Increased ethanol production during growth of electric-field stimulated Kluyveromyces marxianus IMB3 during growth on lactose-containing media at 45°C

Summary Following treatment of the thermotolerant ethanol producing yeast strain Kluyveromyces marxianus IMB3 with single electric field pulses of 2.4kV/cm, ethanol production was found to increase from 25 to 35% of the theoretical maximum yield within 20 hours, during growth on lactose-containing media (4% [w/v]). Following treatment with electric fields and addition of 1mM MnCl₂ to lactose-containing media, ethanol production almost doubled within a 30 hour period. In addition, low levels of functional -galactosidase activity could be detected in the extracellular culture filtrates, presumably as a result of electropermeabilization.     

Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45°C

Thermotolerant Kluyveromyces marxianus var. marxianus IMB3 yeast strain was immobilized on Kissiris (mineral glass foam derived from lava) in column packed reactors, and used for ethanol production from glucose or molasses under continuous culture conditions at temperatures between 40 and 50°C. Both ethanol yield and fermentation efficiency were highest at 45°C and a dilution rate (D) of 0.15/h. Increasing sugar concentration led to an increase in ethanol yield of up to 68.6 and 55.9 g/l on approx. 200g glucose or molasses, respectively. Optimum fermentation efficiency (experimental yields over theoretical maximum yields) however was at about 15% sugar for both glucose and molasses. Slight aeration (25 ml of air/min) through the medium addition line was found advantageous due to its mixing effect and probable maintenance of activity.     

Partial characterization of β-glucosidase activity produced by Kluyveromyces marxianus IMB3 during growth on cellobiose-containing media at 45°C

Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg²⁺. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect.     

The effects of phosphoric acid pretreatment on conversion of cellulose to ethanol at 45°C using the thermotolerant yeast Kluyveromyces marxianus IMB3

Summary Here we report on the effects of phosphoric acid pretreated cellulose as a substrate for ethanol production by K. marxianus IMB3 using simultaneous saccharification and fermentation systems at 45°C. With untreated, milled filter paper as substrate the maximum amount of ethanol produced was 25% of the maximum theoretical yield. After pre-treatment with 100% phosphoric acid, the yield increased to 42% of the maximum theoretical yield. When untreated microcrystalline cellulose was used as the fermentation substrate, yields of ethanol as 45°C amounted to 16% of the maximum theoretical yield whereas pretreatment of the substrate with phosphoric acid resulted in an increase in ethanol production to 69% of the maximum theoretical yield. This suggests that pretreatment of substrate with phosphoric acid would contribute to a reduction in the amount of exogenous enzyme needed.     

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.     

Ethanol production at 45 °C by Kluyveromyces marxianus IMB3 during growth on molasses pre-treated with Amberlite® and non-living biomass

The use of high concentrations of molasses as a fermentation feed-stock for ethanol production is normally precluded by the presence of inhibitory compounds. Use of the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 in fermentations containing high concentrations of molasses resulted in sub-optimal production of ethanol. The results suggested that this was caused by the presence of inhibitory materials rather than an intolerance to increased concentrations of ethanol. In the current study we describe the pretreatment of molasses preparations with either an Amberlite^® monobed mixed ion-exchange resin or non-living microbial biomass from a local distillery. In the study molasses samples diluted to yield a final sugar concentration of 160?g/l were used as the substrate. Control fermentations using the untreated molasses dilutions yielded a maximum ethanol concentration of 40?g/l, representing 49% of the maximum theoretical yield. Fermentations using molasses samples pre-treated with Amberlite^® or non-living biomass yielded maximum ethanol concentrations of 58 and 54?g/l, representing 71 and 66% of the maximum theoretical yield, respectively. The results suggest that pre-treatment brings about removal of toxic or inhibitory materials from the fermentation feed-stock and we believe that such pre-treatments, particularly using the less expensive non-living biomass preparations may find a role in processes concerned with the commercial production of ethanol from molasses using this microorganism.     

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.     

Continuous ethanol production from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus IMB3 in a continuous-flow bioreactor

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and used in a continuous flow bioreactor to produce ethanol from molasses at 45?°C. The molasses was diluted to yield a number of final sugar concentrations and the effect of molasses sugar concentration on ethanol production by the continuous system was examined. Although maximum ethanol concentrations were obtained using sugar concentrations of 140?g/l, within 10?h of introducing the feed to the column bioreactors, those ethanol concentrations subsequently decreased to lower levels over a 48?h period. Examination of viable yeast cell number within the immobilization matrix indicated a dramatic reduction over this time period. At lower molasses concentrations, ethanol production by the continuous flow system remained relatively constant over this time period. In addition, the effect of residence time on ethanol production by the continuous flow bioreactor was examined at a fixed molasses sugar concentration (120?g/l) and a residence time of 0.66?h was found to be optimal on the basis of volumetric productivity. Efficiencies of the continuous flow bioreactor configuration used in these studies ranged from 31–76%.     

