Very high gravity wort fermentation by immobilised yeast

Using calcium alginate-entrapped yeast, 24% (w/w) wort was successfully fermented within 8 days. This is half the time needed for fermentation by free yeast. The highest ethanol concentration obtained was 10.5% (v/v). When the original wort gravity was increased, the specific rate of ethanol production remained constant 0.16 g gh^–1 and the viability did not fall bellow 95% of living cells. Protection of cell against osmotic stress by gel matrix was also confirmed by trehalose measurement. The maximum intracellular trehalose content in calcium alginate-entrapped yeast was 3 times lower compared to free yeast at 30% (w/w) wort fermentation.     

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain’s performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.     

Optimization of bioethanol production during simultaneous saccharification and fermentation in very high-gravity cassava mash

Hydrolysis and fermentation conditions for production of ethanol from very high-gravity cassava mash by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing were optimized using a statistical methodology. During the first part of the study, Placket–Burman design (PBD) was used to study 19 factors that could potentially influence ethanol production. Gravity, particle size, initial pH, and fermentation temperature were identified as key factors that significantly increased final ethanol concentration. The main and interaction effects of these factors were subsequently evaluated based on a quadratic equation generated by central composite design (CCD) using response-surface methodology (RSM). Under the optimized very high-gravity conditions, the final ethanol concentration obtained from experiment increased from 8.21% (wt.%) to 15.03% (wt.%) and was in good agreement with model prediction. By employing two other commercial Saccharomyces strains, similar results were obtained under the same optimized condition. Therefore, we conclude that final ethanol concentration, ethanol productivity (V _P/max), glucose utilization (Y _G/s, Y _P/s), and fermentation efficiency (η _f) were enhanced or maintained under the optimized condition of 40% gravity, 390 μm particle size, initial pH 5.5, and 27°C fermentation temperature.     

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

The effect of start wort on the initial period of Baker's yeast fermentation

В лабораторных ферментационных танках, в условиях, как можно больше отвечающих производственным, исследовалось влияние субстрата на период взбраживания при дрожжевом брожении. Исследовались влияние концентрации инвертного сахара на ход\(Q_{CO_2 }^{N_2 } \) на мальтозе и глюкозе, а также динамика содержания РНК и ДНК. Далее исследовалась также динамика фосфатных фракций и некоторых других составных частей клеток (трегелёза, полисахариды, белки, свободные аминокислоты). Было установлено, что субстрат быстро метаболизируется и немедленно протекает синтез РНК, всех углеводов и белков, что сопровождается регулярным понижением содержания 7-минутного фосфора. Синтез ДНК осущест-вляется позднее. Изменения составных частей клетки находятся в корреляции с морфологическими изменениями клетки, с процессом роста и деления дрожжей. Из результатов опытов очевидно, что процессы, протекающие в этот период брожения, можно сравнить с кратким циклом роста. С точки зрения технологии дрожжевого брожения констатируется, что 1-часовый период взбраживания слишком долог и что клетки в указанных усповиях страдают от недостатка сахарного субстрата и бывают принуждены с углеводного субстрата перейти на алкоголь.     

Selection of Saccharomyces pastorianus variants with improved fermentation performance under very high gravity wort conditions

Saccharomyces pastorianus FBY0095 was mutated and variants were selected for efficient very high gravity brewing using 15% (w/v) maltose and 15% (w/v) ethanol. Two useful variants were obtained of which one (L6) had growth, wort consumption and ethanol production rates of 0.036, 1.13 and 0.49 g l⁻¹ h⁻¹, respectively. The corresponding results for the wild type were 0.028, 0.98 and 0.4 g l⁻¹ h⁻¹, respectively. The vitality of the variant (expressed as acidification power) was 2.5 while that of the wild type was 2.3. There was also an obvious improvement on flavor of resulting beer when using L6 and the other variant.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active substances, which are vital for the complex flavour of beer. In order to obtain insight into the influence of high-gravity brewing and fermentation temperature on flavour formation, we analysed flavour production and the expression level of ten genes (ADH1, BAP2, BAT1, BAT2, ILV5, ATF1, ATF2, IAH1, EHT1 and EEB1) during fermentation of a lager and an ale yeast. Higher initial wort gravity increased acetate ester production, while the influence of higher fermentation temperature on aroma compound production was rather limited. In addition, there is a good correlation between flavour production and the expression level of specific genes involved in the biosynthesis of aroma compounds. We conclude that yeasts with desired amounts of esters and higher alcohols, in accordance with specific consumer preferences, may be identified based on the expression level of flavour biosynthesis genes. Moreover, these results demonstrate that the initial wort density can determine the final concentration of important volatile aroma compounds, thereby allowing beneficial adaptation of the flavour of beer.     

An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars

Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12° all-malt wort supplemented with sorbitol to reach osmolarity equal to 16° and 20°. Three pitchings were performed in 12°/12°/12°, 16°/16°/12°, and 20°/20°/12° worts. Fermentations in 16° and 20° worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd “back to normal” pitching into 12° wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16° and 20° worts was delayed but increased about two times at fermentation end relative to that in 12° wort. Third “back-to-normal” pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.     

Changes in the rate of fermentation of maltose during propagation of industrial Baker's yeast

The rate of fermentation of glucose and maltose and the maltase activity of cellfree preparations of yeast were investigated during yeast propagation at the individual production stages. It was found that the yeast cells do not change much in their fermentation of glucose but that the level of the maltose-hydrolyzing enzyme undergoes changes, together with the character of the anaerobic fermentation of maltose, depending on the character of cultivation (batch or incremental feeding). After an initial decrease the maltose activity of cell-free preparations is maintained practically on the same level until the expedition phase is reached when it rapidly decreases to low values. The basis of the changes investigated is discussed together with their importance for yeast production technology.