Ethanol and Anaerobic Conditions Reversibly Inhibit Commercial Cellulase Activity in Thermophilic Simultaneous Saccharification and Fermentation (tSSF)

A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study

Response surface methodology (RSM) to evaluate moisture effects on corn stover in recovering xylose by DEO hydrolysis

Response surface methodology (RSM), based on a 2(2) full factorial design, evaluated the moisture effects in recovering xylose by diethyloxalate (DEO) hydrolysis. Experiments were carried out in laboratory reactors (10 mL glass ampoules) containing corn stover (0.5 g) properly ground. The ampoules were kept at 160 degrees C for 90 min.(-) Both DEO concentration and corn stover moisture content were statistically significant at 99% confidence level. The maximum xylose recovery by the response surface methodology was achieved employing both DEO concentration and corn stover moisture at near their highest levels area. We amplified this area by using an overlay plot as a graphical optimization using a response of xylose recovery more than 80%. The mathematical statistical model was validated by testing a specific condition in the satisfied overlay plot area. Experimentally, a maximum xylose recovery (81.2%) was achieved by using initial corn stover moisture of 60% and a DEO concentration of 4% w/w. The mathematical statistical model showed that xylose recovery increases during DEO corn stover acid hydrolysis as the corn stover moisture level increases. This observation could be important during the harvesting of corn before it is fully dried in the field. The corn stover moisture was an important variable to improve xylose recovery by DEO acid hydrolysis. (c) 2011 Elsevier Ltd. All rights reserved.CNPq, Brazil [200702/2006-8

Anaerobic Detoxification of Acetic Acid in a Thermophilic Ethanologen

The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield. We have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum.

Genome-scale resources for Thermoanaerobacterium saccharolyticum

Background Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation. Results Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results. Conclusion These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0159-x) contains supplementary material, which is available to authorized users

Development of Risk Prediction Equations for Incident Chronic Kidney Disease

IMPORTANCE ‐ Early identification of individuals at elevated risk of developing chronic kidney disease  could improve clinical care through enhanced surveillance and better management of underlying health  conditions.  OBJECTIVE – To develop assessment tools to identify individuals at increased risk of chronic kidney  disease, defined by reduced estimated glomerular filtration rate (eGFR).  DESIGN, SETTING, AND PARTICIPANTS – Individual level data analysis of 34 multinational cohorts from  the CKD Prognosis Consortium including 5,222,711 individuals from 28 countries. Data were collected  from April, 1970 through January, 2017. A two‐stage analysis was performed, with each study first  analyzed individually and summarized overall using a weighted average. Since clinical variables were  often differentially available by diabetes status, models were developed separately within participants  with diabetes and without diabetes. Discrimination and calibration were also tested in 9 external  cohorts (N=2,253,540). EXPOSURE Demographic and clinical factors.  MAIN OUTCOMES AND MEASURES – Incident eGFR <60 ml/min/1.73 m2.  RESULTS – In 4,441,084 participants without diabetes (mean age, 54 years, 38% female), there were  660,856 incident cases of reduced eGFR during a mean follow‐up of 4.2 years. In 781,627 participants  with diabetes (mean age, 62 years, 13% female), there were 313,646 incident cases during a mean follow‐up of 3.9 years. Equations for the 5‐year risk of reduced eGFR included age, sex, ethnicity, eGFR, history of cardiovascular disease, ever smoker, hypertension, BMI, and albuminuria. For participants  with diabetes, the models also included diabetes medications, hemoglobin A1c, and the interaction  between the two. The risk equations had a median C statistic for the 5‐year predicted probability of  0.845 (25th – 75th percentile, 0.789‐0.890) in the cohorts without diabetes and 0.801 (25th – 75th percentile, 0.750‐0.819) in the cohorts with diabetes. Calibration analysis showed that 9 out of 13 (69%) study populations had a slope of observed to predicted risk between 0.80 and 1.25. Discrimination was  similar in 18 study populations in 9 external validation cohorts; calibration showed that 16 out of 18 (89%) had a slope of observed to predicted risk between 0.80 and 1.25. CONCLUSIONS AND RELEVANCE – Equations for predicting risk of incident chronic kidney disease developed in over 5 million people from 34 multinational cohorts demonstrated high discrimination and  variable calibration in diverse populations

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a d-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-a dagger 1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. d-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose) l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.CNPq, Brazil[200702/2006-8

Strain And Bioprocess Improvement of a Thermophilic Anaerobe for the Production of Ethanol from Wood

Background: The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported. Results: Here, we describe the highest ethanol titers achieved from T. saccharolyticum during a 4-year project to develop it for industrial production of ethanol from pre-treated hardwood at 51-55 degrees C. We describe organism and bioprocess development efforts undertaken to improve ethanol production. The final strain M2886 was generated by removing genes for exopolysaccharide synthesis, the regulator perR, and re-introduction of phosphotransacetylase and acetate kinase into the methyglyoxal synthase gene. It was also subject to multiple rounds of adaptation and selection, resulting in mutations later identified by resequencing. The highest ethanol titer achieved was 70 g/L in batch culture with a mixture of cellobiose and maltodextrin. In a mock hydrolysate Simultaneous Saccharification and Fermentation (SSF) with Sigmacell-20, glucose, xylose, and acetic acid, an ethanol titer of 61 g/L was achieved, at 92 % of theoretical yield. Fungal cellulases were rapidly inactivated under these conditions and had to be supplemented with cellulosomes from C. thermocellum. Ethanol titers of 31 g/L were reached in a 100 L SSF of pre-treated hardwood and 26 g/L in a fermentation of a hardwood hemicellulose extract. Conclusions: This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates, but additional work is needed to produce the same ethanol titers from pre-treated hardwood