Cultivated meat (CM) scaffolds must be edible and produced with high throughput. This study introduces a novel approach for structured CM production using alginate-based scaffolds prepared using a liquid-liquid extrusion method called Pressurized Apparatus for Striated Tissue Assembly (PASTA). We hypothesized that PASTA scaffolds could enable the creation of tissue constructs exceeding 500 µm thickness with food-scale throughput. Leveraging sub-boiling hydrothermal degradation, we modulated the biophysical properties of alginate, resulting in controlled reductions in viscosity, zeta potential, and hydrogel mechanical properties. PASTA extrusion consistently produced hydrogel fibers 10–42% smaller than the extrusion outlet diameter. Murine C2C12 myoblasts cultured on PASTA scaffolds modified with adhesive peptides adhered, proliferated, and differentiated along the noodle axis. This was confirmed by a 3.5-fold increase in DNA quantity over 4 days, substantial cell viability after 5 days in culture, late-stage myogenic gene expression (Myog, Myh2), and positive immunohistochemistry for myosin heavy chain. PASTA enables rapid, parallelizable production of aligned fiber scaffolds with physiologically relevant dimensions using inexpensive, food-grade alginate. These findings demonstrate a proof-of-concept scalable and modular platform to produce cell-laden tissues for manufacturing structured CM.

Cultivated meat has the potential to mitigate many detrimental effects of conventional meat production on land use and greenhouse gas emissions. However, to meet an increasing demand for sustainable protein sources and to achieve cost parity with conventionally grown meat, animal cells will likely be required to be produced in bioreactors at an order of magnitude larger scale than has been done so far. To help de-risk this scale up, simulations of plausible bioreactor configurations were performed at a series of scales ranging from 200 L to 200,000 L using computational fluid dynamics. Several different bubble drag models were compared, and the one that predicted the lowest mass transfer and highest shear was used in order to be conservative about the predicted cell environment. The distributions of shear stress, oxygen mass transfer coefficient, and Kolmogorov length scale were compared across the bioreactor scales, showing only minor changes with increasing scale. The case of a Rushton and pitched impeller was compared to a case with two Rushton impellers, and the latter was found to have higher mass transfer and only slightly higher shear for a given power input. This study provides a step towards predicting animal cell culture performance at the scales needed for sustainable 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.

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.

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.

A head trained Vitis vinifera L. cv. Zinfandel vine was grafted on St. George rootstock (V. rupestris) then planted in a 1.1 m3 plastic container filled with Yolo County, CA sourced sandy loam.To estimate evapotranspiration, we measured the wind speed, air temperature and relative humidity in vine canopies by mounting each vine with a suite of research grade sensors. We measured wind speed (units m ᐧ s-1) inside the vine canopy using a single needle anemometer (East 30 Sensors; Pullman, WA) that took instantaneous wind speed measurements every 10 seconds and recorded the average of the previous 12 instantaneous measurements for every 2-minute interval. We measured temperature (units oC) and relative humidity (units %) using HMP60L sensors (Campbell Scientific; Logan, UT) mounted both inside and outside of each vine canopy and recorded instantaneous measurements at each 2-minute interval. We filtered all biometeorological data using a 3-hour moving average to remove noise without causing any significant over or under-approximation of daily maxima and minima. We automated all data collection using two CR1000 data loggers (Campbell Scientific; Logan, UT), with 1 or 2 vines and associated sensors per logger, using custom CR1 programs. A single 30W solar cell and 12V lead acid battery powered the entire vine-sensor system. This dataset represents all sensor data from a single vine, as measured in August 2020. Columns are named accordingly and include units. Please Note: The column named 'load_cell_kg' is not named accurately. The values given are in units of millivolts, and need to be translated from millivolts to kilograms. The 2020 calibration coefficient is 0.00330693663 millivolts per kilogram.

