Ethanol tolerance in Saccharomyces cerevisiae is a complex polygenic trait. As a toxin, ethanol damages multiple cell constituents as well as being both a substrate and product of the metabolism of S.cerevisiae. This complexity has made ethanol tolerance difficult to study. Deletion screens have identified hundreds of genes that impair ethanol tolerance by their absence and hence might help survival in high ethanol environments. Similarly, expression studies have revealed genes that respond to ethanol shock, but it is unclear whether those genes are likely targets for improvement of ethanol tolerance in strains growing normally. In addition, those yeasts that are currently commercially exploited for their high ethanol tolerance in the brewing and bioethanol industries are commonly aneuploid or polyploid which makes it difficult to correlate particular features of their genotype with the ethanol tolerant phenotype. Experimental evolution can reveal genetic changes that change competitive fitness. It is practical to run numerous competitions in parallel between isogenic S.cerevisiae strains for hundreds of generations under ethanol stress, after which whole genome sequencing can identify the genetic changes. Fluorescent tagging of those strains can reveal small changes in population dynamics. We propagated 144 populations in batch culture for between 100 and 200 generations under 4 ethanol regimes (0%, 4.5%, 6.5% and ramped 0-10%). We monitored the progress of evolution by using mixtures of two fluorescently tagged, but otherwise isogenic, haploid, hoΔ (site-specific endonuclease deletant) founder strains (DeLuna et al 2008). Population dynamics measured using fluorescently labelled strains indicated that changes had occurred in competitive fitness due to adaptive evolution. Cell-size measurement and flow cytometry showed that evolved populations were diploid or triploid and the transition to higher ploidy occurred more rapidly with increasing ethanol stress. During the experimental evolution three strains evolved the capacity to grow on organic acids. We have sequenced the complete genomes of eight evolved strains. These strains are confirmed as being diploid, but not aneuploid. Sequencing of evolved strains revealed mutations that have not been previously characterised in deletion or expression studies of ethanol or organic acid tolerance in S.cerevisiae. Both increasing ploidy, to produce triploids as well as diploids, and the acquisition of organic acid tolerance under ethanol stress are unexpected outcomes that have implications for future work.

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We explored using de-oiled algal biomass (algal cake) as a low-value substrate for production of lactic acid in fermentations with Lactobacillus casei, and strategies for increasing lactic acid production at low pH. L. casei 12A algal cake (AC) fermentations showed carbohydrate and amino acid availability limit growth and lactic acid production. These nutritional requirements were effectively addressed with enzymatic hydrolysis of the AC using α-amylase, cellulase, and pepsin. Producing 0.075 g lactic acid per g AC from AC digested with all three enzymes. We explored heterologous expression of the cellulase gene (celE) from Clostridium thermocellum and the α-amylase gene (amyA) from Streptococcus bovis in L. casei 12A. Functional activity of CelE was not detected, but low-level activity of AmyA was achieved, and increased > 1.5-fold using a previously designed synthetic promoter. Nonetheless, the improvement was insufficient to significantly increase lactic acid production. Thus, substantial optimization of amyA and celE expression in L. casei 12A would be needed to achieve activities needed to increase lactic acid production from AC. We explored transient inactivation of MutS as a method for inducing hypermutability and increasing adaptability of L. casei 12A and ATCC 334 to lactic acid at low pH. The wild type cells and their ΔmutS derivatives were subject to a 100-day adaptive evolution experiment, followed by repair of the ΔmutS lesion in representative isolates. Growth studies at pH 4.0 revealed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. The greatest increases were observed from the adapted ΔmutS derivatives. Further examination of the 12A adapted ΔmutS derivative identified morphological changes, and increased survival at pH 2.5. Genome sequence analysis confirmed transient MutS inactivation decreased DNA replication fidelity, and identified potential genotypic changes in 12A that might contribute to increased acid lactic acid resistance. Targeted inactivation of three genes identified in the adapted 12A ΔmutS derivative revealed that a NADH dehydrogenase (ndh), phosphate transport ATP-binding protein PstB (pstB), and two-component signal transduction system (TCS) quorum-sensing histidine kinase (hpk) contribute to increased acid resistance in 12A.

