Viosa, MG-Brazil2013

ANNALSAbstracts

EditorsHilrio Cuquetto MantovaniGilberto de Oliveira Mendes

Conrado Augusto VieiraMariana Caroline Tocantins Alvim

Lvia Tavares Colomboder Galinari Ferreira

Graphic design and layout: Gilberto MendesCover design: Conrado Vieira and Gilberto MendesBack cover photo: Conrado Vieira

The content of the articles contained in this publication are the sole responsibility of their respective authors.

Ficha catalogrfica preparada pela Seo de Catalogao e

Classificao da Biblioteca Central da UFV

International Symposium on Microbiology and I61a Biotechnology (2 : 2013 : Viosa, MG) 2013 Annals abstracts [of] II International Symposium on Microbiology and Biotechnology / International Symposium on Microbiology and Biotechnology, December 4-7, 2013, Viosa, MG ; organizing committee Hilrio Cuquetto Mantovani [et al.]. Viosa, MG, UFV, DMB, 2013.

145 p. ; 23 cm. ISBN 978-85-81790-60-2

1. Microbiologia - Congressos. 2. Biotecnologia -

Congressos. 3. Microbiologia agrcola. I. Mantovani, Hilrio Cuquetto. II. Universidade Federal de Viosa. Departamento

de Microbiologia. III. Ttulo. CDD 22. ed. 579

OrganizationGraduate Program in Agricultural Microbiology

Department of MicrobiologyUniversidade Federal de Viosa

December 4-7, 2013Viosa, MG-Brazil

Acknowledgments

FacultyTo all the faculty of the Department of

Microbiology, Universidade Federal de Viosa, whom contributed to the

success of the event.

Employees:To all the Microbiology Department

officials, especially the Secretaries Nilca, Sandra and Letcia for their dedicated efforts in organizing this

Symposium.

Invited Speakers:To all invited speakers for their

valuable contribution to the programme of the II International Symposium on Microbiology and

Biotechnology.

Sponsors:To all of our sponsors who help make this event possible with their support.

Organizing Committee

Prof. Hilrio Cuquetto MantovaniPresident

Prof. Maria Cristina Dantas VanettiVice-President

Prof. Maria Catarina Megumi Kasuya 1st Treasurer

Prof. Miriam Teresinha dos Santos2nd Treasurer

Prof. Wendel Batista da Silveira1st Secretary

Scientific CommitteeProf. Antnio Galvo do NascimentoProf. Clia Alencar de MoraesProf. Cynthia Cando da SilvaProf. Denise Mara Soares BazzolliProf. Flvia Maria Lopes PassosProf. Luciano Gomes FiettoProf. Hilrio Cuquetto MantovaniProf. Marcos Rogrio TtolaProf. Maria Catarina Megumi KasuyaProf. Maria Cristina Dantas VanettiProf. Marisa Vieira de QueirozProf. Maurcio Dutra CostaProf. Miriam Teresinha dos SantosProf. Olinto Liparini PereiraProf. Poliane Alfenas ZerbiniProf. Srgio Oliveira de PaulaProf. Wendel Batista da Silveira

Logistics CommitteeProf. Wendel Batista SilveiraProf. Maria Catarina Mugumi KasuyaProf. Cynthia Cando da SilvaTiago de Souza LeiteHugo Leonardo Andr GenirWemerson de Castro OliveiraJosenilda Carlos dos Santos

Sponsorship / Disclosure CommitteeProf. Hilrio C. MantovaniConrado Augusto VieiraLvia Tavares ColomboMariana C. Tocantins AlvimGilberto de Oliveira Mendes

Committee for Logistics of SpeakersProf. Marisa Vieira QueirozProf. Antnio Galvo do NascimentoProf. Flvia Maria Lopes PassosRobson de Assis SouzaCaroline MouraMarcela CamachoSergio A. Diaz

Committee for the EventSocial ActivitiesProf. Mriam T. SantosProf. Poliane Alfenas ZerbiniProf. Denise M. S. BazzoliProf. Maria Cristina Dantas VanettiFernando Augusto da SilveiraJosicelli Souza CrispimPricila Palla Costa

Secretariat Comissionder Galinari FerreiraDeborah Romaskevis G. LopesTaides Tavares dos SantosFbia Giovana V. de AssisCludia Vieira PrudncioCleriane AndrElsa Fernandes da SilvaRenato Pedroso Menicucci

Foreword

The International Symposium on Microbiology and Biotechnology (SIMB) has been developed to become a discussion forum that provides new data and stimulates debates on the latest advances in basic and applied research with microorganisms. Fol-lowing the most recent trends in these fields of studies is often challenging to students and professionals from the biological and agricultural sciences. In this regard, the Sym-posium aimed to bring together internationally renowned experts to deliver formal pre-sentations on recent observations and theories in topics within these fields of study. The broad programme of the symposium intended to challenge participants to push the boundaries of their comfort zone, finding innovative ideas and strategies for current and future research.

A significant number of abstracts were submitted and those selected by the Scien-tific Committee of the Conference were scheduled for presentation in poster sessions during the Symposium. The representative role of microorganisms in industry, agricul-ture and environmental and food sciences are presented in the abstracts you are about to read.

We hope SIMB 2013 will provide a stimulating and interesting forum!

Organizing Committee - SIMB 2013

SIMB 2013 | 8

EVALUATION OF THE AMMONIUM REMOVAL OF NITRIFYING SLUDGE ACCLIMATED TO DIFFERENT SALT

CONCENTRATIONS

SILVA L C F1, DE PAULA S O2, OLIVEIRA V M3, SOUSA M P4, DE SOUZA R S4, TORRES A P R4, SILVA C C1

1Departamento de Microbiologia/UFV,36570-000,Viosa,MG,Brazil. 2Departamento de Biologia Geral/UFV,36570-000,Viosa,MG, Brazil. 3CPQBA/UNICAMP, 13081-970,Campinas, SP,Brasil. 4PETROBRAS/CENPES,Cidade Universitria,Rio de Janeiro,RJ, 21949-915,Brasil.

The extraction of petroleum generates large amounts of oil and saline wastewa-ter (production water). Additionally, this effluent contains high concentrations of toxic compounds, such as ammonium, that when dumped in the environment may be harmful to aquatic life. Among treatments available for ammonium removal, the biological treatment has been choose because is efficient and presents low costs. However, this biological removal of ammonium can be affect by salinity, once that damages the nitrifying bacterial metabolism. Thus, the aim of the study was to eval-uate the ammonium removal rate of nitrifying sludge acclimated to salt when sub-jected to different salt concentrations. The nitrifying sludge aliquot was inoculated into two saline media, MOD and R2A,containing 6% NaCl, and every six days, more 2% NaCl were added to reach the concentration of 20%. After acclimation process, 2.5 mL of MOD e R2A media from each acclimation, 6% to 20% of NaCl, was mixed and used as inoculum to nitrifying medium with and without addition of NaCl, and after 60 days the ammonium removal rate was measured by colorimetric assay. The results showed a high rate of ammonium removal in nitrifying medium with and without NaCl. In nitrifying medium without NaCl was observed high removal rates, including the concentrations 18 and 20% from acclimation. However, after addition of the salt, ammonium removal rate decreases, but reached approximately 70% of remova lin 6, 10 e 12% of NaCl. Thus, we can conclude that after acclimation process the nitrifying sludge was able to remove ammonium in high salt concentrations.

Financial support: Petrobrs

SIMB 2013 | 9

SANITARY QUALITY OF CURD CHEESE SOLD IN THE CITY OF RIO LARGO, ALAGOAS

SILVEIRA,A.J.S.S; SANTOS,T.M.C; SILVA,S.G.M; SILVA,J.M; ARAUJO,B.F.O; TENORIO,F.A; MONTALDO,Y.C

Universidade Fededal de Alagoas;Unidade Academica Centro de Ciencias Agrarias; Campus Delza Gitai, BR 104 norte KM 85 - Mata do Rolo Rio Largo 57100000 - Rio Largo, AL - Brasi

The rennet cheeseis one of the typical culinary delicacies of the Northeastern and its production, is basically the coagulation of milk and press the dough. The quality and safety of milk are of utmost importance for the manufacture of cheese. According to current legislation, the cheeses in general should be obtained from pasteurized milk, whole or standardized,coagulated by rennet enzymes, may be in-creased by lactic ferment, salt and other substances permitted. The cheese made from milk that is not of good quality and is not processed within the minimum requirements of hygiene poses a risk to the consumer health. Therefore, good manu-facturing practices are essential and should be adopted to obtain a good product . This study aimed to analyze the indicators of hygienic-sanitary. Tensamples of curd cheese sold in supermarkets and street fair in the city of Rio Largo. The experiments were performed in the Microbiology Laboratory of the Center for Agricultural Sci-ences, Universidade Federal de Algoas. The results ranged from 2x106to 2x1010for mesophilic microorganisms and 2.3 x102to > 2.4 x104for total and thermotolerant coliforms. In the analysis of dirtiness were detected woven fibers, carbonized mate-rial and wood, meaning inattention during manipulation of the raw material. The maximum allow limits for thermotolerant coliform, and total mesophilic bacteria are respectively: 5x102,

SIMB 2013 | 10

MICROBIOLOGICAL CHARACTERISTICS OF RAW MILK SAMPLES COLLECTED IN SATUBA, AL

LINS,D.T; SANTOS,T.M.C; SILVA,S.G.M; SILVA,J.M; MELO, A.L.S; TENORIO,F.A; SANTOS, S.J.

Universidade Federal de Alagoas;Centro de Ciencias Agrarias;Campus Delza Gitai, BR 104 norte KM 85 - Mata do Rolo Rio Largo 57100000 - Rio Largo, AL - Brasil

The refrigerated storage of raw milk at the source of production reduces eco-nomic losses as well as the presence of microorganisms responsible for intoxica-tions. This study aimed to evaluate the microbiological quality of raw milk samples from refrigerated tanks collected in Satuba,Alagoas, through the determination of indicator microorganisms. The samples were subjected to determination of the most probable number (MPN) of total and thermotolerant coliforms and Salmonella. In determining the MPN of total and thermotolerant coliforms, three dilutions of each sample were subjected to presumptive and confirmatory tests, using a series of three tubes per dilution. In Salmonelladetection, aliquots of 25 mL were subjected to enrichment in 10 mL of selenite cystine broth. After incubation, a loopful was transferred from the sample to a petri dish containing Salmonella Shigella agarin order to obtain isolated colonies. Were selected 3-5 colonies, which were subjected to biochemical tests morphotinctorial for characterization of bacteria. The num-bers of total and thermotolerantcoliforms ranged from 1.1to 2.9x102x103 and1 to 2.9x103NMP mL-1, respectively. Significant differences were detected (P

SIMB 2013 | 11

ANALYSIS BY ATOMIC FORCE MICROSCOPY OF BIOFILM FORMATION BYSTREPTOCOCCUS MUTANSON STAINLESS

STEELS AISI 304 AND 316

SILVA, N. S.1; DIXINI, P. V.2; RIBEIRO, S. S. S.1; ANDR, C.1; ROMO, W.2,3; NICOLIN, V. A. F.1; VIGAS-AQUIJE, G. M.3; KORRES, A. M. N.1

1. Instituto Federal De Educao, Cincia E Tecnologia Do Esprito Santo - IFES/Campus Vitria; 2. LABPETRO/Universidade Federal Do Esprito Santo; 3. Instituto Federal De Educao, Cincia E Tecnologia Do Esprito Santo - IFES/ Campus Vila Velha.

Streptococcus mutansis commonly found in the oral cavity of humans and is impli-cated in caries formation and also able to form a biofilm. Studies ofS. mutansadhesion to stainless steel by imaging may make important contributions to our understanding of the architecture of biofilms. The Atomic Force Microscope (AFM) permits mapping and analysis of surfaces in nanoscale. Stainless steels AISI 304 and AISI 316 are used in orthodontic appliances. Imaging studies of adhesion of this species to such materials may help to elucidate the occurrence of adherence and corrosion. The objective of this study is to verify and characterize the adhesion ofS. mutanson the surface of the steels AISI 304 and AISI 316 by producing an imaging profile for biofilm formation. Images were measured dry, using the AFM (WITec/ Wissenschaftliche Instrumente und Tech-nologie GmbH) of LABPETRO/UFES, on the stainless steels AISI 304 and 316 without the inoculum (standard), and after growth ofS. mutansfor 15 days/35C in brain-heart infusion medium, without removal of exo-polysaccharides (EPS). All of the material analyzed was prepared in the Microbiology Laboratory of IFES/Campus Vitria. The images were made in non-contact mode, with Si3N4cantilever tips, nominal constant of 42 N.m-1and resonance frequency of around 285 kHz, scan rates of 0.3-1.0 Hz and scan size of 2,500 to 10,000 nm. In addition, analysis was performed of the peak-peak height and of the cross section for each topography image. The topography image confirms the formation of the biofilm. Cross section indicates strong irregularities and the peak-peak height confirms this result for both kinds of steel: 304 peak-peak height: 130.35 nm and 316 peak-peak: 46.61 nm. Peak-peak height for both steel with biofilm was similar: 599.21 nm for AISI 304 and 557.99 for AISI 316. These data contribute to defining the structure of biofilms and for future studies concerning its control.

Acknowledgements: LABPETRO/UFES and Surface Characterization Laboratory/UFRJ for allowing the analyses by AFM Financial support: IFES

SIMB 2013 | 12

MICROBIOLOGICAL QUALITY OF CHICKEN MEAT MARKETED IN THE VALE DO PARABA IN THE STATE OF

ALAGOAS

SANTOS, S. J.1; FEITOZA, ERIKSEN J. B. A.2; TORRES, A. R. S.2; AMARAL, M. F.2; SOARES, K. D. A.3SILVEIRA, A. V. M.4; MOTA, R. A.4; MEDEIROS, E. S.5

Mestrando do PPG em Zootecnia UFAL, Rio Largo/Al, Brasil Graduando em Med. Veterinria UFAL, Viosa/Al, Brasil Mestrando do PPG em Nutrio UFAL, Macei/Al, Brasil 4Prof. da - UFRPE, Recife/Pe,Brasil. 5Prof. da - UFAL, Viosa/Al,Brasil

The feeding within hygienic sanitary standards is an essential condition for the promotion and maintenance of health, so you need to be aware and only consume ani-mal products for correct handling and slaughter, to reduce the risk of outbreaks of dis-eases transmitted by food. The objective of this study was to assess the microbiological quality of chicken meat marketed in the Vale do Paraba which is located in the state of Alagoas. 21 samples were collected in seven municipalities, namely, Quebrangulo, Paulo Jacinto, Viosa, Cajueiro, Capela, Atalaia and Pilar. Were performed research coliforms at 45 C, Salmonella, and Staphylococcus coagulase positive. The values found for coli-forms were 1.1 x10 in all samples analyzed. Observed absence of Coagulase Positive Staphylococcus and Salmonella spp. It concludes with this study that chicken meat mar-keted in the Vale do Paraba can pose risks to consumers by high levels of coliforms at 45 C found. Suggest training up the manipulators to ensure best practices in marketing and minimize the risk of contamination by microorganisms of fecal origin and provide a quality food to consumers.

Key-words: Manipulation, hygiene, microorganisms

SIMB 2013 | 13

BIOFILM FORMATION BY PREVALENT SEROTYPES OFACTINOBACILLUS PLEUROPNEUMONIAEIN

SOUTHEASTERN BRAZIL

PEREIRA, M.F., CRISPIM, J.S., ROSSI, C.C., ARAJO, E.F., QUEIROZ, M.V., BAZZOLLI, D.M.S.

Laboratory Of Microbial Molecular Genetics, Department Of Microbiology, BIOAGRO, Federal University Of Viosa, Viosa, Minas Gerais, Brazil.

Actinobacillus pleuropneumoniaeis the causative agent of porcine pleuropneumo-nia, a costly, severe and highly contagious infectious respiratory disease. To date, 15 serotypes of this bacterium are recognized, and serotypes 2, 7 and 8 are the most preva-lent in southeastern Brazil. Biofilm formation contributes to virulence in many Gram-negative bacteria pathogens. Biofilm provides protection against host immune response and to antibiotics. It has been reported that field isolates ofA. pleuropneumoniaehave the ability to form biofilms and this capacity is variable among different serotypes and growth conditions. This study investigated the potential for biofilm formation of 66 field isolates of serotypes 2, 7 and 8 obtained from pigs with clinical signs of pleuropneumo-nia in the state of Minas Gerais and their respective serotype reference strains. Isolates were initially inoculated at DO 0.1 inpolystyrene microplates andincubated at 37 C for 24h. After washing the plate and staining with crystal violet, the absorbance was read at 600nm. The experiment was completely randomized and data were analyzed by ANOVA (p

SIMB 2013 | 14

FORMATION OF VIRUS-LIKE PARTICLES BY THE CAPSID PROTEIN OFPORCINE CIRCOVIRUS-2 EXPRESSED IN

BACTERIAL SYSTEM

JOSICELLI SOUZA CRISPIM, RAFAEL LOCATELLI SALGADO, LEANDRO LICURSI OLIVEIRA, ABELARDO SILVA JNIOR, MRCIA ROGRIA DE

ALMEIDA

Laboratory Of Animal Molecular Infectology, Department Of Biochemistry And Molecular Biology, BIOAGRO And Nucleus Of Microscopy And Microanalysis, Federal University Of Viosa, Viosa, MG, Brazil.

ThePorcine circovirus2 (PCV-2), a member of theCircoviridaefamily, is a virus composed of circular single strand DNA and a non-enveloped icosahedral capsid with 23 nm diameter. The PCV has three well-characterized open reading frames (ORFs). The ORF1 encodes the Rep and Rep' proteins, which are involved in viral replication; the ORF3 encodes proteins involved in apoptosis, and the ORF2 encodes the capsid protein (Cap), which is the main viral structural protein and is directly involved in the host immune response. Virus-like particles (VLPs) are viral capsid structural proteins arranged in a structure similar to the virion particle (infectious viral particle) highly immunogenic and lacking genetic material. The ability of Cap to form these particles is essential for the development of a recombinant vaccine, since its immunostimulat-ing ability is greater when specific conformational epitopes are exposed. However, the expression system in insect cells used for the production of the commercial vaccines is an onerous method. Alternative forms to express the PCV-2 Cap inEscherichia colihave been developed to make the production of recombinant vaccines against PCV-2 more accessible to producers, but only two studies have reported the formation of VLPs by Cap expressed in prokaryotic systems. This study aimed to verify the formation of VLPs by PCV-2 recombinant protein (rCap-PCV-2) expressed in Escherichia coli. For this, the bacterial extract containing the rcap-PCV-2 was treated with cesium chloride and taken to ultracentrifugation. Aliquots of the density gradient formed were analyzed by SDS-PAGE andWestern blotting. Then, aliquots positive for the presence of rCap-PCV-2 were analyzed by transmission electron microscopy. The micrographs obtained allowed the verification of the formation of VLPs by rCap-PCV-2. This result provides amore credible for the development of diagnostic kits and vaccines using recombinant bacterial protein expression system.

Financial support: CAPES, CNPq and FAPEMIG

SIMB 2013 | 15

FOOD SAFETY RELATED WITH THECONSUMPTION OF CORIANDER (CODIANDRUM SATIVUM) SOLD IN THE

RECNCAVO DA BAHIA

KELLY MENEZES MACEDO 1, ISABELLA DE MATOS MENDES DA SILVA 2, FBIO SANTOS DE OLIVEIRA 2, FERNANDA DE FREITAS VIRGNIO NUNES 2,

VALRIA MACEDO ALMEIDA CAMILO 2, DARCILENE FIUZA 2

1- Universidade Federal do Recncavo da Bahia, Rua Rui Barbosa, 710 - Centro - Cruz das Almas, Bahia, Brasil 2- Universidade Federal do Recncavo da Bahia, Av. Carlos Amaral, 1015 - Cajueiro - Santo Antnio de Jesus, Bahia Brasil

The coliform group consists of members of the Enterobacteriaceae family, includ-ing Klebsiella spp., Enterobacter spp., Citrobacterspp. and Escherichia spp. Its occurrence in plants indicates poor sanitary conditions during production and / or handling of food. This study aimed to evaluate the food safetyrelated with the consumption of coriander (Coriandrum sativum) produced and traded in Santo Amaro, Bahia. Initially, 50 food frequency questionnaires and 50 24-hour recall were applied in family careunits of the city, with previous signature of Informed Consent Term. From the application of these instruments it was possible to identify food eaten more frequently by the population, and it was found that coriander was consideredone of the most consumed vegetables by 90% of respondents, along with mint andchives. The microbiologic analysis was carried out using 10 cilantro samples collected from street market. After that the samples were kept in sterileplastic bags and transported in isothermic boxes to the laboratory and theanalysis was performed within 2 hours. The populations of total coliforms and Es-cherichia coli were estimated by Petrifilm quick count method (3M Company) follow-ing fabricant recommendations. The count of total coliforms ranged from 3.47 x 103to 7.9 x 106 CFU / g and 90% of the samples presented populations greater than 103 CFU / g. The population of Escherichia coli ranged from

SIMB 2013 | 16

SOURCES OF ESCHERICHIA COLICONTAMINATION IN BROILERS SLAUGHTERHOUSE FROM RECONCAVO OF BAHIA

MAYKSON COSTA DE JESUS1 (JESUS, M.C.); ISABELLA DE MATOS MENDES DA SILVA2 (SILVA, I.M.M.); RICARDO MENDES DA SILVA2 (SILVA, R.M.);

VANEZA LEAL CARDOSO2 (CARDOSO, V.L.); JOAQUIM EVNCIO NETO2 (EVNCIO-NETO, J.)

1-Universidade Federal do Recncavo da Bahia, Rua Rui Barbosa, 710 - Centro - Cruz das Almas, Bahia, Brasil 2- Universidade Federal do Recncavo da Bahia, Av. Carlos Amaral, 1015 - Cajueiro - Santo Antnio de Jesus, Bahia Brasil

The objective was to identify the sources of contamination of E. coli in broilers from a poultry slaughter house in the Recncavo of Bahia. It was aseptically collected 10 samples, five samples of cellulitis and five samples of livers from five broilers and samples of water, feed and bedding in rearing house where they were raised. Weve detected the presence of E. coli using rapid counting method Petrifilm (3M Company) usingPetri-film EC plates as recommended by the manufacturer. For analysis of the drinkingwater used in the chicken raising were counted for total coliforms and E. coli in 100mL of the sample using the quick method chromogenic Readycult .E. coli was isolated from 100% of cellulitis samples and 20% of livers analyzed,all without macroscopic changes that would lead to the disposal of the carcass, proving to be a risk to public health because of thepossibility of patogenic dissemination and subsequent enteric or extra-intestinal human infection. In evaluating breeding environment, it wasfound the presence of total coliforms and E.coli on feed, suggesting that the avian is the source of dissemination ofthe bacteria into the chicken carcass. It is therefore necessary to implement acontinu-ous program of hygiene in poultry in order to safe production of broilers consumed by the population and possible changes in the criteria for disposal of carcasses in slaughter houses.

Financial Support: CAPES.

SIMB 2013 | 17

EXOPOLYSACCHARIDES PRODUCTION BY DIAZOTROPHIC BACTERIA ASSOCIATED WITH SISAL(AGAVE

SISALANA PERRINE)

ELIANE DE SOUZA SILVA, ADAILSON FEITOZA DE JESUS SANTOS, JORGE ALBERTO CARDOSO, ANA CRISTINA FERMINO SOARES, LENALDO MUNIZ

DE OLIVEIRA

Universidade Federal do Reconcavo da Bahia, Bahia, Cruz das Almas, Brasil

Exopolysaccharides (EPS) are carbohydrate polymersfound outside the cells. Many diazotrophs produce EPS to interact with plantsas well as to protect the nitrogenase. The aim of this study was to evaluatethe EPS production by diazotrophs under different grow-ing conditions. Eleven diazotrophicbacteria isolated from the rhizosphere and tissues of Agavesisalana were selected. The production capacity of EPS was verified bytransfer-ring 5 ?L of bacterial suspension (OD600nm =0.5) to filter paper discs that were placed on the culture medium specific forEPS production. Three carbon sourceswere tested glucose, lactose and sucrose 1%. The growth conditions were: pH (5.5 and 7.0 ) and temperature (28 C and 37 C). After 48 hours EPS productionwas verified measur-ing the formation of a mucoid layer around the discs. The chemicalmethod, blending a portion of this material in 2 ml of absolute ethanol wasused to confirm the results. Data were analyzed by analysis of variance (ANOVA) , and means were compared by the Scott - Knott test at 5% probability. Five isolatesshowed higher EPS production with the three carbon sources. The best conditionswere: with sucrose, 28 C and pH 7.0, with glucose, 37 C and pH 7 0, withlactose 28 and 37 C and pH 5.5 and 7.0. It was observed a wide variation inEPS production. The next step will be the characterization of these EPS andevaluating the potential use of these isolates and / or EPS production in manyindustrial sectors.

Acknowledgements: UFRB e Capes.

SIMB 2013 | 18

MICROBIOLOGICAL EVALUATION OF PACKAGED DRINKING STRAWS IN FOOD ESTABLISHMENTS AND ATTACHED

TOINDUSTRIALIZEDBEVERAGE BRANDS

REBECCA MENDES FERREIRA; JOO BRUNO COSTA SANTOS; FRANOIS FERNANDEZ; MARTHA ARAJO ALENCAR BRANDO DO VALE

Preciso Metrologia e Qualidade, Aparecida de Goinia, Gois - Brasil; Universidade Federal de Gois, Goinia, Gois - Brasil.

One of the main contributing factors to the occurrence of emerging foodborne diseases is a deficiency in thequality control of the products offered to the population. Contamination of food or their containers may begin in the production of the raw mate-rial and be extended to the stages of transport, reception and storage. Thus, one of the focuses of these related contamination may have origin in the utensils used for feed-ing, since they may not be in appropriate sanitary conditions. This demonstrates the importance of constant monitoring microbiological quality in food, as much as from the utensils used in its manipulation, like drinking straws.This work aimedto evaluate the microbiological packaged drinking straws, distributed in fast food eating establish-ments coming from the city of Goinia and its metropolitan region and also of drink-ing straws provided for use in industrialized beverages for consumption. Microbiologi-cal analyzes were performed as counts of mesophilic count, total coliforms, Salmonella spp and counting of molds and yeasts in 24 drinking straws packed, all from different establishments. In which 10 (41.7%) were collected from fast food snack bars and 14 (58.3%) attached in different beverage brands industrialized box (between: chocolaty, juice, cappuccino and coconut water) - both with national distribution.Results: There wasshownno growth of coliforms and Salmonella spp. However, it was found that 17 (70.8%) of drinking straws showed growth the mesophilic (indicating contamination, > 10 CFU/cm) and 3 (12.5%) reported the growthof fungi, with isolation of Rhodotorula spp, Fusarium and Aspergillus fumigatus. Became evident, therefore, poor sanitary con-ditions in this utensil and possible carelessness of the entities involved in the manufac-turing and quality control, as well as the inspection by the competent authorities.

Acknowledgements: We thank ours families for the comprehension and "Preciso Metrolo-gia e Qualidade" and its employees for their support during the development of this work.

SIMB 2013 | 19

ANTIMICROBIAL RESISTANCE PROFILES OFACTINOBACILLUS PLEUROPNEUMONIAECLINICAL

ISOLATES IN SOUTHEASTERN BRAZIL

SANTOS, C.M.A.*; SILVA, A.K.S.; SEIDE, L.E.; QUEIROZ, M.V.; MANTOVANI, H.C.; BAZZOLLI, D.M.S.

Laboratory Of Microbial Molecular Genetics, Department Of Microbiology, UFV, Viosa, Minas Gerais, Brazil. *Actual Address: Laboratory Of Microbial Ecology And Physiology, Department Of Microbiology, UFMG, Belo Horizonte, Minas Gerais, Brazil.

Actinobacillus pleuropneumoniaeis the causative agent of swine pleuropneumonia, a highly severe and contagious disease responsible for significant economic losses in the swine industry worldwide. Many antimicrobials are used clinically, however, a wide variety of resistant profiles to these agents are observed between strains in different geo-graphical regions, serotypes and time spam. The aim of this study was to determine the antimicrobial susceptibility of 70A. pleuropneumoniae clinical isolates from different farms in the southeast region of Brazil.A. pleuropneumoniaestrains were tested for their susceptibility to various antimicrobials by the microdilution method. Furthermore, the correspondent resistant genes were detected by PCR and located in the genome ofA. pleuropneumoniae isolates. A small number of isolates were resistant to enrofloxacin, danofloxacin, ceftiofur and trimethoprim/sulfamethoxazole. It was verified that a high number of isolates were resistant to ampicillin, penicillin, tylosin tartrate, tilmicosin, chlortetracycline, oxytetracycline and tulathromycin. Our study was the second to report resistance to florfenicol and the presence of thefloRgene inA. pleuropneumo-niae. The tetracycline resistant strains exhibited at least one of thetetgenes:tetB,tetH, tetO, tetA or tetK. This study describes for the first time tetA and tetKgenes in A. pleuropneumoniae, as also the discovery of a new tetracycline resistance gene. Several isolates were multiresistant and one isolate in particular was resistant to seven classes of antimicrobials. Finally, plasmids that contained at least one of the resistance deter-minantssulII,strA,strB, tetB, tetH,tetK, tetO,blaROB1orfloRwere found in 28 iso-lates.Our results suggest the importance of continued monitoring ofA. pleuropneumo-niaeclinical isolates in order to choose the most appropriate treatment of infections and to control the increase of resistance to currently used antimicrobials.

Financial support: FAPEMIG, CNPq and CAPES.

SIMB 2013 | 20

INFLUENCE OF BACTERIAL INOCULANTS ON THE AEROBIC STABILITY OF MAIZE SILAGE

1 FABIA GIOVANA DO VAL DE ASSIS 2 CARLA LUZA DA SILVA VILA 2 ROSANE FREITAS SCHWAN 3 JOS CARDOSO PINTO

1 Department of Microbiology, Federal University of Viosa, 36.570-000, Viosa, Minas Gerais, Brazil. 2 end 3 Department of Biology Department of Animal Science Federal University of Lavras, 37.200-000, Lavras, Minas Gerais, Brazil.

Efficient preservation of forages as silage requires minimizing losses during the aer-obic, fermentation, storage and feedout phases. While quality of the fermentation phase has in general been improved over the past years, the same cannot be said about aerobic stability of silages in the feedout phase. One way to control the aerobic deterioration can be inoculation of lactic acid bacteria (LAB) in silage. This work aimed to evaluate the effect of bacterial inoculants in two inoculating rates on the populations of LAB, yeasts, filamentous fungi of maize silage. The treatments consisted of two inoculating rates (5 and 6 log ufc g-1) for each strain of LAB identified as Lactobacillus buchneri (UFLA SIL 09, 103 e 108), L. plantarum (UFLA SIL 32, 35 e 41), L. hilgardii (UFLA SIL 51 e 52) and commercial strain (L. buchneri). The maize was ensiled in experimental PVC silos and samples were taken for the determination of aerobic stability after 30 or 90 days of fer-mentation. The different inoculants as well as the two studied doses did not significantly modify the maximum temperature or the aerobic stability of the silages. There was an effect only in the period of silage on these variables. The aerobic stability of the silages was greater and the maximum temperature was lower at 90 days of fermentation. A significant interaction of the effects of the inoculants as well as the periods of fermenta-tion on the time parameter to reach the maximum temperature after the opening of the silos (TAMT) was observed. At 30 days of fermentation, the values of TAMT were simi-lar between the silages with different inoculants. However, at 90 days of fermentation, the silages with the UFLA SIL 09 (L. buchneri), 32 (L. plantarum) and 51 (L. hilgardii) inoculants took a longer time to reach the maximum temperature. The recommended inoculation dose is 6 log UFC g-1 of forage.

Financial support: Capes, Fapemig and CNPq

SIMB 2013 | 21

INFLUENCE OF BACTERIAL INOCULANTS ON THE BIOLOGICAL CHARACTERISTICS OF MAIZE SILAGE

1 FABIA GIOVANA DO VAL DE ASSIS 2 CARLA LUIZA DA SILVA VILA 2 ROSANE FREITAS SCHWAN 3 JOS CARDOSO PINTO

1 Department of Microbiology, Federal University of Viosa, 36.570-000, Viosa, Minas Gerais, Brazil. 2 end 3 Department of Biology and Department of Animal Science Federal University of Lavras, 37.200-000, Lavras, Minas Gerais, Brazil.

Ensilage is the most used process of conservation of forages in the whole world and maize is the main forage culture. The main principles of preservation by ensilage are anaerobiosis and a low pH. In successful ensilage processes, lactic acid bacteria (LAB) dominate the fermentation. The growth of (facultative) aerobic microorganisms, espe-cially yeast and filamentous fungi, eventually leads to spoilage of the silage. One way to inhibit the growth of these unwanted microorganisms can be inoculation of LAB in silage. This work aimed to evaluate the effect of bacterial inoculants in two inoculating rates on the populations of LAB, yeasts, filamentous fungi of maize silage. The treat-ments consisted of two inoculating rates (5 and 6 log ufc g-1) for each strain of LAB iden-tified as Lactobacillus buchneri (UFLA SIL 09, 103 e 108), L. plantarum ( UFLA SIL 32, 35 e 41), L. hilgardii (UFLA SIL 51 e 52) and commercial strain (L. buchneri). The maize was ensiled in experimental PVC silos and samples were taken for the determination of the microorganisms populations during ensilage and after 30 or 90 days of fermentation. A significant interaction was observed between the inoculating factors and fermentation time for the LAB population. The strain UFLA SIL 32 provided the highest number of LAB in both time of fermentation. There was a reduction in the count of filamentous fungi with an increase of the inoculation rate from 5 to 6 log ufc.g-1for all other tested strains, except for the UFLA SIL 108 and commercial strains. The silages inoculated with these two strains presented an increase in the population of filamentous fungi from 30 to 90 days of fermentation. Different effects observed on LAB populations and filamentous fungi, which was not observed for the yeast population. The inoculation dose indicated was of 6 UFC g-1 of forage for it provides a higher reduction of filamentous fungi in maize silage, thus decreasing the aerobic deterioration by these microorganisms.

Financial support: Capes, Fapemig and CNPq

SIMB 2013 | 22

IN VITRO INHIBITION OFSCLEROTIUM ROLFSIIBYTRICHODERMASPP

SILVA, J.M.; MELO, A.L.S.; ALBUQUERQUE, L.S.; TENRIO, F.A.; SILVA, S.G.M.; SANTOS, T.M.C.; OLIVEIRA, J.U.L.; MONTALDO, Y.C.

Universidade Federal de Alagoas, Unidade Acadmica CECA/UFAL, CEP 57100-00, Rio Largo, AL, Brasil

Is a plant pathogen Sclerotium rolfisii polyphagous affecting tropical regions, with 500 host species, and their control becomes difficult due to the formation of sclerotia, which are resistance structures which become viable in soil for long periods of time even extreme conditions. Trichodermafungi are commonly found in soil, and demonstrate a good performance of the biocontrol of plant pathogens due to their ability hyperpara-site. This study aimed to evaluate the antagonistic potential of five Trichoderma strains against S.rolfissi. The in vitro assay consisted of tests to evaluate the ability hyperparasite antibiosis and the producing non-volatile and volatile metabolites. The techniques of direct confrontation, cellophane and overlapping plates were used. The interaction of hyphae was observed by microscopy. The process of growth inhibition of the pathogen was evaluated using the scale of Bell. The direct confrontation showed averages 50-80 % growth antagonists that showed speed greater than the pathogen growth. To interact hyphae was observed, intimate contact, the pathogen and the winding forming hook-like structures. Antagonists volatile metabolites excreted with fungistatic effect on the pathogen resulting in a less dense mycelial growth by reducing the size of the colony of the pathogen compared with the control. For the production of volatile metabolites test, was found having fungistatic effect in inhibiting mycelial growth of the pathogen. It can be concluded that the five Trichodermastrains tested have potential as antagonists against S.rolfisii.

SIMB 2013 | 23

TITRATION OF BACTERIOPHAGES WITH LYTIC ACTIVITY FORPSEUDOMONAS FLUORECENSISOLATED FROM EXUDATE

OF FROZEN CHICKENS

RAMOS, M.S.; BATALHA, L.S.; CAL, E.C; LOPEZ, M.E.S.; CARVALHO, M.M.; MENDONA, R.C.S.

Universidade Federal De Viosa- UFV- Brazil

Bacteriophages or phages are viruses relatively specific to a particular host, and this specificity allows the biocontrol of the bacterium of interest. They represent one of the most abundant forms of life found in nature, in aquatic environmentits title may vary from 104to 108PFU/mL. All bacteriophages are obligatory parasites and do not have their own metabolism thus require ahost to multiply and infect only prokaryotic organisms, this being the bacteriophages differentiation with respect to other viruses.In the absence of a host in the existence of the phage isrestricted to a metabolically inert state. The aim of this study was to obtain count of bacteriophages isolated from exudate of frozen chickens in Forming Units Plates (PFU mL-1) between 10 to 100 lysis plates per mL of suspension. Bacteriophages dilutions were incubated for 5 min with bacterial culture ofPseudomonas fluorecens(ATCC 13525) incubated at 30 C for 14-16 h. Then it was added 5 mL of overlay of agar (TSAs).This mixture was poured onto plates contain-ing agar base(TSAb) and incubated at 30 C for 16-18 h. There after, it was proceeded to count the number of plaques of lysis and was calculated the title of bacteriophages in PFU mL-1considering that each forming unit of plaque represents the lytic of the phage. The title of phage reached the order of 1010PFU ml-1thus, the phage titration showed good results enabling to obtain a suspension of phage lytic activity effective forP. flu-orecens.

Acknowledgements: FAPEMIG

SIMB 2013 | 24

GENE EXPRESSION RESPONSE OF KLUYVEROMYCES MARXIANUS UFV-3 CULTURED IN LACTOSE AND SUBJECTED

TO ETHANOL STRESS

SOUZA, R. A.1; DINIZ, R. H. S.1; MACEIRAS, M. L.2; VILLANUEVA, M. E. C.2; SISO, M. I. G.2; SILVEIRA, W. B.1; PASSOS, F. M. L.1

1Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil 2Departamento de Bioloxa Celular e Molecular, Facultade de Ciencias, Universidade da Corua, A Corua, Galicia, Spain

Kluyveromyces marxianus is a yeast specie able to ferment lactose, capability shared by few yeasts. In addition to lactose fermentation, K. marxianus has other desirable at-tributes for industrial fermentation processes, such as thermotolerance, a high growth rate and metabolic diversity, often fermenting a wide variety of carbohydrates, such as hexoses, pentoses, and disaccharides. However, this yeast is susceptible to lower ethanol concentrations thanSaccharomyces cerevisiae, showing limited growth over 6% (v/v) of this alcohol. The objective of this work was to determine the gene expression induction when K. marxianus culture was subjected to ethanol.K. marxianus UFV-3 was activated overnight in YNB medium with 2% lactose at 37 C. Subsequently, the yeast was re-in-oculated into fresh medium with 2% lactose. Exponential phase cells were collected and divided in two fractions. One was submitted to RNA extraction while another was in-oculated into YNB with 2% lactose and 6% ethanol. Samples were removed after 4 hours of culture for RNA extraction, which was done in triplicate using specific kits for RNA sequencing. The RNA sequencing reactions were performed in SOLiD 5500 XL. It was observed that 948 genes were differentially expressed (402 upregulated and 546 down-regulated). Functional categories genes such energy metabolism, protein synthesis, fatty acid metabolism and metabolism of purine, pyrimidine, nucleotides and nucleosides were mainly downregulated. Otherwise, in stress response and protein fate categories the most part of differentially expressed genes were upregulated. In conclusion, it could be observed that genes related to anabolism reactions were repressed while genes re-lated to protein degradation and stress response were activated. This is the first step to enhance the ethanol tolerance of the yeastK. marxinaus UFV-3 for industrial purposes.

Acknowledgements: FAPEMIG, CAPES, CNPq, Xunta de Galicia, and FEDER.

SIMB 2013 | 25

PRODUCTION AND PARTIAL PURIFICATION OF ENDOGLUCANASES IN ASPERGILLUS NIGER SP. GROWN IN

SUGAR CANE AND STRAW PAPER AS CARBON SOURCES

BIANCA LANA DE SOUSA; GABRIEL CABRAL BARROS; IZADORA LORRANY ALVES RABELO;GABRIELLA CHRISTINA GONALVES MANINI DE PAULA; PEDRO HENRIQUE SCARPELLI PEREIRA; JEFFERSON VKTOR DE PAULA

BARROS BATA; ISADORA OLIVEIRA PATRA; JOS HUMBERTO DE QUEIROZ.

Universidade Federal de Viosa, UFV Viosa, Minas Gerais, Brasil.

The lignocellulosic biomasses are the major sources of carbohydrates on the planet. Due to the depletion of fossil fuel sources, several research centers have been developing technologies for processing these biomass and bioethanol production. In this context, hydrolysis of cellulose is an essential step. Cellulases and hemicellulases enzymes have been suggested as the best alternative for this process. The objective of this work was to produce and partially purify endoglucanases of the fungus Aspergillus niger sp., using two inducing sources. The microrganism was cultured at 28 C for 8 days under shaking at 180 rpm in a culture medium containing paper or sugar cane straw and aliquots were removed at times of 72, 96, 120, 144, 168 and 192 hours for the evaluation of the produc-tion of endoglucanases. Enzyme assays were conducted using 2 % carboxymethylcellu-lose (m/v) by the method of 3,5-dinitro-salicylic acid reagent. After 144 h of incubation, the sugar cane straw induced higher values ??of enzyme activity compared to the paper substrate. In the first purification step was used (NH4)2SO4 40-80 % saturation. The 80 % precipitate was subjected to size exclusion chromatography using Sephadex G-25. The fractions of highest activity were selected to the "pool", which was submitted to ion ex-change chromatography. After elution of the sample on DEAE-Sephadex A-50, were observed two peaks of enzyme activity with low protein content, being called "pool" and 01 "pool" 02, indicating that the endoglucanases showed two isoforms.

Acknowledgements: CNPq, FAPEMIG e CAPES

SIMB 2013 | 26

MICROBIOLOGICAL QUALITY OF HONEYBEES (APIS MELIFERAS) SOLDIN THE STATE OF ALAGOAS, BRAZIL

ALVES,T.P;SANTOS,T.M.C;NETO,C.E.R;MONTALDO,Y.C;TENORIO,F.A;SILVA,S.G.M;GUEDES,E.L.F; SILVA,J.M.

Universidade Federal de Alagoas, CENTRO DE CINCIAS AGRRIAS. Campus Delza Gitai, BR 104 norte KM 85 - Mata do Rolo Rio Largo 57100000 - Rio Largo, AL - Brasil

The present work aimed to evaluate the quality of honey sold in the State of Alagoas. Were acquired 15 samples of Apis mellifera L. honey marketed in supermarkets, free trade, and cooperative located in the State of Alagoas, in the period from November to December 2012.All samples were conducted at the Laboratory of Microbiology at the Academic Unit Center for Agricultural Sciences, Universidade Federal deAlagoas, which were carried out to analyze microbiological and physical-chemical, to establish a standard microbiology and investigate possible tampering. Regarding the microbio-logical standard, 26.6% of samples showedcounts of mesophilic aerobic bacteria stand-ard. Valuesfound for yeasts and molds only samples (MC5, MM8and MM10) was ob-served contamination with the respective values2.2 x107, 3.4 x107 and 2.5 x107CFUg-1. PMN.g-1 at 35 C and coliforms at 45 C, we found that only two of the samples (13.3%),had results lower than 3.0 PMN.g-1. However in most of the samples86.7%was detected a high rate of contamination. The results of this study indicatedthe presence of sporulating bacteria in 13.3% of the samples, identified by smear slide and stained by the Gram method, both under aerobic as anaerobic. With the physical-chemical analysis it became clear that all samples had pH valuesranging between 2.3 and acids 4.4. Ana-lyzing enzymatic activity,two of the samples of honeys(13.3%),MC7 and MC10 were positive. The lugol reaction obtained positive results indicating the presence of starch and dextrin in three (20%) samples.Regarding theFiehe reaction, 73.3% of samples (11) had the salmon-colored cherry-red positive reaction to the test, indicating that they may have been subjected to conditions of overheating, stored in high temperature or suffered adding sugary syrups (CANO, 2005). In conclusion, noneof the honeys examined in this studywere within the parameters established by the legislation (BRAZIL, 2000).

