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Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

In this work, we applied a multi-information source modeling technique to solve a multi-objective Bayesian optimization problem involving the simultaneous minimization of cost and maximization of growth for serum-free C2C12 cells using a hyper-volume improvement acquisition function. In sequential batches of custom media experiments designed using our Bayesian criteria, collected using multiple assays targeting different cellular growth dynamics, the algorithm learned to identify the trade-off relationship between long-term growth and cost. We were able to identify several media with >100% more growth of C2C12 cells than the control, as well as a medium with 23% more growth at only 62.5% of the cost of the control. These algorithmically generated media also maintained growth far past the study period, indicating the modeling approach approximates the cell growth well from an extremely limited data set.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Culture media used in industrial bioprocessing and the emerging field of cellular agriculture is difficult to optimize due to the lack of rigorous mathematical models of cell growth and culture conditions, as well as the complexity of the design space. Rapid growth assays are inaccurate yet convenient, while robust measures of cell number can be time-consuming to the point of limiting experimentation. In this study, we optimized a cell culture media with 14 components using a multi-information source Bayesian optimization algorithm that locates optimal media conditions based on an iterative refinement of an uncertainty-weighted desirability function. As a model system, we utilized murine C2C12 cells, using AlamarBlue, LIVE stain, and trypan blue exclusion cell counting assays to determine cell number. Using this experimental optimization algorithm, we were able to design media with 181% more cells than a common commercial variant with a similar economic cost, while doing so in 38% fewer experiments than an efficient design-of-experiments method. The optimal medium generalized well to long-term growth up to four passages of C2C12 cells, indicating the multi-information source assay improved measurement robustness relative to rapid growth assays alone.

Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop.

BackgroundMetabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains.ResultsIt was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production.ConclusionsOverall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.

Genetic background and environmental conditions affect the production of sensory impact compounds by Saccharomyces cerevisiae. The relative importance of the strain-specific metabolic capabilities for the production of volatile organic compounds (VOCs) remains unclear. We investigated which amino acids contribute to VOC production and whether amino acid-VOC relations are conserved among yeast strains. Amino acid consumption and production of VOCs during grape juice fermentation was investigated using four commercial wine yeast strains: Elixir, Opale, R2, and Uvaferm. Principal component analysis of the VOC data demonstrated that Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. Biomass formation was similar for all strains, pointing to metabolic differences in the utilization of nutrients to form VOCs. Partial least-squares linear regression showed that total aroma production is a function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters. Mechanistic modeling of the yeast metabolic network via parsimonious flux balance analysis and flux enrichment analysis revealed enzymes with crucial roles, such as transaminases and decarboxylases. Our work provides insights in VOC production in wine yeasts. IMPORTANCE Saccharomyces cerevisiae is widely used in grape juice fermentation to produce wines. Along with the genetic background, the nitrogen in the environment in which S. cerevisiae grows impacts its regulation of metabolism. Also, commercial S. cerevisiae strains exhibit immense diversity in their formation of aromas, and a desirable aroma bouquet is an essential characteristic for wines. Since nitrogen affects aroma formation in wines, it is essential to know the extent of this connection and how it leads to strain-dependent aroma profiles in wines. We evaluated the differences in the production of key aroma compounds among four commercial wine strains. Moreover, we analyzed the role of nitrogen utilization on the formation of various aroma compounds. This work illustrates the unique aroma-producing differences among industrial yeast strains and suggests more intricate, nitrogen-associated routes influencing those aroma-producing differences.

Language policy and planning (LPP) has always drawn on research and scholarship in education as well as the social sciences in general (in particular sociology). Social theory has also figured as an important source of ideas and concepts, and critical LPP has arisen as a distinct strand of inquiry since the 1980s (Tollefson, in Planning language, planning inequality: language policy in the community. Longman, London, 1991; Tollefson (ed) Language policies in education: critical issues. Routledge, London, 2013). More recently, critical LPP researchers have begun to turn to political economy, as a source discipline, and neoliberalism, as a baseline concept, in the study of LPP-related phenomena and practices in a range of contexts (Ricento 2015; Tollefson & Pérez-Milans 2018). This paper examines how a critical political economy-oriented approach may be applied in a specific context, that of Catalonia, where most would agree that there has been a relatively successful recovery of a minority language situated in a larger nation-state structure traditionally dominated by a monolingual (Spanish) polity. This critical approach explores, on the one hand, how political economy--which examines the power relations that mutually constitute the production, distribution, and consumption of resources and the class relations that emerge within these processes--may be brought to bear on issues arising in the ongoing development of LPP in Catalonia. In addition, this critical approach is attentive to issues around race and ethnicity which inevitably arise in societies experiencing high levels of immigration, as has been the case in Catalonia over the past 25 years. This paper aims to bring these two strands together, examining how two key matters of interest in political economy today--inequality and class--intersect with race and ethnicity in the ongoing development of language policy in Catalonia, focussing specifically on the Barcelona metropolitan area. And further to this, it aims to understand how this intersectionality is, at the same time, intersected by the nexus of a Catalan national, cultural and linguistic identity emerging from the aforementioned relatively successful recovery of Catalan over the past several decades.

This paper explores how three Science, Technology, Engineering and Mathematics (STEM) lecturers working in English-medium instruction (EMI) grapple with the prospect of self-positioning as English-language-teachers (ELTs), drawing on interviews in which they explicitly deny acting in this way. It begins with essential background, first discussing key concepts such as EMI, internationalization, Englishization in higher education and 'CLIL-ised EMI', the latter understood as what happens when EMI is reframed as sharing key characteristic with Content and Language Integrated Learning (CLIL) -- language teaching. The paper then outlines the main focus -- the notion that STEM specialist EMI lecturers might, on occasion, act as ELTs -- examining selected findings from previous research exploring this topic. This discussion is followed by further background information about the context and the methodological framework adopted here, a revised version of Positioning Theory. These preliminaries aside, the paper presents a series of excerpts from interviews with informants, which then serve to construct a narrative about EMI lecturers as ELTs. In the face of informants' resistance to this identity, the paper ends with some thoughts on what has been learned, both in this context and further afield.

The quantitative and qualitative impacts of anthocyanins on proanthocyanidin adsorption to grape-derived cell wall material were investigated in fifteen unique systems of varying temperatures, ethanol concentrations, and proanthocyanidin concentrations. Proanthocyanidin solutions were exposed to cell wall material and monitored for changes in concentration over 24 h. Increases in both temperature and ethanol resulted in a larger retention of proanthocyanidins in solution and typically faster adsorption kinetics. Analysis of the solution after exposure to cell wall revealed a significant reduction in the molecular weight of proanthocyanidins present in solution, suggesting that anthocyanins do not alter a previously described mechanism of preferentially binding large molecular weight molecules. Additionally, a reduction in polymeric pigment abundance was noted in most conditions, suggesting rapid formation of polymeric pigment in the model solution and preferential adsorption of the polymeric pigment to cell wall material. Compared to a previous study of proanthocyanidin adsorption in the absence of anthocyanins, a significantly larger percentage of proanthocyanidin material was lost via adsorption-up to 70% of available material. In a winemaking context, this may suggest a preferential loss of polymeric pigment via adsorption to cap cell wall material compared to non-pigmented proanthocyanidins and free anthocyanins.

Establishment of the [GAR +] prion in Saccharomyces cerevisiae reduces both transcriptional expression of the HXT3 hexose transporter gene and fermentation capacity in high sugar conditions. We evaluated the impact of deletion of the HXT3 gene on the expression of [GAR +] prion phenotype in a vineyard isolate, UCD932, and found that changes in fermentation capacity were observable even with complete loss of the Hxt3 transporter, suggesting other cellular functions affecting fermentation rate may be impacted in [GAR +] strains. In a comparison of isogenic [GAR +] and [gar -] strains, localization of the Pma1 plasma membrane ATPase showed differences in distribution within the membrane. In addition, plasma membrane lipid composition varied between the two cell types. Oxygen uptake was decreased in prion induced cells suggesting membrane changes affect plasma membrane functionality beyond glucose transport. Thus, multiple cell surface properties are altered upon induction of the [GAR +] prion in addition to changes in expression of the HXT3 gene. We propose a model wherein [GAR +] prion establishment within a yeast population is associated with modulation of plasma membrane functionality, fermentation capacity, niche dominance, and cell physiology to facilitate growth and mitigate cytotoxicity under certain environmental conditions. Down-regulation of expression of the HXT3 hexose transporter gene is only one component of a suite of physiological differences. Our data show the [GAR +] prion state is accompanied by multiple changes in the yeast cell surface that prioritize population survivability over maximizing metabolic capacity and enable progeny to establish an alternative adaptive state while maintaining reversibility.

Desorption of proanthocyanidins (PA) from grape cell wall material (CWM) was investigated in solutions of varying ethanol concentrations and increasing temperature. The results reveal the reversibility of PA-CWM interactions and the role that temperature and ethanol concentration play in the extent of PA desorption. Sequentially raising temperature from 15 to 35 °C resulted in desorption of up to 48% of the initial adsorbed PA. A comparison to a phenolic extraction model showed significant differences between the predicted and actual amount of PA that desorbed from the CWM. This suggests that the initial conditions of temperature and ethanol concentration must be considered when estimating PA extraction in red wine production. Under typical winemaking conditions, a significant amount of PA may be irreversibly adsorbed if exposed to CWM at low temperature (i.e., cold soak). A compositional analysis suggests the selective desorption of large molecular weight PA from CWM under all experimental conditions. Additionally, a preferential desorption of skin-derived PA over seed-derived PA was noted in the absence of ethanol.

