Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Engineering a native homoethanol pathway in Escherichia coli B for ethanol production

A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase (frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter. The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90% under anaerobic conditions.     

Re-engineering Escherichia coli for ethanol production

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160 (KO11, Δfrd::celY _Ec ΔadhE ΔldhA, ΔackA lacA::casAB _Ko rrlE::(pdc _Zm -adhA _Zm -adhB _Zm -FRT-rrlE) pflB ⁺ ). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.     

Quebrachitol-induced gastroprotection against acute gastric lesions: Role of prostaglandins, nitric oxide and KATP channels

The effect of Quebrachitol (2-O-methyl-l-inositol), a bioactive component from Magonia glabrata fruit extract was investigated against gastric damage induced by absolute ethanol (96%, 0.2 ml/animal) and indomethacin (30 mg/kg, p.o.), in mice. Quebrachitol at oral doses of 12.5, 25, and 50 mg/kg markedly attenuated the gastric lesions induced by ethanol to the extent of 69%, 64%, and 53% and against indomethacin by 55%, 59%, and 26%, respectively. While pretreatment with TRPV1 antagonist capsazepine (5 mg/kg, i.p.) failed to block effectively the gastroprotective effect of quebrachitol (25 mg/kg) against ethanol damage, the non-selective cyclooxygenase inhibitor indomethacin (10 mg/kg, p.o.), almost abolished it. Furthermore, quebrachitol effect was significantly reduced in mice pretreated with l-NAME, or glibenclamide, the respective inhibitors of nitric oxide synthase and K⁺_ATP channel activation. Thus we provide the first evidence that quebrachitol reduces the gastric damage induced by ethanol and indomethacin, at least in part, by mechanisms that involve endogenous prostaglandins, nitric oxide release, and or the activation of K⁺_ATP channels.     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

Construction of ethanol-tolerant yeast strains with combinatorial library-selected peptides

Combinatorial yeast libraries were constructed by transformation of expression plasmids containing artificially synthesized random sequences into Saccharomyces cerevisiae MT8-1 and IFO10150. Approximately 200 yeast strains with enhanced ethanol tolerance were obtained from yeast libraries by incubation in 10% ethanol for 24 h. Following separate evaluation of their ethanol tolerance, the 10 clones with the highest values were selected. After 3 h incubation in 12.5% ethanol, whereas most of the control cells died, the clone with the highest tolerance from the MT8-1 library, M-1, showed approximately 40% cell viability, and the corresponding clone from the IFO10150 library, I-12, 48% viability. The half-life of M-1 cells was 20 times greater than that of control cells. Three of the library-selected peptides endowing with ethanol tolerance were identified as Gly-Thr-Arg-Leu-His pentapeptides. Four seemed to be extremely hydrophobic, and three of these were predicted to be transmembrane peptides. The three other peptides seemed to be more hydrophilic than standard yeast proteins. The results of the study show that yeast strains with fairly high ethanol tolerance can be successfully constructed by directed selection from yeast libraries expressing combinatorial peptides.     

Analysis of ethanol–glucose mixtures by two microbial sensors: application of chemometrics and artificial neural networks for data processing

Although biosensors based on whole microbial cells have many advantages in terms of convenience, cost and durability, a major limitation of these sensors is often their inability to distinguish between different substrates of interest. This paper demonstrates that it is possible to use sensors entirely based upon whole microbial cells to selectively measure ethanol and glucose in mixtures. Amperometric sensors were constructed using immobilized cells of either Gluconobacter oxydans or Pichia methanolica. The bacterial cells of G. oxydans were sensitive to both substrates, while the yeast cells of P. methanolica oxidized only ethanol. Using chemometric principles of polynomial approximation, data from both of these sensors were processed to provide accurate estimates of glucose and ethanol over a concentration range of 1.0–8.0 mM (coefficients of determination, R²=0.99 for ethanol and 0.98 for glucose). When data were processed using an artificial neural network, glucose and ethanol were accurately estimated over a range of 1.0–10.0 mM (R²=0.99 for both substrates). The described methodology extends the sphere of utility for microbial sensors.     

Adaptive strategies in natural populations of Drosophila

Summary Adult tolerance of ethanol vapour in a closed system containing 12% ethanol in solution, decreases in a cline from southern to northern Australia. However a Darwin population is more tolerant than predicted from its latitude. Ethanol tolerance races in Australia have almost certainly evolved within the last 100–150 years, because of resource unavailability prior to that time. Within populations, variation among isofemale strains is lowest in the climatically extreme southern Melbourne (37°S) and northern Darwin and Melville I. (11–12°S) populations. This suggests low resource diversity within extreme populations compared with the climatically less extreme Brisbane (28°S) and especially Townsville (19°S) populations. For desiccation resistance, the population rankings are: Darwin Melbourne > Townsville > Brisbane Melville I. and for development time, rankings are similar: Darwin Melbourne < Townsville < Brisbane Melville I.Therefore resource utilization heterogeneity is greatest in populations not greatly stressed by desiccation and where development times are extended. In total therefore, the utilization of a diversity of resources is a feature of populations tending somewhat towards a K-strategy; this is emphasized by the relative heterogeneity among isofemale strains of these populations for desiccation resistance and to a lesser extent development times.The D. melanogaster gene pool can be viewed as made up of climate-associated races. Since the ethanol tolerances of adjacent (and climatically similar) Darwin and Melville I. are very different, resource utilization races may occur within climatic races. Such a mosaic of resource utilization races are more likely in climatically extreme than in optimal habitats.