PRODUCTION OF A BIOSURFACTANT FROM CANDIDA LIPOLYTICA CULTIVATED IN INDUSTRIAL WASTES USING A BIOREACTOR

SANTOS, D.K.F.1,2*, FARIAS, C.B.B.3,4, RUFINO, R.D.1,2, LUNA, J.M.1,2 & SARUBBO, L.A.1,6

1_{Mestrado em Desenvolvimento de Processos Ambientais, UNICAP; – Rua Nunes Machado, Bloco J, térreo,} Boa Vista, CEP: 50050-590, Recife-PE; 2 Núcleo de Pesquisas em Ciências Ambientais, UNICAP, Recife-

PE; 3Centro de Ciências e Tecnologia, UNICAP; Recife-PE; 4Mestrado em Engenharia Química – UNICAMP, Campinas-SP

*dany_khadydja@hotmail.com

Keywords: Biosurfactants, Candida, industrial residues, bioreactor.

Surfactants constitute an important class of chemical compounds widely used in various industrial sectors. These compounds are formed by molecular structures containing hydrophilic and hydrophobic portions that tend to distribute at the interfaces between fluid phases with different degrees of polarity (oil / water and water / oil). The hydrophilic portion consists of anionic groups, cationic, or amphoteric, whereas the hydrophobic part is usually a linear or branched hydrocarbon or showing no double bonds and / or aromatic groups. Biosurfactants are microbial metabolites produced specially by bacteria and yeasts, although some fungi also produce them. These compounds, due to its biodegradability and compatibility with the environment, unlike similar petrochemicals (synthetic), have been increasingly studied. Thus, this paper describes the production of biosurfactants in bioreactor by Candida lipolytica grown in low-cost waste in order to apply the biomolecule in the environmental area. The following wastes were used as substrates: 5% animal fat and 2.5%. corn steep liquor. Fermentations were performed in a bioreactor of 2.0 L capacity, which operated at a temperature of 28 º C with values of agitation, aeration and at intervals set according to a factorial design. A 23 factorial design with four repetitions and eight tests at the central point was used to evaluate the influence of the independent variables aeration (0,1 and 2 vvm), agitation (200, 300 and 400 rpm) and time of cultivation (48, 96 and 144hours) on the response variables surface tension, yield of biosurfactant and biomass. According to the Pareto charts of standardized effects it could be observed for a confidence level of 95%, that the variable agitation has shown a negative effect on both surface tension reduction, yield and biomass. Moreover, the cultivation time showed a positive and statistically significant influence on surface tension reduction, on the yield and on the biomass. For the variable aeration, it showed a negative influence on surface tension reduction and yield, although not statistically significant, and a positive and statistically significant effect on the biomass increase. The biosurfactant produced by C.

lipolytica grown in a low cost medium showed potential to be applied in the environmental

area once it was able to reduce the water surface tension at 28 mN/m after 144 hours at 200 rpm with an yield of 7 g/L.

Financial support: Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), CNPq e TERMOPE/ANEEL.

PRODUCTION OF CHITIN AND CHITOSAN BY Rhizopus oryzae USING, CASSAVA WASTEWATER SUPPLEMENTED WITH CORN STEEP LIQUOR

LIMA, J.M.N1* , SILVA, G.K.B1 , BRANDÃO, R.M.O1 , OKADA, K1 , CAMPOS- TAKAKI, G.M1 1

Núcleo de Pesquisas em Ciências Ambientais NPCIAMB – UNICAP, Rua Nunes Machado, 42, Boa Vista, Bloco J. Recife –PE. CEP:50.050-590/FAX: 81 2119 – 4043. *Jaceline_maria@yahoo.com.br

Keywords: chitin, chitosan, Rhizopus oryzae, biopolymers, agroindustrial residues

