Prospecting and engineering yeasts for ethanol production under inhibitory conditions: an experimental design analysis.

The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.

Analysis of glucose and xylose metabolism in new indigenous Meyerozyma caribbica strains isolated from corn residues.

Aiming to broaden the base of knowledge about wild yeasts, four new indigenous strains were isolated from corn residues, and phylogenetic-tree assemblings on ITS and LSU regions indicated they belong to Meyerozyma caribbica. Yeasts were cultivated under full- and micro-aerobiosis, starting with low or high cell-density inoculum, in synthetic medium or corn hydrolysate containing glucose and/or xylose. Cells were able to assimilate both monosaccharides, albeit by different metabolic routes (fermentative or respiratory). They grew faster in glucose, with lag phases ~ 10 h shorter than in xylose. The hexose exhaustion occurred between 24 and 34 h, while xylose was entirely consumed in the last few hours of cultivation (44-48 h). In batch fermentation in synthetic medium with high cell density, under full-aerobiosis, 18-20 g glucose l-1 were exhausted in 4-6 h, with a production of 6.5-7.0 g ethanol l-1. In the xylose medium, cells needed > 12 h to consume the carbohydrate, and instead of ethanol, cells released 4.4-6.4 g l-1 xylitol. Under micro-aerobiosis, yeasts were unable to assimilate xylose, and glucose was more slowly consumed, although the ethanol yield was the theoretical maximum. When inoculated into the hydrolysate, cells needed 4-6 h to deplete glucose, and xylose had a maximum consumption of 57%. Considering that the hydrolysate contained ~ 3 g l-1 acetic acid, it probably has impaired sugar metabolism. Thus, this study increases the fund of knowledge regarding indigenous yeasts and reveals the biotechnological potential of these strains.

Biochemical analysis of cellobiose catabolism in Candida pseudointermedia strains isolated from rotten wood.

We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.

Higienização de lodo de esgoto em reator com aquecimento solar: inativação de coliformes totais e Escherichia coli / Sanitisation of sewage sludge in a solar heated reactor: inactivation of total coliforms and Escherichia coli

RESUMO O uso de lodo de esgoto na agricultura se tornou prática corrente em diversos países, sendo atrativa em muitos aspectos, principalmente no fornecimento de matéria orgânica e nutrientes ao solo. Embora os benefícios ambientais e agrícolas do uso de lodo sejam consideráveis, tal prática deve ser realizada de forma sanitariamente segura. Nesse contexto, o objetivo deste estudo foi avaliar a eficiência de um processo de higienização térmica de lodo de esgoto utilizando energia solar. O lodo era aquecido através de um trocador de calor, construído com tubos de cobre e instalado dentro do reator, no qual circula água aquecida em coletores solares planos. Foram realizados 16 ensaios experimentais em diferentes condições de irradiação solar. A inativação térmica da Escherichia coli foi avaliada através do modelo cinético de primeira ordem em condições não isotérmicas. O processo mostrou-se eficiente em ensaios realizados com irradiação solar média do período acima de 500 W.h.m-2, com redução de E. coli entre 4,2 e 7,1 log10 e de coliformes totais entre 4,8 e 7,4 log10. Os ensaios realizados em dias com menores índices de irradiação solar tiveram a eficiência de higienização comprometida, devido às baixas temperaturas atingidas pelo lodo.

ABSTRACT The application of sewage sludge in agriculture has become a common practice in many countries, which is attractive in many aspects, especially for the input of organic matter and nutrients in the soil. Despite the benefits of sludge use in agriculture and for the environment , this practice needs to be conducted considering safety aspects regarding sanitary conditions. In this context, the aim of this study was to evaluate the efficiency of a thermal disinfection process of sewage sludge using solar energy. The sludge was heated through a heat exchanger built with copper pipes and installed inside the reactor, in which water heated in flat plate solar collectors circulates. Sixteen experimental tests were performed under different solar irradiation conditions. The thermal inactivation of Escherichia coli in the reactor was evaluated using the first order kinetic model in non-isothermal conditions. The process proved effective in those tests with an average solar irradiation period above 500 W.h.m-2, and the reduction of E. coli was between 4.2 and 7.1 units log10; and between 4.8 and 7.4 units log10 of total coliforms. When tests were conducted in days with lower levels of solar irradiation, the efficiency of sanitisation was compromised, due to the low temperatures reached by the sludge.

Determinação de parâmetros cinéticos da inativação térmica de Escherichia coli em lodo de esgoto / Determining kinetic parameters for thermal inactivation of Escherichia coli in sewage sludge

O presente trabalho objetivou determinar parâmetros cinéticos da inativação térmica de Escherichia coli em lodo de esgoto. Os ensaios foram realizados em laboratório pelo método do frasco de três bocas nas temperaturas de 45, 50, 55, 60 e 65ºC. Os resultados indicaram que a cinética de inativação térmica deste microrganismo pode ser descrita por um modelo de primeira ordem. A resistência da bactéria é reduzida consideravelmente em temperaturas acima de 55ºC. A energia de inativação encontrada foi 2,48x10(5) J.mol-1. O tempo de redução decimal D55ºC foi de 3,61 minutos e o coeficiente térmico z foi 8,3ºC.

The present study aimed to determine the kinetic parameters of thermal inactivation of Escherichia coli in sewage sludge. The tests were performed in the laboratory using the three-neck flask method at temperatures of 45, 50, 55, 60 and 65ºC. The results indicated that the thermal inactivation kinetic of this microorganism can be described by a first order model. The resistance of bacteria is greatly reduced at temperatures above 55ºC. The inactivation energy was found 2.48x10(5) J.mol-1. The decimal reduction time D55ºC was 3.61 minutes and the thermal coefficient z was 8.3ºC.