Crucial aspects of metabolism and cell biology relating to industrial production and processing of Saccharomyces biomass.

The multitude of applications to which Saccharomyces spp. are put makes these yeasts the most prolific of industrial microorganisms. This review considers biological aspects pertaining to the manufacture of industrial yeast biomass. It is proposed that the production of yeast biomass can be considered in two distinct but interdependent phases. Firstly, there is a cell replication phase that involves reproduction of cells by their transitions through multiple budding and metabolic cycles. Secondly, there needs to be a cell conditioning phase that enables the accrued biomass to withstand the physicochemical challenges associated with downstream processing and storage. The production of yeast biomass is not simply a case of providing sugar, nutrients, and other growth conditions to enable multiple budding cycles to occur. In the latter stages of culturing, it is important that all cells are induced to complete their current budding cycle and subsequently enter into a quiescent state engendering robustness. Both the cell replication and conditioning phases need to be optimized and considered in concert to ensure good biomass production economics, and optimum performance of industrial yeasts in food and fermentation applications. Key features of metabolism and cell biology affecting replication and conditioning of industrial Saccharomyces are presented. Alternatives for growth substrates are discussed, along with the challenges and prospects associated with defining the genetic bases of industrially important phenotypes, and the generation of new yeast strains."I must be cruel only to be kind: Thus bad begins, and worse remains behind." William Shakespeare: Hamlet, Act 3, Scene 4.

An Electro-Microbial Process to Uncouple Food Production from Photosynthesis for Application in Space Exploration.

Here we propose the concept of an electro-microbial route to uncouple food production from photosynthesis, thereby enabling production of nutritious food in space without the need to grow plant-based crops. In the proposed process, carbon dioxide is fixed into ethanol using either chemical catalysis or microbial carbon fixation, and the ethanol created is used as a carbon source for yeast to synthesize food for human or animal consumption. The process depends upon technologies that can utilize electrical energy to fix carbon into ethanol and uses an optimized strain of the yeast Saccharomyces cerevisiae to produce high-quality, food-grade, single-cell protein using ethanol as the sole carbon source in a minimal medium. Crops performing photosynthesis require months to mature and are challenging to grow under the conditions found in space, whereas the electro-microbial process could generate significant quantities of food on demand with potentially high yields and productivities. In this paper we explore the potential to provide yeast-based protein and other nutrients relevant to human dietary needs using only ethanol, urea, phosphate, and inorganic salts as inputs. It should be noted that as well as having potential to provide nutrition in space, this novel approach to food production has many valuable terrestrial applications too. For example, by enabling food production in climatically challenged environments, the electro-microbial process could potentially turn deserts into food bowls. Similarly, surplus electricity generated from large-scale renewable power sources could be used to supplement the human food chain.

Techno-economic implications of improved high gravity corn mash fermentation.

The performance of Saccharomyces cerevisiae MBG3964, a strain able to tolerate >18% v/v ethanol, was compared to leading industrial ethanol strain, Fermentis Ethanol Red, under high gravity corn mash fermentation conditions. Compared to the industrial ethanol strain, MBG3964 gave increased alcohol yield (140g L(-1) vs. 126g L(-1)), lower residual sugar (4g L(-1) vs. 32g L(-1)), and lower glycerol (11g L(-1) vs. 12g L(-1)). After 72h fermentation, MBG3964 showed about 40% viability, whereas the control yeast was only about 3% viable. Based on modelling, the higher ethanol tolerant yeast could increase the profitability of a corn-ethanol plant and help it remain viable through higher production, lower unit heating requirements and extra throughput. A typical 50M gal y(-1) dry mill ethanol plant that sells dried distiller's grain could potentially increase its profit by nearly $US3.4M y(-1) due solely to the extra yield, and potentially another $US4.1M y(-1) if extra throughput is possible.

Use of population genetics to derive nonrecombinant Saccharomyces cerevisiae strains that grow using xylose as a sole carbon source.

According to scientific dogma, Saccharomyces cerevisiae cannot grow utilizing xylose as a sole carbon source. Although recombinant DNA technology has overcome this deficiency to some degree, efficient utilization of xylose appears to require complex global changes in gene expression. This complexity provides a significant challenge to the development of yeasts suitable for the utilization of xylose-rich lignocellulosic substrates. In contrast to the dogma, we have found that native strains of S. cerevisiae can grow on xylose as a sole carbon source, albeit very slowly. This observation provided the basis for a new approach using natural selection to develop strains of S. cerevisiae with improved ability to utilize xylose. By applying natural selection and breeding over an extended period, we have developed S. cerevisiae strains that can double in less than 6 h using xylose as a sole carbon source. Strains with improved growth rate possessed increased xylose reductase and xylitol dehydrogenase activities, with the latter showing the greater improvement. This unique, completely nonrecombinant approach to developing xylose-utilizing strains of S. cerevisiae opens an alternative route to the development of yeast that can fully utilize lignocellulosic substrates.

Involvement of oxidative stress response genes in redox homeostasis, the level of reactive oxygen species, and ageing in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.

A flow-cytometric method for determination of yeast viability and cell number in a brewery.

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.

Inducible gene expression by nonculturable bacteria in milk after pasteurization.

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.