Biorefining Twin Transition: Digitalisation for Bio-based Chemicals/Materials - Discovery, Design and Optimisation.

The article discusses the production of platform chemicals from various biological sources, including glycerol, lignin, cellulose, bio-oils, and sea products. It presents the results of catalytic and downstream processes involved in the conversion of these biomass-derived feedstocks. The experimental approaches are complemented by numerical descriptions, ranging from density functional theory (DFT) calculations to kinetic modellingof the experimental data. This multi-scale modelling approach helps to understand the underlying mechanisms and optimize the production of platform chemicals from renewable resources.

Biodegradation of polysaccharide-based biocomposites with acetylated cellulose nanocrystals, alginate and chitosan in aqueous environment.

Biocomposite films from renewable sources are seen to be viable candidates as sustainable, zero-waste packaging materials. In this study, biocomposites films using chitosan and alginate as matrices, and pristine or acetylated cellulose nanocrystals (CNCs) as reinforcement agents, were fabricated, thoroughly characterized in terms of structure (with ATR-FTIR and XRD), morphology (SEM), thermal stability (TGA coupled with FTIR), water content and solubility and mechanical properties and subjected to controlled biological degradation in aqueous environment with added activated sludge. Biodegradation activity was followed through respirometry by measurement of change in partial O2 pressure using OxiTop® system. While the initial rate of biodegradation is higher in chitosan-based films with incorporated CNCs (both pristine and modified) compared to any other tested biocomposites, it was observed that chitosan-based films are not completely degradable in activated sludge medium, whereas alginate-based films reached complete biodegradation in 107 h to 112 h. Additional study of the aqueous medium with in situ FTIR during biodegradation offered an insight into biodegradation mechanisms. Use of advanced statistical methods indicated that selection of material (ALG vs CH) has the highest influence on biodegradability, followed by solubility of the material and its thermal stability.

Study of Chitosan-Stabilized Ti3C2Tx MXene for Ultrasensitive and Interference-Free Detection of Gaseous H2O2.

The development of sensitive, selective, and reliable gaseous hydrogen peroxide (H2O2) sensors operating at room temperature still represents a remaining challenge. In this work, we have investigated and combined the advantageous properties of a two-dimensional Ti3C2Tx MXene material that exhibits a large specific surface area and high surface activity, with favorable conducting and stabilizing properties of chitosan. The MXene-chitosan membrane was deposited on the ferrocyanide-modified screen-printed working carbon electrode, followed by applying poly(acrylic acid) as an electrolyte and accumulation medium for gaseous H2O2. The sensor showed highly sensitive and selective electroanalytical performance for detecting trace concentrations of gaseous H2O2 with a very low detection limit of 4 µg m-3 (4 ppbv), linear response in the studied concentration range of 0.5-30.0 mg m-3, and good reproducibility with an RSD of 1.3%. The applicability of the sensor was demonstrated by point-of-interest detection of gaseous H2O2 during the real hair bleaching process with a 9 and 12% H2O2 solution.

Effect of Environment on Acetylated Cellulose Nanocrystal-Reinforced Biopolymers Films.

Cellulose nanocrystals (CNCs) were acetylated to the various parametrised degrees of substitution (DS), determined through attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and incorporated into alginate (ALG) and chitosan (CH) film-forming solutions. An investigation of morphology with scanning electron microscopy (SEM) revealed increased chemical compatibility with the CH matrix after acetylation, producing a smooth surface layer, while ALG mixed better with pristine CNCs. The ATR-FTIR analysis of films demonstrated inter-diffusional structural changes upon the integration of pristine/modified CNCs. Films were evaluated in terms of water contact angle (WCA), which decreased upon CNC addition in either of the biocomposite types. The H2O barrier assessed through applicative vapour transmission (WVT) rate increased with the CNC esterification in CH, but was not influenced in ALG. To evaluate the relationship between environmental humidity and mechanical properties, conditioning was applied for 48 h under controlled relative humidity (33%, 54% and 75%) prior to the evaluation of the mechanical properties and moisture content. It was observed that tensile strength was highest upon specimens being dry (25 ± 3 MPa for ALG, reinforced with neat CNCs, or 16 ± 2 MPa in the CH with CNCs, reacting to the highest DS), lowering with dewing, and the elongation at break exhibited the opposite. It is worth noting that the modification of CNCs improved the best base benchmark stress-strain performance. Lastly, (thermal) stability was assessed by means of the thermogravimetric analysis (TGA) technique, suggesting a slight improvement.

Antioxidant and Antimicrobial Biofoil Based on Chitosan and Japanese Knotweed (Fallopia japonica, Houtt.) Rhizome Bark Extract.

