Production of ethanol by the thermotolerant yeastKluyveromyces marxianus IMB3 during growth on lactose-containing media

Summary The thermotolerant yeast strain,Kluyveromyces marxianus IMB3 was shown to be capable of growth and ethanol production on lactose containing media at 45°C. On media containing 4% (w/v) lactose, ethanol production increased to 6.0g/l within 50h and this represented 29% of theoretical yield. During growth on lactose containing media the organism was shown to produce a cell-associated β-galactosidase and no significant enzyme could be detected in the extracellular culture filtrate. Addition of β-galactosidase, released fromKluyveromyces marxianus IMB3 cells, to active fermentations, resulted in increasing ethanol production to 53% of theoretical yield at 45°C.     

Improved ethanol production from whey with Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65–70 g/l lactose) by Saccharomyces cerevisiae CCY 10–13–14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/l × h), compared to the fermentation in which the lactose was directly fermented by K. marxianus.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Effects of elevated solute concentrations on the fermentation of lactose by Kluyveromyces marxianus Y-113

Summary The effect of increased solute concentrations on the fermentation of lactose to ethanol by Kluyveromyces marxianus Y-113 was investigated in batch culture. Elevated concentrations of lactose, maltose, NaCl or ethanol all inhibited the fermentation but to varying extents. Maltose was the least inhibitory of the solutes added while ethanol, and in particular the combination of high ethanol and high lactose concentrations, had the greatest inhibitory action. A maximum concentration of 45–52 g/l ethanol was achieved before growth and ethanol production ceased.     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Selection of yeast strain and fermentation conditions for high-yield ethanol production from lactose and concentrated whey

A total of 65 yeast strains were screened for their ability to grow and ferment lactose in a standard DURHAM tube test at 30 °C. Based on the kinetic parameters for lactose and whey lactose fermentations in shake flask cultures, the strain Candida psedotropicalis 65 was chosen for further studies. Some of the cultural parameters affecting ethanolic fermentations on lactose were standardized. At an initial lactose concentration of 100–120 g/l in the medium containing concentrated whey or lactose, at 40 °C and within 48 h, the selected strain reached an ethanol concentration of 41–59 g/l, an ethanol productivity of 1.3–3.0 g/l/h, a lactose consumption of 99%, an ethanol yield 0.4–0.49 g/g and a biomass yield of 0.027 g/g.     

Control of ethanol production and monitoring of membrane performance by mass-spectrometric gas analysis in the coupled fermentation-pervaporation of whey permeate

A coupled fermentation-pervaporation process was operated continuously with on-line mass spectrometric gas analysis monitoring of product accumulation on both the upstream and the downstream sides of the membrane. Efficient coupling of the fermentation with pervaporation was attained when a steady state of ethanol production and removal was achieved with whey permeate containing high concentrations of lactose (>8%) or by controlled lactose additions that also compensated for loss of liquid due to pervaporation. The combined system consists of a tubular membrane pervaporation module, directly connected to a stirred fermentor to form one circulation loop, kept at 38°C, with both units operating under computer control. Mass spectrometric gas analysis of the CO₂ gas evolved in the fermentor and the ethanol and water in the pervaporate on the downstream side of the membrane enabled us to follow the production of ethanol and its simultaneous removal. Membrane selectivity was calculated on-line and served to monitor the functioning of the membrane. Batch-wise-operated fermentation-pervaporation with Candida pseudotropicalis IP-513 yielded over 120 gl^–1 of concentrated ethanol solution using supplemented whey permeate containing 16% lactose. A steady state lasting for about 20 h was achieved with ethanol productivity of 20 g h^–1 (approx. 4 g l^–1 h^–1). Membrane selectivity was over 8. Controlled feeding of concentrated lactose suspension in the whey permeate (350 g l^–1) resulted in the continuous collection of 120–140 g l^–1 of ethanol pervaporate for 5 days, by which time salt accumulation hampered the fermentation. Medium refreshment restored the fermentative activity of the yeast cells and further extended the coupled process to over 9 days (200 h), when reversible membrane fouling occurred. The membrane module was exchanged and the combined process restarted. Correspondence to: Y. Shabtai     

Ethanol production from deproteinized whey byβ-galactosidase coimmobilized cells ofSaccharomyces cerevisiae

Summary The performance of -galactosidase coimmobilized cells ofSaccharomyces cerevisiae was evaluated during shake flask fermentation of deproteinized cheese whey lactose to ethanol. The performance of the coimmobilized enzyme treatment was compared to that of a treatment using acid prehydrolyzed whey lactose (a readily available substrate). Enzyme coimmobilization resulted in a slower rate and a lower extent of substrate utilization, thus giving a lower maximum ethanol concentration (13.5 versus 16.7 g/l). It did result, however, in a better ethanol yield (95% vs. 89% theoretical). It appears that, compared to acid prehydrolysis of whey lactose, through -galactosidase coimmobilization we could succeed in obtaining substantial process simplifications, thus saving in equipment and operating cost, while gaining in ethanol yield at the cost of some reasonable loss in the rate and the extent of lactose utilization.     

