Ilse Van De Voorde

Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production

Saccharomyces cerevisiae is the organism of choice for many food and beverage fermentations because it thrives in high-sugar and high-ethanol conditions. However, the conditions encountered in bioethanol fermentation pose specific challenges, including extremely high sugar and ethanol concentrations, high temperature, and the presence of specific toxic compounds. It is generally considered that exploring the natural biodiversity of Saccharomyces strains may be an interesting route to find superior bioethanol strains and may also improve our understanding of the challenges faced by yeast cells during bioethanol fermentation. In this study, we phenotypically evaluated a large collection of diverse Saccharomyces strains on six selective traits relevant for bioethanol production with increasing stress intensity. Our results demonstrate a remarkably large phenotypic diversity among different Saccharomyces species and among S. cerevisiae strains from different origins. Currently applied bioethanol strains showed a high tolerance to many of these relevant traits, but several other natural and industrial S. cerevisiae strains outcompeted the bioethanol strains for specific traits. These multitolerant strains performed well in fermentation experiments mimicking industrial bioethanol production. Together, our results illustrate the potential of phenotyping the natural biodiversity of yeasts to find superior industrial strains that may be used in bioethanol production or can be used as a basis for further strain improvement through genetic engineering, experimental evolution, or breeding. Additionally, our study provides a basis for new insights into the relationships between tolerance to different stressors.

Complexation Behavior of Iron(III) with aloxime 800, D(2)EHPA, CYANEX 272, CYANEX 302, and CYANEX 301

Abstract The extraction of iron(III) has been studied from chloride solutions with di(2‐ethylhexyl)phosphoric acid (D2EHPA), bis(2,4,4‐trimethylpentyl)phosphinic acid (CYANEX 272) and its sulfur‐substituted analogs, called CYANEX 302, and CYANEX 301, and 5‐dodecylsalicylaldoxime (Aloxime 800). Jobs method was applied for the characterization of the iron(III) complexes dissolved in hexane. In the case of D2EHPA and CYANEX 272, a 1:1 ligand‐to‐metal ratio was observed, thus inferring the coordination of additional compounds. No chloride transport occurred during extraction, therefore suggesting the formation of [Fe(OH)2L] complexes. With CYANEX 302, a ratio of 2:1 was obtained, whereas for CYANEX 301, the results of Jobs method indicated the presence of four extractant molecules around the metal ion. Less hydrolysis or the possible oxidation of the sulfur‐substituted organophosphinic acids and the corresponding reduction of Fe(III) towards Fe(II) may explain this behavior. In the case of Aloxime 800, the formation of the [FeL3] species is suggested. A comparative study was carried out to identify the ligand‐to‐metal ratio of the iron(III) complexes in anhydrous circumstances. These studies showed that 1:1 ligand‐metal complexes are easily formed in the case of the organophosphoric‐ and organophosphinic‐acid extractants. A higher ligand‐metal ratio may be possible, but is not always a necessary condition for iron(III) extraction. Even the coexistence of [FeCl2L], [FeClL2] and to a smaller extent [FeL3] is quite presumable in anhydrous media. Finally, FT‐IR spectra as well as UV‐VIS spectra of the hexane phases make it possible to gain a better insight into the complexation characteristics of iron(III).

Overexpression of L-Arabinose Isomerase for Production of the Low-Calorie Bulk Sweetener D-Tagatose

High-Cell-Density Cultivations show a huge potential to produce recombinant proteins to amounts greatly exceeding the availability in natural resources. An interesting example of a recombinant protein is an L-arabinose isomerase, which is able to convert D-galactose to the low-caloric and low-glycaemic bulk sweetener D-tagatose. Within this study, the L-arabinose isomerase from Geobacillus stearothermophilus was expressed intracellularly in Escherichia coli. The cultivation medium contained glucose, yeast extract and various macro- and micronutrients. The effect of air flow rate on E. coli growth and expression of L-arabinose isomerase was studied. After 52 hours, an Optical Density and Dry Cell Weight of 154 ± 4 and 54.8 ± 1.3 g L-1 were reached, respectively by regulating the air flow rate between 0.2 and 30 L min-1. A corresponding L-arabinose isomerase activity of 6.99 ± 0.46 U mL-1 was reached. A drawback of High-Cell-Density Cultivation is the production of the by-product acetic acid which may inhibit growth. However, the acetic acid concentration was maintained as low as possible during fermentation to avoid inhibitory effects inherent to this compound. With the L-arabinose isomerase produced, a conversion percentage of 37.1 ± 1.5% was achieved, corresponding to 94.9 ± 3.7 g L-1 D-tagatose. Thus, the implementation of a High-Cell- Density Cultivation led to an efficient expression of the L-arabinose isomerase enzyme and D-tagatose production. Also the storage stability of the cells was investigated during several months at 4°C. A stable L-arabinose isomerase enzyme was noticed during at least 8 months storage at 4°C.

Evaluation of the Extraction Efficiency of Enzymatically Treated Flax Fibers

This study evaluates the efficiency of the extraction of enzymatically treated flax fibers for application in composite materials. Three extraction parameters were introduced. Fiber Efficiency (FE) represents the yield of long fibers extracted after enzymatic treatment. Time Efficiency (TE) is introduced in order to evaluate the ease of extraction. Taking into account both Fiber Efficiency and Time Efficiency results in the introduction of the overall Extraction Efficiency (EE). The Extraction Efficiency is determined for flax fibers treated with pectate lyase, pectin lyase, polygalacturonase, endoxylanase and Viscozyme L as reference while comparing with green and dew retted fibers. Moreover, fiber fineness and transverse properties of the resultant composite materials were investigated. Polygalacturonase treatment appears to be the most promising. Results demonstrate the potential of enzymatic extraction of flax fibers with an increased transverse bending strength of the composite reinforced with polygalacturonase treated fibers (26.4 ± 3.8 MPa) compared to green flax fiber composite (12.1 ± 1.4 MPa) and dew retted fiber composite (16.9 ± 2.2 MPa).