Fábio Coelho Sampaio

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.

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.

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.

Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L−1 oNP min−1 g−1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.

In vitro leishmanicidal, antibacterial and antitumour potential of anhydrocochlioquinone A obtained from the fungus Cochliobolus sp

The bioassay-guided fractionation of the ethyl acetate extract of the fungus Cochliobolus sp. highlighted leishmanicidal activity and allowed for anhydrocochlioquinone A (ANDC-A) isolation. MS, 1D and 2D NMR spectra of this compound were in agreement with those published in the literature. ANDC-A exhibited leishmanicidal activity with EC50 value of 22.4 µg/mL (44 μM) and, when submitted to the microdilution assay against Gram-positive and Gram-negative bacteria, showed a minimal inhibitory concentration against Staphylococcus aureus ATCC 25295 of 128 μg/mL (248.7 μM). It was also active against five human cancer cell lines, showing IC50 values from 5.4 to 20.3 μM. ANDC-A demonstrated a differential selectivity for HL-60 (SI 5.5) and THP-1 (SI 4.3) cell lines in comparison with Vero cells and was more selective than cisplatin and doxorubicin against MCF-7 cell line in comparison with human peripheral blood mononuclear cells. ANDC-A was able to eradicate clonogenic tumour cells at concentrations of 20 and 50 μM and induced apoptosis in all tumour cell lines at 20 μM. These results suggest that ANDC-A might be used as a biochemical tool in the study of tumour cells biochemistry as well as an anticancer agent with durable effects on tumours.

Bioenergetic Aspects of Xylitol Production from Lignocellulosic Materials

With the aim of identifying the best experimental conditions able to optimize the industrial production of xylitol from lignocellulosic materials, this chapter provides a review about the present knowledge on the use of material and bioenergetic balances implied in xylose-to-xylitol bioconversion by yeasts. To this purpose, xylose metabolism was investigated in three different pentose-metabolizing yeasts, namely, Pachysolen tannophilus, Candida guilliermondii and Debaryomyces hansenii, using different lignocellulosic hydrolyzates as carbon and energy sources. The main hypotheses on which material and bioenergetic balances were based are (a) fermentative assimilation of xylose, (b) semi-aerobic xylose-to-xylitol bioconversion, (c) biomass growth from pentoses, (d) catabolic oxidation of xylose and (e) NADH regeneration by the electron transport system. Similar approaches could be proposed to investigate and model other semi-aerobic processes.