A Quantitative Re-Assessment of Microencapsulation in (Pre-Treated) Yeast.

Most hydrophobes easily diffuse into yeast cells, where they experience reduced evaporation and protection from oxidation, thus allowing inherently biocompatible encapsulation processes. Despite a long-standing industrial interest, the effect of parameters such as how is yeast pre-treated (extraction with ethanol, plasmolysis with hypertonic NaCl, depletion to cell walls), the polarity of the hydrophobes and the process conditions are still not fully understood. Here, we have developed thorough analytical protocols to assess how the effects of the above on S. cerevisiae's morphology, permeability, and encapsulation efficiency, using three differently polar hydrophobes (linalool, 1,6-dihydrocarvone, limonene) and three separate processes (hydrophobes as pure 'oils', water dispersions, or acetone solutions). The harsher the pre-treatment (depleted > plasmolyzed/extracted > untreated cells), the easier the diffusion into yeast became, and the lower both encapsulation efficiency and protection from evaporation, possibly due to denaturation/removal of lipid-associated (membrane) proteins. More hydrophobic terpenes performed worst in encapsulation as pure 'oils' or in water dispersion, but much less of a difference existed in acetone. This indicates the specific advantage of solvents/dispersants for 'difficult' compounds, which was confirmed by principal component analysis; furthering this concept, we have used combinations of hydrophobes (e.g., linalool and α-tocopherol), with one acting as solvent/enhancer for the other. Our results thus indicate advantages in using untreated yeast and-if necessary-processes based on solvents/secondary hydrophobes.

Transmission electron microscopy assessment of a novel method for isolating pure exosomes from serum.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Occupational exposure assessment with solid substances: choosing a vehicle for in vitro percutaneous absorption experiments.

Percutaneous occupational exposure to industrial toxicants can be assessed in vitro on excised human or animal skins. Numerous factors can significantly influence skin permeation of chemicals and the flux determination. Among them, the vehicle used to solubilize the solid substances is a tricky key step. A "realistic surrogate" that closely matches the exposure scenario is recommended in first intention. When direct transposition of occupational exposure conditions to in vitro experiments is impossible, it is recommended that the vehicle used does not affect the skin barrier (in particular in terms of structural integrity, composition, or enzymatic activity). Indeed, any such effect could alter the percutaneous absorption of substances in a number of ways, as we will see. Potential effects are described for five monophasic vehicles, including the three most frequently used: water, ethanol, acetone; and two that are more rarely used, but are realistic: artificial sebum and artificial sweat. Finally, we discuss a number of criteria to be verified and the associated tests that should be performed when choosing the most appropriate vehicle, keeping in mind that, in the context of occupational exposure, the scientific quality of the percutaneous absorption data provided, and how they are interpreted, may have long-range consequences. From the narrative review presented, we also identify and discuss important factors to consider in future updates of the OECD guidelines for in vitro skin absorption experiments.

A comparison of the energy use of in situ product recovery techniques for the Acetone Butanol Ethanol fermentation.

The productivity of the Acetone Butanol Ethanol (ABE) fermentation can be significantly increased by application of various in situ product recovery (ISPR) techniques. There are numerous technically viable processes, but it is not clear which is the most economically viable in practice. There is little available information about the energy requirements and economics of ISPR for the ABE fermentation. This work compares various ISPR techniques based on UniSim process simulations of the ABE fermentation. The simulations provide information on the process energy and separation efficiency, which is fed into an economic assessment. Perstraction was the only technique to reduce the energy demand below that of a batch process, by approximately 5%. Perstraction also had the highest profit increase over a batch process, by 175%. However, perstraction is an immature technology, so would need significant development before being integrated to an industrial process.

Acetone-butanol-ethanol fermentation of corn stover: current production methods, economic viability and commercial use.

Biobutanol is a next-generation liquid biofuel with properties akin to those of gasoline. There is a widespread effort to commercialize biobutanol production from agricultural residues, such as corn stover, which do not compete with human and animal foods. This pursuit is backed by extensive government mandates to expand alternative energy sources. This review provides an overview of research on biobutanol production using corn stover feedstock. Structural composition, pretreatment, sugar yield (following pretreatment and hydrolysis) and generation of lignocellulose-derived microbial inhibitory compounds (LDMICs) from corn stover are discussed. The review also discusses different Clostridium species and strains employed for biobutanol production from corn stover-derived sugars with respect to solvent yields, tolerance to LDMICs and in situ solvent recovery (integrated fermentation). Further, the economics of cellulosic biobutanol production are highlighted and compared to corn starch-derived ethanol and gasoline. As discussed herein, the economic competitiveness of biobutanol production from corn stover largely depends on feedstock processing and fermentation process design.

Impact of zinc supplementation on the improved fructose/xylose utilization and butanol production during acetone-butanol-ethanol fermentation.

Lignocellulosic biomass and dedicated energy crops such as Jerusalem artichoke are promising alternatives for biobutanol production by solventogenic clostridia. However, fermentable sugars such as fructose or xylose released from the hydrolysis of these feedstocks were subjected to the incomplete utilization by the strains, leading to relatively low butanol production and productivity. When 0.001 g/L ZnSO4·7H2O was supplemented into the medium containing fructose as sole carbon source, 12.8 g/L of butanol was achieved with butanol productivity of 0.089 g/L/h compared to only 4.5 g/L of butanol produced with butanol productivity of 0.028 g/L/h in the control without zinc supplementation. Micronutrient zinc also led to the improved butanol production up to 8.3 g/L derived from 45.2 g/L xylose as sole carbon source with increasing butanol productivity by 31.7%. Moreover, the decreased acids production was observed under the zinc supplementation condition, resulting in the increased butanol yields of 0.202 g/g-fructose and 0.184 g/g-xylose, respectively. Similar improvements were also observed with increasing butanol production by 130.2 % and 8.5 %, butanol productivity by 203.4% and 18.4%, respectively, in acetone-butanol-ethanol fermentations from sugar mixtures of fructose/glucose (4:1) and xylose/glucose (1:2) simulating the hydrolysates of Jerusalem artichoke tubers and corn stover. The results obtained from transcriptional analysis revealed that zinc may have regulatory mechanisms for the sugar transport and metabolism of Clostridium acetobutylicum L7. Therefore, micronutrient zinc supplementation could be an effective way for economic development of butanol production derived from these low-cost agricultural feedstocks.

Assessment of morphological changes of Clostridium acetobutylicum by flow cytometry during acetone/butanol/ethanol extractive fermentation.

Acetone/butanol/ethanol (ABE) fermentation by Clostridium acetobutylicum was investigated in extractive fed-batch experiments. In conventional fermentations, metabolic activity ceases when a critical threshold products concentration is reached (~21.6 g solvents l(-1)). Solvents production was increased up to 36.6 and 37.2 g l(-1), respectively, using 2-butyl-1-octanol (aqueous to organic ratio: 1:0.25 v/v) and pomace olive oil (1:1 v/v) as extraction solvents. The morphological changes of different cell types were monitored and quantified using flow cytometry. Butanol production in extractive fermentations with pomace olive oil was achieved mainly by vegetative cells, whereas the percentage of sporulating cells was lower than 10%.

1H-NMR-based metabolic analysis of human serum reveals novel markers of myocardial energy expenditure in heart failure patients.

OBJECTIVE: Elevated myocardial energy expenditure (MEE) is related with reduced left ventricular ejection fraction, and has also been documented as an independent predictor of cardiovascular mortality. However, the serum small-molecule metabolite profiles and pathophysiological mechanisms of elevated MEE in heart failure (HF) are still lacking. Herein, we used 1H-NMR-based metabolomics analysis to screen for potential biomarkers of MEE in HF. METHODS: A total of 61 subjects were enrolled, including 46 patients with heart failure and 15 age-matched controls. Venous serum samples were collected from subjects after an 8-hour fast. An INOVA 600 MHz nuclear magnetic resonance spectrometer with Carr-Purcell-Melboom-Gill (CPMG) pulse sequence was employed for the metabolomics analysis and MEE was calculated using colored Doppler echocardiography. Metabolomics data were processed using orthogonal signal correction and regression analysis was performed using the partial least squares method. RESULTS: The mean MEE levels of HF patients and controls were 139.61±58.18 cal/min and 61.09±23.54 cal/min, respectively. Serum metabolomics varied with MEE changed, and 3-hydroxybutyrate, acetone and succinate were significantly elevated with the increasing MEE. Importantly, these three metabolites were independent of administration of angiotensin converting enzyme inhibitor, ß-receptor blockers, diuretics and statins (P>0.05). CONCLUSIONS: These results suggested that in patients with heart failure, MEE elevation was associated with significant changes in serum metabolomics profiles, especially the concentration of 3-hydroxybutyrate, acetone and succinate. These compounds could be used as potential serum biomarkers to study myocardial energy mechanism in HF patients.

Acetone-butanol fermentation of marine macroalgae.