Keywords: Characteristics of honey. Contamination. Physical-chemical. microbiological

SIMB 2013 | 27

CHARACTERIZATION OF BACTERIAL CONSORTIUM AND BACTERIAL ISOLATES WITH POTENTIAL FOR MANGANESE

BIOREMEDIATION

NATLIA ROCHA BARBOZA, SORAYA SANDER AMORIM, NAYARA THAIS BARBOSA SACRAMENTO, PRICILA ALMEIDA SANTOS, FLVIA DONRIA

REIS, MNICA MENDES CORDEIRO, RENATA GUERRA S, VERSIANE ALBIS LEO

Laboratrio De Bioqumica E Biologia Molecular, Universidade Federal De Ouro Preto Laboratrio De Biohidrometalurgia, Universidade Federal De Ouro Preto Faculdade Unileste

Manganese (Mn) is a common contaminant in wastewaters and drainages pro-duced by Brazilian mining operations, due to the solubilization of Mn containing min-erals.The concentration of Mn in these waters usually are not in accordance with the current Brazilian environmental regulations and removing the metal is notoriously difficult because the high stability of the Mn(II) ion in aqueous solutions. One way to remove the contaminant is by the use of microorganisms that can oxidize the Mn(II) ion. Although there are several studies on the biological removal of Mn(II), the identity of such organisms,the mechanism of the reactions and their benefits to microorgan-isms themselves are not well understood. In the present study, a bacterial consortium was obtained from Brazilian mine waters with a high Mn(II) level using a K medium. Small-scale bacth experiments with different Mn(II) concentrations in a 2 week-long period,showed that the bacterial consortium removed on average 80% Mn(II) from syn-thetic solutions. Preliminary analyses of this consortium by BOX-PCR showed that the amount of Mn present in the culture medium seems to modulate the microbial diversity. In addition, we have also selected 22 colonies from K agar medium contained Mn(II). A comparative analysis of Gram stained and carbohydrate fermentations tests allowed to identified preliminary some strains asPaenibacillus polymyxa,Serratia plymuthica, Tatumella ptyseos,Stenotroplomonas maltophiliaand Klebsiella sp. The isolated identified asP.polymyxaandT. ptyseoswere tested for their ability to grow in high Mn(II) con-centrations supporting up to 1200mg/L of Mn(II). Experiments carried out for testing Mn(II) removal with these two isolated showed that T. ptyseos removed 95% Mn(II) from a solution containing 50mg/L, whereasP. polymyxaremoved on overage 57%, in a week long experiment. Thus, the present study showed for the first time bothT. pty-seosandP. polymyxaability to remove Mn.Further studies are needed to understand the genetics involved in bacterial Mn(II) oxidation. The biotechnological potential of these isolated will also beinvestigated.

Financial support: CNPq, Vale, FAPEMIG, FINEP and UFOP

SIMB 2013 | 28

MORPHOLOGICAL ANALYSIS OF BACTERIOPHAGES ISOLATED FROM EXUDATE OF FROZEN CHICKENS

RAMOS, M.S.; CAL, E.C; BATALHA, L.S.; LOPEZ, M.E.S.; CARVALHO, M.M.; MENDONA, R.C.S.

Universidade Federal De Viosa - UFV - Brazil

Bacteriophages are one of the most abundant life forms in nature. They are ubiq-uitous viruses in the environment and are not harmful to humans and animals. They are obligate parasites and do not have their own metabolism, thus, they need a host to multiply. Bacteriophages are divided into 13 families, the most common being:Myoviri-dae, PodoviridadeandSiphoviridae. They have capsids (head) which can be icosahe-dral, cubic, filamentous shape or pleomorphic. Most bacteriophages belong to the order Caudovirales, with isometric head and tail.The aim of this study was to evaluate the morphology of bacteriophages isolated from exudate of frozen chickens. For the mor-phologic examination of a sample of 1 mL of suspension 1010PFU/mL was centrifuged at 9000 x g for 10 min. The supernatant was discarded and the pellet washed with a solu-tion of ammonium acetate 0,1 mol L-1and again centrifuged at 9000 x g for 10 min. The supernatant was discarded and the pellet resuspended in 1 mL of distilled and microfil-tered water.A volume of 8 microlitres of this suspension was deposited on the surface of a fabric of electron scanning microscopy, coated with Formvar (200 mesh, 3 mm) resin. The sample excess was removed with blotting paper and then added a drop of aqueous solution of uranyl acetate (2 % v/v) on the screen surface, leavingin contact for 15 sec. The excess of uranyl acetate was removed with blotting paper the screen rinsed with a drop of distilled water and then dried at room temperature, approximately 24 C for 24 h.It was later made the observation in a transmission electron microscope at 80 kV and increased 85000 times. From the results obtained, it can be observed by transmission electronic microscopy that the isolated bacteriophage can be classified as belonging to the order ofCaudoviralesand familyPodoviridae.

Acknowledgements: FAPEMIG

SIMB 2013 | 29

THE EFFECTS OF PHYTOHORMONE PRODUCING DIAZOTROPH INOCULATION ON THE ROOT OF MAIZE

SEEDLINGS

BIANCA BRAZ MATTOS; IVANILDO EVDIO MARRIEL; CHRISTIANE ABREU DE OLIVEIRA; RENATA RIBBAS; MICHELE CHRISTINA BASTOS LEAL;

VITRIA PALHARES RIBEIRO; ALINE GONALVES DA SILVA & TBATA CHRISTINA ABREU.

EMBRAPA Maize And Sorghum, Sete Lagoas, MG, Brasil. UNIFEMM, Sete Lagoas, MG, Brazil.

Biological nitrogen fixation (BNF) has emerged as an important tool for the devel-opment of grasses sustainable agriculture.Currently,it is known thatthe advantagesof usingmicrobial inoculants with diazotrophic bacteria are not explainedonly byBNF. The production of auxins by these organisms has been associated to stimulatory effects on plant growth and root development. Therefore, the aim of this study was to select indole acetic acid (IAA) producing diazotrophs and evaluate their effects in maize seed-lings roots. For this work, 93 diazotrophic strains were tested for IAA production. The IAA production ranged from 12 to 144 mg ml-1in culture medium supplemented with tryptophan (500 mg ml-1) and 9 to 55 mg ml- 1in culture medium without tryptophan. To evaluate the effects of bacterial inoculation in roots of maize seedlings, germinated seeds were inoculated withcontrastingstrains (3 of high IAA production and 3 of low IAA production)and grown under hydroponic controlled conditions. After 15 days of growth, samples were collected and analyzed. The parameters used for analysis were dry weight of roots and shoots and other root parameters (projected area, volume, length, diameter, apex length) measured by the WinRhizo program. The assessment of these parameters indicated that the strains capable of producing high concentrations of IAA induced a negative effect on root development and, consequently, seedling growth. Simi-lar results were found by other authors, where strains that produce IAA at concentra-tions above 40 mg ml-1showed suppressive effects on seedling growth of rice and wheat, under hydroponic conditions. From these results, we can conclude that IAA production increased with the availability of tryptophan and that the benefits of the inoculation with IAA producing diazotrophs are associated withphytohormone concentrationproduced bybacterialstrains,under hydroponic conditions.

Financial support: Embrapa Maize and Sorghum, CNPq and FAPEMIG

SIMB 2013 | 30

PHOSPHATE SOLUBILING BACTERIA: VIABILITY AND SURVIVAL IN DIFFERENT INOCULATION VEHICLES AND

STORAGE CONDITIONS

BIANCA BRAZ MATTOS, CHRISTIANE ABREU DE OLIVEIRA, ELIANA APARECIDA GOMES, VITRIA PALHARES RIBEIRO, EVELINE CRISTELLI

SOARES, IVANILDO EVDIO MARRIEL & SIMONE SANTOS

EMBRAPA MAIZE AND SORGHUM, SETE LAGOAS, MG, BRASIL UNIFFEMM, SETE LAGOAS, MG, BRASIL.

The use of phosphorus solubilizing microorganisms (PSM) associated with the natural rock fertilization has been shown to be a promising alternative for conventional phosphate fertilization. However, in order to have a high microorganism populations and longer survival time, it is necessary to combine physical, chemical and biological characteristics to produce bio inoculants commercially. Technological applications of biopolymers usually require improvements in their mechanical properties, in order to develop the most appropriate vehicle for product formulation. Hence this study aimed to evaluate the survival time of two PSM strains in different vehicles and storage condi-tions. Four vehicles, starch, carboxymethyl cellulose (CMC), coal and peat, were used to formulate bio inoculants of two PSM strains that belong to Embrapa Maize and Sor-ghum Collection of Multifunction Microorganisms (B32 and B70). The viability os PSM in the inoculants was assessed monthly by viable count for six months in two condi-tions os storage (4C and room temperature). The starch vehicle presented the high-est number of living bacteria, with values higher than 8.5 and 9.0 logcolony-forming unit(CFU) g substrate-1for B70 and B32, respectively, at room temperature. B70-based formulations, using CMC and starch vehicle, had a better performance (p

SIMB 2013 | 31

ISOLATION, IDENTIFICATION AND SCREENING OF THE MICROBIOTA FROM COTTONSEED MEAL AND PALM KERNEL

CAKETO PRODUCE HYDROLASES.

ALMEIDA, J. V. S., MENDES, S. F., SOUZA, I. F., VANZELA, A. P. F. C., SANTOS, A. S., PANTOJA, L. A.

Federal University of the Valleys Jequitinhonha and Mucuri - UFVJM, Institute of Science and Technology - ICT, Department of Pharmacy - DF, Department of Basic Sciences - DCB, Diamantina, Minas Gerais, Brazil.

