Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain.

Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L(-1) h(-1) (corresponding to a feed lactose concentration of 50 g L(-1) and a dilution rate of 0.55 h(-1)) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.     

Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation

Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity.     

Kinetics of lactose fermentation using a recombinant Saccharomyces cerevisiae strain

This work presents a multi-route, non-structural kinetic model for interpretation of ethanol fermentation of lactose using a recombinant flocculent Saccharomyces cerevisiae strain expressing both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces lactis. In this model, the values of different metabolic pathways are calculated applying a modified Monod equation rate in which the growth rate is proportional to the concentration of a key enzyme controlling the single metabolic pathway. In this study, three main metabolic routes for S. cerevisiae are considered: oxidation of lactose, reduction of lactose (producing ethanol), and oxidation of ethanol. The main bioprocess variables determined experimentally were lactose, ethanol, biomass, and dissolved oxygen concentrations. Parameters of the proposed kinetic model were established by fitting the experimental data obtained in a small lab-scale fermentor with the initial lactose concentrations ranging from 5 g/dm3 to 50 g/dm3. A very good agreement between experimental data and simulated profiles of the main variables (lactose, ethanol, biomass, and dissolved oxygen concentrations) was achieved.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Construction of a flocculent Saccharomyces cerevisiae fermenting lactose

A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for β-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. Received: 2 October 1998 / Received revision: 15 January 1999 / Accepted: 17 January 1999     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

酿酒酵母 L-阿拉伯糖转化乙醇代谢工程的研究进展简

L-阿拉伯糖是木质纤维素原料中一种重要的五碳糖组分,但传统的乙醇生产菌株酿酒酵母（ Saccharomyces cerevisiae）不能利用L 阿拉伯糖。通过代谢途径工程手段,在酿酒酵母中引入L 阿拉伯糖初始代谢途径可以获得能利用L 阿拉伯糖乙醇发酵的重组菌株。并且,通过代谢途径的疏通以及吸收系统的优化可以强化重组菌株代谢L 阿拉伯糖的能力。笔者从以上角度综述了近年来酿酒酵母转化L 阿拉伯糖生产乙醇的研究进展。     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Growth and growth estimation of Saccharomyces cerevisiae in solid-state ethanol fermentation

The anaerobic growth of a respiration-deficient mutant of Saccharomyces cerevisiae on solid medium was estimated by the CO₂ evolution rate (CER). The cell growth and ethanol production were calculated by a growth-model associated with CER. The estimated cell growth agreed with the observed data. The calculation and the observed CER suggested that the maximum ethanol production and maximum cell groth are restricted by the initial moisture content of the solid medium.     

Accelerated ethanol fermentation by Saccharomyces cerevisiae with addition of activated carbon

Fermentation with the addition of activated carbon at 100 g l^–1 promoted the glucose consumption and ethanol production rates of Saccharomyces cerevisiae by 1.3 and 1.1 times, respectively. With fermentation using spent medium, the consumption rate was maintained at 90% of that in the fresh medium with the addition of activated carbon, while the rate without any addition decreased to about 70%.     

The role of nisin in fuel ethanol production with Saccharomyces cerevisiae

Aims: To investigate the effects of nisin on lactobacilli contamination of yeast during ethanol fermentation and to determine the appropriate concentration required to control the growth of selected lactobacilli in a YP/glucose media fermentation model. Methods and Results: The lowest concentration of nisin tested (5 IU ml^?1) effectively controlled the contamination of YP/glucose media with 10⁶ CFU ml^?1 lactobacilli. Lactic acid yield decreased from 5·0 to 2·0 g l^?1 and potential ethanol yield losses owing to the growth and metabolism of Lactobacillus plantarum and Lactobacillus brevis were reduced by 11 and 7·8%, respectively. Approximately, equal concentrations of lactic acid were produced by Lact. plantarum and Lact. brevis in the presence of 5 and 2 IU ml^?1 nisin, respectively, thus demonstrating the relatively higher nisin sensitivity of Lact. brevis for the strains in this study. No differences were observed in the final ethanol concentrations produced by yeast in the absence of bacteria at any of the nisin concentrations tested. Conclusions: Metabolism of contaminating bacteria was reduced in the presence of 5 IU ml^?1 nisin, resulting in reduced lactic acid production and increased ethanol production by the yeast. Significance and Impact of the Study: Bacteriocins represent an alternative to the use of antibiotics for the control of bacterial contamination in fuel ethanol plants and may be important in preventing the emergence of antibiotic‐resistant contaminating strains.     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

Reconstruction of ethanol fermentation in permeabilized cells of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae cells from a stationary culture were permeabilized with 1% toluene, 4% ethanol and 0.075% Triton X-100. Not only sugars but also ATP, NAD⁺, magnesium and inorganic phosphate must be simultaneously added to initiate the ethanol fermentation. The optimal pH for the fermentation was between 6.9 to 7.0. Sucrose was a better substrate than glucose. Ethanol fermentation was greatly stimulated by the addition of 1 mM arsenate. Under this condition, permeabilized cells continued to produce ethanol for more than one hour at the rate of 0.141 mmol ethanol/min/mg protein. Methanol inhibited the fermentation with intact cells but did not inhibit the one using permeabilized cells. In contrast, propanol inhibited fermentations both with intact and permeabilized cells.     

Control of an ethanol fermentation carried out with alginate entrapped Saccharomyces cerevisiae

Process control of different reactor models for continuous production of ethanol from sucrose with immobilized yeast has been studied. An enzyme thermistor with immobilized invertase recorded the concentration of sucrose continuously. Ethanol was recorded by a membrane gas sensor with a SnO(2) semiconductor used as detector. A process computer controlled the substrate feed to keep substrate as well as ethanol concentration at preset values by using algorithms of varying complexity. It was thereby demonstrated that PID regulators as well as more advanced algorithms (Otto-Smith regulator, state feedback from a Kalman filter, and cascade control) are useful alternatives to maintain a constant concentration in the fermentor effluents. The time required for the system to return to predetermined conditions after various kinds of disturbances has been especially studied. It was shown that the more advanced regulator used the shorter time.     

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae

 The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 °C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast. Received: 11 June 1999 / Received last revision: 12 November 1999 / Accepted: 28 November 1999     

Kissiris: A mineral support for the promotion of ethanol fermentation by Saccharomyces cerevisiae

Ethanol production using Saccharomyces cerevisiae, promoted by the mineral kissiris, is reported on. A three-fold increase of ethanol productivity in the fermentation of molasses was achieved. An ethanol yield factor 0.43 g/g and conversion of 93.3% at an initial sugar concentration (ISC) 208.5 g/l were obtained in the presence of this mineral in molasses fermentation, compared to 0.21 g/g and 44.2% in its absence. It is also shown that the fermentation of molasses takes place even at relatively higher ethanol levels, with kissiris contributing to a 35% reduction of the energy demand in grade-fuel and potable ethanol production. The proposed mineral was shown to have a smaller effect in fermentations carried out in synthetic media containing glucose or saccharose.