INDUSTRIAL WASTE

RIBEIRO, D. L. R.1*,5, FREITAS SILVA, M. C.²,5, SOUZA, P. M.²,5, ANTUNES, A. A.³,5, ANDRADE, R. F. 4,5, CAMPOS-TAKAKI, G. M.5

1

Graduanda em Química, Universidade Católica de Pernambuco,Recife PE. dafne.luana@hotmail.com 2

PNPD-CAPES /FACEPE/ Universidade Católica de Pernambuco, Recife-PE. . 3 _{Pós-Doutoranda CNPq/ Universidade Católica de Pernambuco, Recife-PE.} 4_{Doutoranda em Ciências Biológicas, Universidade Federal de Pernambuco, Recife-PE} 5_{Núcleo de Pesquisas em Ciências Ambientais, Universidade Católica de Pernambuco, Recife-PE}

Keywords: Rhodotorula glutinis, biosurfactant, industrial residue

Biosurfactants constitute an important class of chemical compounds used in various industrial sectors. The biosurfactant are compounds of microbial origin that exhibit properties of reduction of surface tension or emulsifier capacity. Due to their ability to concentrate at the air-water interface, they are commonly used to separate oily materials from a given medium. Surfactants increase the aqueous solubility of hydrophylic molecules by reducing their surface/interfacial tension at air-water and water-oil interfaces The aim of this work was the biosurfactant production by Rhodotorula glutinis using industrial residues cassava wastewater, corn steep liquor and ice cream effluent as substrates. Rhodotorula glutinis (UCP 1555) isolated from soil in semi-arid (Serra Talhada, PE, Brazil), identified using biochemical and molecular methods. The strain was supplied from the Culture Collection of “Universidade Católica de Pernambuco” (UCP), maintained in Sabouraud dextrose agar at 5 °C. The substrates used “manipueira” (obtained from cassava effluent), corn steep liquor (residue from the manufacture of corn), and effluent from ice cream. The medium composition using the agroindustrial residues for biosurfactant production was established according to factorial design, and variable response was surface tension and emulsifier index. The pre-inoculum was prepared using Sabouraud dextrose broth. The Erlenmeyer’s flasks were inoculated with 5% of the pre- inoculums, and incubated under orbital shaker at 150 rpm for 72 hours at 28 º C. After this period the liquid metabolic free cells was used to determination of surface tension, emulsification index, and pH. The best results was obtained in the assay 17, containing 20% cassava, corn steep liquor 6% and effluent from ice cream to 20% indicated the reduction of surface tension of water 72 to 24.92 mN/m, and emulsifier index showed 86.36% using burned motor oil. The results obtained suggest that the cassava wastewater, ice cream effluent, and corn steep liquor as carbon and nitrogen sources, influencing the reduction of the surface tension during the biosurfactant production. In addition, the biosurfactant produced by R. glutinis showed high activity and emulsifying ability to stabilize oil/water emulsions, and the results showed 95% of confidence about the increased rates of emulsification and surface tension.

THE EFFECT OF TEMPERATURE ON Trichoderma sp LIPASE PURIFIED BY REVERSE MICELLAR EXTRACTION

FILIPPI, R.M1*, LIMA, V. M. G.

Depto. de Ciências Biológicas, Universidade Estadual Paulista “Júlio de Mesquita Filho” Av. Dom Antônio, nº 2100, Pq. Universitário. Assis – SP. *lela_mf@hotmail.com

Keywords: Lipase, reverse micellar extraction, Trichoderma sp.

Introduction: Lipases are triacylglycerol hydrolases enzymes (EC 3.1.1.3) that catalyze the hydrolysis of oils and fats, releasing free fatty acids and glycerol. They may also act in reverse reactions such as esterification and transesterification. Due to its high selectivity, the versatility of their properties and their easy availability, they can be widely used in food and biotechnological industries, such as biodiesel production and processing of dairy foods. The lipase can be produced by submerged fermentation by involving microorganisms. In this present study, the fungus Trichoderma sp was involved. The downstream process was composed by precipitation with ammonium sulfate and reverse micelles system, a purification technique for liquid-liquid extraction, which allows separating the cellular material and simultaneously concentrating the protein of interest. It is a technique with less difficulty and reduced cost compared to traditional chromatographic techniques. The present research work is focused on studying the effect of temperature on the lipase produced by Trichoderma sp and purified by reverse micelles. Materials and methods: The filamentous fungus Trichoderma sp was cultivated in PDA (potato dextrose agar (39 g/L)) for 7 days at 28°C. A suspension of 108 spores/mL was used to prepare the pre -inoculum. Fermentation of 3600 mL containing 1% (v/v) of olive oil was performed in a shaker at 28°C, 180 rpm for 96 h. The fermented broth was filtered with gauze and cotton. Later, precipitation with ammonium sulfate to 80% saturation was performed. The supernatant with the salt was kept at 4°C for 12 h and centrifuged for 20 minutes at 3500 rpm and 4 ° C. The pellet was suspended in a minimum volume of phosphate buffer 0.05 mol/L pH 7.0. This suspension was dialyzed against this same buffer and the extract was stored at 4°C. In the purification by reverse micelles, pH was adjusted to pH 4. For forward extraction, we used 5 mL of crude extract and 5 mL of 0.1 mol/L of AOT in n-heptane. The solution was kept in strong agitation for 30 minutes and then was centrifuged. For the backward extraction, equal volumes of micellar solution and phosphate buffer 0.2 mol/L pH 7 containing KCl 1.2 mol/L were mixed and incubated under the same agitation, and then centrifuged. The aqueous phase containing the enzyme was used to study the effect of temperature on lipolytic activity, accompanied by hydrolysis of p-nitrophenyl palmitate (pNPP) at 410 nm between 20 and 70 º C.

Results and discussion: The crude extract obtained showed 1.36U/mL of activity and 1.06U/mg of specific activity. After precipitation, centrifugation and dialysis of the fermented broth, the activity recovery and the purification factor were found to be 25% and 3.77, respectively. After reverse micelles purification, the activity recovery was 0.17% and purification factor was 4.84. In literature are similar results regarding to purification factors after performing precipitation and reverse micelles extraction. In the temperature assay, it was found: 1.64U/mL at 20ºC;1.35U/mL at 30ºC;0.53U/mL at 37ºC;0.32U/mL at 40ºC; and no activity above 50ºC. Higher activity at 20ºC is unusual for lipases. In previous studies with crude enzyme showed higher activity at 50°C. It can be inferred that micellar extraction could have changed the properties of this lipase. Still, the characterization of the

THE EFFECTS OF DIFFERENT CARBON SOURCES IN LIPASES