Enzymes are important biocatalysts presenting several advantages when compared to the chemical ones. Microbial enzymes are well suited for industrial applications, and one of the current challenges to wise their use is to decrease the cost of production by utilizing alternative, cheaper, carbon-substrates. This study aimed to isolate microor-ganisms from cotton seed meal and palm kernel cake to evaluate their production of amylase, cellulase, lipase and xylanase. Samples of plant biomasses were homogenized with water, and aliquots of the filtrates were inoculated in selective medium. Micro-bial colonies were primarily classified as bacterial, yeast or filamentous fungal strains. Bacterial strains were kept for further studies. Micro and macroscopic morphologies of the filamentous fungi were analyzed, as well as their ability to degrade carboxy-me-thyl cellulose, birchwood xylan, starch, and soy oil. Enzyme activity indexes (IEA) were calculated for each strain by the ratio: substrate degradation zone/ colony diameter. A number of 42 strains were isolated. Among these, 20 bacterial and 9 filamentous fungal strains were obtained from cotton seed meal, while 2 bacterial and 11 fungal strains were isolated from palm kernel cake. No yeast strain was isolated. A range of enzyme activi-ties were produced by 17 fungal strains, whereas 4 produced an IEA above 2, which is indicative of a good producer. Three amylolytic strains produced IEAs of 2.42, 3.25, 2.08. The fungal strain with higher IEA belongs to the genusAspergillus,which is in agree-ment with the recognized industrial importance of a variety of Aspergillus species. One cellulolytic strain produced an IEA of 2.0. These results indicate that trials from natural substrates might end up with the discovery of strains which secrete hydrolytic activities to degrade substrates more than twice their own growth. It is concluded that screening the saprophytic microorganisms in vegetal biomasses may be a valuable tool to search new potential enzyme producers.

Acknowledgements: CNPq and FAPEMIG

SIMB 2013 | 32

DISTRIBUTION OF HEPARIN-BINDING PROTEINS IN LEISHMANIA CHAGASIPROMASTIGOTES ACESSED BY

TRANSMISSION ELECTRONIC MICROSCOPY

THAS VIANA FIALHO MARTINS1; THAS VIEIRA DE CARVALHO1; PRISCILA RAMOS VASCONCELOS1; BIANCA MEIRELLES MIRANDA1; CLUDIA

MIRANDA DE OLIVEIRA2; LEANDRO LICURSI DE OLIVEIRA1; EDUARDO DE ALMEIDA MARQUES DA SILVA1

1 Laboratrio de Imunoparasitologia e Glicobiologia, Departamento de Biologia Geral, UFV, Viosa, Brasil 2 Laboratrio de Infectologia Molecular Animal, Departamento de Bioqumica e Biologia Molecular, UFV, Viosa, Brasil

Visceral leishmaniasis is a fatalhuman disease caused by the intracellular proto-zoan parasite Leishmania infantum/chagasi. The uptake of Leishmaniapromastigotes by host cells is a process mediated by classics receptors that initiate phagocytosis. The search formolecules that are involved in the infection process of the parasite to the host cell is important to design strategies to disease control. Among these molecules is the Heparin Binding Protein (HBP), a lectin of a group of ubiquitous proteins, whose main characteristic is to bind to carbohydratespresent in glycoproteins or glycolipids. The presence of these molecules in Leishmania species is poorly studied. Therefore, in this work, the lectins distribution in L.chagasi was assessed. Initially, promastigotes forms of the parasite were grown in Graces medium supplemented with 10%SFB, pH 6,5 and disrupted by sonication, producing a parasite crude extract that was subjected to af-finity chromatography on Heparin-agarose and D-Salting columns in FPLC automated system. The protein was collected to perform analysis on polyacrylamide gel electropho-resis, revealing bands indicativeof HBP. Later, using polyclonal antibody antiHBPLc, we checked the localization of HBP in promastigote forms of the parasite performing immunolabelling test and transmission electronic microscopy. The results of this test showed a wide distribution of this protein in parasite surface and next to the kinetoplast. This study represent an important step to future studies related to the involvement this proteins in the process of Leishmania infection.

Financial support: FAPEMIG and CNPq are the funding agency for this research.

SIMB 2013 | 33

CHARACTERIZATION OF YEASTS WITHPOTENCIAL FOR MANGANESE BIOREMOVAL

AMORIM, S. S.(1), DUARTE, K. K. S.(1), BARBOSA, R. N.(1), LEO,V. A.(2), GUERRA-S, R.(1)

(1) Departamento de Cincias Biolgicas, Universidade Federal de Ouro Preto UFOP, Ouro Preto, Minas Gerais, Brasil(2) Departamento de Metalurgia, Universidade Federal de Ouro Preto UFOP, Ouro Preto, Minas Gerais, Brasil

Manganese (Mn) is a common contaminant of mine water. Due its high solubil-ity over a wide pH range, it is notoriously difficult to remove from contaminated wa-ters. One way of Mn removal is by using the manganese-oxidizing microorganisms. Although this process mediated by fungi and bacteria have been known since the last century, if yeast also participate in Mn removal are not well known yet. To investigate this hypothesis, yeasts were isolated from Brazilian mine waters with a high Mn(II) concentration. The isolates were characterized by their biochemical profile of carbohy-drates assimilation (Auxanogram) and fermentation (Zymogram). These results suggest that among eight yeast isolates, three were identified as Candida guilliermondii and five as Rhodotorula mucilaginosa.These species were confirmed by PCR, sequencing and phylogenetic analysis of ITS15.8SrRNA-ITS2 gene. These isolates were tested for their ability to grow in high Mn(II) concentrations supporting up to 1800mg/L of these ion. We also observed browning of the medium and/or darkening of the colony, suggest-ing the ability to catalyze the oxidation of Mn(II) ion, in addition to change in colony morphology. Small-scale batch assays for the Mn removal were performed using YPD medium containing 50mg/L of Mn(II) ion and the decay, was measured using atomic emission spectrometry with plasma source. C. guilliermondiiand R. mucilaginosawere able to remove an average of 100% and 60% of the Mn(II) in a one week-long period, re-spectively. We not observed an increase in pH medium during the assay, suggesting that biological Mn(II) removal detected is not affected by chemistry mechanisms. The yeast species isolated are known to participate in bioremediation of pollutants, and this study is the first report showing that yeasts can play an important role in the biogeochemical cycling of Mn.

Acknowledgements: UFOP, Vale, FAPEMIG, CAPES and FINEP

SIMB 2013 | 34

MANGANESE BIOLOGICAL OXIDATION AS AN ALTERNATIVE TREATMENT FOR INDUSTRIAL EFFLUENTS

PRICILA ALMEIDA SANTOS NATLIA ROCHA BARBOZA MNICA MENDES CORDEIRO VERSIANE ALBIS LEO AND RENATA GUERRA S

Laboratrio de Bioqumica e Biologia Molecular, Universidade Federal de Ouro Preto Laboratrio de Biohidrometalurgia, Universidade Federal de Ouro Preto Faculdade Unileste

Brazil isone of the largest producers of manganese (Mn) ore in the world, and Mi-nas Gerais state has the biggest reserves of this metal. The solubilization of minerals con-taining Mn produces a high concentration of Mn(II) in effluents and drainage in most mines. Usually, the strategy adopted is to remove chemical ion Mn(II) by the addition of white wash, however this procedure has high cost and low efficiency. Another way for Mn removal is by using bacteria that can oxidize Mn (II) which are more efficient than chemical processes. Therefore, the goal of this study wasto identify microorganisms capable of oxidizing the Mn(II) to Mn (IV). We haveisolated several bacteria from the mine water. Biochemical tests suggest the presenceof Corynebacterium sp,Klebsiella oxy-toca, Escherichia coli, Stenothrophomonas maltophiliaand Enterobacter cloaceae.Among these strains, four were able to remove Mn(II) and grow in high concentration of this ion. The ability to remove the Mn(II) ions fluctuated between 77.35% and 94.44%. Fur-thermore, K.oxytocawas more efficient than the other strains tested to remove Mn(II) ion. It was also observed that the removal of Mn(II) ion has a direct relationship with medium pH increase. These results shown for the first time the potentialfor bioremedia-tion of Mn(II) ion by K.oxytoca.

Keywords: Manganese; biorremediation; manganese oxidationFinancial support: CNPq, Vale, FAPEMIG, FINEP and UFOP

SIMB 2013 | 35

A NEW ESCOVOPSISSTRAIN ISOLATED FROM THE FUNGUS GARDEN OF A LOWER ATTINE ANT

MEIRELLES, L. A.1;SOLOMON, S. E.2;RODRIGUES, A.1

1.UNESP - So Paulo State University, Department of Biochemistry and Microbiology, Rio Claro, SP, Brazil. 2.Rice University, Department of Ecology and Evolutionary Biology, Houston, TX, USA.

The filamentous fungus Escovopsis is a specialized parasite of the fungus cultivated by attine ants. Although much is known about the wide genetic diversity of this genus, surprisingly, only two species have been formally described: Escovopsis weberi and E. aspergilloides (both associated with higher attine ants). In a previous study, we sur-veyed Escovopsis associated with higher and lower attine ants from several Brazilian biomes. Here we report a newEscovopsisstrain isolated from the fungus garden of Cy-phomyrmexsp. sampled in Florianpolis, Santa Catarina State, Brazil. Morphological and phylogenetic analyses using partial sequences of the transcription elongation factor 1-alfa(tef1-alfa) were carried out to determine the novel status of this strain. Sequenc-es belonging to closest relatives were queried in the GenBank database. After multiple alignments, the tef1-alfa data set was analyzed under the maximum likelihood algo-rithm in RAxML. Our strain showed only 95% similarity with its closest relative, an undescribed species isolated fromCyphomyrmex faunulus (accession # AY172626) and formed a distinct branch within an Escovopsis clade associated with the lower attine ant generaCyphomyrmex and Myrmicocrypta. Morphological characteristics examined in potato-dextrose agar (at 25 C) differ from previously described species: the new strain has pink-colored colony, non-vesiculated conidiophores and ampuliform conidioge-nous cells producing solitary conidia. Chlamydospore-like cells were also observed after 7 days of incubation. The new Escovopsis strain is an indicative that several putative new species of the parasite associated with lower attine ants await discovery.

Acknowledgements: FAPESP and CNPq

SIMB 2013 | 36

COFFEE CROP MANAGEMENT: EFFECT ON THE DYNAMICS OF THE ARBUSCULAR MYCORRHIZAL FUNGI

PRATES JNIOR, P1; MOREIRA, B. C1; FERNANDES, R. B. A1; KASUYA, M. C. M1 MENDONA, E. S2

1. Universidade Federal De Viosa (UFV), Viosa, Minas Gerais, Brazil. 2. Universidade Federal Do Esprito Santo (UFES), Alegre, Esprito Santo, Brazil.

The Agroecology aims mainly to reduce the impact ofcropping systems, the eco-nomic dependence of agricultural to the financialsector, beyond enhance ecosystem ser-vices, by better nutrient management andincrease the use of biological potential. The ar-buscular mycorrhizal fungi (AMF)form a mutualistic symbiosis with the roots of plants and plays an important rolein enhancing plant nutrient use efficiency, plant health and soil quality.The aim of this study was to evaluate the managementpractices and season-ality on mycorrhizal colonization and abundance of sporesin a forest fragment, agro-forestry (coffee, banana and inga) and coffee monoculturesystem in Araponga, Minas Gerais, Brazil. Soil samples were collected in threesites for each area under the canopy projection, at 0-20 cm depth, root systemwere collected for assessing mycorrhizal. AMF spores were extracted from a 100cm3of each soil sample using the wet-sieving tech-nique, centrifugationin water and 50% sucrose solution and quantified. The roots were bleached inKOH 10%, washed in water, immersed in HCl 1% and staining in 0.05% trypan bluein lactoglycerol. Root colonization was quantified by using thegridline-in-tersect method. The forest fragment exhibited temporal stability forspores number and mycorrhizal colonization, possibly associated with a greaterdiversity of plant species and minor disturbances. In the coffee systems therewere temporal variation in mycorrhizal colonization and spores numbers, which shouldbe related to the phenological state of the plant that can be able to modulatethe AMF community. During the grain filling oc-curred decrease in mycorrhizalcolonization and increase in sporulation, in both crop systems. In the harvestperiod there was a tendency to increase colonization, without changes in thenumber of spores. For greater understanding of the dynamics and com-munitycomposition FMA additional tests will be performed based on molecular biolo-gyand species composition.

