The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Mitigating stress in industrial yeasts.

The yeast, Saccharomyces cerevisiae, is the premier fungal cell factory exploited in industrial biotechnology. In particular, ethanol production by yeast fermentation represents the world's foremost biotechnological process, with beverage and fuel ethanol contributing significantly to many countries economic and energy sustainability. During industrial fermentation processes, yeast cells are subjected to several physical, chemical and biological stress factors that can detrimentally affect ethanol yields and overall production efficiency. These stresses include ethanol toxicity, osmostress, nutrient starvation, pH and temperature shock, as well as biotic stress due to contaminating microorganisms. Several cell physiological and genetic approaches to mitigate yeast stress during industrial fermentations can be undertaken, and such approaches will be discussed with reference to stress mitigation in yeasts employed in Brazilian bioethanol processes. This article will highlight the importance of furthering our understanding of key aspects of yeast stress physiology and the beneficial impact this can have more generally on enhancing industrial fungal bioprocesses.

Enhancing Yeast Alcoholic Fermentations.

The production of ethanol by yeast fermentation represents the largest of all global biotechnologies. Consequently, the yeast Saccharomyces cerevisiae is the world's premier industrial microorganism, which is responsible not only for the production of alcoholic beverages, including beer, wine, and distilled spirits, but also for the billions of liters of bioethanol produced annually for use as a renewable transportation fuel. Although humankind has exploited the fermentative activities of yeasts for millennia, many aspects of alcohol fermentation remain poorly understood. This chapter will review some of the key considerations in optimizing industrial alcohol fermentations with a particular emphasis on enhancement opportunities involving cell physiology and strain engineering of the major microbial ethanologen, the yeast S. cerevisiae.

Stimulation of bioprocesses by ultrasound.

Ultrasound (US) has become a ubiquitous technological process in a large variety of scientific disciplines. However, little information exists on the use of ultrasound to enhance biological processes and/or processing and consequently this paper provides an overview of work reported to date on this topic. This review provides a brief introduction to ultrasound and the history of ultrasound as applied to bioprocesses. This is followed by a discussion of the influence of US on discrete enzyme systems, enzymes used in bioremediation, microbial fermentations and enzymatic hydrolysis of biopolymers. Augmentation of anaerobic digestion by US is then considered along with enhancement of enzymes in food science and technology. The use of ultrasonically stimulated enzymes in synthesis is then considered and other relevant miscellaneous topics are described. It is concluded that the precise mechanism of action of US in bio-processing remains to be elucidated though a variety of plausible suggestions are made.

Pichia anomala: cell physiology and biotechnology relative to other yeasts.

Pichia anomala is a most interesting yeast species, from a number of environmental, industrial and medical aspects. This yeast has been isolated from very diverse natural habitats (e.g. in foods, insects, wastewaters etc.) and it also exhibits wide metabolic and physiological diversity. Some of the activities of P. anomala, particularly its antimicrobial action, make it a very attractive organism for biological control applications in the agri-food sectors of industry. Being a 'robust' organism, it additionally has potential to be exploited in bioremediation of environmental pollutants. This paper provides an overview of cell physiological characteristics (growth, metabolism, stress responses) and biotechnological potential (e.g. as a novel biocontrol agent) of P. anomala and compares such properties with other yeast species, notably Saccharomyces cerevisiae, which remains the most exploited industrial microorganism. We await further basic knowledge of P. anomala cell physiology and genetics prior to its fuller commercial exploitation, but the exciting biotechnological potential of this yeast is highlighted in this paper.

Williopsis saturnus yeast killer toxin does not kill Streptococcus pneumoniae.

