Low-temperature Fermentation Research Articles

New alternatives for the fermentation process in the ethanol production from sugarcane: Extractive and low temperature fermentation

Ethanol is produced in large scale from sugarcane in Brazil by fermentation of sugars and distillation. This is currently considered as an efficient biofuel technology, leading to significant reduction on greenhouse gases emissions. However, some improvements in the process can be introduced in order to improve the use of energy. In current distilleries, a significant fraction of the energy consumption occurs in the purification step – distillation and dehydration – since conventional fermentation systems employed in the industry require low substrate concentration, which must be distilled, consequently with high energy consumption. In this study, alternatives to the conventional fermentation processes are assessed, through computer simulation: low temperature fermentation and vacuum extractive fermentation. The aim of this study is to assess the incorporation of these alternative fermentation processes in ethanol production, energy consumption and electricity surplus produced in the cogeneration system. Several cases were evaluated. Thermal integration technique was applied. Results shown that the ethanol production increases between 3.3% and 4.8% and a reduction in steam consumption happens of up to 36%. About the electricity surplus, a value of 85 kWh/t of cane can be achieved when condensing – extracting steam turbines are used.

Design of Green Processing Chain for Processing of Ginseng Fruit Vinegar

Using ginseng, wild wine and acetic acid to the orthogonal experiment method to determine the fermentation process, low temperature and cryogenic grinding fermentation brewing vinegar. The results showed that: cryogenic grinding with a significant impact on the quality of the wine, the best ginseng vinegar fermentation parameters for initial dosage of 6% alcohol, fermentation temperature was 31 °C, fermentation time was 96h.By mixed fermentation Changbai Mountain forest berries and ginseng significantly alter the flavor of vinegar.

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast

Low-temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low-temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.

Fungal Diversity of Rice Straw for Meju Fermentation

Rice straw is closely associated with meju fermentation and it is generally known that the rice straw provides meju with many kinds of microorganisms. In order to elucidate the origin of meju fungi, the fungal diversity of rice straw was examined. Rice straw was collected from 12 Jang factories where meju are produced, and were incubated under nine different conditions by altering the media (MEA, DRBC, and DG18), and temperature (15°C, 25°C, and 35°C). In total, 937 strains were isolated and identified as belonging to 39 genera and 103 species. Among these, Aspergillus, Cladosporium, Eurotium, Fusarium, and Penicillium were the dominant genera. Fusarium asiaticum (56.3%), Cladosporium cladosporioides (48.6%), Aspergillus tubingensis (37.5%), A. oryzae (31.9%), Eurotium repens (27.1%), and E. chevalieri (25.0%) were frequently isolated from the rice straw obtained from many factories. Twelve genera and 40 species of fungi that were isolated in the rice straw in this study were also isolated from meju. Specifically, A. oryzae, C. cladosporioides, E. chevalieri, E. repens, F. asiaticum, and Penicillium polonicum (11.8%), which are abundant species in meju, were also isolated frequently from rice straw. C. cladosporioides, F. asiaticum, and P. polonicum, which are abundant in the low temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at 15°C and 25°C, whereas A. oryzae, E. repens, and E. chevalieri, which are abundant in the high temperature fermentation process of meju fermentation, were frequently isolated from rice straw incubated at 25°C and 35°C. This suggests that the mycobiota of rice straw has a large influence in the mycobiota of meju. The influence of fungi on the rice straw as feed and silage for livestock, and as plant pathogens for rice, are discussed as well.

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10°C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10°C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry

Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD+ synthesis.

Production of aroma compounds by cryotolerantSaccharomycesspecies and hybrids at low and moderate fermentation temperatures

Among the most important factors influencing wine quality are yeast strains and fermentation temperature. Fermentation at low temperature is presently used in winemaking to improve both aroma formation and retention. In this study, we have analysed the oenological characteristics of Tempranillo wines produced at 12 and 28°C by different Saccharomyces species and hybrids. Low temperature had a strong influence on yeasts fermentation kinetics, increasing fermentation times to more than 2 weeks. In some strains, glycerol production seemed to be positively influenced by low fermentation temperature. Analysis of the aroma composition of wines produced by different Saccharomyces species and hybrids revealed large differences depending on fermentation temperature. Higher alcohols production seemed to be dependent on the strain. Production of acetate esters seemed to be favoured at 28°C, whereas production of ethyl esters was apparently preferred at low fermentation temperatures. The best aroma producers at 28°C were Saccharomyces cerevisiae strains, whereas Saccharomyces uvarum and some hybrids excelled at 12°C. Our results suggest that fermentation temperature and yeast species are of crucial importance for production of metabolites influencing wine aroma.

Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: Implication in growth at low temperatures

The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10–15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.

The aroma profile of wheat bread crumb influenced by yeast concentration and fermentation temperature

The consumers of today have an increasing interest in high quality bread with appealing aroma. The scope of this work is to investigate how aroma in wheat bread crumb is influenced by different fermentation conditions: amount of yeast (20, 40 and 60 g/kg flour) and fermentation temperature (5, 15 and 35 °C). Dough samples were fermented to equal height and baked, and the aroma compounds from the bread were extracted by dynamic headspace extraction and analyzed by gas chromatography–mass spectrometry. Quantification of the aroma compounds was performed by multiple headspace extraction. The most aroma active compounds identified were 3-methylbutanal, (E)-2-nonenal, 3-methyl-1-butanol, and 2,3-butanedione. Increasing the yeast concentration was found to increase formation of the majority of the compounds formed from the yeast metabolism, with 2,3-butanedione and phenylacetaldehyde as the most aroma active compounds. High fermentation temperature (15 and 35 °C) increased formation of many lipid oxidation compounds, with hexanal and heptanal having the highest odor activity values. Low fermentation temperature (5 °C) was found to increase formation of the three esters ethyl acetate, ethyl hexanoate, and ethyl octanoate, with ethyl hexanoate having the highest odor activity value. The odor activity values of the esters were generally low.

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent.

Zygomycota associated with traditional meju, a fermented soybean starting material for soy sauce and soybean paste

Various zygomycota species were detected during a study of the mycobiota of meju, a brick of dried fermented soybeans, used in Korean cuisine. Two hundred and sixty-eight strains were isolated from 98 finished meju products collected in various regions of Korea from 2009 to 2011, and 96 strains were isolated from in-process meju on various farms from 2010 to 2011. The isolated zygomycota were identified using phenotypic characteristics combined with DNA sequences of the internal transcribed spacer regions of ribosomal DNA and the D1/D2 nuclear ribosomal large subunit. Of 364 zygomycota strains, 108 were identified as Mucor circinelloides, 96 as M. racemosus, 60 as Lichtheimia ramosa, 22 as Rhizopus stolonifer, 16 as Lichtheimia corymbifera, and the other 62 strains comprised 10 other species. The psychrotrophic species, Mucor circinelloides and M. racemosus were predominantly present during low temperature fermentation (LTF) and the thermotolerant species Lichtheimia ramosa and Rhizomucor species were predominant during high temperature fermentation (HTF). The results suggest that temperature has a large influence on the zygomycota composition during the fermentation process of meju.

Two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (−30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at −20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.

Improving bioethanol production – Comparison between extractive and low temperature fermentation

One of the key issues that must be addressed in the biofuel production based on sugarcane industry is the energy consumption of the process. Process energy demand has direct impact on the amount of lignocellulosic material available for use as feedstock for second generation ethanol production. A significant fraction of the energy consumption in bioethanol production occurs in the purification step, since conventional fermentation systems employed in the industry require low substrate concentration and, consequently, produce wine of low (around 8.5°GL) ethanol content that must be distilled in order to meet product specifications. In this study alternatives to the conventional fermentation processes employed in the industry (low temperature fermentation and vacuum extractive fermentation) were assessed, in the context of a large scale sugarcane autonomous distillery, through computer simulation. Electricity consumption and lignocellulosic material surplus on each case were evaluated. It is shown that the alternative fermentation processes allow a significant reduction on vinasse generation and increases ethanol production when compared with conventional fermentation, but increases electricity consumption (for the extractive fermentation) or steam consumption (for low temperature fermentation); when vinasse concentration is considered in the conventional process, steam consumption in the extractive fermentation is also significantly smaller.

Functional analysis to identify genes in wine yeast adaptation to low-temperature fermentation

To identify genes and proteins involved in adaptation to low-temperature fermentations in a commercial wine yeast. Nine proteins were identified as representing the most significant changes in proteomic maps during the first 24 h of fermentation at low (13°C) and standard temperature (25°C). These proteins were mainly involved in stress response and in glucose and nitrogen metabolism. Transcription analysis of the genes encoding most of these proteins within the same time frame of wine fermentation presented a good correlation with proteomic data. Knockout and overexpressing strains of some of these genes were constructed and tested to evaluate their ability to start the fermentation process. The strain overexpressing ILV5 improved its fermentation activity in the first hours of fermentation. This strain showed a quicker process of mitochondrial degeneration, an altered intracellular amino acid profile and laxer nitrogen catabolite repression regulation. The proteomic and transcriptomic analysis is useful to detect key molecular adaptation mechanisms of biotechnological interest for industrial processes. ILV5 gene seems to be important in wine yeast adaptation to low-temperature fermentation. This study provides information that might help improve the future performance of wine yeast, either by genetic modification or by adaptation during industrial production.

Nano-tubular cellulose for bioprocess technology development.

Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in “cold pasteurization” processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc.

Effect of pressure and temperature on alcoholic fermentation by Saccharomyces cerevisiae immobilized on γ-alumina pellets

Saccharomyces cerevisiae was immobilized on γ-alumina pellets and used for repeated batch fermentations in glucose medium (16.5g/100mL) at various temperatures and pressures. An increase in pressure from 3 to 7atm and a decrease in temperature from 30 to 20°C reduced the ethanol productivity by about 50% and 70%, respectively. Increasing concentrations of volatile by-products were observed at lower fermentation temperatures, while the pressure influence on the concentrations of these by-products was proved to be more complex. Mathematical expressions were established to allow the calculation of the fermentation rate at various pressures and sugar concentrations when the corresponding rate at atmospheric pressure is known. The study showed that the height of bioreactors has to be limited to 19.5m due to hydrostatic pressure shock at higher fill levels.

