Applied Biochemistry and Biotechnology (v.89, #1)

Continuous wine making by γ-alumina-supported biocatalyst by P. Loukatos; M. Kiaris; I. Ligas; G. Bourgos; M. Kanellaki; M. Komaitis; A. A. Koutinas (1-13).
The main objective of the present work was the removal of aluminum from wines produced by γ-alumina-supported yeast cells. Reagents such as Na2CO3, NH4OH, albumin, and Ca(OH)2 were used. Calcium in the presence of albumin was effective, whereas other reagents were not so effective. Because of the improved aroma and taste of distillates produced by γ-alumina-supported biocatalyst, volatile byproducts of distillates were analyzed. They were also assessed by sensory tests. Methanol, acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol, and amyl alcohols were determined in distillates. It was noted that the amounts of higher alcohols and amyl alcohols decreased as the temperature of fermentation dropped, leading to a product of improved quality and reduced toxicity.
Keywords: Wine; distillates; aluminum; volatiles; quality

Bioconversion of cellulose into ethanol by nonisothermal simultaneous saccharification and fermentation by Kyeong-Keun Oh; Seung-Wook Kim; Yong-Seob Jeong; Suk-In Hong (15-30).
The kinetic characteristics of cellulase and β-glucosidase during hydrolysis were determined. The kinetic parameters were found to reproduce experimental data satisfactorily and could be used in a simultaneous saccharification and fermentation (SSF) system by coupling with a fermentation model. The effects of temperature on yeast growth and ethanol production were investigated in batch cultures. In the range of 35–45°C, using a mathematical model and a computer simulation package, the kinetic parameters at each temperature were estimated. The appropriate forms of the model equation for the SSF considering the effects of temperature were developed, and the temperature profile for maximizing the ethanol production was also obtained. Briefly, the optimum temperature profile began at a low temperature of 35°C, which allows the propagation of cells. Up to 10 h, the operating temperature increased rapidly to 39°C, and then decreased slowly to 36°C. In this nonisothermal SSF system with the above temperature profile, a maximum ethanol production of 14.87 g/L was obtained.
Keywords: Kinetic modeling; temperature profile; nonisothermal simultaneous saccharification and fermentation

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.
Keywords: Trichoderma reesei RUT C30; cellulase; steam-pretreated willow; inhibition; acetic acid; furfural

Immobilization of Zymomonas mobilis by different methods was investigated. Experiments were performed order to choose the most appropriate support for the immobilization of the cells. The most advantageous option was to use permeabilized cells in the bore of microporous hollow fibers. Whereas the reaction rate was about 33 g of gluconate/ (g of protein·h) using hollow fibers, which is comparable to that observed by using free cells, the calcium alginate immobilized cells presented a reaction rate of 4 g of gluconate/ (g of protein·h). These results can be explained by the mass transfer resistance effect, which, indeed, was much lower in the case of hollow-fiber membranes than in the alginate gel beads. A loss of enzymatic activity during the reaction was observed in all experiments, which was attributed to the lactone produced as an intermediate of the reaction.
Keywords: Hollow fibers; Zymomonas mobilis ; sorbitol; gluconic acid; GFOR

A mediated modified carbon paste and renewable surface electrode for fructose amperometric measurement based on d-fructose dehydrogenase (FDH) was prepared and optimized. Commercially available ferrocene (FcH) and hydroxymethyl ferrocene (FcCH2OH) were used as mediators. The substituted FcH showed better linearity and higher sensitivity. The influence of different experimental parameters was studied for optimum analytical performance. The final FDH-modified electrode showed good analytical performance for batch mode measurements of fructose.
Keywords: Renewable surface electrode; modified carbon paste electrode; fructose dehydrogenase; fructose

Retrofit of CD-6 (smith) impeller in fermentation vessels by Beth H. Junker; Zorina Mann; George Hunt (67-83).
We extended prior studies on the influence of impeller type on fermentation performance to include a novel low-power-number, high-efficiency radial flow impeller, the CD-6, possessing six curved blades on a disk turbine. Dual impeller combinations of CD-6/CD-6, CD-6/Maxflo T, and CD-6/HE-3 were compared with Rushton/Rushton and Maxflo T/Maxflo T base cases. Qualitative comparisons of unaerated and aerated power draw in both water and glycerol were conducted. These suggested minimal power drops with aeration for dual CD-6 impellers and hybrids containing the CD-6 impeller design. We also examined fermentation performance for Streptomyces and Glarea secondary metabolite fermentations. A qualitative comparison of the data suggested that dual CD-6 impellers and hybrids containing the CD-6 impeller design resulted in reasonable power draws, improved mass transfer rates with airflow increases, and acceptable peak titers. These arrangements may warrant further study under a wider range of production conditions.
Keywords: CD-6; hydrofoil; impeller, axial; impeller, radial; impeller, power; fermentation

A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development by Bernd Sojka; Paul A. E. Piunno; Christopher C. Wust; Ulrich J. Krull (85-103).
Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 1010 strands/mm2. This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 1010/mm2 and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.
Keywords: Covalent immobilization; glass supports; solid-phase synthesis; phosphoramidite chemistry; linker; hexaethylene glycol; hybridization; biosensor; oligonucleotides; nucleic acids