Naringenin and caffeic acid increase ethanol production in yeast cells by reducing very high gravity fermentation-related oxidative stress.

Very high gravity (VHG) fermentation is an industrial-scale process utilizing a sugar concentration above 250 g/L to attain a significant ethanol concentration, with the advantages of decreased labor, production costs, water usage, bacterial contamination, and energy consumption. Saccharomyces cerevisiae is one of the most extensively employed organisms in ethanol fermentation through VHG technology. Conversely, high glucose exposure leads to numerous stress factors that negatively impact the ethanol production efficiency of this organism. Here, the impact of various phytochemicals added to the VHG medium on viability, glucose consumption, ethanol production efficiency, total antioxidant-oxidant status (TAS and TOS), and the response of the enzymatic antioxidant system of yeast were investigated. 2.0 mM naringenin and caffeic acid increased ethanol production by 2.453 ± 0.198 and 1.261 ± 0.138-fold, respectively. The glucose consumption rate exhibited a direct relationship with ethanol production in the naringenin-supplemented group. The highest TAS was determined as 0.734 ± 0.044 mmol Trolox Eq./L in the same group. Furthermore, both phytochemical compounds exhibited robust positive correlations with TAS (rnaringenin = 0.9986; rcaffeic acid = 0.9553) and TOS levels (rnaringenin = -0.9824; rcaffeic acid = -0.9791). While naringenin caused statistically significant increases in glutathione reductase (GR) and thioredoxin reductase (TrxR) activities, caffeic acid significantly increased TrxR and superoxide dismutase (SOD). Both phytochemicals seem to impact the ethanol production ability by regulating the redox status of the cells. We believe that the incorporation of particularly cost-effective antioxidants into the fermentation medium may serve as an alternative way to enhance the efficiency of bioethanol production using VHG technology.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Avoiding burning practice and its consequences on the greenhouse gas emission in sugarcane areas southern Brazil.

There is a growing need of sustainable solutions for balancing agricultural production with the reduction of its environmental impacts. The rapid increase in sugarcane cultivation and the progressive conversion of pre-harvest burning (BH) to green harvest (GH) have brought into debate the contribution of agricultural sector to the greenhouse gas (GHG) mitigation. This study focused on the estimated GHG emission from sugarcane cultivation during years in which sugarcane areas in southern Brazil expanded and passed throughout an important transition, from 2006 to 2012, when harvest adopted was changed from burned to not-burned based. Sugarcane management and harvest were mapped through visual interpretation of Landsat-type satellite images, and the areas under sugarcane cultivation were distinguished according to each agricultural phase and harvest regime (i.e., manual harvest with burning vs. green mechanized harvest). Based on a broad data review and applying the IPCC (2006) methodologies, the results were expressed in terms of kilograms of carbon dioxide equivalent (kg CO2eq ha-1). Avoiding burn prior to harvest, even during expansion of sugarcane areas, promoted a mean reduction of GHG emission from 901 to 686 kg CO2eq ha-1 relative to harvest phase (24% lower) and an increase from 1418.3 to 1507.9 kg CO2eq ha-1 related to the ratoon maintenance phase (6% higher). Analyzing the total GHG emission per unit of cultivated sugarcane area (hectare), it was observed a decrease from 2275 to 2034 kg CO2eq ha-1 (11% reduction). The gradual transition of pre-harvest burning on that period has contributed to the reduction of GHG emission associated with sugarcane production being an important step towards GHG mitigation while still providing more sustainable sugar and ethanol production in southern Brazil.

Ethanol yield calculations in biorefineries.

The ethanol yield on sugar during alcoholic fermentation allows for diverse interpretation in academia and industry. There are several different ways to calculate this parameter, which is the most important one in this industrial bioprocess and the one that should be maximized, as reported by Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91). On the one hand, the various methods currently employed in industry provide dissimilar results, and recent evidence shows that yield has been consistently overestimated in Brazilian sugarcane biorefineries. On the other hand, in academia, researchers often lack information on all the intricate aspects involved in calculating the ethanol yield in industry. Here, we comment on these two aspects, using fuel ethanol production from sugarcane in Brazilian biorefineries as an example, and taking the work of Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91.) as a starting point. Our work is an attempt to demystify some common beliefs and to foster closer interaction between academic and industrial professionals from the fermentation sector. Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91).

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Hybrid neural network modeling and particle swarm optimization for improved ethanol production from cashew apple juice.

