Bioprocess Research Articles

Microbial Surfactants to the Rescue

Microbial Surfactants to the Rescue

Potential Impact of Battery Electric Vehicle Penetration and Changes in Upstream Process Emissions Assuming Night‐Charging on Summer O3 Concentrations in Japan

AbstractA regional meteorology–chemistry model was used to evaluate the impact of battery electric vehicles (BEV) penetration on summer O3 concentrations in Kanto (Japan's most populous region). When all passenger cars shifted to BEVs, daytime ozone (O3) concentrations decreased over a wide area. The reduction of vehicle exhaust reduced O3 in inland suburban areas (NOx‐limited) and the reduction of gasoline fuel evaporation from vehicles and gas stations reduced O3 in urban areas (volatile organic compound (VOC)‐limited). The maximum location of maximum daily 8‐hr average O3 (MDA8hO3) sensitivity (−5%; −5 ppb) was at midway between urban and suburban areas, to which the reduction of exhaust and fuel evaporation contributed equally, respectively. The additional emissions from thermal power plants due to BEV's night‐charging could cause up to ±5% sensitivity to next‐day surface O3 concentrations topically. Depending on the O3 sensitivity regime (NOx‐ or VOC‐limited), additional NOx‐rich plumes from rural (urban) power plants tended to increase (decrease) the next day's O3. However, the spatial distribution of the regime varies temporally depending on such as NOx/VOC emission ratios and meteorological conditions. This study indicated that the distribution of negative (positive) O3 sensitivity by NOx‐rich plume is consistent with that of small (large) H2O2/HNO3 concentration ratios, indicating that H2O2/HNO3 works well as an indicator to discriminate O3 sensitivity regimes. Utilization of the H2O2/HNO3 indicator would enable regime distribution predictions without sensitivity simulations with varying NOx and VOC emissions, which would contribute to reducing computational costs.

High-Temperature Co-Electrolysis and Degradation by Oxidized Trace Contaminants in Feed Gases

For transforming our energy system, the indirect electrification of the sectors heating, mobility and chemical industry seems to be partially necessary. The high-temperature co-electrolysis for producing synthesis gas is very well suited as upstream process for power-to-fuel and power-to-chemicals processes. Besides the intrinsic degradation processes of the cells (e.g. nickel migration) and stacks (e.g. interconnect oxidation) any additional processes triggered by impurities in the feed gases are of relevance for any technical applications. The following manuscript summarizes the investigations performed to elucidate the impact of oxidized trace contaminants in feed gases, in particular of sulfur dioxide and silicon species.

Life cycle assessment of reciprocating engine component in Finland: a case study

ABSTRACT This paper presents the environmental impacts of reciprocating engine component (REC) made of cast iron and low-alloy steel in Finland using life cycle assessment (LCA). The study uses primary data provided by a machining shop in Finland covering cradle-to-gate with a functional unit of 1 tonne of REC. The assessment was carried out using CML-baseline, resulting in eleven impact categories emphasising global warming potential (GWP). The results show that the upstream process, including forging and foundry, to manufacture the material inputs contributes the highest among all impact categories, making up about 52–97% of the total impacts. For GWP, the total emission to manufacture 1 tonne REC is about 2926 kg CO2 eq. Sensitivity analysis indicates that material inputs of cast iron and low-alloy steel are the most critical parameters making process efficiency and technological advance prominent. This study highlights the importance of selecting suppliers, energy sources, and transportation modes.

Integrative processing of untargeted metabolomic and lipidomic data using MultiABLER

Mass spectrometry (MS)-based untargeted metabolomic and lipidomic approaches are being used increasingly in biomedical research. The adoption and integration of these data are critical to the overall multi-omic toolkit. Recently, a sample extraction method called Multi-ABLE has been developed, which enables concurrent generation of proteomic and untargeted metabolomic and lipidomic data from a small amount of tissue. The proteomics field has a well-established set of software for processing of acquired data; however, there is a lack ofa unified, off-the-shelf, ready-to-use bioinformatics pipeline that can take advantage of and prepare concurrently generated metabolomic and lipidomic data forjoint downstream analyses. Here we present an R pipeline called MultiABLER as a unified and simple upstream processing and analysis pipeline for both metabolomics and lipidomics datasets acquired using liquid chromatography-tandem massspectrometry. The code is available via an open-source license at https://github.com/holab-hku/MultiABLER.

LCA Sensitivity Analysis of an Energy-Biochar Chain from an Italian Gasification Plant: Environmental Trade-offs Assessment

Due to its potential applications in bioenergy production, coproducts (bio-oil and syngas), mitigation of global warming, sustainable agriculture, pollutant removal, and other uses, biochar has drawn interest from all over the world. Producing and using soil-based biochar as a method of carbon sequestration could help reduce emissions while benefiting the soil and opening up possibilities for bioenergy production. However, to characterize the production cycle’s environmental and energy loads and confirm all of the advantages of biochar, Life Cycle Assessment (LCA) represents a reliable tool for evaluation. This work is based on continuing the study of Marzeddu and Cappelli (Marzeddu, Cappelli, et al., 2021) to understand the environmental impact of an energy-biochar chain involving a gasification plant in Italy. In the LCA carried out in the previous paper for the characterization of biochar, which is used as a soil conditioner, soil carbon sequestration, nitrous oxide emissions, fertilizer use, and water use for irrigation were considered. The results showed that the use of gasification for energy and biochar is an attractive strategy for mitigating the environmental impact analysis, especially climate change, with a net decrease of about ‒8.3·103 kg CO2, eq. The previous study was lacking a sensitivity analysis. For this reason, a sensitivity analysis is proposed in this study to consistently assess the environmental trade-offs of the biochar and the amended soil. In specific for the upstream processes the sensitivity is addressed to the selection of a different type of woodchips, for the core process in terms of selection of different packing material, and to the entire cradle-to-grave perspective by improving the logistics of the transportation, the distances within the supply chain and the choice of BAT technology for the transportation vehicles. This study highlights strategic research developments that combine to find potential environmental trade-offs and thresholds towards using biochar and its final use as a soil conditioner.

Production of bioethanol from pretreated rice straw, an integrated and mediated upstream fermentation process

Production of bioethanol from pretreated rice straw, an integrated and mediated upstream fermentation process

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

Using the Product Social Impact Life Cycle Assessment (PSILCA) database for product comparison: Confetti case study.

Social Life Cycle Assessment (S-LCA) is a methodology that can help companies administer their supply chain and promote both social responsibility and sustainability through the assessment of both positive and negative social and socio-economic aspects of products during their entire life cycle. Here, a case study was performed to test an S-LCA database as a comparative assessment tool of two products, i.e. the traditional almond sugared confetti and Tenerelli sugared almonds, made by the same company. The S-LCA case study was carried out by using the Product Social Impact Life Cycle Assessment (PSILCA) database, for the two products on openLCA software. The risk assessment helped to identify the most affected impact categories and which product influences the most on them as well as the categories on which the foreground processes have the greatest impact. For both the analysed products, the category "Contribution of the sector to economic development" is marginally affected. Overall, the production of Confetti entails higher medium risk hours regarding some categories (e.g. "Public sector corruption", "Promoting social responsibility", "Migration flows"), whilst for others, it is the Tenerelli that affect the most (e.g. "Biomass consumption", "Trade unionism"), depending on the different inputs used. Finally, the results are not very influenced by foreground processes, and the social risks are therefore related to upstream processes for both products. The comparison can become more useful for products of the same or similar functions and the same audience but of different inputs and therefore different supply chains. Indeed, it could be useful for decision-making with the aim of selecting between various possible options the one that entails lower social risks.

A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca PLX image cytometer

Abstract Cellular therapies such as chimeric antigen receptor (CAR) T or NK cells undergo phenotypic characterization during discovery and development of novel therapies, biomanufacturing, and upstream patient cell processing for clinical use. Samples are routinely analyzed via flow cytometry for T cell, NK cell, B cell, and monocyte surface marker expression. Image cytometry systems perform cell-based assays that can be used as a convenient alternative to flow cytometry. Recently, a new high-throughput image cytometer, the Cellaca PLX system (Nexcelom, Lawrence, MA) was developed for immunophenotyping, transfection/transduction efficiency, and cell health assays. This new instrument can assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization that focuses on cellular therapeutic products. Here we demonstrate a rapid and high-throughput immunophenotyping and viability detection in peripheral blood mononuclear cells (PBMCs) using the Cellaca PLX. PBMCs underwent red blood cell (RBC) lysis and CD3 enrichment, were stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead surface marker kits and measured on the Cellaca PLX and three different flow cytometers for comparison. Results showed highly comparable cell populations between the flow cytometers and the Cellaca PLX suggesting that the image cytometer may provide a rapid and convenient alternative method for immunophenotyping. The proposed method can streamline workflows from sample staining to data analysis, quickly moving precious patient samples into downstream processes.

Cardiomyocyte electrophysiology and its modulation: current views and future prospects.

Normal and abnormal cardiac rhythms are of key physiological and clinical interest. This introductory article begins from Sylvio Weidmann's key historic 1950s microelectrode measurements of cardiac electrophysiological activity and Singh & Vaughan Williams's classification of cardiotropic targets. It then proceeds to introduce the insights into cardiomyocyte function and its regulation that subsequently emerged and their therapeutic implications. We recapitulate the resulting view that surface membrane electrophysiological events underlying cardiac excitation and its initiation, conduction and recovery constitute the final common path for the cellular mechanisms that impinge upon this normal or abnormal cardiac electrophysiological activity. We then consider progress in the more recently characterized successive regulatory hierarchies involving Ca2+ homeostasis, excitation-contraction coupling and autonomic G-protein signalling and their often reciprocal interactions with the surface membrane events, and their circadian rhythms. Then follow accounts of longer-term upstream modulation processes involving altered channel expression, cardiomyocyte energetics and hypertrophic and fibrotic cardiac remodelling. Consideration of these developments introduces each of the articles in this Phil. Trans. B theme issue. The findings contained in these articles translate naturally into recent classifications of cardiac electrophysiological targets and drug actions, thereby encouraging future iterations of experimental cardiac electrophysiological discovery, and testing directed towards clinical management. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Ultra-high throughput microfluidic concentrator for harvesting of Tetraselmis sp. (Chlorodendrophyceae, Chlorophyta)

Microalgae are of commercial interest for their ability to produce high-value compounds and their potential to be used as feedstock across numerous industries. The production of algal biomass and other algal-based products involves different upstream and downstream processing steps, including cultivation, harvesting, and extraction. In particular, the harvesting process involves the extraction of a condensed algal slurry from a watery growth medium. This process accounts for 20 to 30 % of the biomass production costs, creating technological and economic barriers. The conventional harvesting methods suffer from high costs, cross-contamination, and low yield. This study explores the use of a microfluidic technique, in particular, the rigid spiral microchannel, for ultra-high-throughput algae harvesting. We have designed a novel spiral channel that enables microalgae separation at high flow rates and spatial resolution, taking advantage of inertial microfluidic principles. The results from trials with surrogate microparticles and algal cells reveal that the microchannel has the potential to operate at 12 mL/min with separation efficiency >99 %. Our inertial microfluidic device operates at high flow rates as a single channel, can be multiplexed, and shows excellent potential for large-scale processing of microalgae cultures. In addition, the lower number of loops provides the system with reduced back pressure, making it more desirable for operation using a range of different pumps. Since the overall cost of microalgae dewatering is substantial at the industrial scale, the design and fabrication of low-cost devices are of great importance, similar to the one we have developed in this study.

Natural Carotenoids: Recent Advances on Separation from Microbial Biomass and Methods of Analysis.

Biotechnologically produced carotenoids occupy an important place in the scientific research. Owing to their role as natural pigments and their high antioxidant properties, microbial carotenoids have been proposed as alternatives to their synthetic counterparts. To this end, many studies are focusing on their efficient and sustainable production from renewable substrates. Besides the development of an efficient upstream process, their separation and purification as well as their analysis from the microbial biomass confers another important aspect. Currently, the use of organic solvents constitutes the main extraction process; however, environmental concerns along with potential toxicity towards human health necessitate the employment of "greener" techniques. Hence, many research groups are focusing on applying emerging technologies such as ultrasounds, microwaves, ionic liquids or eutectic solvents for the separation of carotenoids from microbial cells. This review aims to summarize the progress on both the biotechnological production of carotenoids and the methods for their effective extraction. In the framework of circular economy and sustainability, the focus is given on green recovery methods targeting high-value applications such as novel functional foods and pharmaceuticals. Finally, methods for carotenoids identification and quantification are also discussed in order to create a roadmap for successful carotenoids analysis.

Microfluidic systems as a novel approach for microalgal bioprocess

Challenges such as controlling parameters for microalgal bioprocess optimization, increased need for inoculum and chemicals with the increasing number of experiments, long procedures and high production costs make it difficult to commercialize high value-added bioactive compounds from microalgae. Furthermore, downstream processes comprise the majority of the total production costs and increase the amount of energy consumed, depending on the desired purity percentage and the biochemical features of the material. So, it is important to maximize the effectiveness, quality, and economic factors of both upstream and downstream processes. Due to their micro-scale size, diluted cultures and controlled operational parameters, reduced sample and material utilization, increased mass and heat transfer, better species interaction, and high-throughput cell/reagent processing capabilities, microfluidic devices are becoming increasingly used in microalgal bioprocess studies for both fundamental research and industrial applications. Additionally, microfluidic devices outperform conventional techniques in their capacity to carry out a lot of functions, including the separation and more sensitive analysis of small amounts of samples. In this review, diverse microfluidic platforms for microalgal cultivation, purification and separation processes, sorting and high-throughput screening of microalgae, and emerging studies of microfluidic technologies in various microalgal processes are discussed.

