Production and biochemical characterization of α-glucosidase from Aspergillus niger ITV-01 isolated from sugar cane bagasse.

Aspergillus niger ITV-01 presents amylolytic activity, identified as α-glucosidase, an enzyme that only produces α-d-glucose from soluble starch and that presents transglucosylase activity on α-d-glucopyranosyl-(1-4)-α-d-glucopyranose (maltose) (200 gL-1). Biochemical characterization was performed on A. niger ITV-01 α-glucosidase; its optimum parameters were pH 4.3, temperature 80 °C but stable at 40 °C, with an energy of activation (Ea) 176.25 kJ mol-1. Using soluble starch as the substrate, Km and Vmax were 5 mg mL-1 and 1000 U mg-1, respectively. As α-glucosidase is not a metalloenzyme, calcium and EDTA did not have any effect on its activity. The molecular weight was estimated by SDS-PAGE to be about 75 kDa. It was also active in methanol and ethanol. When ammonium sulfate (AS) and yeast extract (YE) nitrogen sources and calcium effect were evaluated, the greatest activity occurred using YE and calcium, as opposed to AS media where no activity was detected. The results obtained showed that this enzyme has industrial application potential in the processes to produce either ethanol or malto-oligosaccharides from α-d-glucopyranosyl-(1-4)-α-d-glucopyranose (maltose).

Effect of carbon sources on the growth and ethanol production of native yeast Pichia kudriavzevii ITV-S42 isolated from sweet sorghum juice.

The importance of non-Saccharomyces yeast species in fermentation processes is widely acknowledged. Within this group, Pichia kudriavzevii ITV-S42 yeast strain shows particularly desirable characteristics for ethanol production. Despite this fact, a thorough study of the metabolic and kinetic characteristics of this strain is currently unavailable. The aim of this work is to study the nutritional requirements of Pichia kudriavzevii ITV-S42 strain and the effect of different carbon sources on the growth and ethanol production. Results showed that glucose and fructose were both assimilated and fermented, achieving biomass and ethanol yields of 0.37 and 0.32 gg-1, respectively. Glycerol was assimilated but not fermented; achieving a biomass yield of 0.88 gg-1. Xylose and sucrose were not metabolized by the yeast strain. Finally, the use of a culture medium enriched with salts and yeast extract favored glucose consumption both for growth and ethanol production, improving ethanol tolerance reported for this genre (35 g L-1) to 90 g L-1 maximum ethanol concentration (over 100%). Furthermore Pichia kudriavzevii ITV-S42 maintained its fermentative capacity up to 200 g L-1 initial glucose, demonstrating that this yeast is osmotolerant.

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient.

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (g ethanol g(-1) biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82 g g(-1 )h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80 g L(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5 g L(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22 %, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

Using high pressure processing (HPP) to pretreat sugarcane bagasse.

High pressure processing (HPP) technology was used to modify the structural composition of sugarcane bagasse. The effect of pressure (0, 150 and 250 MPa), time (5 and 10 min) and temperature (25 and 50 °C) as well as the addition of phosphoric acid, sulfuric acid and NaOH during the HPP treatment were assessed in terms of compositional analysis of the lignocellulosic fraction, structural changes and crystallinity of the bagasse. The effect of HPP pretreatment on the bagasse structure was also evaluated on the efficiency of the enzymatic hydrolysis of bagasse. Results showed that 68.62 and 45.84% of the hemicellulose fraction was degraded by pretreating at 250 MPa with sulfuric and phosphoric acids, respectively. The removal of lignin (54.10%) was higher with the HPP-NaOH treatment. The compacted lignocellulosic structure of the raw bagasse was modified by the HPP treatments and showed few cracks, tiny holes and some fragments flaked off from the surface. Structural changes were higher at 250 MPa and 50 °C. The X ray diffraction (XRD) patterns of the raw bagasse showed a major diffraction peak of the cellulose crystallographic 2θ planes ranging between 22 and 23°. The distribution of the crystalline structure of cellulose was affected by increasing the pressure level. The HPP treatment combined with NaOH 2% led to the higher glucose yield (25 g/L) compared to the combination of HPP with water and acids (>5 g/L). Results from this work suggest that HPP technology may be used to pretreat sugarcane bagasse.

Use of sugarcane molasses "B" as an alternative for ethanol production with wild-type yeast Saccharomyces cerevisiae ITV-01 at high sugar concentrations.

Molasses "B" is a rich co-product of the sugarcane process. It is obtained from the second step of crystallization and is richer in fermentable sugars (50-65%) than the final molasses, with a lower non-sugar solid content (18-33%); this co-product also contains good vitamin and mineral levels. The use of molasses "B" for ethanol production could be a good option for the sugarcane industry when cane sugar prices diminish in the market. In a complex medium like molasses, osmotolerance is a desirable characteristic for ethanol producing strains. The aim of this work was to evaluate the use of molasses "B" for ethanol production using Saccharomyces cerevisiae ITV-01 (a wild-type yeast isolated from sugarcane molasses) using different initial sugar concentrations (70-291 g L(-1)), two inoculum sizes and the addition of nutrients such as yeast extract, urea, and ammonium sulphate to the culture medium. The results obtained showed that the strain was able to grow at 291 g L(-1) total sugars in molasses "B" medium; the addition of nutrients to the culture medium did not produce a statistically significant difference. This yeast exhibits high osmotolerance in this medium, producing high ethanol yields (0.41 g g(-1)). The best conditions for ethanol production were 220 g L(-1) initial total sugars in molasses "B" medium, pH 5.5, using an inoculum size of 6 × 10(6) cell mL(-1); ethanol production was 85 g L(-1), productivity 3.8 g L(-1 )h(-1) with 90% preserved cell viability.

Modelling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations.

AIM: To study the effect of glucose concentrations on the growth by Brettanomyces bruxellensis yeast strain in batch experiments and develop a mathematical model for kinetic behaviour analysis of yeast growing in batch culture. METHODS AND RESULTS: A Matlab algorithm was developed for the estimation of model parameters. Glucose fermentation by B. bruxellensis was studied by varying its concentration (5, 9.3, 13.8, 16.5, 17.6 and 21.4%). The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol and biomass production; at a substrate concentration of 50-138 g l(-1), the ethanol and biomass production were 24, 59 and 6.3, 11.4 g l(-1), respectively. However, an increase in glucose concentration to 165 g l(-1) led to a drastic decrease in product formation and substrate utilization. CONCLUSIONS: The model successfully simulated the batch kinetic observed in all cases. The confidence intervals were also estimated at each phase at a 0.95 probability level in a t-Student distribution for f degrees of freedom. The maximum ethanol and biomass yields were obtained with an initial glucose concentration of 138 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments illustrate the importance of using a mathematical model applied to kinetic behaviour on glucose concentration by B. bruxellensis.

Effect of Hibiscus sabdariffa L. dried calyx ethanol extract on fat absorption-excretion, and body weight implication in rats.

The effect of Hibiscus sabdariffa L. (Hs) calyx extract on fat absorption-excretion and body weight in rats, was investigated. Rats were fed with either a basal diet (SDC = Control diet) or the same diet supplemented with Hs extracts at 5%, 10% and 15% (SD(5), SD(10) and SD(15)). Only SD(5) did not show significant increases in weight, food consumption and efficiency compared to SD(C). The opposite occurred in SD(15) group which showed a significant decrease for these three parameters. The SD(10) responses were similar to SD(15), with the exception of food consumption. In both SD(C) and SD(5) groups, no body weight loss was observed; however, only in the latter group was there a significantly greater amount of fatty acids found in feces. A collateral effect emerging from the study is that components of Hs extract at the intermediate and greater concentrations used in this experiment could be considered possible antiobesity agents.

Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production.

The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1). In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.

Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures.

The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.