A hybrid neural model (HNM) and particle swarm optimization (PSO) was used to optimize ethanol production by a flocculating yeast, grown on cashew apple juice. HNM was obtained by combining artificial neural network (ANN), which predicted reaction specific rates, to mass balance equations for substrate (S), product and biomass (X) concentration, being an alternative method for predicting the behavior of complex systems. ANNs training was conducted using an experimental set of data of X and S, temperature and stirring speed. The HNM was statistically validated against a new dataset, being capable of representing the system behavior. The model was optimized based on a multiobjective function relating efficiency and productivity by applying the PSO. Optimal estimated conditions were: S0 = 127 g L-1, X0 = 5.8 g L-1, 35 °C and 111 rpm. In this condition, an efficiency of 91.5% with a productivity of 8.0 g L-1 h-1 was obtained at approximately 7 h of fermentation.

Improvement of ethanol production in fed-batch fermentation using a mixture of sugarcane juice and molasse under very high-gravity conditions.

Ethanol fermentation in very high gravity (VHG) saves energy consumption for ethanol distillation. As the technology offers high ethanol yield and low waste generation and it can be operated at low cost, it could be more efficient at an industrial scale than other ethanol production methods. This work studied ethanol production using a fed-batch bioreactor with a working volume of 1.5 L. The main objective of this research was evaluate the effects of temperature, sugar concentration, and cellular concentration using a Central Composite Design (CCD). Experimental conditions were selected using the surface response technique obtained from the CCD, and the results were validated to test the reproducibility. The following operating conditions were selected: temperature of 27.0 °C, sugar concentration 300.0 g/L, and cell concentration 15.0% (v/v). Under these conditions, after 30 h of fermentation the ethanol concentration, productivity and yield were 135.0 g/L, 4.42 g/(L·h) and 90.0%, respectively. All sugar was completely consumed.

Biomass Sorghum: Effect of Acid, Basic and Alkaline Peroxide Pretreatments on the Enzymatic Hydrolysis and Ethanol Production

Abstract This study evaluated the effects of three chemical pretreatments of biomass sorghum (BS): dilute alkaline (PTA1 and PTA2), dilute acid (PTB1 and PTB2) and alkaline hydrogen peroxide (PTC1 and PTC2) in the enzymatic hydrolysis and ethanol production. Among the six investigated conditions, the pretreatment with 7.36% H2O2 (PTC2) was the most efficient in the lignin removal and preservation of the polysaccharide fraction. After the enzymatic hydrolysis, increases in the glucose and xylose concentrations were observed in the pretreated BS hydrolysates, mainly in PTB1 and PTC1. All the hydrolysates obtained low concentrations of inhibitors. In the alcoholic fermentations with Pichia stiptis, the greatest ethanol yield was obtained in PTB1 hydrolysate (3.84 g L-1), corresponding to 16.15% of yield. The highest ethanol yield in PTB1 hydrolysate can be justified by the maximum concentration of xylose obtained in this hydrolysate, demonstrating the potential of P. stiptis in the fermentation of pentose to ethanol. The results indicated that biomass sorghum is an alternative lignocellulose source with potential for the production of second generation ethanol, opening up prospects for additional studies.

Influence of the sugarcane vinasse of the balance of charges in high weathered oxide soil of subtropical region in Brazil / Influência da vinhaça no balanço de cargas em Latossolo Veemelho distrófico da região subtropical do Brasil

Brazil is one of the largest sugarcane producers around the world, however the effects concerning environment issues in soil are still no well described. Soil electrochemical can provide important information about residues uses and environmental contamination, and then zero point of charge (ZPC) is a parameter that may demonstrate this variation. In this study, It was analyzed the soil behavior when submitted to sugarcane vinasse application in different doses simulating high and low doses application as regularly done by the manufactures in typical tropical weathered oxisols. To procedure ZPC test, microcosmsexperiment was managed with all parameters measured and controlled in a factorial design:2 soil depths (superficial group – 0.0 – 20.0 cm and sub-superficial group – 60.0 – 80.0 cm), 3 salt concentration (0.002; 0.02 and 0.2 mL NaCl), and 4vinasse doses (Without application; Low – 164.28 mL L-1; Intermediate – 328.57 mL L-1 and High 657.14 mL L-1), after incubation all the samples were measured electrochemically.Based on these analyses, data rised two main highlights: 1. vinasse low doses behaved like a buffer solution, 2. High doses in sub-superficial layer disturbed ZCP. Then, vinasse seems correlate with lixiviation capacity, and then over-applications can disrupt soil solution on this soil.

