2, or CH4) remains elusive. Hence, a novel process for CO2 removal from a mixture of gases using hollow fiber membrane contactors is investigated theoretically and experimentally. A theoretical model is constructed to show that the dissolved residual CO2 hinders the capacity of the absorbent when it is regenerated. This model, backed up by experimental investigation, proves that achieving a purity > 99% without consuming excessive chemicals or energy remains challenging in a closed-loop system. As a solution, a novel strategy is proposed: the pH Swing Absorption which consists of manipulating the acido-basic equilibrium of CO2 in the absorption and desorption stages by injecting moderate acid and base amount. It aims at decreasing CO2 residual content in the regenerated absorbent, by converting CO2 into its ionic counterparts (HCO3- or CO32-) before absorption and improving CO2 degassing before desorption. Therefore, this strategy unlocks the theoretical limitation due to equilibrium with CO2 residual content in the absorbent and increases considerably the maximum achievable purity. Results also show the dependency of the performance on operating conditions such as total gas pressure and liquid flowrate. For N2/CO2 mixture, this process achieved a nitrogen purity of 99.97% with a N2 recovery rate of 94.13%. Similarly, for H2/CO2 mixture, a maximum H2 purity of 99.96% and recovery rate of 93.96% was obtained using this process. Moreover, the proposed patented process could potentially reduce energy or chemicals consumption.

Effectiveness of photocatalysis of MMT-supported TiO2 and TiO2 nanotubes for rhodamine B degradation.

The aim of this paper is to synthesize montmorillonite/TiO2-nanoparticles (MMT/TiO2 and montmorillonite/TiO2-nanotubes (MMT/TiO2-NTs) photocatalysts through a simple wet agitation method based on TiO2 nanoparticles and MMT. They are likely to accumulate the effect of adsorption and photodegradation. Then, the photocatalysts are applied to degrade the rhodamine B in dye effluents. The structural characterizations of photocatalysts are investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDX). The photocatalytic activities and effectiveness of photocatalysts are evaluated through rhodamine B degradation at different concentrations under dark and UV-C irradiation conditions. The results show that the synthesized TiO2-NTs have an average tube diameter of 5 nm and a tube length at least about 110 nm, which are intercalated into MMT sheets in MMT/TiO2-NTs photocatalyst. Meanwhile, TiO2 nanoparticles are immobilized on the surface of MMT sheets in the MMT/TiO2 photocatalyst. The photocatalytic effectiveness of rhodamine B degradation of TiO2-NTs shows a significantly enhance compared to that of TiO2 nanoparticles. However, photocatalytic performance of MMT/TiO2-NTs is lower than that of MMT/TiO2. The degradation effectiveness of MMT/TiO2 photocatalyst reaches to 100% for 3 ppm and 90% at 10 ppm of rhodamine B, while these values are 97.5% and 85.5%, respectively, recorded for MMT/TiO2-NTs.

Multiscale investigation on the chemical and anatomical changes of lignocellulosic biomass for different severities of hydrothermal treatment.

The chemical changes sustained by lignocellulosic biomass during hydrothermal treatment are reflected at multiple scales. This study proposes to benefit from this multiscale nature in order to provide a global understanding of biomass alterations during hydrothermal treatment. For this purpose, complementary imaging techniques-confocal Raman microscopy and X-ray nano-tomography-analysed by image processing and coupled to chemical measurements were used. This unique combination of analyses provided valuable information on topochemical and morphological changes of poplar samples, without the artefacts of sample preparation. At the cell wall level, holocellulose hydrolysis and lignin modifications were observed, which corresponded to anatomical modifications observed at higher scales. Overall, after treatment, samples shrank and had thinner cell walls. When subjected to more severe pre-treatments, cells were disrupted and detached from adjacent cells. Anatomical changes were then used to obtain quantitative indicators of the treatment severity. The effects of treatment at different scales can thus be quantitatively connected in both directions, from micro to macro and from macro to micro.

Shear stress computation in a millimeter thin flat panel photobioreactor: Numerical design validated by experiments.