Production of ethanol at 45°C on starch-containing media by mixed cultures of the thermotolerant,ethanol-producing yeast Kluyveromyces marxianus IMB3 and the thermophilic filamentous fungus Talaromyces emersonii CBS 814.70

 The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, was shown to produce ethanol at 45°C on starch-containing media supplemented with a crude amylase preparation derived from the thermophilic, filamentous fungus Talaromyces emersonii CBS 813.70. Ethanol production on media containing 4% (w/v) starch increased to a maximum of 15 g/l with 40 h, and this represented 74% of the maximum theoretical yield. Subsequent experimentation involving growth of both organisms in fermentations on starch-containing media (4% w/v) demonstrated that the mixed-culture system was capable of ethanol production at 45°C with maximum yields at 12 g/l obtained with 65 h. The advantages associated with ethanol production by this system are discussed. Received: 16 May 1994/Accepted: 22 October 1994     

Continuous ethanol fermentation at 45 °C using Kluyveromyces marxianus IMB3 immobilized in Calcium alginate and kissiris

The thermotolerant ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and a 1:1 mixture of calcium alginate and the porous volcanic mineral, kissiris. Immobilized preparations were placed in fixed-bed column bioreactors and continuous ethanol production by systems containing both immobilized preparations was examined at 45?°C with a 100?g/l glucose feed. The effect of residence time on product concentration, bioreactor efficiency and volumetric productivities have been examined and these were all higher in systems containing the alginate/kissiris mixed immobilization matrix. Maximum ethanol concentrations produced by the continuous system ranged between 46 and 48?g/l representing efficiencies of 90–94%.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Production of ethanol from molasses at 45 °C using Kluyveromyces marxianus IMB3 immobilized in calcium alginate gels and poly(vinyl alcohol) cryogel

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 has been immobilized in calcium alginate gel and poly(vinyl alcohol) cryogel (PVAC) beads. The immobilized preparations were used as biocatalyst in fed-batch reactor systems for prolonged periods. The substrate utilized in each case consisted of sugar cane molasses diluted to yield a sugar load of 140?g/l. During the first cycle the maximum ethanol concentration produced by the alginate system was 57?g/l, representing 80% of the maximum theoretical yield. In the system employing the PVAC-immobilized biocatalyst, ethanol production increased to a maximum of 52–53?g/l, representing 73% of the maximum theoretical yield. In both cases, maximum ethanol concentration was achieved within a 72-hour period. When each system was operated on a fed-batch basis for a prolonged period of time the average ethanol concentrations produced in the alginate- and the PVAC-immobilized systems were 21 and 45?g/l, respectively. The results suggest that the PVAC-based immobilization system may provide a more practical alternative to alginate for the production of ethanol by K. marxianus IMB3 in continuous or semi-continuous fermentation systems.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part II – Use of Kluyveromyces marxianus IMB3

It is clear that only a small proportion of all micro-organisms have been isolated and identified. The simple technique of seeking a thermotolerant fermentative yeast from a suitable hot environment has yielded a number of strains. These organisms, identified as strains of Kluyveromyces marxianus var. marxianus, have been shown to have a wide range of metabolic capabilities that could be used in industrial applications. Not only have the metabolic capabilities been elucidated but possible bioreactor configurations and process application options have been investigated. It appears that there are a number of specific situations where this thermotolerant yeast could find industrial applications. A full-scale industrial ethanol production trial using this yeast was successfully carried out in India. K. marxianus IMB3's performance in terms of the ethanol concentrations achieved was comparable to that obtained using the distillery's own yeast strain with an added advantage of eliminating cooling.     

The growth of yeasts at 37° C

Summary More than 2,300 strains of 70 species of yeasts have been tested on yeast autolysate agar at 37° C. Of these, all strains of 15 species grew at this elevated temperature while no strains of 13 species grew well. The remaining 42 species, represented by 2 or more strains each, included strains both capable and incapable of growth at 37° C. It is suggested that such species include two groups of strains, one capable of adaptation to growth conditions at elevated temperatures. In sewage-polluted waters such strains may be indicative of fecal pollution.U.S. Department of Health, Education, and Welfare, Bureau of State Services, Public Health Service.     

The growth of dermatophytes at 4° C and 37° C; The relation of this character to others

Summary The ability of the generaEpidermophyton, Microsporon andTrichopyton to grow on some media at 4° C and 37° C was studied. It has been shown that specific differences exist among these fungi in the capability or rapidity of the growth at extreme temperatures.There is high positive correlation among perfect state production, isolation from the soil and growth at 4° C (group of characters A) and between pathogenicity and growth at 37° C (group of characters B). Between the groups A and B of characters exists a slighter negative correlation. Some prognosis about the five characters by certain species of dermatophytes may be given.     

Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and β-hydroxybutyrate

Pseudomonas AM1 grows on beta-hydroxybutyrate and methanol at similar rates. beta-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C(1) compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.     

The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4°C

This study evaluated the protective effects of supplementation with three different sugars on the motility, morphology and DNA integrity of rat epididymal sperm chilled and stored at 4°C Epididymides were obtained from each donor. Rat epididymal sperm was diluted in Ham's F10 plus raffinose, Ham's F10 plus trehalose, Ham's F10 plus fructose, and Ham's F10 medium for control purposes. Thereafter, the extended sperm were chilled and stored in liquid form at 4°C. Sperm motility, morphological abnormalities and DNA damage were determined at 0 and 12h after chilling. No significant difference was observed in any of the parameters evaluated at 0h, before storage (P>0.05). After 12h of storage, all sugar additives led to statistically higher motility, normal sperm morphology and DNA integrity in comparison to the control group. Raffinose gave the best motility percentages (32.86±1.84%) after 12h of storage at 4°C, compared to the other groups (P<0.001). In conclusion, Raffinose, trehalose and fructose provided a better protection of sperm functional parameters against chilling injury, in comparison to the control group.     

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.     

Effect of media composition and growth conditions on production of cellulase and β-glucosidase by a mixed fungal fermentation

The effect of growth temperature, medium composition and pH were examined in shake-flask-scale studies to determine the optimum conditions for cellulase production in mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis. The optimum temperature and pH were 27°C and pH 4.6. Decreases in medium complexity and cost were achieved through reductions in the concentration of Ca, Mg, and K salts. Ten litre fermentations were implemented to study the kinetics of substrate utilization and product formation. The pH at which the fermentation was controlled appeared to be the critical parameter for batch growth of mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis.