Flávia Maria Lopes Passos

The influence of presaccharification, fermentation temperature and yeast strain on ethanol production from sugarcane bagasse.

Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50°C than at 37 or 42° C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process.

Xylose metabolism in Debaryomyces hansenii UFV-170. Effect of the specific oxygen uptake rate.

The new yeast Debaryomyces hansenii UFV‐170 was tested in this work in batch experiments under variable oxygenation conditions. To get additional information on its fermentative metabolism, a stoichiometric network was proposed and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. The yeast metabolism resulted to be practically inactive under strict oxygen‐limited conditions ( qO2 = 12.0 mmolO2 C‐molDM−1 h−1), as expected by the impossibility of regenerating NADH2+. Significant fractions of the carbon source were addressed to both respiration and biomass growth under excess oxygen levels ( qO2 ≥ 55.0 mmolO2 C‐molDM−1 h−1), thus affecting xylitol yield ( YP/S = 0.41–0.52 g g−1). Semi‐aerobic conditions ( qO2 = 26.8 mmolO2 C‐molDM−1 h−1) were able to ensure the best xylitol production performance ( Pmax = 76.6 g L−1), minimizing the fractions of the carbon source addressed either to respiration or biomass production and increasing YP/S up to 0.73 g g−1. An average P/ O ratio of about 1.0 molATP molO−1 allowed estimation of the main kinetic‐bioenergetic parameters of the biosystem. The overall ATP requirements of biomass were found to be particularly high and dependent on the oxygen availability in the medium as well as on the physiological state of the culture. Under semi‐aerobic and aerobic conditions, they varied in the ranges 13.5–15.4 and 9.74–10.2 molATP C‐molDM−1, respectively, whereas during the best semi‐aerobic bioconversion they progressively increased from 5.68 to 24.7 molATP C‐molDM−1. After a starting phase of adaptation to the medium, the cell achieved a phase of decelerated growth during which its excellent xylose‐to‐xylitol capacity kept almost constant after 112 h up to the end of the run.

A mechanistical mathematical model to predict lactose hydrolysis by β-galactosidase in a permeabilized cell mass of Kluyveromyces lactis: validity and sensitivity analysis

Abstract The kinetics of lactose hydrolysis by intracellular β-galactosidase in a preparation of Kluyveromyces lactis cells permeabilized with ethanol was assessed in the presence of lactose and its hydrolysis reaction products galactose and glucose. Enzyme inhibition by the reaction products was tested using different concentrations of galactose (1, 5, 10, 20, 30 and 40 mM) and glucose (10, 30 and 50 mM) and a combination of both. Galactose was a competitive inhibitor. The competitive or non-competitive nature of the inhibition was defined by the smallest value presented by the residual sum of squares of regression as determined using the Marquardt iterative method of the non-linear regression (NLIN) program of the statistical analysis system (SAS). The kinetic constants V max , K m and K i , with values corresponding to 0.291±0.01 mM min −1 ; 2.536±0.412 mM and 10.88±8.86 mM, respectively, were also estimated by the same program. Enzyme activity was not affected by glucose at the concentrations tested. Glucose affected enzyme activity only when galactose was present. A mechanistic mathematical model was developed to describe lactose hydrolysis, taking into consideration the inhibitory effect of galactose. Sensitivity analysis of the coefficients of the model proposed was performed. Varying estimated K m and K i values by ±20% had no effect on lactose hydrolysis kinetics. However, with a 20% increase in the estimated V max value, the model more closely approximated the experimental data for initial concentrations of 30 mM glucose and 10 mM galactose. Model simulation closely approximated experimental data from lactose hydrolysis in a 5% phosphate-buffered lactose solution as well as in skim milk.

Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer

Penicillium griseoroseum was grown in bioreactors on mineral medium supplemented with yeast extract and sucrose. The influence of inoculum and carbon source concentrations, aeration and pH on pectin lyase (PL) production, as well as the capacity of P. griseoroseum to produce PL when grown on sugar cane syrup as carbon source were evaluated. Inoculum concentration did not influence PL production. Production was higher in non-aerated than in aerated medium. The best results were obtained using 60 mM sucrose at pH 6.3-7.2. Production using cane syrup 25% (v/v), without yeast extract supplement, was equal to that obtained under the conditions cited above.

Screening of filamentous fungi for production of xylitol from D-xylose

Eleven filamentous fungi were screened for xylitol production in batch cultures. Production was generally low under the growth conditions used in this study. Penicillium crustosum presented the highest production, 0.52 g L-1 from 11.50 g L-1 of D-xylose, representing consumption of 76% of the original D-xylose.

Use of response surface methodology to evaluate the extraction of Debaryomyces hansenii xylose reductase by aqueous two-phase system

Xylose reductase (XR) from Debaryomyces hansenii was extracted by partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) 4000 in the presence of different salts, specifically sodium sulfate, lithium sulfate and potassium phosphate. Batch extractions were carried out under different conditions of temperature (25-45 degrees C) and tie-line length (TLL) for each system, according to a central composite design face-centered of 36 tests, and the response surface methodology was used to evaluate the results. Quadratic polynomial models were adjusted to the data to predict the behavior of four responses, namely the XR partition coefficient (K(XR)), the selectivity (S), the purification factor (PF(T)) and the activity yield (Y(T)) in the top phase. The optimal extraction conditions were found using the PEG 4000/sodium sulfate system at 45 degrees C and TLL=25.1, which ensured PF(T)=3.1 and Y(T)=131%. The ATPS proved effective for partial purification of D. hansenii xylose reductase in cell-free crude extract, and the response surface methodology revealed to be an appropriate and powerful tool to determine the best dominion of temperature and ATPS composition.

The activity of β‐galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims:  Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells.

Debaryomyces hansenii UFV-1 intracellular α-galactosidase characterization and comparative studies with the extracellular enzyme.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Influence of inhibitory compounds and minor sugars on xylitol production by Debaryomyces hansenii.

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.

Computational analysis of the interaction between transcription factors and the predicted secreted proteome of the yeast Kluyveromyces lactis

BackgroundProtein secretion is a cell translocation process of major biological and technological significance. The secretion and downstream processing of proteins by recombinant cells is of great commercial interest. The yeast Kluyveromyces lactis is considered a promising host for heterologous protein production. Because yeasts naturally do not secrete as many proteins as filamentous fungi, they can produce secreted recombinant proteins with few contaminants in the medium. An ideal system to address the secretion of a desired protein could be exploited among the native proteins in certain physiological conditions. By applying algorithms to the completed K. lactis genome sequence, such a system could be selected. To this end, we predicted protein subcellular locations and correlated the resulting extracellular secretome with the transcription factors that modulate the cellular response to a particular environmental stimulus.ResultsTo explore the potential Kluyveromyces lactis extracellular secretome, four computational prediction algorithms were applied to 5076 predicted K. lactis proteins from the genome database. SignalP v3 identified 418 proteins with N-terminal signal peptides. From these 418 proteins, the Phobius algorithm predicted that 176 proteins have no transmembrane domains, and the big-PI Predictor identified 150 proteins as having no glycosylphosphatidylinositol (GPI) modification sites. WoLF PSORT predicted that the K. lactis secretome consists of 109 putative proteins, excluding subcellular targeting. The transcription regulators of the putative extracellular proteins were investigated by searching for DNA binding sites in their putative promoters. The conditions to favor expression were obtained by searching Gene Ontology terms and using graph theory.ConclusionA public database of K. lactis secreted proteins and their transcription factors are presented. It consists of 109 ORFs and 23 transcription factors. A graph created from this database shows 134 nodes and 884 edges, suggesting a vast number of relationships to be validated experimentally. Most of the transcription factors are related to responses to stress such as drug, acid and heat resistance, as well as nitrogen limitation, and may be useful for inducing maximal expression of potential extracellular proteins.