ANNALS

Abstracts

Editors
Hilrio Cuquetto Mantovani
Gilberto de Oliveira Mendes
Conrado Augusto Vieira
Mariana Caroline Tocantins Alvim
Lvia Tavares Colombo
der Galinari Ferreira

Viosa, MG-Brazil
2013

Graphic design and layout: Gilberto Mendes

Cover design: Conrado Vieira and Gilberto Mendes
Back 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

I61a
2013

International Symposium on Microbiology and

Biotechnology (2 : 2013 : Viosa, MG)
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

Organization
Graduate Program in Agricultural Microbiology
Department of Microbiology
Universidade Federal de Viosa
December 4-7, 2013
Viosa, MG-Brazil

Acknowledgments
Faculty
To 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 Mantovani
President
Prof. Maria Cristina Dantas Vanetti
Vice-President
Prof. Maria Catarina Megumi Kasuya
1st Treasurer
Prof. Miriam Teresinha dos Santos
2nd Treasurer
Prof. Wendel Batista da Silveira
1st Secretary
Scientific Committee
Prof. Antnio Galvo do Nascimento
Prof. Clia Alencar de Moraes
Prof. Cynthia Cando da Silva
Prof. Denise Mara Soares Bazzolli
Prof. Flvia Maria Lopes Passos
Prof. Luciano Gomes Fietto
Prof. Hilrio Cuquetto Mantovani
Prof. Marcos Rogrio Ttola
Prof. Maria Catarina Megumi Kasuya
Prof. Maria Cristina Dantas Vanetti
Prof. Marisa Vieira de Queiroz
Prof. Maurcio Dutra Costa
Prof. Miriam Teresinha dos Santos
Prof. Olinto Liparini Pereira
Prof. Poliane Alfenas Zerbini
Prof. Srgio Oliveira de Paula
Prof. Wendel Batista da Silveira
Logistics Committee
Prof. Wendel Batista Silveira
Prof. Maria Catarina Mugumi Kasuya
Prof. Cynthia Cando da Silva
Tiago de Souza Leite
Hugo Leonardo Andr Genir
Wemerson de Castro Oliveira
Josenilda Carlos dos Santos

Sponsorship / Disclosure Committee

Prof. Hilrio C. Mantovani
Conrado Augusto Vieira
Lvia Tavares Colombo
Mariana C. Tocantins Alvim
Gilberto de Oliveira Mendes
Committee for Logistics of Speakers
Prof. Marisa Vieira Queiroz
Prof. Antnio Galvo do Nascimento
Prof. Flvia Maria Lopes Passos
Robson de Assis Souza
Caroline Moura
Marcela Camacho
Sergio A. Diaz
Committee for the Event
Social Activities
Prof. Mriam T. Santos
Prof. Poliane Alfenas Zerbini
Prof. Denise M. S. Bazzoli
Prof. Maria Cristina Dantas Vanetti
Fernando Augusto da Silveira
Josicelli Souza Crispim
Pricila Palla Costa
Secretariat Comission
der Galinari Ferreira
Deborah Romaskevis G. Lopes
Taides Tavares dos Santos
Fbia Giovana V. de Assis
Cludia Vieira Prudncio
Cleriane Andr
Elsa Fernandes da Silva
Renato 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. Following 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 Symposium aimed to bring together internationally renowned experts to deliver formal presentations 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 Scientific Committee of the Conference were scheduled for presentation in poster sessions
during the Symposium. The representative role of microorganisms in industry, agriculture 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

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
Departamento 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.
1

The extraction of petroleum generates large amounts of oil and saline wastewater (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 evaluate the ammonium removal rate of nitrifying sludge acclimated to salt when subjected 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 | 8

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 pressthe 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 increased 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 manufacturing 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 Sciences, 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 material and wood, meaning inattention during manipulation of the raw material. The
maximum allow limits for thermotolerant coliform, and total mesophilic bacteria
are respectively: 5x102, <3x10 and 8x104. The results characterize the samples as unfit
for consumption, being outside of the minimum standards required by law, and may
thus be contamination for consumers.
Key-words: Mesophilic microorganisms; Pasteurized milk; Total and thermotolerant coliforms

SIMB 2013 | 9

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 economic losses as well as the presence of microorganisms responsible for intoxications. 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 Salmonella detection, 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 numbers of total and thermotolerantcoliforms ranged from 1.1to 2.9x102x103and1 to
2.9x103 NMP mL-1, respectively. Significant differences were detected (P<0.05) for
thermotolerant coliform in the fourth session that had the lowest mean. Sixty colonies were isolated and 56.7% of the cultures had cells that were rod-shaped and
stainedGram-negative intriple sugar iron agar (TSI), which are morphotinctorial
and biochemical evidenceconsistent withSalmonella. These results show an indication of impairment in the cleaning of equipment, serious flaws in handling, personal
hygiene of food handlers and asepsis or antisepsis cows.
Key-words: Microorganisms;Salmonella;Total and thermotolerantcoliforms

SIMB 2013 | 10

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 implicated 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 Technologie 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 | 11

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 animal products for correct handling and slaughter, to reduce the risk of outbreaks of diseases 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 coliforms 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 marketed 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 | 12

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 pleuropneumonia, 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 prevalent in southeastern Brazil. Biofilm formation contributes to virulence in many Gramnegative 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 pleuropneumonia 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<0.01) followed by Dunnet test to compare the treatments with the control
(p<0.05) using the R 3.0.2 software. The results showed that 71% of the field isolates
display potential for biofilm formation, and this potential differs significantly between
serotypes. Among field isolates of serotype 2, 87.5% form biofilm, whereas in serotype 7,
41.3% present this capacity. The isolates of serotype 8 showed a profile of biofilm formation statistically superior to the others, and 95.8% of the isolates form biofilm. Our results show that the biofilm formation is a prevalent phenotype among field isolates ofA.
pleuropneumoniaeand is particularly strong for serotype 8, which not only contributes
to the colonization and pathogenesis, but may have also played an important role in its
spreading throughout the region analyzed.
Financial support: FAPEMIG, CNPq and CAPES.

SIMB 2013 | 13

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 immunostimulating 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 | 14

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 theEnterobacteriaceaefamily, includingKlebsiella spp.,Enterobacter spp.,Citrobacterspp. andEscherichia 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 andEscherichiacoli were estimated by Petrifilm quick count method (3M Company) following fabricant recommendations. The count of total coliforms ranged from 3.47 x 103to
7.9 x 106CFU / g and 90% of the samples presented populations greater than 103CFU
/ g. The population ofEscherichia coliranged from <10 to 103UFC / g and40% of the
samples were above the limit allowed by Brazilian law (102CFU / g). The high population
of Escherichia coli demonstrates that the vegetables were grown and marketed under
inadequate sanitary conditions, providing food insecurity for consumers. Thus,there is
a need for preventive and corrective actions by the Health Surveillance to provide improved hygienic quality of that food.
Acknowledgements: CAPES

SIMB 2013 | 15

SOURCES OFESCHERICHIA 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 ofE. coliusing rapid counting method Petrifilm (3M Company) usingPetrifilm EC plates as recommended by the manufacturer. For analysis of the drinkingwater
used in the chicken raising were counted for total coliforms andE. coliin 100mL of the
sample using the quick method chromogenic Readycult .E. coliwas 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 acontinuous 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 | 16

EXOPOLYSACCHARIDES PRODUCTION BY DIAZOTROPHIC

BACTERIA ASSOCIATED WITH SISAL(AGAVE
SISALANAPERRINE)
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 growing conditions. Eleven diazotrophicbacteria isolated from the rhizosphere and tissues
of Agavesisalanawere selected. The production capacity of EPS was verified bytransferring 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 measuring 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 | 17

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 material 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 feeding, 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 establishments coming from the city of Goinia and its metropolitan region and also of drinking straws provided for use in industrialized beverages for consumption. Microbiological analyzes were performed as counts of mesophilic count, total coliforms,Salmonella
sppand 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 andSalmonella 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 ofRhodotorula
spp, FusariumandAspergillus fumigatus. Became evident, therefore, poor sanitary conditions in this utensil and possible carelessness of the entities involved in the manufacturing and quality control, as well as the inspection by the competent authorities.
Acknowledgements: We thank ours families for the comprehension and "Preciso Metrologia e Qualidade" and its employees for their support during the development of this work.

SIMB 2013 | 18

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 geographical regions, serotypes and time spam. The aim of this study was to determine the
antimicrobial susceptibility of 70 A. 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. pleuropneumoniae. 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 determinantssulII,strA,strB,tetB,tetH,tetK,tetO,blaROB1orfloRwere found in 28 isolates.Our results suggest the importance of continued monitoring ofA. pleuropneumoniaeclinical 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 | 19

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 aerobic, 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 asLactobacillus 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 fermentation. 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 fermentation 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 similar 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-1of forage.
Financial support: Capes, Fapemig and CNPq

SIMB 2013 | 20

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, especially 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 treatments consisted of two inoculating rates (5 and 6 log ufc g-1) for each strain of LAB identified asLactobacillus 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 | 21

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 pathogenSclerotium rolfisiipolyphagous 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 hyperparasite. This study aimed to evaluate the antagonistic potential of fiveTrichodermastrains
againstS.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 hooklike 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 fiveTrichodermastrains tested have potential as antagonists
againstS.rolfisii.

SIMB 2013 | 22

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 containing 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. fluorecens.
Acknowledgements: FAPEMIG

SIMB 2013 | 23

GENE EXPRESSION RESPONSE OFKLUYVEROMYCES

MARXIANUSUFV-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
Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viosa, Viosa,
Minas Gerais, Brazil2Departamento de Bioloxa Celular e Molecular, Facultade de
Ciencias, Universidade da Corua, A Corua, Galicia, Spain
1

Kluyveromyces marxianusis a yeast specie able to ferment lactose, capability shared

by few yeasts. In addition to lactose fermentation,K. marxianushas other desirable attributes 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
whenK. marxianusculture was subjected to ethanol.K. marxianusUFV-3 was activated
overnight in YNB medium with 2% lactose at 37 C. Subsequently, the yeast was re-inoculated 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 inoculated 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 downregulated). 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 related to protein degradation and stress response were activated. This is the first step to
enhance the ethanol tolerance of the yeastK. marxinausUFV-3 for industrial purposes.
Acknowledgements: FAPEMIG, CAPES, CNPq, Xunta de Galicia, and FEDER.

SIMB 2013 | 24

PRODUCTION AND PARTIAL PURIFICATION OF

ENDOGLUCANASES INASPERGILLUS NIGERSP. 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 fungusAspergillus nigersp., 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 production of endoglucanases. Enzyme assays were conducted using 2 % carboxymethylcellulose (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)2SO440-80 % 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 exchange 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 | 25

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 microbiological standard, 26.6% of samples showedcounts of mesophilic aerobic bacteria standard. Valuesfound for yeasts and molds only samples (MC5, MM8and MM10) was observed contamination with the respective values2.2 x107, 3.4 x107and 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. Analyzing 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 | 26

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 produced by Brazilian mining operations, due to the solubilization of Mn containing minerals.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 microorganisms 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 synthetic 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) concentrations 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. ptyseosandP. 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 | 27

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 ubiquitous 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:Myoviridae, PodoviridadeandSiphoviridae. They have capsids (head) which can be icosahedral, 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 morphologic 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 solution 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 microfiltered 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 | 28

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 development of grasses sustainable agriculture.Currently,it is known thatthe advantagesof
usingmicrobialinoculants 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 seedlings 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. Similar results were found by other authors, where strains that produce IAA at concentrations 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 | 29

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 conditions. 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 Sorghum 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 conditions os storage (4C and room temperature). The starch vehicle presented the highest 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<0.05) than
B32-based on viability, except for starch under 4C. In general, all strains had higher
or equal viable cell density than the initial inoculation (8.0 logCFU) at different storage
conditions. Thus, the microrganisms and vehicles studied here are promising for bio
inoculants development.
Financial support: EMBRAPA MAIZE AND SORGHUM, FAPEMIG AND CNPQ.

SIMB 2013 | 30

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 microorganisms 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. Microbial 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-methyl 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 activities 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 agreement with the recognized industrial importance of a variety ofAspergillusspecies. 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 | 31

DISTRIBUTION OF HEPARIN-BINDING PROTEINS

INLEISHMANIA 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 protozoan 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 carbohydrates present in glycoproteins or glycolipids. The
presence of these molecules inLeishmaniaspecies is poorly studied. Therefore, in this
work, the lectins distribution inL.chagasiwas 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 affinity chromatography on Heparin-agarose and D-Salting columns in FPLC automated
system. The protein was collected to perform analysis on polyacrylamide gel electrophoresis, revealing bands indicative of 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 ofLeishmaniainfection.
Financial support: FAPEMIG and CNPq are the funding agency for this research.

SIMB 2013 | 32

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 solubility over a wide pH range, it is notoriously difficult to remove from contaminated waters. 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 carbohydrates assimilation (Auxanogram) and fermentation (Zymogram). These results suggest
that among eight yeast isolates, three were identified asCandida guilliermondiiand 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, suggesting 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. guilliermondiiandR. mucilaginosawere
able to remove an average of 100% and 60% of the Mn(II) in a one week-long period, respectively. 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 | 33

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 Minas Gerais state has the biggest reserves of this metal. The solubilization of minerals containing 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 presenceofCorynebacterium sp,Klebsiella oxytoca,Escherichia coli,Stenothrophomonas maltophiliaandEnterobacter 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%. Furthermore,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 bioremediation of Mn(II) ion byK.oxytoca.
Keywords:Manganese; biorremediation; manganese oxidation
Financial support: CNPq, Vale, FAPEMIG, FINEP and UFOP

SIMB 2013 | 34

A NEWESCOVOPSISSTRAIN 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 fungusEscovopsisis 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 surveyed Escovopsis associated with higher and lower attine ants from several Brazilian
biomes. Here we report a newEscovopsisstrain isolated from the fungus garden ofCyphomyrmex sp. 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. Sequences belonging to closest relatives were queried in the GenBank database. After multiple
alignments, the tef1-alfa data set was analyzed under the maximum likelihood algorithm 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 anEscovopsisclade 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 conidiogenous cells producing solitary conidia. Chlamydospore-like cells were also observed after
7 days of incubation. The newEscovopsisstrain is an indicative that several putative new
species of the parasite associated with lower attine ants await discovery.
Acknowledgements: FAPESP and CNPq

SIMB 2013 | 35

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 economic dependence of agricultural to the financialsector, beyond enhance ecosystem services, by better nutrient management andincrease the use of biological potential. The arbuscular 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 seasonality on mycorrhizal colonization and abundance of sporesin a forest fragment, agroforestry (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 100cm3 of each soil sample using the wet-sieving technique, 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-intersect 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 occurred 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 communitycomposition FMA additional tests will be performed based on molecular biologyand species composition.
Acknowledgements: The CTA-ZM and the farmers who participated in this research.
Thanks also to CAPES Brazilian Research Agency.

SIMB 2013 | 36

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 levels (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 interaction between inoculants and Nitrogen levels was detected. However, analysis indicated 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 | 37

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 anthracnose, is one of the most frequently observed pathogens and is responsible for significant
damage to common bean crops. Understanding the genetic and physiological mechanisms 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 pSilentDual1-egfpvector. This vector is responsible for producing double-stranded RNA (dsRNA) 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 functional 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 present 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 | 38

THE CHAPERONE HFQ IS IMPORTANT FOR

BIOFILM FORMATION INACTINOBACILLUS
PLEUROPNEUMONIAESEROTYPE 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 anaerobic, belonging to thePasteurellaceaefamily 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 asFrancisella novicida,Neisseria meningitidesand even a serotype 1 reference strain ofA. 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 ofA. pleuropneumoniae. To this purpose, wild type (WT),hfqmutant andhfqmutant 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 complemented 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 inA.
pleuropneumoniaeand 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 Kingdom Agency: BBSRC.
SIMB 2013 | 39

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 performance. This work aimed to characterize a yeast strain obtained from the rumen of crossbred Holstein cattle (dairy cows). Batch cultures were performed in YPD medium containing 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 inhibition 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 thatP.kudriavzeviiEF-1
was not autochthonous to the rumen ecosystem.
Acknowledgements: CNPq, CAPES, FAPEMIG

SIMB 2013 | 40

RESEARCH OF ANTIVIRAL ACTIVITY OF EXTRACTS

FROMARISTOLOCHIA 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 ofDengue virus, is transmitted to humans by vectors from genusAedes.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 research on medicinal plants.Aristolochiaspp. (Aristolochiaceaefamily) is a well-known
genre used in folk medicine, commonly applied in the treatment of diarrhea and abdominal pain. Phytochemical studies revealed the presence of alkaloids, flavonoids
and aristolochic acids, which may be promising compounds in antiviral activity. Aerial 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 cytotoxicconcentration50(CC50) and effective concentration 50 (EC50) of the extracts by methylthiazol-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 hexnico 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 EC50and
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 | 41

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 researchers. 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 methodology 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 activity 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,Aspergillussp. LEMI 2 andAspergillussp. 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 distilled 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 maximum activity occurred 20 days after fermentation with an enzymatic production of 4,78;
6,19; 5,55 U.g-1(Aspergillussp. LEMI 1,Aspergillussp. LEMI 2 andAspergillussp. 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 | 42

AVICELASE PRODUCTION BYASPERGILLUSSPP. 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) processes. 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 glucose. 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 production. 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 coffeehuskas 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 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 forAspergillussp. LEMI 1 (2,13 UI g-1) and at 20 days, forAspergillussp.
LEMI 2 (2,08 UI g-1) andAspergillussp. 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 | 43

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 regulation of hundreds of genes, including genes of virulence and stress response.Little is
described about the functionality of these proteins inA. pleuropneumoniae. In this context, 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 genome ofA. pleuropneumoniaewas analyzed by PCR. Real time quantitative PCR (qRTPCR) was used to determine the expression of genesihfA, 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 | 44

FILAMENTOUS FUNGIPRODUCTION 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 solidstate fermentation (SSF) process to produce value-added products such as enzymes. The
SSF process offers numerous advantages including high volumetric productivity, relativelyhigher 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 andcoffeehuskas 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 enzyme 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 withAspergillussp. 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, especially. TheAspergillussp. LEMI 3 was the best strain tested in the study for production
of amylase and xylanase by solid-state fermentation (SSF) using coffeehuskas substrate.
Financial support: Capes, Fapesb, CNPq and UFBA

SIMB 2013 | 45

XYLOPODIUMJACARATIA CORUMBENSISAS SUBSTRATE

INSOLID STATE FERMENTATION FOR PRODUCTION OF
XYLANASE BY FILAMENTOUS FUNGI
ANGRA P. B. REGO; APARECIDO A. CONCEIO; LUCAS M. ALCANTRA;
EDVALDO X. F. FILHO; INGRID A. L. TEIXEIRA; ROBERT O. GUSMO;
PATRCIA L. LEAL; FELIX G. SIQUEIRA;
Federal University of Bahia, Campus Ansio Teixeira, Vitria da Conquista/BA,
Brasil University of Braslia, Braslia/DF, Brasil Embrapa Agroenergia, Laboratory of
Biochemical Processes, Braslia/DF, Brasil
Wild papaya (Jacaratia corumbensisO. kuntze)is a shrub occurring in the Brazilian
semi-arid region. Its fruits and tuber are consumed by wild animals and used as a baking
ingredient by small farmers. The application ofJ. corumbensisas a substrate for growing filamentous fungi in solid state fermentation (SSF) may be a promising strategy for
the production of enzymes such as xylanases. The objective of this study was to quantify
the enzymatic activity of xylanase produced under solid-state fermentation, using fungi
isolates from the semi-arid of Bahia (LEMI 4, LEMII 5, LEMI 6 and LEMI 7) and different parts ofJ. corumbensisas substrate. Solid-state fermentation was carried out using a
500 ml Erlenmeyer flask containing 50g of xylopodium crumb (T1) or husk (T2), previously ground, autoclaved and inoculated with filamentous fungi strains by deposition of
fungal mycelium in agar discs. The cultivation was carried out at 30C for 15 days. 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 enzyme solution for enzyme activity measurements by the dinitrosalicylic acid
method. The enzymatic activity of xylanase showed significant values ??for T1 and T2
treatments. The mean values and standard deviation for each fungal isolates tested were:
T1) [LEMI 4 (2,356 0,111); LEMI 5 (2,218 0,081); LEMI 6 (0,119 0,024); LEMI
7 (2,777 0,103)] and T2) [LEMI 4 (4,629 0,468); LEMI 5 (3,629 0,460); LEMI 6
(1,482 0,331); LEMI 7 (3,704 0,681)]. The xylanase activity was significant for all
fungal isolates when grown in both carbon sources, except the isolated LEMI 6 when
cultived with xylopodium crumb. These results indicate that the xylopodium fromJacaratia corumbensishas biotechnological potential and therefore can be used as substrate
for the production of xylanase through solid-state fermentation.
Financial support: Capes, Fapesb, CNPq and UFBA

SIMB 2013 | 46

PATHOGENICITY OFSYNCEPHALASTRUM RACEMOSUMTO

LEAF CUTTER ANT GARDENS
BARCOTO, M. O.1; BUENO, O. C.2; RODRIGUES, A.1
1. Department of Biochemistry and Microbiology, UNESP - So Paulo State University,
Rio Claro, SP, Brazil. 2. Center for the Study of Social Insects, UNESP-So Paulo State
University, Rio Claro, SP, Brazil.
Previous studies indicated thatSyncephalastrum racemosumis an antagonist of the
mutualistic fungus cultivated by leaf-cutting ants. To determine thein vivopathogenicity ofS.racemosum, spore suspensions of four strains were inoculated on fungus gardens
kept in the presence or absence of workers. In both treatments, garden pieces were removed and inoculated in MA2% to determine the presence ofS. racemosum. For comparison, spore suspensions ofEscovopsis weberi,Trichoderma harzianumandFusarium
solaniwere also applied. In gardens tended by workers, the proportion of infected garden
pieces was significantly lower than pieces recovered from ant-deprived gardens, when
spores of threeS.racemosumstrains (A105, A101 and H1a) andT. harzianum(17BFIII)
were applied. In such treatments, ants removed alien fungi within 48 hours. On the other hand, when spores ofS. racemosum(A086b),E. weberi(A086a) andF. solani(AR037)
were applied, the proportion of infected garden pieces was similar between gardens
tended and unattended by workers. This result indicates that ants could not completely
remove such fungi from their gardens. Infected pieces were more likely recovered in
dumps than in the fungus garden. Interesting, after spore inoculation, ants removed
healthy garden fragments in all treatments using S. racemosum spores. This weeding
behavior was previously described as a response toEscovopsisinfections, and now is also
described forS. racemosum. Overall, our results highlight the pathogenic potential of
fungi other thanEscovopsisto fungus gardens of leaf-cutting ants.
Acknowledgements: FAPESP and CNPq

SIMB 2013 | 47

PHOMOPSIS SP. AS ENDOPHYTIC TURNERA SUBULATA:

ISOLATION, IDENTIFICATION AND BIOLOGICAL ACTIVITY
OF ITS EXTRACTS
SANTOS,G.B.L; CAETANO,L.C; SANTOS,T.M.C; MONTALDO,Y.C; SANTOS,R.M;
SOBRINHO, R.R; SILVA,S.G.M; SILVA, R.A.S
Universidade Federal de Alagoas. Campus A. C. Simes - Av. Lourival Melo Mota, s/n,
Cidade Universitria - Macei - AL, CEP:57072-900. Macei-Brasil | Universidade
Federal de Alagoas, CENTRO DE CINCIAS AGRRIAS.Campus Delza Gitai, BR 104
norte KM 85 - Mata do RoloRio Largo57100000 - Rio Largo, AL - Brasil
TurnerasubulataL. is a plant belonging to the Turneraceae family and is popularly
known in Brazil as chanana. The genusTurnera has about 120 species distributed in the
Americas and Africa, being the most representative ofthe family Turneraceae. Some species of Turnera are highly used in popular medicine for different types of inflammatory
diseases. The association betweenliving beings is a vital condition for species unable to
achieve alone, as ameans of survival for obtaining nutrients and defenses against predatory species. Among the microorganisms, the fungi are those who are most often associated with plants. Endophytic are microorganisms that inhabit the bodies of plants that,
at some period of their life cycle, colonize internal tissues of the plant, without causing
any apparent damage to its host. From the fragments of leaves fromTurnera subulataL.
could be isolated a filamentous fungus that was identified as the fungusPhomopsissp.
through traditional microscopy and molecular techniques. In a statistical evaluation it
was noted that the endophyticPhomopsissp. presents a higher growth in BDA and BSA
media, as well as in the presence of light. The steroid ergosterol could be isolated from the
hexane extract of this fungus and identified by the NMR techniques. In tests of antagonism of the endophyticPhomopsissp.against phytopathogens, it was observed halum of
inhibition againstRhizoctoniasp.,Colletotrichumsp. andPestalotiopsissp. Concerning
the antioxidant activity it was observed that the chloroform extract was more effective
than the hexane one. On the other hand all the extracts from the mycelium of Phomopsis
sp. and its ethylacetate extract from the culturedfiltrate showed no antimicrobial activity against strains ofEscherichiacoli, Staphylococcus aureus, Candida albicans, Candida
tropicalisandCandidaglabrata.Thereforeit was concluded thatPhomopsissp.may act as
endophyticofT. subulata.ithas shownto grow bestin BDA and BSA medium, as well as
under continuous lightining. It producesand accumulatesergosterol in his micelium.It
promotes inhibition zone of growthwhen tested agains tphytopathogens. His hexanic
and chloroform extrat has shownlittle antioxidant activity.
Keywords: Extracts, Antagonism, Endophytic microorganisms

SIMB 2013 | 48

PERMEABILITY AND ULTRASTRUCTURE OF THE CELL WALL

OFPISOLITHUS MICROCARPUSBASIDIOSPORES
1SILVRIO, MERIELLE ANGLICA MARTINES; 2SERRO, JOS EDUARDO;
3TTOLA, MARCOS ROGRIO; 4MENDES, GILBERTO OLIVEIRA; 5DUARTE,
JOSIANE LEAL; 6CARVALHO, MARA MARIZ; 7ANASTACIO, THALITA
CARDOSO; 8GALLO, SRGIO ALBERTO DIAS;9COSTA, MAURCIO DUTRA

1Universidade Federal de Viosa (UFV), Viosa, Minas Gerais, Brasil; 2CNPq Researcher,
General Biology Department, UFV; 3CNPq Researcher, Departamento de Microbiologia,
UFV; 4UFV; 5UFV; 6UFV; 7UFV; 8UFV;9CNPq Researcher, Departamento de
Microbiologia, UFV
Basidiospores of the ectomycorrhizal fungusP. microcarpuspossess impermeable,
hydrophobic cell walls. Thesefeatures are possibly related to the low percentage of germination of thesepropagules and make it difficult the isolation of monokaryons and the
use ofthese spores as inoculants. Sodium hypochlorite can be used as a permeabilizingagent of the cell wall of fungal spores.The aim of this study was to evaluate the effects
of permeabilization treatmentswith commercial bleach on the cell wall ultrastructure
and hydrophobicity, andon the viability and germination basidiospores ofP. microcarpus. For this, fungal basidiospores, from fruitingbodies associated withEucalyptusspp.
were collected and permeabilized using different concentrations of bleach andtimes
of exposure. After permeabilization, the basidiospores were analyzed byscanning and
transmission electron microscopy. Surface hydrophobicity,viability, and germination of
these propagules were also analyzed. Thepercentage of permeabilized basidiospores ofP.
microcarpuswas proportional to the increases in bleach concentrationand the exposure
time. Basidiospores from different fruiting bodies differedsignificantly in their susceptibility to the permeabilization treatments withbleach and in the decrease of cell surface
hydrophobicity after permeabilization.Changes in the ultrastructure of permeabilized
basidiospores were observed at bleachconcentrations of 15 and 50 %, with an exposure
time of 40 s. For one basidiocarp,after permeabilization with bleach at 5 % for 40 s, 80%
of the permeabilized basidiosporeswere viable. The plating of basidiospores permeabilized with 10% bleach for 40s resulted in the production of colonies ofP.microcarpus.
The colonies appeared from the 15thof incubation of the permeabilized basidiospores
inthe presence of the host plant, E.citriodora. The germination percentage obtained,
0,001 %, was similar tothose reported for non-permeabilized basidiospores.
Keywords:Cell wall; ectomycorrhiza; cell wall hydrophobicity, electron microscopy.
Acknowledgements: Ncleo de Microscopia e Microanlise - UFV Financial support:
CAPES, FAPEMIG, CNPq

OBTAINING BIOETHANOL FROMSWEET SORGHUM

SIMB 2013 | 49

(SORGHUM BICOLOR L. MOENCH) BY CONTINUOUS

FERMENTATION USING IMMOBILIZED YEAST IN CALCIUM
ALGINATE
RAPHAEL S E ROCHA, PHILIPE LUAN BRITO, LLIAN DE ARAJO PANTOJA,
ALEXANDRE SOARES DOS SANTOS
Federal University Of Jequitinhonha And Mucuri Valleys - UFVJM, Institute Of Science
And Technology - ICT, Diamantina, Minas Gerais, Brazil.
Bioethanol can be obtained from various raw materials, sugary, starchy or lignocellulosic. Among the many possible sources for obtaining bioethanol, sweet sorghum
(Sorghum bicolorL. Moench), a kind of grass that focuses sucrose in stems, is a biomass
that can use the existing infrastructure for production of ethanol from sugar cane.One
of the steps that can be worked to the optimization of bioethanol production is the fermentative process. In this work was studied a bench scale process for obtaining bioethanol from sweet sorghum using the fixed bed reactor employing yeast (Saccharomyces
cerevisiae) immobilized in calcium alginate. The continuous fermentation of the juice of
sweet sorghum was evaluated regarding the effects of dilution rates (0.1 to 0.6 h-1) over
the volumetric productivity (QP), conversion factor of substrate to product (YP/S) and
consumption of sugars. The temperature of the must was kept constant (28 C), and each
worked dilution rate was maintained by two residence times. The continuous process
was carrying out for 60 hours, with scheduled changes in thefeed flow system. Samples
were collected at intervals of time corresponding to residence times determined for each
tested dilution rate. The samples were characterized for pH, total soluble solids, reducing
sugars and ethanol concentration. On the condition with the dilution rate of 0.5 h-1was
obtained fermentation broth with final ethanol concentration of 62.1 g L-1(7.9% v/v),
QP36.5 3.2 g L-1h-1and consumption of 86.2% of total reducing sugars (TRS). In the
dilution rate of 0.4 h-1was obtained fermentation broth with final ethanol concentration
of 49.0 g L-1(6.2% v/v), QP20.2 1.2 g L-1h-1, YP/Sof 0.45 and consuming 96.4% ofthe
TRS. The next challenge will be the scale up.
Acknowledgements: FAPEMIG and CNPq Financial support: CNPq e FAPEMIG

SIMB 2013 | 50

EFFECT OF ANTIOXIDANT AGENTS ON BACTERICIDAL

ACTIVITY OF SILVER NANOPARTICLES AND SILVER NITRATE
FERNANDES, P. E; COSTA, D. S.; SILVEIRA, M. P.; ANDRADE, N. J.;
MANTOVANI, H. C.; BERNARDES, P.C.; ALVES, R.B.T.
Universidade Federal de viosa, Viosa, MG, Brasil Universidade Federal do Esprito
Santo, Alegre, ES, Brasil
Previous studies indicated that free-radicals might interfere with the antibacterial
activity of silver nanoparticles (AgNPs) and the interaction between reactive oxygen
species (ROS) and bacterial cell death has been reported. Scavengers such as n-acetyl
cysteine (NAC) and ascorbic acid (AA) have been used to scavenge the ROS produced
by AgNPs and to show the involvement of ROS affecting the antibacterial activity of AgNPs. This work aimed to evaluate theeffect of antioxidants on the antimicrobial activity
of AgNPs and silver nitrate.AgNPs were synthesized by reducing 1 mM silver nitrate
(AgNO3) with 36 mM of sodium citrate. The appearance of yellow color indicated the
formation of nanoparticles. Approximately 106 CFU/mL of P. aeruginosawere inoculated into a microtiter plate well containing Mueller Hinton broth added with AgNPs or
AgNO3at a final concentration of 25 g/mL. Ascorbic acid, sodium thiosufate (TS) or
L-cisteine (CS) were added as antioxidants at 10 mM and 20 mM. The microtiter plate
was incubated at 37 C for 24 h and the optical density (DO) was measured at 600 nm.
Controls were made with antioxidants (at 10 mM and 20 mM) without nanoparticles
or without antioxidants and AgNPs.Microbial growth was completely inhibited when
culture media was added with 25 g/mL of the AgNO3 or AgNPs. However, bacterial
growth could be restored if the media containing 25 g/mL of the AgNO3or AgNPs was
added with 10 mM of AA, TS or CS. When used alone at 10 mM, the antioxidants did
not affect the growth ofP. aeruginosa. If the concentration of AA or CS was increased
to 20 mM in the absence of AgNO3 or AgNPs growth of P. aeruginosa was no longer
observed. When used at a final concentration of 10 mM, all antioxidants tested were
able to protect the cells from the toxicity of AgNPs and AgNO3without interfering with
microbial growth. This result suggests that ROS are involved on bactericidal mechanism
of AgNPs and AgNO3.
Acknowledgements: The authors thank CNPQ and FAPEMIG for financial support.

