In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation. © 2012 Silva et al.21113Agarry, S.E., Solomon, B.O., Kinetics of batch microbial degradation of phenols by indigenous Pseudomonas fluorescenc (2008) Int J Environ Sci Tech, 5, pp. 223-232Ahmed, Z., Cho, J., Lim, B.-R., Song, K.-G., Ahn, K.-H., Effects of sludge retention time on membrane fouling and microbial community structure in a membrane bioreactor (2007) J Membrane Sci, 287, pp. 211-218. , doi:10.1016/j. memsci.2006.10.036Amann, R.I., Ludwig, W., Schleifer, K., Phylogenetic identification and in sit detection of individual microbial cells without cultivation (1995) Microbiol Rev, 59, pp. 143-169Arai, H., Akahira, S., Ohishi, T., Maeda, M., Kudo, T., Adaptation of Comamonas testosteron TA441 to utilize phenol: Organization and regulation of the genes involved in phenol degradation (1998) Microbiology, 144, pp. 2895-2903. , doi:10.1099/00221287-144-10-2895Aziz, A.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Zagnitko, O., The RAST server: Rapid annotation using subsystems technology (2008) BMC Genomics, 9, p. 75. , doi:10.1186/1471-2164-9-75Bae, H.S., Lee, J.M., Kim, Y.B., Lee, S.T., Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteron CPW301 (1996) Biodegradation, 7, pp. 463-469Basu, A., Apte, A.K., Phale, P.S., Preferential utilization of aromatic compounds over glucose by Pseudomonas putid CV86 (2006) Appl Environ Microbiol, 72, pp. 2226-2230. , doi:10.1128/AEM.72.3.2226-2230.2006Barrios-Martinez, A., Barbot, E., Marrot, B., Moulin, P., Roche, N., Degradation of synthetic phenol-containing wastewaters by MBR (2006) Journal Membrane Science, 281, pp. 288-296. , doi:10.1016/j.memsci.2006.03.048Billy, K., Viau, E., Peccia, J., Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids (2010) Water Res, 44, pp. 4252-4260. , doi:10.1016/j.watres.2010.05.039Braile, P.M., (1979) Manual of Industrial Wastewater Treatment, , CETESB, São Paulo, 1Breinig, S., Schiltz, E., Fuchs, G., Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium Thauera aromatic (2000) J Bacteriol, 182, pp. 5849-5863. , doi:10.1128/JB.182.20.5849-5863.2000Brown, M.V., Philip, G.K., Bunge, J.A., Smith, M.C., Bissett, A., Lauro, F.M., Fuhrman, J.A., Donachie, S.P., Microbial community structure in the North Pacific Ocean (2009) ISME J, 3, pp. 1374-1386. , doi:10.1038/ismej.2009.86Chang, B.V., Chiang, F., Yuan, S.Y., Biodegradation of nonylphenol in sewage sludge (2005) Chemosphere, 60, pp. 1652-1659. , doi:10.1016/j.chemosphere.2005.02.042Gonzalez, G., Herrera, G., García, M.T., Peña, M., Biodegradation of phenolic industrial wastewater in a fluidized bed bioreactor with immobilized cells of Pseudomonas putid (2001) Biores Technol, 80, pp. 17-142Handelsman, J., Metagenomics: Application of Genomics to Uncultured microorganisms (2004) Microbiol Mol Biol Rev, 68, pp. 669-685. , doi:10.1128/MMBR.68.4.669-685.2004Harayama, S., Kok, M., Neidle, E.L., Functional and evolutionary relationships among diverse oxygenases (1992) Annu Rev Microbiol, 46, pp. 565-601. , doi:10.1146/annurev.mi.46.100192.003025Henze, M., Harremoes, P., la Cour, J.J., Arvin, E., (1997) Wastewater treatment, , Berlin: Springer, 2 Environmental EngineeringHuson, D.H., Auch, A.F., Qi, J., Schuster, S.C., MEGAN analysis of metagenomic data (2007) Genome Res, 17, pp. 377-386. , doi:10.1101/gr.5969107Jiang, Y., Wen, J.P., Li, H.M., Yang, S.L., Hu, Z.D., The biodegradation of phenol at high initial concentration by the yeast Candida tropicali (2005) Biochem Eng J, 24, pp. 243-247. , doi:10.1016/j.bej.2005.02.016Kim, M.