This paper examines the emergent identities of three STEM (science, technology, engineering and mathematics) lecturers, focusing especially on how they construct themselves with regard to their disciplines and how researchers construct them as potential English language teachers. It begins with essential background information before discussing identity as a key construct. The paper then moves to the presentation of a Foucauldian-inflected version of positioning theory which is employed as a means of understanding the construction of English-medium instruction (EMI) STEM lecturer identities. In the second half of the paper, there is a discussion of three case-studies of EMI STEM lecturers, which shows how each orientates to his or her subject discipline, and the prospect of acting, on occasion, as an English language teacher. Specifically, there is a detailed consideration of how EMI lecturers position themselves in their contacts with researchers and in their teaching; how their self-positionings are consistent or not with how researchers position them; and how self-positionings are similar or different across EMI lecturers. The paper ends with a discussion of what conclusions can be drawn from the research findings.

In the shift to English-medium instruction (EMI) in European higher education, policy often runs ahead of research and curricular decisions are taken independent of evidence regarding their suitability for achieving broader educational goals, which may range from internationalisation as a general strategy to English language learning as a more specific one. Where English language learning is a goal, EMI may be CLIL"ised," that is, it is adopted not only for content delivery, but also as a means through which students might improve their English. Drawing on interviews and classroom observations, and employing a Membership Categorisation Analysis methodology, this paper examines how a lecturer in agronomic engineering at a Catalan university experiences CLIL"ised" EMI. It documents how the lecturer positions himself as a content lecturer and how categories are produced and negotiated in interviews and classroom practices. Among other things, the paper shows that while the lecturer refuses to inhabit an English-language teacher identity, he nonetheless acts in this capacity when he focusses on vocabulary learning through the provision of glossaries and translation. These and other findings point to a more general disjuncture between policy and practice, which, it is argued, needs to be addressed by university administrators and EMI lecturers.

The effects of temperature and ethanol concentration on the kinetics of anthocyanin adsorption and desorption interactions with five cell wall materials (CWM) of different composition were investigated. Using temperatures of 15 °C and 30 °C and model wine with ethanol concentrations of 0% and 15% (v/v) over 120 min, the adsorption and desorption rates of five anthocyanin-glucosides were recorded in triplicate. Small-scale experiments were conducted using a benchtop incubator to mimic a single berry fermentation. Results indicate that more than 90% of the adsorption occurs within the first 60 min of the addition of anthocyanins to CWM. However, desorption appears to occur much faster, with maximum desorption being reached after 30 min. The extent of both adsorption and desorption was clearly dependent not only on temperature and ethanol concentration but also on the CWM composition.

Phenolic extraction is a critical part of red wine making. Though empirical models of phenolic extraction kinetics exist, the current level of mechanistic understanding does not allow for accurate predictions. In this work, we propose a mechanistic model for the extraction of phenolics from grape skins and seeds as a function of temperature and ethanol. This model examines the release of phenolics, the adsorption of phenolics onto grape material, and the disappearance of anthocyanins from solution. Additionally, we performed epifluorescence microscopy to explore our finding that seed tannins' release rate appears independent of concentration, and found that the grape seed appears to ablate over fermentation. We also determined the activation energy of anthocyanin disappearance, in good agreement with similar systems. The proposed model results in an excellent fit, and increases the understanding of phenolic extraction and the ability to predict and optimize product outcome in red wine making.

More than half of global water use can be attributed to crop irrigation, and as the human population grows, so will the water requirements of agriculture. Improved irrigation will be critical to mitigating the impact of increased requirements. An ideal irrigation system is informed by measurements of water demand—a combination of water use and water status signals—and delivers water to plants based on this demand. In this review, examples of methods for monitoring water status are reviewed, along with details on stem and trunk water potential measurements. Then, methods for monitoring evapotranspiration (ET), or water use, are described. These methods are broken into coarse- and fine-scale categories, with a 10 m spatial resolution threshold between them. Fourteen crop ET technologies are presented, including examples of a successful estimation of ET in research and field settings, as well as limitations. The focus then shifts to water distribution technologies, with an emphasis on the challenges associated with the development of systems that achieve dynamic single plant resolution. Some attention is given to the process of choosing ET and water status sensing methods as well as water delivery system design given site characteristics and agronomic goals. This review concludes with a short discussion on the future directions of ET research and the importance of translating findings into useful tools for growers. [ABSTRACT FROM AUTHOR]

In recent years, a discourse of Spanish exceptionalism has arisen, whereby Spain has managed to integrate over five million immigrants in a very short period of time without the kind of social upheaval witnessed in other European countries over the past several decades. Overtly racist and xenophobic discourses have generally been, with notable exceptions, absent in the public domain. However, under the radar, visible minorities frequently suffer 'racial micro-aggressions', that is, racially-loaded verbal and other behaviours that are seemingly innocuous but are uptaken as offensive and insulting by "the micro-aggressed." More specifically, these racial micro-aggressions are often raciolinguistic in nature, that is, they reflect the confluence of language and race in the unequal positioning of individuals in interactions. In this article, we focus on how young people from immigrant backgrounds tell 'school stories', embedded in normative Catalan/Spanish bilingualism in Catalonia, which are raciolinguistic in nature and relate to racial micro-aggressions experienced by tellers. We do this with a view to showing how all is not as well in Spain as many might think as regards the integration of immigrants, and that greater attention to racism is in order.