Sensor technologies for quality control in engineered tissue manufacturing.

The use of engineered cells, tissues, and organs has the opportunity to change the way injuries and diseases are treated. Commercialization of these groundbreaking technologies has been limited in part by the complex and costly nature of their manufacture. Process-related variability and even small changes in the manufacturing process of a living product will impact its quality. Without real-time integrated detection, the magnitude and mechanism of that impact are largely unknown. Real-time and non-destructive sensor technologies are key for in-process insight and ensuring a consistent product throughout commercial scale-up and/or scale-out. The application of a measurement technology into a manufacturing process requires cell and tissue developers to understand the best way to apply a sensor to their process, and for sensor manufacturers to understand the design requirements and end-user needs. Furthermore, sensors to monitor component cells' health and phenotype need to be compatible with novel integrated and automated manufacturing equipment. This review summarizes commercially relevant sensor technologies that can detect meaningful quality attributes during the manufacturing of regenerative medicine products, the gaps within each technology, and sensor considerations for manufacturing.

Strategies to achieve high productivity, high conversion, and high yield in yeast fermentation of algal biomass hydrolysate.

The conversion of carbohydrates in biomass via fermentation is an important component of an overall strategy to decarbonize the production of fuels and chemicals. Owing to the cost and resources required to produce biomass hydrolysates, the economic and environmental sustainability of these fermentation processes requires that they operate with high yields, sugar conversion, and productivity. Immobilized-cell technology in a continuous bioprocess can achieve significantly higher volumetric productivities than is possible from standard batch fermentation using free cells. Here, we demonstrate approaches for improvement of ethanol yield from algal hydrolysates and a mock hydrolysate medium. Saccharomyces cerevisiae was immobilized in alginate and incorporated into a two-column immobilized cell reactor system. Furthermore, the yeast quorum-sensing molecule, 2-phenylethanol, was added to improve ethanol yield by restricting growth and diverting sugar to ethanol. The bioreactor system could achieve high ethanol volumetric productivity (>20 g/Lreactor·h) and high glucose conversion (>99%) in mock hydrolysate, while the addition of 0.2% 2-phenylethanol resulted in 4.9% higher ethanol yield. With an algal hydrolysate of <10 g/L sugar, the ethanol volumetric productivity reached 9.8 g/Lreactor·h, and the addition of 0.2% 2-phenylethanol increased the ethanol yield by up to 7.4%. These results demonstrate the feasibility of novel strategies to achieve sustainability goals in biomass conversions.

Quorum-sensing molecules increase ethanol yield from Saccharomyces cerevisiae.

One strategy to increase the yield of desired fermentation products is to redirect substrate carbon from biomass synthesis. Nongenetic approaches to alter metabolism may have advantages of general applicability and simple control. The goal of this study was to identify and evaluate chemicals for their ability to inhibit the growth of Saccharomyces cerevisiae while allowing ethanol production with higher yields. Eight potential growth-inhibitory chemicals were screened for their ability to reduce cell growth in 24-well plates. Effective chemicals were then evaluated in cultivations to identify those that simultaneously reduced biomass yield and increased ethanol yield. The yeast quorum-sensing molecules 2-phenylethanol, tryptophol and tyrosol were found to increase the ethanol yield of S. cerevisiae JAY 270. These molecules were tested with seven other yeast strains and ethanol yields of up to 15% higher were observed. The effects of 2-phenylethanol and tryptophol were also studied in bioreactor fermentations. These findings demonstrate for the first time that the ethanol yield can be improved by adding yeast quorum-sensing molecules to reduce the cell growth of S. cerevisiae, suggesting a strategy to improve the yield of ethanol and other yeast fermentation products by manipulating native biological control systems.

Practical monitoring technologies for cells and substrates in biomanufacturing.