O Brasil é um dos maiores produtores de cana-de-açúcar do mundo, porém os efeitos relativos às questões ambientais do uso dos resíduos desta atividade no solo ainda não estão bem descritos. A eletroquímica do solo pode fornecer informações importantes sobre o impacto do uso de resíduos e contaminação ambiental, sendo o ponto de carga zero (ZPC) um parâmetro que pode demonstrar esta variação. Neste estudo, foi analisada a resposta do solo quando submetido à aplicação de vinhaça de cana-de-açúcar em diferentes doses, utilizando a aplicação de altas e baixas doses deste resíduo normalmente feita pelas indústrias sucroalcooleira em Latossolos tropicais intemperizados típicos. Para o teste de ZPC, o experimento laboratorial foi realizado com todos os parâmetros medidos e controlados em um planejamento fatorial: 2 profundidades de solo (grupo superficial - 0,0 - 20,0 cm e grupo subsuperficial - 60,0 - 80,0 cm), 3 concentração de solução salina (0,002; 0,02 e 0,2 mL NaCl) e 4 doses de vinhaça (Sem aplicação; Baixa - 164,28 mL L-1; Intermediário - 328,57 mL L-1 e Alta 657,14 mL L-1), após a incubação todas as amostras foram medidas eletroquimicamente e calculado o ZPC. Com base nessas análises, observou-se: 1. A dose mais baixa de vinhaça apresentou-se como uma solução tampão, 2. As doses altas na camada subsuperficial influenciaram a ZPC. Assim, a vinhaça parece estar correlacionada com a capacidade de lixiviação, desta forma, aplicações de altas doses de vinhaça nestes solos podem afetar eletronicamente a solução dos mesmos.

Influence of the sugarcane vinasse of the balance of charges in high weathered oxide soil of subtropical region in Brazil / Influência da vinhaça no balanço de cargas em Latossolo Veemelho distrófico da região subtropical do Brasil

Brazil is one of the largest sugarcane producers around the world, however the effects concerning environment issues in soil are still no well described. Soil electrochemical can provide important information about residues uses and environmental contamination, and then zero point of charge (ZPC) is a parameter that may demonstrate this variation. In this study, It was analyzed the soil behavior when submitted to sugarcane vinasse application in different doses simulating high and low doses application as regularly done by the manufactures in typical tropical weathered oxisols. To procedure ZPC test, microcosmsexperiment was managed with all parameters measured and controlled in a factorial design:2 soil depths (superficial group 0.0 20.0 cm and sub-superficial group 60.0 80.0 cm), 3 salt concentration (0.002; 0.02 and 0.2 mL NaCl), and 4vinasse doses (Without application; Low 164.28 mL L-1; Intermediate 328.57 mL L-1 and High 657.14 mL L-1), after incubation all the samples were measured electrochemically.Based on these analyses, data rised two main highlights: 1. vinasse low doses behaved like a buffer solution, 2. High doses in sub-superficial layer disturbed ZCP. Then, vinasse seems correlate with lixiviation capacity, and then over-applications can disrupt soil solution on this soil.(AU)

O Brasil é um dos maiores produtores de cana-de-açúcar do mundo, porém os efeitos relativos às questões ambientais do uso dos resíduos desta atividade no solo ainda não estão bem descritos. A eletroquímica do solo pode fornecer informações importantes sobre o impacto do uso de resíduos e contaminação ambiental, sendo o ponto de carga zero (ZPC) um parâmetro que pode demonstrar esta variação. Neste estudo, foi analisada a resposta do solo quando submetido à aplicação de vinhaça de cana-de-açúcar em diferentes doses, utilizando a aplicação de altas e baixas doses deste resíduo normalmente feita pelas indústrias sucroalcooleira em Latossolos tropicais intemperizados típicos. Para o teste de ZPC, o experimento laboratorial foi realizado com todos os parâmetros medidos e controlados em um planejamento fatorial: 2 profundidades de solo (grupo superficial - 0,0 - 20,0 cm e grupo subsuperficial - 60,0 - 80,0 cm), 3 concentração de solução salina (0,002; 0,02 e 0,2 mL NaCl) e 4 doses de vinhaça (Sem aplicação; Baixa - 164,28 mL L-1; Intermediário - 328,57 mL L-1 e Alta 657,14 mL L-1), após a incubação todas as amostras foram medidas eletroquimicamente e calculado o ZPC. Com base nessas análises, observou-se: 1. A dose mais baixa de vinhaça apresentou-se como uma solução tampão, 2. As doses altas na camada subsuperficial influenciaram a ZPC. Assim, a vinhaça parece estar correlacionada com a capacidade de lixiviação, desta forma, aplicações de altas doses de vinhaça nestes solos podem afetar eletronicamente a solução dos mesmos.(AU)

Physiological characterization of a new thermotolerant yeast strain isolated during Brazilian ethanol production, and its application in high-temperature fermentation.