Streptococcus pneumoniae is an important human bacterial pathogen, and the increase in antibiotic resistance demands the development of new antimicrobial compounds. Several reports have suggested that yeast killer toxins show activity against bacteria and we therefore investigated the activity of K9 killer toxin from the yeast Williopsis saturnus var. mrakii NCYC 500 against S. pneumoniae. However, no inhibition of bacterial growth was observed with concentrated K9 preparations in agar diffusion assays and in liquid culture. Although cell morphology was slightly affected by K9 treatment, no effect on cellular viability was detectable, and K9 had no stimulatory effect on cell lysis induced by ß-lactams or Triton X-100. This indicated that K9 did not contribute to cell wall damage. Moreover, flow cytometry was used as a sensitive assessment of integrity of cells exposed to killer toxin. No significant damage of S. pneumoniae cells was evident, although minor changes in fluorescence suggested that K9 killer toxin may interact with bacterial surface components.

Influence of cell surface characteristics on adhesion of Saccharomyces cerevisiae to the biomaterial hydroxylapatite.

The influence of the physicochemical properties of biomaterials on microbial cell adhesion is well known, with the extent of adhesion depending on hydrophobicity, surface charge, specific functional groups and acid-base properties. Regarding yeasts, the effect of cell surfaces is often overlooked, despite the fact that generalisations may not be made between closely related strains. The current investigation compared adhesion of three industrially relevant strains of Saccharomyces cerevisiae (M-type, NCYC 1681 and ALY, strains used in production of Scotch whisky, ale and lager, respectively) to the biomaterial hydroxylapatite (HAP). Adhesion of the whisky yeast was greatest, followed by the ale strain, while adhesion of the lager strain was approximately 10-times less. According to microbial adhesion to solvents (MATS) analysis, the ale strain was hydrophobic while the whisky and lager strains were moderately hydrophilic. This contrasted with analyses of water contact angles where all strains were characterised as hydrophilic. All yeast strains were electron donating, with low electron accepting potential, as indicated by both surface energy and MATS analysis. Overall, there was a linear correlation between adhesion to HAP and the overall surface free energy of the yeasts. This is the first time that the relationship between yeast cell surface energy and adherence to a biomaterial has been described.

Nanoscopic morphological changes in yeast cell surfaces caused by oxidative stress: an atomic force microscopic study.

Nanoscopic changes in the cell surface morphology of the yeasts Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354), due to their exposure to varying concentrations of hydrogen peroxide (oxidative stress), were investigated using an atomic force microscope (AFM). Increasing hydrogen peroxide concentration led to a decrease in cell viabilities and mean cell volumes, and an increase in the surface roughness of the yeasts. In addition, AFM studies revealed that oxidative stress caused cell compression in both S. cerevisiae and Schiz. pombe cells and an increase in the number of aged yeasts. These results confirmed the importance and usefulness of AFM in investigating the morphology of stressed microbial cells at the nanoscale. The results also provided novel information on the relative oxidative stress tolerance of S. cerevisiae and Schiz. pombe.

Bioconversion of brewer's spent grains to bioethanol.

Spent grains (SG), the residue remaining after extraction of wort, are a major by-product of brewing. This lignocelluose-rich biomass may provide a source of sugars for fuel ethanol fermentations. Dilute acid and enzyme treatments were developed to convert the hemicellulose and cellulose fractions to glucose, xylose and arabinose. Pretreatment of dried, milled grains with 0.16 N HNO(3) at 121 degrees C for 15 min was chosen as the most suitable method for solubilizing grains before enzymatic digestion with cellulase and hemicellulase preparations. Solids loading concentrations (10%, 15% and 20% w/v) were compared and reducing sugar between 40 and 48 g (100 g SG)(-1) was extracted. Hydrolysate, prepared from 20% SG, pretreated with 0.16 N HNO(3), partially neutralized to pH 5-6 and digested with enzymes for 18 h, contained 27 g L(-1) glucose, 16.7 g L(-1) xylose and 11.9 g L(-1) arabinose. Fermentation of this hydrolysate for 48 h by Pichia stipitis and Kluyveromyces marxianus resulted in 8.3 and 5.9 g L(-1) ethanol corresponding to ethanol conversion yields of 0.32 and 0.23 g ethanol (g substrate)(-1), respectively. Substrate utilization efficiency was less when compared with glucose/xylose mixtures in synthetic media, suggesting that yeast inhibitory compounds derived from SG were present in the hydrolysate.

Physiological and transcriptional responses of Saccharomyces cerevisiae to zinc limitation in chemostat cultures.

Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium), zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified.

Correlating yeast cell stress physiology to changes in the cell surface morphology: atomic force microscopic studies.

Atomic Force Microscopy (AFM) has emerged as a powerful biophysical tool in biotechnology and medicine to investigate the morphological, physical, and mechanical properties of yeasts and other biological systems. However, properties such as, yeasts' response to environmental stresses, metabolic activities of pathogenic yeasts, cell-cell/cell-substrate adhesion, and cell-flocculation have rarely been investigated so far by using biophysical tools. Our recent results obtained by AFM on one strain each of Saccharomyces cerevisiae and Schizosaccharomyces pombe show a clear correlation between the physiology of environmentally stressed yeasts and the changes in their surface morphology. The future directions of the AFM related techniques in relation to yeasts are also discussed.

Atomic force microscopic study of the effects of ethanol on yeast cell surface morphology.

The detrimental effects of ethanol toxicity on the cell surface morphology of Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354) were investigated using an atomic force microscope (AFM). In combination with culture viability and mean cell volume measurements AFM studies allowed us to relate the cell surface morphological changes, observed on nanometer lateral resolution, with the cellular stress physiology. Exposing yeasts to increasing stressful concentrations of ethanol led to decreased cell viabilities and mean cell volumes. Together with the roughness and bearing volume analyses of the AFM images, the results provided novel insight into the relative ethanol tolerance of S. cerevisiae and Sc. pombe.

Atomic force microscopic study of the influence of physical stresses on Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Morphological changes in the cell surfaces of the budding yeast Saccharomyces cerevisiae (strain NCYC 1681), and the fission yeast Schizosaccharomyces pombe (strain DVPB 1354), in response to thermal and osmotic stresses, were investigated using an atomic force microscope. With this microscope imaging, together with measurements of culture viability and cell size, it was possible to relate topological changes of the cell surface at nanoscale with cellular stress physiology. As expected, when the yeasts were exposed to thermostress or osmostress, their viability together with the mean cell volume decreased in conjunction with the increase in thermal or osmotic shock. Nevertheless, the viability of cells stressed for up to 1 h remained relatively high. For example, viabilities were >50% and >90% for the thermostressed, and >60% and >70% for the osmostressed S. cerevisiae and Schiz. pombe, respectively. Mean cell volume measurements, and bearing and roughness analyses of atomic force microscope images of stressed yeasts indicate that Schiz. pombe may be more resistant to physical stresses than S. cerevisiae. Overall, this study has highlighted the usefulness of atomic force microscope in studies of yeast stress physiology.

Anti-Candida activity of a novel killer toxin from the yeast Williopsis mrakii.

A screening of putative killer yeast strains showed that spore-forming ascomycetous yeasts of the genera Pichia and Williopsis displayed the broadest range of activity against sensitive strains of Candida spp. and Saccharomyces cerevisiae. Williopsis mrakii (NCYC 500) showed extensive anti-Candida activity against strains isolated from clinical specimens. W. mrakii killer factor was produced in minimal media as a function of growth and its activity reached constant levels as cells entered stationary phase. The proteinaceous killer toxin was found to be unstable without a specific range of temperature and pH (above 30 degrees C and pH 4.0), and further analysis showed that the active toxin molecule was an acidic polypeptide with a relative molecular mass between 1.8-5.0 kDa. At critical concentrations the killer factor exerted a greater effect on stationary phase cells of Candida than cells from an exponential phase of growth. At low concentrations, the killer toxin produced a fungistatic effect on sensitive yeasts but at higher concentrations there was evidence to suggest that membrane damage accounted for the zymocidal effects of the killer factor. the cidal nature of the toxin was reflected in a rapid decrease in sensitive cell viability. Findings presented suggest that W. mrakii killer toxin has potential as a novel antimycotic agent in combatting medically important strains of Candida.