SIMB 2013 | 51

ASSESSMENT OF THE VIABILITYOF FUNGI HOLD AT THE

UNESP- MICROBIAL RESOURCE CENTER
ANA MARIA LIMA CORREIA; LARA DURES SETTE; ANDR RODRIGUES
Departament of Biochemistry and Microbiology, UNESP So Paulo State University,
Rio Claro, SP, Brazil
The UNESP- Microbial Resource Center (UNESP-MRC) harbors several strains of
filamentous fungi, yeasts and bacteria from diverseunderexplored environments. From
2002 to 2009, 539 fungal strains were identified, cryopreserved in glycerol 10% at - 80 C
and deposited at UNESP-MRC. Such strains comprise ascomycetous, basidiomycetous
and zygomycetous genera isolated from leaf-cutting ant nests. Here, we evaluated the
viability and the identification authenticity of the strains stored in the culture collection. Strains were revived in Sabouraud Dextrose Agar supplemented with antibiotic.
We checked the viability, purity and the correct identification of the strains through
macroscopic and microscopicaspects of the colony. Overall, a total of 228 fungal strains
were revived, but only 4% lost their viability during long-termpreservation. Such strains
belong toEscovopsis, Fusarium, MortierellaandThermoascusgenera. Regarding to the
29 fungal genera successfully revived, Escovopsis (21%) Cryptococcus (13%) Penicillium(12%) andTrichoderma(10%) were the most abundant. Overall, the strains grew in
two to three days of incubation, except forEscovopsisstrains, which grew only after ten
days. Morphological analysis showed that the strains kept their initial macroscopic and
microscopic aspects. Our results support available data from other culture collections,
which indicates that ascomycetous, basidiomycetous and zygomycetous fungi stored
over ten years maybe successfully revived. The results demonstrate the efficiency of
the cryopreservation technique and the relevance of such long-term maintenance performed at UNESP-MRC for identification and exploitation of the microbial resources as
well as preservation of brazilian microbial genetic diversity.
Acknowledgements: CNPq

SIMB 2013 | 52

CELLULOLYTIC FILAMENTOUS FUNGI FROM HIGH

ALTITUDE ENVIRONMENT INITATIAIANATIONAL PARK
EUTIZIO LUCA D'OTTAVIO LONGO1; MATHEUS UCHOA OLIVEIRA1;
MARIANA MENEZES QUADROS OLIVEIRA2; ANDRE LUIZ GRIGOREVSKILIMA2; ROSALIE REED RODRIGUES COELHO2; RODRIGO PIRES DO
NASCIMENTO1;
1 Departamento de Engenharia Bioquimica, Escola de Quimica, UFRJ, Rio de Janeiro,
Brasil; 2 Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de
Goes, UFRJ, Rio de Janeiro, Brasil
The Brazilian Atlantic Forest has a great and rich biodiversity, making it a promising source for studies involving bioprospection of filamentous fungi with biotechnological potential. Among microbial enzymes, cellulases have a great biotechnological importance, and could be applied in a wide variety of industrial processes such as bioenergy,
pulp and paper, detergents and textiles. The investigation of new areas for bioprospecting fungi with biotechnological application is a very interesting approach. In this sense,
the high altitude soils from the Brazilian Tropical Forest could be a special environment.
Therefore, our research group have isolated 13 filamentous fungi from soil and litter of
the Itatiaia National Park in Rio de Janeiro, Brazil, at a very high altitude (above 2.000m)
using agar cellulose medium, pH 5.5. After 10 days incubation at 28oC, the isolates were
collected and conserved by Castellanis method. Endoglucanase (CMCase) production
was analyzed using Mandels medium, modified by supplementation with 1% (w/v) filter
paper as carbon source, after incubation for 7 days/28 C/180 rpm. The CMCase activities were determined by measuring the reducing sugars released after carboxymethylcellulose degradation, which were quantified by DNS method. The enzymatic assay was
performed by the incubation of 0.5mL supernatant (crude enzyme extract) and 0.5mL of
substrate (CMC) in sodium citrate buffer pH 4.8 at 50 C for 15 minutes. The strains I75, I7-6, I7-7 and I8-1 were selected as the most promising endocellulolytic strains. The
higher endoglucanase activities were 1.884, 1.032, 1.043 and 1.037 U/mL, respectively.
The results shown in this work indicate the potential of high altitude environments, like
Itatiaia National Park in Rio de Janeiro, Brazil, as a new source for searching new cellulolytic fungi strains.
Acknowledgements: Marta Ferreira de Sousa for laboratory technical support; IBAMA for
sample's collection and transport authorization; Parque Nacional de Itatiaia for the access
to park; CGEN/CNPq for property genetic access authorization. Financial support: Conselho Nacional de Desenvolvimento Cientfico e Tecnologia - CNPq; Universidade Federal
do Rio de Janeiro - UFRJ

SIMB 2013 | 53

ADHESION OF SPORES AND VEGETATIVE CELLS

OFBACILLUS CEREUSENTEROTOXIGENIC GENOTYPES IN
DIFFERENT STORAGE TEMPERATURES ON STAINLESS STEEL
S JOO PAULO NATALINO DE SILVA DAIENE DA COSTA ANDRADE NLIO
JOSE DE ARAJO EMILIANE ANDRADE PENNA WILMER EDGAR LUERA
BERNARDES PATRICIA CAMPOS
Universidade Federal de Viosa, Av. P H Rolfs, s/n - Campus Universitrio, Viosa - MG,
36570-000; Minas Gerais. Brasil ( Todos autores exceto Patricia Campos Bernardes,
Universidade Federal do Espirito Santo, Espirito Santo. Brasil
Among the various micro-organisms of concern inthe food industry,Bacillus cereusisa food pathogen that has acquired increasing importance in relation to publichealth. Most strains of B. cereus arecapable of producing a wide variety of extracellular
metabolites, which areconsidered virulence factors, leading to outbreaks of foodborne
illness. Inthis study, we evaluated the adhesion of enterotoxigenic genotypes of B. cereusvegetative and spore forms onthe surface of stainless steel 304#4 at 10 C and 32 C.
Adhesion was notsignificantly different (p>0.05) among genotypes at a same temperature.However, significant effect was observed (p<0.05) for the physiologicalstructures
between genotypes at the same temperature (10 C and 32 C). It wasobserved that at 32
C, the vegetative form adhered more. This may have occurred,by the fact that at 10 C,
the bacteria do not produce cellular appendageswhich contribute to increased adhesion.
However, at 32 C, appendices can beproduced by the vegetative cells, and microbiological factors overlapphysicochemical factors, since the spores are more hydrophobic than
thevegetative cells, resulting in increased cell adhesion. The effect oftemperature onB.
cereusvegetativecells and spores adhesion can also be explained, among other factors,
byhydrophobic interactions. These interactions are governed by the entropy of thesystem, thus increasing the temperature favors the entropic component. So, asthe temperature increases, hydrophobic interactions become strong, favoringadhesion. Although
there are no specific studies that quantify the adhesion ofB. cereusenterotoxigenic genotypes onstainless steel surface, these seem not to influence the adhesion process.
Acknowledgements: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
(CNPq)

SIMB 2013 | 54

MODELING OF THE GROWTH KINETICS OFBACILLUS

CEREUSENTEROTOXIGENIC GENOTYPES
S JOO PAULO NATALINO DE ANDRADE NLIO JOSE DE COSTA DAIENE
SILVA DA PIRES ANA CLARISSA DOS SANTOS BERNARDES PATRICIA
CAMPOS FERNANDES PATRCIA RICA
Universidade Federal de Viosa, Viosa, Minas Gerais . Brasil ( todos autores, exceto
Patricia Campos Bernardes : Universidade Federal do Esprito Santo, Esprito Santo.
Brasil
Among the various contaminating microorganismsin the food industry, Bacillus
cereusis a pathogen that can cause intoxication and also deteriorate food. In thisresearch,
the kinetic parameters related to multiplication rate (mi), and the lagtime (l) of four
genotypes of this microorganismwere evaluated. Multiplication was monitored using a
spectrophotometer andreadings of optical density at 600 nm. The DMFit program was
used to fit thedata curves to the model of Baranyi and Roberts. The maximum multiplicationrate (/max) was determined by the model Ratkowsky et al. modified. Nodifferences were observed by the F test (p> 0.05) in the values ??of themultiplication rate (?)
between the B.cereus genotypes at the same temperature. However, there was significantdifference (p <0.05) between genotypes at the same temperature, in the lag time(?),
showing that in the same temperature, the genotypes have differentconditions of adaptation. Probably, the lower temperatures (10 C, 15 C and 25C) were one of the possible
causes of greater permanence in this stage, since,at temperatures of 32 C and 42 C,
there was no significant difference (p>0.05) in the adaptation time. The low multiplication rate and high lag time at10 C obtained by B. cereus genotypes,regardless of the
enterotoxin type which may be produced, shows that the refrigerationtemperature used
in the food industry, can be an important tool to ensure themicrobiological quality and
food safety to consumers.
Acknowledgements: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
(CNPq)

SIMB 2013 | 55

SELECTION OF ENDOPHYTIC BACTERIA ISOLATED FROM

SUGARCANE WITH POTENTIAL FOR SOLUBILIZATION OF
INORGANIC PHOSPHATE
SILVA, J.M.; MELO, A.L.S.; SILVA, S.G.M.; MONTALDO, Y.C.; SILVA,T.F.; SANTOS,
T.M.C.
Universidade Federal De Alagoas, Unidade Acadmica CECA/UFAL, CEP 57100-00, Rio
La
Various micro-organisms, such as fungi and bacteria have the ability to solubilize
phosphorus in inorganic form available to plants in organic form. These organisms can
be found in soil, water, or live epiphytic or endophytic state. The present work aimed
to select endophytic bacteria with potential to solubilize inorganic phosphate. Samples
of leaf, stem and root of sugarcane were collected. The samples were subjected to serial dilutions inoculated in a solid medium for solubilizing phosphate (NBRIP) and
characterized by forming a translucent halo around the colonies. The evaluation of the
ability to solubilize phosphate in vitro was performed using the solubilization index.
It wasobtainedtwenty three isolateswhich were inoculated in a Petri dish containing
culturemedium NBRIPand incubated at room temperature. The evaluations were conducted in three intervals (five, ten and fifteen days after hatching), where the growth of
the colony and the diameter to calculate the solubilization index were observed. In the
first evaluation the phosphate solubilization by seventeen of the twenty three isolates
were observed. In the second and third assessment was verified by solubilization nineteen isolates, two more than the first observation. In conclusion, it could be observed
that the ability to solubilize phosphate was higher among bacteria isolated from roots,
followed by those isolated from stems and leaves.

SIMB 2013 | 56

INHIBITIONIN VIVOOFSCLEROTIUM ROLFISII IN COWPEA

BYTRICHODERMASPP.
MELO, A.L.S.; SILVA, J.M.; SANTOS, J.M.C.; SANTOS, R.B.; MONTALDO, Y.C.;
SANTOS, T.M.C.
Universidade Federal de Alagoas, Unidade acadmica CECA/UFAL, CEP 57100-000, Rio
Largo, Alagoas, Brasil
The pathogenSclerotium rolfisiiis causingdumping offin legumes. TheTrichoderma spp. is a fungus commonly found in soil, with high potential for biocontrol. This
study aimed to evaluate the potential biocontrol of S. rolfisii by strains of Trichodermaspp. For inhibitionin vivowas done planting cowpea seeds in plastic cups of 400
mL with sterilized vermiculite for two cycles 1h in autoclave. The isolates (pathogen
and antagonist) were inoculated directly on the substrate, which was placed in each
cup, a disk of each fungus at a depth of approximately 1 cm. The treatments used were:
T1(Trichoderma1+S. rolfisii), T2(Trichoderma2 +S. rolfisii), T3(Trichoderma3 +S. rolfisii), T4(Trichoderma 4 + S. rolfisii), T5(Trichoderma 5 + S.rolfisii), T6(S. rolfisii) and
T7(Witness). Fifteen days later, the plants were removed from the cups and washed to
remove the substrate from the roots to the present analysis, which assessed the action
of the pathogen and the control exercised by antagonist and root colonization isolates.
Was observed in plants if there was rot in theroots, plant height, leaf area, stem diameter
and root length. All plants were inoculated with the pathogen colonization suffered the
same, however, no damage, presenting healthy development, compared to the control.
This can be considered because of the potential antagonist fungi of the genusTrichoderma. The seeds that received inoculation ofTrichodermagrew better, this is due to
the potential for stimulating the production of hormones and production of secondary
metabolites by the fungus. The T2 and T5 showed the best results of germination and
growth compared to the other treatments, including the witness.

SIMB 2013 | 57

GROWTH AND PHYSIOLOGY OFASPERGILLUS

TUBINGENSISAN1257 IN SUBMERGED BIOPROCESSES
VERON, N. W.(1); SILVA,T. J.(1); MAIA, A. C. F.(1); VANZELA, A. P. C.(1)
Universidade Federal dos Vales do Jequitinhonha e Mucuri Diamantina, MG, Brasil.

(1)

Enzymes are applied in several industrial sectors, whereas most of them are obtained in microbial processes, with a particular role for fungi producing carbohydrate
active enzymes. Besides having a good producer, development of bioprocesses also relies
on studying the biological characteristics of the strains. This work aimed to evaluate
growth and physiology of an isolate of the black fungiAspergillus tubingensisAN1257,
former selected as a cellulase producer. Liquid media (pH 6.0) supplemented with glucose, starch or carboxy-methylcellulose (CMC) were inoculated (106conidia .mL-1) and
incubated for 8 days at 30 C and 150 rpm. Biomass,?-amylase and endoglucanase activities, reducing sugars, glucose, extracellular protein, pH and starch were determined daily. Growth was higher in media supplemented with glucose, reaching 0.99 g . L-1after 24
h. At the same time, pH decreased to 2.81. Glucose concentration decreased gradually.
A low amylolitic activity (6.174 1.321 U.mL-1) was detected after 24 h in media supplemented with starch, whereas it was observed a complete consumption of the substrate,
a low reducing sugar (0.499 0.367 mg.mL-1) and glucose (0.576 0,392 mg.mL-1) concentrations, biomass formation (1.2499 0.1169 mg.mL-1) and a decrease in pH (2.88
0.00). Supplementation with CMC avoided pH decreasing, and yielded a scarce growth.
There was no production of cellulase, nor liberation of reducing sugars or glucose in
this medium. Diminishing pH to 2.62 0,03 by acid addition increased growth and reducing sugar concentrations in cultures supplemented with CMC. Extracellular protein
coincided with growth in all cultures. It was concluded that the soluble CMC was not a
good endoglucanase inducer, even for a fungus that had previously shown to produce
cellulases on solid natural substrates. It was also concluded that a sharp decrease in pH
supports A. tubingensis AN1257 growth,and its synthesis of ?-amylase and endoglucanase requires substrate induction.
Acknowledgements: UFVJM, CNPq

SIMB 2013 | 58

PHYSIOLOGY OF GROWTH AND ENZYME PRODUCTION

BYTALLAROMYCES TRACHYSPERMUST10.5
VERON, N. W.(1); SILVA,T. J.(1); MAIA, A. C. F.(1); VANZELA, A. P. C.(1)
Universidade Federal dos Vales do Jequitinhonha e Mucuri Diamantina, MG, Brasil.

(1)

Penicillium species and their teleomorphs areamong the important fungi for the
commercial production of enzymes, whereas physiologicaland biochemical studies
on such microorganisms may result in a discovery of anew producer or an improved
bioprocess. This work aimed to analyzephysiological and biochemical characteristics
ofTallaromyces trachyspermusT10.5 grown on polymeric carbon sources.Bioprocesses
were conducted in liquid media supplemented with glucose (control)starch and carboxy-methylcellulose (CMC) for 8 days at 30 C under 150 rpm. InitialpH and inoculum were 6.0 and 106conidia mL-1, respectively.Endoglucanaseand ?-amylase activities,
reducing sugars, glucose, extracellular protein, pHand starch were determined daily in
the filtrates. Extracellular proteinremained almost constant after biomass formation in
each culture. Cultivationon glucose resulted in pH decrease to 4.25 0.02 after 48 h,
when all thesubstrate was consumed. After 96 h, pH increased to 8.92 0.08 in thesecultures. No amylase or endoglucanase were produced. In media supplemented withstarch,
a maximum amylase activity (239.2 11 U min-1mL-1)was produced after 48 h, when
pH reached a minimum (4.54 0.07); reducingsugars and glucose were kept very low.
As all glucose and starch were consumedafter 48 h, the progressive basification in older
cultures might be due to celldeath, or loss of pH regulation since enzyme activity was no
longer needed. Inmedia supplemented with CMC, there was no endoglucanase production, andreducing sugars and glucose were not detectable. Besides, pH did not decrease,
butit increased gradually to 8.87 0.25 (8 days). Data allowed conclude thatamylase
production byT. trachyspermusT10.5 is repressible by glucose, and its production correlates with mediaacidification. It was also concluded that T10.5 is not able to utilize CMCunder the described conditions, but this strain is highly efficient for starchhydrolysis
and rapid sugar assimilation.
Acknowledgements: UFVJM, CNPq

SIMB 2013 | 59

PERFORMANCE ASSESSMENT OFSACCHAROMYCES

CEREVISIAEIN ALCOHOLIC FERMENTATION OF PINEAPPLE
MUST
POLIANA BERGAMIN ATHAYDE DE SOUZA1, KEILANE FIOROTTI
POLTRONIERI2, LIVIA DE SOUZA SIMES1, CRISTINA BOLLIS
CAMPAGNARO2, MATEUS DA SILVA JUNQUEIRA3.
1 Federal University of Viosa-UFV, Brazil. 2 Federal University of Esprito Santo-UFES,
Brazil. 3 Federal University of Sete Lagoas-UFSL, Brazil.
Fermented beverages are produced from the conversion of the sugars contained
in a must by the action of yeast. The must to be fermented may be originated from the
maceration of fruits, cereals, and stem. This study aimed to evaluate the yeastSaccharomyces cerevisiaein alcoholic fermentation of pineapple must. Initially there was the
conditioning of yeasts in three different musts containing 5 Brix, 8 Brix and 11 Brix
successively. The yeast already conditioned to high concentrations of soluble solids was
used as inoculum to ferment 1L of whole pineapple juice (pH 4.35 and 11.5 Brix) stored
for 24 hours at 30 C. The development of the fermentation process and the yeast performance were monitored by measuring the soluble solids concentration and pH throughout the process. After 24 hours the concentration of soluble solids reached a value of 3
Brix and the fermentation was terminated. The soluble solids concentration showed a
considerable drop, indicating that the pH of the must, even below conventional was not
able to inhibit the action of the yeast. The must had nice aroma and characteristic color
of pineapple juice. The final product had an alcohol content of 4.167 GL allowing its
inclusion in the category of fermented beverage.
Keywords: fruit processing, alcoholic beverages, pineapple, fermented beverages.

SIMB 2013 | 60

PARTIAL PURIFICATION OF AN AMYLASE PRODUCED

BYTALLAROMYCES TRACHYSPERMUS
MAIA1, A. C. F.; VERON1, N. W.; SILVA1, T. J.; PANTOJA1, L. A.; SANTOS1, A. S.;
REIS1, A. P.; VANZELA1, A. P. F. C.
1

Universidade Federaldos Vales do Jequitinhonha e Mucuri, Diamantina-MG, Brazil

Amylases are among the most used industrial biocatalysts, and knowledgeabout
their purification is essential for commercial uses. Salting-out is oftenemployed for an
efficient, feasible and cost-effective purification. This workaimed the partial purification
of the ?-amylase produced byTallaromyces trachyspermusT10.5, by meansof ammonium sulfate precipitation. Cultures in liquid media (2g L-1starch, 2.5g L-1yeast extract; pH
6) were incubated at 30 C (150 rpm, 72 h). Filtrates wereseparated and utilized to determine the amylase precipitation range underdifferent ammonium sulfate concentrations
(25-80% saturation). Afterdetermining the suitable range, 50 mL of filtrate were submitted to twoconsecutive precipitations: ammonium sulfate was first added to a finalsaturation of 45%, and the sample was centrifuged; supernatant was recoveredand submitted
to a new precipitation (65% saturation). After centrifugation,supernatant was discarded.
The precipitate was dissolved in 10 mL of 100 mM sodiumphosphate buffer pH 6, and
further purified by dialysis for salt elimination.Amylase activity (starch hydrolysis) and
total protein content (Lowry) weredetermined after each step. A good enzyme activity
was determined in theinitial filtrates (189 U min-1mL-1). The precipitationrange was 4565% saturation. After two precipitations, enzyme recovery was 41%.The initial specific
activity was 85.4 U mg-1, and afterprecipitation the specific activity reached 1832.4 U mg1
, yieldinga purification factor of 21 times. Results were promising, once it was obtaineda
good recovery of amylase and a single purification step was enough to attainan elevated
purification factor. It is concluded that precipitation withammonium sulfate may largely
contribute for purification of the amylase producedby strain T10.5. New steps of purification will allow a better comprehension ofthe biochemical and kinetic properties of this
enzyme, and its potential forfuture applications.

SIMB 2013 | 61

DOUBLE-STRANDEDRNA AFFECT GROWTH OFFUNGAL

PLANT PATHOGENIC
ANA PAULA OLIVEIRA DE BARROS; ANDR DA SILVA XAVIER; POLIANE
ALFENAS ZERBINI
Universidade FederaldeViosa,Av. P.H. Rolfs, s/n - Campus Universitrio, Viosa/MG Brasil
Phytopathogenic fungi are responsible for most of the agricultural losses, generating negative impacts on both qualities as quantity of products. A phenomenon associated with attenuation of aggressiveness in fungi, known as hypovirulence, has been
consistently associated with viruses (mycovirus), with most showing dsRNA genomes
. Increase knowledge about the virus -fungus interactions is essential to accelerate the
status virocontrole that traces the landmark use of these new agents for biological control of plant diseases. In order to investigate the presence of mycovirus in fungal populations, and the overall impact of infections in morphology, we performed a screening
of a total of 18 isolates (10 ofSclerotinia sclerotiorumand 8Pseudocercospora griseola),
obtained from bean plants (Phaseolus vulgaris), displaying symptoms of white mold and
angular leaf spot, respectively. The fungi were grown on PDA medium containing cellophane sterilized under controlled conditions at 25C in the dark for 12 days. Total RNA
from 18 specimens was extracted and subjected to DNase I digestion individual, RNase
and S1 nuclease, intending to explore the diversity of viral genomes in the samples. The
colony morphology and the growth rate were evaluated with standard established as the
typical characteristics of free virus isolates for each species. In this test, all isolates were
separately cultured on PDA for 8 days under the same conditions described above. In
only one isolate of S. sclerotiorum two dsRNA segments were detected , corresponding to approximately 0.85 Kb and 2.4 Kb , respectively. InP. griseolaeight segments of
dsRNA could be observed, with lengths varying in the range from 0.8 to 5.0 kb (0.9, 1.0,
1.2, 1.65, 2.2 and 4.0 Kb). The nature of the molecules of dsRNA was confirmed by resistance to degradation by enzymes DNase I and S1 nuclease. The growth rate of the isolate
of S. sclerotiorum infected is slightly lower compared with the isolated non-infected,
about 15%, moreover, a decrease quantitative and qualitative production was reported
as sclerotia. The interference of the biology of the infection isolates ofP. griseolaseems
to be more disturbing because there were observed significant differences in growth
rate. The results indicate that the presence of these dsRNAs is strongly associated with
the modulation of the characteristics of the host, which may compromise the fitness of
these species in the field.

SIMB 2013 | 62

OPTIMIZATION OF AMYLASE PRODUCTION

BYTALLAROMYCES TRACHYSPERMUS
MAIA1, A. C. F.; VERON1, N. W.; REIS1, A. P.; SANTOS1, A. S.; PANTOJA1, L. A.;
VANZELA1, A. P. F. C.
1

Universidade Federal dos Vales do Jequitinhonha e Mucuri,Diamantina-MG, Brazil.

Amylases are among the most important biocatalysts and, together with other industrial and special enzymes, represented USD 140 million for Brazilian importation
and USD 49.6 million for exportation in 2012. Fungal strains are good sources for industrial production of amylolytic enzymes with interesting properties. This work aimed to
determine the optimum conditions for amylase production by an isolate ofTallaromycestrachyspermusT10.5. A central composite design (CCD) 23was generated withStatistica7.0 by combining high and low values of three independent variables: starch (1-3
g L-1), yeast extract (1.5-3.5 g L-1) and pH (4-8) in 8 planned assays. Axial (, -1.41 and
1.41) and central values (2g L-1starch, 2.5g L-1yeast extract; pH 6) of each variable were
also combined. All incubations were carriedout at 30 C and 150 rpm for 96 h. Biomasses were separated, and -amylase was determined in the filtrates. One unit of activity was defined as the amount of enzyme which hydrolyzes 10 ?g of starch per min per
mL. Amylase activity increased progressively from 24 to 72h. The highest activity (338.3
6.8 U min-1 mL-1) was determined after 72 h in bioprocesses adjusted for the central
values. A similar activity (338 U min-1mL-1) was produced in a bioprocess supplemented
with 2g L-1 starch; 4.18 g L-1 yeast extract at pH 6. The lowest concentration of yeast
extract resulted in a decreased enzyme production (148.6 U min-1 mL-1). Elevated and
diminished amounts of starch or extreme pH values were also prejudicial. The results
were indicative of optimization, since the highest values for -amylase production were
obtained in the central points. Analysis allowed to conclude that -amylase production
byTallaromycestrachyspermusis strongly influenced by pH. It was also concluded that
strain T10.5 is suitable for -amylase production, because by adjusting external conditions it was possible to obtain an elevated enzyme activity in a short period of time (72h).

SIMB 2013 | 63

IDENTIFICATION OF THE VOLATILE COMPOUND 6-PENTYLALPHA-PYRONE PRODUCED BYTRICHODERMASPP.

DIAS, M. C. F.1, ACOSTA, J. A. M.1, LUZ, J. M. R.1; DEMUNER, A. J.1; KASUYA, M.
C. M.1.
1Universidade Federal De Viosa, Campus Viosa, Avenida Peter Henry Rolfs, S/N.
Campus Universitrio. Viosa / MG. Brasil.
Biological control has been an alternative to the use of chemicals to control pests
and diseases. Fungi of the genus Trichoderma are of great economic importance for
agriculture and has been quoted to produce secondary metabolites with antimicrobial
activity. One of theses metabolites belong to 6-pentyl-alpha-pyrone (6-PP), which present antifungal activity potential, and can be used to control phytopathogenic fungi. The
aim of this study was to investigate the production of lactone 6-PP by five species of the
genusTrichoderma:T. virede, T. harzianum, T. virens,T. pseudokonigiiandT. asperullum.The fungi weregrown in test tubes containing potato dextrose agar, for seven days
at temperature 25 C. The volatile compounds was adsorbed in fiber coated by PDMS/
DVB in the headspace of the vials containing the fungi, for 30 min.Then, the volatile
compounds were desorbed for 2 min in split-/splitless injector, and analysed by a gas
chromatograph coupled with a mass detector. Only fungi T. harzianum and T.asperullumproduced the 6-PPat 15% and 39,52%, respectively. As abiotic and biotic stress
can affect secondary metabolites production, new conditions for fungi growth has been
investigated for obtaining this coumpound in higher quantities in allTrichodermaspecies.
Acknowledgements: CAPES UVF

SIMB 2013 | 64

HEAT-TOLERANT FUNGI ON COMPOSTING HEAPS OF

SUGARCANE FILTER CAKE: DIVERSITY AND POPULATION
DYNAMICS
TSSIO BRITO DE OLIVEIRA1; ELENI GOMES2; ANDR RODRIGUES1
1 Department of Biochemistry and Microbiology, UNESP So Paulo State University,
Rio Claro, SP, Brazil. 2 Department of Biology, UNESP So Paulo State University, So
Jos do Rio Preto, SP, Brazil
The exothermic metabolism of microorganisms leads to self-heating of compost
heaps, providing an ideal environment for the development of heat-tolerant fungi. The
present work aimed to evaluate the succession of thermophilic and thermotolerant fungi
on composting heaps made of residues from the sugarcane juice filtration process (i.e.
filter cake). We sampled 10 g of substrate from three composting piles during the mesophilic I, thermophilic and mesophilic II phases over 60 days. Samples were ten-fold
diluted and inoculated in Yeast Glucose Agar, Yeast Starch Agar and Malt Agar 2% supplemented with antibiotic and incubated at 45 C for five days. Fungal isolates were purified and, subsequently identified using morphological and molecular markers (DNA
sequencing of barcode regions). A total of 332 filamentous fungi were obtained comprehending seven genera and 10 species. Five ascomycetous fungi,Aspergillus fumigatus,Myceliophthorasp.,Myceliophthora thermophila,Scytalidium thermophilaandThermomyces lanuginosusaccounted for 91.6% of the isolates. In addition, five zygomycetous
species,Lichtheimia ramosa, Rhizomucor pusillus, Rhizomucorsp. 1,Rhizomucorsp. 2
andRhizopus microsporusrepresented 8.4% of the isolates.A. fumigatusandT. lanuginosuswere the prevalent species representing 66.3% and 16% of the isolates, respectively.
These species were observed in all heaps over the whole composting process.Myceliophthorasp.,M. thermophila,R. pusillusandRhizomucorsp. 2 were observed in all phases
of the composting process, but they were not found in all phases in each heap.L. ramosawas restricted to the mesophilic I phase and was the first taxon to disappear in the
succession.S. thermophilumwas present in the first phase and the mesophilic II phase.
Rhizomucorsp. 2 andR. microsporuswere observed at first in the thermophilic and the
mesophilic II phase. According to the Shannon and Simpson diversity indices, except
to one heap, fungal diversity increased over the succession, and heaps shared a high
number of species. The heat-tolerant fungi community dynamic was different among
the three heaps, suggesting that several factors such as humidity and aeration in addition
to temperature, may influence the species composition over the composting process of
sugarcane filter cake.
Acknowledgements: Usina Santa Lcia (Araras, SP) Financial support: FAPESP

SIMB 2013 | 65

SOIL MICROBIAL ACTIVITY SUBMITTED TO HERBICIDE

AND GROWTH OF CASSAVA
DIAMANTINA DA COSTA S.S.(1); FERREIRA E.A. (1); VALADO SILVA D. (2);
SANTOS J.B. (1)
(1) Federal University Of The Jequitinhonha And Mucuri; Diamantina-MG; Brazil; (2)
Federal University Of Viosa, Viosa-MG; Brazil
The response of cassava to apply herbicides ranges from full selectivity for
some products until the full commitment of production because of poisoning caused to
culture. Considering also the effect of these products on soil microbiota usually small
changes in soil quality associated with changes in its microbiological properties, which
exhibit high sensitivity to perturbations arising from the management. Thus, the aim
of this study was to evaluate the effect of post emergence herbicides in total area and
directed the microbial activity of soil cultivated with cassava, as well as its effect on crop
growth, through the respiratory rate through the respirometric method for evaluating
the CCO2 evolved soil and biomass carbon; visual evaluations of intoxication and dry
weight of root, stem, leaf and total. The experimental design was a randomized block
with four replications and the treatments consisted of a attestant weeded a control grown
without weed control (weeds) and plots subjected to herbicide application: fluazifopp-butyl; fomesafen; glyphosate; paraquat e a mistura fluazifop-p-butyl + fomesafem.
Herbicide application fomesafen mixture and fluazifop-p-butyl + fomesafen treatments
that are most negatively affect microbial biomass carbon. Increased soil stability was observed for plots where cassava plants grown without weeding (in the bush), which shows
the lowest metabolic coefficient, and, fluazifop-p-butyl, and the mixture fomesafen these
herbicides are those that most affect metabolic coefficient, reducing the stability of the
soils where these herbicides were applied. The mixture fluazifop-p-butyl + fomesafen
provides greater toxicity in cassava plants, and greater reductions in dry mass of plants
treated with this product.

SIMB 2013 | 66

DETECTION OFSALMONELLASPP.AND PHYSICALCHEMICAL QUALITY OF FRESH-CUT PARSLEY MARKETED IN

NORTHWEST FLUMINENSEREGION
BRANDO VEIGA, E. O.; RABELO, E. R.; TONINI, C. B.; RIBEIRO, M. C. B.;
CHAVES, C. R.
Instituto Federal Fluminense CampusBom Jesus, Bom Jesus do Iabapoana, RJ, Brasil
The consumption of fresh vegetables has increased significantly due to the change
in the populations life style. Thus, theminimal processing appears to provide convenience and time savings in preparation of daily food. Fresh fruit and vegetables, usually
consumed raw, is being increasingly associated with the outbreaks food. In some ways,
the aim of this study was research for the presence ofSalmonellaspp.andevaluate the
physical-chemical quality of fresh-cut parsley marketed in Northwest Fluminense region. The samples (n= 6) were obtained in the retail trade of the region and analyzed,
in quadruplicate, in the Microbiology and Physical-chemical laboratories of the Fluminense Federal Institute, during the autumn of 2013.The microbiological analysis consisted in theSalmonellaresearch, as described in the normative instruction n62/2003
(MAPA). The physical-chemistry evaluation was performed by the measurement of pH
and titratable acidity according to the Adolfo Lutz Institutes methodology. The microbiological results were confronted with parameters established by ANVISA (Resolution
n12/01), that is, absence in 25g of food.Salmonellawas found in 33% of samples analyzed. This result may be associated with the lack of good manufacturing practices and
possible cross-contamination. The physical-chemical analysis showed that the pHof the
samples ranged between 5.4 and 5.8, pH favorable to the development of microorganisms per benear neutrality. The titratable acidity ranged between 6.4% and 17.5%, showing that the fresh-cut parsley presents an acidity higher thanin natura, as described in
the literature. This may be due tominimal processing. The results reveal that the freshcut parsley minimally processed sold in the Northwest Fluminense are in disagreement
with the Brazilian Legislation because it showed the presence ofSalmonella, a potentially
pathogenic microorganism that representsa risk to consumers health.

SIMB 2013 | 67

SOIL MICROBIAL ACTIVITY IN DIFFERENT DEPTHS

WITH CULTIVATED SPECIES ASBRACHIARIA
BRIZANTHAREMEDIATION OF PICLORAM
DIAMANTINA DA COSTA S.S.(1); FERREIRA E.A. (1); BRAGA R.R. (2); SANTOS
J.B. (1)
(1) Federal University of the Jequitinhonha and Mucuri; Diamantina-MG; Brazil; (2)
Federal University of Viosa, Viosa-MG; Brazil
The hormonal herbicide exhibit high persistence in soil, they have to promote the
effect of weed control for long periods, plus the ability to leach depending on soil and climatic conditions. The principal molecules have been used 2,4-D and Picloram, comprising almost all commercial formulations suitable for grazing. Thus, the aim of this study
was to evaluate the effect ofB. brizanthaused as remedial species in microbial activity of
soil contaminated with picloram and in different conditions regarding their pH values.
For it was mounted two experiments (one at pH 4.5 and another at pH 5.6) in a protected environment. The experimental design for both studies was a randomized block
design with four replications in a split plot design 2x2x4 being the first level with and
withoutB. brizantha, the second level of the herbicide doses (0, 240 g ha-1 picloram in
Padron commercial formulation) and third level as the evaluated depths (0 to 10, 10 to
20, 20 to 30 and 30 to 40 cm). B. plantaginea affected differently TR, and the CBM qCO2
both in soils of pH 4.5 as at pH 5.6 in all cases, two levels of acidity in the soils treated
with picloram evaluated had higher values for these variables except at pH 5.6 where the
upper layers of 0-20 cm was found that the soils without applying the product showed
higher qCO2. Importantly, soils cultivated with forage in the presence of the herbicide
showed the highest values of TR and CBM may indicate the action of microorganisms
in the degradation of picloram.

SIMB 2013 | 68

ASSOCIATION OF THE FUNGUSPIRIFORMOSPORA

INDICAWITH'IMPERIAL' PINEAPPLE PLANT AND
HERBICIDE APPLICATION
LANA IVONE BARRETO CRUZ;MARIA DO CU MONTEIRO CRUZ;AMANDA
MIRANDA SOUZA;GUILHERME DUMB MONTEIRO DE CASTRO
Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina
-Minas Gerais- Brazil

Mycorrhization with pineapple plant favors growth nursery by improving nutrient

uptake allowing its early production. However, weed management is an indispensable
practice after the nursery planting in field, since they negatively affect the crop growth.
The work was carried out to evaluate the association mycorrhizal, growth and nutrient uptake in 'Imperial' pineapple nursery inoculated with Piriformospora indica and
sulfentrazone application. The experiment was carried out in a greenhouse of UFVJM
using 'Imperial' pineapple nursery. The factorial 2 x 4 was used, referring to two inoculations:P. indicaand without inoculation and four sulfentrazone doses 0, 0.4, 0.8 and 1.6
L ha-1, distributed in a completely randomized design with three replications. Inoculation was done at planting. Herbicide application was taken 24 hours before planting
nursery. At 150 days after inoculation was made the analysis of colonization, levels N,
P and K and growth nursery. In the colonization analysis was found to associated ofP.
indicawith the pineapple roots at all herbicide tested doses. Increase of more than 29%
in the levels of N and P was observed in inoculated nursery with theP. indicawhen under the effect of the mean dose of 0.8 L ha-1and for K the increase was proportional to
the dose applied, while those without inoculation no differences were found in relation
to N and P, verifying linear decrease of the K levels. The associated nursery with theP.
indicashowed significant increases in size, the estimated with mean dose of 0.74 L ha1
, while in the non-inoculated nursery decreased linearly with increasing doses of the
herbicide. The association ofP.indicawith Imperial pineapple nursery was observed
with the application sulfentrazone. Imperial pineapple nursery associated withP.indicashowed higher levels of nutrients and growth. The sulfentrazone at doses greater than
0.8 L ha-1interfered in growth and nutrient uptake by mycorrhizal pineapple nursery.
Financial support: CAPES, CNPq, FAPEMIG

SIMB 2013 | 69

FILM BIODEGRADABLE PRODUCTION FROM MICROALGAL

BIOPOLYMER
IGOR SEVERO GONALVES, ROBERTA GUIMARES MARTINS, MICHELE
GREQUE DE MORAIS, JORGE ALBERTO VIEIRA COSTA
Laboratory of Biochemical Engineering -School of Food and Chemistry -Federal
University of Rio Grande (FURG) - Street Eng. Alfredo Huck - 475 - Center -Rio Grande
-RS - 96201-900 -Brazil
Microalgae are microorganisms that synthesize biopolymers, which belong to
the group of polyhydroxyalkanoates (PHA). A type of PHA is poly-?-hydroxybutyrate
(PHB) which is stored as asource of energy and reserves, besides presenting biocompatibility and biodegradation. In view of the disposability of plastics from petrochemical
origin are having different environmental impacts, emerge as an alternative,the use of
biopolymers for reducing pollution. The aim of the work was to produce film from biopolymers extracted and purified from Spirulina sp. LEB 18.The microalga was cultivated in Zarrouk medium. The extraction of PHB wasperformed by differential digestion
and subsequent washing with distilled waterand acetone. The product resulting from the
extraction was defatted with hexaneand further purified by solubilization in propylene
carbonate and precipitationin acetone. The purified PHB (0.25 g) was dissolved in 25
ml of chloroform for 2 hours under stirring at 50 C. The solution was placed in a petri
dish and allowed to air-saturated chloroform until complete solvent evaporation. Film
of PHB dried in less than 24 h, forming a homogeneous film, showing that the method
of atmospheric saturation is efficient for this type of film. The biopolymer produced by
Spirulina sp. LEB 18 was efficient for the production offilms which can be used for food
packaging, for presenting antimicrobial and antifungal characteristics.

SIMB 2013 | 70

SELECTION OF XYLOSE-FERMENTATION YEAST WITH

POTENTIAL TO PRODUCE ETHANOL SECOND GENERATION
SILVEIRA, D. A. F.1; FIETTO, L. G.2; PASSOS, F. M. L.1; SILVEIRA, W. B.1
Laboratrio de Fisiologia de Micro-organismos, Departamento de Microbiologia/
BIOAGRO, Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil.2Laboratrio
de Biotecnologia Molecular, Departamento de Bioqumica e Biologia Molecular,
Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil.
1

Fossil fuels are sources of non-renewable primary energy that cause environmental problems due to their combustion, and subsequently emission of greenhouse gases
(GHG). Biofuels are an interesting strategy to reduce these GHG. Ethanol, produced
from renewable sources, such as plants and organic matter, is the most produced biofuel worldwide. Second generation ethanol is produced from agricultural and forestry
by-products as feedstock (lignocellulosic biomass). Xylose is the second most abundant
sugar in the lignocellulosic biomass hydrolysate. Nevertheless, Saccharomyces cerevisiae, the yeast commonly used in the industrial fermentation processes, does not assimilate xylose. The aim of this work was to select xylose-fermenting yeasts capable of
producing ethanol from xylose. Initially, yeast strains belonging to the culture collection
of the Laboratory of Physiology of Microorganisms, Universidade Federal de Viosa,
were selected for their fermentation potential in minimal medium Yeast Nitrogen Base
(YNB) containing xylose as sole carbon and energy source. Subsequently, fermentation
experiments were conducted with the selected strain H4, in fermenter Bioflo310 NEWBRUNSWICK equipped with dissolved oxygen (DO) probe, in YPX medium with 2%
xylose (w/v), 10% of DO at 30 oC and pH 6.0. After 48 hours, the H4 strain produced
27.82 g L-1of ethanol. It reached an ethanol yield (YP/S) of 1.32 g g-1and a volumetric productivity (QP) of 0.58 g L-1h-1. These results demonstrated that this strain has potential
for second generation ethanol production from lignocellulosic feedstock.
Acknowledgements: FAPEMIG, CAPES, CNPq.

SIMB 2013 | 71

TRICHODERMA SPP.EXTRACTS WITH ALLELOPATHIC

ACTIVITY
MARTNEZ ACOSTA, J.A.1,2; DEMUNER, A.J.1; ALMEIDA BARBOSA, L.C.1,3;
ALVARES MALTHA, C.R.1; MEGUMI KASUYA, M.C.4; RODRIGUES DA LUZ,
J.M.4; MOURA, G.1
1. Laboratrio De Anlise E Sntese De Agroqumicos, Dpto De Qumica, UFV, Viosa
(MG), BRA; 2. UTP, Pereira (Ris), COL; 3. UFMG, Belo Horizonte (MG), BRA; 4.
Laboratorio De Associaes Micorrzicas, Dpto De Microbiologia, UFV, Viosa (MG),
BRA.
The genusTrichodermacomprises a large number of fungal strains that present biological control potential. Activation of these mechanisms are related to the production
of specific compounds: plant growth regulators, enzymes or antibiotics. Some of these
secondary metabolites are pyrones. In this work we evaluated the allelopathic activity
of ethyl acetate extract of five species ofTrichodermaonSorghum bicolorandCucumis
sativus. TheTrichodermaspecies were:T. harzarium(Th),T. viride(Tv),T. reesei(Tr),T.
longibrachiatum(Tl) andT. aureoviride(Ta), This fungi were grown in strength PDB
for 20 d at 25 C by shaking at 100 rpm. The substances were extracted with ethyl acetate
and analyzed by GC-MS using a column HP5-MS 30 m0.25 mm0.25 mm; oven temperatures of 40 C (2 min), 10 C/min to 200 C, 25 C/min to 260 C (5 min), acopled
to mass detector EI 70 eV, source 230 C. The extracts were tested in the Petri dish assay
to determine the effect on the germination of cucumber and sorghum with concentrations of 1000, 500, 250, 125, 62.5 and 10 mg L-1in 0.5% DMSO at 25 C for 7 d. The
most representative compounds were: 2-Phenylethanol (in all fungus) and the pyrones
Dehydroacetic acid and Dehydromevalonic lactone (not produced by Ta). The contents
of the compounds ranged from 10-60%, 3-18% and 5-15%, respectively. The extracts
of Tr and Tv, showed the greater root growth inhibition of sorghum and cucumber at
concentrations of 500 mg L-1, inhibiting 65-75% of growth with Tv and 125 and 250 mg
L-1inhibiting 40 and 60%, respectively with Tr. The strains of Tv and Tr tested show to
have high potential to produce metabolic compound to control weeds.
Acknowledgements: UFV, LASA, MIC, CNPq, FAPEMIG, CAPES.

SIMB 2013 | 72

CHARACTERIZATION OF A BACTERIOPHAGE ASSOCIATED

WITH NOSOCOMIAL INFECTIONS CAUSED BY MULTIDRUGRESISTANT BACTERIA
JONAS SILVA TEIXEIRA, VINCIUS DA SILVA DUARTE, ROBERTO SOUSA
DIAS, CYNTHIA CANDO DA SILVA, EDUARDO RESENDE HONDA, SRGIO
OLIVEIRA DE PAULA
Universidade Federal De Viosa,Laboratrio de Imunovirologia
The emergence of Multi-Drug Resistant Bacteria is consequence of the high selective pressure coming from the indiscriminate use of antibiotics, especially in hospital environment. The study of mobile genetic elements in the horizontal transfer of
antibiotic resistance genes in a given environment, called "resistome", has given focus
mainly plasmids,transposons and pathogenicity islands with few reports about the real
role of bacteriophages as carriers of resistance by transduction mechanisms. The aim
of this study was to characterize a phage carrier of antibiotic resistance gene belonging to the aminoglycosides. Analysis by transmission electron microscopy allowed us
to characterize the virus as belonging to the order Caudovirales, family Podoviridae.
The molecular weight of the phage genome it was estimated in approximately 22,380 kb
by conventional electrophoresis. Searching to find possible resistance genes to different
classes of antimicrobial agents in bacteria and phage, PCR was performed with specific
primers related to different classes of antimicrobials such as Aminoglycosides , Betalactams, Cephalosporins and Sulfas.Ampliconswere observed only related to aminoglycosides such as streptomycin, for example. A polyacrylamide gel electrophoresis (SDSPAGE) showed that the phage has three major proteins (43 kDa , 40 kDa and 23 kDa)
.The N-terminal sequencing of the 43 kDa protein showed that this protein is found in
other bacteriophages belonging to the orderCaudovirales, such asPelagibacter,Bacillus
subtilisBacteriophage SPO1andSynechococcus. Access numbers in NCBI, respectively
: YP_007517785.1 , YP_002300330.1 and YP_007518261.1 . The sequencing of the viral
genome is being performed. It is concluded that a podovirus carries genes for resistance
to the aminoglycosides, allowing its transfer to other bacteria.
Acknowledgements: Ncleo de Anlise de Biomolculas (NuBioMol)

SIMB 2013 | 73

ENDOPHYTIC FUNGI FROMPHASEOLUS VULGARISL.

INHIBITIN VITROGROWTH OF PHYTOPATHOGENIC FUNGI
AND EXHIBIT ANTIMICROBIAL ACTIVITY
TAIDES TAVARES DOS SANTOS1; AUGUSTO VELOZO GONALVES1; ELZA
FERNANDES DE ARAJO1; MARISA VIEIRA DE QUEIROZ1
1

Universidade Federal de Viosa, Viosa - MG, Brazil

Endophytic fungi have been studied for their capacity to produce secondary bioactive metabolites with potential applications in different areas, and also for their capacity for biological control of phytopathogens. The aim of this study was to investigate the antimicrobial activity of endophytic fungi from Phaseolus vulgaris L. and the
potential of these fungi in inhibiting the mycelial growth of phytopathogens. In vitro
microbial antagonism by dual culture assay was assessed between ninety-two endophytic (twenty-seven different species) and four phytopathogenic fungi (Colletotrichum
lindemuthianum, Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum).The isolates with the best antagonistic activities were submitted to solid state fermentation, using wheat bran as organic substratum, and crude extract was obtained
with ethyl acetate. Antifungal activity was evaluated by the poisoned food assay against
the same phytopathogenic fungi and anti-bacterial activity was evaluated by the agar
diffusion method against Bacillus cereus, B. subtilis, Escherichia coli, Listeria innocua,
L. monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus. Among the
results, it was found that 45 endophytes significantly inhibited the growth of C. lindemuthianum (22.6% - 62.7%), 33 inhibited F. oxysporum (34.9% - 53.6%), 15 inhibited
R. solani(30.4% - 49.1%) and 25 inhibited S. sclerotiorum (27.7% - 46.9%).The isolates
Bipolaris sp. (CMT 68), Cochliobolus sativus (CMON 25 and CMON 27), Cochliobolus
sp. (CMON 53) and Fusarium sp. (CMT 37) showed good antagonistic activity in the
dual culture assay and their extracts were assessed.The extracts from CMON 25, CMON
27, CMON 53 and CMT 37 significantly inhibited growth of the tested phytopathogenic
fungi, while the extracts from CMON 27, CMON 53 and CMT 37 inhibited growth of all
Gram-positive bacteria. Bean endophytic fungi show potential for phytopathogen biological control and produce promising antimicrobial metabolites, which can and should
be explored in terms of biotechnology.
Acknowledgements: CAPES, CNPq and FAPEMIG for financial support.

SIMB 2013 | 74

CHARACTERIZATION OF A PARTIALLY PURIFIED

ENDOGLUCANASE FROMASPERGILLUS NIGER SP.
JEFFERSONVIKTOR DE PAULA BARROS BATA; GABRIEL CABRAL
UFV,Campus Viosa,Avenida Peter Henry Rolfs, s/n,CampusUniversitrio,Viosa MG
- Brasil,CEP: 36570-000
Beyondits use for energy purposes, cellulases have been widely usedintextile, lapping
and processing of cellulosic fibers.Amongtheseenzymes, endoglucanases have applicability in the production offruitjuices, beer filtration, detergent production and extraction
ofoil,among others. These applications suggest the necessity of awiderange of specific
and thermostable endoglucanases, with differentpHvalues and optimal temperatures.
The purpose of this study wastocharacterize a partially purified endoglucanasefromAspergillusniger sp.This microorganism wascultured for six days (28 C, 180 rpm) and
theobtained enzyme waspartially purified by precipitation with(NH4)2SO4,molecular
exclusion and ion exchange chromatographies. Theinfluenceof temperature and pH on
the endoglucanasic activity wasevaluated atpH's between 3.0 and 8.0 and temperatures
between 30 and80 C. Forthe evaluation of thermal stability, the enzyme sampleswereincubated for 6 h at temperatures of 60, 70 and 80 C.Kineticconstants Kmand Vmaxwere
calculated by determining enzymaticactivities at differentconcentrations of carboxymethylcellulose. Theinfluence of metal ionsand reagents was evaluated by adding them
tothe buffer used in the enzyme assays. Enzyme assays were performed bythe method
of3,5-dinitro-salicylic acid reagent. The pH andtemperature of theenzyme optimum activity were 4.0 and 50 Crespectively. At thistemperature, the endoglucanase showed
lowthermostability, losing approximately 80% of its activity during 2.5 hof incubation. Thekinetic constants were Vmax 0.288 ?mol.min-1and Km0.018 g.mL-1.The ions
Ca2+,Mg2+,and Zn2+and reagents SDS and EDTAinfluenced positively, while the presence of Mn2+and Cu2+decreasedthe enzymes activity. Thus, this partialcharacterization becomesuseful to infer the viability of thisendoglucanase for industrialapplications.
Acknowledgements: CNPq, FAPEMIG e CAPES

SIMB 2013 | 75

KINETIC PARAMETERS AND CARBOHYDRATES CONTENT

DETERMINATION INSCENEDESMUS ACTUSANDCHLORELLA
FUSCAMICROALGAE STRAINS
ANA PAULA AGUIAR CASSURIAGA; ETIELE GREQUE DE MORAIS;
BRBARA CATARINA FREITAS; EDUARDA HOLZ BRACHERE JORGE
ALBERTO VIEIRA COSTA*
FederalUniversity of Rio Grande - School of Chemical and Food - Laboratory of
Biochemical Engineering - Rio Grande, RS Brazil
Microalgae are photoautotrophic, unicelular, eukaryotes or prokaryotesmicroorganisms, which have the ability to grow in different environmentalconditions and can
modify their metabolism, becoming attractive for specificcompounds synthesis such as
carbohydrates. Due to high productivity and the factthat no fertile land for cultivation,
microalgae has been used for biomass productionwith a view to preparing food and getting bioproducts. The aim of this studywas to determine kinetic parameters of growth
and quantify carbohydrate inScenedesmus actusandChlorella fuscabiomass. The cultureswere performed in triplicate with BG-11 modified media in closed 2L erlemeyer
photobioreactors(working volume of 1.8 L) under optimal cultivationconditions and
initial cellconcentration of 0.2 g L-1. The temperature was set at 30 C,illuminance of
3200 lx, photoperiod 12 h light / dark and stirring wasperformed by pneumatic pump.
Every day cell concentration was determined, byspectrophotometry at 670nm, and pH
by digital pHmeter. Maximum cellconcentration, productivity, specific growth rate and
generation timeparameters were determined. The carbohydrates were determined by
Dubois (1956)method. The highest cell concentration was obtained in Scenedesmus
actusculture (0.62 g.L-1) which wassignificantly different (p> 0.05) fromChlorellavulgarismicroalga (0.48 g.L-1). Maximum productivity, specificgrowth rate and generation
time showed no significant difference (p> 0.05)between the study strains.Chlorellavulgaris showed carbohydrate content of 20.0% and Scenedesmus actus obtained 17.3%.
Thestudiedstrains can be applied as a carbohydratesource with biotechnologicalapplication inbiofuels production such as bioethanol.
Acknowledgements: The authors acknowledge CAPES and FAPERGS for financial support

SIMB 2013 | 76

PLACKETT-BURMAN DESIGN AS A TOOL TO IMPROVE LIPID

PRODUCTION BYRHODOTORULA MUCILAGINOSA
BOEIRA, C. Z.1; SPIER, F.1; UEBEL, L. S.1; BURKERT C. A. V.1
Federal University of Rio Grande, School of Chemistry and Food - FURG, Rio Grande,
RS, Brazil
1

The Plackett- Burman designs are extremely useful when you want to choose one
or two most important factors of many factors used . Thus work aimed to select variables
that exert major effects on lipid production from raw glycerol derived from biodiesel
synthesis. The wild yeast Rhodotorula mucilaginosa was cultivated in shaken flasks at
180 rpm in 500 ml Erlenmeyer flasks containing 180 ml of medium whose composition
was determined according to the experimental design that presented 16 trials over 4 central points, where 10 variables were evaluated at 3 levels each. The responses evaluated in
PlackettBurman design were maximum biomass, biomass productivity, lipid content,
total lipids and lipid productivity. The software Statistica (Statsoft, v. 7.0) was used to analyze the results. In the assay that stood out from the others, parameters like concentration of MgSO4.7H20 , Na2HPO4 , yeast extract , CaCl2 . 2H2O and ZnSO4.7H2O were
at the highest levels (+1), while KH2PO4 , glycerol, pH, temperature and FeCl3.6H2O
were at the lower levels (-1). Besides presenting high lipid content, 59.27 % , this condition showed the higher biomass ( 9.29 g.L- 1 ) , higher biomass and lipid productivity (
0.038 g.L - 1.h - 1 and 0.023 g.L - 1.h -1, respectively ) and higher total lipids produced
(5.50 g.L -1). According to the results the greatest effects were found for yeast extract and
MgSO4.7H20, these being fixed at the level +1 (1,2 e 3 g.L-1, respectively) . With respect
to temperature, which showed a significant negative effect on biomass , it was set at - 1
(25C). How other variables showed no significant effects, they were fixed on level -1 ,
thus the salts of calcium, iron and zinc were excluded of the medium . The cultivation
condition established by PlackettBurman design was tested , showing an increase of 8.9
% in lipid content and 28.4 % in total lipid. These parameters are extremely important
because they are related directly to the yield of the process.
Acknowledgements: CAPES, CNPq and FAPERGS.

SIMB 2013 | 77

VALIDATION OF MOLECULAR CHARACTERIZATION

METHOD BY ELECTROPHORESIS
MARIA AUGUSTA DE CARVALHO SILVELLO, ROSANA BASSO KRAUS,
TIAGO SILVEIRA LIMA, JLIO CSAR FLORES JOHNER, JAQUELINE GARDABUFFON.
Federal University Of Rio Grande, FURG, School Of Chemistry And Food, Laboratory Of
Mycotoxins, Street Eng. Alfredo Huch 475 Center, 96201-900 Rio Grande (RS), Brazil
SDSPAGE analysis has supported protein characterization of microorganisms including the biomarkers study. The validation by SDS-PAGE electrophoresis in photo
documentation steps and images analysis using software ImageJ was tested using albumin standard and molecular weight marker.The protein standard was diluted to a concentration of 0.4 mg / mL for determination of detection limit and method linearity by
the estimation of the image pixes variations (peak area), correlated with the concentration of six dilutions which were tested as minimum and maximum range of resolution.
The molecular mass marker with seven fragments was used to evaluate the efficiency of
the program ImageJ as protein characterization and to construct the curve relating Rf
and molecular weight in electrophoresis, comparing statistically manual and automated
measures of three analysts. The validation set the linearity method, between 0,03 and 1,5
g of protein, and 60 ng was fixed as the lower quantification limit and 1,5 g as the upper quantification limit. While there are no significant differences between manual and
automated measurements, the benchmarks made by program may have less subjectivity
and express a response with greater precision compared to manual measurement. The
equation generated by the points linearization was y= - 60,6x +251 (R2 = 0,990) for the
manual measurement and y = - 259.1 X 246 (R2 = 0.982 ) for automated measurement,
and the answer was the molecular weight (kDa). Thus, the Electrophoresis SDS PAGE
method validation by photo documentation and images treatment by ImageJ program
provide subsidies for the molecular characterization by electrophoresis, including biomarkers detection.
Acknowledgements: CAPES, FURG e ImageJ

SIMB 2013 | 78

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION

OFSEIRIDIUMSPP.ASSOCIATED WITH CYPRESS CANKER IN
PORTUGAL
MILAGRE, J.C.1,2; RAMOS, A.P.1;ROCHA, M.1; BELCHIOR, S.1; MORAIS JUNIOR,
V.T.M.2;CAETANO, M.F.1
CEER, ISA, Universidade de Lisboa, 1349-017 Lisbon, Portugal 2Universidade
FederalViosa, 36570-000 Viosa, MG, Brazil;
1

Cypress canker is a serious disease that has devastatedCupressusspp. in Mediterranean basin.Seridium cardinaleandSeridium unicorne represented twodistinct species
of fungi that cause cypress canker in Portugal, threatening the occurrence ofCupressus
sempervirens, both as forest and as ornamental or hedge tree.The aim of this study was
to evaluate possible variations among the populations of the causal agents from different
regions of Portugal.A sanitary survey was conducted in clonal plantations in Tavira, Lisbon and Coimbra. Isolates were obtained of plants bearing canker. Assessments of morphological and cultural characteristics were performed for each isolate grown at 22, 24,
26, 28 and 30 C. The DNA was extracted and purified from freeze-dried mycelium and
then the products were submitted for sequencing of rDNA ITS region (ITS1 and ITS4).
The results showed that the optimal growth temperature was isolate-dependent occurring between 24C and 28C forS. cardinaleisolates. Colonies ofS. unicorneshowed a
faster radial growth at 22C than at 26C. Colonies were dense, cottony, white at first,
then turning grey to dark brown inS. cardinalean pale grey-green inS. unicorne. The
BLAST similarity search confirmed the identity of all the isolates (100% homology with
sequences ofS. cardinaleandS. unicorneisolates, from Italy and New Zealand, respectively). Morphological and molecular analysis showed that the populations of both species are very homogenous which is in agreement with the evidence of clonal reproduction described for the fungi in the country.This work contributes to the development
studies of new clones to control the disease in Mediterranean countries under the scope
of EU projects.
Financial support: EU/MED Programme/ERDF & CNPq.

SIMB 2013 | 79

CHARACTERIZATIONOF AN ENDOGLUCANASE
FROMASPERGILLUS NIGERSP
GABRIELLA CHRISTINA GONALVES MANINI DE PAULA; ISADORA
OLIVEIRA PATRA;BIANCA LANA DE SOUSA; GABRIEL CABRAL BARROS;
PEDRO HENRIQUE SCARPELLI PEREIRA; JEFFERSON VKTOR DE PAULA
BARROS BATA; IZADORA LORRANY ALVES; RABELOJOS HUMBERTO DE
QUEIROZ.
Universidade Federal de Viosa, MG, Brasil.
Not only the depletion of fossil fuel reserves, but also the environment damages
have been encouraging the search for new renewable energy sources, such as biomass
wastes. In this scenery, cellulases and hemicellulases enzymes play an important role,
because they can hydrolyze lignocellulosic biomass sources to produce bioethanol, biodegradable alcohol and far less toxic than fossil fuels.Therefore, to try find new industrial enzymes out with high specificity and efficiency, the intuit of this work was to produce
and to characterize an endoglucanase from Aspergillus niger sp regarding optimum pH,
ideal temperature and thermal stability. After six days of the microorganism incubation at 28 C and 180 rpm, the enzymatic extract was partially purified by precipitation
with ammonium sulfate followed by chromatography of molecular exclusion and, subsequently, of ion exchange.The enzymatic activity of endoglucanase was valued in a pH
range of 2,0 to 8,0 and the optimum pH achieved was approximately 5,0. In addition,
to establish the ideal temperature, enzymatic activity was performed in a temperature
range among 40C and 80C; however no significant difference among activities was
found. Then, the assays were performed in 60C, 70C and 80C, until lack of activity, to
determine the thermal stability. As a result, at 60C, none significant difference in enzymatic activity was found. Nevertheless, after 3 hours of incubation at 80Cand 2 hours at
90C,the endoglucanase activity dropped more than 90%, suggesting that this enzymeis
not stable in temperatures above 60C.

SIMB 2013 | 80

LIGNIN RECOVERY OPTIMIZATION FROMBLACK LIQUOR

RESIDUES OF SUGARCANE BAGASSE CELLULIGNIN
ALKALINE HYDROLYSIS
ANTNIO AVELAR XAVIER1;SLVIO SILVRIO DA SILVA2
UniversidadeFederal de Juiz de Fora, Juiz de Fora - MG, Brazil 2Escolade Engenharia de
Lorena - Universidade de So Paulo, Lorena - SP, Brazil
1

Ligninis a natural organic polymer with major occurrence in the world and can
befound in all vascular plants. Next to cellulose and hemicellulose is one of themost
abundant components of the fibers of all kinds of woody plants and grassescell walls.
This polymer is composed of the alcohols coniferyl, ?-coumaryl and sinapyl in different proportionsaccording to the plant species and has been used in industrial processes
insteadof synthetic polymers such as phenolic foams and bio fertilizers production.
Thisstudy aimed to contribute to the optimization of lignin recovery from theresidual
black liquor obtained from the sugarcane bagass celulignin alkalinehidrolisis. for that,
2 kg of cellulignin (product of sugarcane bagasse acidhydrolysis) underwent alkaline
hydrolysis (NaOH concentration of 1.5%, solid/liquidratio 1:20, reactor rotation of 1.72
RPM and temperature of 120 C for 60minutes). The lignin recovery from black liquor
(200 mL samples) was testedusing different pH (1, 4 and 7), temperatures (0, 25 and 50
C) and mixing times(0, 12 and 24 h). for recovery yields evaluation, the liquor was
filtered andthe dry masses of lignin were recorded. Among the results, tests showed
that,regarding the recovery of lignin, the most important factor was the pH. pHvalues
??beneath 4 had the best yields. The temperature has become an importantfactor in order to facilitate the filtration process. The mixing times had noinfluence in the recovery.
It was concluded that the lignin recovery from blackliquor can be performed immediately after alkaline hydrolysis, with pH values??below 4 and a temperature of 50 C, fact
that speeds up the recoveringprocess.
Acknowledgements: CAPES, CNPq and FAPESP for financial support.

SIMB 2013 | 81

OPTIMIZATION OF ROCK PHOSPHATE SOLUBILIZATION

BYASPERGILLUS NIGERIN A SOLID STATE FERMENTATION
SYSTEM
GILBERTO DE OLIVEIRA MENDES1, NINA MORENA RGO MUNIZ DA SILVA1,
THALITA CARDOSO ANASTCIO1, NIKOLAY BOJKOV VASSILEV2, JOS IVO
RIBEIRO JR.3, IVO RIBEIRO DA SILVA4, MAURCIO DUTRA COSTA1
1. Department of Microbiology, Federal University of Viosa, Av. PH Rolfs, s/n, BIOAGRO,
Campus UFV, 36570-000, Viosa, MG, Brazil 2. Department of Chemical Engineering,
Faculty of Sciences, University of Granada, Spain 3.Department of Statistics,Federal
University of Viosa, Brazil 4.Department of Soil Science,Federal University of Viosa, Brazil
The use of phosphate-solubilizing microorganisms (PSM) is considered an attractive
alternative for the management of phosphorus (P) in agriculture. PSM can be applied directly to the soil together with poorly soluble P sources, such as rock phosphates (RP), or
used in in vitro systems to produce high-solubility P fertilizers from RP. The latter option
allows the control of the variables that govern microbial RP solubilization, something difficult to attain in the soil. An interesting strategy proposed for RP solubilization is the use
of agro-industrial wastes as a support for microbial growth. Recently, our group obtained
anAspergillus nigerisolate (FS1) able to solubilize RP in a solid state fermentation (SSF)
system using sugarcane bagasse as substrate. Thus, the objective of the present work was to
optimize the SSF cultivation conditions that allow maximum RP solubilization by FS1. The
experiments were done in 250-mL Erlenmeyer flasks with 5 g of sugarcane bagasse ground
into 2-mm fragments. Initially, a 2-level fractional factorial design was used to screen the
factors with significant effect on the concentration of solubilized P. The following variables
were screened (low/high levels): initial pH [unadjusted (4.6)/7.0], temperature (25/30 C),
incubation time (7/13 days), inoculum (105/107conidia flask-1), methanol [0/5 % (v/w)],
mineral solution (0/100 % of the minerals of Czapeks medium), RP [1.5/4.5 % (w/w)],
biochar [0/10 % (w/w)], sucrose [2.5/5 % (w/w)], and moisture [570/800 % (v/w)]. Only
the last four variables showed significant effects on the process and were submitted to optimization. The levels of these variables were increased gradually in successive experiments
aiming to achieve a near-stationary region. At this point, a composite central design was
used to fit a response surface with optimized values of the variables. The highest concentration of solubilized P was obtained with 86.5 % of biochar, 25 % of RP, 27 % of sucrose,
and 620 % of moisture content (% based on sugarcane bagasse mass). At the optimal values, the concentration of solubilized P was increased from 3.6 to 8.6 g P per kg of dry bagasse, representing a gain of 139 %. These results are of great interest for the management
of P fertilization given that the process is based on the use of inexpensive waste materials
that contribute to lower the costs of the final product.
Financial support: CNPq and FAPEMIG (CAG-APQ-00712-12)
SIMB 2013 | 82

ENVIRONMENTAL CONDITIONS INFLUENCE THE

ACTION OF BOVICIN HC5 ASSOCIATED WITH EDTA
ONSALMONELLATYPHIMURIUM
PRUDNCIO, C. V.1; MANTOVANI, H.C.1; VANETTI, M. C. D.1
Department of Microbiology, Federal University of Viosa, Viosa, Minas Gerais, Brazil.

Bovicin HC5 is a lantibiotic with wide spectrum of action on Gram-positive bacteria. Gram-negative bacteria are naturally resistant to the action of these bacteriocins,
because the outer membrane acts as a barrier. Nevertheless, it has been shown that the
combination of membrane destabilizing agents, such as EDTA, allows the use of this
bacteriocin on Gram-negative bacteria. However, Salmonella can alter the membrane
composition according to environmental conditions, which influences the permeability of outer membrane and can change the resistance to antimicrobial peptides, such
as bacteriocins. In this study, we evaluated the effect of environmental conditions of
temperature and pH on the sensitivity ofSalmonellato action bovicin HC5 combined
with EDTA. To do this, a culture ofSalmonellaenterica serovar Typhimurium ATCC
14028 was grown in brain heart infusion broth (BHI) at 37 C for 24 h,centrifuged at
1742g, washed, and resuspended in saline 0.85%. The culture was inoculated at a density
of 105 CFU.mL-1in BHI broth with a pH between 5.0 and 7.2, with bovicin HC5 (200
AU.mL-1) and EDTA (1.5 mM) and incubated at different temperatures between 10 and
45 C, according to the experimental design with two-factor central composite. To assess
the viability of the cells at different time intervals for 12 h, aliquots were plated on agar
for standard count (PCA) by the drop method. The plates were incubated at 37 C for
10 h. It was observed that bactericidal effect was only verified on specific conditions of
temperature and pH (>30 C and >6.0, respectively). In the remaining conditions, there
was a bacteriostatic action. These differences in mechanism of action may be caused by
changes in pathogen growth and/or changes in the permeability of the outer membrane
of the bacteriocin.
Financial support: CAPES and CNPq.

SIMB 2013 | 83

GENOME MINING REVEALS BACTERIOCINOGENIC

POTENTIAL IN RUMINAL BACTERIA
AZEVEDO, A. C1; RUIZ, J.C2.; QUEIROZ, M; V1.; MANTOVANI, H. C1
Departament of Microbiology/Universidade Federal de Viosa, Viosa, MG- Brasil
The rumen represents a complex microbial ecosystem and several ecological interactions occur among rumen microbes, including predation, substrate competition
and antagonism. Previous studies indicated that some species ofruminal bacteria are
able to produce ribosomally-synthesized proteinaceous compounds (bacteriocins) that
often inhibit other related organisms. Considering the increasing availability of ruminal
bacterial genomes and the potential use of ruminal bacteriocins for commercial applications, this work aimed to identify bacteriocins clusters among bacterial genomes publically available at the Hungate 1000 database. We mined 77 genomes of ruminal bacteria
for genes related to the production and/or regulation of bacteriocin biosynthesis using
BAGEL and antiSMASH algorithms. Protein sequences of the genes identified in the
bacteriocin clusters were further analyzed using BLAST. The BACTIBASE was used to
identify similarities among precursor peptides. Although previousin vitroandin silicostudies had reported only four species of bacteriocinogenic ruminal bacteria, in the
present study a total of 94 putative bacteriocin gene clusters were identified distributed
in 42 ruminal bacteria. Bacteriocin genes were abundant in the genusButyrivibrio, and
the majority of the strains (62 %) showed at least one cluster related to bacteriocin production. AlltheStreptococcusandRuminococcusstrains examined in this study exhibited bacteriocin genes in their genomes. Among the clusters that were related to known
bacteriocins, the ones belonging to class III (n=17), sactipeptide(n=13) and lantipeptide
(n=13) were the dominant groups. Five strains harbored complete lantibiotic biosynthetic clusters. These results indicate that bacteriocin production is a widespread ability
among ruminal bacteria. Further studies of these antimicrobial peptides might be useful
to develop new strategies to manipulate ruminal fermentation and improve ruminant
productivity.
Financial support: CAPES, FAPEMIG, CNPq

SIMB 2013 | 84

DEVELOPMENT OF MIXED REFERENCE MATERIAL FOR

DETECTION OFSALMONELLAAND COLIFORM BACTERIA
SILVA, M. R.1; MARQUES, L. F. M.1; ALMEIDA, F. A.1; COELHO, A. I. M.2;
VANETTI, M. C. D.1
Department of Microbiology, Federal University of Viosa, Viosa, Minas Gerais, Brazil.
Department of Nutrition and Health, Federal University of Viosa, Viosa, Minas
Gerais, Brazil.
1
2

Reference materials (RMs) are important tools for determining the quality of the
analyzes. RMs have to fulfill requirements including homogeneity, stability and similarity to the samples examined in a laboratory routine. The aim of this study was to
produce mixed RMs containingSalmonella entericaand coliform bacteria:Escherichia
coli,Citrobacter freundiiandCronobacter sakazakii. To increasing the resistance of bacteria to desiccation two strategies were used: (1) bacteria growth to stationary phase in
minimal salts medium plus NaCl and MnSO4and (2) lyophilizing the cells in reconstituted skim milk plus trehalose as osmoprotectant. The lyophilized cells were stabilized
at 4 ?C for 90 to 95 days. It was found that there was no significant difference between
the number of log cycles reducedSalmonellaand other bacterial cultures after stabilization. However, the survival ofC. sakazakiiwas significantly higher thanE. coliandC.
freundii. Two types of RMs were produced in matrix milk powder using freeze-dried
stabilized cells ofSalmonella,E. coli,C. freundiiandC. sakazakii: a RM containing 101102CFU/0.3 gSalmonellaand 103-104CFU/0.3 g coliforms and a second MR containing
103-104CFU/0.3 gSalmonellaand 103-104CFU/0.3 g coliforms. It was found that only
the mixed RM containing low counts ofSalmonellawas homogeneous and that it was
possible to detect Salmonella in this RM using the conventional method (ISO 6579),
the immunomagnetic separation and PCR. The analysis ofE. coliin RMs homogeneous
showed that counts may fluctuate, suggesting a higher sensitivity of this bacterium to
the steps of RMs production. Furthermore, it was observed that the use of enumeration
methods that do not involve a step of recovery of bacteria, possibly injured, can lead to
underestimation of true number of cells. These results will contribute to the development of mixed microbiological RM.
Acknowledgements: CAPES

SIMB 2013 | 85

SECRETOME ANALYSIS OF THE INTERACTION

BETWEENPHASEOLUSVULGARIS ANDTRICHODERMA
SPP.FROM BRAZILIAN CERRADO SOIL
SILVA, F. L.1, SANTOS,T. B. M.1,AQUINO, E. N.1,2, CUNHA, D.
C.1,STEINDORFF, A. S.1, JUNQUEIRA, M.3, DOMONT, G. B.3,ULHOA, C.
J.2,NORONHA, E. F.1
1Enzimology Laboratory, Department of Cellular Biology,Braslia University, Braslia,
DF, Brazil.2Biological Sciencies Institute, Federal University ofGois, Goinia, GO,
Brazil.3Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ,
Brazil
Fungaldiseases inPhaseolus vulgaris(commonbean) can be controlled using biological control agents in alternative tochemical fungicides, such as the mycoparasitic
fungi from Trichoderma genus. Species of Trichodermain intimate association with a
host plant may trigger defense response inducingits resistance against subsequent fungal infections. The present study aimed to:a) analyze the interaction between species
ofTrichodermaisolated fromCerrado soils andP. vulgaris,evaluating the expression of
defensegenes in the common bean after 24, 48 and 72 hours of interaction withT.harzianum(303/02 and 457/02) andT. asperellum(468/02) by RT - PCR q and b) identify the
secretome of the interaction amongT. harzianum(303/02) and the host plantP. vulgaris.
A hydroponic growingsystem was used to obtain the protein samples and the secretome
was analyzed byLC-MS/MS technique. Common bean plants challenged by the mycoparasite isolatespresented increasing in the expression levels of the defense related genes
(ch1,chitinase, ?-1,3-D-Glucanase,peroxidase 3 and Lipoxygenase) ina time course
dependence. Chitinase (chit 1) and 1,3-?-D-Glucanase encoding genes presented increasedexpression in the first hours of the interaction, decreasing along the interactiontime, while the expression levels of the genes encoding Peroxidase 3 and showedhigher
expression levels after 48 hours.The secretome in the presence orabsence of T. harzianum (303/02), presented102 and 92 proteins, respectively, using P.vulgarisDatabase.
It was also detected, 227 and 180 proteins using T. harzianum Database,respectively.
Among the secretome proteins detected exclusively in the presenceofT. harzianumwere
identifiedGlycoside hydrolases family 17, pathogenesis-related proteins and proteases.
This workcould help to elucidate the mechanism by whichTrichodermaspp can modulate the defense response of common beanplants and induces its resistance against phytopathogenic fungi.
Acknowledgements: CAPES, CNPQ, UnB, UFG e UFRJ

SIMB 2013 | 86

ENZYMATIC POTENTIAL TEST OF BACTERIAL STRAINS

ISOLATED FROM LETTUCE (LACTUCA SATIVA) ROOTS WITH
4 DIFFERENT CARBON SOURCES
FORZANI, M. V. A.1; MILHOMENS, J.1;OLIVEIRA, B. F. R.1; NETO, P. J. F.1;
CARRIM, A. J. I.1; VIEIRA, T. M.1; LIMA, J. M. S.1; REIS, L. G. V.1; SADOYAMA,
G.2;LEO-VASCONCELOS, L. S. N. O.1; VIEIRA, J. D. G.1
Instituto de Patologia Tropical e Sade Pblica, Universidade Federal de Gois (UFG),
Goinia, Gois, Brazil. 2Campus Catalo, Universidade Federal de Gois (UFG), Catalo,
Gois, Brazil. mforzani@gmail.com
1

Lettuce, Lactuvia sativa, is an ordinary vegetable in human feeding from about

500 b.C. It is largely cultivated worldwide and it has a lot of varieties. Its rhizosphere
is colonized by several genera of bacteria, highlighting Bacillus, Klebsiella, Citrobacter and Clostridium, that are broadly explored with biotechnological and commercial
goals. Fermentative processes are an important catabolic pathway of energetic obtainment of anaerobic organisms, furthermore produce alcohol and CO2 as byproducts.
Alcohols are largely used as green fuels, whereas they are less pollutants than the fossil
fuels and they are obtained through biological processes, fermentation. The aim of this
study was to isolate, cultivate and identify bacterial strains from lettuce root to biofuel
production. The isolated strains were tested preliminarily for their fermentative capacity
front four carbon sources. Seven morphspecies were isolated. To test the morphspecies
fermentative ability, they were inoculated in minimum media with saccharose (2% b/v),
cane bagasse (1% b/v), glycerol (1% b/v) and carboximetilcellulose (0,2% b/v) as single carbon source. The visualization of fermentation was done by gas collection, using
Dhuran tubes in the fermentative tubes. They were incubated at 20C and the reading
of gas production was done in 24/24 hours for 7 days. Four isolated, AG1, AG3, AG5
and AG7, fermented saccharose. Fermentation was not seen for the rest of the carbon
sources. However, these isolated and AG2 and AG6 were capable to reduct the viscosity
of media with carboximetilcellulose and all they have grown in cane bagasse. These results suggest that these strains have cellulolytic activity. More tests are being performed
to determinate the fermentation byproducts of saccharose and the products of cellulose
sources degradation.
Financial support: FAPEG (CH 05-2012)

SIMB 2013 | 87

COAGULANT AND ANTIBACTERIAL PROPERTIES

OFBOTHROPS LEUCURUS(WAGLER, 1824) POISON
JAQUELINE RAMOS MACHADO BRAGA, DIEGO DOS SANTOS PEREIRA,
NORMA SUELY EVANGELISTA BARRETO, CARLA SILVA DA SILVEIRA, IRANA
PAIM SILVA, JOANA ANGELI B. LIMA
Universidade Federal do Recncavo da Bahia, Centro de Cincias Agrrias, Ambientais e
Biolgicas. Rua Rui Barbosa, 710 Centro Cruz das Almas-Ba, Brasil.
Snakes ofBothrops leucurusspecie are responsible for about 70% of snakebites occurred in Bahia, Brazil. Its venom is a complex biological mixture of toxins that can
cause pain, necrosis, hemorrhage, and in severe cases, death. However, several studies
describe that the venom can present a strong antimicrobial action. This study aimed to
analyze the antibacterial effect ofBothrops leucurusvenom, in strains of Gram-negative
bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), as well
as evaluate theircoagulant action in horse blood. The susceptibility of bacterial strains
was evaluated using the agar diffusion method, for determining the antimicrobial potential in different poison concentrations, while the coagulation was measured in minutes
through the method of Lee-White, in the same concentrations of the poison. The results
were negative for antimicrobial activity onE. coliand onS. aureusat all concentrations
tested (5 to 1000 g/ml). However, the poison proved active in the coagulation assay at
all concentrations tested, with maximum coagulation time in about 3 min, even when
tested at a concentration ten times less than the maximum, which confirmed its activity
when compared to the control time (16 min). The results suggest that changes in the
chemical composition of the venom ofB. leucurus, which are related to age, diet, climate
or region where the snake lives, could interfere with their microbicide potential.

SIMB 2013 | 88

USE OF ALTERNATIVE SUBSTRATE FOR ISOLATION OF

MICROORGANISMS PRODUCING PHB
R. SALOMONI1,2, E. S. SILVA2, C.M.S. RIBEIRO3, M.F.A. RODRIGUES2*
1 University of Sao Paulo USP Biotechnology Interunits Post Graduation Program
USP, Butantan Institute, IPT Sao Paulo, SP, Brazil. 2Industrial Biotechnology
Laboratory, NanoBiomanufacture Nucleus, Institute for Technological Research IPT,
Sao Paulo, SP, Brazil. 3Petrobrs Cenpes Rio de Janeiro, RJ, Brazil. *Corresponding
author: filomena@ipt.br
1

The development of new strains and the search for cheaper production processes
and recovery of PHB has required much effort by research groups from different parts
of the world. The metabolic pathways used for both glycerol and the fatty acids are quite
widespread in the nature, there is, in principle, inconvenient use of these substances
as carbon source. The selection of new strains producing P3HB from Waste Biodiesel
Production becomes therefore quite interesting, both from a technical and an economic aspects.Two soil samples have been used, both with apparent contamination of the
residue containing glycerol. Nutrient broth and mineral medium containing different
concentration of nitrogen and carbon source were used for cultivation, selection of microorganisms or PHB production. PHB analysis was performed by gas chromatography
of propyl ester.A total of 25 microorganisms were isolated in the first phase of work. The
colonies were examined under UV light on plates with Nile Red dye and fluorescence
was noted in some of them, indicating the possibility of accumulation of polymers.
Among 11 strains obtained in the second phase, five strains were selected for evaluation
of the ability of polymer accumulation. In the third phase of the experiment, which
involved the cultivation of five isolates in shaking flasks containing residue, two cultures showed potential for polymer production. The results of the fourth phase showed
different behaviors of the two colonies, resulting in accumulation of P3HB of 40% for
culture 7B and 1.0% for culture 7A after 48 hours of cultivation.A strain of soil bacteria
capable of producing the polymer P3HB from glycerol as a carbon source was isolated
and selected. The strategy used in the isolation of strains producing P3HB from glycerol
was effective, despite the small number of microorganisms obtained. The use of a larger
number of samples from different sources would be recommended in order to obtain a
larger number of microorganisms producing this biopolymer.

SIMB 2013 | 89

EFFECT OF IRRADIATION ON THE INACTIVATION OF

MICROORGANISMS IN STRAWBERRY
RONDINELLI MOULIN LIMA;TARCSIO LIMA FILHO;SUZANA MARIA
DELLA LUCIA; NAYARA BENEDITO MARTINS DA SILVA;RAQUEL VIEIRA
DE CARVALHO,PATRCIA CAMPOS BERNARDES;WILMER EDGARD
LUERA PEA
Universidade Federal do Esprito Santo, Centro de Cincias Agrrias, Alegre,Esprito
Santo, Brasil. Universidade Federal de Viosa, Viosa, Minas Gerais, Brasil.
Strawberry has a short shelf life due to microbial spoilage. Irradiation has been
suggested as a possible solution to increase the shelf life of food due to the reduction
of microbial load. Recently, it was shown that by applying doses higher than 0.405 kGy
noticeable changes begin to occur in sensory characteristics of irradiated strawberry
and that in doses higher than 3.6 kGy it begins to occur sensory rejection of irradiated
strawberry. Thus, this study aimed to investigate the effect of irradiation, at those doses,
on the inactivation of micro-organisms present in strawberry. Analyses were conducted
to determine filaments fungi and yeast, aerobic mesophilic, total and thermotolerant
coliforms in non-irradiated (control) and irradiated strawberries (at 0.405 kGy and 3.5
kGy), stored at 8C on the first, eighth and fifth day of storage. The filaments fungi and
yeast were the most resistant microorganisms to irradiation. On the first day of storage, the doses of 0.405 kGy and 3.6 kGy caused a reduction of approximately 1 and 2
log cycles, respectively, comparing to the control sample. The irradiated samples also
presented a reduction in the count of aerobic mesophilic. The dose of 3.6 kGy reduced
in 4 log cycles the count of these microorganisms, on the first day. On the last day of
storage, analyzes of the control and irradiated (0.405 kGy) samples were not performed
due to high fungal rot. The irradiated sample at 3.6 kGy presented no signs of advanced
deterioration and it was observed that the count of aerobic mesophilic and filaments
fungi and yeast were lower than in the control sample on the 8th day. Thermotolerant
coliforms were not detected in all the analyzed samples. The irradiation of strawberries
at 3.6 kGy dose was effective in reducing the microbial load of all studied microorganisms throughout the storage period, and the dose of 0.405 kGy did not show the same
efficiency as the 3.6 kGy dose.
Acknowledgements: The authors would like to acknowledge the CNPq (National Council
for Scientific and Technological Development) and CAPES (Coordination for the Improvement of Higher Education Personnel), Brazil, for their financial support and the Gamma
Radiation Laboratory of the Center for Development of Nuclear Technology (Belo Horizonte - MG, Brazil).

SIMB 2013 | 90

VALIDATION OF A STATISTICAL MODEL TO PREDICT LIPASE

PRODUCTION BYPENICILLIUMSP.T9.1
EUSTACHIO1, P. F. P.;SIMES1, F, A, O.; BALTAZAR1, P. O. J.; PANTOJA1, L. A.;
SANTOS1, A. S.; VANZELA1, A. P. F. C.
1

Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina-MG, Brazil

Lipases are special enzymes that hydrolyze ester linkages and present a variety of
applications, ranging from food manufacturing and incorporation in detergents, to esterification of fat acids with simple alcohols like methanol or ethanol (biodiesel production). Considering the global perspective of increasing lipase applications, and the
importance of prospecting new producers and bioprocesses, the objective of this work
was to validate a statistical model generated by central composite design (CCD) to predict lipase production by a new isolate ofPenicilliumsp.T9.1. By means of experimental
data analyzed withStatistica7.0, it was generated the equation: Lipase activity = 0.19
+ 0.55 x 0.09 x2+ 0.21 y 0.05 y2+ 0.03 xy, where x, ammonium sulphate; y, soy oil.
According to the model, an activity of 1.14 U mL-1min-1would be obtained by employing a medium (NaCl 0,01%, MgSO4.7H2O 0.02%, KH2PO40.04%, K2HPO40.01%) supplemented with 3.5% soy oil and 3.5 g L-1ammonium sulphate. In order to test the model
under experimental conditions, bioprocesses were conducted in triplicate in media supplemented with carbon and nitrogen sources as describe above, and adjusted to pH 5.0.
Cultures were inoculated to a final concentration of 105conidia mL-1and incubated for 5
days at 30 C under 150 rpm. Lipase activity in each culture was determined after 24-120
h at 24 h intervals. One unit was defined as the amount of enzyme that releases 1 Eq of
fat acid .min-1.mL-1from soy oil under the adjusted reaction conditions (pH 6.0, 37 C, 5
min). Lipase production byPenicilliumsp.T9.1 reached 0.44 U mL-1min-1after 48 h. A
maximum activity of 1.15 U. mL-1min-1was obtained after 96 h, as predicted by the statistical model. According to these data, it was concluded that CCD was useful to plan the
conditions for lipase production byPenicilliumsp.T9.1 and for predicting lipase production with a high degree of confidence, whereas the statistical model was validated.

SIMB 2013 | 91

ANTIMICROBIAL ACTION OF ACTINOBACTERIA ISOLATED

FROM GOIANO CERRADO SOIL AGAINST DIVERSE
STANDARD STRAINS OF GRAM-NEGATIVE BACTERIA
OLIVEIRA, B. F. R.1; NETO, P. J. F.1; CARRIM, A. J. I.1; VIEIRA, T. M.1,
FORZANI, M. V. A.1; MILHOMENS, J.1; REIS, L. G. V.1; SADOYAMA, G.2; LEOVASCONCELOS, L. S. N. O.1; VIEIRA, J. D. G.1
Instituto de Patologia Tropical e Sade Pblica, Universidade Federal de Gois (UFG),
Goinia, Gois, Brazil. 2Campus Catalo, Universidade Federal de Gois (UFG), Catalo,
Gois, Brazil.
1

Actinobacteria form an enormous group of gram-positive bacteria characterized

by the production of half of the discovered bioactive secondary metabolites, especially
antimicrobial agents. Generally, in screening programs for new antibiotic biomolecules,
the gram-positive bacteria show greater susceptibility to these compounds when compared to gram-negative bacteria. This fact can be explained by the existent morphological differences between these micro-organisms and, wherefore, to the probable action sites of these molecules. Multi-drug resistant gram-negative bacterial infections
constitute a challenging and crescent problem in health-care institutions globally, and
the actual conjunct of available antimicrobials drugs is already very limited. This study
aimed to isolate actinobacteria from the goiano Cerrado soil with potential antimicrobial activities against gram-negative bacteria. The chemotactic method described by Palleroni (1980) was applied to isolation. The plug technique was used in the primary tests
for antibacterial activity against the following standard strains:Escherichia coliATCC
25922, Enterobacter aerogenesATCC 13048, Klebsiella pneumoniae ATCC 70063, Serratia marcescens ATCC 14576, Salmonella typhimurium ATCC 14028, Pseudomonas
aeruginosaATCC 27853. From the total of isolated 20 morphospecies, only the isolate
A22 exhibited bactericidal activity against the standard strains ofE. coli,E. aerogenes,S.
marcescensandS. typhimurium, and bacteriostatic action toP. aeruginosaandK. pneumoniae. Other 4 isolates showed bacteriostatic activity against P. aeruginosa. The results indicate that the isolate A22 synthesize biomolecules with specific action to certain
gram-negative bacteria, thus it is a candidate to production, purifying and cytotoxic
determination testing of these generated substances.
Financial support: Fundao de Amparo a Pesquisa do Estado de Gois (FAPEG).

SIMB 2013 | 92

DETECTION OF ANTIBACTERIAL ACTIVITY OF ISOLATES

OF ACTINOBATERIA FROM CERRADO SOIL AGAINST
GRAM-POSITIVE BACTERIA AND METHICILLINRESISTANTSTAPHYLOCOCCUS AUREUS(MRSA)
OLIVEIRA, B. F. R.1; NETO, P. J. F.1; CARRIM, A. J. I.1; VIEIRA, T. M.1, ARAUJO, M.
V. F.1; MILHOMENS, J.1; REIS, L. G. V.1; SADOYAMA, G.2; LEO-VASCONCELOS,
L. S. N. O.1; VIEIRA, J. D. G.1
Instituto de Patologia Tropical e Sade Pblica, Universidade Federal de Gois (UFG),
Goinia, Gois, Brazil. 2Campus Catalo, Universidade Federal de Gois (UFG), Catalo,
Gois, Brazil.
1

The phylum Actinobacteria involves a broad conjunct of gram-positive bacteria

with high genomic content of G+C. They are extremely valuable biotechnologically, considering that the greatest part of current antibiotics in clinical use was synthesized by
them. The gradual depletion of antimicrobials for the treatment of multi-drug resistant
bacterial infections, may characterize a return to the pre-antibiotic era. Methicillin-resistantStaphylococcus aureus(MRSA) excels itself from these bacterial pathogens, being
known to cause a wide of serious infections in hospital and community environments.
Thereby, it is needful the search for new antibiotics. The aim of this study was to isolate actinobacteria from the Cerrado soil and evaluate their antibacterial against various
gram-positive bacteria, including MRSA. The chemotactic method of Palleroni (1980)
was adopted for the isolation of actinobacteria. 20 isolated morfospecies showed different characteristics in ISP-2 Agar. These isolates were tested using the plug methodology, against five standard strains of gram-positive bacteria,Staphylococcus aureusATCC
25923,Micrococcus luteusATCC 9341,Enterococcus faecalisATCC 29212,Corynebacterium glutamicum ATCC 13032, Bacillus cereus ATCC 14579 and B. subtilis ATCC
6633, and four clinical strains of MRSA. From the screened isolates, 8 (40%) inhibited
the growth of all standard strains and all MRSA strains. The zones of inhibition varied
between the isolates and the indicator organisms. The isolate A22 stood out with the
biggest zones of inhibition. The results imply that the 8 isolates are promising to the
production of new antimicrobials, mainly against MRSA. Essays of increase production
scale, extraction, cytotoxicity of these bioactive metabolites are being executed, and they
may come in a near future to be prototypes of new drugs.
Acknowledgements: Fiocruz. Financial support: Financial support: Fundao de Amparo
a Pesquisa do Estado de Gois (FAPEG).

SIMB 2013 | 93

EVALUATION OF A DRYING PROCESS BY GENETICALLY

MODIFIED STRAINS OFSACCHAROMYCES CEREVISIAEFOR
OENOLOGYS FIELD
LUMMY MARIA OLIVEIRA MONTEIRO1,2; LUCAS ALVES TAVARES3; GEMMA
LPEZ-MARTNEZ.2; RICARDO CORDERO-OTERO2.
Universidade Federal de Viosa - UFV, Viosa MG, Brasil. 2Universitat Rovira i Virgili URV, Tarragona, Espanha. 3Universidade Federal de Pelotas UFPel, Pelotas RS, Brasil.
lummymonteiro@hotmail.com
1

Progresses in microbial biotechnology in the field of oenology in recent years have

been made in development of new strains of yeast vinous. Advances in different stages
of the production of ethanol are required, and the production of genetically modified
strains for industrial use can validate observations obtained in previous studies. The
yeastSaccharomyces cerevisiaeis able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. Some of the proteins
termed hydrophilins participate in the cellular tolerance to this stress condition. The objective of this study was to evaluate the tolerance of genetically modified strains ofSaccharomyces cerevisiaeto the drying process. Control strains 1, 2, 3 and 4, and also the
genetically modified strains 5, 6, 9 and 10 were inoculated in the medium synthetic complete drop-out and incubated for 24 hours at 37C. Five aliquots of 1 mL were collected
from each solution. The aliquots were centrifuged and the supernatants were discarded.
An aliquot for each strain was used in test oxidative stress ROS before drying. The remaining aliquots were added Trehalose 10%, 50L, and were taken oven for 20 hours at
28C. After the drying process, the rehydration was performed and aliquots were incubated for 30 minutes at37C. Then,, an aliquot of each strain was taken for testing ROS,
and the remaining three aliquots of each strain were added 0.5L of Peopidium iodide
(PI) and Acetoxymethye ester, followed by dilution to be led to the flow cytometer. The
ROS test result shows that the control strain 4 has viability of 58.23%, while the genetically modified strain 10 showed 79.28%; and the control strain 1 presents viability 10%
lower than the strain genetically modified 5. Flow cytometry showed the viability of the
control strain 1 was 23%, while the genetically modified strain 5 was around 93%. Hydrophilin proteins are necessary for the cells to overcome dehydration stress. Therefore,
considering the cell viability results strains genetically modified 5 and 10 are promising
for future studies, by showing higher cell viability than their control strains after drying.
Financial support: CNPq, Ministerio de Ciencia e Innovacin (Spain)

SIMB 2013 | 94

INVITROANTAGONISM OFSCLEROTIUM ROLFISIIBY

STRAINS OFTRICHODERMASPP
SILVA, J.M.; MELO, A.L.S.; ALBUQUERQUE, L.S.; GUEDES, E.L.F.; SANTOS,
T.M.C.
Universidade Federal de Alagoas, Unidade Acadmica CECA-UFAL, BR 104 km 85, CEP
57100-000, Rio Largo, Alagoas, Brasil
The biocontrol has been considered as an alternative to the use of chemicals for
the control of plant diseases. The fungi of the genusTrichodermaare of great economic
importance for agriculture, since they are able to act as agents to control diseases of various cultivated plants, growth promoters and inducers of plant resistance to diseases. This
study aimed to evaluate the antagonistic potential of fiveTrichodermastrains againstSclerotuim rolfissi. For the experiment we used the method of direct confrontation,
where the fungi were inoculated in Petri dishes containing PDA medium with a sterile
cover slip which was previously placed a disc of each fungus, pathogen and antagonist,
one at each end of the board. After seven days, we observed the growth of fungal hyphae and interaction, making sure there was growth inhibition of the pathogen by the
antagonist, and the type of interaction between hyphae of the isolates. The interaction
of hyphae was observed by microscopy. The cover slips were removed and stained with
methylene blue, and placed on a slide and brought under the microscope, then analyzing the type of interaction between the hyphae. The process of growth inhibition of the
pathogen was evaluated using a scale of Bell. The interaction between hyphae showed
that the pathogen was parasitism by all Trichoderma isolates. The confrontation showed
averages 50-80 % growth of the antagonist. Thus, it shows that fungi of the genusTrichodermahave good potential in biocontrol ofS. rolfisii. These data can be considered due
to the fact that fungi of the genusTrichodermahave hyperparasite capacity.

SIMB 2013 | 95

SENSITIVITY OF 'IMPERIAL' PINEAPPLE NURSERY

ASSOCIATED WITH THE FUNGUSPIRIFORMOSPORA
INDICATHE APPLICATION OF THE DIURON

LANA IVONE BARRETO CRUZ, MARIA DO CU MONTEIRO CRUZ, AMANDA

SOUZA MIRANDA, GUILHERME DUMB MONTEIRO CASTRO

Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Diamantina,

Minas Gerais, Brazil
The low supply of healthy and good nursery has been an obstacle to the pineapple
crop growth in Brazil. The use of mycorrhizal fungi may be an alternative in order to
reduce the time of nursery production, because they can optimize their root system to
uptake water and nutrients. The study was carried out to evaluate the association of the
fungusPiriformospora indicaon the growth of micropropagated pineapple nursery in
cultivation with application of diuron. The research was conducted in a greenhouse at
UFVJM, using micropropagated plants of 'Imperial' pineapple, in factorial 2 x 4, referring to two inoculations:P. indicaand without inoculation and four diuron doses: 0, 1.6,
3.2, 6.4 L ha-1, distributed in a completely randomized design with 3 replications. Inoculation with theP. indicait was made at the time of planting, placing in the substrate mycelial discs of 5 mm. Herbicide application was taken 24 hours before planting nursery.
At 150 days after inoculation, the number of tillers, number of leaves per plant, shoot
height, crown diameter, root length, shoot dry weight, leaf area, specific leaf area and leaf
dry mass were evaluated. The roots were separated and washed in water to evaluate colonization. Data were submitted to analysis of variance and regression. The association of
fungusP.indicawith the Imperial pineapple nursery was observed with the application
of all diuron doses. The pineapple nursery inoculated showed increases in height from
7.8%, in diameter of 14.1%, the number of leaves of 13.58%; leaf area of 7.17%, the specific leaf area of 53.3 %, dry mass of 129.1%, tiller number of 119.39% and 17.39% of the
length of the root system with the application of diuron. The increases in the size of the
seedlings were estimated doses that did not exceed 3.72 L ha-1. The 'Imperial' pineapple
plants associated with the P. indica showed higher growth. The application diuron in
high doses interfered in the growth of mycorrhizal pineapple nursery.
Financial support: CAPES, CNPq, FAPEMIG

SIMB 2013 | 96

ULTRAFILTRATION SYSTEM AND SUBSTRATE INFLUENCE

ON THE PRODUCTION OF CYCLODEXTRINS BY PURIFIED
CGTASE FROMBACILLUS FIRMUSSTRAIN 37.
FENELON, V.C.1; AGUIAR, M. F. A.1; MIYOSHI, J. H.1; MARTINEZ, C. O.2;
MATIOLI, G.1
1Departamento de Farmcia, Universidade Estadual de Maring (UEM), Maring,
Paran, Brasil. 2Programa de Ps-graduao em Cincias de Alimentos, Universidade
Estadual de Maring (UEM), Maring, Paran, Brasil.
Cyclodextrins (CDs) are cyclic oligosaccharides produced from starch cyclization
reaction, catalyzed by the enzyme cyclodextrin glycosyltransferase (CGTase). The most
common are -CD, -CD and -CD, showing ability to encapsulate a wide range of
molecules, considerably increasing their stability and/ or solubility. Because of the large
increase of the applicability and CDs use, several researchers have searched technological innovations to produce these molecules. This study aimed to improve the CDs yield
obtained by the purified CGTase enzyme fromBacillus firmusstrain 37. An ultrafiltration system with a 10kDa NMWL membrane was used to remove the inhibitory products accumulated in the reaction medium and, at the same time, recovering the CGTase
enzyme along several consecutive batch cycles of 24 h. Maltodextrin 10% (w/v) and
corn starch 5% (w/v) were used as substrate. The ultrafiltration system for CDs production batch, in a simple way and without requiring sophisticated reactor, allowed CDs
production practically constant in the initial cycles, with good productivity until the
eighth production cycle. The -CD yield obtained using the purified enzyme and corn
starch substrate was 0.54 mmol/L/h, 36% greater than that observed with the maltodextrin substrate (0.35 mmol/L/h). This behavior may be related to a higher affinity of the
CGTase active site by the corn starch substrate. The ultrafiltration model used in this
study, combined with the use of corn starch substrate, proved to be an innovative system
for efficient and economic batch CDs production. Corn starch is a raw material of wide
availability and low cost in Brazil, thus, the possibility of industrial application of this
system is promising.
Acknowledgements: CNPq, CAPES and Fundao Araucria.

SIMB 2013 | 97

SELECTION OF LIGNIN PEROXIDASE PRODUCERS FUNGI

FOR DEGRADATION OF TEXTILE DYES
SABRINA FELICIANO OLIVEIRA1 JOS MARIA RODRIGUES DA LUZ2
MARIANA RACHEL NASCIMENTO CARVALHO2 MARIA CATARINA MEGUMI
KASUYA2 ARY CORREA JUNIOR1
1- UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE, MINAS
GERAIS 2- UNIVERSIDADE FEDERAL DE VIOSA, VIOSA, MINAS GERAIS
Textile effluents present different contaminants, including hydroxides, surfactants
and dyes. In textile processing approximately 50,000 tons of dyes are discarded annually which results in serious environmental problems due to the recalcitrance of these
dyes. Bioremediation using microorganisms and microbial enzymes is a viable alternative for these effluents treatment. Unlike microbial cells, enzymes present lower
rate of inhibition by toxic substances and work in various environmental conditions.
Among the enzymes used in the textile effluents treatment highlight fungi lignin peroxidase (LiP) that can be produced from lignocellulosic residue colonization by whiterot fungi. Our aim was to select LiP producers fungi using agro-industrial residues.
Strains of Pleurotus spp (PLO2, PLO6, PLO9, PLO13, PLOA2 and PLE04), Pycnoporus sanquineus (PYC02),Phanerochaete chrysosporium (PC), Ganoderma sp. (GR117)
andTrametessp. (J5) were used. Initially, each isolate was transferred to Petri dishes containing PDA supplemented or not with 0.05 % dye Remazol Brilliant Blue R. Cultures
were incubated in the dark for 14 d at 25 C. The strains selected were GR117 because
it removes all dye and, PLO9 and PLO13 that present the largest discoloration zones
compared to other tested fungi. The selected fungi mycelium was used as inoculum in
substrate containing 10g ofJatropha curcasseed cake and 10g ofAgave sisalanaresidues
plus 140 mL water and 10 mL of Kirk solution. The flasks were incubated at 100 rpm
at 25 C for 20 d. Independent of the substrate and the fungal strains, we observed the
highest specific activity after 20 d. In both substrates PLO9 was the fungus that presents
highest specific activity. However,J. curcas seed cake presents highest potential to be
used as substrate for the production of LIP. Therefore, the previously selected isolates
present the capacity of dyes degradation and LiP industrial production using agro-industrial residues.
Financial support: CNPq, FAPEMIG, CAPES

SIMB 2013 | 98

ISOLATION AND CHARACTERIZATION OF THE MICROBIOTA

FROM AGRO-INDUSTRIAL RESIDUES: MACAUBA CAKE AND
SWEET SORGHUM BAGASSE
MENDES, S. F., ALMEIDA, J. V. S., SOUZA, I. F., VANZELA, A. P. F. C., SANTOS, A.
S., PANTOJA, L. A.
Federal University of the Valleys of Jequitinhonha and Mucuri - UFVJM, Diamantina,
Minas Gerais, Brazil.
Enzymes are widely used in a range of industrial sectors due to their feasible production by biotechnological processes and the increased quality of their products, both
economically and environmentally. The aim of this study was to isolate and characterize microorganisms from agro-industrial residues (macauba cake and sweet sorghum
bagasse) to select potential enzyme producers. Strains were isolated after homogenizing
cake or bagasse with distilled water. Aliquots of the filtrate were inoculated into the selective medium Manachini supplemented with different carbon sources: starch, olive oil,
carboxy-methylcellulose and xylan. Bacterial isolates were stored in glycerol, at freezing
temperatures for later studies. The macroscopic morphology of fungal strains grown on
potato-dextrose and oatmeal agar was characterized by evaluating texture, pigmentation, surface, edges, topography, color, appearance and pigment. The microscopic morphology was analyzed by the micro-culture technique of Riddell. Filamentous fungi were
also evaluated for the production of amylase, cellulase, xylanase and lipase in screening
culture media. An index of enzyme activity (IEA) was calculated by the ratio: diameter
of substrate degradation / radial growth of the colony. Thirty bacterial strains and twenty
filamentous fungi were isolated, among which, five were classified to genus by means of
macro and microscopic analysis, along with a Mycology database. Screening for enzyme
producers revealed nine fungal strains that presented IEA above 2.0. It were determined
IEAs of 3.65, 3.54, 3.06, 3.28 and 2.17 (amylase); 3.92 and 2.69 (cellulase); 2.82 (xylanase) and 2.00 (lipase). It was possible to conclude that the microbiota of common
agro-industrial residues is rich in potential enzyme producers. A better understanding
of these microorganisms will open new prospective studies on biotechnological production and future applications for the enzymes synthesized by the strains isolated.

SIMB 2013 | 99

ETHANOL EFFECT ON THE YIELD OF

-CYCLODEXTRIN PRODUCED BY THE CGTASE FROM
ALCALOPHILICMICROORGANISM
FENELON, V.C.;AGUIAR, M. F. A.;MIYOSHI, J. H.;MORIWAKI, C.; VALENTINI,
S. R.; MATIOLI, G.
Departamento de Farmcia, Universidade Estadual de Maring (UEM), Maring,
Paran, Brasil.
Cyclodextrin glycosyltransferase (CGTase) is an important enzyme of industrial
interest, unique in its ability to convert starch into cyclodextrins (CDs). CDs are cyclic
maltooligosaccharides of six ( -CD), seven ( -CD), eight ( -CD) or more units of
glucose attached by -(1,4) bonds. These molecules are able to form inclusion complexes
and to stabilize a large number of substances, making them attractive for several applications in pharmaceutical, cosmetic, food, textile, chemistry and otherindustries. Considering the importance of reducing the production costs of CDs, this study aimed to assay
the effect of ethanol 10% in the yield of -CD produced by the CGTase fromBacillus
firmusstrain 37. The crude extract obtained from the enzyme was used and corn starch
5% (w/v) as substrate. Eight batch production cycles of 24 h were carried out, and an ultrafiltration system was used to remove the inhibitory products accumulated in the reaction medium and recovering the CGTase enzyme. The -CD yield reached for the assay
conducted in the presence of ethanol 10% was 0.43 mmol/L/h, 12% higher if compared
to the use of the crude extract and the corn starch substrate without ethanol, in which
the yield was 0.38 mmol/L/h. These results show the possibility of a satisfactory -CD
yield from crude CGTase, which is much easier to obtain than the pure enzyme. The
presence of ethanol 10% in the reaction medium proved to be an inexpensive alternative
to improve the -CD yield, using crude extract of CGTase and corn starch substrate.
Acknowledgements: CNPq, CAPES and Fundao Araucria.

SIMB 2013 | 100

COMPATIBILITY BETWEEN BRAZILIAN ISOLATES OF

ECTOMYCORRHIZAL FUNGI ANDACACIA MANGIUM
BARROS C. S.; CAMPOS A. N. R.
Agroecology Student at IF Sudeste MG Campus Rio Pomba/MG Brazil. Professor
at IF Sudeste MG Campus Rio Pomba/MG Brazil.
Acaciamangium is a leguminous tree which features rusticity, rapid growth and
ability to deposit large amount of organic material of good quality in the soil. Although
the benefits of rhizobia and arbuscular mycorrhizal fungi for this plant are well known,
information about its ability to interact with ectomycorrhizal fungi strains isolated
in Brazil is needed. The objective of this study was to investigate the interaction betweenA.mangiumand Brazilian strains of ectomycorrhizal fungi. Initially, we performed
the collection of fruiting bodies belonging to generaPisolithussp. andSclerodermasp.
inEucalyptusspp. plantation to isolate these fungi in Petri dishes. Also, we evaluated
the effect of sterilization and dormancy break forin vitrogermination ofA.mangium.
After, we carried out thein vitrosynthesis of mycorrhizae betweenA.mangiumand ectomycorrhizal isolates. Finally, a greenhouse study was conducted with inoculation ofA.
mangiumwith isolates ofPisolithusspp. andSclerodermasp. Initially, we established a
protocol forin vitrogermination ofA. mangiumincluding sterilization with hydrogen
peroxide for 5 min followed by dormancy break with water at 100 C for 1 min. We observed that all strains tested were able to establishin vitroectomycorrhizal association
withA.mangiumand that the isolates PIS 2, PIS 10 and PT441 induced lateral root formation. The greenhouse experiment showed increased fresh and dry mass of roots and
total dry mass in inoculated treatments. Also, we observed that the isolate Pis 2 showed
increased total dry matter compared to isolates Dic 3 and Pis 10. We concluded that Brazilian ectomycorrhizal strains andAcacia mangiumare compatible and that these strains
have potential to be used for production of seedlings of this species.
Acknowledgements: The authors are grateful to Conselho Nacional de Desenvolvimento
Cient?co e Tecnolgico (CNPq; Project 407082/2012-3) and to Fundao de Amparo Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial support.

SIMB 2013 | 101

EVALUATION OF DIFFERENT CARBON SOURCES FOR LFAAMYLASE PRODUCTION BYBACILLUS AMYLOLIQUEFACIENS

SILVA, I.F1., VANZELA, A. P. F. C2., SANTOS, A. S2.
1- Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil.2 Universidade
Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, Minas Gerais, Brazil.
Amylases represent approximately 30% of the worldwide industrial enzyme production, ranking second after proteases. They have applications in various industry
sectors, such as food, textile, chemical, pharmaceutical. The -amylase secreted by the
genusBacillus, highlighting the bacteriumBacillus amyloliquefaciens, is responsible for
much of the industrial production of this enzyme. However, the industrial production is
limited by the high costs of substrates. Thus, the use of lignocellulosic residues as inducers of amylase synthesis emerges as a promising alternative to decrease cost and to obtain a composition of the enzyme extract that is differentiated. Our aim was to evaluate
the influence of different lignocellulosic raw materials in the production of -amylase
byBacillus amyloliquefaciens. The residues evaluated were: peach palm flour, palm macauba, castor bean and lupine cakes. The fermentation media were prepared in Erlenmeyer flasks containing 10 grams of the respective residues, 100 mL of salt solution with
the following composition: 1gL-1 MgSO4.7H2O, 2gL-1 KH2PO4, 10 g L-1 NH4NO3 and
1gL-1 NaCl. The pH of the media was adjusted to 4.0. Inoculation was performed to
obtain 1.06 x108UFC/mLBacilluscells and the flasks were incubated at 37 C for 72
hours under agitation (120 rpm). Experiments were performed in duplicate. One unit
of enzyme activity was defined as the amount of enzyme capable of consuming 10 mg of
starch/minute/mL. In all experiments maximum activity was observed at 24 hours of incubation. According to the results, the best carbon sources for enzyme production were
macauba and peach palm flour with maximum values ??of 1165 UmL-1and 946.5 UmL-1,
respectively, followed by palm oil residue with 856.5 UmL-1, lupine with 791 UmL-1and
castor bean with 610 UmL-1. Considering the high enzyme activities produced, it was
concluded that alternative carbon sources may be successfully employed for -amylase
production byBacillus amyloliquefaciens.
Acknowledgements: CNPq Fapemig

SIMB 2013 | 102

ANALYSIS OF PHAGE USE AS TREATMENT OF MASTITIS

CAUSED BYESCHERICHIA COLI
FLVIA DE OLIVEIRA SOUZA, SRGIO OLIVEIRA DE PAULA, ROBERTO
SOUSA DIAS, VINCIUS DA SILVA DUARTE
Universidade Federal de Viosa,Avenida Peter Henry Rolfs, s/n,Campus
Universitrio,Viosa MG, Brasil
Bovine mastitis is the main disease afflicting dairy cattle in Brazil and all over the
world. The clinical and subclinical infections are ordinarily caused by a large number of
microorganisms, asEscherichia coli. Nowadays the most used procedure for treatment
is the antibiotic therapy, but alternate ways are being envisaged due to the increase of
multi-drug-resistant-bacteria and antibiotic residue present on the milk, which presents
risk to public health and interferes on dairy products production. Therefore, the use of
bacteriophages as a natural tool, nontoxic and viable for pathogens control has become
an effective alternative to mastitis treatment, since it is very specific, it replicates in the
infection area, it presents no side effects, and the bacteriophage resistant bacteria remain
susceptible to others.The objective of this study was to evaluate the efficacy of bacteriophages in the control of mastitis induced in an animal model, for assessing inflammatory and histological analysis of breast tissue. The samples used for viral isolation have
been gathered from the SAAEs (Servio Autnomo de gua e Esgoto) sewage system in
Viosa-MG. A wistar rat breast feeding was used as a model, being made inoculations
in duplicates (right and left teat). In the first pair of teats there was no inoculation as the
negative control, were inoculated in the second pair medium LB, which is used for the
propagation of theE. coli; the third pair PBS buffer was used, in the fourthE.coli(positive control), in the fifth were inoculated E coli and phage with 2 days an interval between them, and finally at the sixth pair, only the phage. After one week of the start of
inoculations was the sacrifice of the animal and histological analysis of the breast tissue
of all teats. It was found that the teats of negative control and those treated with PBS and
LB, glands were normal; however, the ducts were more closed when subjected to the
latter two treatments. In the teat where there was bacterial inoculation was noted the
presence of large numbers of inflammatory cells, confirming the presence of inflammation with little change in the morphology of the gland and milk production. However,
the roof subjected to phage therapy although indicates inflammatory infiltrate, exhibit
milk production.

SIMB 2013 | 103

INFLUENCE OF MS SALTS CONCENTRATION ON THE INITIAL

DEVELOPMENT OF SOYBEAN EMBRYOSIN VITRO
LOPES, F. S.1, LIMA, A. M.1, SANTOS, C. A.1, BARROS, E. G.1,2, MOREIRA, M. A.1
Instituto de Biotecnologia Aplicado Agropecuria (BIOAGRO), Universidade Federal
de Viosa, Viosa, Minas Gerais, Brasil 2Programa de Ps-Graduao em Cincias
Genmicas e Biotecnologia, Universidade Catlica Braslia, Braslia, DF Brasil
1

Soybean (Glycine maxL. Merrill) is the most important vegetable cultivated worldwide, due to its high protein (40%) and oil (20%) contents, high productivity and low
production cost. The soybean breeding programs have been increasingly concerned with
the development of high yielding cultivars, with high nutritional quality and other desirable characteristics in the grain. Among these characteristics, we highlight the efforts to
increase the oil content and improve its quality using classical breeding techniques and
modern biotechnological methods. The aim of this study was to determine the salt concentration of Murashige and Skoog medium (MS) that favors the initialin vitrodevelopment of embryos of different soybean varieties. Explants of varieties CAC-1, Msoy-8400,
Conquista and CD-219 were sterilized with chlorine gas for 6 h, excised after immersion
for 16 h in water sterile, and inoculated in MS medium containing different concentrations of MS salts (4.4, 2.2 and 1.1 g.L-1). The experimental design was randomized with
three replications. The explants were kept in the dark for 5 days and under light (36mol
m-2s-1irradiance) for 2 days, at 25 2 C, 75% relative humidity and a photoperiod of 16
h of light. We evaluated the following developmental parameters: hypocotyl diameter,
total length, fresh weight and dry weight. Statistical analysis was performed by the SAEG
program, version 9.1. No significant interaction among the treatments was observed. At
concentration of 2.2 g.L-1MS salts significantly higher values ??for diameter of the hypocotyl, total length and dry matter were obtained. The initial development of embryos
of different varieties is favored by the concentration of 2.2 g.L-1MS salts, although other
factors may also increase the efficiency ofin vitrocultivation of soybean explants.
Financial support: CNPq, CAPES and FAPEMIG.

SIMB 2013 | 104

ECOLOGICAL ASPECTS OF PARABA DO SUL RIVER IN

THE STATE OF RIO DE JANEIRO AS HYDROCHEMISTRY
AND MICROBIOTA ASSOCIATED WITH FLOATING
MACROPHYTESALVINIA AURICULATAAUBL.
GRANATO, T.M.1, GOMES, P.H.1, LEITE, T.C.1,ESTEVES, B.S.1, SUZUKI, M.S.1,
INTORNE, A.C.2
Laboratrio de Cincias Ambientais (LCA),2Laboratrio de Fisiologia e Bioqumica de
Microrganismos (LFBM), Centro de Biocincias e Biotecniologia (CBB), Universidade
Estadual do Norte Fluminense Darcy Ribeiro (UENF), Avenida Alberto Lamego, 2000 Parque Califrnia -Campos dos Goytacazes-RJ, Brazil
1

The industrialization, urban sprawl and increasing agricultural areas have led to a
rapid deterioration of environmental quality. Aquatic pollution is considered a major
problem in both developed and developing countries. Therefore, the protection of these
ecosystems is urgent. The Paraba do Sul River extends through the southeast region
of Brazil and is considered one of the country's major rivers. The lotic ecosystems have
extensive marginal areas colonized by aquatic macrophytes, which play many important roles, including the detoxification of these environments. The bacteria have different metabolic pathways for the decontamination of compounds. However, very little is
known about the microbiota associated with aquatic plants. In this work, our goal was
to determine ecological aspects of the Paraba do Sul River regarding their hydrochemistry and microbial community associated with floating aquatic macrophyte Salvinia
auriculataAubl. Therefore, samples were collected in two cities: So Joo da Barra - SJB
(agricultural area) and Resende - RES (industrialized area), both located in the State
of Rio de Janeiro. It was evaluated variables related with water: temperature - SJB: 22.9
C and RES: 21.2 C, pH - SJB: 6.4 and RES: 6.8 and electrical conductivity - SJB: 105.1
S.cm- 1and RES: 88.9 S.cm- 1(portable equipment), alkalinity - SJB: 0:48 meq.L- 1and
RES: 0.38 meq.L- 1 (titration) , dissolved oxygen - SJB : 8.6 mg L- 1 and RES: 6.4 mg.
L- 1 (Winkler method) and suspended particulate matter - SJB: 6 mg L- 1 and RES : 1
mg L- 1(gravimetry). A total of 37 isolates were identified from leaves and roots ofS.
auriculatacollected in the Paraba do Sul River by plating in DYGS and NYDA media.
The most of isolates were observed in the two studied areas. After sequencing of these
strains will be possible to assess whether the differences observed in the microbiota of
these plants is related to the different impacts suffered in these areas of study.
Acknowledgements: CAPES, FAPERJ and UENF

SIMB 2013 | 105

ISOLATION OF BACTERIA FROM FLOATING AQUATIC

MACROPHYTESALVINIA AURICULATAAUBL.
GRANATO, T.M.1, GOMES, P.H.1, SOUZA, S.A.2, SILVA, N.D.3, LOPES. P.S.3,
OLIVARES, F.L.3, DE SOUZA FILHO, G.A.2, SUZUKI, M.S.1, INTORNE, A.C.4
Laboratrio de Cincias Ambientais (LCA),2Laboratrio de Biotecnologia
(LBT),3Laboratrio de Biologia Celular e Tecidual (LBCT),4Laboratrio de Fisiologia e
Bioqumica de Microrganismos (LFBM),Centro de Biocincias e Biotecniologia (CBB),
Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Avenida Alberto
Lamego, 2000 - Parque Califrnia -Campos dos Goytacazes-RJ, Brazil
1

The increase in human population has spurred the production of effluents and the
most of it is discarded improperly in water bodies. Some macrophytes have great ability to remove substances present in water, and they are indicated for maintenance of
environmental quality.Salvinia auriculataAubl. (Salvinaceae) is a floating aquatic macrophyte which has potential for wastewater treatment, due to its high rate of growth. The
genusSalviniaalso demonstrates ability to accumulate large quantities of metals. During
evolution, microorganisms have developed a variety of resistance mechanisms to attenuate the toxicity of compounds in the environment. The coexistence of bacteria and plants
occurred over of years. Therefore, it is believed that the bioremediation can be provided
efficiently by the result of the interaction between plants and bacteria. However, very
little is known about the microbiota associated with these plants. This study aimed to
isolate, characterize and identify bacteria associated withS.auriculata. For this purpose,
in order to establish an isolation protocol for the initiation of studies, 0.5g of grown
plants in the greenhouse were placed in 50mL tubes and washed twice for 1min in 10mL
of distilled water. Then the plants were transferred to tubes containing 10mL of saline
solution (0.85% NaCl) and incubated for 10min on ultrasound. Subsequently, the plants
were macerated in the same solution, diluted and plated on the DYGS and NYDA. The
plates were incubated for 24, 48 and 72h, when bacterial colonies were isolated. By the
time we obtained 20 strains whose sequencing of 16S ribosomal RNA identified the genera:Agrobacterium,Bacillus,Curtobacterium,Enterobacter,Pantoea, andPseudomonas.
Some bacteria were positive for nitrogen biological fixation and nutrient solubilization.
Then, we intend to explore the potential application of these microorganisms for bioremediation of contaminated environments.
Acknowledgements: CAPES, FAPERJ and UENF

SIMB 2013 | 106

XANTHAN GUM PRODUCTION BY

XANTHOMONASCAMPESTRIS PV. CAMPESTRISFROM SUGAR
CANE JUICE
TARGINO, BRENDA NERES1; OLTVARO, JUANITA PEREZ1; COELHO,
JUSSARA MOREIRA1; RODRIGUES, VIVIAN CAROLINA1; SILVA, PAULO
HENRIQUE ALVES1
Biomolecules and Bioprocesses Laboratory, Food Technology Department, Federal
University of Viosa, Av PH Rolfs, s/n 36570-000, Viosa, Minas Gerais, Brazil
1

Xanthan gum is an exopolysaccharide, with high molecular weight, synthesized by

fermentation of Xanthomonas campestris. These polymers have a lot of industrial applications: foods, toiletries and cosmetics, as well oil recovery and others. The rheological properties allows it to be used as thickening and stabilizer agents in emulsions and
suspensions. It can be obtained from different carbon sources such as sucrose, glucose,
fructose and lactose. In order to find alternatives medias of cultivation, profitable and
viable, we have evaluated the production of xanthan gum by Xanthomonas campestris
pv campestris IBSBF 629 using sugar cane juice as carbon source. Five different compositions of growing media were evaluated for gum production, after addition of the
inoculum (10% v/v). The fermentation conditions were 250 rpm / 28 C / 72 h. The gum
rheological properties were determined using a Brookfield rheometer by monitoring
the shear rate and the corresponding shear stress applied. The growing media that have
shown the best performance was compared to a standard growing media and a commercial xanthan gum. The medium 4 (sugar cane juice, hydrolyzed casein, potassium
phosphate and magnesium sulfate) have shown the highest gum production, but poor
rheological properties. The medium 5 (sugar cane juice, hydrolyzed casein, yeast extract,
potassium phosphate and magnesium sulfate) have shown a lower yield, however, its apparent viscosity was 201.55 mPa.s, being the growing medium that have shown the best
rheological behavior. It was possible to produce xanthan gum by using the strain IBSBF
629 and growing medium made with sugar cane juice and other micronutrients in the
conditions tested in this experiment. However, the apparent viscosity obtained was very
low when it was compared to the viscosity of the commercial gum, being necessary to
test other concentrations of sugar cane juice and micronutrients, which could improve
the rheological characteristics of this product.
Acknowledgements: Thanks to CAPES and FAPEMIG by the fellowships

SIMB 2013 | 107

MATHEMATICAL MODELING AND DYNAMIC

BEHAVIORANALYSIS OF ANINDUSTRIAL FERMENTATION
PROCESS FOR ETHANOL PRODUCTION
DECHECHI, E. D.1; OLIVEIRA, S. C.2,*
CECE/PTI-Department of Engineering and Sciences at Itaipu Research Park (PTI),
Western ParanState University (UNIOESTE), Foz do Iguau, PR, Brazil, E-mail:
eduardo.dechechi@unioeste.br 2Department of Bioprocesses and Biotechnology, School
of Pharmaceutical Sciences (FCF), SoPaulo State University (UNESP), Araraquara, SP,
Brazil, E-mail: samueloliveira@fcfar.unesp.br *Corresponding Author
1

In this study, the dynamic behavior of a continuous process of alcoholic fermentation in four stirred tank reactors connected in series with cell recycle is analyzed. From a
phenomenological mathematical model was possible to simulate the dynamic behavior
of the main bioprocess output variables (substrate, product, and cell concentrations in
the reactors) when step type disturbances were imposed on the input variables (volumetric flow rate of feed medium, total reducing sugars concentration in feed medium,
recycle ratio, inlet temperature of the feed medium and coolant inlet temperature). The
mathematical model is represented by 28 ordinary differential equations (ODEs) corresponding to the balances of mass and energy at each stage, and 15 algebraic equations
including kinetic expressions and complementary equations. The specific growth rate
is based on Monods model and includes additional terms that take into account the
inhibitory effects linked to the ethanol and to the biomass itself. The specific substrate
consumption and ethanol production rates are given as a function of the specific growth
rate, using appropriate yield coefficients. The kinetic parameters were representative of
typical industrial conditions and the dependence of these parameters on temperature
was also considered. Numerical integration of the ODEs was carried out by a fourth-order Runge-Kutta method. The simulation results showed that the fermentation process
is highly sensitive to changes in certain process input variables, exhibiting linear, asymmetric or inverse responses according to the variable disturbed. Among the investigated
input variables, the volumetric flow rate of feed medium and the recycle ratio showed
to be those that exert more effects on the dynamic behavior of the fermentation process,
setting up as potential manipulated variables to control purposes.

SIMB 2013 | 108

ADVANCED PREDICTIVECONTROL OF AN
INDUSTRIALFERMENTATION PROCESSFOR ETHANOL
PRODUCTION
DECHECHI, E. D.1; OLIVEIRA, S. C.2,*
CECE/PTI - Department of Engineering and Sciences at Itaipu Research Park (PTI),
Western ParanState University (UNIOESTE), Foz do Iguau, PR, Brazil, E-mail:
eduardo.dechechi@unioeste.br 2Department of Bioprocesses and Biotechnology, School
of Pharmaceutical Sciences (FCF), SoPaulo State University (UNESP), Araraquara, SP,
Brazil, E-mail: samueloliveira@fcfar.unesp.br *Corresponding Author
1

The predictive control algorithm DMC (Dynamic Matrix Control) is frequently

reported in the literature as a robust and efficient tool for the control of a variety of industrial chemical processes. However, this control algorithm has been little applied in
bioprocesses. The main contribution of this study is the application of the multivariable
DMC predictive control strategy to a fermentation process operating under real conditions. The DMC control algorithm was applied to a multivariable system of a higher
dimension than the traditional 2x2 dimension commonly found in the literature. The
system analyzed was a continuous industrial process of ethanol production from sugarcane juice usingSaccharomyces cerevisiaeyeasts. The alcoholic fermentation process
is carried out in a cascade of four reactors with cell recycling for the first stage. The
disturbances that actually occur in an alcohol distillery are variations in the concentration of TRS (Total Reducing Sugars) in the feed medium (S0) and oscillations in ambient temperature, which directly affect the inlet temperature of the feed medium (T0)
and the temperature of the cooling water (Tw). To simulate real operating conditions,
disturbances of different degrees of magnitude were imposed on the variables S0, T0and
Tw. The operating conditions simulated were aimed at reproducing real operating conditions in an industrial ethanol production unit. Even when subjected to varying operating conditions and to a long delay time of the TRS measurements, the DMC-MIMO
(Multiple Input Multiple Output) controller kept the fermentation process operating
stably at high levels of performance. However, a significant improvement in the controllers action was found when the non-manipulated input variables (measured disturbances) were included in the control algorithm. The good performance displayed by the
DMC-MIMO controller recommends its application in industrial units resembling the
one simulated in this study.

SIMB 2013 | 109

INFLUENCE OF SONICATION PROCESS ON THE VIABILITY

OFAGROBACTERIUM TUMEFASCIENS
LOPES, F. S.1, PRUDNCIO, C. V.2, SANTOS, C. A.1, MOREIRA, M. A.1
Instituto de Biotecnologia Aplicada Agropecuria (BIOAGRO), Universidade Federal
de Viosa, Viosa, Minas Gerais, Brasil. 2Departamento de Microbiologia, Universidade
Federal de Viosa, Viosa, Minas Gerais, Brasil.
1

Agrobacterium tumefasciens has been used for genetic transformation of many

plant species, due to its ability to transfer information in their plasmid ADN to infected
plant. This infection occurs mainly in injury in plant tissue. The sonication has been
used in transformation protocols, by its capacity to promote the opening of pores in
tissue, in order to enhance the infection and promote a large efficiency of the process.
However, there are no reports in the literature of the impact of the process of sonication
on the viability of the bacteria. Therefore, the objective of our work was to evaluate the
influence of sonication on the survival ofA. tumefasciens. For this, the culture ofA. tumefaciensKYRT1, with plasmid resistance to rifampicin and kanamycin, was grown in
YEP broth with kanamycin and rifampicin for 12 h. The cells were centrifuged, washed
and the pellet resuspended in Murashige and Skoogs medium with OD600 of 0.7. The
sonication treatments were performed in tubes Eppendorf tubes at 47 kHz, 5, 10, 30, and
60 seconds. The viability was assessed by plating on agar for standard count (PCA), by
the drop method. The counting of microcolonies was performed after 12 h of incubation
at 37 C. Statistical analysis was effected by SAEG software, version 9.1. It was noted that
under the conditions used the process of sonication had no effect on cellular viability,
even with the higher time utilized (60 sec.) These data show that the transformation
protocols can extend the sonication time without loss of viability of the cells. However,
it should suit the optimum time in accordance with the plant tissue to be transformed.
Financial support: CNPq, CAPES and FAPEMIG.

SIMB 2013 | 110

EVALUATION OF THE USE OF BACTERIOPHAGES TO

CONTROL BIOFILMS ON REVERSE OSMOSIS SYSTEM USED IN
OIL REFINERIES BY SCANNING ELECTRON MICROSCOPY
LARISSAASSISBARONYVALADARES FONSECA, ROBERTOSOUSA DIAS2;
JAMILLEMANSUR ALBANESE3, CYNTHIACANEDO SILVA4,MAIRA P.
SOUSA5, RODRIGO S. DE SOUSA5, ANA PAULA TORRES5,SRGIOOLIVEIRA
DEPAULA6
Departamento de Biologia Geral/UFV - Viosa, Brasil2Doutorando do
Departamento de Biologia Geral/UFV - Viosa, Brasil;3Estudante do curso de
Bioqumica da UFV- Viosa, Brasil;4Departamento de Biologia Geral/UFV- Viosa,
Brasil;5PETROBRAS-Brasil;6Professor do departamento de Biologia Geral da UFV
-Viosa, Brasil
The current trend in wastewater management by industries focuses on reduction of
the environmental impacts. In this context, industries with high consumption of water
make use of reverse osmosis technology in wastewater treatment, since this technology provides high productivity and efficiency in removing a variety of contaminants.
Nevertheless, the adhesion and growth of microorganisms, and subsequent biofouling,
difficult the use of this technology. Biofilms are microbial communities that develop
adhered to a surface and surrounded by extracellular polysaccharides substances (EPS).
Biofilms are very difficult to eradicate. The utilization of phages is a good way to control
the biofilm formation once they have high specificity, affecting only the target bacterium. There is some phage which have a simple, rapid and relatively inexpensive production, that produce depolymerases, enzymes that affects bacterial cell wall. In order to
isolate the phage, was used a bacteria, which was isolated using activated sludge from
Gabriel Passos Refinery (REGAP), located in the city of Betim, Brazil. In result, four
bacteriophages were isolated from activated sludge and named UFVhalophageR2, R3,
M3 and M4. All the isolated showed the same morphological characteristics according
to electronic microscopy. The phage have an average size of 33 nm, a 3 nm-short tail,
and an isometric head approximately 25 nm in diameter, belonging to Podoviridae family, of Caudovirales order. In addition, the technique RAPD was performed in order to
evaluate the genetic diversity/polymorphism in 4 isolated phages . After analysis, it was
observed that the size of the obtained fragments ranged from 500 bp to 5 kb, with 14 polymorphic bands for P2 primer and 12 bands for OLP5 primer. The four isolates showed
an identical polymorphism, indicating that they're same phage. Subsequent tests will be
made in order to evaluate the use of these bacteriophages in biofilm formation control.
Acknowledgements: Fapemig Petrobras

SIMB 2013 | 111

DYNAMIC OF NITROGEN-FIXING BACTERIA IN FOREST

PLANTATIONS OFEUCALYPTUS UROGRANDISUNDER
FERTILIZING NITROGEN
MARLIANE DE CSSIA SOARES DA SILVA(a); IGOR RODRIGUES MENDES(a);
THIAGO DE ALMEIDA PAULA(a); ROBERTO SOUSA DIAS(b); SRGIO
OLIVEIRA DE PAULA(b); CYNTHIA CANEDO SILVA(a); DENISE MARA SOARES
BAZZOLLI(a); MARIA CATARINA MEGUMI KASUYA(a*)
Departamento de Microbiologia, Federal University of Viosa, Viosa, Minas Gerais,
36570-000, Brazil. bDepartamento de Biologia Geral, Federal University of Viosa,
Viosa, Minas Gerais, 36570-000, Brazil.
a

In Brazil, the demand for forest products has led to an increase in planted forests,
and these have now reached seven million hectares (ha), of which 74.8% are eucalypt
plantations. A large proportion of eucalypt plantations in Brazil are located in areas with
low soil fertility. The action of microorganisms is of great importance for the cycling
of nutrients, including nitrogen (N), which are essential for plant metabolism. In this
study, the DGGE was used to monitor and identify active microorganisms involved in
the N cycle, in both soil and root system from forest ofEucalyptus urograndis, fertilized
or not with N. Quantitative real time PCR was used to examinenifHgene expression in
N-fixing bacteria. ThenifHgene DGGE profiles of the populations of active N-fixing
bacteria are influenced by the winter and summer seasons and/or N fertilization. DGGE
band sequencing from RNA revealed that Nostoc, Hyphomicrobium and Bradyrhizobium genera appeared in higher amounts. The quantitative real time PCR revealed
thatNifHexpression was higher in soil, especially in those that did not receive N fertilization. The detection of diazotrophs active populations in soil and roots shows the
effective population responsible for Biological Nitrogen Fixation.
Acknowledgements: The Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES) and
Fundao de Amparo Pesquisa do estado de Minas Gerais (FAPEMIG) for their financial
support.

SIMB 2013 | 112

ISOLATION OF ENDOPHYTIC BACTERIA FROM LEAVES, SAP

AND ROOTS OF MAYZE (ZEA MAYS)
VIEIRA, J. A. C.; ALVES, N. L. S.; LIMA, R. R.; SANTOS, V. L.
Universidade Federal de Minas Gerais, Instituto de Cincias Biolgicas, Departamento de
Microbiologia, Av. Antnio Carlos, 6627 - Pampulha - Belo Horizonte, MG
One of the greatest challenges of this century is the development of more efficient
farming methods combined to environmentally sustainable techniques. Endophytic microorganisms are able to naturally colonize the tissues of plants without causing harm,
and can be a solution to this challenge, since several studies have demonstrated their
ability to produce growth factors and antagonist compounds against pathogens. In this
work, we isolated endophytic microorganisms from leafs, roots and sap of maize (Zea
mays), with the initial goal of studying their diversity in the plant. The isolation was
performed in Petri dish containing Trypticase Soy Agar, followed by phenotypic characterization of the different morphotypes. 301 isolates were obtained and grouped into
108 morphotypes, in which only six were simultaneously observed in leaves, roots and
sap. The root presented the highest diversity of unique morphotypes, 33, while in sap
and leaves we observed 13 and 10 unique morphotypes, respectively. Also, it was noted
that the number of microorganisms common in sap and leafs and in sap and roots was
lower when compared with the exclusives of each one of this regions. The largest source
of endophytic bacteria is the soil, which explains the greater diversity of these in the
roots. Studies showing the specificity of relations plant/endophytic microorganisms corroborate with the low number of morphotypes observed simultaneously in more than
one region of the maize. Our results open doors for exploring the potential of the isolates
to promote plant growth and development, enabling future applications in grain crops
such as corn, to increase productivity or as a biological control agent.
Acknowledgements: CAPES, Fapemig

SIMB 2013 | 113

MICROBIOLOGICAL AND SENSORY CHARACTERISTICS

IN CONSERVATIONOF 'FORMOSA' PAPAYA AND 'PEARL'
PINEAPPLE MINIMALLY PROCESSEDSTORED AT 4C
LIMA, P.C.C.1; SOUZA, B.S.2; BORGES, S.S.2; ASSIS, M.D.O.2
Federal University of Viosa, Minas Gerais, Brazil. 2Federal Institute of Education,
Science and Technology of South of Minas Gerais Muzambinho campus, Minas Gerais,
Brazil.
1

The minimal processing is the process that modifies physically fruits or vegetables without altering their natural state. The consumption of pineapple and papaya is
not convenient because it requires a laborious peeling. The degree of convenience for
the consumption of these fruits can be increased if they were sold already peeled, cut
into pieces and packaged. In order to increase the consumption of these fruits and add
value to the product, this paper aims to present sensory and microbiological aspects of
the conservation of 'Formosa' papaya and 'Pearl' pineapple together in the same package, minimally processed and stored at 4 C. The fruits were selected, washed, sanitized
(0.2 Kg/m Cl) and were stored for 12 hours at 10C. After this period, at 12 C, the
pineapple fruits were peeled, cut into slices (1.5-2 cm of thickness), and these slices
were cut in half or into four parts, the papaya fruits were cut longitudinally, seeds and
the ends have been removed, the pieces were cut in the transverse direction (1.5-2 cm
of thickness) and, then, cut into four parts. The pieces were rinsed (0.02 kg/m Cl),
drained and packed in PET (Poly Ethylene Terephthalate) or polystyrene coated with
PVC film (Polyvinyl Chloride) and stored at 4C. During storage were evaluated counts
of Coliforms,Salmonella and Staphylococcus and sensory evaluation by the consumer
for texture, taste and preference. The sensory analysis 'Perola' pineapple did not differ
significantly from the 'fresh' product. Already in the sensory analysis of 'Formosa' papaya was observed an increase in product texture and decreased quality due to storage
time, differing significantly from the 'fresh' product.The presence of microorganisms
was not detectedduring storage. The products stored at 4 C showed adequate quality
for consumption and marketing, indicated by sensory and microbiological analysis, for
up to six days.
Acknowledgements: FAPEMIG by the financing of project (APQ-00926-09).

SIMB 2013 | 114

ANTIFUNGAL ACTIVITY OF ACTINOBACTERIA ISOLATED

FROMCERRADO SOILS OF GOIS, BRAZIL
OLIVEIRA, B. F. R.1; FERREIRA-NETO, P. J.1; CARRIM, A. J. I.1; VIEIRA, T. M.1,
ARAUJO, M. V. F.1; MILHOMENS, J.1; REIS, L. G. V.1; SADOYAMA, G.2; LEOVASCONCELOS, L. S. N. O.1; VIEIRA, J. D. G.1
Instituto de Patologia Tropical e Sade Pblica, Universidade Federal de Gois (UFG),
Goinia, Gois, Brazil. 2Campus Catalo, Universidade Federal de Gois (UFG),
Catalo, Gois, Brazil.
1

The actinobacteria are generally filamentous, soil-dwelling prokaryotes with a

complex life cycle. They constitute one of the most economically exploited groups of
micro-organisms, in as much as they are producers of secondary metabolites which
can exhibit a wide variety of biological activities. Thus, they represent a rich source of
new pharmacological agents, notably antibiotic drugs. The incidence of fungal infections drastically increased in the last decades. It was followed by an elevation of innate
and acquired resistance to several antifungal classes by these pathogens, especially for
treatment of systemic mycosis in patients in risk groups, such as those immunocompromised ones. The aim of this study was to isolate actinobacteria from Cerrado soil and to
verify their antifungal action. Isolation of actinobacteria was conducted by the chemotaxis methodology of Palleroni (1980). The antifungal activity was determined using the
plug technique. The following tested microorganisms were applied: the standard strain
ofCandida albicansATCC 10231, and the clinical isolates ofAspergillus flavus1218,A.
niger1218 andPenicilliumsp. 1218. From the 20 morphospecies of isolated actinobacteria, 9 (45%) showed antifungal action againstC. albicans, 1 (5%) againstA. flavusand
3 (15%) againstA. niger. The isolate A22 presented antimicrobial activity against these
three fungi; however no antagonism against Penicillium sp. 1218 was observed. The
achieved data suggest the potential of these isolates against different types of tested fungi
to obtain specific biomolecules for these microorganisms. Studies of production and
isolation of these compounds are being conducted for characterizing them.
Acknowledgements: Laboratrio de Micologia - IPTSP/UFG.
Financial support: FFundao de Amparo a Pesquisa do Estado de Gois (FAPEG).

SIMB 2013 | 115

ACTINOBACILLUS PLEUROPNEUMONIAESEROTYPES
MOST COMMOLY ASSOCIATED TO DISEASE IN DIFFERENT
BRAZILIAN AREAS
REIS, KLEDNA CONSTANCIA PORTES; BOUILLET, LEONEIDE ERIKA
MADURO; HENRIQUES, MAYKA RABELO; VANNUCCI, FABIO AUGUSTO;
GUIMARAES, WALTER VIEIRA; PEREIRA, DANIELA MENDES; SANTOS,
DANIEL LUCIO; SANTOS, LUCAS FERNANDO; SANTOS, JOSE LUCIO.
Microvet Microbiologia Veterinria Especial Ltda, Av. Joaquim Lopes de Faria, 730 Santo Antonio, Viosa, Minas Gerais, Brazil
Pleuropneumonia due toActinobacillus pleuropneumoniae (App)is one of the most
important respiratory diseases of swine, because it causes economical losses by animal
death, decrease of production, and increase wastes associated with therapeutic and
prophylaxis. Presently, there are 15 different serotypes of App identified according to
their capsular antigens. Different serotypes are usually present in different countries and
regions. The serotyping is important for epidemiological studies and control of the disease by vaccination programs. This work shows the occurrence of App serotypes by the
polysaccharide capsule identification using gel immunodiffussion or PCR. The samples
(243) analyzed were isolated from lung lesions from 2006 to 2013 at different Brazilians regions by Microvet. In this period the most prevalent serotypes were 8 (31,28%),
5 (22,23%), 7 (18,11%) and 3 (13,58%). Twelve (4,94%) samples were not serotypables
(NS). We observed a gradual increase of the serotype 3 in this period. Furthermore, the
other serotypes 1, 2, 4, 6 and 9 occurred in low prevalence. These results show that the
occurrence and prevalence of serotypes evolves and changes over time. A clear shift in
predominant serotypes within a specific region compared to previous years can be observed. These results give further support for monitoring of the Brazilian herds and may
assist in developing the better strategies for vaccination programs.
Acknowledgements: We thank all colleagues in Microvet

SIMB 2013 | 116

BACTERIOCINS AND LACTIC ACID BACTERIA TO CONTROL

BIOFILM FORMATION AND ENTEROTOXIN PRODUCTION
BYSTAPHYLOCOCCUS AUREUS
ANDRE, C.1, SALIVE, A.V1,2, PIMENTEL-FILHO, N. J.1& VANETTI, M. C. D.1
Universidade Federal de Viosa, Viosa, Minas Gerais, Brazil 2Universidad del Tolima,
Ibagu, Tolima Colombia
1

Staphylococcus aureusis an important pathogen due to a combination of toxin-mediated virulence, invasiveness, antibiotic resistance and biofilm formation. In response
to certain environmental cues, bacteria can leave biofilms and return to the planktonic
state in which sensitivity to antimicrobials is regained. Some strains are able to produce
thermostable enterotoxins that are the causative agents of staphylococcal food poisonings. This work aimed to evaluate the effect of the bacteriocin bovicin HC5 against biofilm formation byS. aureusand the combined interference of nisin and lactic acid bacteria (LAB) on staphylococcal enterotoxin production.S. aureusFRI 722, an enterotoxin A
producer, was grown in Mueller-Hinton broth with bovicin HC5 and the biofilm assays
were performed in microtiter plates using violet crystal methodology. The enterotoxin
production was evaluated after 48 h of cultivation at 37 C by the immunoenzymatic
test VIDAS Staphenterotoxin System II in trypticase soy broth added of 20 AU/ml of
nisin and inoculated with 108CFU/ml ofLactococcus lactisATCC 19435 and 106CFU/
ml ofS. aureus. The minimal inhibitory concentration of bovicin HC5 onS. aureuswas
4.3 M and, low dosages of bovicin HC5 seems to induce the biofilm formation by the
pathogen. The enterotoxin production byS. aureusFRI 722 was not affected by the presence of low dosages of the bacteriocin nisin and LAB. Although used in the foodborne
growth control, low dosages of the tested bacteriocins did not prevent biofilm formation
and enterotoxin production byS. aureusFRI 722 even when associated withL. lactis.
Acknowledgements: We thank the CNPq, Capes and Fapemig for the financial support.

