Rosane Rech

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks.

A growth kinetic model of Kluyveromyces marxianus cultures on cheese whey as substrate

This work presents a multi-route, non-structured kinetic model for determination of microbial growth and substrate consumption in an experimental batch bioreactor in which β-galactosidase is produced by Kluyveromyces marxianus growing on cheese whey. The main metabolic routes for lactose, and oxygen consumption, cell growth, and ethanol production are derived based on experimental data. When these individual rates are combined into a single growth rate, by rewriting the model equations, the model re-interpretation has a complexity similar to that of the usual variations of the Monod kinetic model, available in the literature. Furthermore, the proposed model is in good agreement with the experimental data for different growth temperatures, being acceptable for dynamic simulations, processes optimization, and implementations of model-based control technologies.

Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

Saccharomyces cerevisiae strain W303 was transformed with two yeast integrative plasmids containing Kluyveromyces lactis LAC4 and LAC12 genes that codify β-galactosidase and lactose permease respectively. The BLR030 recombinant strain was selected due to its growth and β-galactosidase production capacity. Different culture media based on deproteinized cheese whey (DCW) were tested and the best composition (containing DCW, supplemented with yeast extract 1 %, and peptone 3 % (w/v)) was chosen for bioreactor experiments. Batch, and fed-batch cultures with linear ascending feeding for 25 (FB25), 35 (FB35), and 50 (FB50) hours, were performed. FB35 and FB50 produced the highest β-galactosidase specific activities (around 1,800 U/g cells), and also the best productivities (180 U/L.h). Results show the potential use of fed-batch cultures of recombinant S. cerevisiae on industrial applications using supplemented whey as substrate.

Ultrasound as an alternative technology to extract carotenoids and lipids from Heterochlorella luteoviridis

The present work evaluated the use of ultrasound as a pre-treatment to lipid and carotenoid extraction from the microalgae Heterochlorella luteoviridis. The pre-treatment was performed in the presence of ethanol (25%, v/v) with the ultrasound intensity varying from 0 to 100% (435kJkg-1). After the pre-treatment, a diffusive step was performed in order to evaluate different ethanol concentrations (50-75%, v/v). The results regarding carotenoid extraction showed that there is an optimal extraction region: 40-80% of ultrasound intensity and 60-75% of ethanol concentration. The lipid extraction was not influenced by the ultrasound pre-treatment and increased with the increase of ethanol concentration.

Chemical composition of microalgae Heterochlorella luteoviridis and Dunaliella tertiolecta with emphasis on carotenoids

BACKGROUND Microalgae have been used as food supplements owing to their high protein, polyunsaturated fatty acid and carotenoid contents. As different carotenoids have distinct properties and the carotenoid composition of microalgae has been poorly explored in the literature, this study determined the complete carotenoid composition of two microalgae species, Heterochlorella luteoviridis and Dunaliella tertiolecta, using high-performance liquid chromatography coupled with diode array detection and tandem mass spectrometry (HPLC-DAD/MS2 ). Additionally, the proximate composition and major minerals were evaluated. RESULTS The carotenoid composition of the two microalgae was similar, with 13 carotenoids being found in H. luteoviridis and 12 in D. tertiolecta. The major carotenoids were all-trans-lutein (1.18 mg g-1 in H. luteoviridis and 1.59 mg g-1 in D. tertiolecta), all-trans-violaxanthin (0.52 mg g-1 in H. luteoviridis and 0.45 mg g-1 in D. tertiolecta) and all-trans-β-carotene (0.50 mg g-1 in H. luteoviridis and 0.62 mg g-1 in D. tertiolecta). All-trans-lutein was the predominant carotenoid in both microalgae, representing around 40% (mass fraction) of the total carotenoids. The lutein content found in these microalgae was significantly higher (2-40 times) than that in other important food sources of lutein (e.g. parsley, carrot, red pepper and broccoli). CONCLUSION The microalgae H. luteoviridis and D. tertiolecta are excellent sources of lutein that could be commercially exploited by the food and pharmaceutical industries. Moreover, it was confirmed that both microalgae are good sources of protein, lipids and calcium.

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1-butyl-3-methylimidazolium acetate and their bioconversion to ethanol.

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1‐butyl‐3‐methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second‐generation ethanol production and other fermentation processes.

Avaliação sensorial de pães de fermentação natural a partir de culturas starters inovadoras

The sourdough fermentation in bread making is strongly motivated due to its benefi cial aspects on fl avor, texture, shelf life, and the nutritional properties of the obtained products. In this research, the yeasts Kluyveromyces marxianus and Dekkera bruxellensis, and the bacterium Lactobacillus plantarum were used as alternative starter cultures in bread making. Experimental designs generated from simplex-centroid mixture were used in the sensorial analyses of breads. Results have shown that bread obtained with pure cultures of L. plantarum presented the smallest specifi c volume, while the mixture of K. marxianus and L. plantarum positively infl uenced the formation of good specifi c volume of products. Sensorial analysis showed good acceptability for breads obtained with D. bruxellensis and K. marxianus mixture, as well as for the mixture of the three microorganisms. The results obtained in this research suggest the possibility of using defi ned starter cultures for sourdough fermentation, which is traditionally carried out without any process controls.

Exponential Fed-Batch Cultures of Klebsiella pneumoniae under Anaerobiosis Using Raw Glycerol as a Substrate to Obtain Value-Added Bioproducts

In this study, Klebsiella pneumoniae BLh-1 was grown under exponential fed-batch mode in cultures using raw glycerol from biodiesel synthesis under anaerobiosis to produce value-added products. Specific growth rates of 0.035, 0.07, and 0.105 h were tested and 1,3-propanediol (1,3-PDO) and ethanol were produced in all cultures, whereas the formation of 2,3-butanediol was not observed. The highest concentrations of 1,3-PDO (38.5 g L) and ethanol (13.2 g L) were achieved under the specific growth rate of 0.105 h. Acetic and lactic acids were also produced at high concentrations (17.3 and 7.8 g L, respectively) under these conditions. The yields of 1,3-PDO and ethanol increased over time, reaching final values of 0.56 and 0.19 g g, respectively. Overall productivities were 1.43 g L h for 1,3-PDO and 0.49 g L h for ethanol. Compared to batch results, fed-batch operation favored K. pneumoniae metabolism towards the production of 1,3-PDO and ethanol, being an interesting process to add value to the ever-growing availability of raw glycerol from biodiesel industry.

The effect of temperature and moderate electric field pre-treatment on carotenoid extraction from Heterochlorella luteoviridis

Moderate electric field (MEF) was evaluated as a pre-treatment (0–180 V) to carotenoid extraction at four different temperatures (30, 40, 50 and 60 °C) followed by a extraction step using ethanol/water as solvent (75% of ethanol, v/v). During the extraction step, samples were collected at 1, 5, 30, 40 and 50 min. Results showed that MEF allowed higher carotenoid concentration at the beginning of the extraction step at 30, 40 and 50 °C, leading to better extraction results along the extraction time. A different behaviour was observed at 60 °C probably due to the thermal effects on the cell and to carotenoids thermal degradation. The highest extraction yield, 86% of the total carotenoid content (compared to exhaustive extraction), was achieved at both 40 and 50 °C with the MEF pre-treatment, indicating that a high extraction yield can be obtained even at relatively low temperatures (40 °C).