Zymomonas mobilis is one of the most promising microorganisms for bioethanol production. However, its practical use on industrial scale is impeded by its high sensitivity to acetate, which is present in high concentration in pretreated biomass. This research develops an adaptive mutation method for generating acetate-tolerant strains for bioethanol production. The goal is to obtain Zymomonas mobilis strain capable of growing and producing ethanol in the presence of acetate at a concentration typical of a pretreated biomass (2-3%). The interplay between the ability of fermentative production of ethanol and acetate tolerance will be investigated through careful fermentation studies. The potential cross-tolerance to other inhibitors, commonly present in pretreated biomass will be evaluated. A preliminary study on the mechanism of acetate tolerance at the cell membrane level will be conducted. The strain developed through this research will be useful in bioethanol production from biomass. The insights into tolerance mechanisms gained through this study will allow a more rational approach to further engineer a better producing strain.

Listeria monocytogenes represents a high risk for consumers, because it causes severe illness. This work studied in-vitro growth and survival of L. monocytogenes in media acidified with malic acid, lactic acid or blueberry extract. The growth of L. monocytogenes and shelf life extension of fresh blueberries were evaluated after storage at 4 deg C or 12 deg C under different controlled atmosphere conditions, including air (control); 5% O2 and 15% CO2, 80% N2 (CAS); or ozone gas (O3) 4ppm at 4 deg C or 2.5ppm at 12 deg C, at high relative humidity (90-95%) for a total of 10 days. L. monocytogenes growth in tryptic soy broth with yeast extract (TSB+YE) mixed with different acid solutions (malic acid, lactic acid and blueberry extract) and incubated at 25 deg C for 24h, was calculated measuring optical density. Complete inhibition occurred in the presence of treatments including malic acid pH 2.0 and pH 3.0; lactic acid pH 2.0, pH 3.0 and pH 4.0; and with blueberry extract pH 2.0 in the mixture. After 6h, there were significant differences among growing treatments. At 18h, there were no significant differences in turbidity among media mixed with blueberry extract at pH 3.0, 4.0 and 5.0, and their optical density values were higher than treatments including media mixed with malic acid 5.0 or lactic acid 5.0. Blueberry extract was not an effective acidifying media and acid adapted L. monocytogenes grew in acidified media. Fresh blueberries inoculated with L. monocytogenes were stored at 4 deg C or 12 deg C under different controlled atmosphere conditions (Air, CAS or O3 4ppm at 4 deg C or 2.5ppm at 12 deg C) and sampled on day 0, 1, 4, 7 and 10 for bacterial growth, weight loss, firmness and yeast and molds counts. CAS did not delay or inhibit Listeria monocytogenes, yeast, or molds by day 10. Storage at 4 deg C showed lower weight loss values compared to 12 deg C. Ozone controlled weight loss and firmness loss. Moreover, gaseous ozone achieved 3 and 2 log reductions when compared with air at 4 deg C and 12 deg C, respectively. / Ph. D.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Durch die Flutung stillgelegter Tagebaue entstehen zahlreiche Seen in den ostdeutschen Braunkohlerevieren. Sie sind überwiegend groß, oligo- bis mesotroph, und ihr Litoral ist nur in geringem Umfang durch Makrophyten strukturiert. Viele von ihnen sind versauert oder versauerungsgefährdet. In einer Feldstudie wurden (1) die Auswirkung von Seemorphologie und Habitatstruktur auf die Fischgemeinschaft des Senftenberger Sees, eines 1050 ha großen mesotrophen Tagebausees, und (2) die Säuretoleranz einheimischer Fischarten unter den hydrochemischen Bedingungen der geogen versauerten Tagebauseen untersucht. Die Fischgemeinschaft des Senftenberger Sees wurde von Barschen (Perca fluviatilis) und Plötzen (Rutilus rutilus) dominiert, wobei die verschiedenen Barsch- und Plötzengrößenklassen ihre Habitate durch Nutzung verschiedener Tiefenbereiche des Litorals segregierten. Innerhalb der flachen Litoralbereiche waren juvenile Plötzen und juvenile Güstern deutlich stärker auf die wenigen Makrophytenbestände fixiert als juvenile Barsche. Die Habitatwahl der Barsche zeigt, dass diese in mesotrophen Seen wahrscheinlich eher von den guten Sichtverhältnissen als von der strukturellen Komplexität submerser Makrophytenbestände profitieren. Als Ursache für die unterschiedliche Habitatwahl juveniler Barsche, Plötzen und Güstern wird postuliert, dass diese generell verschiedene Habitatwahlstrategien verfolgen. Während der entscheidende Parameter für die Habitatwahl juveniler Barsche die Optimierung der Energieaufnahme ist, folgen juvenile Plötzen und Güstern vorrangig einer Räubervermeidungsstrategie und sind daher sehr viel stärker auf strukturreiche Bereiche angewiesen. Die kritischen pH-Untergrenzen für die einzelnen Fischarten entsprachen weitgehend den publizierten Ergebnissen aus regenversauerten Weichwasserseen. Vermutlich wurde in den Tagebauseen der Säurestress, den die erhöhte Al-Konzentration von bis zu 0,6 mg/L verursachte, durch die ebenfalls erhöhte Ca-Konzentration kompensiert. / Numerous post-mining lakes are created by flooding the pits remaining from open-cast lignite mining in eastern Germany. They are typically large, oligo- or mesotrophic and only sparsely structured by macrophytes. Many of them are acidified or at risk of acidification. The aim of this study was (1) to determine the effects of lake morphology and habitat structure on the fish community of Lake Senftenberg, a large mesotrophic post-mining lake, and (2) to determine the acid tolerance of indigenous fish species under the hydrochemical conditions of geogenically acidified post-mining lakes. The most abundant fish species in Lake Senftenberg were Eurasian perch (Perca fluviatilis) and roach (Rutilus rutilus). Different size classes of both species segregated along a depth gradient within the littoral zone. Unvegetated shallow littoral areas (depth 1-3 m) were mainly used by juvenile perch, whereas juvenile roach and juvenile white bream (Blicca bjoerkna) preferred macrophyte stands. This indicates that the structural complexity of submersed macrophytes is not the key factor favouring perch over roach under mesotrophic conditions. Instead, low turbidity is probably the most important factor. The habitat choice of juvenile perch, roach and white bream is suggested to reflect different habitat selection strategies. Whereas habitat selection of juvenile perch is predominantly driven by maximisation of food consumption rates, juvenile roach and juvenile white bream aim at minimising predation risk. Consequently, they are much more dependent on structurally complex habitats. The critical lower pH-thresholds of the fish species investigated in this study were similar to the values derived from soft water lakes affected by acid rain. The deleterious effects of elevated Al-concentrations (up to 0.6 mg/L) in the post-mining lakes were probably mitigated by high Ca-concentrations.

The aim of present work is cultivation of probiotic bacteria L. acidophilus, B. breve and B. longum in such a way that the culture forms cells clusters or comprehensive biofilm on the variety of free carriers. Biofilm formation of L. acidophilus on the silica from point of view bile and acid tolerance in gastrointestinal tract was studied. While the number of living cells in planktonic form (planktonic form) at pH 1 fell by 30 %, the viability of the biofilm cells was maintained to 90 % under the same environmental conditions. The biofilm culture showed also the protection against environment contained bile. Furthermore, the possibilities of drying procedures of biofilm cultures used as commercial technologies in pharmaceutical industry were studied. The comparison of freeze-drying and fluidization bed drying showed, that freeze-drying is more suitable method, which is able to achieve higher amount of viable cells after drying than fluidization bed drying. The effectivity of freeze-drying method is dependent on the selection of suitable cryprotective medium. In this case, about 90 % higher viability after freeze drying was achieved in comparison with fluidization bed drying. Finally, the industrial processing of probiotic strains into the solid dosage form was studied. Tablets should be produced at hardness between 70 and 90 N and water activity of tablet mixture can be maintained below 0.3. Consequently, the drying step of the tablets in a hermetically closed space with at least 10 % of silica gel must be ensured. Thereafter, the tablets contain (5.4 ± 0.7)109 viable cells after 6 months of drying process. Capsule production technology has no significant effect on the cell‘s viability during production. The triplex blistering foil for primary blistering of probiotic capsules was chosen. The triplex foil, which has low values of water vapour transition rate (0.07 g H2O / (m2 × day) and oxygen transition rate (0.01 cm3/m2 × day), was chosen. Other studied blistering foils commonly used in the pharmaceutical industry are not suitable for long storage of solid dosage forms contained probiotics.