SIMB 2013 | 117

INFLUENCE OF CARBON SOURCES ON INDUCTION OF

CELLULASES AND HEMICELLULASES INCHRYSOPORTHE
CUBENSISGROWNIN SUBMERGED CULTURE.
DUTRA, THIAGO RODRIGUES1, FIALHO, LLIAN DA SILVA1, FALKOSKI,
DANIEL LUCIANO2, DE REZENDE, SEBASTIO TAVARES1.
Universidade Federal de Viosa Viosa-MG, Brazil,2Fungal Biodiversity Center,
Utrecht, Netherlands.
1

Our research has demonstrated that Chrysoporthe cubensis, phytopathogen fungus, has potential for producing enzymes involved in hydrolysis of biomass. This work
aimed to cultivateC. cubensisin carbon sources to observe the production of cellulases
and hemicellulases in each source. Complex carbon sources (CCS) such as wheat bran
(WB), carboxymethilcellulose (CMC), birchwoodxylan (BX), locust bean gum (LBG),
citrus peel (CP) and also simpler carbon sources (SCS) such as glucose (Glc), cellobiose
(Cb), lactose (Lac), galactose (Gal), mannose (Man), xylose (Xyl) and arabinose (Ara)
were tested as enzymatic production inducers. Zimograms containning 4-Methylumbelliferyl-glucopyranoside (MUG), BX and CMC as substrate were made to analize
qualitatively the enzymes present in extracts. When cultivated in WBC. cubensisproduced activity of 0.4 U/mL of b-glucosidase, 4.0 U/mL of endoglucanase and 14.0 U/mL
of xylanase. When grown in CMC, the fungus produced activity of 0.5 U/mL of b-glucosidase, 3.0 U/mL of endoglucanase and 10.7 U/mL of xylanase. The extract produced
by the fungus, when cultivated in BX, presented xylanase activity of 9.9 U/mL, however
cellulases was not presented. SCS presented low inducing capacity in C. cubensis. Zimogram containning MUG has demonstrated thatC. cubensisproduced b-glucosidase
activity when cultivated in all sources of carbon, except in Man and Gal. Zimogram
containing BX, has demonstrated that the fungus produced a xylanase constitutive of
19 kDa when grown in all sources tested. Extracts produced by fungus when grown in
WB, CMC and BX, presented a well defined region activity of xylanase between 97 and
39 kDa. Zimogram containing CMC as substrate has demonstrated that only in extract
produced byC. cubensiscultivated in Man and Xyl, was not noted the activity of endoglucanase. WB, CMC and BX induced the production of two endoglucanases (38 kDa
and 56 kDa). WhenC. cubensisgrew in Glc, LBG, CP, Lac, Gal, Ara and Cb it was produced one endoglucanase of 38 kDa. Analizyng results of enzymatic activities together
with zimogram analysis, it is clear that CCS have high inducing potential in relation to
the production of enzymes involved with biomass scarification. However SCS do not
induced meaningly the cellulases and hemicellulases production in this fungus.
Acknowledgements: We thank Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES) for providing scholarships.
SIMB 2013 | 118

THE TRANSLATIONALY CONTROLLED TUMOUR PROTEIN

IS A HOST FACTOR NECESSARY TOPEPPER YELLOW
MOSAICVIRUSINFECTION IN SUSCEPTIBLE HOSTS
FERNANDA PRIETO BRUCKNER1; ANDR DA SILVA XAVIER2; RENAN DE
SOUZA CASCARDO3; FRANCISCO MURILO ZERBINI2; POLIANE ALFENASZERBINI1
UNIVERSIDADE FEDERAL DE VIOSA; DEPARTAMENTO DE MICROBIOLOGIA;
CAMPUS UNIVERSITRIO s/n. VIOSA - MG; BRASIL 2UNIVERSIDADE FEDERAL
DE VIOSA; DEPARTAMENTO DE FITOPATOLOGIA; CAMPUS UNIVERSITRIO
s/n. VIOSA - MG; BRASIL 3UNIVERSIDADE FEDERAL DO RIO DE JANEIRO;
DEPARTAMENTO DE GENTICA; RIO DE JANEIRO - RJ; BRASIL
1

Translationaly controlled tumor protein (TCTP) is a ubiquitous distributed protein of eukaryotes, involved in regulation of several processes as cell cycle progression,
growth, stress protection, anti-apoptotic effect and maintenance of genomic integrity. It
was identified as a gene induced during the early stages of tomato infection by the potyvirusPepper yellow mosaic virus(PepYMV). To understand the functional role of this
protein during virus infection, complete cDNA sequence was cloned, sequenced and
characterizedin silico. It codifies a protein of 168 aminoacids, which contains conserved
domain of TCTP family. Tomato TCTP has four putative sites of phosphorylation, two
by kinases C and two by casein kinases type 2, one putative site of miristoylation and
typical signature sequence TCTP2. Moreover, a putative leucine-rich nuclear export signals and a putative weak nuclear localization signal were identified. To study the effect of
TCTP silencing in PepYMV infection,N. benthamianaplants were silenced by VIGS and
tomato transgenic plants silenced to TCTP were obtained and the effect of TCTP silencing in viral infection was analyzed. In the early stages of infection both tomato andN.
benthamianasilenced plants accumulate fewer viruses than control plants. Besides the
reduced virus accumulation, systemically infected tomato transgenic plants showed a
drastic reduction of symptoms and viral accumulation. Sub-celular localization of TCTP
was determined in health and systemically infectedN. benthamianaleaves by confocal
microscopy. TCTP is observed both in nucleus and cytoplasm of health plants and only
in cytoplasm of infected leaves. Thus, we showed that TCTP is a host factor necessary to
PepYMV infection and it localization is altered by the infection, probably favoring virus
establishment. Further studies have to be performed to elucidate the exact role of this
protein in PepYMV infection.
Financial support: CAPES; FAPEMIG; CNPq, INCTPP, IFS

SIMB 2013 | 119

BIOCHEMISTRY AND MOLECULAR CHARACTERIZATION

OF MICROORGANISM ASSOCIATED WITHLANGSDORFFIA
HYPOGAEA-HOST-RHIZOSPHERE INTERACTIONS
RICA BARBOSA FELESTRINO1, LEANDRO MARCIO MOREIRA1,2
Programa de Ps-Graduao em Biotecnologia, Ncleo de Pesquisas em Cincias
Biolgicas, Universidade Federal de Ouro Preto UFOP, Ouro Preto, Minas Gerais, Brasil
2
Departamento de Cincias Biolgicas, Universidade Federal de Ouro Preto UFOP,
Ouro Preto, Minas Gerais, Brasil
1

Microbial populations are directly related to different niches. The soil is an important ecosystem which continuous interactions between plant, roots and microorganisms
are observed. Based on these interactions,Langsdorffia hypogaea,a holoparasite plant
not well studied, was used as model. This study aimed bioprospect the microbiota of this
model and its possible interactions with its host plant and corresponding rhizosphere.
For this, microbiological techniques were used.Initially the bacteria weregrown and isolated, followed by molecular assays for the identification. Were also performed biochemical assays that allow verifying the ability of these 81 isolates to produce hydrocyanic acid
(HCN), indoleacetic acid (IAA), siderophores and ability to fix nitrogen. Tests to verify
the potential for degradation of starch, pectin, cellulose and proteins were also incorporated in this study. We have found bacteria of genusBacillus, Enterobacter, Lysinibacillus,
Paenibacillus,Serratia, Klebsiella, Ewingella, Citrobacter, Pseudomonas, Viridibacillus,
and a set of similar microorganisms deposited in the database as "uncultivated". All the
biochemical parameters showed positive results for some of the isolates. Highlight for
bacteria that were able to produce siderophores and IAA, respectively totaling 50% and
80% of isolates investigated. This feature allows understanding the complementarities
relationship between plant and microbiota that justify the adaptation of holoparasite
plant and host survive in a region where the soil is extremely hard and metal-rich, especially ferric iron, with low water availability. Features widely found at Quadriltero
Ferrfero (Brigidas Mountain - MG), where the plants were collected. These results help
to understand better the interaction between microorganisms and plants, which could
partly explain the survival of these species in an environment with several peculiarities.
Finally this work has identified a set of plant growth promoting bacteria that will now be
tested for potential use as biofertilizers.

SIMB 2013 | 120

CO2FLUX FROM THE SOIL AFTER PHOSPHATE

BIOFERTILIZER APPLICATION
NINA MORENA RGO MUNIZ DA SILVA1, THALITA CARDOSO ANASTCIO1,
VICTOR HUGO ARAJO BONDUKI1,GILBERTO DE OLIVEIRA MENDES1, IVO
RIBEIRO DA SILVA2, MAURCIO DUTRA COSTA1
1. Department of Microbiology, Federal University of Viosa, Av PH Rolfs, s/n,
BIOAGRO, Campus UFV, 36570-000, Viosa, MG, Brazil 2. Department of Soil
Science,Federal University of Viosa, Brazil
The use of organic residues in agricultural systems has beneficial effects on the
soil and on crop production. Residues can be applied to soilin naturaor be previously
processed by microbial treatment, such as composting. One interesting microbial treatment of residues that has been proposed is the production of a phosphate biofertilizer
based on the solubilization of low-grade rock phosphates (RP) by fungi in solid state fermentation (SSF) systems. However, the application of organic-residue-based products
to soil can trigger the emission of greenhouse gases by increasing soil microbial activity,
which can mobilize stable carbon from soil organic matter. The objective of this work
was to evaluate the emission of CO2following the application of three formulations of
a phosphate biofertilizer produced in a SSF system with sugarcane bagasse as substrate.
The biofertilizer was produced in 250-mL Erlenmeyer flasks with 5 g of sugarcane bagasse supplemented with 86.5 % of biochar, 25 % of RP, 27 % of sucrose, and 620 % of
moisture content (% based on sugarcane bagasse mass). The flasks were inoculated with
1 mL of a conidial suspension ofAspergillus nigerFS1 and incubated at 30 C during 7
days. The final product was further processed in three formulations: drying at 70 C and
incineration at 350 and 500 C. The formulations were applied to pots containing 1 kg of
soil at the concentration of 300 mg P per pot. The control treatments were: triple superphosphate, RP, and non-amended soil. The experiment consisted of pots planted with
beans (Phaseolus vulgaris) and pots with only soil. The CO2evolved was collected from
PVC chambers installed on the soil and injected in an isotopic carbon dioxide analyzer.
The highest CO2flux (16 mg min-1m-2) was observed in the treatment that received the
biofertilizer dried at 70C. However, in the presence of the plants, the CO2flux was decreased to 6.3 mg min-1m-2. The CO2flux of the other biofertilizer formulations (1.3 mg
min-1m-2) did not differ from the control.
Financial support: CNPq and FAPEMIG (CAG-APQ-00712-12)

SIMB 2013 | 121

PROCESS OPTIMIZATION FOR PRODUCTION OF

ENDOGLUCANASE BYASPERGILLUS TUBINGENSISUSING
COTTON SEED CAKE AS A CARBON SOURCE
RICARDO SALVIANO DOS SANTOS1, FLVIA KNIA PINHEIRO ALVES1,
ILVA DE FTIMA SOUZA1, LLIAN ARAJO PANTOJA1, ANA PAULA DE
FIGUEIREDO CONTE VANZELA1, ALEXANDRE SOARES DOS SANTOS1
Universidade Federal dos Vales do Jequitinhonha e Mucuri UFVJM, Diamantina, MG,
Brasil
1

The second generation bioethanol has been produced by hydrolysis and fermentation of lignocellulosic biomass since the second half of the nineteenth century, but only
in the last 20 years this technology has been scaled to meet the fuel market. Cellulases
comply important role in the production of second generation ethanol, being applied in
saccharification step of cellulose. This study aimed to optimize the production of cellulolytic enzyme with - 1, 4 - endoglucanasic activity using the microorganismAspergillus
tubingensisand cottonseed cake as carbon source in submerged fermentation process.
The optimization of the production of the - 1, 4 - endoglucanase was performed by factorial design and, after selecting the variables with significant effects, by use of response
surface methodology with central composite rotational design. Finally, the enzymatic
extract obtained in optimized condition was submitted to biochemical characterization, which were determined the optimum pH and temperature, as well as evaluate the
stability of the studied activity under different conditions of temperature and pH. The
optimum condition for the production of endoglucanase enzyme was 1.25% cottonseed
cake in a liquid medium containing 0.9 g L-1urea. Under these conditions the extract
obtained showed values of 3.12 U mL- 1for endoglucanasic activity after 101 hours of
cultivation. The optimum temperature and pH values were respectively 60 C and 4.0.
Studies for molecular characterization and kinetics of enzyme are in progress, the same
way that the application of obtained enzyme extract in saccharification of lignocellulosic
biomass.
Acknowledgements: FAPEMIG and CNPq

SIMB 2013 | 122

OPTIMIZATION OF XYLANASE PRODUCTION

BYASPERGILLUS TUBINGENSISUSING COTTONSEED CAKE
AS CARBON SOURCE IN SUBMERGED FERMENTATION
PROCESS
RICARDO SALVIANO DOS SANTOS1, FLVIA KNIA PINHEIRO ALVES1,
ILVA DE FTIMA SOUZA1, LLIAN ARAJO PANTOJA1, ANA PAULA DE
FIGUEIREDO CONTE VANZELA1, ALEXANDRE SOARES DOS SANTOS1
Universidade Federal dos Vales do Jequitinhonha e Mucuri UFVJM, Diamantina, MG,
Brasil
1

Enzymes degrading plant cell wall have various applications such as the bleaching
of cellulose pulp, ethanol second generation production, manufacture of animal feed,
beverages clarification and processes of textile industry. Among these enzymes, xylanases are responsible for the hydrolysis of xylan, the major component of hemicellulose and
the second most abundant polysaccharide existing in nature. Xylanolytic enzymes are
produced mainly by filamentous fungi, including Aspergillus genus, commonly touted
as a great producer of xylanases. This study aimed to optimize the production process of
xylanases in submerged fermentation usingAspergillus tubingensisstrain and cottonseed
cake as carbon source. The optimization of enzyme production above was performed using factorial design followed by the application of response surface methodology with
central composite rotational design. Finally, the extract was characterized for the parameters related to temperature and pH, as well as stability under different conditions of
temperature and pH. The optimum condition presented for the production of xylanase
was 1.25% cottonseed cake in a liquid medium containing 0.9 g L-1NH4NO3after 101
hours of fermentation. Under these conditions the extract showed values of 76 U mL-1of
xylanolytic activity. Regarding the biochemical characterization was possible to observe
an optimum temperature of activity at 55 C and optimum pH close to 4.0. The use of
cottonseed cake as a carbon source in this study was also relevant for the production of
xylanase by adding value to a coproduct underutilized, as well as its efficiency when used
as substrate in the fermentation process.
Acknowledgements: FAPEMIG and CNPq

SIMB 2013 | 123

ACCLIMATIZATION AND FERTILIZATION OF PLANTS

MYCORRHIZALHADROLAELIA JONGHEANA(ORCHIDACEAE)
IN GRAVEL SUBSTRATE
SILVA, J.A; BOCAYUVA, M. F.; VELOSO, T.G.R.; FREITAS, E.F.; VIEIRA, C.A.,
KASUYA, M.C.M.
Department of Microbiology, Laboratory of Mycorrhizal Association, Viosa Federal
University, Avenida Peter Henry Rolfs, s/n Campus Universitrio 36570-000, Viosa,
Minas Gerais, Brazil.
Acclimatization, which involves the transfer ofin vitrocultivated plants toex vitro conditions, is a critical stage in endangered orchids plan conservation. The aim of
this work was to evaluate the vegetative development ofHadrolaelia jongheanaplants
in the acclimatization phase with different fertilizing frequencies. Six months after symbiotic germination withTulasnellasp., HJ21B isolate,Hadrolaelia jongheanaseedlings
were transferred to polypropylene vases containing gravels as substrate. These seedlings
were kept in screen-shadowed nursery by a 50 % of lighting, under four levels of B&G
Orchides mineral fertilizer (0; 0.06; 0.12; 0.24; 0.48 g L-1). After one year, we evaluated
the survival of seedlings, length of the longest leaf (LLL), aerial part height (APH), dry
and fresh weight and mycorrhizal colonization. The survival rate was 100 % for all treatments except for 0.48 g L-1, which had no survival. The best growth and development of
seedlings were observed in treatment that were fertilized with 0.24 g L-1. However, this
concentration of nutrient inhibited mycorrhizal colonization. In 0.12 g L-1treatment, the
mycorrhizal colonization was present and the development variables analysed were also
high, sometime similar to 0.24 g L-1treatment. We can conclude that it is important to
define the best nutrient concentration that allow a better plant growth assuring mycorrhizal colonization for guarantee plant survival after reintroduction in the field.
Financial support: AOS, CAPES,CNPq, FAPEMIG

SIMB 2013 | 124

ARBUSCULAR MYCORRHIZAL FUNGI AND PHOSPHORUS

FERTILIZATION OFSEEDLINGS OFSCHIZOLOBIUM
PARAHYBAVAR.AMAZONICUM (HUBER EX DUCKE)
BARNEBY
VANESSA NASCIMENTO BRITO,2LETCIA CLIA HEITOR,2FERNANDO
REYNEL FUNDORA TELLECHEA, 2MARCO ANTONIO MARTINS,3SRGIO
HEITOR SOUSA FELIPE
1

Universidade Federal de Viosa UFV, Viosa, MG, Brazil. 2Universidade Estadual

do Norte Fluminense Darci Ribeiro UENF, Campos dos Goytacazes, RJ, Brazil.
3
Universidade Federal Rural da Amaznia UFRA, Belm, PA, Brazil.
1

The production of seedlings of native forest essences with quality is crucial to meet
the growing demand in the forestry sector, mainly species of great commercial interest. The present study aimed to evaluate the effect of arbuscular mycorrhizal fungi and
different doses of phosphorus on the quality of seedlings of forest speciesSchizolobium
parahybavar.amazonicum(Huber ex Ducke) Barneby. The experiment was conducted
in a greenhouse at the Universidade Estadual do Norte Fluminense Darcy Ribeiro (21
19'23"S, 41 19'41"W), in a factorial 4x4 (inoculation of arbuscular mycorrhizal fungi
(AMF)Glomus clarum, Gigaspora margarita, mixed inoculum (G. clarum + G. margarita) and control (without AMF ) and four levels of phosphorus (P): 0, 60, 120 and 180
mg kg- 1P), with 4 replicates. The parameters evaluated were: height/stem diameter (H/
DC), relative shoot/root (PA/R) and Dickson quality index (IQD). Regarding the H/DC
in the absence of phosphate fertilizer inoculated seedlings did not differ significantly
from control. At 60 mg dm- 3of P, seedlings inoculated withG. margaritahad higher H/
DC compared to other treatment and to dose 180 mg dm- 3of P, only seedlings inoculated with mixed inoculum differed from control. For the ratio PA/R seedlings indicated
relationships in applied dose of 60 and 180 mg dm- 3of P statistically similar for all treatments, except for the dose 0 and 120 mg dm- 3of P, whereG. margaritaand control were
higher. For the IQD was recorded the highest valuesat the dose 0 mg dm- 3P for seedlings inoculated with G. clarum and mixed inoculum, which were statistically different from seedlings inoculated withG. margaritaand control.Therefore, toincreasethe
quality of seedlingsS. amazonicuminoculation of mycorrhizal fungiG.clarumor mixed
inoculum without the need for phosphate fertilizers in the soil is recommended.
Acknowledgements: CNPq

SIMB 2013 | 125

ANTIMICROBIAL RESISTANCE OFCAMPYLOBACTERSPP.

STRAINS ISOLATED FROM CHICKEN CARCASSES IN MINAS
GERAIS BRAZIL
HUMBERTO MOREIRA HUNGARO;VINCIUSORNELAS ROSA;ANDRA
CTIALEALBADAR ANDREGINACLIA SANTOSMENDONA.
Federal University of Viosa - UFV,DepartmentofFoodScience andTechnology,Av. PH
Rolfs, s/n, 36.570-000,Viosa, Minas Gerais, Brazil.
Campylobacterhas been recognized as the major cause of foodborne disease worldwide. The ingestion of contaminated chicken and by-products is known as the main
source of human campylobacteriosis. Many studies reported variations inCampylobacter prevalence in chicken carcasses, and the increasing of its antimicrobial resistance
is a growing concern around the world. Here we enumerated and isolated Campylobacter from chicken carcasses, and characterized the species and its phenotypic and
genotypic antimicrobial resistance profiles. The samples were collected from nineteen
slaughterhouses from Minas Gerais Brazil, and they were analyzed by the Most Probable Number Method (MPN) associated with the plating onCampylobacterAgar and
morphological and biochemical confirmation. Typical colonies were selected and subjected to polymerase chain reaction (PCR) and antimicrobial resistance testing.Campylobacter were present in 35.6% from a total of 45 samples from nine slaughterhouses
with microbial load ranging from 3.0to 9.2MPN g-1. Twenty specimens were isolated
and confirmed asCampylobacter, of which two wereC. coliand the remainingC. jejuni.
Eight isolates (40%) showed multi-drug resistance to ampicillin, tetracycline and ciprofloxacin. None of the isolates were resistant to erythromycin. High-level resistance to
ciprofloxacin was conferred by mutation ingyrAgene. Six isolated resistant to tetracycline showed the plasmid-encodedtet(O)gene, while four isolated resistant to ampicilin
showed the -lactamaseblaOXA-61gene. The resistance of other isolates that did not
showed the evaluated genes may be explained by the existence of other mechanisms
such as multidrug efflux pump. The results reported here indicate a low prevalence
ofCampylobacterin chicken carcasses samples; meanwhile the antimicrobial resistance
of isolated strains represents an important concern and emphasizes the need for continuous monitoring of this bacterium in the poultry production chain.
Acknowledgements: This investigation was supported by CNPq and FAPEMIG, Brazil, and
the students were supported by fellowships from CAPES.

SIMB 2013 | 126

L-ASPARAGINASE PRODUCING ACTINOBACTERIA IN

ISOLATED GROUND OF CERRADO
FERREIRA-NETO, P. J.1; OLIVEIRA, B. F. R.1; CARRIM, A. J. I.1; VIEIRA, T. M.1,
FORZANI, M. V. A.1; LIMA, J. M. S.1; REIS, L. G. V.1; SADOYAMA, G.2; LEOVASCONCELOS, L. S. N. O.1; VIEIRA, J. D. G.1
Instituto de Patologia Tropical e Sade Pblica, Universidade Federal de Gois (UFG),
Goinia, Gois, Brazil. 2Campus Catalo, Universidade Federal de Gois (UFG), Catalo,
Gois, Brazil.
1

The neoplasms usually smite children and adolescents, affecting embryonic cells,
therewith the patients body accepts and recovers better by therapeutic treatment to
combat this unusual growing. Leukemia and lymphoma are the most common type of
cancer until 15 years old. The government expenses with diseases like Acute Lymphoblastic Leukemia (ALL) are really high. This malignancy has a production deficiency in
some amino acids, like asparagine. Therefore, the neoplasic cells take these amino acids
from the bloodstream and use for their own metabolism. L-asparaginase is the enzyme
most implicated for the treatment of ALL and it is produced by microorganisms. Asparaginase catalyzes the hydrolysis of L-asparagine in ammonia and aspartate, thereby
generating a deficiency of this amino acid in the blood which leads to neoplastic cell
to death. The aim of this study was to identify strains of actinobacteria from a Cerrado
soil that are producers of L-asparaginase. The selected strains were taken by Paleronni
(1985) method and subsequently tested in a specific medium for the enzyme activity. 21
morph species were isolated from the soil sample; they were named A.1 to A.22, A.14
except the ones that were not a L-asparaginase producer. The activity of L-asparaginase
was determined with 3 days of growth of the isolated samples. The positivity was determined by the formation of pink halo around the colonies. The diameter of the halos and
the growth of the colonies were measured and used for calculate the Index Enzyme (IE)
for each sample. 19 examples were positive for the enzymatic activity. The IE ranged 1,1
to 4,1, for the strains A.10 and A.1, respectively. The outcomes suggests the morph species isolated have biotechnological potential to L-asparaginase production. Additional
tests are being conducted to characterize the parameters of production and enzyme activity for future biotechnological applications.
Acknowledgements: to the Programa de Ps-graduao da Relao Parasito-Hospedeiro
(PPGRBH/IPTSP/UFG)

SIMB 2013 | 127

CHARACTERIZATION OF PROTEASES FROM CELLULOLYTIC

MICROBIAL COMMUNITY
JOSENILDA CARLOS DOS SANTOS1;ROBSONDEASSISSOUZA1; HUGO
LEONARDO ANDR GENIER1AND FLVIA MARIA LOPES PASSOS1
Laboratrio de Fisiologia de Micro-organismos, Departamento de Microbiologia/
BIOAGRO,Universidade Federal de Viosa, Viosa, MG, Brazil.
Proteases are multifunctional enzymes that catalyze the hydrolysis of peptide
bonds and have commercial importance, for detergent, pharmaceutical, medical and
food industries. There are several studies on the production and characterization of proteases, but none of those evaluate the production of proteases by cellulolytic microbial
communities. Thus, the objective of this study was to verify the protease secretion by a
cellulolytic microbial community culture and to characterize their activity as function
of temperature and pH. An aliquot of cell suspension of the community was inoculated
into 500 mL flasks containing 300 mL of the enrichment medium Peptone-Cellulose
Solution (PCS), with 1% (w/v) sugar cane bagasse and kept under static conditions at 50
C for 72 h. The culture supernatant was concentrated by ultrafiltration. The proteolysis
activity were assayed on casein as substrate and hydrolyzed products measured spectrophotometrically at 280 nm, using a standard curve with different concentrations of
tyrosine. One unit of protease activity was defined as the amount of enzyme required to
liberate 1.0 mg of tyrosine per minute at 43 C and pH 7.0. No proteolysis activity was
detected at 30 C, except at pH 4.0 where the highest activity was recorded (728.17 U
mL-1). Also, it was observed no activity at pH 2.0 and 10.0 at 40 C. However, there were
high activities in other pH values ??at that temperature. At 50 C were detected higher
values ??of activity at pH 4.0, 5.0 and 9.0 (716.8, 252.7 and 109.3 U mL-1, respectively).
At the highest temperature tested (60 C), all pH ranges showed activity, with maximum
of 699.24 U mL-1at pH 4.0. These results revealed that enzymatic cocktails secreted by
cellulolytic communities also present proteases with maximum activity at high temperatures and pH 4.0, which differ from extracellular microbial proteases that commonly
present optimal activity at pH 8.0.
Acknowledgements: CNPq, CAPES and FAPEMIG

SIMB 2013 | 128

ISOLATION OF BACTERIOCIN PRODUCING BACTERIA FROM

RAW MILK TO CONTROLLISTERIA MONOCYTOGENES
SALIVE, A.V ,QUIONES, R.N, GUERRERO, O.M.L
Universidad del Tolima, Ibagu, Tolima Colombia. Grupo de Investigacin: Gentica y
Biotecnologa Vegetal y Microbiana-GEBIUT
1

Listeria monocytogenes causes disease in high-risk groups. In Colombia has detected the presence ofL. monocytogenesin variety of meat, dairy and vegetables. With
the purpose to find new ways to control this organism was evaluate the effect of bacteriocin-producing strains possibly corresponding to Lactobacillus buchneri and native
lactic acid bacteria from raw milk. For the extraction of native strains, 10 ml of raw milk
were mixed with90 ml of distilled water and 3.4 ml of NaCl. Peptone (0.4 g)was added
andserial dilutions were made from the stock solution until dilution 10-10. To extract
bacteriocins, three different extraction protocols were used foreach of the strains. After
extraction, antimicrobial activity tests wereperformedusing the method of diffusion
wells in tryptone soya agar (ATS). A total of5 wells were distributedper plate,as follows:
1)purebacteriocin extract, 2) The 10-2dilution of the bacteriocin, 3) The 10-4dilution of
the bacteriocin, 4) positive control which was a broad-spectrum antibiotic (ampicillin),
and 5) negative control (sterile distilled water). None of the extracs obtainedwith three
different protocols evidenced inhibition ofListeria monocytogenes IS742.However, bacterialantagonism wasevident in the native strains dilution 10-3and 10-5. Because bacteriocin production was not quantified, we could not establishif bacteriocin production
was producedby the lactic acid bacteria under study. Therefore, it can not be established
ifL.monocytogenes IS742presented a resistance to bacteriocins.However evidence an
unusual pH resistance present on one of the three extraction protocols. These results
and those obtained from the tests for potential probiotic represent a important challenge
in order to know the resistance mechanism of lactic acid bacteria to antibiotics andL.
monocytogenesIS742 at acidic pH.

SIMB 2013 | 129

BOVICIN HC5 ASSOCIATED WITH TREATMENTS WITH

POTENTIAL APPLICATION IN FOOD TECHNOLOGY: A
STRATEGY FOR INACTIVATION OFSALMONELLA
GALVO, M. F.1; PRUDNCIO, C. V.2; VANETTI, M. C. D2.
Department of Microbiology, Federal University of Minas Gerais, Belo Horizonte, Minas
Gerais, Brasil 2Department of Microbiology, Federal University of Viosa, Viosa, Minas
Gerais, Brazil.
1

Bovicin HC5 is a lantibiotic produced byStreptococcus bovisHC5 and has potential

for use in food preservation. Often, Gram-negative bacteria are resistant to the action of
lantibiotics due to the presence of the outer membrane. However, if the outer membrane
permeability is reduced, Gram-negative bacteria can show bacteriocins sensitivity. In
this study, we evaluated the effect of association of bovicin HC5 to physical and chemical
agents on the inactivation ofSalmonella.SalmonellaTyphimurium ATCC 14028 was inoculated (108CFU.mL-1) into Eppendorf tubes with bovicin HC5 (200 AU.mL-1), EDTA
(1.6 mM) and diluents (saline 0.85% pH 5,5 and 7,0; distilled water; BHI broth and
sodium phosphate solution), and submitted to 30 min of ultrasound (45 kHz). We also
analyzed the influence of the order of agents: ultrasound followed by EDTA and bovicin
HC5, and EDTA followed by ultrasound and bovicin HC5, each step lasting of 30 min
at 37 C. To evaluate the effect of temperature, tubes containing BHI broth with bovicin
HC5 (50 AU.mL-1) and EDTA (1.6 mM) were inoculated withSalmonella(105CFU.mL1
) and incubated under different temperature (5, 15, 25, 37 C). The combination of
ultrasound with bovicin HC5 did not affect the viable cell number ofSalmonellain any
of the suspension media, including those containing EDTA. The order of treatments also
had no effect on cell viability. There was a reduction of about 3 log cycles at 37 C, when
the cells were treated with bovicin HC5 and EDTA, after 30 min of treatment. After 300
min, the counts were below the detection limit of the method. This same activity was not
observed at temperatures less than or equal to 25 C. These results show that ultrasound
does not destabilize the outer membrane ofSalmonellain order to allow the action of
bovicin HC5; and that bovicin HC5 associated with EDTA has higher bactericidal activity whenSalmonellais in their optimum growth temperature.
Financial support: CNPq and CAPES

SIMB 2013 | 130

EVALUATION OF THE LYTIC ACTIVITY OF BACTERIOPHAGES

IN THE CONTROL OFSALMONELLAENTERITIDIS
BATALHA, L. S. ;FONTES, E. A. F. ;MENDONA, R. C. S. ;RAMOS, M. S;
CAL, E. C.
UNIVERSIDADE FEDERAL DE VIOSA -DEPARTAMENTO DE TECNOLOGIA DE
ALIMENTOS-VIOSA-MG, BRASIL
Bacteriophages are viruses that host bacteria, they are obligatory intracellular parasites due to the lack of a metabolism of their own. They are capable of infecting specific
species of bacteria, using their cellular structure to reproduce. The purpose of this research was to evaluate the lytic activity of bacteriophages that were isolated from cattle
manure onSalmonellaEnteritidis ATCC 13076. To isolate these bacteriophages, 10 g of
the samples were diluted 1:1 in an SM buffer (Tris-HCl, NaCl, MgSO4.7H2O and gelatin)
adding 0,3 g of TSB, which was then kept under constant stirring at 150 rpm and 17 C
for 24 h to allow the elution of bacteriophages to the buffer. Following this stage, 2 mL of
NaCl 5 mol.L-1were added to the samples and maintained under refrigeration at 4 C for
one hour. The solutions were centrifuged at 12 000 x g for 15 min at 4 C and stored under refrigeration for 24 h at 4 C after addition of 10% polyethylene glycol. Subsequently,
the solutions were centrifuged again at 11 000 x g for 20 min at 4 C. The supernatant
was discarded and the pellet was resuspended in 10 mL of SM buffer. After one hour in
room temperature, 10% chloroform (v/v) was added and the sample was centrifuged
again at 5 000 rpm for 15 min at 4 C. To determinate thein vitroactivity of the phages,
microdrops (10 L) of bacteriophage suspension were utilized over a TSA agar plate
with 100 L ofS.Enteritidis. Then, they were incubated at 37 C for 24 h. The presence
of a clear halo in the microdrop region, typical of the infection and the bacterial lysis, revealed that bacteriophages have lytic activity against the target pathogen. Therefore, we
conclude that it is possible to use the lytic bacteriophages in the control ofS. Enteritidis,
which is an effective tool in the biocontrol and in the reduction of this sort of bacterium.
Acknowledgements: The FAPEMIG.