A 70% ethanol(aq) extract of the rhizome bark of the invasive alien plant species Japanese knotweed (JKRB) with potent (in the range of vitamin C) and stable antioxidant activity was incorporated in 1% w/v into a chitosan biofoil, which was then characterized on a lab-scale. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay confirmed the antioxidant activity of the JKRB biofoil upon contact with the food simulants A, B, C, and D1 (measured half-maximal inhibitory concentrations-IC50) and supported the Folin-Ciocalteu assay result. The migration of the antioxidant marker, (-)-epicatechin, into all food simulants (A, B, C, D1, D2, and E) was quantified using liquid chromatography hyphenated to mass spectrometry (LC-MS). Calculations showed that 1 cm2 of JKRB biofoil provided antioxidant activity to ~0.5 L of liquid food upon 1 h of contact. The JKRB biofoil demonstrated antimicrobial activity against Gram-positive bacteria. The incorporation of JKRB into the chitosan biofoil resulted in improved tensile strength from 0.75 MPa to 1.81 MPa, while elongation decreased to 28%. JKRB biofoil's lower moisture content compared to chitosan biofoil was attributed to the formation of hydrogen bonds between chitosan biofoil and JKRB compounds, further confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The JKRB biofoil completely degraded in compost in 11 days. The future upscaled production of JKRB biofoil from biowastes for active packaging may support the fights against plastic waste, food waste, and the invasiveness of Japanese knotweed, while greatly contributing to the so-called 'zero-waste' strategy and the reduction in greenhouse gas emissions.

Functional Nanocellulose, Alginate and Chitosan Nanocomposites Designed as Active Film Packaging Materials.

The aim of the study was to characterize and compare films made of cellulose nanocrystals (CNC), nano-fibrils (CNF), and bacterial nanocellulose (BNC) in combination with chitosan and alginate in terms of applicability for potential food packaging applications. In total, 25 different formulations were made and evaluated, and seven biopolymer films with the best mechanical performance (tensile strength, strain)-alginate, alginate with 5% CNC, chitosan, chitosan with 3% CNC, BNC with and without glycerol, and CNF with glycerol-were selected and investigated regarding morphology (SEM), density, contact angle, surface energy, water absorption, and oxygen and water barrier properties. Studies revealed that polysaccharide-based films with added CNC are the most suitable for packaging purposes, and better dispersing of nanocellulose in chitosan than in alginate was observed. Results showed an increase in hydrophobicity (increase of contact angle and reduced moisture absorption) of chitosan and alginate films with the addition of CNC, and chitosan with 3% CNC had the highest contact angle, 108 ± 2, and 15% lower moisture absorption compared to pure chitosan. Overall, the ability of nanocellulose additives to preserve the structure and function of chitosan and alginate materials in a humid environment was convincingly demonstrated. Barrier properties were improved by combining the biopolymers, and water vapor transmission rate (WVTR) was reduced by 15-45% and oxygen permeability (OTR) up to 45% by adding nanocellulose compared to single biopolymer formulations. It was concluded that with a good oxygen barrier, a water barrier that is comparable to PLA, and good mechanical properties, biopolymer films would be a good alternative to conventional plastic packaging used for ready-to-eat foods with short storage time.

Chitin Deacetylation Using Deep Eutectic Solvents: Ab Initio-Supported Process Optimization.

Chitin is the most abundant marine biopolymer, being recovered during the shell biorefining of crustacean shell waste. In its native form, chitin displays a poor reactivity and solubility in most solvents due to its extensive hydrogen bonding. This can be overcome by deacetylation. However, this process requires a high concentration of acids or bases at high temperatures, forming large amounts of toxic waste. Herein, we report on the first deacetylation with deep eutectic solvents (DESs) as an environmentally friendly alternative, requiring only mild reaction conditions. Biocompatible DESs are efficient in disturbing the native hydrogen-bonding network of chitin, readily dissolving it. First, quantum chemical calculations have been performed to evaluate the feasibility of different DESs to perform chitin deacetylation by studying their mechanism. Comparing these with the calculated barriers for garden-variety alkaline/acidic hydrolysis, which are known to proceed, prospective DESs were identified with barriers around 25 kcal·mol-1 or lower. Based on density functional theory results, an experimental screening of 10 distinct DESs for chitin deacetylation followed. The most promising DESs were identified as K2CO3:glycerol (K2CO3:G), choline chloride:acetic acid ([Ch]Cl:AA), and choline chloride:malic acid ([Ch]Cl:MA) and were subjected to further optimization with respect to the water content, process duration, and temperature. Ultimately, [Ch]Cl:MA showed the best results, yielding a degree of deacetylation (DDA) of 40% after 24 h of reaction at 120 °C, which falls slightly behind the threshold value (50%) for chitin to be considered chitosan. Further quantum chemical calculations were performed to elucidate the mechanism. Upon the removal of 40% N-acetyl groups from the chitin structure, its reactivity was considerably improved.

Hydrophobic functionalization reactions of structured cellulose nanomaterials: Mechanisms, kinetics and in silico multi-scale models.

Nanoscale-interfaced cellulose nanomaterials are extracted from polysaccharides, which are widely available in nature, biocompatible and biodegradable. Moreover, the latter have a potential to be recycled, upcycled, and formulate therefore a great theoretical predisposition to be used in a number of applications. Nanocrystals, nano-fibrils and nanofibers possess reactive functional groups that enable hydrophobic surface modifications. Analysed literature data, concerning mechanisms, pathways and kinetics, was screened, compared and assessed with regard to the demand of a catalyst, different measurement conditions and added molecule reactions. There is presently only a scarce technique description for carbonOH bond functionalization, considering the elementary chemical steps, sequences and intermediates of these (non)catalytic transformations. The overview of the prevailing basic research together with in silico modelling approach methodology gives us a deeper physical understanding of processes. Finally, to further highlight the applicability of such raw materials, the review of the development in several multidisciplinary fields was presented.

Incorporating RNA-Seq transcriptomics into glycosylation-integrating metabolic network modelling kinetics: Multiomic Chinese hamster ovary (CHO) cell bioreactors.

In this work, the kinetic model based on the previously developed metabolic and glycan reaction networks of the ovarian cells of the Chinese hamster ovary (CHO) cell line was improved by the inclusion of transcriptomic data that took into account the values of the RPKM gene (Reads per Kilobase of Exon per Million Reads Mapped). The transcriptomic (RNASeq) data were obtained together with metabolic and glycan data from the literature, and the concentrations with RPKM values were collected at several points in time from two fed-batch processes. First, the fluxes were determined by regression analysis of the metabolic data, then these fluxes were corrected by using the fold change in gene expression as a measure of enzyme concentrations. Next, the corrected fluxes in the kinetic model were used to calculate the concentration profiles of the metabolites, and literature data were used to evaluate the predicted results of the model. Compared to other studies where the concentration profiles of CHO cell metabolites were described using a kinetic model without consideration of RNA-Seq data to correct the fluxes, this model is unique. The additional integration of transcriptomic data led to better predictions of metabolic concentrations in the fed-batch process, which is a significant improvement of the modelling technique used.

Dynamic multiscale metabolic network modeling of Chinese hamster ovary cell metabolism integrating N-linked glycosylation in industrial biopharmaceutical manufacturing.

Experimental and modeling work, described in this article, is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody protein production. CHO cells are one of the primary hosts for monoclonal antibodies production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and CO2 content, and dissolved oxygen. A total of 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional eight reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and one reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions, and membrane transport. With the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested.

Reduction in Spoilage Microbiota and Cyclopiazonic Acid Mycotoxin with Chestnut Extract Enriched Chitosan Packaging: Stability of Inoculated Gouda Cheese.

Active chitosan-based films, blended with fibrous chestnut (Castanea sativa Mill.) tannin-rich extract were used to pack Gouda cheese that has been contaminated with spoilage microflora Pseudomonas fluorescens, Escherichia coli, and fungi Penicillium commune. A comprehensive experimental plan including active chitosan-based films with (i) chestnut extract (CE), (ii) tannic acid (TA), and (iii) without additives was applied to evaluate the film's effect on induced microbiological spoilage reduction and chemical indices of commercial Gouda cheese during 37 days while stored at 4 °C and 25 °C, respectively. The cheese underwent microbiology analysis and chemical assessments of ultra-high-performance liquid chromatography (UHPLC) (cyclopiazonic acid), pH, and moisture content. The biopackaging used for packing cheese was characterized by mechanical properties before food packaging and analyzed with the same chemical analysis. The cheese microbiology showed that the bacterial counts were most efficiently decreased by the film without additives. However, active films with CE and TA were more effective as they did not break down around the cheese and showed protective properties against mycotoxin, moisture loss, and pH changes. Films themselves, when next to high-fat content food, changed their pH to less acidic, acted as absorbers, and degraded without plant-derived additives.

Chitin isolation from crustacean waste using a hybrid demineralization/DBD plasma process.