-K., Harwood, C.S., Regulation of benzoate-CoA ligase in Rhodopseudomonas palustri (1991) FEMS Microbiol Let, 83, pp. 199-203Kraigher, B., Kosjek, T., Heath, E., Kompare, B., Mandic-Muleca, I., Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors (2008) Water Res, 42, pp. 4578-4588. , doi:10.1016/j.watres.2008.08.006Lack, A., Fuchs, G., Evidence that phenol phosphorylation to phenylphosphate is the first step in anaerobic phenol metabolism in a denitrifying Pseudomona sp (1994) Arch Microbiol, 161, pp. 132-139Li, X., Ma, H., Wang, Q., Matsumoto, S., Maeda, T., Ogawa, H.I., Isolation, identification of sludge-lysing strain and its utilization in thermophilic aerobic digestion for waste activated sludge (2009) Bioresource Technology, 100, pp. 2475-2481. , doi:10.1016/j.biortech.2008.12.019Liu, Z., Lozupone, C., Hamady, M., Bushman, F.D., Knight, R., Short pyrosequencing reads suffice for accurate microbial community analysis (2007) Nucleic Acids Res, 35, pp. e120. , doi:10.1093/nar/gkm541Madigan, M., Martinko, J., Dunlap, P., Clark, D., (2008) Brock Biology of Microorganisms, , New York, USA: Pearson Higher Education, 12Manefield, M., Whiteley, A.S., Griffiths, G.I., Bailey, M.J., RNA stable isotope probing, a novel means of linking microbial community function to phylogeny (2002) Applied Environment and Microbiology, 68, pp. 5367-5373. , doi:10.1128/AEM.68.11.5367-5373.2002Mariano, J.B., (2001) Environmental impacts of petroleum refining, , COPPE/UFRJ, Rio de Janeiro, RJ, Brazil: MSc TheseMerimaa, M., Heinaru, E., Liivak, M., Vedler, E., Heinaru, A., Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol-and pcresol-degrading Pseudomonas species and biotypes (2006) Arch Microbiol, 186, pp. 287-296. , doi:10.1007/s00203-006-0143-3Marques, S., Ramos, J.L., Transcriptional control of the Pseudomonas putid TOL plasmid catabolic pathways (1993) Mol Microbiol, 9, pp. 923-929. , doi:10.1111/j.1365-2958.1993.tb01222.xMechichi, T., Stackebrandt, E., Gadon, N., Fuchs, G., Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions, and description of Thauera phenylacetic sp. nov., Thauera aminoaromatic sp. nov., and Azoarcus buckeli sp. nov (2002) Arch Microbiol, 178, pp. 26-35. , doi:10.1007/s00203-002-0422-6Meyer, F., Paarmann, D., D'Souza, M., Olson, R., Glass, E.M., Kubal, M., Paczian, T., Edwards, R.A., The Metagenomics RAST server-A public resource for the automatic phylogenetic and functional analysis of metagenomes (2008) BMC Bioinformatics, 9, p. 386. , doi:10.1186/1471-2105-9-386Miura, Y., Hiraiwa, M.N., Ito, T., Itonaga, T., Watanabe, Y., Okabe, S., Bacterial community structures in MBRs treating municipal wastewater: Relationship between community stability and reactor performance (2007) Water Res, 41, pp. 627-637. , doi:10.1016/j.watres.2006.11.005Nair, C.I., Jayachandran, K., Shashidhar, S., Biodegradation of phenol (2008) African J Biotechnol, 7, pp. 4951-4958Ojumu, T.V., Bello, O.O., Sonibare, J.A., Solomon, B.O., Evaluation of microbial systems for bioremediation of petroleum refinery effluents in Nigeria (2005) African J Biotechnol, 4, pp. 31-35Oliveira, G.S.S., Araújo, C.V.M., Fernandes, J.G.S., Activated sludge system microbiology and its relationship with the industrial effluents treatment: The experience of the Cetrel (2009) Eng Sanit Ambient, 14, pp. 183-192Roesch, L.F., Fulthorpe, R.R., Riva, A., Casella, G., Hadwin, A.K.M., Kent, A.D., Daroub, S.H., Triplett, E.W., Pyrosequencing enumerates and contrasts soil microbial diversity (2007) ISME J, 1, pp. 283-290Sanapareddy, N., Hamp, T.J., Gonzalez, L.C., Hilger, H.A., Fodor, A.A., Clinton, S.M., Molecular diversity of a north carolina wastewater treatment plant as revealed by pyrosequencing (2009) Appl Environ Microbiol, 175, pp. 1688-1696Schlüter, A., Krause, L., Szczepanowski, R., Goesmann, A., Pühler, A., Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant (2008) J Biotechnol, 136, pp. 65-72. , doi:10.1016/j.jbiotec.2008.03.017Sercu, B., Boon, N., Verstraete, W., van Langenhove, H., H2S degradation is reflected by both the activity and composition of the microbial community in a compost biofilter (2006) Appl Microbiol Biotechnol, 72, pp. 1090-1098. , doi:10.1007/s00253-006-0382-xShinoda, Y., Sakai, Y., Uenishi, H., Uchihashi, Y., Hiraishi, A., Yukawa, H., Yurimoto, H.K.N., Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauer sp. strain DNT-1 (2004) Appl Environ Microbiol, 70, pp. 1385-1392. , doi:10.1128/AEM.70.3.1385-1392.2004Silva, C.C., Jesus, E.C., Torres, A.P.R., Sousa, M.P., Santiago, V.M.J., Oliveira, V.M., Investigation of bacterial diversity in membrane bioreactor and conventional activated sludge processes from petroleum refineries using phylogenetic and statistical approaches (2010) J Microbiol Biotechnol, 20, pp. 447-459Silva, C.C., Viero, A.F., Dias, A.C.F., Andreote, F.D., Jesus, E.C., De Paula, S.O., Torres, A.P.R., Oliveira, V.M., Monitoring the bacterial community dynamics in a petroleum refinery wastewater membrane bioreactor fed with a high phenolic load (2010) J Microbiol Biotechnol, 20, pp. 17-25Sleator, R.D., Shortall, C., Hill, C., Under the microscope: Metagenomics (2008) Let Appl Microbiol, 47, pp. 361-366. , doi:10.1111/j.1472-765X.2008.02444.x(1998) American Public Health Association and American Water Works Association and Water Pollution Control Federation, , Standard Methods for the Examination of Water and Wastewater, USA: Washington, 20Steele, H.L., Jaeger, K.-E., Daniel, R., Streit, W.R., Advances in recovery of novel biocatalysts from metagenomes (2009) J Mol Microbiol Biotechnol, 16, pp. 25-37. , doi:10.1159/000142892Stepnowski, P., Siedlecka, E.M., Behrend, P., Jastorff, B., Enhanced photodegradation of contaminants in petroleum refinery wastewater (2002) Water Res, 36, pp. 2167-2172. , doi:10.1016/S0043-1354(01)00450-XSzczepanowski, R., Bekel, T., Goesmann, A., Krause, L., Krömeke, H., Kaiser, O., Eichler, W., Schlüter, A., Insight into the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to antimicrobial drugs analyzed by the 454-pyrosequencing technology (2008) J Biotechnol, 136, pp. 54-64. , doi:10.1016/j.jbiotec.2008.03.020Torsvik, V., Goksoyr, J., Daae, F.L., High diversity in DNA of soil bacteria (1990) Appl Environ Microbiol, 56, pp. 782-787Valle, A., Bailey, M.J., Whiteley, A.S., Manefield, M., N-acyl-L-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge (2004) Environmental Microbiology, 6, pp. 424-433. , doi:10.1111/j.1462-2920.2004.00581.xViero, A.F., Melo, T.M., Torres, A.P.R., Ferreira, N.R., Sant'Anna Jr., G.L., Borges, C.P., Santiago, V.M.J., The effects of long-term feeding of high organic loading in a submerged membrane bioreactor treating oil refinery wastewater (2008) J Membrane Sci, 319, pp. 223-230. , doi:10.1016/j.memsci.2008.03.038Yamanishi, Y., Hattori, M., Kotera, M., Goto, S., Kanehisa, M., E-zyme: Predicting potential EC numbers from the chemical transformation pattern of substrateproduct pairs (2009) Bioinformatics, 25, pp. 179-182. , doi:10.1093/bioinformatics/btp223Zang, K., Kurisu, F., Kasuga, I., Furumai, H., Yagi, O., Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization (2008) Systematic Appl Microbiol, 31, pp. 206-214. , doi:10.1016/j.syapm.2008.03.005Zhang, H., Banaszak, J.E., Parameswaran, P., Alder, J., Krajmalnik-Brown, R., Rittmann, B.E., Focused-pulsed sludge pre-treatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester (2009) Water Res, 43, pp. 4517-4526. , doi:10.1016/j.watres.2009.07.034Zhou, J., Xia, B., Treves, D.S., Wu, L.-Y., Marsh, T.L., O'Neill, R.V., Palumbo, A.V., Tiedje, J.M., Spatial and resource factors influencing high microbial diversity in soil (2001) Appl Environ Microbiol, 68, pp. 326-334