MICROORGANISMS IN CONCRETE A THESIS SUBMITTED FOR THE DEGREE OF ENVIRONMENTAL ENGINEERING BY ÁNGELA MARCELA QUINTERO MARTÍNEZ DIRECTOR MAURICIO SÁNCHEZ SILVA, PH.D. ADVISOR AIDA JULIANA MARTÍNEZ LEÓN, MSC. UNIVERSIDAD DE LOS ANDES FACULTY OF ENGINEERING DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING BOGOTÁ, COLOMBIA 2011 Dedicated to my parents and my sister… Acknowledgements The author would like to acknowledge the supervision, suggestions and ideas about the investigation, provided by the engineer Mauricio Sánchez Silva and the collaboration, contributions, explanations and advices of the microbiologist Juliana Martínez. Many thanks are extended to the proofreader, the philologist Laura Ontibón; the people that collaborated in the field work (transportation and photographs), Mr. José Manuel Quintero and Mrs. Rocío Martínez; the company which realized the sequencing of DNA samples, Macrogen –Korea Inc.; and the persons of the laboratory of electrophoresis of Universidad de los Andes Abstract This study reports the use of culture dependent and independent techniques to identify indigenous and naturally occurring bacteria in deteriorate concrete surfaces. In the field work was sampled a total of 6 bridges located in via Bogotá – Villeta. Genomic DNA was isolated to the samples in two different ways: indirect and direct. The indirect way involves the cultivation of microorganisms, while the direct way does not. PCR amplification of bacterial ribosomal DNA (16S rDNA) was conducted with subsequent DNA sequencing to identify the microbes. Three bacteria genus was recognized Pseudomonas, Rahnella and Buttiauxella corresponding to four distinct DNA samples (Pseudomonas was found twice) occurring at two bridges. Of these genuses, only Pseudomonas was reported in the scientific articles related to deterioration of concrete. But in this study, Rahnella and Buttiauxella were associated indirectly with acid attack of concrete. Key words: molecular techniques, cultivation of microorganisms, deterioration of concrete, biodeterioration. Resumen Este estudio presenta el uso de técnicas dependientes e independientes del cultivo de microorganismos para identificar bacterias autóctonas que se encuentren naturalmente en superficies de concreto deterioradas. En el trabajo de campo se muestrearon un total de 6 puentes ubicados en la vía Bogotá – Villeta. El ADN de las muestras fue extraído en dos formas: indirecta y directa. La forma indirecta involucra cultivo, mientras que la directa no. Se realizó PCR con amplificación del ADN ribosomal bacteriano (16S rADN) y subsecuentemente se hizo secuenciación del AND para identificar los microbios. Tres géneros bacterianos se reconocieron, Pseudomonas, Rahnella y Buttiauxella de cuatro muestras de ADN (Pseudomonas se encontró dos veces) tomadas de dos puentes. De estos géneros, sólo las Pseudomonas fueron reportadas en artículos científicos relacionados con el deterioro del concreto. Sin embargo en este estudio, Rahnella y Buttiauxella se asociaron indirectamente con el ataque ácido en el concreto. Palabras claves: técnicas moleculares, cultivo de microorganismos, deterioro del concreto, biodeterioro. Content Acknowledgements ................................................................................................................................. 3 Abstract ................................................................................................................................................... 4 Resumen.................................................................................................................................................. 4 1. Introduction ..................................................................................................................................... 7 2. Field visits......................................................................................................................................... 8 2.1. Places visited ............................................................................................................................ 9 Railway bridge in the ‘Alto de la Tribuna’ .............................................................................. 10 Vehicular bridge located 580m (approx.) after ‘Alto de la Tribuna’ ........................................ 12 Vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession. ..... 13 Vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto) ................... 14 Concrete structures located near to weighing scale for heavy vehicles ................................. 15 Vehicular bridge located at 200 m (approx.) after ‘Jalisco’ Toll Road ..................................... 17 Railway bridge located at the entrance of the Vereda ‘Santa Ana’ ........................................ 19 Vehicular bridge at the edge of the ‘Seco’ River .................................................................... 21 2.2. Bridges were to be sampled.................................................................................................... 21 3. Materials and methods................................................................................................................... 21 3.1. Sampling technique in bridges – removal of microorganisms .................................................. 21 3.2. Culture mediums .................................................................................................................... 22 PYGV medium....................................................................................................................... 22 Brain Heart Infusion Broth .................................................................................................... 24 3.3. Culture techniques ................................................................................................................. 24 3.4. DNA isolation.......................................................................................................................... 25 3.5. PCR (Polymerase Chain Reaction) ........................................................................................... 27 3.6. Electrophoresis ....................................................................................................................... 29 3.7. Spectrophotometric measurement of DNA concentration. ..................................................... 30 3.8. DNA sequencing and purification ............................................................................................ 30 3.9. Gram staining ......................................................................................................................... 30 3.10. Culture collection of isolated microorganisms......................................................................... 32 3.11. Assembly of DNA sequences ................................................................................................... 32 4. Results ........................................................................................................................................... 33 4.1. DNA isolation and classic microbiology (culture mediums) ...................................................... 33 DNA isolation and PCR .......................................................................................................... 34 Classic microbiology and samples surfaces............................................................................ 35 4.2. DNA sequences and NCBI blast search .................................................................................... 45 Blast results of DNA sequences ............................................................................................. 45 Blast results of consensus sequences .................................................................................... 46 5. Discussion ...................................................................................................................................... 47 6. Conclusions and recommendations ................................................................................................ 49 7. References ..................................................................................................................................... 50 8. Annexes ......................................................................................................................................... 54 Annex A: Classification scheme of weathering forms .......................................................................... 54 Annex B: DNA indirect isolation .......................................................................................................... 62 Annex C: Bacteria morphology ........................................................................................................... 63 Cultural characteristics ......................................................................................................... 63 Annex D: Results of DNA sequencing .................................................................................................. 64 Quintero-Martínez, A.M. [2011] IAMB 201120 30 1. Introduction Concrete surfaces are susceptible to some kind of microbiological attack, called biodeterioration. All microorganisms may be of importance in biodeterioration – phototrophic, chemolithotrophic and chemoorganotrophic bacteria, cyanobacteria, algae, fungi and lichens. The higher plants may contribute to weathering of concrete due to root growth and