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.

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.

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.

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.

Isolation and characterization of lignin-degrading bacteria from rainforest soils.

The deconstruction of lignin to enhance the release of fermentable sugars from plant cell walls presents a challenge for biofuels production from lignocellulosic biomass. The discovery of novel lignin-degrading enzymes from bacteria could provide advantages over fungal enzymes in terms of their production and relative ease of protein engineering. In this study, 140 bacterial strains isolated from soils of a biodiversity-rich rainforest in Peru were screened based on their oxidative activity on ABTS, a laccase substrate. Strain C6 (Bacillus pumilus) and strain B7 (Bacillus atrophaeus) were selected for their high laccase activity and identified by 16S rDNA analysis. Strains B7 and C6 degraded fragments of Kraft lignin and the lignin model dimer guaiacylglycerol-ß-guaiacyl ether, the most abundant linkage in lignin. Finally, LC-MS analysis of incubations of strains B7 and C6 with poplar biomass in rich and minimal media revealed that a higher number of compounds were released in the minimal medium than in the rich one. These findings provide important evidence that bacterial enzymes can degrade and/or modify lignin and contribute to the release of fermentable sugars from lignocellulose.

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.

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).

Phosphoproteomics and molecular cardiology: techniques, applications and challenges.

Protein phosphorylation has been widely documented as a key regulatory and signaling mechanism associated with many cardiac diseases. Recent advances in phosphoproteomic technologies such as phosphopeptide enrichment, novel mass spectrometry applications, and bioinformatic tools have resulted in high-throughput identification and quantitation of protein phosphorylation in a global manner. This review summarizes mainstream phosphoproteomic workflows and highlights the most recent applications of phosphoproteomics used in a range of molecular cardiology research.

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.

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.

Testosterone-mineralizing culture enriched from swine manure: characterization of degradation pathways and microbial community composition.

Environmental releases and fate of steroid sex hormones from livestock and wastewater treatment plants are of increasing regulatory concern. Despite the detection of these hormones in manures, biosolids, and the environment, little attention has been paid to characterization of fecal bacteria capable of hormone degradation. The enrichments of (swine) manure-borne bacteria capable of aerobic testosterone degradation were prepared and the testosterone mineralization pathway was elucidated. Six DNA sequences of bacteria from the Proteobacteria phylum distributed among the genera Acinetobacter, Brevundimonas, Comamonas, Sphingomonas, Stenotrophomonas, and Rhodobacter were identified in a testosterone-degrading enriched culture with testosterone as the sole carbon source. Three degradation products of testosterone were identified as androstenedione, androstadienedione, and dehydrotestosterone using commercially available reference standards, liquid chromatography-UV diode array detection, and liquid chromatography-time-of-flight mass spectrometry (LC-TOF/MS). Three additional degradation products of testosterone were tentatively identified as 9α-hydroxytestosterone, 9α-hydroxyandrostadienedione or 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, and 9α-hydroxydehydrotestosterone or 9α-hydroxyandrostenedione using LC-TOF/MS. When (14)C-testosterone was introduced to the enriched culture, 49-68% of the added (14)C-testosterone was mineralized to (14)CO(2) within 8 days of incubation. The mineralization of (14)C-testosterone followed pseudo-first-order reaction kinetics in the enriched culture with half-lives (t(1/2)) of 10-143 h. This work suggests that Proteobacteria play an important environmental role in degradation of steroid sex hormones and that androgens have the potential to be mineralized during aerobic manure treatment or after land application to agricultural fields by manure-borne bacteria.

Culturing and investigation of stress-induced lipid accumulation in microalgae using a microfluidic device.

There is increasing interest in using microalgae as a lipid feedstock for the production of biofuels. Lipids used for these purposes are triacylglycerols that can be converted to fatty acid methyl esters (biodiesel) or decarboxylated to "green diesel." Lipid accumulation in most microalgal species is dependent on environmental stress and culturing conditions, and these conditions are currently optimized using slow, labor-intensive screening processes. Increasing the screening throughput would help reduce the development cost and time to commercial production. Here, we demonstrated an initial step towards this goal in the development of a glass/poly(dimethylsiloxane) (PDMS) microfluidic device capable of screening microalgal culturing and stress conditions. The device contained power-free valves to isolate microalgae in a microfluidic growth chamber for culturing and stress experiments. Initial experiments involved determining the biocompatibility and culturing capability of the device using the microalga Tetraselmis chuii. With this device, T. chuii could be successfully cultured for up to 3 weeks on-chip. Following these experiments, the device was used to investigate lipid accumulation in the microalga Neochloris oleabundans. It was shown that this microalga could be stressed to accumulate cytosolic lipids in a microfluidic environment, as evidenced with fluorescence lipid staining. This work represents the first example of microalgal culturing in a microfluidic device and signifies an important expansion of microfluidics into the biofuels research arena.

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.

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.

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.

On-line infrared spectroscopy for bioprocess monitoring.

One of the major aims of bioprocess engineering is the real-time monitoring of important process variables. This is the basis of precise process control and is essential for high productivity as well as the exact documentation of the overall production process. Infrared spectroscopy is a powerful analytical technique to analyze a wide variety of organic compounds. Thus, infrared sensors are ideal instruments for bioprocess monitoring. The sensors are non-invasive, have no time delay due to sensor response times, and have no influence on the bioprocess itself. No sampling is necessary, and several components can be analyzed simultaneously. In general, the direct monitoring of substrates, products, metabolites, as well as the biomass itself is possible. In this review article, insights are provided into the different applications of infrared spectroscopy for bioprocess monitoring and the complex data interpretation. Different analytical techniques are presented as well as example applications in different areas.

Environmental proteomics: applications of proteome profiling in environmental microbiology and biotechnology.

In this review, we present the use of proteomics to advance knowledge in the field of environmental biotechnology, including studies of bacterial physiology, metabolism and ecology. Bacteria are widely applied in environmental biotechnology for their ability to catalyze dehalogenation, methanogenesis, denitrification and sulfate reduction, among others. Their tolerance to radiation and toxic compounds is also of importance. Proteomics has an important role in helping uncover the pathways behind these cellular processes. Environmental samples are often highly complex, which makes proteome studies in this field especially challenging. Some of these challenges are the lack of genome sequences for the vast majority of environmental bacteria, difficulties in isolating bacteria and proteins from certain environments, and the presence of complex microbial communities. Despite these challenges, proteomics offers a unique dynamic view into cellular function. We present examples of environmental proteomics of model organisms, and then discuss metaproteomics (microbial community proteomics), which has the potential to provide insights into the function of a community without isolating organisms. Finally, the environmental proteomics literature is summarized as it pertains to the specific application areas of wastewater treatment, metabolic engineering, microbial ecology and environmental stress responses.

Fenton's oxidation of pentachlorophenol.

The combination of H(2)O(2) and Fe(II) (Fenton's reaction) has been demonstrated to rapidly degrade many organics via hydroxyl radicals. However, few studies have related hydroxyl radical generation rates with measured organic chemical degradation data. The goals of this work were to investigate the kinetics, stoichiometry, and intermediates of pentachlorophenol (PCP) degradation in the Fenton's reaction and to develop a mathematical model of this reaction system. Batch reaction experiments were performed to assess both initial transients and steady states, and special attention was given to the analysis of intermediates. Solutions of PCP (55 microM) and Fe(II) (200 microM) were treated with variable levels of H(2)O(2) (<850 microM), and the concentrations of these reactants and their products were measured. Partial PCP degradation and near stoichiometric dechlorination were observed at low initial H(2)O(2) concentrations. Higher H(2)O(2) doses achieved at most 70% dechlorination even though nearly all of the PCP was degraded. The reaction intermediates tetrachlorohydroquinone and dichloromaleic acid accounted for up to 5% of the PCP degraded. Organic carbon mineralization (transformation to CO(2)) was not observed. The ()OH scavenging effects of the PCP-by-products mixture were characterized as a lumped parameter in the reaction kinetics model, which provided reasonable predictions of experimental results at different oxidant concentrations and reaction time.