Acknowledgements: The CTA-ZM and the farmers who participated in this research. Thanks also to CAPES Brazilian Research Agency.

SIMB 2013 | 37

ASSESSMENT OF ARGINASE AND UREASE ENZYMES IN RHIZOSPHERE OF MAIZE

PLANTS (ZEA MAYSL.) INOCULATED WITH AZOSPIRILLUMDIAZOTROPHIC BACTERIA

FONSECA, L.M.F.; REIS, D.P.; GUIEIRO, C.S.M.; RIBAS, R.N.R.; MATTOS, B.B.; OLIVEIRA,C..A.; MARRIEL, I.E.

CNPMS - Embrapa Milho E Sorgo (MG 424, Km 65, Zona Rural, Sete Lagoas, Minas Gerais - MG), 2 UFSJ / CSL - Universidade Federal De So Joo Del-Rei ( Rodovia MG 424 Km 47, Zona Rural, Sete Lagoas - MG)

Enzyme activities can play a role as a sensitive bioindicator for detecting changes due to soil use and management. The rhizosphere environment stimulates the growth and the activity of microorganisms in the soil, while arginase activity represents the microbial community metabolically active. This reflects the nitrogen fraction potentially available to plants, while the urease activity becomes cummulative in the soil. This study aimed to evaluate the impact ofAzospirillum sp.inoculation to under the nitrogen (N) dynamics in the rhizosphere of maize plants by determining the activity of these two enzymes. The experiment was carried out by 28 treatments consisted of sixAzospirillum spstrains (E0, without inoculation, E1; E2; E3; E4; E5 and E6) under four nitrogen lev-els (0; 40; 80; 160 kg.ha-1of N), as urea font, applied to soil surface . The experimental design adopted was a Randomized Blocks Design (RBD) with treatments in a split plot arrangement with four replications. The plot was the Nitrogen levels, and the subplot each kind of inoculation. The arginase and urease activities were estimated by the own hydrolysis rate, respectively. As results, there was no significant correlation among the enzymes activity analyzed. Similarly, no significant difference (P>0.05) for the inter-action between inoculants and Nitrogen levels was detected. However, analysis indi-cated that the urease activity increased on rhizospheric soil according to inoculation in three of the seven tested strains independently of the nitrogen levels. Finally, the results showed that the inoculation impact on the microbes activity in the rhizospheric soil of maize plants depends of the strain adopted.

SIMB 2013 | 38

RNA INTERFERENCE: A SILENCING MECHANISM FOR THE EVALUATION OF GENE FUNCTION INCOLLETOTRICHUM

LINDEMUTHIANUM

NOGUEIRA, GB; SANTOS, LV; MENICUCCI, RP; BAZZOLLI, DMS; ARAJO, EF; QUEIROZ, MV.

Federal University Of Viosa - Department Of Microbiology, Viosa, MG - Brazil

The fungusColletotrichum lindemuthianum, which is the causal agent of anthrac-nose, is one of the most frequently observed pathogens and is responsible for significant damage to common bean crops. Understanding the genetic and physiological mecha-nisms involved in the plant/pathogen interaction may be useful in devising new and efficient methods for controlling this disease because the use of resistant cultivars has limitations arising from the high genetic variability present in this fungus. In the present study, we demonstrated, for the first time, the occurrence of RNA interference-mediated gene silencing inC. lindemuthianumby means of genetic transformation with a pSilent-Dual1-egfpvector. This vector is responsible for producing double-stranded RNA (dsR-NA) from the target gene through transcription from two strong constitutive promoters fromAspergillus nidulans(PtrpC and Pgpd) in opposite orientations. Furthermore, we demonstrated the potential applicability of this method as a molecular tool for the func-tional characterization of genes expressed during the interaction between a pathogen and its host. To this end, we built a vector named pSilentDual1-slnCl, which silences a gene encoding a histidine kinase (slnCl) that belongs to a two-component system pre-sent in fungi. In addition to demonstrating that this gene is essential for pathogenicity, this work also implicated this gene in the adaptive response to osmotic stress.

Financial support: FAPEMIG

SIMB 2013 | 39

THE CHAPERONE HFQ IS IMPORTANT FOR BIOFILM FORMATION IN ACTINOBACILLUS

PLEUROPNEUMONIAE SEROTYPE 1, 8 AND 15

(1) JOSICELLI SOUZA CRISPIM, (1) MONALESSA FBIA PEREIRA, (1) CIRO CSAR ROSSI, (1) ELZA FERNANDES DE ARAJO, (1) MARISA VIEIRA DE

QUEIROZ, (2) YANWEN LI, (2) JANINE T. BOSS, (2) PAUL R. LANGFORD, (1) DENISE MARA SOARES BAZZOLLI

1 Laboratory of Microbial Molecular Genetics, Department of Microbiology, BIOAGRO and Nucleus of Microscopy and Microanalysis, UFV, Viosa, MG, Brazil. 2 Section of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom

Actinobacillus pleuropneumonie is a Gram-negative coccobacillus, facultative an-aerobic, belonging to the Pasteurellaceae family and is the main causative agent of swine pleuropneumonia. Currently there are 15 recognized serotypes of this bacterium spread worldwide and the serotype 8 is the most widely distributed in the farms in southeastern Brazil. The adherence in the form of biofilm is an important determinant of virulence of this bacterial pathogen. In many pathogenic bacteria such as Francisella novicida, Neis-seria meningitides and even a serotype 1 reference strain of A. pleuropneumoniae, the chaperone Hfq is shown to have a critical role in virulence. Hfq acts mainly together with small regulatory RNAs, playing regulatory roles by base pairing with mRNA targets to attenuate or stop their translation.This study aimed to determine the involvement of the Hfq on the ability of biofilm formation by different serotypes of A. pleuropneu-moniae. To this purpose, wild type (WT), hfq mutant and hfq mutant complemented strains for serotypes 1, 8 and 15 were used. The mutant strains were obtained by natural transformation and gene replacement. The WT, hfq mutant and hfq mutant comple-mented strains were analyzed by biofilm formation assay on polystyrene microplates stained with crystal violet and scanning microscopy after bacterial growth on stainless steel coupons immersed in broth. Data from both trials allowed us to observe that all the wild type strains investigated are able to form more biofilm than mutant strains, result clearly displayed in the clinical isolate MIDG_2331 serotype 8 that has a higher ability to form biofilm than reference isolates Shope4074 sorotype 1 and HS143 sorotype 15. Our results confirm the great importance of Hfq as a virulence determinant in A. pleuropneumoniae and indicate the need for deeper investigations on other features, as different serotypes react differently to the lack of the protein, which are our object of further studies by our group.

Financial support: Brazilian Agencies: CNPq, CAPES, FAPEMIG and FINEP. United King-dom Agency: BBSRC.

SIMB 2013 | 40

PHENOTYPIC CHARACTERIZATION AND IDENTIFICATION OF A YEAST STRAIN ISOLATED FROM THE BOVINE RUMEN

ELSA FERNANDES DA SILVA; CLUDIA BRAGA PEREIRA BENTO; ANALICE CLUDIA AZEVEDO; DBORAH ROMASKEVIS GOMES LOPES; SOFIA MAGALHES MOREIRA; ISABELA MARIA FERNANDES DE OLIVEIRA; WENDEL BATISTA DA SILVEIRA; HILRIO CUQUETTO MANTOVANI

Laboratrio de Microbiologia de Anaerbios, Departamento de Microbiologia, Universidade Federal de Viosa - UFV Av. Ph Rolfs, s/n, CEP:36.570-900,Brasil

Yeasts have high metabolic diversity and can be found in soil, foods and in the gastrointestinal tract of animals. The use of yeast as a feed additive has been suggested to manipulate ruminal fermentation, with potential beneficial effects on ruminant perfor-mance. This work aimed to characterize a yeast strain obtained from the rumen of cross-bred Holstein cattle (dairy cows). Batch cultures were performed in YPD medium con-taining different mono and disaccharides at a final concentration of 4 g.l-1. Yeast growth was tested in a temperature range of 10 C to 60 C. The yeast strain was grown in NaCl concentrations ranging from 0 to 10 % and ethanol concentrations ranging from 0 to 16 %. Identification of the yeast strain was carried out at CBS-KNAW Fungal Biodiversity Center,Utrecht, Netherlands. Total DNA was extracted from over night cultures and the regions D1 and D2 that refer to the larger subunit of the gene coding the 26S rRNA was amplified using primers LR0R and LR5A. Out of 14 sources of carbohydrates tested, the yeast was able to ferment only glucose, mannose and fructose. Concentrations >2.0 % ethanol and>1.5 % NaCl reduced yeast growth in YPD medium, but complete inhibi-tion was only achieved when 7.5 % ethanol and 6.0 % NaCl was added to the growth medium. The yeast strain (named EF-1) was able to grow at temperatures ranging from 20 C to 40 C, but greater biomass production (O.D. 600 nm = 6.82) was observed at 20 C. Sequencing of the 26SrRNA indicated that the yeast isolated from bovine rumen was Pichia kudriavzevii. Because the yeast population was low in the rumen and the range of substrates used for growth was very limited, it appears that P. kudriavzevii EF-1 was not autochthonous to the rumen ecosystem.

Acknowledgements: CNPq, CAPES, FAPEMIG

SIMB 2013 | 41

RESEARCH OF ANTIVIRAL ACTIVITY OF EXTRACTS FROM ARISTOLOCHIA CYMBIFERAAGAINSTDENGUE

VIRUS2

SAMYRA GIAROLA CECILIO; ANTNIO HELVCIO TTOLA; CINTIA LOPES DE BRITO MAGALHES; JAQUELINE MARIA SIQUEIRA FERREIRA; JOS

CARLOS DE MAGALHES

Laboratrio de Microbiologia Geral e Enzimologia e Laboratrio de Bioqumica e Imunologia Celular e Molecular, Departamento de Qumica, Biotecnologia e Engenharia de Bioprocessos (DQBIO/UFSJ), Ouro Branco, MG., Brasil.