SIMB 2013 | 131

ENDOPHYTIC FUNGI FROM THREE ORCHID SPECIES

OCCURRING IN THE CERRADO
1

LETCIA MIRANDA;1MARLON CORRA PEREIRA;1HERIKA PAULA

PESSOA;1ANITA FERNANDES DOS SANTOS TEIXEIRA

(1) Universidade Federal de Viosa, campus Rio Paranaba; Rio Paranaba, MG, Brasil.
(2) Universidade Federal de Lavras; Lavras, MG, Brasil.
Orchids establish symbioses with endophytic fungi.Rhizoctonia-like fungi are the
endophytic most studied in orchids, since they are the main mycorrhizal fungi of orchid.
The observation of fungal hyphae coiled (pelotons) cells in the root cortex and base of
the embryo during germination is characteristic of mycorrhizal establishment in orchid.
However, other groups of fungi are isolated from the roots orchids. The aim of this work
was to isolate and identify endophytic fungi from roots of orchid speciesCattleya walkeriana, Epidendrum difforme and Oeceoclades maculatta, sampled within the Cerrado
biome. 177 strains were isolated from the cortex fragments root containing pelotons.
Isolates were separated into 26 morphotypes based on the rate growth, aerial mycelium,
margin, appearance and color of the colony. FromC. walkeriana, 12 morphotypes were
obtained, identified asRhizoctonia-like,Fusariumsp.,Trichodermasp. andXylariasp.
From E. difforme, 6 morphotypes were obtained and identified as Rhizoctonia-like.
Nine morphotypes were isolated from O. maculate and identified as basidiomycetes
andRhizoctonia-like. The analysis of ITS region confirmed the identification of isolates
ofTrichodermaandXylaria. We confirmed the ability ofRhizoctonia-like to induce symbiotic seed germination ofC. walkeriana,E. difformeand other orchid species in latter
experiments. There wasnt observed seeds germination in the presence ofFusarium,TrichodermaorXylaria, suggesting that they colonize the roots of these orchids after germination and embryo development. InO. maculata, the basidiomycetes were more efficient
thanRhizoctonia-like to promote seed germination and seedling development. Farther
studies should be performed to reveal the role ofFusarium,TrichodermaandXylariain
endophytic interactions.

SIMB 2013 | 132

OXALIC ACID PRODUCTION BY UV-INDUCED MUTANTS

OFASPERGILLUS NIGER
THALITA CARDOSO ANASTCIO, NINA MORENA RGO MUNIZ DA SILVA,
GILBERTO DE OLIVEIRA MENDES, UBIANA DE CSSIA SILVA, MAURCIO
DUTRA COSTA
Universidade Federal de Viosa, UFV,Avenida Peter Henry Rolfs, s/n, Viosa, Minas
Gerais, Brasil.
Organic acids are key components for a series of reactions in nature and organisms.
The oxalic acid is produced by various species of fungi and it is involved in their pathogenicity and ecology in the soil environment. Oxalic acid also presents a high potential
to release phosphorus from rock phosphate (RP) and soil particles, which qualifies it as
a promising alternative for use in microbial systems aiming at RP solubilization. Under
optimized and controlled conditions, some fungi, such asAspergillus niger, are able to
produce high quantities of oxalic acid which is synthesized from oxaloacetate through
the activity of the cytoplasmic enzyme oxaloacetase. In this study, the potential of oxalic
acid production by two UV-induced mutantsA. niger, FS1-331 and FS1-555, was evaluated. The fungal isolates were cultivated in 125-mL Erlenmeyer flasks with 50 mL of a
medium optimized for oxalic acid production: sucrose 100 g, NaNO31.5 g, KH2PO40.5
g, MgSO4.7H2O 0.025 g, KCl 0.025 g, yeast extract 1.6 g, distilled water, 1 L. The pH was
buffered at 6.0 with 1 M MES. Each flask was inoculated with two disks of mycelium and
incubated for 6 days in an orbital shaker at 250 rpm and 30 C. After the incubation, the
spent medium was filtered through a 20-m filter and analysed for oxalic acid by HPLC.
The mutant FS1-555 produced the highest concentration of oxalic acid at the conditions
evaluated, achieving 10.3 mM. The mutant FS1-331 and the wild type produced, approximately, 1 mM. Despite medium buffering, the pH dropped to 4.8 during the cultivation
of FS1-555. As the oxaloacetase activity is decreased under pH 6, this result indicates
that the production of oxalate could be improved by using a system with a more efficient
control of pH. Furthermore, the mutant FS1-555 presents a high potential for application in RP solubilization systems based on the production of oxalic acid.
Financial support: CNPq and FAPEMIG (CAG-APQ-00712-12)

SIMB 2013 | 133

IMPROVEMENT OF ENDANGERED ORCHIDS SYMBIOTIC

PROPAGATION: STUDY CASE OFHADROLAELIA JONGHEANA
EMILIANE FERNANDA SILVA FREITAS, MELISSA FAUST
BOCAYUVA,JULIANA APARECIDA SILVA, TOMS GOMES REIS VELOSO,
CONRADO AUGUSTO VIEIRA, MARIA CATARINA MEGUMIKASUYA
Department of Microbiology, Laboratory of Mycorrhizal Association, Viosa Federal
University, Avenida Peter Henry Rolfs, s/n Campus Universitrio 36570-000, Viosa,
Minas Gerais, Brazil.
The orchid family is the most diverse of all angiosperm families, and is distributed
across all continents. The members of this family produce thousands of seeds per capsule but without reserve tissue, a fact that makes them dependent on the association with
mycorrhizal fungi for germination and assisted migration/reintroduction.The objective
of this study was to develop new symbiotic propagation protocol to increase seedling
growth, assuring mycorrhizal colonization. Seedlings of Hadrolaelia jongheana were
obtained byin vitrogermination, in oat meal agar medium (OMA) inoculated byTulasnella spp., inoculated with M65, HC3E or HB7G isolates, and growth for 80 d in
growth chamber (25 C, 16 h light). After this period, the seedlings were divided in two
treatments: one group was transferred to the flasks containing OMA and other group
OMA + macro and micronutrients(0.2 gL-1of B&G Orchides). These seedlings were
maintained at the same growth chamber for one year, when mycorrhizal colonization,
the fresh and dry weight and nutrient contents were evaluated. Mycorrhizal colonization were observed in all treatments.The isolates have no influence on plant growth in
both medium treatments. The fresh and dry weight and nutrient contents were higher
in seedlings growing on OMA added with nutrients, especially to HB7G treatment. Addition of macro and micronutrients in the tested condition is recommendable for symbiotic propagation of H.jongheana, assuring mycorrhization and efficient plant grow/
development.
Acknowledgements: At Fernanda Schulthais for help in analyzis the results.
Financial support: AOS, CAPES,CNPq, FAPEMIG

SIMB 2013 | 134

MORPHOLOGICAL CARACTERIZATION OF LYTIC

BACTERIOPHAGES WITH ESPECIFICITY FORESCHERICHIA
COLIO157:H7
SOTO L., M.1; BENEVENUTO O., T.1; ROSA, V. O.1; MENDOZA C., J.1;
MENDONA S., R. C.1
Universidade Federal de Viosa; Departamento de Tecnologia de Alimentos; Viosa,
Minas Gerais, Brazil.
1

Bacteriophages are viruses that infect bacteria. As they are obligate intracellular
parasites, they resort to the hosts metabolism and material to replicate, and the infection
occurs through the injection of their genetic material. Phages may be found in various
ecological niches, sharing a common environment with their host. Phages exhibit specificity for one specie or group inside a specie. This specificity is related to specific receptors present on the bacterial cell wall, to which the phage attaches.Phages may be classified based on their morphology. They have double or single-stranded DNA or RNA.
Tailed phages constitute three families,Myoviridae,PodoviridadeeSiphoviridae,which
represents the majority of phages.Phages may be classified based on their replication
cycle as well: lytic phages and lysogenic phages. The lytic cycle occurs in four steps:
attachment, penetration, integration and replication. In the end of this cycle, the cell
lyses, releasing the new phages. These lytic phages are used as biocontrol agents in food
industry. The aim of this project was to characterize bacteriophages that have specificity forE. coliO157:H7 as their morphology. The methodology employed includes a
centrifugation of phages solution; the supernatant was dropped and the sediment was
rinsed with ammonium acetate solution 0,1 M; after this, a second centrifugation was
done, where the sediment was suspended in distilled water, filtered with a cellulose acetate membrane and was placed on a screen surface with uranyl acetate for visualization
in electronic microscopy. 24h after the application of the contrast on that screen surface,
it was possible to observe their morphology under 80 kV of transmition equipment.
Four bacteriophages isolated of different sources have showed similar morphology: head
rigid, icosahedral, long tail and size between 100 and 200 nm. These bacteriophages may
be classified in the orderCaudoviralesand in the familySiphoviridae(dsDNA).

SIMB 2013 | 135

PRODUCTION OF A RECOMBINANT PROTHROMBIN

ACTIVATOR PROTEASE INPICHIA PASTORI: PURIFICATION,
STABILITY EVALUATION AND STRUCTURAL
CHARACTERIZATION
FERNANDES, S.1,2; PICCOLI, R.A.M.2; RODRIGUES, M.F.A.2; SCIANI, J.M.3;
CARVALHO, C.3; CARRIJO-CARVALHO, L.C.3; CHUDZINSKI-TAVASSI, A.M.3*
1University of So Paulo USP Biotechnology Interunits Post Graduation Program
USP, Butantan Institute, IPT So Paulo, SP, Brazil;2Industrial Biotechnology
Laboratory, NanoBiomanufacture Nucleus, Institute for Technological Research IPT,
So Paulo, SP, Brazil;3Biochemistry and Biophysics Laboratory, Butantan Institute, So
Paulo, SP, Brazil. *Corresponding author: ana.chudzinski@butantan.gov.br
Lopap is a protein isolated from the bristles extract ofLonomia obliquamoth caterpillar. It is a prothrombin activator which shows serine protease-like activity with procoagulant effect, and also has induced cytokine secretion and antiapoptotic pathways in
human cultured endothelial cells. This work studied the production of recombinant Lopap
(rLopap) in yeast (Pichia pastoris), by using a previously modified method, purified the
obtained protein, evaluated the protein stability, the enzymatic and cytoprotection activities. The protein structural characterization was also accomplished. The production of
rLopap was held in bioreactors enhanced with automatic control of the pH, temperature,
aeration and stirring. Dissolved Oxygen (DO) and dry biomass were also analyzed. A preseed stage was developed during 48h in shaker (OD600> 2 and < 6). Then, an inoculum of
10% of the final volume of bioreactor was obtained, beginning a batch step, which lasted
almost 20h. After, a fed batch of 3-5h was carried out. These steps have used glycerol as
carbon source. The lasting phase was a fed batch with methanol as carbon source, during
86-120h, to grow the biomass (160g/L) and produce the rLopap, excreted into the fermentation broth. Then, centrifugation and filtration were carried out, and the biomass was
discarded. The supernatant undergone two chromatographic purification steps, an ionic
exchange and, then, size exclusion, both steps interchanged with a spin concentration of
molecular weight selective membranes. Protein content, SDS-PAGE, enzymatic activity
(prothrombin activation), enhancing cell culture viability, and circular dichroism (CD)
were taken into account for the final purified materials.Two batches of protein production procedure (10L bioreactor) and purification were performed, and the concentration
of rLopap (21kDa) was 0.316 and 0.714 mg/L. It was not detected any prothrombin activation for the rLopap obtained in both processes. Nevertheless, an enhancing effect on
HUVEC (Human Umbilical Vein Endothelium Cells) culture was observed. Regarding the
findings, the protein was expressed byPichiayeast with correct size and sequence, but the
rLopap would not be built up properly considering the secondary structure, which is a determinant for the enzymatic activity. That was supported by the CD results, which showed
a quite different profile in comparison to the predicted secondary structure.
SIMB 2013 | 136

PRESENCE ANALYSIS OF LUXS GENE IN ISOLATED BACTERIA

FROM FEED WATER OF REVERSE OSMOSIS SYSTEMS
JAMILLE MANSUR ALBANESE;ROBERTO SOUSA
DIAS;LARISSA ASSIS BARONY VALADARES FONSECA;DAIANE
RODRIGUES;BARBOSABELGINI;SIQUEIRA, VM;SUHETTE, R;TORRES,
APR;SOUSA, MP;CYNTHIA CANEDO SILVA;SERGIO OLIVEIRA DE PAULA
Universidade Federal de Viosa, UFV, Viosa, Brasil
Biofilm formation in the membranes of reverse osmosis systems(RO) utilized in
the treatment of residual waters by different kind of industries, including oil refineries,
represents a disadvantage in the use of this technology. Several genus of bacteria, grampositive and gram-negative, utilizes quorum sensing to control different procedures,
among them the adhesion and consequent formation of biofilm, through the production
and secretion of signaling molecules. One of these molecules, AI-2, is a self-inductor
expressed by luxS, a highly conserved gene in bacteria. The goal of this study was to
investigate the presence ofluxSin bacterial isolates and correlate it with the capacity of
biofilm formation in the samples analyzed. This analysis is interested in which bacteria
will be the targets to phage therapy. 27 isolated bacteria were analyzed from feed water
samples of RO systems from the Gabriel Passo Refinery (REGAP), Betim, Brazil. DNA
extractions were made, followed by PCR usingluxSspecific primers (5AACGGGCAGA
e 5ACGCAGGCAC). The presence of positive amplicons was analyzed by agarose gel
electrophoresis. Seven out of the 27 bacteria havent presented the gene, comparing with
the biofilm formation assay using the crystal violet technique, we observed that the bacteria that have theluxSgene were the main producers of the biofilm. In this study, we
analyzed only the presence of the gene that codifies AI-2; therefore, the bacterial isolate
7,Pseudomonas sp., which presented high biofilm formation capacity and was negative
to the presence ofluxSgene, can express other self-inductors of less influence, also related to the biofilm formation.

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MICROBIOLOGICAL ACTIVITY EVALUATION THROUGH THE

RESPIRATION RATE IN SOIL WITH DIFFERENT VEGETATION
COVER
LULLA RODRIGUES FINZER, HRIKA PAULA PESSOA, MARLON CORREA
PEREIRA
Laboratrio de Microbiologia - Universidade Federal de Viosa - Campus de Rio
Paranaba
The microbial activity has been used as an important indicator of soil quality.
Among the indicators the soil respiration rate analysed by CO2emission by microorganism during the organic material degradation. The main purpose of this soil analysis
is compare the microbiota in different soil type of vegetation, through the microbial
respiration. We analysed three types of environment for soil, the first one without the
vegetation cover, other has a graminea cover and the last one has the typical cerrado
vegetation. The soil samples were taken, kept in plastic bags and taken to the Microbiological Federal University of ViosaRio ParanabaCampus, of its processing. The soil
was sieved and after that we weight 2 samples soils of 100gr each, it was watered with distilled water, the sufficient quantity to identify this humity in 60% of soil capacity, as soon
as possible it was kept in glass bottles.Inside each one of these bottles, it was inserted a
test tube and a plastic glass was put on it with 20ml of NaOH 0,5mol.L- after, the bottle
was closed. The same was repeated in a tube without the sample soil. It was developed
through titulometric evaluation with HCl 0,25mol.L- 24 hours per 7 days. After the
anasysis, plastic glass were substituted. So it was possible to know the exact CO2quantity
produced during this time interval. The results shows CO2production of the soil was
decreasing in the following sequence: cerrado>graminea>uncovered soil. The difference
between CO2 production of cerrado soil and the one covered of graminea was lower
than the difference observes between CO2production of this soil than the uncovered
soil. Its believed that the respiration rate was greater in the closed ground, due to the
high diversity of plants and also of substrate.The decrease of the soil substrate quantity
caused decreasing in microbial respiration. We could observe that higher the vegetation
diversity and the substrate available are, higher the microbial soil activity gets.

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TOLERANCE OF PISOLITHUS SP. ISOLATES TO GLYPHOSATECONTAINING HERBICIDES

ANTUNES, L.A.(1); GRAZZIOTTI, P.H.(2); FONSECA, A.J.(3); GOMES, A.L.F.(4);
AVELAR, D.C.S.(1); SANTOS, J.B. (2); SOARES, E.C(4)
(1)Undergraduate Agronomy in UFVJM, (2) PhD Professor in UFVJM; (3) PHD
student in Plant Science in UFLA; (4) Undergraduate Forest Engineering in UFVJM.
UFVJM Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina,
Minas Gerais, Brasil; UFLA Universidade Federal de Lavras, Lavras, Minas Gerais,
Brasil. li_antunescvo@hotmail.com; grazziot@yahoo.com.br.
The glyphosate is the active principle most used in weed control in eucalypt culture. The herbicides with basis of this product are nonselectives, post emergents and of
systemic action, with wide use and have inhibitory effects on ectomycorrhizal fungi.
The objective of this study was to evaluate whether glyphosate-containing herbicides
have different toxicity on ectomycorrhizal fungi. The growth of Pisolithus sp. isolate
D51, obtained from eucalypt plantations in the Alto Vale do Jequitinhonha region, was
evaluated in solid modified Melin-Norkrans (MNM) medium, added of 92,6 e 185,2 g
L-1 of glyphosate obtained from Roundup Original (Isopropylamine salt), Roundup
Ready (Isopropylamine salt), Roundup Ultra (Ammonium salt), Zapp (Potassium
salt), Scout (Ammonium salt) and a control without herbicides, with nine repetitions.
The Roundup Ready has environmental and toxicological classification II and III respectively and for all other hebicides the both classifications are III. For obtaining concentrations, the herbicides (stock solutions) were added to the growing culture medium
sterilized and cooled to 48 oC. Then, 20 mL of medium were poured in Petri dishes of
100 mm diameter. After solidification of the growing medium, a 5 mm diameter disc
containing mycelia removed from the edge of grown colonies in the same medium and
pre-grown for three days, was placed in the center of each Petri dish with mycelium
facing up and incubated at 25 C for 30 days. The diameters of colonies of Pisolithus sp.
were smaller in the medium containing the highest glyphosate concentration (185,2 g
L-1) obtained from Scout, and the two concentrations obtained from Roundup Original. The Glyphosate-containing herbicides have differentiated toxicity on ectomycorrhizal and this may be dependent on their concentration. Roundup Original and Scout
were the most toxic to Pisolithus and this effect is related to the ion accompanying or
other additive not specified in the formula.
Acknowledgements: To Universidade Federal dos Vales do Jequitinhonha e Mucuri, for the
infrastructure needed to perform the study, to FAPEMIG for financial support and CNPq
for the granting of scholarship.

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ACCLIMATIZATION OF MYCORRHIZED SEEDLINGS OF

HADROLAELIA AND HOFFMANNSEGGELLA (ORCHIDACEAE)
1VELOSO, T.G.R.; 1BOCAYUVA, M.; 1SILVA, J.A.; 1FREITAS, E.F.; 1VIEIRA, C.A;
1OLIVEIRA, S.F.; 2OTONI, W.C., 1KASUYA, M.C.M.
Universidade Federal de Viosa, 1Departmento de Microbiologia e 2Departamento de
Biologia Vegetal/BIOAGRO, Avenida Peter Henry Rolfs, s/nCampus Universitrio, ,
36570-000, Viosa, Minas Gerais, Brasil.
Hadrolaelia jongheana, Hoffmannseggella caulescens and Hoffmanseggella cinnabarina are species on the List of Threatened Brazilian Flora Species. These orchids
occurs in the Atlantic Forest and, an important global hotspot constantly threatened by
anthropogenic impacts. This study aimed evaluate the acclimatization of these three orchid species inoculated with different mycorrhizal fungi. Seedlings were obtained from
in vitro symbiotic propagation with some Tulasnella spp. The isolates was transferred to
gravel and cultivated in green house. After 14 months of transplantation we evaluated
the number of leaves (NL), the length of the longest leaf (CMF), the survival and the mycorrhizal colonization status in the root system. The survival varied from 46% to 100%.
There were observed increments for NL and CMF for the three orchid species inoculated
with the fungi, except for H. cinnabarina inoculated with M65. All root samples presented intact and degraded pelotons, showing that the initial inoculation is still present
even after this long period of acclimatization. The proposed methodology is successful
for acclimatization of these orchids, proving seedling development without application
of any fertilizer and assuring high percentages of survival.

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PRODUCTION OF PLEUROTUS SPP. MUSHROOMS IN AGROINDUSTRIAL RESIDUES

DENHAMA, T. B.; PAULA, T. A.; SILVA, M. C. S.; KASUYA, M. C. M.
a University of Kentucky, Kentucky, USA b Departamento de Microbiologia, Federal
University of Viosa, Viosa, Minas Gerais, 36570-000, Brazil.
Pleurotus mushroom can be cultivated in agro-industrial residues with high yield
potential or biological efficiency (BE). The use of these residues in mushrooms production avoids their direct release into the environment, increases the familys income
and leads to food production with high nutritional quality. Mushroom yields and their
chemical composition can be affected by the substrates used in their growth. In this
present study, it was attempted to evaluate the influence of three substrates: sugar cane
bagasse, sisal and banana waste to Pleurotus mushrooms production. The substrates
were immersed in solution containing 0.5% hydrated lime for 12 h and subsequently
centrifuged for 5 min at 1500 x g. Samples of 300 g of substrate were placed in polypropylene bags. Three isolates of Pleurotus were tested: Plo 02 and PC 98 (P. ostreatus) and
Plo 13 (P. ostreatoroseus). The collected mushrooms were weighted, dried and nutrient
content determinate. Plo 02 isolate grew in all agro-industrial residues with BE between
31 - 36 %. Highest BE was observed to Plo 13 isolate in sisal residue (42%). The PC 98
isolate did not produce any mushroom in sugar cane bagasse. High concentration of N,
P and S were determinate in mushrooms grown in sisal. These results demonstrate the
great potential of the use of these residues for mushrooms production. Sisal residues is
an efficient agroindustrial residue for Pleurotus mushroom production.
Financial Support: CNPq, Capes and Fapemig

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DIAZOTROPHIC BACTERIA ARE ASSOCIATED WITH

PLEUROTUS OSTREATUS
MENDES, I. R.; DA LUZ, J. M. R.; SILVA, M. C. S.; PAULA, T. A.; BAZZOLLI, D. M.
S.; KASUYA, M. C. M.
Departamento de Microbiologia, Universidade Federal de Viosa, Viosa, Minas Gerais,
36570-000, Brazil.
Pleurotus ostreatus is able to grow using, as source of carbon and energy, oxo-biodegradable plastics and other residues with low relative concentration of N. This capacity could be due to high efficiency of their enzymatic system and the presence of
endomycotic diazotrophic bacteria on fungal hyphae. Thus, the aim this work was to
identify the diazotrophic bacteria on mycelium of P. ostreatus. It was used the P. ostreatus PLO6 (GenBank acess KC782771) which belongs to the collection of Laboratory of
Mycorrhizal Associations/DMB/BIOAGRO/UFV. After DNA extraction, amplification
was performed using primers for nifH gene, and the amplified product was subjected
to DGGE. Bands were selected, sequenced and analyzed by Blast (NCBI, GenBank). It
observed the presence of the nifH gene on mycelium of PLO6. The bands observed were
similar those bands found in different species of diazotrophic bacteria, including Burkholderia cepacia and Rizhobium tropici. The sequencing showed the predominance of
group Betaproteobacteria and Alphaproteobacteria. The presence of endomycotic diazotrophic microorganisms can be important for adaptation of the fungus in substrates
with low nitrogen concentration. Interactions between diazotrophic microorganisms
and P. ostreatus can be an important factor in the processes of bio-bleaching and bioremediation applications using this fungus.
Financial Support: CNPq, Capes and Fapemig

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SPECIATION OF SELENIUM IN PROTEIN EXTRACTS OF

SELENIUM-ENRICHED MUSHROOMS PLEUROTUS OSTREATUS
AND LENTINULA EDODES
ASSUNO, L. S.A; FERNANDEZ, M. G.B; GARCA-BARRERA, T.B; GOMZARIZA, J. L.B; SILVA, M. C. S.A; PALOMAS, J. D. B.C; KASUYA, M. C. M.A
aDepartment of Microbiology, University Federal of Viosa, Viosa, Minas Gerais, 36570000, Brazil. bDepartment of Chemistry and Material Sciences, University of Huelva,
Huelva, 21007, Spain. cDepartment of Biochemistry and Molecular Biology, University of
Sevilla, Sevilla, 41012, Spain.
Lentinula edodes and Pleurotus ostreatus are the two most commercialized mushrooms species in the world and present potential to accumulate selenium in agro-industrial residues. The objective of this study was to evaluate the species of Se in the protein
extract of P. ostreatus and L. edodes to obtain a protein extract with high concentration
of selenomethionine (SeMet). P. ostreatus mushrooms were cultivated on coffee husk
substrates added or not with of 25 mg kg-1 Se and L. edodes were grown on wood sawdust added or not with 50 mg kg-1 Se. The protein extract containing Se was extracted
with phosphate buffer, borate buffer and lysis buffer. Proteins were purified by acetone
precipitation. The protein extracts were analyzed by reverse phase liquid chromatography coupled plasma mass spectrometry. The extraction efficiency of compounds of
Se varied with type of extractant tested and of the species of fungus, reaching higher
value in protein extracts of P. ostreatus obtained with lysis buffer, approximately 60% for
both Se-enriched and non Se-enriched mushrooms. The Se compounds found in greater
quantities in most of the eluates was SeMet, and extractant more efficient was lysis buffer
for both mushroom species evaluated. Protein extracts of P. ostreatus with lysis buffer
presented a great potential for production of protein biomass with high concentrations
of the amino acid SeMet on industrial scale. The nutritional value of this protein extracts
containing SeMet can be an interesting topic for future studies.

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DEGRADATION OF GREEN PLASTIC BY PLEUROTUS

OSTREATUS
DA LUZ, J.M.R, PAES, S.A, KASUYA, M.C.M
Departamento de Microbiologia, Universidade Federal de Viosa, Viosa, Minas Gerais,
Brasil
We studied the biodegradation of green polyethylene (GP) by Pleurotus ostreatus.
The GP was developed from renewable raw materials to help to reduce the emissions
of greenhouse gases. However, there is no information on the biodegradation of GP
when discarded in the environment. P. ostreatus is a lignocellulolytic fungus that has
been used in bioremediation processes of agroindustrial residues, pollutants and recalcitrant compounds and oxo-biodegradable polyethylene. GP plastic bags were exposed
to sunlight for up to 120 d to induce an initial photodegradation of the polymers, and
fragments of these bags were used as substrate for growth of P. ostreatus. After exposed
to sunlight, it was not observed any crack, pit, nor formation of new functional groups
in the structure of GP. However, alterations in the mechanical properties of GP caused
by sunlight were important for fungal growth and for biotic degradation of the green
polyethylene. After 90 d of incubation, a significant reduction of the dry mass of substrate and, physical and chemical alterations in the structure of GP were observed. We
conclude that the exposure of GP to sunlight and subsequent incubation in presence of
P. ostreatus can be an alternative to decrease the half-life of GP, facilitating the mineralization of these polymers.
Acknowledgements: FUNARBE, CAPES, CNPq, FAPEMIG and Ncleo de Microcospia.

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EVALUATION OF THE POTENTIAL FOR BACTERIAL

EXOPOLYSACCHARIDE PRODUCTION USING CRUDE
GLYCEROL AS MAIN CARBON SOURCE
VANESSA AMARAL RIBEIRO, MYRIAN TEIXEIRA, CARLOS ANDR VEIGA
BURKERT
Federation University of Rio Grande, FURG, School of Chemistry and Food Bioprocess
Engineering Laboratory. E-mail: vanessa.amaralribeiro@gmail.com
The demand for renewable energy has been encouraged new studies by renewable
fuels being fossil fuels expected to exhaustion in the future. Biodiesel is an example of
biofuel produced from renewable sources. Thus, with the expansion of biofuel industry
has created a surplus of glycerol. So, the bacterial exopolysaccharide using the residual
glycerol as carbon source would be an alternative to the use of this byproduct since the
exopolysaccharide is used as stabilizers, emulsifiers, coagulants and suspending agent
in variety industries. The objective of this work was to evaluate different bacterias of
the genus Rhizobium (R) with the potential to produce expolisaccharide using crude
glycerol as the main carbon source. We tested five different species of bacteria R. japocium SEMIA 580, R. leguminosarum bv vicae SEMIA 344, R. eguminosarum bv trifolii
SEMIA 2050, R. tropici SEMIA 4077 and R. huatlense SEMIA 6450 from Fepagro (State
Foundation for Agricultural Research). The cultures were performed in shake flasks in
triplicate conducted in rotary incubator at 30 C and 180 rpm using the culture medium
as the following composition ( g / L ): 10 crude glycerol , 0.4 KH2PO4 , 0.1 K2HPO4
, 0 2 MgSO4.7H2O , 0.1 NaCl , 0.4 yeast extract , 0.12 and 0.15 MnCl2.4H2O CaCl2.2
H2O , pH was adjusted to 7.0. In the experiment was accompanied biomass and pH over
time and at the end of cultivation, the concentration of exopolysaccharide was determined. The bacterium R. leguminosarum bv vicae SEMIA 344 showed the most promising among the bacteria studied for production of exopolysaccharide , reaching at 48 h ,
biomass, pH and concentration of exopolysaccharide 1.31 0.11 g / L, 5.70 0 01 and
2.32 0.07 g / L, respectively. Therefore, the use of glycerol derived from biodiesel production has shown to be a promising source of carbon for the production of exopolysaccharide by Rhizobium bacteria.
ACKNOWLDGMENTS: CAPES, CNPq, Fapergs

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