Abstract
The active sludge process is one of the most-used techniques for the biodegradation of organic compounds present in effluents from an assortment of wastewaters. This study investigated the bacterial community structure of a petroleum industry’s activated sludge and its physical and chemical parameters using high-throughput sequencing. Samples were collected over one year: autumn 2015 (C1), winter 2015 (C2), spring 2015 (C3), and summer 2016 (C4). Total DNA was extracted, and the primers targeting the V4 region of the 16S rRNA gene were used for amplicon sequencing. The majority of the detected microorganisms were considered rare microbiota, presenting a relative abundance below 1% of the total sequences. All of the sequences were classified at the phylum level, and up to 55% of the ASVs (Amplicon Sequence Variants) were associated with known bacterial genera. Proteobacteria was the most abundant phylum in three seasons, while the phylum Armatimonadota dominated in one season. The genus Hyphomicrobium was the most abundant in autumn, winter and summer, and an ASV belonging to the family Fimbriimonadaceae was the most abundant in the spring. Canonical Correspondence Analysis showed that physicochemical parameters of SS, SD and TSS are correlated, as well as ammoniacal nitrogen. Sample C3 presented the highest values of COD, AN and solids (SS, SD and TSS). The highest COD, AN, and solids values are correlated to the high frequency of the phylum Armatimonadota in C3.
Keywords:
bacterial community; high throughput sequencing; wastewater sludge
Resumo
O processo de lodo ativo é uma das técnicas mais utilizadas para biodegradação de compostos orgânicos presentes nos efluentes de uma variedade de águas residuais. A estrutura da comunidade bacteriana do lodo ativado de uma indústria de petróleo e sua relação com parâmetros físicos e químicos foram investigadas por meio de sequenciamento de alto rendimento. As amostras foram coletadas durante um período de um ano: outono de 2015 (C1), inverno de 2015 (C2), primavera de 2015 (C3) e verão de 2016 (C4). O DNA total foi extraído e para amplificação foram utilizados primers específicos para região V4 do gene 16S rRNA. A maioria dos microrganismos detectados foi considerada microbiota rara, apresentando abundância relativa abaixo de 1% do total de sequências. Em geral, quase a totalidade das sequências (99,9%) foi classificada em nível de filo, mas apenas algumas ASVs (23,7%) foram associadas a gênero bacteriano conhecido. As proteobactérias foram o filo mais abundante em três das estações, enquanto o filo Armatimonadota dominou em uma estação. O gênero Hyphomicrobium foi o gênero mais abundante no outono, inverno e verão, e uma ASV pertencente à família Fimbriimonadaceae (filo Armatimonadetes) foi o microrganismo mais abundante na primavera. A Análise de Correspondência Canônica (CCA) indica uma diferença consistente da comunidade bacteriana da primavera quando comparada com amostras de outras estações. Os resultados mostram uma correlação entre o filo Armatimonadota e a alta concentração de DQO, NA e sólidos.
Palavras-chave:
comunidade bacteriana; lodo ativado; sequenciamento de alto rendimento
1. INTRODUCTION
Biological and industrial wastewater treatment plants (WWTP) are standout biotechnological processes in operation worldwide (Figuerola and Erijman, 2007FIGUEROLA, E. L.; ERIJMAN, L. Bacterial taxa abundance pattern in an industrial wastewater treatment system determined by the full rRNA cycle approach. Environmental Microbiology, v.9, p.1780-1789, 2007. https://doi.org/10.1111/j.1462-2920.2007.01298.x
https://doi.org/10.1111/j.1462-2920.2007...
), whose significance is increasing in a consistently developing human society. Most wastewater treatment processes use the natural self-depuration limit of aquatic conditions, which is the effect of microbial activity (Heidenwag et al., 2001HEIDENWAG, I.; LANGHEINRICH, U.; LÜDERITZ, V. Self Purification in upland and lowland streams. Acta Hydrochimica at Hydrobiologica, v. 29, n. 1, p. 22-33, 2001.). It is crucial to recognize the relationship between microbial communities and their performance in the full-scale installations, since bacterial metabolism is essential for effective biological treatment of wastewater (Kwiatkowska and Zielinska, 2016KWIATKOWSKA, A. C.; ZIELINSKA, M. Bacterial communities in full-scale wastewater treatment systems. World Journal Microbiology Biotechnology, v. 32, p. 66, 2016. https://dx.doi.org/10.1007/s11274-016-2012-9
https://dx.doi.org/10.1007/s11274-016-20...
).
Biological treatment by the active-sludge process is well known. This most-used technique for the biodegradation of organic compounds in effluents from a variety of wastewaters and their microbial community has been studied in urban, industrial, and petrochemical wastewaters (Zhang et al., 2011ZHANG, T.; SHAO, M. F.; YE, L. 454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. The ISME Journal, v. 6, p. 1137-1147, 2011. https://doi.org/10.1038/ismej.2011.188
https://doi.org/10.1038/ismej.2011.188...
; Sánchez et al., 2013SÁNCHEZ, O.; FERRERA, I.; GONZÁLEZ, J.M.; MAS, J. Assessing bacterial diversity in a seawater-processing wastewater treatment plant by 454-pyrosequencing of the 16S rRNA and amoA genes. Microbial Biotechnology, v. 6, n. 4, p. 435-442, 2013. https://dx.doi.org/10.1111/1751-7915.12052
https://dx.doi.org/10.1111/1751-7915.120...
; Ye and Zhang, 2013YE, L.; ZHANG, T. Bacterial communities in different sections of a municipal wastewater treatment plant revealed by 16S rDNA 454 pyrosequencing. Applied Microbiology Biotechnology, v. 97, p. 2681, 2013. https://doi.org/10.1007/s00253-012-4082-4
https://doi.org/10.1007/s00253-012-4082-...
). These studies have demonstrated that the most prevalent microorganisms in these samples are Betaproteobacteria, Alphaproteobacteria, Nitrobacteria, Bacteroidetes, Firmicutes, and Actinobacteria.
High-throughput sequencing technologies provide deep insights into the bacterial populations (Ibarbalz et al., 2013IBARBALZ, F. M.; FIGUEROLA, E. L. M.; ERIJMAN, L. Industrial activated sludge exhibit unique bacterial community composition at high taxonomic ranks. Water research, v. 47, p. 3854-3864, 2013. https://doi.org/10.1016/j.watres.2013.04.010
https://doi.org/10.1016/j.watres.2013.04...
) and have been used to reveal the bacterial range of some complex environments, including activated sludge samples (Claesson et al., 2010CLAESSON, M. J.; WANG, Q.; O'SULLIVAN, O.; GREENE-DINIZ, R.; COLE J. R.; ROSS, R. P.; O'TOOLE, P. W. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Research, v. 38, p. e200, 2010. https://doi.org/10.1093/nar/gkq873
https://doi.org/10.1093/nar/gkq873...
; Zhang et al., 2011ZHANG, T.; SHAO, M. F.; YE, L. 454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. The ISME Journal, v. 6, p. 1137-1147, 2011. https://doi.org/10.1038/ismej.2011.188
https://doi.org/10.1038/ismej.2011.188...
; Yang et al., 2014YANG, Y.; YU, K.; XIA, Y.; LAU, F. T.; TANG, D. T.; FUNG, W. C.; FANG, H. H. Metagenomic analysis of sludge from full-scale anaerobic digesters operated in municipal wastewater treatment plants. Applied Microbiology Biotechnology, v. 98, p. 5709, 2014. https://doi.org/10.1007/s00253-014-5648-0
https://doi.org/10.1007/s00253-014-5648-...
; Gwin et al., 2018GWIN, C. A.; LEFEVRE, E.; ALITO, C. L.; GUNSCH, C. K. Microbial community response to silver nanoparticles and Ag+ in nitrifying activated sludge revealed by ion semiconductor sequencing. The Science of the Total Environmental, v. 616-617, p. 1014-1021, 2018. https://doi.org/10.1016/j.scitotenv.2017.10.217
https://doi.org/10.1016/j.scitotenv.2017...
). Some microorganisms have not been completely identified (Krishnan et al., 2016KRISHNAN, M.; SUGANYA, T.; PANDIARAJAN, J. Bacterial community exploration through Ion Torrent sequencing from different treatment stages of CETP for tannery. Expert Opinion Environmental Biology Journal, v. 5, p. 3, 2016. https://dx.doi.org/10.4172/2325-9655.1000136
https://dx.doi.org/10.4172/2325-9655.100...
; Abe et al., 2017ABE, T.; USHIKI, N.; FUJITANI, H.; TSUNEDA, S. A rapid collection of yet unknown ammonia oxidizers in pure culture from activated sludge. Water Research, v. 108, p. 169-178, 2017. https://doi.org/10.1016/j.watres.2016.10.070
https://doi.org/10.1016/j.watres.2016.10...
), showing that there is much more to discover about the biodiversity of activated sludge. In this study, we accessed the microbial community diversity present in activated sludge from the petrochemical industry using amplicon sequencing based on the 16S rRNA gene.
2. MATERIAL AND METHODS
2.1. Active sludge samples collection
Activated sludge samples were collected from a wastewater treatment plant (WWTP) located in Triunfo, Rio Grande do Sul, Brazil (29°51’01.1” S 51°22’50.9” W) previously described by Antunes et al. (2018)ANTUNES, T. C.; BALLARINI, A. E.; VAN DER SAND, S. Temporal variation of bacterial population and response to physical and chemical parameters along a petrochemical industry wastewater treatment plant. Annals of the Brazilian Academy of Sciences, v. 91, n. 2, 2018. https://doi.org/10.1590/0001-3765201920180394
https://doi.org/10.1590/0001-37652019201...