Human Metabolome Reference Database in a Biracial Cohort across the Adult Lifespan.

As one of the OMICS in systems biology, metabolomics defines the metabolome and simultaneously quantifies numerous metabolites that are final or intermediate products and effectors of upstream biological processes. Metabolomics provides accurate information that helps determine the physiological steady state and biochemical changes during the aging process. To date, reference values of metabolites across the adult lifespan, especially among ethnicity groups, are lacking. The "normal" reference values according to age, sex, and race allow the characterization of whether an individual or a group deviates metabolically from normal aging, encompass a fundamental element in any study aimed at understanding mechanisms at the interface between aging and diseases. In this study, we established a metabolomics reference database from 20-100 years of age from a biracial sample of community-dwelling healthy men and women and examined metabolite associations with age, sex, and race. Reference values from well-selected healthy individuals can contribute to clinical decision-making processes of metabolic or related diseases.

Process economics evaluation and optimization of adeno-associated virus downstream processing.

Adeno-associated virus (AAV) manufacturing has traditionally focused upon lab-scale techniques to culture and purify vector products, leading to limitations in production capacity. The tool presented in this paper assesses the feasibility of using non-scalable technologies at high AAV demands and identifies optimal flowsheets at large-scale that meet both cost and purity targets. The decisional tool comprises (a) a detailed process economics model with the relevant mass balance, sizing, and costing equations for AAV upstream and downstream technologies, (b) a built-in Monte Carlo simulation to assess uncertainties, and (c) a brute-force optimization algorithm for rapid investigation into the optimal purification combinations. The results overall highlighted that switching to more scalable upstream and downstream processing alternatives is economically advantageous. The base case analysis showed the cost and robustness advantages of utilizing suspension cell culture over adherent, as well as a fully chromatographic purification platform over batch ultracentrifugation. Expanding the set of purification options available gave insights into the optimal combination to satisfy both cost and purity targets. As the purity target increased, the optimal polishing solution moved from the non-capsid purifying multimodal chromatography to anion-exchange chromatography or continuous ultracentrifugation.

Photolysis of chloramines in vacuum-UV and vacuum-UV/chlorine advanced oxidation processes for removal of 1,4-dioxane: Effect of water matrix, kinetic modeling, and implications for potable reuse

Advanced oxidation processes (AOPs) are a key step in eliminating persistent micropollutants in potable reuse trains. Under such conditions, chloramines are an inevitable component in the AOP feed water given their application as an antifouling agent for the upstream membrane processes. In cases when other oxidants, such as free chlorine, are to be used in the AOP treatment, the effect of background chloramines and any potential interplays between the oxidants should be considered. In this study, vacuum-UV (VUV) and VUV/Cl2 have been proposed as promising AOP alternatives for potable reuse and the effect of chloramine photolysis has been considered on the removal of 1,4-dioxane. Results indicated that while presence of chloramine reduces the treatment efficiency in the VUV AOP, coexistence of free chlorine and chloramine oxidants significantly improves 1,4-dioxane degradation rates. Experimental data and kinetic modeling both confirmed the roles of OH• and Cl2•- in 1,4-dioxane removal with 62.5% and 32.5% contribution in the VUV/Cl2/chloramines, respectively. Among the other water matrix conditions, Cl- was shown to improve the degradation rates while HCO3- suppressed the reactions by scavenging radical species. Overall, the findings of this research are informative for the design and development of VUV AOPs at small scale potable reuse facilities.