Dengue, the most important arthropod borne viral disease in Brazil, caused by four serotypes of Dengue virus, is transmitted to humans by vectors from genus Aedes. In last decades, dengue was intensified with severe signs of expansion and adaptation of these vectors. There is no vaccine or therapy available yet. The scientific community aspires to the discovery of antiviral drugs against the virus, which can be obtained through re-search on medicinal plants. Aristolochia spp. (Aristolochiaceaefamily) is a well-known genre used in folk medicine, commonly applied in the treatment of diarrhea and ab-dominal pain. Phytochemical studies revealed the presence of alkaloids, flavonoids and aristolochic acids, which may be promising compounds in antiviral activity. Aer-ial parts (stem /wood) were macerated with ethanol, dichloromethane and hexane for three weeks. Ethanol and hexane were removed on a rotary evaporator (35-40C/60-110 mmHg) and dichloromethane. Mosquitoes cells (C6/36) were used for multiplication and titration of the virus and mammalian cells (VERO) for the search of cytotoxiccon-centration50 (CC50) and effective concentration 50 (EC50) of the extracts by methyl-thiazol-tetrazolium (MTT) colorimetrics method. As a result, the less toxic extract was the ethanolic (CC50 = 59.09 2.49 g /mL) followed by hexanic (CC50 = 55.27 4.93 g/mL) and dichloromethanic (CC50 = 54.10 10.62 g/mL). The extracts showed antiviral activity against 500 TCID 50 of DENV-2, with EC50values of 9.10 0.57 g /mL for ethanolic, 3.32 1.47 g/ml for dichloromethanic and 10.05 2.21 g/mL for hexni-co extract. The Selectivity Indexes (CC50/EC50) were 6.49, 16.26 and 5.50, respectively. The dichloromethanic extract was found to be the most effective, with lower EC50 and higher Seletivity Index. Chromatographic studies may not only elucidate the bioactive (s) component (s) of the plant as well as allow the continuity research of antiviral actions mechanisms.

Financial support: Fapemig, CAPES e PET

SIMB 2013 | 42

PECTINASE AND CMCASE PRODUCTION BYFILAMENTOUS FUNGI UNDER SOLID STATE FERMENTATION USING

AGRICULTURAL WASTES

ROBERT O. GUSMO; INGRI A. L. TEXEIRA; LUTHIANE M. FERRAZ; ANGRA P. B. RGO; MATHEUS F. ANDRADE; PATRCIA L. LEAL

Federal University of Bahia , Campus Ansio Teixeira, CEP:45.029-094, Vitria da Conquista, Bahia, Brazil

Commercial applications at a low cost production of new enzymes with desirable biochemical and physicochemical characteristics have been the focus of several re-searchers. Use of agro-industrial wastes as carbon sources aiming enzyme production through solid-state fermentation (SSF) process reduces costs and helps solve problems regarding its disposal. Moreover the SSF can be considered an advantageous methodol-ogy for growing microorganisms such as filamentous fungi, since it simulates the habitat of such microorganisms. The objective of this study was to quantify the enzymatic activ-ity of CMCase (endoglucanase) and pectinase produced under SSF, using fungi isolated from semi-arid of Bahia e coffee huskas substrate. Solid-state fermentation was carried out using a 500 ml Erlenmeyer flask containing 100g of sterilized substrate inoculated with strains filamentous fungi (Aspergillussp. LEMI 1, Aspergillus sp. LEMI 2 and As-pergillus sp. LEMI 3) by deposition of fungal mycelium in agar discs. After inoculation, nutrient solution was added to each flask. The substrates were mixed in proportions of 50%. The cultivation was carried out at 30C for 20 days. At 36h intervals, the solid fermented material corresponding to one Erlenmeyer flask was mixed with 40 ml dis-tilled water, stirred, filtered under vacuum and centrifuged. The supernatant was used as crude enzyme solution for enzyme activity measurements. All strains showed a peak of production for the CMCase at first day of fermentation, while for pectinase the maxi-mum activity occurred 20 days after fermentation with an enzymatic production of 4,78; 6,19; 5,55 U.g-1 (Aspergillus sp. LEMI 1, Aspergillus sp. LEMI 2 and Aspergillus sp. LEMI 3, respectively). The data presented herein suggests a potential application of coffee huskfor production of CMCase and pectinase byfilamentous fungi strains. However, the adequacy of fermentation conditions should be better studied and adjusted.

Financial support: Capes, Fapesb, CNPq and UFBA

SIMB 2013 | 43

AVICELASE PRODUCTION BY ASPERGILLUS SPP. UNDER SOLID STATE FERMENTATION

ROBERT O. GUSMO; INGRID A. L. TEIXEIRA; LUTHIANE M. FERRAZ; ANGRA P. B. RGO; CARINE N. RAMOS; PATRCIA LOPES LEAL

Federal University of Bahia , Campus Ansio Teixeira, CEP:45.029-094, Vitria da Conquista, Bahia, Brazil

The biomass originated from industrial residues, such as coffeehusk, are abundant carbon sources that can be used as a substrate in solid state fermentation (SSF) process-es. Filamentous fungi are important producers of cellulolyticenzyme (exoglucanases, endoglucanases and -glucosidases). These enzymes are highly specific biocatalysts that act in synergy during the degradation of cellulose fibers to soluble sugars, especially glu-cose. The trading profit of these enzymes for the future of energy agribusiness, mainly in terms of ethanol production from biomass, essentially depends on the economic pro-duction. Thus, the selection of the microorganisms with high enzyme synthesis ability is required, as well as the application of an economically feasible production process. The objective of this study was to quantify the enzymatic activity of avicelase produced under SSF, using fungi isolates from the semi-arid region of the Bahia Stateand cof-feehuskas substrate. Solid-state fermentation was carried out using a 500 ml Erlenmey-er flask containing 100g of sterilized substrate inoculated with three strains filamentous fungi (Aspergillus sp. LEMI 1, Aspergillus sp. LEMI 2 and Aspergillus sp. LEMI 3) by deposition of fungal mycelium in agar discs. After inoculation, nutrient solution was added to each flask. The substrates were mixed in proportions of 50%. The cultivation was carried out at 30 C for 18 days. At 36 h intervals, the solid fermented material cor-responding to one Erlenmeyer flask was mixed with 40 ml distilled water, stirred, filtered under vacuum and centrifuged. The supernatant was used as crude enzyme solution for enzyme activity measurements. All fungi strains showed a capacity for production of the avicelase at all times of fermentation. A peak of activity occurred on the16 days of fermentation for Aspergillus sp. LEMI 1 (2,13 UI g-1) and at 20 days, for Aspergillus sp. LEMI 2 (2,08 UI g-1) and Aspergillus sp. LEMI 3 (1,7 UI g-1) . The data presented herein suggests a potential application of strains fungal tested for avicelase production by SSF using coffeehuskas substrate

Financial support: Capes, Fapesb, CNPq and UFBA

SIMB 2013 | 44

EXPRESSION PROFILE OF NUCLEOID-ASSOCIATED PROTEINS (NAPS) INACTINOBACILLUS PLEUROPNEUMONIAE

OLIVEIRA, L.V.N.*; SILVA, A.K.S.; QUEIROZ, M.V.; NASCIMENTO, A.G.; VANETTI, M.C.D.; BAZZOLLI, D.M.S.

Laboratory Of Microbial Molecular Genetics, Department Of Microbiology, UFV, Viosa, Minas Gerais, Brazil. *Actual Address: Laboratory Of Mycology, Department Of Microbiology, UFMG, Belo Horizonte, Minas Gerais, Brazil.

Actinobacillus pleuropneumoniaeis the causative agent of swine pleuropneumonia, responsible for great economic losses in pig production worldwide.Nucleoid-associated proteins (NAPs) are DNA binding proteins, capable of influencing transcription on a global scale. Many NAPs such as H-NS, IHF, Fis, Lrp and HU are essential in the regu-lation of hundreds of genes, including genes of virulence and stress response.Little is described about the functionality of these proteins inA. pleuropneumoniae. In this con-text, the aims of this study wereto identify and analyze the expression of these proteins inA.pleuropneumoniae. The presence of NAPs H-NS, IHF, Fis, Lrp and HU in the ge-nome ofA. pleuropneumoniaewas analyzed by PCR. Real time quantitative PCR (qRT-PCR) was used to determine the expression of genes ihfA, ihfBandfis. As result, the genesihfA, ihfB, fis, lrp, hns andhupAwere identified in the 15 serotypes reference strains and in 20 clinical isolates ofA. pleuropneumoniae,and distinct groups based on specific species of members of thePasteurellaceaefamily were verified. Furtherin silicoanalyzes indicated that numerous genes in the genome ofA. pleuropneumoniaeserotype 3 (JL03) are supposedly regulated by IHF and Fis, due to the presence of 184 and 68 binding motifs, respectively.Our data from qRT-PCR revealed thatfis, ihfAandihfBgenes are constitutively expressed, but there was an overexpression offisgene during exponential phase, while ihfA and ihfB genes were slightly overexpressed on stationary phase for both serotype 1 (ATCC 27088) and clinical isolate 010. According to the results obtained inA.pleuropneumoniaeas like others pathogenic bacteria, the Fis and IHF proteins can to regulate several genes in a growth dependent manner.

Financial support: FAPEMIG, CNPq and CAPES.

SIMB 2013 | 45

FILAMENTOUS FUNGI PRODUCTION OF AMILASE AND XYLANASE UNDER SOLID-STATE FERMENTATION

ROBERT O. GUSMO; INGRID A. L. TEIXEIRA; LUTHIANE M. FERRAZ; ANDRESSA P. CORDEIRO; PATRCIA L. LEAL

Federal University of Bahia , Campus Ansio Teixeira, CEP:45.029-094, Vitria da Conquista, Bahia, Brazil.

The southwest region of the Bahia is considered the fourth largest producer of coffee in Brazil. Coffeeruskis a major waste generated by this crop and many researches show the usefulness of this residue as a carbon source for microorganims cultivated in solid-state fermentation (SSF) process to produce value-added products such as enzymes. The SSF process offers numerous advantages including high volumetric productivity, rela-tivelyhigher concentration of the products and less effluent generation. The objective of this study was to quantify the enzymatic activity of amylase and xylanase produced way of the fermentation in solid state, using fungi isolates from semi-arid region of the Bahia State andcoffee huskas substrate. Solid-state fermentation was carried out using a 500 ml Erlenmeyer flask containing 100g of sterilized substrate inoculated with three strains filamentous fungi (Aspergillus sp. LEMI 1, Aspergillus sp. LEMI 2 and Aspergillus sp. LEMI 3) by deposition of fungal mycelium in agar discs. After inoculation, nutrient solution was added to each flask. The substrates were mixed in proportions of 50%. The cultivation was carried out at 30 C for 18 days. At 36 h intervals, the solid fermented material corresponding to one Erlenmeyer flask was mixed with 40 ml distilled water, stirred, filtered under vacuum and centrifuged. The supernatant was used as crude en-zyme solution for enzyme activity measurements. All strains of fungi showed a capacity for production of amylase at all times of fermentation. The maximum activity occurred after 10 days of fermentation with Aspergillus sp. LEMI 3 (10,3 UI g-1). Moreover, not all the strains were able to produce xylanase. The peak of production for this enzyme occurred in the first day by Aspergillus sp. LEMI 3 (0,42 UI g-1). The data presented herein suggests a potential application of husk coffee for production of amylase, espe-cially. The Aspergillus sp. LEMI 3 was the best strain tested in the study for