Low carbon fuels and commodity chemicals from waste gases – systematic approach to understand energy metabolism in a model acetogen

Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains.

Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

Tailoring texture of γ-Y2Si2O7 by strong magnetic field alignment and two-step sintering

In this study, a well-dispersed γ-Y2Si2O 7 ethanol-based suspension with 30 vol% solid loading was prepared by adding 1 dwb% polyethylene imine dispersant, which allows feeble magnetic γ-Y2Si2O7 particles with anisotropic magnetic susceptibility to rotate in a 12 T strong magnetic field during slip casting, resulting in the development of a strong texture in green bodies. Pressureless sintering gives rise to more pronounced grain growth in the textured sample than in the untextured sample prepared without the magnetic field due to the rapid migration of the grain boundaries of the well-oriented grains, which was revealed by constant-heating-rate sintering kinetics. It was found that the use of two-step sintering is very efficient not only for inhibiting the grain growth but also for enhancing the texture. This implies that controlled grain growth is crucial for enhancing texture development in γ-Y2Si2O7.

Sugarcane-Based Biofuels and Bioproducts

Sugarcane has garnered much interest for its potential as a viable renewable energy crop. While the use of sugar juice for ethanol production has been in practice for years, a new focus on using the fibrous co-product known as bagasse for producing renewable fuels and bio-based chemicals is growing in interest. The success of these efforts, and the development of new varieties of energy canes, could greatly increase the use of sugarcane and sugarcane biomass for fuels while enhancing industry sustainability and competitiveness. Sugarcane-Based Biofuels and Bioproducts examines the development of a suite of established and developing biofuels and other renewable products derived from sugarcane and sugarcane-based co-products, such as bagasse. Chapters provide broad-ranging coverage of sugarcane biology, biotechnological advances, and breakthroughs in production and processing techniques. This text brings together essential information regarding the development and utilization of new fuels and bioproducts derived from sugarcane. Authored by experts in the field, Sugarcane-Based Biofuels and Bioproducts is an invaluable resource for researchers studying biofuels, sugarcane, and plant biotechnology as well as sugar and biofuels industry personnel.

Breath Testing by Fourier Transform Infrared Spectroscopy for Solvent Intoxication Diagnostics

Technical or contaminated ethanol products are sometimes ingested either accidentally or on purpose. Typical misused products are black-market liquor and automotive products, e.g., windshield washer fluids. In addition to less toxic solvents, these liquids may contain the deadly methanol. Symptoms of even lethal solvent poisoning are often non-specific at the early stage. The present series of studies was carried out to develop a method for solvent intoxication breath diagnostics to speed up the diagnosis procedure conventionally based on blood tests. Especially in the case of methanol ingestion, the analysis method should be sufficiently sensitive and accurate to determine the presence of even small amounts of methanol from the mixture of ethanol and other less-toxic components. In addition to the studies on the FT-IR method, the Dräger 7110 evidential breath analyzer was examined to determine its ability to reveal a coexisting toxic solvent. An industrial Fourier transform infrared analyzer was modified for breath testing. The sample cell fittings were widened and the cell size reduced in order to get an alveolar sample directly from a single exhalation. The performance and the feasibility of the Gasmet FT-IR analyzer were tested in clinical settings and in the laboratory. Actual human breath screening studies were carried out with healthy volunteers, inebriated homeless men, emergency room patients and methanol-intoxicated patients. A number of the breath analysis results were compared to blood test results in order to approximate the blood-breath relationship. In the laboratory experiments, the analytical performance of the Gasmet FT-IR analyzer and Dräger 7110 evidential breath analyzer was evaluated by means of artificial samples resembling exhaled breath. The investigations demonstrated that a successful breath ethanol analysis by Dräger 7110 evidential breath analyzer could exclude any significant methanol intoxication. In contrast, the device did not detect very high levels of acetone, 1-propanol and 2-propanol in simulated breath. The Dräger 7110 evidential breath ethanol analyzer was not equipped to recognize the interfering component. According to the studies the Gasmet FT-IR analyzer was adequately sensitive, selective and accurate for solvent intoxication diagnostics. In addition to diagnostics, the fast breath solvent analysis proved feasible for controlling the ethanol and methanol concentration during haemodialysis treatment. Because of the simplicity of the sampling and analysis procedure, non-laboratory personnel, such as police officers or social workers, could also operate the analyzer for screening purposes.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2 minutes), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3 minutes), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (~40% of corneal surface at 5 weeks), corneal neovascularization (2 to 3 quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.

N,N′-Bis(aryl)pyridine-2,6-dicarboxamide complexes of ruthenium: Synthesis, structure and redox properties

Reaction of five N,N′-bis(aryl)pyridine-2,6-dicarboxamides (H2L-R, where H2 denotes the two acidic protons and R (R = OCH3, CH3, H, Cl and NO2) the para substituent in the aryl fragment) with [Ru(trpy)Cl3](trpy = 2,2′,2″-terpyridine) in refluxing ethanol in the presence of a base (NEt3) affords a group of complexes of the type [RuII(trpy)(L-R)], each of which contains an amide ligand coordinated to the metal center as a dianionic tridentate N,N,N-donor along with a terpyridine ligand. Structure of the [RuII(trpy)(L-Cl)] complex has been determined by X-ray crystallography. All the Ru(II) complexes are diamagnetic, and show characteristic 1H NMR signals and intense MLCT transitions in the visible region. Cyclic voltammetry on the [RuII(trpy)(L-R)] complexes shows a Ru(II)–Ru(III) oxidation within 0.16–0.33 V versus SCE. An oxidation of the coordinated amide ligand is also observed within 0.94–1.33 V versus SCE and a reduction of coordinated terpyridine ligand within −1.10 to −1.15 V versus SCE. Constant potential coulometric oxidation of the [RuII(trpy)(L-R)] complexes produces the corresponding [RuIII(trpy)(L-R)]+ complexes, which have been isolated as the perchlorate salts. Structure of the [RuIII(trpy)(L-CH3)]ClO4 complex has been determined by X-ray crystallography. All the Ru(III) complexes are one-electron paramagnetic, and show anisotropic ESR spectra at 77 K and intense LMCT transitions in the visible region. A weak ligand-field band has also been shown by all the [RuIII(trpy)(L-R)]ClO4 complexes near 1600 nm.

Solubilities of Hexadecanoic and Octadecanoic Acids in Supercritical CO2 With and Without Cosolvents

The solubilities of hexadecanoic acid (palmitic acid) and octadecanoic acid (stearic acid) in supercritical carbon dioxide without cosolvents and with two cosolvents, namely, ethanol and 3-methyl-1-butanol, were determined at (308 and 318) K at pressures varying from (12.8 to 22.6) MPa. The solubility data, in both the absence and presence of cosolvents, were correlated by a model proposed by Mendez-Santiago and Teja.

Chain structures in alkali metal borophosphates: synthesis and characterization of K3[BP3O9(OH)3] and Rb3[B2P3O11(OH)2]

Two new alkali metal borophosphates, K-3[BP(3)o(9)(OH)(3)] and Rb-3[B2P3O11(OH)(2)], were synthesized by applying solvothermal techniques using ethanol as solvent. The crystal structures were solved by means of single-crystal X-ray diffraction (K-3[BP3O9(OH)(3)], monoclinic, C2/c (No. 15), a = 2454.6(8) pm, b = 736.3(2) pm, c = 1406.2(4) pm, beta = 118.35(2)degrees, Z = 8; Rb-3[B2P3O11(OH)(2)], monoclinic, P2(1)/c (No. 14), a = 781.6(2) pm, b:= 667.3(2) pm, c = 2424.8(5) pm, beta = 92.88(1)degrees, Z = 4). Both crystal structures comprise borophosphate chain anions. While for the rubidium compound a loop-branched chain motif is found as common for most of the chain anions in alkali metal borophosphates, the crystal structure of the potassium phase comprises the first open-branched chain with the highest phosphate content found so far in this group of compounds. Both chain anions are Closely related to known anhydrous or hydrated phases, and the structural relations are discussed in terms of how the presence of OH groups and hydrogen bonds as well as number, charge, and size of charge balancing cations influence the 3D structural arrangement. The anionic entities are classified in terms of general principles of structural systematics for borophosphates.