Chitin is a natural linear polymer found in the shells of crustaceans, insects, fungi and yeasts. Chitosan is a natural polysaccharide originated from the deacetylation of chitin. It is soluble in acidic due to the presence of amino groups in the form of free radicals along the polymer chain. The fungal class Zygomycetes, in particular Rhizopus oryzae present in their cell walls as chitin and chitosan. This work was analyzed the production of chitin and chitosan by R. oryzae using low cost substrate, such as cassava wastewater, supplemented with corn steep liquor. The strain of Rhizopus oryzae (UCP 1506), was kindly supplied to the Culture Collection Universidade Católica de Pernambuco (UCP), and maintained on Potato Dextrose Agar, at 5 ° C. The experiments were carried out using cassava wastewater (10%, 7.5% and 5%), supplemented with corn steep liquor (6%, 4% and 2%), at pH 5.5 to 6.0, incubated in orbital shaker at 150 rpm, at 38, 33 C and 28°C for 96. After this period the cultures were collected using nylon filter (Silkscreen, 120F). The biomass was washed with distilled water, dried and maintained in desiccator until constant weight. The extraction of chitin and chitosan were carried out by deproteinization of biomass with 2% NaOH (30:1 v / g) at 60 ° C for 2 hours. Then was separated alkali-insoluble fraction by centrifugation. The residue obtained was treated with 1% sulfuric acid (40:1 v / g) in an autoclave at 121 ° C for 15min. Chitosan was obtained by neutralization to pH 10 and precipitation overnight at 5°C. The copolymers obtained chitin and chitosan, were washed with distilled water, dried and kept in a desiccator until constant weight. Chitin after 96h of culture showed higher in amount in 5% and 2% of cassava and corn steep liquor, at 38°C showed a yield of 51.90 mg / g and 22.01 mg/g dry of dry biomass, respectively. The best chitosan production was found in the condition 10% cassava supplemented with 2% corn steep liquor at 28°C showing a yield of 44.67 mg/g and 24.49 mg/g dry weight of chitin. The agro-industrials substrates used in these studies indicated the biotechnological potential of manipueira supplemented with corn steep liquor as rich components for mirobiological production of chitin and chitosan by R. oryzae.

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PRODUCTION OF CHITINASE, LIPASE AND PROTEASE OF Beauveria bassiana GROWN IN THE PRESENCE AND ABSENCE OF THE PULVERIZED Alphitobius

diaperinus (LESSER MEALWORM)

PAPI, M.C.1*, DANIEL, J.F.S.2*, NEVES, P.M.O.J.3*, REZENDE, M.I.1*

1*_{Depto. de Bioquímica e Biotecnologiamirezende@uel.br,} 3*

Depto. de Agronomia, Universidade Estadual de Londrina

Rodovia Celso Garcia Cid, PR-445 Km380, Campus Universitário, Cx postal 6001, CEP 86051-980 2*_{Universidade Tecnológica Federal do Paraná Av. dos Pioneiros, 3131, Londrina-PR, CEP 86036-370}

ABSTRACT

Alphitobius diaperinus (Panzer), also known as lesser mealworm, is considered an

important world poultry plague. This beetle is known as a potential reservoir for poultry pathogens causing several diseases due to transmission of bacteria, virus, fungi and protozoa, resulting in serious veterinary and economical problems. Usually its control is performed using chemical insecticides (organophosphates and pyrethroids). Laboratory tests demonstrated that the use of these insecticides cause a temporary reduction of the insect population, further this control practice becomes ineffective due to habits of the beetle and the continuing presence of birds in the poultry houses. Extracellular enzymes such as chitinases, lipases, and proteases as well secondary metabolites with insecticide capability were already extracted from Beauveria bassiana and demonstrated potential for use in biological control. The objective of this work was to evaluate the production capacity of chitinases, lipases and proteases from four strains of B. bassiana (UNI-4, UNI- 40, CG-71 e CG-152). The cultures were performed in 125 mL Erlenmeyer flasks with 25 mL of Minimal Vogel´s media containing 2% (w/v) of pulverized A. diaperinus or 2% (w/v) of glucose as carbon source. All cultures were carried out in triplicate and incubated under agitation (180 rpm) for 7 days at 28 ± 2 °C. The supernatants (enzyme extracts) were used to determine the activities of chitinases, lipases and proteases.Moreover, the content of total sugars, reducing sugars, protein, initial pH and biomass, were also determined, contributing for a better assessment about microorganism growth under these conditions. All strains were able to produce chitinases, lipases and proteases when grown in presence of pulverized A. diaperinus as sole carbon source and in this condition showed greater activity compared to those strains that grown in Vogel's medium containing glucose as carbon source. This result demonstrates that the presence of pulverized A. diaperinus in the medium induced the production of extracellular enzymes. It was also observed that the increasing order of enzyme activity produced by four strains studied was always chitinases < lipases < proteases under these conditions studied. The strain CG-152 was the best enzyme producer, which showed chitinase activity corresponding to 0.05 U/mL, lipase 3.36 U/mL and protease 346 U/mL. The four strains showed a higher capacity to produce proteases when cultured in presence of pulverized A. diaperinus making them potential biological control agents, since these enzymes are essential in the process of pathogenicity. Keywords: biological control, Beauveria bassiana, Alphitobius diaperinus, extracellular enzymes

PRODUCTION OF LIPASE FROM Fusarium sp. USING CORN OIL AND SURFACTANTS THROUGH EXPERIMENTAL DESIGN AND SURFACE