Production of ethanol and biomass from various carbohydrates byKluyveromyces fragilis

Summary Growth ofKluyveromyces fragilis NRC 2475 and the production of ethanol by the yeast were studied in the media containing one of the following sugars: glucose, lactose, galactose, or a glucose-galactose (50% 50%) mixture as a carbon source.The largest biomass yield and the lowest yield of ethanol were obtained in the medium containing glucose. The medium containing galactose gave the lowest yield of biomass and the largest yield of ethanol. When lactose was used for the growth and production of ethanol the obtained results for both biomass and ethanol were between those obtained with glucose and galactose.The ethanol productivities, expressed in terms of ethanol produced either per unit of cells, or per unit of cells and time, were the highest in the system with galactose and the lowest in that with glucose.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Enzymatic production of α-<Emphasis Type="SmallCaps">d</Emphasis>-galactose 1-phosphate by lactose phosphorolysis

α-d-Galactose 1-phosphate (αGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of αGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells. After purification of the product from the reaction mixture by anion-exchange chromatography and ethanol precipitation, 9.5 grams of highly pure αGal1P were obtained from a 1 l reaction volume.     

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.     

Ethanol production from concentrated whey permeate using a fed-batch culture ofKluyveromyces fragilis

Summary Fed-batch fermentation of non-supplemented concentrated whey permeate resulted in high ethanol productivity for feeds of lactose for which batch fermentation had a poor performance. At an initial lactose concentration of 100 g/L and a constant lactose feeding rate of 18 g/h we have obtained: ethanol concentration 64 g/L, ethanol productivity 3.3 g/Lh, lactose consumption 100%, ethanol yield 0.47 g/g, and biomass yield 0.058 g/g.Nomenclature St total lactose fed per medium volume in the bioreactor, g/L - Si initial lactose concentration, g/L - F lactpse feeding rate, g/h - P final ethanol concentration, g/L - Y_p/s ethanol yield, g ethanol/g lactose - Y_x/s biomass yield, g biomass/g lactose - X_S lactose consumption, % - Qp overall ethanol volumetric productivity, g/Lh - m maximum specific growth rate, h - qsm maximum specific lactose consumption rate, g/gh - qpm maximum specific ethanol production rate, g/gh     

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.     

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.

     

Inhibition of alcoholic fermentation by substrate and ethanol

The effect of ethanol and sugars on rates of fermentation was studied. We used a strain of Canadida pseudotropicalis. The specific rate of fermentation was determined by using the Warburg manometer. The effect of ethanol was formulated as an exponential function of ethanol concentration, but the empirical constant was different when glucose or lactose was used as a substrate. The effects of both ethanol and substrate were formulated. It was demonstrate that when lactose and glucose were present in the medium with a small amount of alcohol, a synergistic effect on the rate of fermentation appeard. This phenomenon considerably limits the rate of fermentation.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Whey disposal by deproteinization and fermentation

The non-pollutant plant support material of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) was used for the entrapment of living yeast cells (Kluyveromyces fragilis) which hydrolyse lactose with the subsequent fermentation of glucose and galactose at high cell densities (up to 7.0 × 10⁸/ml support). The stabile yeast-plant cell immobilizates are able to produce ethanol from lactose-containing media (e.g. whey) by batch fermentation (on a rotary shaker) or continuous fermentation (in a turbulence reactor) for several days (at a pH below 4.2 and a temperature of 30°C). The removal of whey proteins by a preceding heat denaturation of whey, high dilution rates, C_So values of 50 to 60 g lactose per litre whey and the preferential use of the K. fragilis strain DSM 7238 were determined as the prerequisites for an optimum continuous fermentation. Economically interesting productivities (P_max ? 15 g ethanol/1 · h, D = 0.72 h^?1) with an actual lactose turnover of 90% were obtained by using these parameters.     

High ethanol productivity from lactose by immobilized cells of Kluyveromyces fragilis and Zymomonas mobilis

Ethanol production from 200 g lactose/l by Kluyveromyces fragilis immobilized in calcium alginate was 63 g/l whereas with co-immobilized K. fragilis and Zymomonas mobilis 72 g ethanol/l was attained. With free cells of K. fragilis, only 52 g ethanol/l was obtained. The beads were relatively stable without significant reduction in activity for about six batches of fermentation.The authors are with the Department of Microbiology and Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.This paper is dedicated to Professor M. Lakshmanan, Vice-Chancellor, Madurai Kamaraj University, in commemoration of his 60th birthday.     

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.