ISMOS-2
MOLECULAR BIOLOGY TOOLS IN THE STUDY OF BIODIESEL IN BRAZIL M.T.S M T S Lutterbach-INT L tt b h INT G. de Souza Pimenta-PETROBRAS L.S. Contador-INT M. Galvão-INT V.Oliveira-INT
BIOFUELS IN BRAZIL •In In the 70´s 70 s, the Pro-Alcohol Pro Alcohol program was launched to reduce dependence on fossil fuels •In the early 80's, 90% % off the cars produced in Brazil run on alcohol, but by the end of decade this sector fell f ll iinto crisis i i •From From 2000 on, on a resumption in ethanol production happened •In I 2003 th the fl flexible-fuel ibl f l car engines, i called ll d “fl “flex”, ” started to be sold •Today, almost 90% of the cars in Brazil are flex
INT AND THE BIOFUELS IN BRAZIL Since 1925, severall studies t di employing alcohol as a fuel f l were being performed and the ProAlcohol program was based on INT researches
The INT was also a pioneer in the area of vegetable oil as fuel, which in 1929 published the work Coconut, babassu, and the fuel problem In the decade of 80 studies 80, di with ih INTOL mixture diesel mixed with peanut oil or palm oil or babassu were performed performed.
In 2005 the National Program of Production and Use of Biodiesel was l launched. h d It provided id d th the production d ti of fuels from renewable sources It was also enacted a law which determines the addition of biodiesel to diesel fuel derived from oil
Brazilian law n° 11097 of January 2005 • The blend of biodiesel and diesel will be y on 2008 mandatory • In 2013 the mandatory proportion of biodiesel will be of five percent (B5) (B5). The B5 requirement will probably be moved up to 2010 • Nowadays, diesel sold to a final consumer must contain two percent of biodiesel (B2)
Raw material research • There are many species of plants in Brazil which can be used to produce biodiesel • Brazil wants to diversify the raw material used in the production of biodiesel. For each region of the country, a variety of vegetable oils and animal fats are being investigated to develop the national bi di biodiesel l production. d ti
Castor Oil/ Babassu Oil / Palm
P l / Soybeans Palm/ S b
North
Northeast
Midwest Cottonseed / Soybeans / Castor Oil
Southeast
Soybeans / Cottonseed / Sunflower
South
Soybeans / Cottonseed / Sunflower
It’s important to emphasize that the diesel that are used in B il iis S 500 Brazil
• The Laboratory of Biocorrosion and g , LABIO,, analyses y biodiesel Biodegradation, samples since 2004, from pilot scale production and more recently from industrial production.
EXPERIMENTS
DNA extraction and PCR
SAMPLE
Sequencing of the PCR product
Identification of the microbial strain
Isolation of strains
Microbial Strain Collection ((-80°C) 80 C)
Cultivable bacteria quantification f ((MPN) PN)
Bioassays
Denaturing Gradient Gel Electrophoresis El t ph sis Analysis A l sis (DGGE) Total DNA extraction and PCR
Analysis of Microbial Community Diversity
Analysis of Target Mi Microbial bi l G Groups
Single-Strand Conformation Polymorphism Analysis by Capillary Electrophoresis (CE-SSCP)
Detection of target microorganisms
PCR (specific primers)
Quantification of target microorganisms
Real-time PCR quantification
FILTRATION
SAMPLE
1
2
Total DNA extraction
3
Denaturing D t i G Gradient di t Gel Electrophoresis Analysis (DGGE)
Analysis of Microbial Community Diversity
PCR
SAMPLE
Cultivable bacteria q quantification (MPN)
Isolation of strains
DNA extraction
PCR
Gram stain
Sequencing of the PCR product
Identification of the microbial strain
Microbial Strain Collection (-80 ( 80°C) C)
CULTURE COLLECTION
Why Cupriavidus sp sp.? ? •M More than h 90% off the h strains i iisolated l d from biodiesel samples p were identified as Cupriavidus sp. • It was isolated from biodiesels produced from different raw materials • Habitat soil • High EPS producer • Biocides • Biocorrosion
Environmentally Friendly biocides x THPS Controls
E.Friendly
THPS
BIODEGRADATION
CONCLUSION • Although there are many international p , standards for biodiesel specifications, none of the standards specify limits or analytical methods for microbial contamination of biodiesel, even though there are several studies reporting p y to microorganisms g biodiesel susceptibility and its high biodegradability
• Although the use of biodiesels is a global y, the interaction of microorganisms g reality, with this substrate needs to be more investigated
NATIONAL INSTITUTE OF TECHNOLOGY WWW INT GOV BR WWW.INT.GOV.BR
Má i T Márcia Teresa Soares S Lutterbach L tt b h
[email protected] i l tt b h@i t b