Developing low-cost technology for custom water delivery to individual or small groups of plants is a critical next step to advance precision irrigation. Current systems for estimating evapotranspiration (ET), or plant water use, work on the scale of a full vineyard (e.g., 3–5 acres) or the scale of a single vine, but at a cost that prohibits monitoring past a small number of representative vines. To develop and evaluate low-cost ET sensors for individual grapevines, we used three head-pruned Zinfandel vines in pots and placed them on load cells to collect continuous weights indicative of actual ET. We mounted research-grade sensors for humidity, temperature, and wind speed on each vine and saved data at 2-minute intervals during three growing seasons. We developed three models based on first principles (Convective Mass Transfer or Mass Balance approaches) or simple correlations to predict actual single-plant ET from these data. We present here the results of a multi-year trial at the UC-Davis RMI vineyard to illustrate the performance of each of the models for ET estimation. Relative model performance was assessed by comparing model predictions to ground truth data provided by measurements from load cells–including assessments of estimated instantaneous ET rate, estimated cumulative water use over a one-hour window surrounding solar noon, and estimated cumulative water use over a full 24-hour period. The three algorithms developed consistently performed well, with single vine ET rate predictions showing a strong linear relationship with ground truth (range in r2 over three seasons CMT r2 = 0.61–0.86; MB r2 = 0.07–0.91; EM r2 = 0.57–0.92). The MB approach, which includes two measurements of relative humidity and temperature, was the most variable, likely due to the impact of sensor placement. In all seasons, we also examined the trend in the plant scaling factor found in each model, deemed As, which, based on model theory, is a function of vine size. Taken together, these results suggest that high-resolution irrigation (HRI) models are a promising new method for ET estimation at the single plant level.

Background and goals During wine fermentation, grape berry components, including seeds, undergo extensive physical and chemical changes that result in the release of flavonoids, such as tannins, from seeds into wine. Understanding changes in seed morphology during fermentation is crucial for aiding the development of accurate prediction models for flavonoid extraction during winemaking, which enhances fermentation management and ensures consistency in wines from year to year. Methods and key findings High-resolution x-ray microcomputed to-mography (x-ray mu CT) was used to investigate the effect of red wine fermentation on changes in grape seed morphology. Using a PyTorch-based implementation of a fully con-volutional network with a Resnet-101 back-bone for semantic segmentation of x-ray mu CT images, we quantified extensive alteration to grape seed structure during fermentation. Image analyses revealed the development of a pore network breaking apart the seed endosperm by the end of fermentation, leading to an increase in surface area. Conclusions and significance Fermentation significantly altered grape seed morphology. Such alterations could enable transport of seed flavonoids from inside the endosperm and integument to outside the seed. Further research on the physical processes occurring in seeds during wine fermentation is necessary to build better physiochemical models.

Successful commercialization of cultivated meat products as alternative sources of dietary protein for a growing global population will require development of efficient and inexpensive serum-free cell culture media. It is known that serum-free media elicit dissimilar cell growth behavior compared to serum-based media, but data comprehensively exploring the effects of serum-free culture on the nutrient requirements of cultivated meat-relevant cell types have yet to be reported. We performed spent media analysis of C2C12 cells growing in Essential 8 serum-free media and in conventional serum-containing media. Data indicated that although the cell growth rates were similar in the two media over seven days, there were significant differences in the utilization rates of some key nutrients such as glucose, glutamine, glycine, and cystine. We extrapolated from the data that roughly 250–275 g of amino acids and 1100–1500 g of glucose would be required to produce 1 kg of C2C12 cells, though these requirements may change when using more optimized cells, media, and feed strategies. Our results highlight the influence of serum and serum replacements on cell metabolism, and indicate that previous data from metabolic studies performed using serum-containing media may not directly translate to serum-free systems.

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, water consumption, and farm animal welfare. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell culture and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg of cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modeled to create a comprehensive analysis to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; ~42,000 L stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $35/kg, ~211,000 L STR with a COGS of $25/kg, and ~262,000 L airlift reactor (ALR) with a COGS of $17/kg. This study outlines how advances in scaled up bioreactors, alternative bioreactor designs, and decreased media costs are necessary for commercialization of cultured meat products.

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.

