Multi-information source Bayesian optimization of culture media for cellular agriculture.

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.

Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts.

BACKGROUND: Metabolomics 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. RESULTS: It 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. CONCLUSIONS: Overall, 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.

Considerations for the development of cost-effective cell culture media for cultivated meat production.

Innovation in cultivated meat development has been rapidly accelerating in recent years because it holds the potential to help attenuate issues facing production of dietary protein for a growing world population. There are technical obstacles still hindering large-scale commercialization of cultivated meat, of which many are related to the media that are used to culture the muscle, fat, and connective tissue cells. While animal cell culture media has been used and refined for roughly a century, it has not been specifically designed with the requirements of cultivated meat in mind. Perhaps the most common industrial use of animal cell culture is currently the production of therapeutic monoclonal antibodies, which sell for orders of magnitude more than meat. Successful production of cultivated meat requires media that is food grade with minimal cost, can regulate large-scale cell proliferation and differentiation, has acceptable sensory qualities, and is animal ingredient-free. Much insight into strategies for achieving media formulations with these qualities can be obtained from knowledge of conventional culture media applications and from the metabolic pathways involved in myogenesis and protein synthesis. In addition, application of principles used to optimize media for large-scale microbial fermentation processes producing lower value commodity chemicals and food ingredients can also be instructive. As such, the present review shall provide an overview of the current understanding of cell culture media as it relates to cultivated meat.

A combined phenolic extraction and fermentation reactor engineering model for multiphase red wine fermentation.

Red wine production begins with a simultaneous fermentation and solid-phase extraction process. Red wine color and mouthfeel is the result of the extraction of phenolics from grape skins and seeds during fermentation, where extraction is a strong function of temperature and ethanol concentration. During fermentation, grape solids form a porous "cap" at the top of the fermentor, resulting in a heterogeneous fermentation system with significant temperature and concentration gradients. In this work, we present a spatial, time-variant reactor engineering model for phenolic extraction during red wine fermentation, incorporating fermentation kinetics, mass transfer, heat transfer, compressible fluid flow, and phenolic extraction kinetics. The temperature and ethanol concentration profiles predicted by this model allow for the calculation of phenolic extraction rates over the course of fermentation. Phenolic extraction predictions were validated against prior experimental data to good agreement and compared to a well-mixed model's predictions to show the utility of a spatial model over well-mixed models.

Creation and validation of a reactor engineering model for multiphase red wine fermentations.

Red wine fermentations are performed in the presence of grape skins and seeds to ensure the extraction of color and other phenolics. The presence of these solids results in two distinct phases in the fermentor, as the solids float to the top to form a "cap." Modeling of red wine fermentation is, therefore, complex and must consider spatial heterogeneity to predict fermentation kinetics. We have developed a reactor-engineering model for red wine fermentations that includes the fundamentals of fermentation kinetics, heat transfer, diffusion, and compressible fluid flow. To develop the heat transfer component of the model, the heat transfer properties of grapes were experimentally determined as a function of fermentation progression. COMSOL was used to solve all components of the model simultaneously utilizing a finite element analysis approach. Predictions from this model were validated using prior experimental work. Model prediction and experimental data showed excellent agreement. The model was then used to predict spatial profiles of active yeast cell concentration and ethanol productivity, as well as liquid velocity profiles. Finally, the model was used to predict how these gradients would change with differences in initial bioavailable nitrogen concentration, a key parameter in predicting fermentation outcome in nitrogen-limited wine fermentations.

A Mechanistic Model for the Extraction of Phenolics from Grapes During Red Wine Fermentation.

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.

Impact of Temperature, Ethanol and Cell Wall Material Composition on Cell Wall-Anthocyanin Interactions.

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.

Impact of cold soak duration on Cabernet Sauvignon fermentation and phenolic composition.