BACKGROUND: The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. RESULTS: We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-coenzyme A synthetase 2 (ACS2). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature. CONCLUSION: The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids.

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L-1 ) and lipid production (1.04 g·L-1 ), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C16:1 , 9.4%), oleic acid (C18:1 , 47.2%), linoleic acid (C18:2 n-6 , 9.6%), and linolenic acid (C18:3 n-3 , 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L-1 of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

Abstract High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Native yeasts for alternative utilization of overripe mango pulp for ethanol production / Levaduras nativas para la utilización alternativa de pulpa de mango senescente para la producción de etanol

Mango fruits (Mangifera indica L.) are highly perishable, causing postharvest losses and producing agroindustrial waste. In the present work, native yeasts were used to evaluate ethanol production in overripe mango pulp. The two isolated strains showed similar sequences in the 18S rDNA region corresponding to Kluyveromyces marxianus, being different to the data reported in the NCBI database. Values of up to 5% ethanol (w/v) were obtained at the end of fermentation, showing a productivity of 4g/l/day, a yield of up to 49% of ethanol and a process efficiency of 80%. These results represent a viable option for using the surplus production and all the fruits that have suffered mechanical injury that are not marketable and are considered as agroindustrial waste, thus achieving greater income and less postharvest losses.

Las frutas de mango (Mangifera indica L.) son altamente perecederas, lo cual causa pérdidas poscosecha y produce desechos agroindustriales. En el presente trabajo, se utilizaron 2 levaduras nativas para evaluar la producción de etanol en pulpa de mango senescente. Las 2 cepas aisladas mostraron similitud en la región 18S ADNr, correspondiente a Kluyveromyces marxianus, la cual es diferente a lo reportado en la base de datos del NCBI. Se obtuvieron valores de hasta el 6% de etanol (v/v) al final de la fermentación, con una productividad de hasta 4g/l/día, un rendimiento de hasta 49% de etanol y una eficiencia en el proceso fermentativo del 80%. Esto representa una opción viable para utilizar excedentes de producción o frutos que han sufrido daño mecánico y no son comercializables, al lograr más ingresos y menos pérdida poscosecha.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.(AU)

Improvement of Brazilian bioethanol production - Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process.

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15-25 % of wet weight/v) and high sugar concentration (18-22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.

Ethanol production from sweet sorghum by Saccharomyces cerevisiae DBKKUY-53 immobilized on alginate-loofah matrices.

Ethanol production from sweet sorghum juice (SSJ) using the thermotolerant Saccharomyces cerevisiae strain DBKKUY-53 immobilized in an alginate-loofah matrix (ALM) was successfully developed. As found in this study, an ALM with dimensions of 20×20×5mm3 is effective for cell immobilization due to its compact structure and long-term stability. The ALM-immobilized cell system exhibited greater ethanol production efficiency than the freely suspended cell system. By using a central composite design (CCD), the optimum conditions for ethanol production from SSJ by ALM-immobilized cells were determined. The maximum ethanol concentration and volumetric ethanol productivity obtained using ALM-immobilized cells under the optimal conditions were 97.54g/L and 1.36g/Lh, respectively. The use of the ALM-immobilized cells was successful for at least six consecutive batches (360h) without any loss of ethanol production efficiency, suggesting their potential application in industrial ethanol production.

Native yeasts for alternative utilization of overripe mango pulp for ethanol production.

Mango fruits (Mangifera indica L.) are highly perishable, causing postharvest losses and producing agroindustrial waste. In the present work, native yeasts were used to evaluate ethanol production in overripe mango pulp. The two isolated strains showed similar sequences in the 18S rDNA region corresponding to Kluyveromyces marxianus, being different to the data reported in the NCBI database. Values of up to 5% ethanol (w/v) were obtained at the end of fermentation, showing a productivity of 4g/l/day, a yield of up to 49% of ethanol and a process efficiency of 80%. These results represent a viable option for using the surplus production and all the fruits that have suffered mechanical injury that are not marketable and are considered as agroindustrial waste, thus achieving greater income and less postharvest losses.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production.

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51g/L and 33.84g/L at 37°C and 40°C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42°C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.