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