BACKGROUND: Cold soak is a prefermentative maceration technique believed to enhance grape skin extraction. Studies show variable results depending on cold soak and winemaking conditions. To investigate the effect of cold soak more fully, systematic and highly reproducible Cabernet Sauvignon fermentations with increasing cold-soak durations were performed. RESULTS: Phenolic extraction during cold soak and fermentation showed significant differences among all treatments for monitored phenolics at the end of the cold soak. At the end of alcoholic fermentation only gallic acid, (-)-epicatechin, and the flavonols were significant, and only (-)-epicatechin was significant after bottle ageing. Descriptive analysis of the bottled wines showed that the 4- and 7-day treatments were significantly higher in caramelized/vanilla/browned flavor compared to the 1-day treatment and lower levels of bitterness were observed up to 2 days of cold soak. While oligosaccharide content increased with increasing cold-soak duration, differences were not large enough to result in sensory differences. CONCLUSION: While increased cold soak duration led to differences in phenolic extraction during early fermentation, these differences did not last through to the end product. Thus, under the conditions of this study, cold-soak duration had little overall impact on Cabernet Sauvignon wine composition and style. © 2018 Society of Chemical Industry.

Conversion of cassava leaf to bioavailable, high-protein yeast cell biomass.

BACKGROUND: Cassava leaves are an abundant global agricultural residue because the roots are a major source of dietary carbohydrates. Although cassava leaves are high in protein, the protein is not bioavailable. This work aimed to convert cassava leaves to a bioavailable protein-rich animal feed ingredient using high-protein yeasts. RESULTS: The structural proteins (ca 200 g kg-1 d.b.) from sundried cassava leaves were solubilized by mild alkali pretreatment, and the resulting cassava leaf hydrolysate (CLH) was used to screen for growth of 46 high-protein yeasts from 30 species. Promising candidates from the initial screen cultivated at a 10 mL scale demonstrated increases in relative abundance of essential amino acids over that of CLH. In particular, lysine, growth-limiting for some livestock, was increased up to 226% over the CLH content. One yeast, Pichia kudriavzevii UCDFST 11-602, was grown in 3 L of CLH in a bioreactor to examine the scale-up potential of the yeast protein production. While glucose was completely consumed, yeast growth exited log phase before depleting either carbon or nitrogen, suggesting other growth-limiting factors at the larger scale. CONCLUSIONS: High-value animal feed with enriched essential amino acid profiles can be produced by yeasts grown on agricultural residues. Yeasts convert structural protein solubilized from cassava leaves to essential amino acid-enriched, digestible protein. The low carbohydrate content of the leaves (ca 200 g kg-1 d.b.), however, necessitated glucose supplementation for yeast growth. © 2018 Society of Chemical Industry.

Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by Rhodotorula babjevae and Rhodotorula aff. paludigena.

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L-1 PEFA and produced 8.8 ± 1.0 g L-1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L-1 PEFA and 18.5 ± 1.7 g L-1 TG. The overall glucose conversion was 0.24 and 0.22 g(total lipid) g (glucose)-1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

UNLABELLED: Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-ß-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE: It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.

Investigating the Effect of Cold Soak Duration on Phenolic Extraction during Cabernet Sauvignon Fermentation.

The impact of increasing cold soak (CS) duration (0, 1, 4, 7, and 10 days at 10 °C) on the extraction of phenolic compounds during the CS period and primary fermentation as well as the final composition of Cabernet Sauvignon wine was investigated. The results showed that CS duration had no effect on hydroxycinnamate and flavonol extractions. Greater amounts of gallic acid, (+)-catechin, (-)-epicatechin, and total tannins were extracted with increasing CS duration, with differences maintained during bottle aging. Anthocyanin extraction and color density increased with longer periods of CS; however, by the end of primary fermentation, as well as three months' bottle aging, there were no significant differences due to CS duration. The wines made with seven and 10 days of CS had higher seed tannin contributions and total tannin compared to the non-CS wine, which could potentially result in increased astringency.

Examining the role of membrane lipid composition in determining the ethanol tolerance of Saccharomyces cerevisiae.

Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated.

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production.

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.

Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains.

Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R(2) = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R(2) = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.

Fermentation temperature modulates phosphatidylethanolamine and phosphatidylinositol levels in the cell membrane of Saccharomyces cerevisiae.

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at "normal" temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates.

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding ß-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant ß-galactosidases from B. infantis ATCC 15697 displayed different preferences for ß-galactosides such as 4' and 6'-galactobiose, and four ß-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host.

Multi-objective Bayesian algorithm automatically discovers low-cost high-growth serum-free media for cellular agriculture application.

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.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

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.

Spent media analysis suggests cultivated meat media will require species and cell type optimization.

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.