Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Solubilization of sugarcane bagasse by mono and cocultures of thermophilic anaerobes with and without cotreatment.

Cotreatment, mechanical disruption of lignocellulosic biomass during microbial fermentation, is a potential alternative to thermochemical pretreatment as a means of increasing the accessibility of lignocellulose to biological attack. Successful implementation of cotreatment requires microbes that can withstand milling, while solubilizing and utilizing carbohydrates from lignocellulose. In this context, cotreatment with thermophilic, lignocellulose-fermenting bacteria has been successfully evaluated for a number of lignocellulosic feedstocks. Here, cotreatment was applied to sugarcane bagasse using monocultures of the cellulose-fermenting Clostridium thermocellum and cocultures with the hemicellulose-fermenting Thermoanaerobacterium thermosaccharolyticum. This resulted in 76 % carbohydrate solubilization (a 1.8-fold increase over non-cotreated controls) on 10 g/L solids loading, having greater effect on the hemicellulose fraction. With cotreatment, fermentation by wild-type cultures at low substrate concentrations increased cumulative product formation by 45 % for the monoculture and 32 % for the coculture. These findings highlight the potential of cotreatment for enhancing deconstruction of sugarcane bagasse using thermophilic bacteria.

Enhancing anaerobic digestion of lignocellulosic biomass by mechanical cotreatment.

BACKGROUND: The aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanical cotreatment: milling during fermentation, as an alternative to conventional pretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the 'status quo' control. RESULTS: The results indicate 0.5 - 10 min of cotreatment increased sugar solubilization by 5- 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production. CONCLUSIONS: Cotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.

Unmasking of a Giant Coronary Aneurysm by Chronic Total Coronary Occlusion Percutaneous Coronary Intervention Techniques.

A young female patient presenting with a non-ST-segment elevation myocardial infarction underwent invasive coronary angiography, revealing a total occlusion of the right coronary artery. During percutaneous coronary intervention with dual catheter access, a retrograde tip injection and peculiar retrograde wiring unmasked a giant coronary aneurysm, which noninvasive imaging confirmed.

The Search for an Outcome Variable That Measures Both Quality and Processes in Cardiac Surgery: Comparing the Quality Process Index and Mortality.

BACKGROUND: The translation of a large quantity of data into valuable insights for daily clinical practice is underexplored. A considerable amount of information is overwhelming, making it difficult to distill and assess quality and processes at the hospital level. This study contributes to this necessary translation by developing a Quality Process Index that summarizes clinical data to measure quality and processes. METHODS: The Quality Process Index was constructed to enable retrospective analyses of quality and process evolution from 2011 to 2021 for various surgery types in the Amsterdam Cardiosurgical Database (n = 5497). It is presented alongside mortality rates, which are the golden standard for quality measurement. The two outcome variables are compared as quality and process measurement options. RESULTS: Results showed that the mean Quality Process Index appeared rather stable, even though analysis of variance found that the mean Quality Process Index differed significantly over the years (p < 0.001). The 30-day and 120-day mortality rates appeared to fluctuate more, but interestingly, we failed to reject the null hypothesis of equal means. The Quality Process Index and mortality rates were statistically negatively correlated, and the extent of correlation was more pronounced with the 120-day mortality rate, as computed using the Pearson correlation coefficient r (30-day rQPI,30 = -0.07, p < 0.001 and 120-day mortality rates rQPI,120 = -0.12, p < 0.001). CONCLUSIONS: The Quality Process Index seeks to address the need to translate data for quality and process improvement in healthcare. While mortality remains the most impactful outcome measure, the Quality Process Index provides a more stable and comprehensive measurement of quality and process improvement or deterioration in healthcare. Therefore, the Quality Process Index as a quantification reinforces the understanding of the definition of quality and process improvement.

Metaproteomics reveals enzymatic strategies deployed by anaerobic microbiomes to maintain lignocellulose deconstruction at high solids.

