Improvement of inulin hydrolysis yeast cell reactor by mutants selection

In a previous publication, we described a continuous production ofd-fructose from enzymatic hydrolysis of inulin with immobilized permeabilized cells (1).Kluyveromyces fragilis ATCC 12424 have been shown to possess inulase activity. The half life of the reactor was at least 1300 h, but productivity was relatively low (around 40 g/L/day). A selection of 50 mutants was tested on liquid medium for a possible increase of productivity. In relation to the improvement of the reactor, the most important factor is intracellular inulase activity, and this activity was increased with the KF 28 mutant. Productivity reached 2000 g/L/day with the increase (of the productivity), proportional to the increase of intracellular inulase activity.     

Improved Ethanol Production by Mixed Immobilized Cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from Cheese Whey Powder Solution Fermentation

Ethanol productions from cheese whey powder (CWP) solution were investigated by using free or immobilized cells of Kluyveromyces marxianus in monocultures or mixed cultures with free or immobilized cells of K. marxianus and Saccharomyces cerevisiae. K. marxianus free cells produced 3.8% v/v ethanol in monocultures, while S. cerevisiae immobilized cells produced 5.3% v/v ethanol in mixed cultures. The percentage of theoretical yield was found to be higher in mixed cultures than that in monocultures. The maximum ethanol fermentation efficiency was achieved (79.9% of the theoretical value) using mixed cultures of immobilized cells of K. marxianus and S. cerevisiae. The beads were relatively stable without significant reduction in activity for about eight batches of fermentation.     

Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation

Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

Difructose anhydride-forming bacterial inulinase II and fructogenic fungal inulinase I

The reserve polymeric inulin from dahlia tuber (>12% or >60% yield, wet or dry basis, respectively) follows as an attractive source for both free fructose or difructofuranose anhydride (DFA III). Although DFA biological activity is not completely understood, there is interest in characterizing other DFA III-producers besidesArthrobacter ureafaciens. The inulinolytic bacterial isolate named “YLW,” owing to the yellow hue in agar slants, is such a producer. Its biochemical characterization showed the presence of galactosylated and mannosylated glycolipids associated with the bacterial cells. Immobilization of fungal inulinase I and bacterial inulinase II, the respective enzymatic catalysts for the production of fructose and DFA III by inulin hydrolysis, was attempted using controlled-pore silica (CPS). The effects of pH, temperature, and incubation time was analyzed and compared for both enzymes in the free and immobilized forms.

     

Inulin-containing biomass for ethanol production

The use of stalks instead of tubers as a source of carbohydrates for ethanol production has been investigated. The inulin present in the stalks of Jerusalem artichoke was extracted with water and the effect of solid-liquid ratio, temperature, and acid addition was studied and optimized in order to attain a high-fructose fermentable extract. The maximum extraction efficiency (corresponding to 35 g/L) of soluble sugars was obtained at 1/6 solidliquid ratio. Fermentations of hydrolyzed extracts by baker's yeast and direct fermentation by an inulinease activity yeast were also performed and the potential to use this feedstock for bioethanol production assessed. The results show that the carbohydrates derived from Jerusalem artichoke stalks can be converted efficiently to ethanol by acidic hydrolysis followed by fermentation with Saccharomyces cerevisiae or by direct fermentation of inulin using Kluyveromyces marxianus strains. In this last case about 30 h to complete fermentation was required in comparison with 8–9 h obtained in experiments with S. cerevisiae growth on acid extracted juices.     

Optimization of inulinase production by Kluyveromyces marxianus using factorial design

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (2^5–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K₂HPO₄), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K₂HPO₄ had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K₂HPO₄. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.     

Kluyveromyces marxianus CBS 6556 growing cells as a new biocatalyst in the asymmetric reduction of substituted acetophenones

The use of microorganism growing cells is a well recognized methodology in biocatalyzed organic reactions. A non-conventional thermotolerant Kluyveromyces marxianus yeast strain was used for the bio-reduction of different arylketones. Differently substituted ketones were converted into the corresponding (S)-alcohols with up to 96% enantiomeric excess under very mild reaction conditions. Kluyveromyces marxianus represents a promising biocatalyst for the production of optically active 1-arylethanols.     

High-temperature wine making using the thermotolerant yeast strain Kluyveromyces marxianus IMB3

Kluyveromyces marxianus IMB3 yeast cells were immobilized on delignified cellulosic material, apple, and quince separately. Both immobilized and free cells were used in high-temperature wine making, and their fermented grape must contained 3 to 4% alcohol. Semisweet wines were produced by the addition of potable alcohol to the fermented must. Preliminary sensory evaluation of the produced semisweet wines showed good flavor and aroma. The final product contained extremely low levels of higher and amyl alcohols while ethyl acetate was at levels usually present in wines. The ferment produced may be blended with other products to improve their quality.     

Cell Disruption Optimization and Covalent Immobilization of β-D-Galactosidase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YW-1 for Lactose Hydrolysis in Milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.     

Immobilized microorganisms in the reduction of ethyl 4-chloro acetoacetate

Microorganisms were used to reduce ethyl 4-chloroacetoacetate (CAAE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Mucor ramannianus provided 98% conversion with 84% ee. Free cells of Kluyveromyces marxianus led to 95% conversion with 81% ee. After a fractionary factorial design to study the reaction conditions, calcium alginate immobilized cells of K. marxianus furnished the product with 99% conversion with 91% ee.     

