Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress.

BACKGROUND: Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. RESULTS: Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. CONCLUSIONS: Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach.

Production of bioethanol from brewers spent grains (BSG) using consolidated bioprocessing (CBP) is reported. Each CBP system consists of a primary filamentous fungal species, which secretes the enzymes required to deconstruct biomass, paired with a secondary yeast species to ferment liberated sugars to ethanol. Interestingly, although several pairings of fungi were investigated, the sake fermentation system (A. oryzae and S. cerevisiae NCYC479) was found to yield the highest concentrations of ethanol (37 g/L of ethanol within 10 days). On this basis, 1 t of BSG (dry weight) would yield 94 kg of ethanol using 36 hL of water in the process. QRT-PCR analysis of selected carbohydrate degrading (CAZy) genes expressed by A. oryzae in the BSG sake system showed that hemicellulose was deconstructed first, followed by cellulose. One drawback of the CBP approach is lower ethanol productivity rates; however, it requires low energy and water inputs, and hence is worthy of further investigation and optimisation

Physiological and transcriptomic response of Saccharomyces pastorianus to cold storage

Removal of yeast biomass at the end of fermentation, followed by a period of storage before re-inoculation into a subsequent fermentation, is common in the brewing industry. Storage is typically conducted at cold temperatures to preserve yeast quality, a practice which has unfavourable cost and environmental implications. To determine the potential for alleviating these effects, the transcriptomic and physiological response of Saccharomyces pastorianus strain W34/70 to standard (4°C) and elevated (10°C) storage temperatures was explored. Higher temperatures resulted in increased expression of genes associated with the production and mobilisation of intracellular glycogen, trehalose, glycerol and fatty acids, although these observations were limited to early stages of storage. Intracellular trehalose and glycerol concentrations were higher at 4°C than at 10°C, as a consequence of the cellular response to cold stress. However, significant changes in glycogen degradation or cellular fatty acid composition did not occur between the two sets of populations, ensuring that cell viability remained consistent. It is anticipated that this data may lead to changes in standard practice for handling yeast cultures, without compromising yeast quality. This work has significance not only for the brewing industry, but also for food and biofuel sectors requiring short term storage of liquid yeast

111In-BnDTPA-F3: an Auger electron-emitting radiotherapeutic agent that targets nucleolin

INTRODUCTION: The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy. METHODS: F3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks. RESULTS: Membrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023). CONCLUSION: 111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation

Rapid analysis of formic acid, acetic acid, and furfural in pretreated wheat straw hydrolysates and ethanol in a bioethanol fermentation using atmospheric pressure chemical ionisation mass spectrometry.

Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) offers advantages as a rapid analytical technique for the quantification of three biomass degradation products (acetic acid, formic acid and furfural) within pretreated wheat straw hydrolysates and the analysis of ethanol during fermentation. The data we obtained using APCI-MS correlated significantly with high-performance liquid chromatography analysis whilst offering the analyst minimal sample preparation and faster sample throughput

Maximising high solid loading enzymatic saccharification yield from acid-catalysed hydrothermally-pretreated brewers spent grain

Enzyme saccharification of pretreated brewers spent grains (BSG) was investigated, aiming at maximising glucose production. Factors investigated were; variation of the solids loadings at different cellulolytic enzyme doses, reaction time, higher energy mixing methods, supplementation of the cellulolytic enzymes with additional enzymes (and cofactors) and use of fed-batch methods. Improved slurry agitation through aerated high-torque mixing offered small but significant enhancements in glucose yields (to 53 ± 2.9 g/L and 45% of theoretical yield) compared to only 41 ± 4.0 g/L and 39% of theoretical yield for standard shaking methods (at 15% w/v solids loading). Supplementation of the cellulolytic enzymes with additional enzymes (acetyl xylan esterases, ferulic acid esterases and α-L- arabinofuranosidases) also boosted achieved glucose yields to 58 – 69 ± 0.8 - 6.2 g/L which equated to 52 - 58% of theoretical yield. Fed-batch methods also enhanced glucose yields (to 58 ± 2.2 g/L and 35% of theoretical yield at 25% w/v solids loading) compared to non-fed-batch methods. From these investigations a novel enzymatic saccharification method was developed (using enhanced mixing, a fed-batch approach and additional carbohydrate degrading enzymes) which further increased glucose yields to 78 ± 4.1 g/L and 43% of theoretical yield when operating at high solids loading (25% w/v)

Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation.

Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid

A Bayesian approach to analyzing phenotype microarray data enables estimation of microbial growth parameters

Biolog phenotype microarrays enable simultaneous, high throughput analysis of cell cultures in different environments. The output is high-density time-course data showing redox curves (approximating growth) for each experimental condition. The software provided with the Omnilog incubator/reader summarizes each time-course as a single datum, so most of the information is not used. However, the time courses can be extremely varied and often contain detailed qualitative (shape of curve) and quantitative (values of parameters) information. We present a novel, Bayesian approach to estimating parameters from Phenotype Microarray data, fitting growth models using Markov Chain Monte Carlo methods to enable high throughput estimation of important information, including length of lag phase, maximal ``growth'' rate and maximum output. We find that the Baranyi model for microbial growth is useful for fitting Biolog data. Moreover, we introduce a new growth model that allows for diauxic growth with a lag phase, which is particularly useful where Phenotype Microarrays have been applied to cells grown in complex mixtures of substrates, for example in industrial or biotechnological applications, such as worts in brewing. Our approach provides more useful information from Biolog data than existing, competing methods, and allows for valuable comparisons between data series and across different models

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol.

BACKGROUND: During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. RESULTS: The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. CONCLUSIONS: Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained