The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Galactose oxidase production by immobilized cells ofDactylium dendroides

Summary Dactylium dendroides cells were immobilized with calcium alginate, calcium pectate and k-carrageenan. Alginate immobilized cells produced relatively small amounts of (D-galactose: O₂ oxidoreductase, EC 1.1.3.9, GOase). Pectate immobilized cells gave the best yield of GOase, which was comparable with that obtained with free cells, and productivity could be extended up to 28 days (7 cycles). Controlled dosage of phosphate to the medium markedly improved GOase production with higher yields per cycle than with free cells.     

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.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

The effect of Mn2+ on ethanol production from lactose using Kluyveromyces marxianus IMB3 immobilized in magnetically responsive matrices

The thermotolerant, ethanol producing yeast strain, K. marxianus IMB3 was immobilized in calcium alginate containing magnetically responsive Fe₃O₄ particles. In these studies the β-galactosidase derived from K. marxianus IMB3 was immobilized onto the Fe₃O₄ particles prior to inclusion into the alginate matrix. Ethanol production by the immobilized microorganism in the presence of Fe₃O₄ reached a maximum of 16?g/L on 40?g/L lactose whereas prior immobilization of the enzyme to the particles and inclusion into the alginate matrix increased ethanol production to a maximum concentration of 18 g/L. When Mn²⁺ was incorporated into fermentations containing the immobilized enzyme in the alginate matrix, ethanol production increased further to a maximum concentration of 20?g/L. In addition, the behaviour of the magnetically responsive biocatalyst containing the co-immobilized enzyme was examined in a batch-fed system in the presence and absence of Mn²⁺.     

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.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Soil O2 and CO2 effects on root respiration of cacti

Through use of a recently developed technique that can measure CO₂ exchange by individual attached roots, the influences of soil O₂ and CO₂ concentrations on root respiration were determined for two species of shallow-rooted cacti that typically occur in porous, well-drained soils. Although soil O₂ concentrations in the rooting zone in the field were indistinguishable from that in the ambient air (21% by volume), the CO₂ concentrations 10 cm below the soil surface averaged 540 μLL⁻¹ for the barrel cactusFerocactus acanthodes under dry conditions and 2400 μLL⁻¹ under wet conditions in a loamy sand. For the widely cultivated platyopuntiaOpuntia ficus-indica in a sandy clay loam, the CO₂ concentration at 10 cm averaged 1080 μLL⁻¹ under dry conditions and 4170 μLL⁻¹ under wet conditions. For both species, the respiration rate in the laboratory was zero at 0% O₂ and increased to its maximum value at 5% O₂ for rain roots (roots induced by watering) and 16% O₂ for established roots. Established roots ofO. ficus-indica were slightly more tolerant of elevated CO₂ than were those ofF. acanthodes, 5000 μLL⁻¹ inhibiting respiration by 35% and 46%, respectively. For both species, root respiration was reduced to zero at 20,000 μLL⁻¹ (2%) CO₂. In contrast to the reversible effects of 0% O₂, inhibition by 2% CO₂ was irreversible and led to the death of cortical cells in established roots in 6 h. Although the restriction of various cacti and other CAM plants to porous soils has generally been attributed to their requirement for high O₂ concentrations, the present results indicate that susceptibility of root respiration to elevated soil CO₂ concentrations may be more important.     

2,3,5-triphenyltetrazolium chloride as a viability assay for immobilized plant cells

Grape (V. vinifera L. cv Gamay Fréaux) cells were entrapped in calcium alginate beads. The effect of bead diameter and gel concentration on the viability of the cells was checked. The reduction of 2,3,5-triphenyltetrazolium chloride as well as O₂ consumption were used as viability test, and their results compared. The existence of diffusional limitations for O₂ introduces an unaccuracy as high as 25% for the O₂ consumption method. The reduction assay is more simple, precise and reliable.     

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl₂ concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl₂ concentrations higher than 0.4 M were utilised for bead preparation.     

Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Enhancement of yeast ethanol tolerance by calcium and magnesium

Ethanol-induced changes of CO₂ production were compared in three strains ofSaccharomyces cerevisiœ. CaCl₂ and MgCl₂ exerted protective effects against the action of ethanol. Optimal concentrations ensuring maximum of CO₂ production at 10% (V/V) of ethanol under non-growing conditions were 3 mmol/L Ca²⁺ and 2 mmol/L Mg²⁺. Yeast growth with and without ethanol addition was stimulated by Mg²⁺ more than by Ca²⁺ during fermentation, whereas ethanol production was more efificient when both Ca²⁺ and Mg²⁺ were added.     