Biotechnology and BioengineeringVolume 120, Issue 4 p. 861-864 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 120, Number 4, April 2023 First published: 14 March 2023 https://doi.org/10.1002/bit.28137AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume120, Issue4April 2023Pages 861-864 RelatedInformation

The composition of skin cell wall material from Pinot noir and Cabernet Sauvignon grape berries from four different regions in California was investigated to determine relationships between cell wall composition and phenolic extractability observed under winemaking conditions. Multiple vineyards (sites) per region were studied. Cell wall composition analysis included determining total soluble sugars , proteins , noncellulosic glucose , cellulose , lignin , lipids , total polyphenolic content , soluble polysaccharides , and uronic acid , as well as isolation efficiency. Results indicated that cell wall material (CWM) composition is mainly site-specific , with some effect due to variety and little to no effect from the growing region. Grape phenolics analyzed included monomeric flavan-3-ols , antho-cyanins , polymeric phenols , and polymeric pigments and could be used to distinguish between varieties. Grapes grown in the same region exhibited similar phenolic extractability , indicating a significant effect of the growing area , as well as a synergistic effect between CWM composition and grape phenolics. CWM composition analysis indicated that demethylation of pectin favored the release of phenolics , while lignin content correlated negatively with the phenolics extracted. Proteins showed a negative correlation with polymeric phenols , whereas no correlation was found with polymeric pigments. Anthocyanin extractability was highly affected by its content within grape skins, more so than other phenolics that are present in both the skins and seeds.

White wine fermentations are typically performed in an entirely batchwise manner, with yeast nutrients only added at the beginning of fermentation. This leads to slow (2+ weeks) fermentation cycle times, with large capital expenditures required to increase winery processing capacity. Prior attempts to speed fermentations via increasing temperature have resulted in unpalatable wine, and continuous fermentation processing is uneconomical and impractical in the winery setting. In this work, we measured yeast nutrient consumption as a function of fermentation progression at the 300 mL scale, and from this derived an equation to optimize yeast nutrient concentration as a function of fermentation progression. These findings were applied at the pilot scale in 150 L fermentors, which resulted in a 60% cycle time reduction versus “best practices” control fermentations. The resultant wines were compared via GC-MS as well as by a trained sensory panel. Organoleptic analysis found statistically significant, but overall, small differences in sensory characteristics between the control and process intensified wines. This intensified fermentation process shows great promise for fermented beverage producers wishing to maximize equipment utilization and debottleneck wineries or other beverage fermentation facilities.

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.

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.

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.

In this study, phenolic extractability of Cabernet Sauvignon grapes from two California regions (Sonoma County and Central Coast) and its relation with skin cell wall composition was investigated. Phenolic grape composition, wine phenolic content as well as berry and pomace cell wall composition of three sites per region were determined. Grape cell wall material (CWM) composition, and thus pomace CWM composition, was impacted by the growing region. The process of fermentation modified CWM composition, solubilizing some of the compounds such as pectin and polysaccharides making pomace CWM composition from different sites more similar in the case of Sonoma County and more different for the samples grown in the Central Coast. Growing region had a significant impact on grape phenolics, particularly on flavan−3-ols and polymeric phenols, whereas polymeric pigments and anthocyanin contents were more similar among samples. Wines made from Sonoma County grapes showed higher anthocyanin and polymeric phenol content when compared to wines made from Central Coast grapes. Comparing wine to grape phenolic composition suggests a large difference in extractability based on region. Of all the CWM components analyzed, only lignin and the amount of cell wall isolated were found to have a significant impact on phenolic extractability.

Experimental optimization of physical and biological processes is a difficult task. To address this, sequential surrogate models combined with search algorithms have been employed to solve nonlinear high-dimensional design problems with expensive objective function evaluations. In this article, a hybrid surrogate framework was built to learn the optimal parameters of a diverse set of simulated design problems meant to represent real-world physical and biological processes in both dimensionality and nonlinearity. The framework uses a hybrid radial basis function/genetic algorithm with dynamic coordinate search response, utilizing the strengths of both algorithms. The new hybrid method performs at least as well as its constituent algorithms in 19 of 20 high-dimensional test functions, making it a very practical surrogate framework for a wide variety of optimization design problems. Experiments also show that the hybrid framework can be improved even more when optimizing processes with simulated noise.