Formation of an oxo-radical of peroxovanadate during reduction of diperoxovanadate with vanadyl sulfate or ferrous sulfate

Formation of oxygen radicals during reduction of H2O2 or diperoxovanadate with vanadyl sulfate or ferrous sulfate was indicated by the 1:2:2:1 electron spin resonance (ESR) signals of the DMPO adduct typical of standard radical dotOH radical. Signals derived from diperoxovanadate remained unchanged in the presence of ethanol in contrast to those from H2O2. This gave the clue that they represent a different radical, possibly radical dotOV(O2)2+, formed on breaking a peroxo-bridge of diperoxovanadate complex. The above reaction mixtures evolved dioxygen or, when NADH was present, oxidized it rapidly which was accompanied by consumption of dioxygen. Operation of a cycle of peroxovanadates including this new radical is suggested to explain these redox activities both with vanadyl and ferrous sulfates. It can be triggered by ferrous ions released from cellular stores in the presence of catalytic amounts of peroxovanadates.

Effect of vitamins A and K on colon lysosomes

1. 1. Colon lysosome were separated by differential centrifugation and lysosomes with three different densities, probably arising from the three layers of colon, were found. 2. 2. Hypervitaminosis A resulted in a significant increase in prothrombin time which was restored to normal on vitamin K1 (20) supplementation. 3. 3. There was no appreciable change in the liver storage of vitamin A between hypervitaminotic rats receiving vitamin A and those rats receiving vitamin K1 (20) in addition to excess vitamin A. 4. 4. The colon lysosomes were unstable in hypervitaminosis A, showing an increased free activity of lysosomal enzymes like β-glucuronidase, acid phosphatase and arylsulphatase. This increase of free activity of lysoso3al enzymes in hypervitaminosis A could be prevented by oral supplementation of vitamin K1 (20). 5. 5. In "mild" vitamin A deficiency the release of arylsulphatase was significantly retarded, whereas the decreased free acid phosphatase activity was not significant. 6. 6. "Severe" vitamin A deficiency resulted in a significantly increased free activity of arylsulphatase and acid phosphatase, thus showing the instability of the lysosomal particles in this condition. 7. 7. Addition of vitamin K1 (20) to the incubation medium in vitro could prevent the vitamin A-induced release of arylsulphatase from liver lysosomes, whereas α-tocopherol was inactive. 8. 8. Retinol and retinoic acid were nearly twice as active as ethanol in the release of arylsulphatase from lysosomes in vitro, whereas 5,6-monoepoxyretinoic acid was inactive. 9. 9. The role of vitamins A and K on the lysosomal membrane structure is discussed.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Hydrolysis of riboflavin in plants

The enzymic hydrolysis of riboflavin to lumichrome and ribitol by extracts of Crinum longifolium bulbs has been demonstrated. The enzyme was purified 48-fold by ZnSO4 treatment and ethanol fractionation, and concentrated by using Sephadex G-25. After establishing the stoichiometry of the reaction, the general properties of the purified enzyme were studied. The enzyme showed maximal activity at pH 7·5, and it had a requirement for reduced glutathione which could be replaced by cysteine or ascorbic acid. Mg2+ and Li+ activated the enzyme. The reaction was highly specific to riboflavin and was competitively inhibited by riboflavin 5′-phosphate.

Nanostructured Copper(II) oxide thin film for alcohol sensing

Nanostructured copper(II) oxide film was deposited using reactive DC magnetron sputtering. It has been characterized using XRD, EDAX, XPS, and FESEM. The grain size of copper oxide film was found to be 40-65 nm with size distribution. The entire study was divided into two parts. In the first part, the film has been studied for its response to alcohol at different temperatures to find the optimum sensing temperature, whereas in the second part, the film sensitivity to different alcohol concentrations were studied at fixed optimum operating temperature. The optimum temperature for the response of ethanol was observed to be 400 C,and the response for different concentrations was found to be almost linear.