Efeitos de parâmetros de fermentação na produção de etanol a partir de resíduo líquido da industrialização da mandioca (manipueira)

“Manipueira”, a liquid residue from the processing of cassava starch and flour, has recognized high pollution potential. Aiming at a possible use of “manipueira” as raw material for ethanol production, this study aimed to evaluate the effect of the percentage of inoculated yeast and fermentation temperature on the chromatographic profile of the components of wine of “manipueira”. The residue was characterized for starch and sugar content, as well as pH and total acidity. The residue was hydrolyzed by the action of enzymes Termamyl 120 L and AMG 300 L. The hydrolysate obtained was fermented under different experimental conditions. A factorial central composite design (22) with two independent variables and the response surface methodology were used to evaluate the results. The results showed a significant effect of variable parameters on the components of wine. The conditions of low fermentation temperature and lower percentages of inoculated yeast were the most appropriate to obtain ethanol from “manipueira”.

Effect of growth temperature on yeast lipid composition and alcoholic fermentation at low temperature

The loss of viability of wine yeast strains due to low-temperature fermentations could be overcome by increasing their stress tolerance and adaptability. Changes in membrane lipid composition are one of the first responses to cold stress. The aim of this study was to analyze the various adaptation mechanisms to low temperatures by comparing the better adapted Saccharomyces species. The viability, vitality, fermentation capacity, and lipid composition of different Saccharomyces species (S. cerevisiae, S. bayanus, S. uvarum, and a hybrid S. cerevisiae/S. uvarum) with different fermentative origins (wine, beer, and baker’s strains together with a laboratory strain) were compared after culturing at low (13 °C) and optimal (25 °C) temperatures. In spite of specific responses of the different strains/species, the results showed that at low temperature, the medium-chain fatty acid and the triacylglyceride content increased, whereas the phosphatidic acid content and the phosphatidylcholine/phosphatidylethanolamine ratio decreased. Only the laboratory strain was not able to ferment the sugars, and after growing at both temperatures, its lipid composition was very different from that of the other strains. The hybrid strain showed the highest sugar consumption at 13 °C and the best vitality whatever the preculture temperature used. The rest of the species needed a preadaptation at low temperature involving a change in their lipid composition to improve their fermentation rate at 13 °C.

Effect of fermentation conditions on yeast growth and volatile composition of wine produced from mango ( Mangifera indica L.) fruit juice

In this study mango juice fermentation at laboratory scale with controlled inoculation using selected yeast strain was performed ( Saccharomyces cerevisiae 101). Effect of fermentation conditions (temperature, pH, SO 2 and aeration) on wine fermentation was evaluated based on yeast growth, duration, fermentation rate and volatile composition. The composition of the major volatile compounds with low boiling points was determined by gas chromatography under the different operating conditions of fermentation temperature (15–35 °C), pH (3.5–6.0), SO 2 (100–300 ppm) and aeration (initial dissolved O 2 and shaking at 30 rpm). Temperature had important effect on yeast growth and on the levels of volatile compounds. It was observed that the final concentrations of ethyl acetate and some of the higher alcohols decreased when fermentation temperature increased to 25 °C (35 mg/l at 15 °C and 27 mg/l at 25 °C). SO 2 stimulated the yeast growth up to certain level and in excess it inhibited the yeast metabolism. Ethanol concentration slightly increased with the addition of 100 ppm SO 2 (8.2 g/l) and decreased with increase in concentration of SO 2 (6.2 g/l in 300 ppm SO 2). Aeration by shaking increased the viable cell count (from 52 × 10 6 in the absence of oxygen to 98 × 10 6 in shaking at 30 rpm) but decreased the ethanol productivity (from 7.2 in initial dissolved O 2 to 6.5 g/l shaking at 30 rpm). With the results obtained it was concluded that the temperature (25 °C), pH (5), SO 2 (100 ppm) and must with initial oxygen were optimum for better quality of wine from mango fruits. The results of the present study considering the traditionally recognized preference for low alcoholic fermentation temperatures in wine making.

Scale-up of extremely low temperature fermentations of grape must by wheat supported yeast cells

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole wheat grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape must at various temperatures. The biocatalyst retained its operational stability for a long period and it was proved capable to produce dry wines of fine clarity even at extremely low temperatures (5 °C). After the completion of these fermentations the new biocatalyst was used in a scale-up system of 80 L for wine making at ambient (20 °C) and extremely low temperatures (2 °C). The scale-up process did not affect the fermentative ability of biocatalyst, even at low temperatures, while the produced wines had almost the same improved aromatic profile compare to free cells as revealed by GC and GC–MS analyses. More specifically the results showed that both systems with immobilized cells (laboratory scale and 80 L bioreactor) increased the formation of esters and produced wines with improved aromatic profile compared to those with free cells. Finally an increase in the percentages of total esters and a decrease in those of higher alcohols was observed in lower fermentation temperatures.