Economically viable production of cellulosic biofuels requires operation at high solids loadings-on the order of 15 wt%. To this end we characterize Nature's ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate solubilization at loadings ranging from 30 g/L to 150 g/L. Metaproteomic interrogation reveals marked increases in the abundance of specific carbohydrate-active enzyme classes. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are ß-glucosidases, xylosidases, carbohydrate-debranching, and pectin-acting enzymes-all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization. Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings, informing the future development of defined cultures for efficient bioconversion.

Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation and diagnostic tests.

BACKGROUND: For economically viable 2nd generation biofuels, processing of high solid lignocellulosic substrate concentrations is a necessity. The cellulolytic thermophilic anaerobe Clostridium thermocellum is one of the most effective biocatalysts for solubilization of carbohydrate harbored in lignocellulose. This study aims to document the solubilization performance of Clostridium thermocellum at increasing solids concentrations for two lignocellulosic feedstocks, corn stover and switchgrass, and explore potential effectors of solubilization performance. RESULTS: Monocultures of Clostridium thermocellum demonstrated high levels of carbohydrate solubilization for both unpretreated corn stover and switchgrass. However, fractional carbohydrate solubilization decreases with increasing solid loadings. Fermentation of model insoluble substrate (cellulose) in the presence of high solids lignocellulosic spent broth is temporarily affected but not model soluble substrate (cellobiose) fermentations. Mid-fermentation addition of cells (C. thermocellum) or model substrates did not significantly enhance overall corn stover solubilization loaded at 80 g/L, however cultures utilized the model substrates in the presence of high concentrations of corn stover. An increase in corn stover solubilization was observed when water was added, effectively diluting the solids concentration mid-fermentation. Introduction of a hemicellulose-utilizing coculture partner, Thermoanaerobacterium thermosaccharolyticum, increased the fractional carbohydrate solubilization at both high and low solid loadings. Residual solubilized carbohydrates diminished significantly in the presence of T. thermosaccharolyticum compared to monocultures of C. thermocellum, yet a small fraction of solubilized oligosaccharides of both C5 and C6 sugars remained unutilized. CONCLUSION: Diminishing fractional carbohydrate solubilization with increasing substrate loading was observed for C. thermocellum-mediated solubilization and fermentation of unpretreated lignocellulose feedstocks. Results of experiments involving spent broth addition do not support a major role for inhibitors present in the liquid phase. Mid-fermentation addition experiments confirm that C. thermocellum and its enzymes remain capable of converting model substrates during the middle of high solids lignocellulose fermentation. An increase in fractional carbohydrate solubilization was made possible by (1) mid-fermentation solid loading dilutions and (2) coculturing C. thermocellum with T. thermosaccharolyticum, which ferments solubilized hemicellulose. Incomplete utilization of solubilized carbohydrates suggests that a small fraction of the carbohydrates is unaffected by the extracellular carbohydrate-active enzymes present in the culture.

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum.

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50-85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose.

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate-1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate-1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 ± 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 ± 0.01 h-1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h-1 Additional introduction of a G-to-V mutation at position 148 in cbpA resulted in immediate growth on fructose at 0.32 ± 0.03 h-1 These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings.IMPORTANCEC. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems in vivo, (iii) allow experiments at >120 g liter-1 soluble substrate concentration, or (iv) reduce costs for labeling studies.

Anatomic Suitability for Branched Thoracic Endovascular Repair in Patients with Aortic Arch Pathological Features.

Background Endovascular repair has become a viable alternative for aortic pathological features, including those located within the aortic arch. We investigated the anatomic suitability for branched thoracic endovascular repair in patients previously treated with conventional open surgery for aortic arch pathological features. Methods and Results Patients who underwent open surgery for aortic arch pathological features at our institution between 2000 and 2018 were included. Anatomic suitability was determined by strict compliance with the anatomic criteria within manufacturers' instructions for use for each of the following branched thoracic stent grafts: Relay Plus Double-Branched (Terumo-Aortic), TAG Thoracic Branch Endoprosthesis (W.L. Gore & Associates), Zenith Arch Branched Device (Cook-Medical), and Nexus Stent Graft System (Endospan Ltd/Jotec GmbH). Computed tomography angiography images were analyzed with outer luminal line measurements. A total of 377 patients (mean age, 64±14 years; 64% men) were identified, 153 of whom had suitable computed tomography angiography images for measurements. In total, 59 patients (15.6% of the total cohort and 38.6% of the measured cohort) were eligible for endovascular repair using at least one of the devices. Device suitability was 30.9% for thoracic aneurysms, 4.6% for type A dissections, 62.5% for type B dissections, and 28.6% for other pathological features. Conclusions The anatomic suitability for endovascular repair of all aortic arch pathological features was modest. The highest suitability rates were observed for thoracic aneurysms and for type B dissections, of which repair included part of the aortic arch. We suggest endovascular repair of arch pathological features should be reserved for high-volume centers with experience in endovascular arch repair.

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. C. thermocellum produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of T. saccharolyticum, ethanol was the main fermentation product. Enzyme assays confirmed that in C. thermocellum, glycolysis proceeds via pyrophosphate (PPi)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In T. saccharolyticum, ATP but not PPi served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in T. saccharolyticum In C. thermocellum, glycolysis via PPi-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PPi → 2 pyruvate + 5 NTP + Pi (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PPi-generating mechanism must be present that operates according to ATP + Pi → ADP + PPi Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type C. thermocellum revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids.IMPORTANCE This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in Escherichia coli but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in Clostridium thermocellum, could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + Pi → ADP + PPi.

Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production.

BACKGROUND: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. RESULTS: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. CONCLUSIONS: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

[Diagnosis and management of vascular malformations : Interdisciplinary teamwork in demand]. / Diagnostik und Management von Gefäßmalformationen : Interdisziplinäres Teamwork gefragt.

Vascular anomalies are very rare, but can occur in children and adults in almost every region of the body. Due to the complexity of this disease, the path to a definitive diagnosis is often difficult. It requires interdisciplinary teamwork with close exchange of information between the individual treatment partners to reach the correct diagnosis and then to start the best therapy. This article provides an overview of the main types of vascular malformations from a clinical, imaging, and histological point of view, following the current classification of the International Society for the Study of Vascular Anomalies (ISSVA).

Correction to: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

[Successful pembrolizumab therapy in metastasized adenosquamous carcinoma of the colon]. / Erfolgreiche Pembrolizumab-Therapie bei metastasiertem adenosquamösem Karzinom des Kolons.

Adenosquamous carcinoma (ASqC) is an exceedingly rare subtype of colorectal cancer without any known special guidelines for treatment. The biological behaviour and molecular background are widely unknown, although a few case studies report a worse prognosis compared to ordinary colorectal adenocarcinoma. We herein report for the first time the successful immune checkpoint inhibitor therapy in a 40-year-old patient suffering from metastasized right-sided colonic ASqC with unique molecular features, after having previously progressed under standard chemotherapy.

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production.

BACKGROUND: Clostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA, for ethanol production. RESULTS: Here, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum. The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes (adhA, nfnA, nfnB, and adhEG544D ) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production. CONCLUSIONS: Here, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.

Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times.