Ethanol Production Using Whole Plant Biomass of Jerusalem Artichoke by Kluyveromyces marxianus CBS1555

Jerusalem artichoke is a low-requirement sugar crop containing cellulose and hemicellulose in the stalk and a high content of inulin in the tuber. However, the lignocellulosic component in Jerusalem artichoke stalk reduces the fermentability of the whole plant for efficient bioethanol production. In this study, Jerusalem artichoke stalk was pretreated sequentially with dilute acid and alkali, and then hydrolyzed enzymatically. During enzymatic hydrolysis, approximately 88 % of the glucan and xylan were converted to glucose and xylose, respectively. Batch and fed-batch simultaneous saccharification and fermentation of both pretreated stalk and tuber by Kluyveromyces marxianus CBS1555 were effectively performed, yielding 29.1 and 70.2 g/L ethanol, respectively. In fed-batch fermentation, ethanol productivity was 0.255 g ethanol per gram of dry Jerusalem artichoke biomass, or 0.361 g ethanol per gram of glucose, with a 0.924 g/L/h ethanol productivity. These results show that combining the tuber and the stalk hydrolysate is a useful strategy for whole biomass utilization in effective bioethanol fermentation from Jerusalem artichoke.     

Effect of surfactants and zeolites on simultaneous saccharification and fermentation of steam-exploded poplar biomass to ethanol

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.     

Permeabilization of Kluyveromyces marxianus with Mild Detergent for Whey Lactose Hydrolysis and Augmentation of Mixed Culture

Cheese whey is a by-product of cheese-manufacturing industries, and the utilization of whey is a challenging problem either to use it or dispose it, because only few microorganisms can metabolize the whey lactose. Enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase is the approach for biotechnological application. Kluyveromyces marxianus cells were permeabilized with non-toxic, biodegradable, anionic detergent N-lauroyl sarcosine (N-LS) for the enzyme activity. The permeabilization process parameters (N-LS concentration, solvent volume, temperature and incubation time) were optimized. The maximum β-galactosidase activity of 1,220 IU/g dry weight was obtained using permeabilized cells under optimized conditions. Moreover, viability of the permeabilized cells was also evaluated, which showed that cells were alive; however, viability was reduced by two log cycles. The permeabilized cells were evaluated for whey lactose hydrolysis. The maximum lactose hydrolysis of 91 % was observed with 600 mg (dry cell weight/100 mL) in whey powder (5 % w/v) solution at 180-min incubation, pH 6.5 and 30 °C. Further, the hydrolyzed whey was evaluated for amelioration of growth of non-lactose-consuming yeast Saccharomyces cerevisiae. S. cerevisiae was able to grow in hydrolyzed whey simultaneously with K. marxianus. The study confirmed that N-LS could be used to permeabilize K. marxianus cells to make available the enzyme activity.     

Uranium removal from acidic aqueous solutions by <Emphasis Type="Italic">Saccharomyces cerevisiae,Debaryomyces hansenii,Kluyveromyces marxianus</Emphasis> and <Emphasis Type="Italic">Candida colliculosa</Emphasis>

The sorption of uranium from acidic aqueous solutions (pH 4.5, C _init = 10 to 1000 mg U/L) by Saccharomyces cerevisiae, Debaryomyces hansenii, Kluyveromyces marxianus and Candida colliculosa was investigated using a batch technique. The U-sorption onto Saccharomyces cerevisiae and Debaryomyces hansenii followed a Langmuir, while that onto Kluyveromyces marxianus and Candida colliculosa a Freundlich isotherm. The results demonstrated that all investigated biomasses could effectively remove uranium from acidic aqueous solutions. From all sorbents, Saccharomyces cerevisiae appeared to be the most effective with a maximum sorption capacity of 127.7 mg U/g dry biomass.     

Growth Inhibition of Thermotolerant Yeast,Kluyveromyces marxianus,in Hydrolysates from Cassava Pulp

In this study, we report the inhibition of Kluyveromyces marxianus TISTR5925 growth and ethanol fermentation in the presence of furan derivatives and weak acids (acetic acid and lactic acid) at high temperatures. Cassava pulp, obtained as the waste from starch processing, was collected from 14 starch factories located in several provinces of Thailand. At a high temperature (42 °C), the cassava pulp hydrolysate from some starch factories strongly inhibited growth and ethanol production of both K. marxianus (strain TISTR5925) and Saccharomyces cerevisiae (strain K3). HPLC detected high levels of lactic acid and acetic acid in the hydrolysates, suggesting that these weak acids impaired the growth of K. marxianus at high temperature. We isolated Trp-requiring mutants that had reduced tolerance to acetic acid compared to the wild-type. This sensitivity to acetic acid was suppressed by supplementation of the medium with tryptophan.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

Economic evaluation of the hydrolysis of lactose using immobilized β-galactosidase

A computer program for preliminary cost estimates of free and immobilized enzyme systems has been developed. The cost for the hydrolysis of lactose by β-galactosidase fromAspergillus oryzae has been calculated for a batch tank reactor, with free (BTRF) and immobilized (BTRI) enzymes, a continuously stirred tank reactor (CSTR) and a plug-flow tubular reactor (PFTR), considering the mass transfer behavior and deactivation of the enzyme.

Enzyme immobilization is economically feasible, compared with a system with free enzymes, despite a very high cost for the enzyme attachment. At a half-life time of 80 d, the PFTR gives the lowest cost (0.48 SEK/kg lactose), but the cost for the BTRI is just slightly higher (0.66 SEK/kg lactose) and still much lower than the BTRF (2.10 SEK/kg lactose).

     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Maximization of β-Galactosidase Production: A Simultaneous Investigation of Agitation and Aeration Effects

In this work, the agitation and aeration effects in the maximization of the β-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2² trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL⁻¹ for enzymatic activity, 1.2 U mL⁻¹ h⁻¹ for productivity in 14 h of process, a cellular concentration of 11 mg mL⁻¹, and a 167.2 h⁻¹ volumetric oxygen transfer coefficient.