This is the first report of a pyrosequencing approach for a broad phylogenetic and metabolic diversity analysis of metagenomic fosmid libraries derived from phenol degrading sludge samples of petroleum refinery WWTP. Sequencing of the metagenomic libraries has allowed us to have deeper insight to the complex metagenome of the phenol degrading sludge samples, with thousands of reads assigned to different taxa and metabolic categories driving the functioning of the microbial community in the membrane bioreactors.
Based on previous studies of the use of short sequence reads (~90 bp) to accurately classify microbial communities (Liu et al. 2007
; Sanapareddy et al. 2009
), this work used MEGAN and RDP Classifier tools for obtaining a microbial phylogenetic profile of the metagenomic data. The results obtained using both tools were consistent and showed that the most abundant phyla in the metagenomic data were similar, despite the fact that the MEGAN analysis is based on all metagenomic reads, while the RDP Classifier is based on only the 16S rRNA gene reads.
Results of RDP classifier were compared to a previous report by Silva and collaborators (2010a), where the authors used the phenol degrading sludge sample (MBR1) to construct a 16S rRNA gene library. The comparison of the two libraries showed that Proteobacteria was the predominant phylum, but the richness of phyla was higher in the metagenomic dataset, which contained 6 different phyla, whereas the 16S rRNA library revealed only 3 phyla for the MBR 1 sample. However, the comparison between deeper levels, such as order and genus, showed that the richness between both libraries is similar, although the groups found were different. Probably, these differences can be explained by the bias inherent to each method, e.g. pyrosequencing and 16S rRNA gene library, used to survey the bacterial diversity. Nonetheless, in this case, the data obtained using both approaches can be considered complementary in order to depict a bacterial diversity scenario of the phenol degrading sludge.
The high abundance of the Proteobacteria group in microbial communities from wastewater treatment samples was also observed in several other studies using PCR-based experiments targeting the 16S rRNA gene (Miura et al. 2007
; Ahmed et al. 2007
; Li et al. 2009
; Sanapareddy et al. 2009
; Silva et al. 2010a
; Silva et al. 2010b
). The predominance of Proteobacteria in such environments could be explained by the fact that this phylum comprises the most phylogenetically diverse group in the Bacteria Domain, known to be metabolically versatile, including aerobic and facultative aerobic bacteria (Madigan et al. 2008
). These are quite interesting characteristics for microorganisms inhabiting wastewater treatment plant, an environment showing great daily variations in the composition and concentration of pollutant compounds. These results demonstrate that phylogenetic studies based on pyrosequencing of metagenomic fosmid libraries can give broad and reliable information about the predominant microbial groups present in the microbial community sampled.
Within the Proteobacteria, a few genera containing important species, e.g. Pseudomonas putida
(Marques and Ramos 1993
, Gonzales et al. 2001
), Thauera aromatica
(Breinig et al. 2000
), Thauera aminoaromatica
, Thauera phenylacetica
(Mechichi et al. 2002
sp. DNT-1 (Shinoda et al. 2004
) and Comamonas testosterone
, are able to utilize different kinds of aromatic compounds, including phenol, polyphenol, toluene and halobenzoate as carbon sources. Functional studies based on SIP-RNA have revealed that members of Thauera
genus dominated the phenol degradation process in bioreactor sludges (Manefield et al. 2002
; Valle et al. 2004
). Additionally, studies based on culturing analyses have showed that Comamonas testosteroni
can be involved with the metabolism of aromatic compounds, such as phenol and 4-clorophenol (Bae et al. 1996
; Arai et al. 1998
). Basu et al. (2006)
verified that Pseudomonas putida
CSV86 is able to degrade preferentially naphthalene over glucose. Agarry and Solomon (2008)
studied batch culture using synthetic phenol and observed that Pseudomonas fluorescens
was able to degrade phenol in the concentration range of 100-500 mg/L.
A considerable fraction of the metagenomic sequence data was not assigned at the genus level, demonstrating the astonishing microbial diversity present in the sludge from wastewater treatment plants. Similar findings were reported by Sanapareddy et al. (2009)
when analyzing sewage sludge from biologic treatment system, and these data corroborate other previous studies of complex environments, such as soil and oceans, in which the sequence classification becomes worse as one moves into deeper taxonomic levels (Roesch et al. 2007
; Brown et al. 2009
The phylogenetic richness observed reflected the wide metabolic diversity present in the metagenomic data from the WWTP sludge. As expected, genes assigned to the metabolism of carbohydrates, amino acids and proteins were more numerous since they are related to the housekeeping functions of all living organisms. The presence of sequences assigned to functions like nitrogen, phosphor, sulfur and aromatic compounds metabolisms are essential for the high performance of wastewater treatment plant, since they are indicative that the microorganisms from the sludge are degrading and/or assimilating such compounds. High concentrations of these organic compounds, such as ammonia, sulfate, phosphate, phenol may be toxic to human health and cause negative environmental impacts (Nair et al. 2008
The metabolic profile revealed a broad set of important genes related to the utilization and mineralization of aromatic and/or xenobiotics compounds, including some key enzymes related to aerobic phenol degradation, such as phenol hydroxylase, catechol 1,2 dioxygenase and catechol 2,3 dioxygenase. A high number of sequences related to benzoate degradation pathway was observed as well, what is coherent with the fact that benzoate is a central intermediary compound in the anaerobic and aerobic metabolism of various aromatic compounds, such as toluene, xylene, fluorene, carbazole and biphenyl (Kim and Harwood 1991
). In addition, the anaerobic phenol degradation via carboxylation of phenol to 4-hydroxybenzoate ends in the anaerobic benzoate pathway (Lack and Fuchs 1994
). These results suggest that the microorganisms of the sludge under study have potential to degrade phenol and derivatives via aerobic and anaerobic pathway. This versatility is actually a great benefit to the performance of the biological treatment.
Finally, the phylogenetic and metabolic diversities observed are an indicative that the phenol enrichment of the sludge did not affect other important functions besides phenol degradation, which are necessary for the efficient performance of biological treatment systems. Additionally, the metagenome dataset generated by pirosequencing may provide useful sequence information for the characterization of whole catabolic pathways, particularly phenol degrading pathway, that support fundamental key processes occurring in the wastewater treatment plants. Future studies will be conducted aiming at the design and use of probes or primers to detect fosmid clones bearing specific target new genes and pathways related to pollutant compound degradation, thus offering efficient tools for the improvement of bioremediation technologies.