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

The photosynthetic linear electron transport rate in excess of that used for CO₂ reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C₄ plant], Amaranthus cruentus L. (NAD-ME-type C₄ plant) and Helianthus annuus L. (C₃ plant) leaves at different CO₂ and O₂ concentrations. The electron transport rate (J _F) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO₂ · mol⁻¹ and 2% O₂. Under high light intensities there was a large excess of J _F/4 at 10–100% O₂ in the C₃ plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C₄ photosynthesis is limited by supply of atmospheric CO₂ to the C₄ cycle, the C₃ cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO₂ fixation in C₄ plants was very sensitive to the presence of O₂ in the gas phase, rapidly increasing between 0.01 and 0.1% O₂, and at 2% O₂ it was about two-thirds of that at 21% O₂. This shows the importance of the Mehler O₂ reduction as an electron sink, compared with photorespiration in C₄ plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C₄ photosynthesis. Received: 8 November 1997 / Accepted: 26 December 1997     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

The effect of soluble alginate and calcium on β-galactosidase activity produced by the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus imb3

Since it has previously been demonstrated that ethanol production by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 is more efficient in calcium alginate-based immobilization systems during growth on lactose-containing media, it was decided to examine the separate effects of soluble alginate and free calcium on the β-galactosidase activity produced by that organism. It was found that the presence of Ca²⁺ significantly increased the thermal stability of the activity at 45?°C, although the pH?and temperature optima remained the same in the presence and absence of that cation. It was also found that the presence of 2% (w/v) sodium alginate (soluble) had a very limited positive effect on the thermal stability of the enzyme at 45?°C, although it was found that activity was very significantly stimulated at that temperature. The activity was found to have an enhanced thermal stability at 30?°C in the presence of sodium alginate. The presence of sodium alginate in assay mixtures had no significant effect on the Km of the activity for the substrate o-nitrophenyl-β-D-galactoside. The results observed in the presence of either free calcium or soluble alginate may at least partially explain enhanced ethanol production by this microorganism in alginate-based immobilization systems.     

Ethanol production at 45°C using preparations of Kluyveromyces marxianus IMB3 immobilized in calcium alginate and kissiris

The thermotolerant yeast, K. marxianus IMB3, was grown in free and immobilized states in batch-fed culture at 45°C and ethanol production was examined over a 61-day period. The organism was grown in the free state, in the free state with mineral kissiris, immobilized in calcium alginate and immobilized in calcium alginate together with kissiris. Initially, reactors were fed every two days with 10% (w/v) glucose-containing media and no significant difference in ethanol production was observed. In subsequent refeeding experiments, reactors were re-fed every two days with 15% (w/v) sucrose-containing media. Although overall ethanol concentrations decreased, production in the immobilized systems was higher. In the final stages fermentations were re-fed every 3 days and although overall ethanol production decreased further, production remained highest in the systems containing calcium alginate and kissiris.     

Light- and CO2-saturated photosynthesis: enhancement by oxygen

Oxygen may enhance CO₂-saturated photosynthesis in intact leaves, which display the Warburg effect when illuminated at the current atmospheric level of CO₂ and O₂, of about 350 μl l⁻¹ and 21%, respectively. The magnitude of the stimulation depends on irradiance. The K _M(O₂) of the stimulation is 128 μM (10.6% O₂). Maximum enhancement in wheat leaves is 6.1 and 5.3 μmol m⁻² s⁻¹ under 27.9 and 18.7 mW cm⁻², respectively, corresponding to a 25–30% increase in the ribulose 1,5-bisphosphate (RuBP) turnover rate if compared with O₂-free ambient gas phase. The stimulation appears in 5–10 s after a sharp increase in O₂. In response to a decrease in O₂, the new stabilized rate is reached in 5–7 min. The stimulation does not involve any increase in the activity of Rubisco. The effect correlates with increased concentration of RuBP. Oxygen enhances CO₂-saturated photosynthesis by acting as a terminal electron acceptor in the photosynthetic electron transport. The magnitude of the effect may be adopted as an index of the pseudocyclic photophosphorylation in vivo.     

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO₂ production without affecting the rate of respiratory O₂ consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939–944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO₂ fixation at saturating irradiance, and almost 100% inhibition of CO₂ fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 μM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO₂ compensation point) resulted in a stimulation in the rate of gross photosynthetic O₂ evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light. Received: 15 September 1999 / Accepted: 14 October 1999     

Cauliflower waste incorporation into cane molasses improves ethanol production using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 178

Diluted cane molasses having total sugar and reducing sugar content of 9.60 and 3.80% (w/v) respectively was subjected to ethanol production by Saccharomyces cerevisiae MTCC 178. Incorporation of dried Cauliflower Waste (CW) in molasses at the level of 15 % increased ethanol production by nearly 36 % compared to molasses alone. Addition of 0.2 % yeast extract improved ethanol production by nearly 49 % as compared to molasses alone. When the medium containing diluted molasses and 0.2 % yeast extract was supplemented with 15 % CW, 29 % more ethanol was produced compared to molasses with 0.2 % yeast extract. Cell biomass, ethanol production, final ethanol concentration and fermentation efficiency of 2.65 mg mL⁻¹, 41.2 gL⁻¹, 0.358 gg⁻¹ and 70.11 % respectively were found to be best at 15% CW supplementation level besides reduction in fermentation time but further increase in CW level resulted in decline on account of all the above parameters. This is probably the first report to our knowledge, in which CW was used in enhancing ethanol production significantly using a small quantity of yeast extract.