Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.     

Food Preference and Appetite after Switching between Sweet and Savoury Odours in Women

Background

Exposure to food odours increases the appetite for congruent foods and decreases the appetite for incongruent foods. However, the effect of exposure to a variety of food odours, as often occurs in daily life, is unknown.

Objective

Investigate how switching between sweet and savoury odours affects the appetite for sweet and savoury products.

Design

Thirty women (age: 18-45y; BMI: 18.5-25kg/m²) intensely smelled the contents of cups filled with banana, meat or water (no-odour) in a within-subject design with four combinations: no-odour/banana, no-odour/meat, meat/banana and banana/meat. Participants received one combination per test day. In each combination, two cups with different fillings were smelled for five minutes after each other. Treatment order was balanced as much as possible. The effects of previous exposure and current odour on the appetite for (in)congruent sweet and savoury products, and odour pleasantness were analysed. A change from meat to banana odour or banana to meat odour was referred to as switch, whereas a change from no-odour to meat odour or no-odour to banana odour was no-switch.

Results

The current odour (P<0.001), as opposed to the previous exposure (P = 0.71), determined the appetite for (in)congruent sweet and savoury products, already one minute after a switch between sweet and savoury odours. The pleasantness of the odour decreased during odour exposure (P = 0.005).

Conclusions

After a switch, the appetite for specific products quickly adjusted to the new odour and followed the typical pattern as found during odour exposure in previous studies. Interestingly, the appetite for the smelled food remained elevated during odour exposure, known as sensory-specific appetite, whereas the pleasantness of the odour decreased over time, previously termed olfactory sensory-specific satiety. This seeming contradiction may result from different mechanisms underlying the odour-induced anticipation of food intake versus the decrease in hedonic value during prolonged sensory stimulation.     

Clostridium beijerinckii Cells Expressing Neocallimastix patriciarum Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.     

Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.     

Substrate-induced production and secretion of cellulases by Clostridium acetobutylicum

Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of beta-1,3- and beta-1,4-linked beta-D-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.     

Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

Background  

The production of hydrogen from biomass by fermentation is one of the routes that can contribute to a future sustainable hydrogen economy. Lignocellulosic biomass is an attractive feedstock because of its abundance, low production costs and high polysaccharide content.     

Production by Clostridium acetobutylicum ATCC 824 of CelG,a cellulosomal glycoside hydrolase belonging to family 9

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon