• Energy Research

Abstract Fuel ethanol is produced by the yeast Saccharomyces cerevisiae mainly from corn starch in the United States and from sugarcane sucrose in Brazil, which together manufacture ∼85% of a global yearly production of 109.8 million m3 (in 2019). While in North America genetically engineered (GE) strains account for ∼80% of the ethanol produced, including strains that express amylases and are engineered to produce higher ethanol yields; in South America, mostly (>90%) non-GE strains are used in ethanol production, primarily as starters in non-aseptic fermentation systems with cell recycling. In spite of intensive research exploring lignocellulosic ethanol (or second generation ethanol), this option still accounts for <1% of global ethanol production. In this mini-review, we describe the main aspects of fuel ethanol production, emphasizing bioprocesses operating in North America and Brazil. We list and describe the main properties of several commercial yeast products (i.e., yeast strains) that are available worldwide to bioethanol producers, including GE strains with their respective genetic modifications. We also discuss recent studies that have started to shed light on the genes and traits that are important for the persistence and dominance of yeast strains in the non-aseptic process in Brazil. While Brazilian bioethanol yeast strains originated from a historical process of domestication for sugarcane fermentation, leading to a unique group with significant economic applications, in U.S.A., guided selection, breeding and genetic engineering approaches have driven the generation of new yeast products for the market.

Selected Saccharomyces cerevisiae strains are used in Brazil to produce the hitherto most energetically efficient first-generation fuel ethanol. Although genome and some transcriptome data are available for some of these strains, quantitative physiological data are lacking. This study investigates the physiology of S. cerevisiae strain PE-2, widely used in the Brazilian fuel ethanol industry, in comparison with CEN.PK113-7D, a reference laboratory strain, focusing on tolerance to low pH and acetic acid stress. Both strains were grown in anaerobic bioreactors, operated as batch, chemostat or dynamic continuous cultures. Despite their different backgrounds, biomass and product formation by the two strains were similar under a range of conditions (pH 5 or pH < 3, with or without 105 mM acetic acid added). PE-2 displayed a remarkably higher fitness than CEN.PK113-7D during batch cultivation on complex Yeast extract - Peptone - Dextrose medium at low pH (2.7). Kinetics of viability loss of non-growing cells, incubated at pH 1.5, indicated a superior survival of glucose-depleted PE-2 cells, when compared with either CEN.PK113-7D or a commercial bakers' strain. These results indicate that the sulfuric acid washing step, used in the fuel ethanol industry to decrease bacterial contamination due to non-aseptic operation, might have exerted an important selective pressure on the microbial populations present in such environments.

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h(-1), which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO2 production and O2 consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.

Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

Abstract Background Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. Results Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. Conclusion The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

Improved biofuels production requires a better understanding of industrial microorganisms. Some wild Saccharomyces cerevisiae strains, isolated from the fuel ethanol industry in Brazil, present exceptional fermentation performance, persistence and prevalence in the harsh industrial environment. Nevertheless, their physiology has not yet been systematically investigated. Here we present a first systematic evaluation of the widely used industrial strains PE-2, CAT-1, BG-1 and JP1, in terms of their tolerance towards process-related stressors. We also analyzed their growth physiology under heat stress. These strains were evaluated in parallel to laboratory and baker's strains. Whereas the industrial strains performed in general better than the laboratory strains under ethanol or acetic acid stresses and on industrial media, high sugar stress was tolerated equally by all strains. Heat and low pH stresses clearly distinguished fuel ethanol strains from the others, indicating that these conditions might be the ones that mostly exert selective pressure on cells in the industrial environment. During shake-flask cultivations using a synthetic medium at 37 °C, industrial strains presented higher ethanol yields on glucose than the laboratory strains, indicating that they could have been selected for this trait-a response to energy-demanding fermentation conditions. These results might be useful to guide future improvements of large-scale fuel ethanol production via engineering of stress tolerance traits in other strains, and eventually also for promoting the use of these fuel ethanol strains in different industrial bioprocesses.

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.

The yeast Kluyveromyces lactis has received attention both from academia and industry due to some important features, such as its capacity to grow in lactose-based media, its safe status, its suitability for large-scale cultivation and for heterologous protein synthesis. It has also been considered as a model organism for genomics and metabolic regulation. Despite this, very few studies were carried out hitherto under strictly controlled conditions, such as those found in a chemostat. Here we report a set of quantitative physiological data generated during chemostat cultivations with the K. lactis CBS 2359 strain, obtained under glucose-limiting and fully aerobic conditions. This dataset serves [corrected] as a basis for the comparison of K. lactis with the model yeast Saccharomyces cerevisiae in terms of their elemental compositions, as well as for future metabolic flux analysis and metabolic modelling studies with K. lactis.