Precise control over bioreactor operation is desired for optimal productivity and product quality, and there is an increased drive to automation in biomanufacturing. All of these goals require sensors, not only of the basic parameters of temperature, pH, and dissolved oxygen, but of the biomass and substrate concentrations, which directly determine the outcome of the bioprocess. While there are many innovative sensing concepts for biomass and substrate concentrations, this review focuses on sensors that are in-line with the bioreactor, providing data continuously without the removal of sample from the system. The discussion emphasizes the requirements of industry for these sensors, including performance, ease of use, and cost. As the bioeconomy grows, advances in sensing technologies will be needed to achieve the automation of the future for a wider array of bioreactors.

Inoculum microbiome composition impacts fatty acid product profile from cellulosic feedstock.

Conversion of organic wastes to fatty acids rather than methane through anaerobic digestion-based technologies has considerable promise. However, the relationships between microbiome structure and fatty acids produced from cellulosic feedstocks are not well understood. This study investigated the nature of those relationships for anaerobic digester sludge, bison rumen, and cattle rumen inocula grown on cellulose. Acetic acid production was highest in anaerobic sludge reactors, while propionic acid production was highest in cattle rumen reactors. Butyric and pentanoic acid were produced at the highest rates in bison rumen before Day 5. Reactor microbiomes remained distinct, despite identical operating conditions. Novel associations linked Alistipes with butyric acid production and Eubacterium nodatum and Clostridiales bacterium with pentanoic acid production. This study provides new insights into the ability of microbiomes to convert cellulose to different fatty acid mixtures and adds impetus for the rewiring of anaerobic digestion to generate high-value products.

Electromagnetically-vibrated solid-phase microextraction for analysis of aqueous-miscible organic compound transport in soil columns.

Current methods of sampling pore water from soil columns to determine solute concentrations are slow and require relatively large volumes. Accordingly, an electromagnetically-vibrated (EMV) solid-phase microextraction (SPME) device was evaluated for determining temporal and spatial distributions of solute pore-water concentrations (solute concentration profiles) for four organic compounds, two polar (2-hexanone, 2,4-dimethyl phenol) and two nonpolar (toluene, 1,4-dichlorobenzene), in columns packed with simulated aquifer sands with different fractions of organic carbon. In batch equilibrium extraction tests, the equilibrium extraction time of the organic compounds in aqueous mixtures decreased from 30 to less than 10 min as the frequency of electromagnetic vibration increased from zero to 250 Hz. Mixture effects were not statistically significant (p > 0.05) in the extraction process using EMV SPME. Comparisons of the solute concentration profiles within the soil columns at different elapsed times measured by pore-water samples and in situ EMV SPME extractions revealed both methods were equally effective. However, EMV SPME extraction removed no solution volume and only 0.6-14% of the solute mass removed by the pore-water sample collections, substantially minimizing disturbances to solute transport and fate. Thus, the equilibrium extraction-based calibration method using EMV SPME offers an effective approach for rapidly and accurately determining solute concentration profiles in column tests with negligible solute mass loss and minimal solution flow disturbance.

Digitalization and Bioprocessing: Promises and Challenges.

The production of pharmaceuticals, industrial chemicals, and food ingredients from biotechnological processes is a vast and rapidly growing industry. While advances in synthetic biology and metabolic engineering have made it possible to produce thousands of new molecules from cells, few of these molecules have reached the market. The traditional methods of strain and bioprocess development that transform laboratory results to industrial processes are slow and use computers and networks only for data acquisition and storage. Digitalization, machine learning (ML), and artificial intelligence (AI) methods are transforming many fields - how can they be applied to bioprocessing to overcome current bottlenecks? What are the challenges, especially for regulatory issues, in the production of biopharmaceuticals? This chapter begins with a discussion of the current challenges for strain and bioprocess development and then considers how digitalization can be used to approach these tasks in completely new ways. Finally, regulatory considerations are addressed, with the goal of incorporating these issues from the outset as new digitalization methods are created.

Meta-proteomic analysis of protein expression distinctive to electricity-generating biofilm communities in air-cathode microbial fuel cells.

BACKGROUND: Bioelectrochemical systems (BESs) harness electrons from microbial respiration to generate power or chemical products from a variety of organic feedstocks, including lignocellulosic biomass, fermentation byproducts, and wastewater sludge. In some BESs, such as microbial fuel cells (MFCs), bacteria living in a biofilm use the anode as an electron acceptor for electrons harvested from organic materials such as lignocellulosic biomass or waste byproducts, generating energy that may be used by humans. Many BES applications use bacterial biofilm communities, but no studies have investigated protein expression by the anode biofilm community as a whole. RESULTS: To discover functional protein expression during current generation that may be useful for MFC optimization, a label-free meta-proteomics approach was used to compare protein expression in acetate-fed anode biofilms before and after the onset of robust electricity generation. Meta-proteomic comparisons were integrated with 16S rRNA gene-based community analysis at four developmental stages. The community composition shifted from dominance by aerobic Gammaproteobacteria (90.9 ± 3.3%) during initial biofilm formation to dominance by Deltaproteobacteria, particularly Geobacter (68.7 ± 3.6%) in mature, electricity-generating anodes. Community diversity in the intermediate stage, just after robust current generation began, was double that at the early stage and nearly double that of mature anode communities. Maximum current densities at the intermediate stage, however, were relatively similar (~ 83%) to those achieved by mature-stage biofilms. Meta-proteomic analysis, correlated with population changes, revealed significant enrichment of categories specific to membrane and transport functions among proteins from electricity-producing biofilms. Proteins detected only in electricity-producing biofilms were associated with gluconeogenesis, the glyoxylate cycle, and fatty acid ß-oxidation, as well as with denitrification and competitive inhibition. CONCLUSIONS: The results demonstrate that it is possible for an MFC microbial community to generate robust current densities while exhibiting high taxonomic diversity. Moreover, these data provide evidence to suggest that startup growth of air-cathode MFCs under conditions that promote the establishment of aerobic-anaerobic syntrophy may decrease startup times. This study represents the first investigation into protein expression of a complex BES anode biofilm community as a whole. The findings contribute to understanding of the molecular mechanisms at work during BES startup and suggest options for improvement of BES generation of bioelectricity from renewable biomass.

Bacterial community changes in an industrial algae production system.

While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

Grain and sweet sorghum (Sorghum bicolor L. Moench) serves as a novel source of bioactive compounds for human health.

Grain sorghum is an important staple food crop grown globally while sweet sorghum is increasingly considered as a promising biofuel feedstock. Biofuels are the major economic products from the processing of large quantities of biomass, which is currently being utilized to make value-added products in the biorefinery approach. To date, these value-added products are typically commodity chemicals and waste materials used in agriculture. However, there are opportunities to generate high-value bioactive compounds from sorghum grain and biomass. Chronic diseases, such as cancers, are the top causes for morbidity and mortality in developed nations and are promoted by inflammation and oxidative stress. Globally, colorectal cancer results in approximately one-half million deaths annually. It is estimated that as much as 80% of colorectal cancer cases can be attributed to environmental and dietary factors. The sorghum grain and ligno-cellulosic biomass generated for biofuel production has been reported to be high in bioactive compounds, including phenolic acids and flavonoids, with antioxidant and anti-inflammatory properties. This review focuses on the bioactive compounds of grain and sweet sorghum (Sorghum bicolor L. Moench), for their anti-inflammatory, antioxidant, anti-colon cancer, and immune modulator functions. The review summarizes previous efforts to identify and quantify bioactive compounds in sorghum and documents their anti-cancer biological activities. Finally, this review discusses bioactive compound extraction methodologies and technologies as well as considerations for incorporating these technologies into current biorefining practices.

Sensors for disposable bioreactors.

Modern bioprocess monitoring demands sensors that provide on-line information about the process state. In particular, sensors for monitoring bioprocesses carried out in single-use bioreactors are needed because disposable systems are becoming increasingly important for biotechnological applications. Requirements for the sensors used in these single-use bioreactors are different than those used in classical reusable bioreactors. For example, long lifetime or resistance to steam and cleaning procedures are less crucial factors, while a requirement of sensors for disposable bioreactors is a cost that is reasonable on a per-use basis. Here, we present an overview of current and emerging sensors for single-use bioreactors, organized by the type of interface of the sensor systems to the bioreactor. A major focus is on non-invasive, in-situ sensors that are based on electromagnetic, semiconducting, optical, or ultrasonic measurements. In addition, new technologies like radio-frequency identification sensors or free-floating sensor spheres are presented. Notably, at this time there is no standard interface between single-use bioreactors and the sensors discussed here. In the future, manufacturers should address this shortcoming to promote single-use bioprocess monitoring and control.