The objective of this study was to subject mannitol, either as a sole carbon source or in combination with glucose, and aqueous extracts of the kelp Saccharina spp., containing mannitol and laminarin, to acetone-butanol fermentation by Clostridium acetobutylicum (ATCC 824). Both mannitol and glucose were readily fermented. Mixed substrate fermentations with glucose and mannitol resulted in diauxic growth of C. acetobutylicum with glucose depletion preceding mannitol utilization. Fermentation of kelp extract exhibited triauxic growth, with an order of utilization of free glucose, mannitol, and bound glucose, presumably laminarin. The lag in laminarin utilization reflected the need for enzymatic hydrolysis of this polysaccharide into fermentable sugars. The butanol and total solvent yields were 0.12 g/g and 0.16 g/g, respectively, indicating that significant improvements are still needed to make industrial-scale acetone-butanol fermentations of seaweed economically feasible.

Economical challenges to microbial producers of butanol: feedstock, butanol ratio and titer.

Butanol is an important solvent and transport fuel additive, and can be produced by microbial fermentation. Attempts to generate a superior microbial producer of butanol have been made through different metabolic engineering strategies. However, to date, butanol bio-production is still not economically competitive compared to petrochemical-derived production because of its major drawbacks, such as, high cost of the feedstocks, low butanol concentration in the fermentation broth and the co-production of low-value by-products acetone and ethanol. Here we analyze the main bottlenecks in microbial butanol production and summarize relevant advances from recently reported studies. Further needs and directions for developing real industrially applicable strains in butanol production are also discussed.

Recent progress on industrial fermentative production of acetone-butanol-ethanol by Clostridium acetobutylicum in China.

China is one of the few countries, which maintained the fermentative acetone-butanol-ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.

Downstream process synthesis for biochemical production of butanol, ethanol, and acetone from grains: generation of optimal and near-optimal flowsheets with conventional operating units.

Manufacturing butanol, ethanol, and acetone through grain fermentation has been attracting increasing research interest. In the production of these chemicals from fermentation, the cost of product recovery constitutes the major portion of the total production cost. Developing cost-effective flowsheets for the downstream processing is, therefore, crucial to enhancing the economic viability of this manufacturing method. The present work is concerned with the synthesis of such a process that minimizes the cost of the downstream processing. At the outset, a wide variety of processing equipment and unit operations, i.e., operating units, is selected for possible inclusion in the process. Subsequently, the exactly defined superstructure with minimal complexity, termed maximal structure, is constructed from these operating units with the rigorous and highly efficient graph-theoretic method for process synthesis based on process graphs (P-graphs). Finally, the optimal and near-optimal flowsheets in terms of cost are identified.

The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures.

Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems. In the case of Escherichia coli, interference involves the Na+(Li+)/H+ antiporter transport system. The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions. The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance. In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs. The ultimate fate of the LiCl was not determined.

The economics of acetone-butanol fermentation: theoretical and market considerations.

Acetone-butanol (AB) fermentation was once run commercially in many countries until these chemicals could be made more cheaply from fossil oil sources. Research into the revitalisation of the process has shown that the process could once again be run economically in niche markets if run in a relatively small industrial scale processing low-grade agricultural products. The following analysis is intended to help identify suitable niche markets.

Assessment of the deamination of aminoacetone, an endogenous substrate for semicarbazide-sensitive amine oxidase.

Methylglyoxal, a toxic aldehyde, has been reported to be increased in diabetes and has been claimed to be related to diabetic complications. Aminoacetone, an intermediate in the metabolism of threonine and glycine, has been proposed to be an endogenous substrate for semicarbazide-sensitive amine oxidase (SSAO). Methylglyoxal is the product. An HPLC procedure for the determination of SSAO activity toward aminoacetone in vitro is described. It was observed in previous assays that methylglyoxal formed via deamination of aminoacetone was quite unstable and led to erroneous results. o-Phenylenediamine (o-PD) was therefore employed for derivatization of methylglyoxal. o-PD does not affect SSAO activity and can be included in the enzyme reaction mixture for continuous trapping of methylglyoxal. This can avoid the loss of methylglyoxal during incubation. Deamination of aminoacetone by human umbilical artery SSAO was confirmed with this improved assay. The values of Km and Vmax, are 125.9 +/- 20.5 microM and 332.2 +/- 11.7 nmol/h/mg protein, respectively. Deamination of aminoacetone was nearly completely inhibited by 1 mM semicarbazide and 1 microM MDL-72974A, a potent selective SSAO inhibitor, whereas MAO inhibitors clorgyline (1 mM) and deprenyl (1 mM) had no inhibitory effect.