Flat panels are the most spread type of photobioreactors for studying light effects on a microalgae culture. Their low thickness, usually between 1 and 3 cm, aims at ensuring light homogeneity across the culture. Yet because optical density has to remain very low, studies are still limited to low cell density cultures. To alleviate this problem, even thinner photobioreactors can be designed. Nevertheless, thin flat panel reactors are very prone to induce high shear stress. This work aimed at designing a new millimeter thin panel photobioreactor to study light effects on Chlorella and Scenedesmus genera. We proposed a numerical workflow that is capable of assessing the shear stress intensity in such a reactor. The numerical predictions were validated at three different levels: 2D preliminary simulations were able to reproduce bubble commonly known behaviors; close to the nozzle, the predictions were successfully confronted to shadowgraphy experimental reference; at the mini bioreactor scale, experimental and numerical mixing were found to be close. After these throughout validations, shear stress intensity in the photobioreactor was calculated over 1000 Lagrangian tracers. The experienced shear stress was agglomerated at the population level. From the computed shear stress, it was possible to choose the minimal reactor thickness that would not hinder cell growth.

Effect of increasing oxygen partial pressure on Saccharomyces cerevisiae growth and antioxidant and enzyme productions.

This study investigated the impact of oxygen partial pressure on yeast growth. Saccharomyces cerevisiae cells were exposed to various hyperbaric air conditions from 1 bar to 9 bar absolute pressure (A). Batch cultures were grown under continuous airflow in a 750 mL (500 mL culture) bioreactor and monitored through growth rate and specific yields of ethanol and glycerol. In addition, the concentrations of antioxidant metabolites glutathione (reduced state, GSH and oxidized state, GSSG) and the activity of antioxidative enzymes superoxide dismutases (SOD) and catalases (CAT) were monitored. The results demonstrated that the different oxygen partial pressures significantly impacted the key growth parameters monitored. Compared with atmospheric pressure, under 2 to 5 bar (A), yeast cells showed higher growth rates (µ = 0.32 ± 0.01 h-1) and higher catalase (CAT) concentrations (214 ± 5 mU/g). GSH/GSSG ratio (6.36 ± 0.37) maintained until 6 bar (A) and total SOD (240 ± 5 mU/g) level significantly increased compared with 2 bar (A) until 7 bar (A). Under 6 to 9 bar (A), cell growth was inhibited, and a pressure of 9 bar (A) led to excessive GSSG accumulation (GSH/GSSG = 0.31 ± 0.06). The inhibition of t-SOD (160 ± 3 mU/g) and CAT (62.73 ± 0.2 mU/g) was observed under 9 bar (A). A reference experiment (8 bar (A) N2 + 1 bar (A) air) confirmed that the observed behaviors were entirely due to O2. In addition to their utility in biotechnological process design, these results showed that growth impairment was solely due to oxidative stress induced by excessive oxygen pressure. KEY POINTS: • Yeast cells were grown in batch mode under 1 to 9 bar (A) air pressures and up to 5 bar (A) promoted then hindered growth. • The GSH/GSSG ratio was stable up to 5 bar (A) then GSSG accumulated to excess. • Complementary investigations of the activity of SOD and CAT validated growth limitations due to oxidative stress.

In situ measurements of viscoelastic properties of biomass during hydrothermal treatment to assess the kinetics of chemical alterations.

This work aimed to use continuous measurements of viscoelastic properties to evaluate the effect of hydrothermal treatment on poplar samples. Different conditions (temperature and pre-soaking liquid: acidic, neutral and alkaline) were tested on wood in both tangential and radial directions. Two viscoelastic properties were determined: the modulus of elasticity and the stress relaxation. The applicability of these properties as indicators of the kinetics of biomass deconstruction was also evaluated, thanks to the chemical analyses performed on the treated solid and the recovered liquid phase. The ultimate goal is to build a macroscopic indicator capable of establishing rules to optimize the hydrothermal treatment before the explosion stage. The joint use of the two parameters succeeded in revealing the effects of chemical degradation, including the coexistence of cleavage and re-condensation and the impact of process conditions (temperature, residence time, and pre-soaking liquid). The monotonous behavior of stress relaxation is a major asset as a possible macroscopic indicator of biomass deconstruction.