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.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

The production of ethanol from lactose in a tubular reactor by immobilized cells of Kluyveromyces fragilis

Summary An immobilized-cell tubular reactor for the continuous fermentation of lactose by Kluyveromyces fragilis was developed. Two types of supporting media were successfully tested; beechwood cubes and activated charcoal pellets. Ethanol productivity of 17.2 g/l/h was achieved from a 15% whey-lactose solution using K. fragilis immobilized on charcoal pellets, with a final ethanol concentration of 18 g/l. The use of two reactors in series demonstrated that it is possible to obtain up to 50 g/l of ethanol in the final product. No decrease in biological activity of the immobilized yeast cells occurred over a period of up to 31 days of continuous operation.     

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.     

Biosynthesis regulation of the β-glucosidase produced by a yeast strain transformed by genetic engineering

The biosynthesis of the -glucosidase enzyme was studied in a transformed yeast obtained by cloning in Saccharomyces cerevisiae the structural gene coding for -glucosidase in Kluyveromyces fragilis. The enzyme biosynthesis was found to be non-adaptative, and repressed by glucose. These features are similar to those observed in K. fragilis. -Glucosidase activity in the transformed yeast was much higher than in K. fragilis. We attempted to ferment cellobiose with the transformed yeast: practically no cellobiose was consumed, growth and ethanol production were negligible. Warburg experiments showed that cellobiose fermentation did not occur when the respiratory chain was not functioning.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

Continous ethyl acetate production by Kluyveromyces fragilis on whey permeate

The synthesis of ethyl acetate by Kluyveromyces fragilis on diluted whey permeate was studied. Ethanol, lactose and O₂ are the direct precursors for ethyl acetate synthesis by this yeast. Ethyl acetate production is affected by many parameters, particularly the carbon/nitrogen (C/N) ratio, Tween 80 and iron. Ethyl acetate synthesis is optimum for C/N = 45. Tween 80 lowered slightly the level of ethyl acetate whereas iron completely stopped ethyl acetate production. The level of ethanol in the feed, the dissolved O₂ (DO) and dilution rate (D) were also optimised. Thus at D = 0.24 h ^–1, for 4 g/l of ethanol in the feed and 40% DO, the productivity of ethyl acetate was optimal (0.7 g/l per hour). Correspondence to: A. Miclo     

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.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Construction, electroporatic transfer and expression of ZpβypGH and ZpβrtGH in zebrafish

Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter geneβ-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 μF capacitance; 8 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNNml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosenF ₀ individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosenF ₁ progenies was screened for germline transformation. Southern analysis of chosenF ₁ progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of theF ₁ progenies. Southern analyses of chosenF ₁ progenies and the frequency (53% ofF ₁ ZpβrtGH and 53% ofF ₁ ZP{β}ypGH) of transmission revealed the degree of mosaicism inF ₀ transformants. Expression was confirmed from the 3–4 times elevated levels of activity of the reporter gene and 30–40% accelerated growth of transgenicF ₀ andF ₁ progenies. Construction of the transgenes was made at the Taiwan National Ocean University, Taiwan and all other work at the Madurai Kamaraj University, Madurai.     

Fermentation ofd-xylose,d-glucose andl-arabinose mixture byPichia stipitis Y 7124: sugar tolerance

Summary The effect of substrate concentration (S ₀) on the fermentation parameters of a sugar mixture byPichia stipitis Y 7124 was investigated under anaerobic and microaerobic conditions. Under microaerobiosisP. stipitis maintained high ethanol yield and productivity when initial substrate concentration did not exceed 150 g/l; ethanol yield of about 0.40 g/g and volumetric productivity up to 0.39 g/l per hour were obtained. Optimal specific ethanol productivity (0.2 g/g per hour) was observed withS ₀=110 g/l. Under anaerobic conditionsP. stipitis exhibited the highest fermentative performances atS ₀=20 g/l; it produced ethanol with a yield of 0.42 g/g, with a specific rate of 1.1 g/g per day. When the initial substrate level increased, specific ethanol productivity declined gradually and ethanol yield was dependent on the degree of utilization of each sugar in the mixture.Abbreviations E _m maximum produced ethanol (g/l) - E ₀ initial ethanol (g/l) - E _v evaporated ethanol (g/l) - Q _p volumetric productivity of ethanol (g ethanol/l per hour or g/l per day) - q _p specific productivity of ethanol (g ethanol/g cells per hour) - q _pm maximum specific productivity of ethanol (g/l per hour) - S ₀ initial substrate concentration (g/l) - t _f time at which produced ethanol is maximum (h) - Y _p/s ethanol yield (g ethanol produced/g substrate utilized) - Y _x/s cell yeild (g cells produced/g substrate utilized) - Y _xo/xy xylitol yield (g xylitol produced/g xylose utilized) - probability coefficient - specific growth rate coefficient (h^-1 or d^-1)     

Ethanol production at 45°C by Kluyveromyces marxianus IMB3 immobilized in magnetically responsive alginate matrices

Summary The thermotolerant yeast, Kluyveromyces marxianus IMB3 produced 11g ethanol/l during growth at 45°C on media containing 4% (w/v) lactose when immobilized in alginate beads whereas the free cells produced 5g ethanol/l. A magnetically responsive biocatalyst, prepared by incorporating Fe₃O₄ into the alginate matrix increased ethanol production to 12g/l in batch-fed reactors. Ethanol concentrations were further increased to a maximum of 18g/l by immobilization of the endogenous K. marxianus -galactosidase to the Fe₃O₄ particles prior to inclusion into the alginate matrix. Maximum ethanol productivity by the system was 87% of the maximum theoretical yield.     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

Kinetics of ethanol production from Jerusalem artichoke juice with some Kluyveromyces species

Summary The kinetics of ethanol production by Kluyveromyces marxianus ATCC 12708 and ATCC 10606, K. cicerisporus ATCC 22295 and K. fragilis 105 have been studied using raw juice of the Jerusalem artichoke in which the carbohydrates were not hydrolysed prior to fermentation. The experiments revealed that this juice contains enough nutrients and can serve as a complete medium without additional nutrients both for growth of the yeasts and for ethanol production. It was found that both specific ethanol productivity and specific glucose uptake rates were the highest with K. marxianus ATCC 12708 (1.68 gg^-1h^-1 and 3.78 gg^-1h^-1 respectively). This microorganism produced an ethanol yield of 87.5% of the theoretical value in 25 hours.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene

Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained.     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Cloning and expression of the structural gene for beta-glucosidase of Kluyveromyces fragilis in Escherichia coli and Saccharomyces cerevisiae

Summary Cellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a -glucosidase. S. cerevisiae does posses the structural gene for a -glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis.We constructed in E. coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the -glucosidase. The structural gene for -glucosidase was identified by its expression in E. coli. The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4).This cloned -glucosidase gene introduced in S. cerevisiae by transformation is expressed at a level of about 500 times that of K. fragilis. We checked by Southern hybridization that the high expression level was not due to a rearrangement of K. fragilis DNA during the cloning experiments. Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.