Spectroscopic sensors for in-line bioprocess monitoring in research and pharmaceutical industrial application.

The use of spectroscopic sensors for bioprocess monitoring is a powerful tool within the process analytical technology (PAT) initiative of the US Food and Drug Administration. Spectroscopic sensors enable the simultaneous real-time bioprocess monitoring of various critical process parameters including biological, chemical, and physical variables during the entire biotechnological production process. This potential can be realized through the combination of spectroscopic measurements (UV/Vis spectroscopy, IR spectroscopy, fluorescence spectroscopy, and Raman spectroscopy) with multivariate data analysis to obtain relevant process information out of an enormous amount of data. This review summarizes the newest results from science and industry after the establishment of the PAT initiative and gives a critical overview of the most common in-line spectroscopic techniques. Examples are provided of the wide range of possible applications in upstream processing and downstream processing of spectroscopic sensors for real-time monitoring to optimize productivity and ensure product quality in the pharmaceutical industry.

Supplementing Blends of Sugars, Amino Acids, and Secondary Metabolites to the Diet of Termites (Reticulitermes flavipes) Drive Distinct Gut Bacterial Communities.

Although it is well known that diet is one of the major modulators of the gut microbiome, how the major components of diet shape the gut microbial community is not well understood. Here, we developed a simple system that allows the investigation of the impact of given compounds as supplements of the diet on the termite gut microbiome. The 16S rRNA pyrosequencing analysis revealed that feeding termites different blends of sugars and amino acids did not majorly impact gut community composition; however, ingestion of blends of secondary metabolites caused shifts in gut bacterial community composition. The supplementation of sugars and amino acids reduced the richness significantly, and sugars alone increased the evenness of the gut bacterial community significantly. Secondary metabolites created the most dramatic effects on the microbial community, potentially overriding the effect of other types of compounds. Furthermore, some microbial groups were stimulated specifically by particular groups of compounds. For instance, termites fed with secondary metabolites contained more Firmicutes and Spirochaetes compared to the other treatments. In conclusion, our results suggest that the termite (Reticulitermes flavipes) can be used as a simple and effective system to test the effects of particular chemical compounds in modulating the gut microbiome.

Monitoring of Microalgal Processes.

Process monitoring, which can be defined as the measurement of process variables with the smallest possible delay, is combined with process models to form the basis for successful process control. Minimizing the measurement delay leads inevitably to employing online, in situ sensors where possible, preferably using noninvasive measurement methods with stable, low-cost sensors. Microalgal processes have similarities to traditional bioprocesses but also have unique monitoring requirements. In general, variables to be monitored in microalgal processes can be categorized as physical, chemical, and biological, and they are measured in gaseous, liquid, and solid (biological) phases. Physical and chemical process variables can be usually monitored online using standard industrial sensors. The monitoring of biological process variables, however, relies mostly on sensors developed and validated using laboratory-scale systems or uses offline methods because of difficulties in developing suitable online sensors. Here, we review current technologies for online, in situ monitoring of all types of process parameters of microalgal cultivations, with a focus on monitoring of biological parameters. We discuss newly introduced methods for measuring biological parameters that could be possibly adapted for routine online use, should be preferably noninvasive, and are based on approaches that have been proven in other bioprocesses. New sensor types for measuring physicochemical parameters using optical methods or ion-specific field effect transistor (ISFET) sensors are also discussed. Reviewed methods with online implementation or online potential include measurement of irradiance, biomass concentration by optical density and image analysis, cell count, chlorophyll fluorescence, growth rate, lipid concentration by infrared spectrophotometry, dielectric scattering, and nuclear magnetic resonance. Future perspectives are discussed, especially in the field of image analysis using in situ microscopy, infrared spectrophotometry, and software sensor systems.

