Clash of Kingdoms: How Do Bacterial Contaminants Thrive in and Interact with Yeasts during Ethanol Production?

Brazilian fuel ethanol production from sugarcane is one of the largest industrial biotechnological processes in the world. However, in view of the complex chemical nature of this feedstock, as well as the non-aseptic conditions of the process, various stress conditions are imposed to the fermenting yeast. In this chapter, we deemed to elaborate a brief overview of the ethanol production process, and to dissect the chemical nature of sugarcane-based worts, as well as their physiological effects on the fermenting yeasts. Finally, the interplay between yeast and lactic acid bacteria, the two main players in the ethanol fermentation process, is generally discussed

A synthetic medium to simulate sugarcane molasses

Abstract\ud \ud Background\ud Developing novel microbial cell factories requires careful testing of candidates under industrially relevant conditions. However, this frequently occurs late during the strain development process. The availability of laboratory media that simulate industrial-like conditions might improve cell factory development, as they allow for strain construction and testing in the laboratory under more relevant conditions. While sugarcane molasses is one of the most important substrates for the production of biofuels and other bioprocess-based commodities, there are no defined media that faithfully simulate it. In this study, we tested the performance of a new synthetic medium simulating sugarcane molasses.\ud \ud \ud Results\ud Laboratory scale simulations of the Brazilian ethanol production process, using both sugarcane molasses and our synthetic molasses (SM), demonstrated good reproducibility of the fermentation performance, using yeast strains, PE-2 and Ethanol Red™. After 4 cycles of fermentation, the final ethanol yield (gp g\ud s\ud −1\ud ) values for the SM ranged from 0.43 ± 0.01 to 0.44 ± 0.01 and from 0.40 ± 0.01 to 0.46 ± 0.01 for the molasses-based fermentations. The other fermentation parameters (i.e., biomass production, yeast viability, and glycerol and acetic acid yield) were also within similar value ranges for all the fermentations. Sequential pairwise competition experiments, comparing industrial and laboratory yeast strains, demonstrated the impact of the media on strain fitness. After two sequential cocultivations, the relative abundance of the laboratory yeast strain was 5-fold lower in the SM compared to the yeast extract-peptone-dextrose medium, highlighting the importance of the media composition on strain fitness.\ud \ud \ud Conclusions\ud Simulating industrial conditions at laboratory scale is a key part of the efficient development of novel microbial cell factories. In this study, we have developed a synthetic medium that simulated industrial sugarcane molasses media. We found good agreement between the synthetic medium and the industrial media in terms of the physiological parameters of the industrial-like fermentations.The authors would like to acknowledge funding from the Novo Nordisk Foundation under the NNF Grant Number: NNF10CC1016517

Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells

Abstract The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates and glucose uptake rates, while commercial AFA had no effect in concentrations relevant for defoaming purposes. Industrial AFA were further tested in laboratory scale simulations of the Brazilian ethanol production process and proved to decrease cell viability compared to the control, and the effects were intensified with increasing AFA concentrations and exposure time. Transcriptome analysis showed that AFA treatments induced additional stress responses in yeast cells compared to the control, shown by an up-regulation of stress-specific genes and a down-regulation of lipid biosynthesis, especially ergosterol. By documenting the detrimental effects associated with chemical AFA, we highlight the importance of developing innocuous systems for foam control in industrial fermentation processes.</jats:p

Evolutionary engineering applied in Trichoderma sp. for the production of cellulase.

O projeto visou aumentar a produção de celulases em fungos T. harzianum IPT 821 e T. reesei QM 9414. Esporos foram submetidos à radiação UV-C. Células das colônias mais capacitadas ao crescimento foram sucessivamente cultivadas em meio solidificado, contendo concentrações progressivamente reduzidas fonte de carbono, de modo a resultar pressão ambiental seletiva e crescente. Após esta etapa as linhagens isoladas foram cultivadas em meio composto por bagaço de cana/farelo de trigo na proporção 80/20, 60% de umidade, 30°C por 72h. Uma linhagem, originária da cepa IPT 821, apresentou atividade de 3,7 FP U/gms, 50% superior em relação à parental: 2,6 U/gms (p=0,001; p<0,05). Análises das frações enzimáticas indicaram uma diferença significativa (p=0,001; p<0,05) na atividade de xilanase: 4,7 U/gms (mutante) e 4 U/gms (parental). Ensaios de hidrólise, Avicel como substrato (1% de sólidos; w/v) indicaram um aumento de quase 70% na hidrólise em 48h, da mutante em comparação à parental (8,7% (mutante) e 4,8% (parental), concentração enzimática de 2,5 FP U/gms).This project aimed to increase cellulase production in fungi T. harzianum IPT 821 and T. reesei QM 9414. Spores were exposed to UV-C radiation. Colonies were plated and those showing best growth were successively cultivated in plates containing increasingly stress conditions reduced concentrations of the carbon source thus creating a progressive selective pressure. After this pre-selection step isolated strains were cultivated in medium comprising sugar cane bagasse and wheat straw, in the proportion of 80/20 respectively, 60% of moisture, 30°C for 72h. One strain, originated from IPT 821 strain, showed a cellulolytic activity of 3,7 U/gdw; 50% superior to the parental strain: 2,6 U/gdw (p=0,001; p<0,05). Analysis of the enzymatic cocktail showed significant difference (p=0,001; for p <0,05) on xylanase activity: 4,7 U/gdw (mutant strain) and 4 U/gdw (parental strain). Hydrolysis assays, using Avicel as substrate (1% w/v) showed an increase on hydrolysis of about 70%, in 48h (8,7% (mutant strain) and 4,8% (parental strain), enzyme load of about 2,5 U/gdw)