SELECTION OF ALTERNATIVE MEDIUM FOR PRODUCTION OF PROTEASE BY Bacillus subtilis (UCP 0999).

BORBA, P. P.1, *, SOARES DA SILVA, R. C. F. 1; ALVES DA SILVA, C.A2.

1*_{Mestrado em Desenvolvimento de Processos Ambientais – UNICAP}

2*_{Núcleo de Pesquisas de Ciências Ambientais (NPCIAMB), Universidade Católica de Pernambuco “Rua do} Príncipe, 526. Boa Vista - Recife – PE”. *[email protected]

Keywords: protease, Bacillus subtilis, selection medium

Proteases (EC 3.4), are enzymes that catalyze the cleavage of peptide bonds in other proteins, resulting in total hydrolysis of protein and are essential constituents of all forms of life on earth, including prokaryotes, fungi, plants and animals. Microbial proteases are among the most important hydrolytic enzymes and have been studied extensively since the advent of enzymology. There is renewed interest in the study of proteolytic enzymes, mainly due to the recognition that these enzymes not only play an important role in the cellular metabolic processes but have also gained considerable attention in the industrial community. It is a well-known fact that extracellular protease production in microorganisms is greatly influenced by media components, especially carbon and nitrogen sources, metal ions, and physical factors such as pH, temperature, inoculum density, dissolved oxygen, and incubation time. Today, proteases account for approximately 40% of the total enzyme sales in various industrial market sectors, such as detergent, food, pharmaceutical, leather, diagnostics, waste management and silver recovery. Microbial proteases are classified into various groups, dependent on whether they are active under acidic, neutral, or alkaline conditions and on the characteristics of the active site group of the enzyme, metallo (EC.3.4.24), aspartic (EC.3.4.23), cysteine or sulphydryl (EC.3.4.22), or serine-type (EC.3.4.21). The genus Bacillus is one of the most ubiquitous and diverse, with representatives being found in the soil and associated water sources such as rivers, coastal waters and estuaries. The metabolic diversity of these organisms has led to members of this group being used for a wide range of industrial processes, including the. antibiotics, fine biochemicals insecticides and production hydrolytic (and other) enzymes. The utilization in medium of production protease by several studies have been made to replace components of traditional means of production for substrates with low cost and high added value. In this sense, the objective of this work was to produce protease by Bacillus species using alternative substrates and low cost. The medium of production used as control was called Horikoshi-I, and the alternative media had the following substitutions: Medium "A" nitrogen sources replaced by corn steep liquor, "B" medium carbon sources replaced by soybean oil, medium " C "Carbon and nitrogen sources replaced by corn steep liquor and soybean oil respectively. The assays occurred in 150 rpm, 37oC, 96 hours. The enzyme assay used was described in the method of Lowry, with the modifications proposed by Petterson, The results indicate that the combination of C and N sources in the middle C, enhanced the production of protease in the middle alternative, obtaining values of alkaline pH (10.9) in 56 hours of growth and enzyme activity of 62 IU/mL. These values show that the association of substrates in alternative means of production, may increase the yield of production in enzymatic processes and reduce production costs of fermentation processes.

SELECTION OF YEASTS IN ORDER TO PRODUCE FUEL ETHANOL BY SMALL PRODUCERS FROM BAHIA

PINHEIRO-RIBEIRO, C.S.1; SILVEIRA, G.G. da 2*; GONÇALVES, C.M. 1; AZEREDO, A.1; ROSA, C.A.1,3; UETANABARO, A.P. T.1,4

1 _{Programa de Pós-Graduação em Biotecnologia UEFS/FIOCRUZ, Laboratório de Pesquisa em Microbiologia,}

Universidade Estadual de Feira de Santana

2* _{Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Ituiutaba - MG} 3

Departamento de Microbiologia, Laboratório de Biotecnologia e Ecologia de Leveduras, Universidade Federal de Minas Gerais

4

Departamento de Ciências Biológicas, Laboratório de Microbiologia da Agroindustrial, Universidade Estadual de Santa Cruz,

Keywords: yeast selection, ethanol, cachaça

Artisanal production of ethanol fuel in a small distillery of cachaça in Brazil is becoming a popular solution within the country, and contributes to sustainable agriculture for small farmers, mainly in Bahia state. This kind of alcohol differs from the ethanol contained in alcoholic beverages only by the type of fuel required for standardization. The starter yeasts have been used with excellent results in the production of ethanol, making it possible to improve the process and the fermentation conditions. Some features are considered desirable in this selection such as tolerance to high concentrations of carbohydrates, high- speed fermentation, high alcohol tolerance, high temperature tolerance and stability. The selection of strains adapted to each type of process is very important to ensure a complete fermentation, and consequently a high production of ethanol. This study selected yeast strains targeting a high yield of ethanol fuel. The yeasts were isolated from two small distilleries in the interior of Bahia and are preserved in the Coleção de Culturas de Microrganismos da Bahia (CCMB / UEFS). 44 isolates were inoculated in modified Sabouraud agar (2% glucose, 1% peptone, 0.5% yeast extract and 2% agar). For stress testing (ethanol tolerance, osmotolerance and thermotolerance), were used a rate of 0.1 mL at a concentration of approximately 1.0 X 107 UFC.mL-1. Counting of viable cells was performed using a Neubauer chamber and methylene blue dye 0.01%. The strain used as control was the Saccharomyces cerevisiae JP1. All tests were performed in triplicate. Analysis of the yeasts, in most cases, the results were similar or superior to the control, indicating a high potential of these isolates to produce ethanol. Of the 44 isolates tested, 13 showed potential as a yeast starter of the process, but other parameters such as ethanol production and growth speed of yeasts are important parameters for selection of strains with desired characteristics for the production of ethanol fuel.

Financial support: Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) Laboratório de Pesquisa em Microbiologia (UEFS)

Programa de Pós-Graduação em Biotecnologia UEFS/FIOCRUZ Coleção de Culturas de Micro-Organismos da Bahia (CCMB)

Soil Biological attributes and Quality of a Oxisol under