The influence of the factors acetic acid, furfural, and p-hydroxybenzoic acid on the ethanol yield (Y EtOH) of Saccharomyces cerevisiae, bakers' yeast, S. cerevisiae ATCC 96581, and Candida shehatae NJ 23 was investigated using a 2 3-full factorial design with 3 centrepoints. The results indicated that acetic acid inhibited the fermentation by C. shehatae NJ 23 markedly more than by bakers' yeast, whereas no significant difference in tolerance towards the compounds was detected between the S. cerevisiae strains. Furfural (2 g L −1) and the lignin derived compound p-hydroxybenzoic acid (2 g L −1) did not affect any of the yeasts at the cell mass concentration used. The results indicated that the linear model was not adequate to describe the experimental data (the p-values of curvatures were 0.048 for NJ 23 and 0.091 for bakers' yeast). Based on the results from the 2 3-full factorial experiment , an extended experiment was designed based on a central composite design to investigate the influence of the factors on the specific growth rate (µ), bio-mass yield (Y x), volumetric ethanol productivity (Q EtOH), and Y EtOH. Bakers' yeast was chosen in the extended experiment due to its better tolerance towards acetic acid, which makes it a more interesting organism for use in industrial fermentations of lignocellulosic hydrolysates. The inoculum size was reduced in the extended experiment to reduce any increase in inhibitor tolerance that might be due to a large cell inoculum. By dividing the experiment in blocks containing fermentations performed with the same inoculum preparation on the same day, much of the anticipated systematic variation between the experiments was separated from the experimental error. The results of the fitted model can be sum-marised as follows: µ was decreased by furfural (0-3 g L − 1). Furfural and acetic acid (0-10 g L − 1) also interacted negatively on µ. Furfural concentrations up to 2 g L −1 stimulated Y x in the absence of acetic acid whereas higher concentrations decreased Y x. The two compounds interacted negatively on Y x and Y EtOH. Acetic acid concentrations up to 9 g L −1 stimulated Q EtOH , whereas furfural (0-3 g L −1) decreased Q EtOH. Acetic acid in concentrations up to 10 g L −1 stimulated Y EtOH in the absence of furfural, and furfural (0-2 g L −1) slightly increased Y EtOH in the absence of acetic acid whereas higher concentrations caused inhibition. Acetic acid and furfural interacted negatively on Y EtOH .

No abstract

No abstract

An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.Sequence-specific nucleic acid quantification in areas such as diagnostic PCR and molecular biology has been greatly improved by the introduction of real-time PCR technology (9). While this technology has tremendous potential for accurate and sensitive quantification, further studies addressing the quantification aspect of this technology are required before it can be widely implemented. Previous results from this laboratory (5), in which the range of detection of real-time PCR was modeled in a pure system using two different DNA polymerases, gave an indication that DNA polymerases and their buffer systems influence the performance of PCR by affecting the detection window and linear range of amplification. The aim of this work was to systematically study the effect of five DNA polymerase-buffer systems on absolute quantification using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany).A primer set, Y1 and Y2, for Yersinia enterocolitica was used (6). To a commercial LightCycler kit (LCTaq) (Roche Diagnostics), a 0.4 mM concentration of each primer was added together with 4 mM MgCl 2 . Sterile Millipore water was added to a volume of 16 l and complemented with 4 l of Y. enterocolitica DNA. The concentration of DNA was fluorimetrically determined using a TD-700 fluorimeter (Turner Designs, Sunnyvale, Calif.), and the DNA was diluted to appropriate concentrations in sterile Millipore water. The four other master mixtures, contained 2.5 U of DNA polymerase and 1ϫ associated buffer, 4 mM MgCl 2 , 0.4 ml of each primer, 0.2 mM (each) deoxynucleoside triphosphate, 10,000-fold-diluted SYBR Green I, and 4 l of Y. enterocolitica DNA in a total volume of 20 l. The following DNA polymerases were used: DyNazyme II (FINNZYMES OY, Espoo, Finland), rTth (Applied Biosystems, Foster City, Calif.), and Taq (Roche Diagnostics) and Tth (Roche Diagnostics). Each amplification started with a denaturation step of 1 min at 95°C, followed by 40 cycles of 0.1 s of denaturation at 95°C, 5 s of annealing at 60°C, and elongation for 15 s at 72°C, followed by a single fluorescence measurement and finally 25 s of final elongation. Amplification was followed by melting curve analysis between 65 and 95°C and finally cooling for 1 min at 40°C. The quantification data, in terms of the crossing point value (ROM) (which is expressed as the fractional cycle number and is the intersection of the log-linear fluorescence curve with a threshold crossing line), were determined using the second derivative method of the LightCycler Software, version 3 (Roche Diagnostics).Amplification efficiency and analytical repea...