Selection of lactose-fermenting yeast for ethanol production from whey

Summary Candida pseudotropicalis ATCC 8619 was selected among nine strains of lactose-fermenting yeasts on the basis of its ability to ferment concentrated whey. In 28% (wt/vol) deproteinized whey solutions it produced an average of 12.4% (vol/vol) ethanol. This yeast could be used in a process for whey treatment.     

Optimization of fermentation conditions for ethanol production from whey

Summary Optimal conditions for ethanol production in 7% whey solutions by the yeast Candida pseudotropicalis ATCC 8619 included initial pH of 4.57 and 30°C. Complete fermentation of the available lactose took place without supplementary nutrients; additions of nitrogen or phosphorus salts, yeast extract or corn steep liquor resulted in increased yeast production and lower ethanol yields. A positive correlation was observed between increases in yeast inocula and lactose utilization and ethanol production rates; 8.35 g/l of ethanol was obtained within 22 h by using yeast inoculum of 13.9 g/l. No differences in fermentation rates or ethanol yields were observed when whole or deproteinized whey solutions were used. Concentrated whey permeates, obtained after removal of the valuable proteins from whey, can be effectively fermented for ethanol production.     

Selection of yeast strains for ethanol production from inulin

Of the many yeast specie scapable of fermenting inulin, some can produce sufficient amounts of ethanol from the substrate, in particularKluyverotmyces fragilis andTorulopsis colliculosa. The results indicate the feasibility of producing ethanol from inulin-rich plants, such as Jerusalem artichoke.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Butanol production from concentrated lactose/whey permeate: Use of pervaporation membrane to recover and concentrate product

In these studies, butanol (acetone butanol ethanol or ABE) was produced from concentrated lactose/whey permeate containing 211 g L^?1 lactose. Fermentation of such a highly concentrated lactose solution was possible due to simultaneous product removal using a pervaporation membrane. In this system, a productivity of 0.43 g L^?1 h^?1 was obtained which is 307 % of that achieved in a non-product removal batch reactor (0.14 g L^?1 h^?1) where approximately 60 g L^?1 whey permeate lactose was fermented. The productivity obtained in this system is much higher than that achieved in other product removal systems (perstraction 0.21 g L^?1 h^?1 and gas stripping 0.32 g L^?1 h^?1). This membrane was also used to concentrate butanol from approximately 2.50 g L^?1 in the reactor to 755 g L^?1. Using this membrane, ABE selectivities and fluxes of 24.4–44.3 and 0.57–4.05 g m^?2 h^?1 were obtained, respectively. Pervaporation restricts removal of water from the reaction mixture thus requiring significantly less energy for product recovery when compared to gas stripping.     

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.     

Ethanol production from lactose by extractive fermentation

Summary The suitability of extractive fermentation as a technique for the production of ethanol from lactose by Candida pseudotropicalis was examined as a potential improvement over conventional methods. A biocompatible solvent was selected through determination of the critical log P (octanol-water distribution coefficient) of the fermentation organism. Using Adol 85 NF, the selected solvent, extractive fed-batch and conventional fed-batch systems were operated for 160 h. The extractive system showed a 60% improvement in lactose consumption and ethanol production, as well as a 75% higher volumetric productivity.     

Hydrolysis of whey lactose using CTAB-permeabilized yeast cells

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to β-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 °C.     

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.     

Recovery of yeast invertase from ethanol fermentation broth

Summary Ethanol fermentation broth produced by an aggregated form ofSaccharomyces uvarum strain contained invertase when sucrose-based raw materials were used. The amount of invertase in the borth was in the range of 1.4 to 4.8 units/ml, which was affected by the dilution rate, the concentration of corn steep liquor, and the type of sugar used. The activity of invertase in the broth could be maintained at 0.8 units/ml over two months. When the broth was passed through DEAE-cellulose beads and eluted with a NaCl-Tris-HCl buffer solution, a 75% recovery yield of invertase with 9-fold purification and 30-fold concentration could be achieved.     

Selection of lactic yeast producing glucosylceramide from cheese whey

From 2150 isolates from raw milk and milk products, yeast strains were surveyed to produce glucosylceramide from cheese whey. Most of the 54 strains that had accumulated a detectable amount of glucosylceramide were identified as Kluyveromyces lactis var. lactis. The cells of K. lactis var. lactis strain M-11 derived from domestic raw milk accumulated glucosylceramide 2.5-fold higher than K. lactis var. lactis NBRC 1267, the reference strain selected from the culture collections. Strain M-16 of K. lactis var. lactis derived from the same origin was found to synthesize a considerable amount of steryl glucoside in addition to glucosylceramide. Sequence analysis of ribosomal DNA intergenic spacer two regions revealed that strains M-11 and M-16 were diverged from a type strain of K. lactis var. lactis in the same species.     