BACKGROUND: Anaerobic fermentation of lignocellulose occurs in both natural and managed environments, and is an essential part of the carbon cycle as well as a promising route to sustainable production of fuels and chemicals. Lignocellulose solubilization by mixed microbiomes is important in these contexts. RESULTS: Here, we report the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times and moderately high (55 °C) temperatures. Anaerobic microbiomes derived from a digester inoculum were incubated at 55 °C and fed semi-continuously with medium containing 30 g/L mid-season harvested switchgrass to achieve residence times (RT) of 20, 10, 5, and 3.3 days. Stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids were achieved for all RTs. Fractional carbohydrate solubilization was 0.711, 0.654, 0.581 and 0.538 at RT = 20, 10, 5 and 3.3 days, respectively, and glucan solubilization was proportional to xylan solubilization at all RTs. The rate of solubilization was described well by the equation r = k(C - C0fr), where C represents the concentration of unutilized carbohydrate, C0 is the concentration of carbohydrate (cellulose and hemicellulose) entering the bioreactor and fr is the extrapolated fraction of entering carbohydrate that is recalcitrant at infinite residence time. The 3.3 day RT is among the shortest RT reported for stable thermophilic, methanogenic digestion of a lignocellulosic feedstock. 16S rDNA phylotyping and metagenomic analyses were conducted to characterize the effect of RT on community dynamics and to infer functional roles in the switchgrass to biogas conversion to the various microbial taxa. Firmicutes were the dominant phylum, increasing in relative abundance from 54 to 96% as RT decreased. A Clostridium clariflavum strain with genetic markers for xylose metabolism was the most abundant lignocellulose-solubilizing bacterium. A Thermotogae (Defluviitoga tunisiensis) was the most abundant bacterium in switchgrass digesters at RT = 20 days but decreased in abundance at lower RTs as did multiple Chloroflexi. Synergistetes and Euryarchaeota were present at roughly constant levels over the range of RTs examined. CONCLUSIONS: A system was developed in which stable methanogenic steady-states were readily obtained with a particulate biomass feedstock, mid-season switchgrass, at laboratory (1 L) scale. Characterization of the extent and rate of carbohydrate solubilization in combination with 16S rDNA and metagenomic sequencing provides a multi-dimensional view of performance, species composition, glycoside hydrolases, and metabolic function with varying residence time. These results provide a point of reference and guidance for future studies and organism development efforts involving defined cultures.

Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum.

BACKGROUND: Milling during fermentation, termed cotreatment, has recently been proposed as an alternative to thermochemical pretreatment as a means to increase the accessibility of lignocellulosic biomass to biological attack. A central premise of this approach is that partial solubilization of biomass changes the slurry's physical properties such that milling becomes more impactful and more feasible. A key uncertainty is the energy required to mill partially fermented biomass. To inform both of these issues, we report rheological characterization of small-particle, corn stover slurries undergoing fermentation by Clostridium thermocellum. RESULTS: Fermented and unfermented corn stover slurries were found to be shear-thinning and well described by a power law model with an exponent of 0.10. Plastic viscosity of a slurry, initially at 16 wt.% insoluble solids, decreased as a result of fermentation by a factor of 2000, with the first eightfold reduction occurring in the first 10% of carbohydrate conversion. Large amplitude oscillatory shear experiments revealed only minor changes to the slurry's rheological fingerprint as a result of fermentation, with the notable change being a reduction in the critical strain amplitude needed for the onset of nonlinearity. All slurries were found to be elastoviscoplastic, with the elastic/viscous crossover at roughly 100% strain amplitude. CONCLUSIONS: Whereas prior biomass rheology studies have involved pretreated feedstocks and solubilization mediated by fungal cellulase, we report results for feedstocks with no pretreatment other than autoclaving and for solubilization mediated by C. thermocellum. As observed in prior studies, C. thermocellum fermentation results in a dramatic decrease in viscosity. The magnitude of this decrease, however, is much larger starting with unpretreated feedstock than previously reported for pretreated feedstocks. LAOS measurements provide a detailed picture of the rheological fingerprint of the material. Viscosity measurements confirm the hypothesis that the physical character of corn stover slurries changes dramatically during fermentation by C. thermocellum, and indicate that the energy expended on overcoming slurry viscosity will be far less for partially fermented corn stover than for unfermented corn stover.