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.

A plastic total internal reflection photoluminescence device for enzymatic biosensing.

A total internal reflection photoluminescence (TIRPh) device employing an easily fabricated PMMA/PDMS waveguide system provides a detection limit comparable to the best reported results but without using an excitation filter. The optical mechanism is similar to total-internal-reflection-fluorescence (TIRF) but uses a ruthenium-based phosphorescent dye (Ru(dpp)3) deposited on the PMMA core, motivating the generalized term of photoluminescence to include both fluorescence and phosphorescence. An enzymatic hydrogen peroxide (H2O2) biosensor incorporating catalase was fabricated on the TIRPh platform without photolithography or etching. The O2-sensitive phosphorescence of Ru(dpp)3 was used as a transduction mechanism and catalase was used as a biocomponent for sensing. The H2O2 sensor exhibits a phosphorescence to scattered excitation light ratio of 76 ± 10 without filtering. The unfiltered device demonstrates a detection limit of (2.2 ± 0.6) µM with a linear range of 0.1 mM to 20 mM. The device is the first total internal reflection photoluminescence based enzymatic biosensor platform, and is promising for cost-effective, low excitation interference, field-portable sensing.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.

Modeling sorption of neutral organic compound mixtures to simulated aquifer sorbents with pseudocompounds.

The feasibility of the ideal adsorbed solution theory (IAST) in reducing the complexity associated with predicting the sorption behaviors of 12 neutral organic compounds (NOCs) contained in complex mixtures as a fewer number (four to six) of pseudocompounds (groups of compounds) to simulated aquifer sorbents was investigated. All sorption isotherms from individual- and multiple-pseudocompound systems were fit reasonably well ( ≥ 0.953) by the Freundlich sorption model over the range of aqueous concentrations evaluated (i.e., ≤200 µmol L). The presence and magnitude of mutual competition among pseudocompounds varied depending on the composition of the mixtures (i.e., concentrations and polarities of pseudocompounds) and the properties of sorbents (i.e., the fraction of organic carbon and the availability of hydrophilic specific sorption sites). Finally, comparisons between the IAST-based predictions with individual-pseudocompound sorption parameters and experimentally measured data revealed that the accuracy in predicting the sorption behaviors of several NOCs in terms of a fewer number of pseudocompounds decreased with increasing deviations from the assumption of equal and ideal competition in the IAST (i.e., differential availability of sorption sites and nonideal competitions among pseudocompounds).

On-line monitoring of large cultivations of microalgae and cyanobacteria.

Large cultivations of microalgae will benefit from on-line monitoring to achieve process control and improved productivity. This monitoring requires reliable sensors for on-line, in situ measurement of both physicochemical and biological process variables. Although standard industrial sensors can be used for many physicochemical variables, monitoring methods for most biological quantities rely on sensors that are currently suitable only for laboratory scale or off-line use. Here, we review these methods and discuss new approaches that could be adapted. We suggest that these new methods should be noninvasive and based on approaches that have already been applied to other bioprocesses; examples discussed here are in situ microscopy, flow cytometry (FC), IR spectroscopy, and software sensors.

Variations in diversity and richness of gut bacterial communities of termites (Reticulitermes flavipes) fed with grassy and woody plant substrates.

Diets shape the animal gut microbiota, although the relationships between diets and the structure of the gut microbial community are not yet well understood. The gut bacterial communities of Reticulitermes flavipes termites fed on four individual plant biomasses with different degrees of recalcitrance to biodegradation were investigated by 16S rRNA pyrosequencing analysis. The termite gut bacterial communities could be differentiated between grassy and woody diets, and among grassy diets (corn stover vs. sorghum). The majority of bacterial taxa were shared across all diets, but each diet significantly enriched some taxa. Interestingly, the diet of corn stover reduced gut bacterial richness and diversity compared to other diets, and this may be related to the lower recalcitrance of this biomass to degradation.