An investigation of ethanol inhibition and other limitations occurring during the fermentation of concentrated whey permeate byKluyveromyces fragilis

Based on the well-known fact thatKluyveromyces fragilis strains show sub-optimal performance when grown in concentrated whey permeate, previously optimized medium was investigated for possible limitations appearing at high concentrations. Shaken flask cultures showed that no additional vitamin or mineral sources were required when the optimized amount of yeast extract was added to the concentrated permeate. Several aspects of the ethanol inhibition of the growth ofK. fragilis NRRL 665 were investigated in continuous culture. The maximum ethanol concentration tolerated by this yeast, i.e. 45 g/l, was much lower than commonly reported for other strains. Ethanol and biomass production were also influenced by the increased ethanol concentration of the medium. At 31 g/l of alcohol product yield was reduced to 0.23 g/g whereas biomass yield was 0.05 g/g. Some evidence suggested that residence time and residual lactose concentration played a significant role in modulating the toxic effect of ethanol.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

Influence of lactose hydrolysis and solids concentration on alcohol production by yeast in acid whey ultrafiltrate

Alcohol yields of 6.5% were obtained with Saccharomyces cerevisiae in lactasehydrolyzed acid whey permeate containing 30–35% total solids. Maximum alcohol yields obtained with Kluyveromyces fragilis were 4.5% in lactase-hydrolyzed acid whey permeate at a solids concentration of 20% and 3.7% in normal permeate at a solids concentration of 10%. Saccharomyces cerevisiae efficiently converted the glucose present in lactase-hydrolyzed whey permeates containing 5–30% total solids (2–13% glucose) to alcohol. However, the galactose, which comprised about half the available carbohydrate in lactase-hydrolyzed whey, was not utilized by S. cerevisiae, so that even though alcohol yields were higher when this organism was used, the process was wasteful in that a substantial proportion of the substrate was not fermented. For the process to become commercially feasible, an efficient means of rapidly converting both the galactose and glucose to alcohol must be found.     

Ethanol production from whey with immobilized living yeast

Summary Living Kluyveromyces fragilis yeast cells were succesfully entrapped in calcium alginate gel beads at cell loadings of 4 to 16 g yeast (0.8 to 3.2 g d.m.) per 1 g of sodium alginate. In batch systems, about 90 % conversion in 48 h was obtained both with free and immobilized yeast using demineralized whey of 5 to 10 % lactose content as substrate. In continuous packed-bed column operation nearly a constant 2 % product ethanol concentration could be maintained at 5 % substrate lactose level for at least one month.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

Simultaneous ethanol production from lactose byKluyveromyces fragilis andZymomonas mobilis

Ethanol production from lactose byKluyveromyces fragilis NRRL 665 in monoculture and coculture with strains ofZymomonas mobilis was studied. One of the strains,Z. mobilis NRRL 1960, when cocultured withK. fragilis, produed 55.2 g/l of ethanol, whereasK. fragilis in monoculture procuded only 36 g/l ethanol from 200 g/l lactose medium. Increased Qp (g ethanol produced/g biomass/h) and Qs (g substrate consumed/g biomass/h) were observed in coculture than in monoculture. However, the residual sugar concentration increased in coculture; this increase might be due to the slow utilization rate of galactose.     

高生物量富铁酵母菌的选育及其发酵条件的研究

对402株不同种属的酵母菌株进行初筛、复筛,筛选到一株生物量较高的二倍体菌株ZY-46(Saccharomyces cerevisiae)和一株铁富集量较高的二倍体菌株ZY-173(Saccharomyces kluyveri)。然后以它们为出发菌,分别进行单倍体分离、硫酸二乙酯(DES)诱变,并通过原生质体融合,得到一株高生物量富铁酵母融合菌株ZYF-15。在优化的发酵条件下,该融合菌株生物量可达11．2g／L,细胞铁含量达24．5mg／g干细胞,细胞总铁含量分别比原始亲株ZY-46和ZY-173提高了2．6倍和1．9倍。