Maria Antonieta Ferrara

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Use of Steam Explosion Liquor from Sugar Cane Bagasse for Lignin Peroxidase Production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.

Kinetics of asparaginase II fermentation in Saccharomyces cerevisiae ure2dal80 mutant: effect of nitrogen nutrition and pH.

Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.

Biomass pretreatment: a critical choice for biomass utilization via biotechnological routes

The necessary biomass pretreatment step, to render the material accessible to the relevant enzyme pool, has been under thorough investigation as the production of biomass syrups, via enzymatic hydrolysis, with high sugars concentrations and yields and low inhibitors concentrations requires the pretreatment to be both efficient and low cost. A good choice for biomass pretreatment should be made by considering: (i) the possibility to use high biomass concentration; (ii) a highly digestible pretreated solid by either increasing the biomass superficial area or decrease in crystallinity or both; (iii) no significant sugar degradation into toxic compounds; (iv) yeast and bacterial fermentation compatibility of the derived sugar syrups; (v) lignin recovery; (vi) operation in reasonably sized and moderately priced reactors and (vii) minimum heat and power requirements [1]. Considering the most known pretreatments, such as diluted acid, hydrothermal processes, steam explosion, milling, extrusion, and ionic liquids, different pretreatment methods produce different effects on the biomass in terms of its structure and composition [2]. For example, the hydrothermal, steam explosion and acidic pretreatments conceptually remove mainly the biomass hemicellulose fraction whereas alkaline pretreatments remove lignin. On the other hand the product of a milling-based pretreatment retains the biomass initial composition. Furthermore, cellulose crystallinity is not significantly reduced by pretreatments based on steam, or hydrothermal, or acidic procedures, whereas ionic liquid-based techniques can shift crystalline cellulose into amorphous cellulose, substantially increasing the enzymatic hydrolysis rates and yields. As such, the choice of pretreatment and its operational conditions as well as the composition of the enzyme blend used in the hydrolysis step, determines the hexose and pentose sugars composition, the concentration and toxicity of the resulting biomass syrups. The activity profile of the enzyme blend and the enzyme load for an effective saccharification may also vary according to the pretreatment. Indeed, a low hemicellulase load can be used for a xylan-free biomass and a lower cellulase load will be needed for the hydrolysis of a low crystalline and highly amorphous pretreated biomass material. As the pretreatment choice will also be affected by the type of biomass, the envisaged biorefinery model will need to consider the main types of biomass that will be used for the biorefinery operation so as to select an appropriate, and versatile pretreatment method [3]. Considering the biorrefinery concept which broadens the biomass derived products, the C6 sugars could be fermented into ethanol, while the C5 stream could be used for the production, via biotechnological routes, of a wide range of chemicals with higher added value. To date, sugarcane and woody biomass, depending on the geographic location, are strong candidates as the main renewable resources to be fed into a biorefinery. However, due to major differences regarding their physical properties and chemical composition, the relevant pretreatments to be used in each case are expected to be selective and customized. Moreover, a necessary conditioning step for wood size reduction, prior to the pretreatment, may not be necessary for sugarcane bagasse, affecting the pretreatment energy consumption and costs. Moreover, the choice of pretreatment should take into account the foreseen utilization of the main biomass molecular components (cellulose, hemicelluloses and lignin). It is important to point out that lignin can be used as a valuable solid fuel or as a source of aromatic structures for the chemical industry. Sugarcane is one of the major agricultural crops when considering ethanol production, especially in tropical countries. In Brazil, sugarcane occupies 8.4 million hectares, which corresponds to 2.4% of farmable lands in Brazil. The gross revenue of this sector is about US

Purification and characterization of asparaginase II from Saccharomyces cerevisiae cloned in Pichia pastoris: a study on a possible antileukemic drug

20 billion (54% as ethanol, 44% as sugar, and 2% as bioelectricity) [4]. In addition, up to 50% of all vehicles in Brazil are flex fuel cars, which corresponds to approximately 15 million cars [5]. Given the above, Brazil is an important player in this scenario, and, consequently, sugarcane bagasse and straw are promising feed stocks for biomass ethanol. Brazil produced, in 2008, 415 million tons of sugar cane residues, 195 million tons of sugarcane bagasse, and 220 million tons of sugarcane straw, whereas the forecast for the 2011 sugarcane production is 590 million tons, which would correspond to 178 million tons of bagasse, and 200 million tons of straw [6]. Currently, in Brazil, R&D on the use of biomass via biotechnological routes has been focused mainly on agricultural residues such as sugarcane residual biomass.

Kinetics of Asparaginase II Fermentation in Saccharomyces cerevisiae ure2dal80 Mutant

Bacterial asparaginase obtained from Escherichia coli and Erwinia chrysanthemi are used as medicine to treat acute lymphocytic leukemia and non-Hodgkin lymphoma. Despite the therapeutic properties of such enzymes there have been reports on adverse reactions, eventually so severe as to impede some patients of using the medicine. Besides, the only drug Brazil used to import is no longer produced. Considering these two factors our proposition is to produce non-bacterial asparaginase.

Biomass Residues in Brazil: Availability and Potential Uses

Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU /L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.