Conventional isolation of chitin from crustacean waste demands the use of high amounts of hazardous chemicals, hence not leading to a sustainable process. Atmospheric-pressure dielectric barrier discharge (DBD) plasma has demonstrated an enhanced ability to remove proteins directly from the biomass without the formation of any waste. Simultaneously, organic acids have proven very efficient in the removal of inorganic minerals from crustacean waste. Therefore, a hybrid process composed of DBD plasma and demineralization using organic acids has been successfully applied for the isolation of chitin. Results showed that the integration of nitrogen-based plasma and lactic acid demineralization allowed the elimination of 90 % of the proteins and ensures the complete removal of minerals from shrimp shells waste. The isolated chitin was further characterized using distinct techniques, namely XPS, ATR-FTIR, XRD and SEM. Chitin degree of deacetylation and molecular weight were also assessed. Hence, this work presents a sustainable and feasible platform for the extraction and purification of chitin from crustacean waste with almost zero waste formed.

Formulation of active food packaging by design: Linking composition of the film-forming solution to properties of the chitosan-based film by response surface methodology (RSM) modelling.

An active chitosan-based film, blended with the hydrolysable tannin-rich extract obtained from fibrous chestnut wood (Castanea sativa Mill.), underwent a simultaneous engineering optimization in terms of measured moisture content (MC), tensile strength (TS), elongation at break (EB), and total phenolic content (TPC). The optimal product formulation for a homogeneous film-forming solution was sought by designing an empirical Box-Behnken model simulation, based on three independent variables: the concentrations of chitosan (1.5-2.0% (w/v)), extracted powder-form chestnut extract (0.5-1.0% (w/v)) and plasticizer glycerol (30.0-90.0% (w/w); determined per mass of polysaccharide). Obtained linear (MC), quadratic (TS or EB), and two-factor interaction (TPC) sets were found to be significant (p < 0.05), to fit well with characteristic experimental data (0.969 < R2 < 0.992), and could be considered predictive. Although all system parameters were influential, the level of polyol played a vital continuous role in defining EB, MC, and TS, while the variation of the chestnut extract caused an expected connected change in affecting TPC. The component relationship formula of chemical mixture fractions (1.93% (w/v) of chitosan, 0.97% (w/v) chestnut extract and 30.0% (w/w) of glycerol) yielded the final applicable material of adequate physico-mechanical properties (MC = 17.0%, TS = 16.7 MPa, EB = 10.4%, and TPC = 19.4 mgGAE gfilm-1). Further statistical validation of the concept revealed a sufficient specific accuracy with the computed maximal absolute residual error up to 22.2%. Herein-proposed design methodology can thus be translated to smart packaging fabrication generally.

Enzymatic conversion reactions of 5-hydroxymethylfurfural (HMF) to bio-based 2,5-diformylfuran (DFF) and 2,5-furandicarboxylic acid (FDCA) with air: mechanisms, pathways and synthesis selectivity.

BACKGROUND: 2,5-Furandicarboxylic acid (FDCA) is one of the top biomass-derived value-added chemicals. It can be produced from fructose and other C6 sugars via formation of 5-hydroxymethilfurfural (HMF) intermediate. Most of the chemical methods for FDCA production require harsh conditions, thus as an environmentally friendly alternative, an enzymatic conversion process can be applied. RESULTS: Commercially available horseradish peroxidase (HRP) and lignin peroxidase (LPO), alcohol (AO) and galactose oxidase (GO), catalase (CAT) and laccase (LAC) were tested against HMF, 2,5-diformylfuran (DFF), 5-hydroxymethyl-2-furoic acid (HMFA) and 5-formyl-2-furoic acid (FFA). Enzyme concentrations were determined based on the number of available active sites and reactions performed at atmospheric oxygen pressure. AO, GO, HRP and LPO were active against HMF, where LPO and HRP produced 0.6 and 0.7% of HMFA, and GO and AO produced 25.5 and 5.1% DFF, respectively. Most of the enzymes had only mild (3.2% yield or less) or no activity against DFF, HMFA and FFA, with only AO having a slightly higher activity against FFA with an FDCA yield of 11.6%. An effect of substrate concentration was measured only for AO, where 20 mM HMF resulted in 19.5% DFF and 5 mM HMF in 39.9% DFF, with a K m value of 14 mM. Some multi-enzyme reactions were also tested and the combination of AO and CAT proved most effective in converting over 97% HMF to DFF in 72 h. CONCLUSIONS: Our study aimed at understanding the mechanism of conversion of bio-based HMF to FDCA by different selected enzymes. By understanding the reaction pathway, as well as substrate specificity and the effect of substrate concentration, we would be able to better optimize this process and obtain the best product yields in the future.

α-Chitin dissolution, N-deacetylation and valorization in deep eutectic solvents.

Chitin displays a highly rigid structure due to the vast intra- and intermolecular hydrogen bonding, thus hindering its dissolution and deacetylation using most solvents. Deep eutectic solvents (DESs) are special and environmentally friendly solvents composed of a hydrogen bond acceptor and a hydrogen bond donor. This allows them to dissolve chitin by disturbing its natural hydrogen bonding while establishing new bonds, hence turning the polymer more susceptible to solvents. Therefore, four distinct DESs (choline chloride-lactic acid ([Ch]Cl:LA), choline chloride:oxalic acid ([Ch]Cl:OA), choline chloride:urea ([Ch]Cl:U) and betaine-glycerol (Bet:G)) were applied in chitin dissolution, being the most performant ones further applied in its homogenous N-deacetylation with NaOH. In this work, a milder and more biocompatible approach was carried out by using 30 wt% NaOH at 80°C, instead of the typical ≥40 wt% NaOH at temperatures ≥100°C. Herein, the reaction process took up to 18 hours, being the results analyzed through ATR-FTIR. Chitin was converted into chitosan with a 70-80% degree of deacetylation (DDA) in a short period while using homogenous conditions. These promising results provide the first proof of concept of the ability of Bet:G and [Ch]Cl:LA-based DESs to be used as a greener approach for the chitin homogeneous N-deacetylation.

Chitosan-based films with incorporated supercritical CO2 hop extract: Structural, physicochemical, and antibacterial properties.

Chitosan-based films with incorporated supercritical CO2 hop extract (HE) were developed and evaluated regarding structural, physicochemical, and antibacterial properties. The morphological and spectroscopic analyses have confirmed successful incorporation of HE into the polymer matrix, which affected films' structure and visual appearance. The presence of HE has caused a reduction in the hydrophilic character of films, but also provided a complete UV light blockage at wavelengths below 350 nm. Furthermore, a declining trend of tensile strength (from 14.4 MPa to 6.4 MPa) and Young's modulus (from 218.8 MPa to 26.9 MPa), as well as an ascending trend of elongation at break (from 10.7% to 35.1%), have been observed after the extract incorporation. The total phenolic content in the films was up to ∼13 mgGAE gfilm-1. Besides, the HE-loaded films exhibited antibacterial activity against foodborne pathogen Bacillus subtilis.

Feature Article: Metabolic Network Modelling of Chinese Hamster Ovary (CHO) Culture Bioreactors Operated as Microbial Cell Factories.

Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower organism viability, protein productivity and manufactured biosimilar, so a careful optimal balance of medium, bioreactor operational parameters and bioprocess is needed. A precise phenomenological knowledge of metabolism's chemical transformations can predict problems that may arise during batch, semi-continuous fed batch and continuous reactor operation. For a better detailed understanding (and relations), future performance optimization and scaling, mechanistic model systems have been built. In this specific work, the main metabolic pathways in mammalian structured CHO cultures are reviewed. It starts with organic biochemical background, controlling associated phenomena and kinetics, which govern the sustaining conversion routes of biology. Then, individual turnover paths are described, overviewing standard mathematical formulations that are commonly applied in engineering. These are the core of black box modeling, which relates the substrates/products in a simplified relationship manner. Moreover, metabolic flux analysis (MFA)/flux balance analysis (FBA), that are traditionally characterizing mechanisms, are presented to a larger portion extent. Finally, similarities are discussed, illustrating the approaches for their structural design. Stated variables' equations, employed for the description of the growth in the controllable environmental conditions of a vessel, the researched reaction series of proliferating dividing CHO population, joint with the values of maximal enzymatic activity, and solutions are outlined. Processes are listed in a way so that a reader can integrate the state-of-the-art. Our particular contribution is also denoted.

Microfluidic droplet-based liquid-liquid extraction: online model validation.

Droplet-based liquid-liquid extraction in a microchannel was studied, both theoretically and experimentally. A full 3D mathematical model, incorporating convection and diffusion in all spatial directions along with the velocity profile, was developed to depict the governing transport characteristics of droplet-based microfluidics. The finite elements method, as the most common macroscale simulation technique, was used to solve the set of differential equations regarding conservation of moment, mass and solute concentration in a two-domain system coupled by interfacial surface of droplet-based flow pattern. The model was numerically verified and validated online by following the concentrations of a solute in two phases within the microchannel. The relative azobenzene concentration profiles in a methanol/n-octane two-phase system at different positions along the channel length were retrieved by means of a thermal lens microscopic (TLM) technique coupled to a microfluidic system, which gave results of high spatial and temporal resolution. Very good agreement between model calculations and online experimental data was achieved without applying any fitting procedure to the model parameters.