Identification of genetic variants that impact gene co-expression relationships using large-scale single-cell data

BackgroundExpression quantitative trait loci (eQTL) studies show how genetic variants affect downstream gene expression. Single-cell data allows reconstruction of personalized co-expression networks and therefore the identification of SNPs altering co-expression patterns (co-expression QTLs, co-eQTLs) and the affected upstream regulatory processes using a limited number of individuals.ResultsWe conduct a co-eQTL meta-analysis across four scRNA-seq peripheral blood mononuclear cell datasets using a novel filtering strategy followed by a permutation-based multiple testing approach. Before the analysis, we evaluate the co-expression patterns required for co-eQTL identification using different external resources. We identify a robust set of cell-type-specific co-eQTLs for 72 independent SNPs affecting 946 gene pairs. These co-eQTLs are replicated in a large bulk cohort and provide novel insights into how disease-associated variants alter regulatory networks. One co-eQTL SNP, rs1131017, that is associated with several autoimmune diseases, affects the co-expression of RPS26 with other ribosomal genes. Interestingly, specifically in T cells, the SNP additionally affects co-expression of RPS26 and a group of genes associated with T cell activation and autoimmune disease. Among these genes, we identify enrichment for targets of five T-cell-activation-related transcription factors whose binding sites harbor rs1131017. This reveals a previously overlooked process and pinpoints potential regulators that could explain the association of rs1131017 with autoimmune diseases.ConclusionOur co-eQTL results highlight the importance of studying context-specific gene regulation to understand the biological implications of genetic variation. With the expected growth of sc-eQTL datasets, our strategy and technical guidelines will facilitate future co-eQTL identification, further elucidating unknown disease mechanisms.

Application of recycled crushed glass in road pavements and pipeline bedding: An integrated environmental evaluation using LCA

The study aims to conduct a comprehensive life cycle assessment (LCA) of mixed glass waste (MGW) recycling processes to quantify the environmental impacts of crushed glass as a partial substitute for virgin aggregate. Upstream washing, crushing, and sorting conducted at material recycling facilities (MRF) are the prime activities to assess whether reprocessed MGW in pavement construction is an alternate feasible solution. None of the previous studies explicitly account for the relative uncertainties and optimization of waste glass upstream processes from an environmental perspective. The study calculates environmental impacts using the LCA tool SimaPro considering design factors attributed to transportation, electricity consumption, use of chemicals, and water for reprocessing glass waste. Relative uncertainties of design variables and the national transition policy (2021−2030) from non-renewable to renewable energy sources have been validated by performing detailed Monte Carlo simulations. The correlation coefficients (r = 0.64, 0.58, and 0.49) of successive variables explain how the higher environmental gains of the glass recycling process are outweighed by diesel, energy consumption, and transportation distances. Compared to natural quarry sand, the recycled glass aggregate produced through crushing and recycling of its by-products reduces CO2eq emissions by 16.2 % and 46.7 %, respectively. The need for a washing line at the plant, in addition to crushing, results in a higher environmental impact over natural sand by 90.1 % and emphasizes the benefits of collecting waste glass through a separate bin, hence avoiding contamination. The result indicates that the benefit of lowering emissions varies significantly when considering waste glass landfilling. Moreover, this study evaluates the potential impacts on asphalt and reinforced concrete pavements (RCP) with 5 %, 10 %, 15 %, and 20 % replacement of natural sand with recycled glass aggregate. The LCA emphasizes the limitations of energy-intensive waste glass reprocessing. The obtained results and uncertainty analysis based on primary MRF data and recycled product applications provide meaningful suggestions for a more fit-for-purpose waste management and natural resource conservation.

Development of an Integrated Continuous Manufacturing Process for the rVSV-Vectored SARS-CoV-2 Candidate Vaccine.

The administration of viral vectored vaccines remains one of the most effective ways to respond to the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. However, pre-existing immunity to the viral vector hinders its potency, resulting in a limited choice of viral vectors. Moreover, the basic batch mode of manufacturing vectored vaccines does not allow one to cost-effectively meet the global demand for billions of doses per year. To date, the exposure of humans to VSV infection has been limited. Therefore, a recombinant vesicular stomatitis virus (rVSV), which expresses the spike protein of SARS-CoV-2, was selected as the vector. To determine the operating upstream process conditions for the most effective production of an rVSV-SARS-CoV-2 candidate vaccine, a set of critical process parameters was evaluated in an Ambr 250 modular system, whereas in the downstream process, a streamlined process that included DNase treatment, clarification, and a membrane-based anion exchange chromatography was developed. The design of the experiment was performed with the aim to obtain the optimal conditions for the chromatography step. Additionally, a continuous mode manufacturing process integrating upstream and downstream steps was evaluated. rVSV-SARS-CoV-2 was continuously harvested from the perfusion bioreactor and purified by membrane chromatography in three columns that were operated sequentially under a counter-current mode. Compared with the batch mode, the continuous mode of operation had a 2.55-fold increase in space-time yield and a reduction in the processing time by half. The integrated continuous manufacturing process provides a reference for the efficient production of other viral vectored vaccines.