. The WWTP handles 450-m³ h-1 of wastewater and is operated as a conventional activated-sludge treatment process, mechanically aerated by blades. One liter of sludge was collected directly from the input aeration tank (Figure 1) during four sampling collections over one year: Autumn 2015 (C1), Winter 2015 (C2), Spring 2015 (C3), and Summer 2016 (C4). Samples were collected using a collection bucket and transported on ice to the laboratory. The samples were thereafter kept at -80ºC until further analysis. Active sludge chemical composition and physical parameters were summarized in Antunes et al. (2018)ANTUNES, T. C.; BALLARINI, A. E.; VAN DER SAND, S. Temporal variation of bacterial population and response to physical and chemical parameters along a petrochemical industry wastewater treatment plant. Annals of the Brazilian Academy of Sciences, v. 91, n. 2, 2018. https://doi.org/10.1590/0001-3765201920180394
https://doi.org/10.1590/0001-37652019201...
.
Schematic representation of the wastewater treatment plant. The black star indicates the sampling point. Arrows represent the effluent pathway.
The following parameters were determined by a certified laboratory, according to the American Public Health Association (APHA et al. , 2012APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p.): total organic carbon (TOC), chemical oxygen demand (COD), dissolved oxygen (DO), total suspended solids (TSS), solids suspended (SS), solids dissolved (SD); and total Kjeldahl nitrogen (TKN). The chemical results are listed in Table 1.
Active sludge chemical parameters. Results are shown in mg L-1. (Modified from Antunes et al., 2018ANTUNES, T. C.; BALLARINI, A. E.; VAN DER SAND, S. Temporal variation of bacterial population and response to physical and chemical parameters along a petrochemical industry wastewater treatment plant. Annals of the Brazilian Academy of Sciences, v. 91, n. 2, 2018. https://doi.org/10.1590/0001-3765201920180394
https://doi.org/10.1590/0001-37652019201... ).
2.2. DNA isolation and 16S rRNA gene fragment sequencing
Total DNA was extracted from 0.25 g of active sludge using the Dneasy PowerSoil Kit (Qiagen) following the manufacturer’s standard protocol. The concentration and purity of the isolated DNA were determined using an ND-100Nanodrop spectrophotometer (Thermo Fisher). Partial 16S rRNA gene sequences were amplified using universal primers 515F and 806R, previously identified as suitable for bacteria and archaea (Bates et al., 2011BATES, S. T.; BERG-LYONS, D.; CAPORASO, W. W. A; KNIGHT, R.; FIERER, N. Examining the global distribution of dominant archaeal populations in soil. The ISME Journal, v. 5, p. 908-17, 2011. https://doi.org/10.1038/ismej.2010.171
https://doi.org/10.1038/ismej.2010.171...
). Amplification was performed in a 25 μL mixture, consisting of 1 μL of genomic DNA, 2 mM MgCl2, 0.2 μM of each primer, 200 μM of each dNTP, 1U Taq DNA polymerase and 1X reaction buffer. These primers amplify 291 bp from the V3-V4 hypervariable region of the prokaryotic 16S rRNA gene. Amplification was carried out in a Mastercycler Personal 5332 Thermocycler (EppendorfR) according to the following program: initial denaturation at 94ºC for 2 min, followed by 25 cycles of 45 s at 94ºC, 45 s at 55ºC, 1 min at 72ºC and a final cycle at 72ºC for 6 min. For library construction, 100 ng of DNA was used as described in the Ion Plus Fragment Library manual kit. Barcode sequences were added to identify each sample from the total sequencing output, since all samples were sequenced in a multiplexed run. Amplicon sequencing was conducted on the Ion PGM System (Thermo Fisher) using an Ion 316 chip, following the manufacturer’s instructions.
Sequences from 16S rRNA amplicon sequencing were processed using DADA2 (Divisive Amplicon Denoising Algorithm) (Callahan et al., 2016CALLAHAN, B. J.; MCMURDIE, P. J.; ROSEN, M. J.; HAN, A. W.; JOHNSON, A. J.; HOLMES, S. P. DADA2: High-resolution sample inference from Illumina amplicon data. Nature Methods, v. 13, p. 581-583, 2016. https://dx.doi.org/10.1038/nmeth.3869
https://dx.doi.org/10.1038/nmeth.3869...
) in R (R Core Team, 2019R CORE TEAM. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2019. ). Filtering, dereplication, sample inference, and chimera identification were performed, and the generated amplicon sequence variants (ASVs) were taxonomically assigned based on the SILVA database v. 138 (Quast et al., 2013QUAST, C.; PRUESSE, E.; YILMAZ, P.; GERKEN, J.; SCHWEER, T.; YARZA, P. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research, v. 41, p. D590-D596, 2013. https://dx.doi.org/10.1093/nar/gks1219
https://dx.doi.org/10.1093/nar/gks1219...
). The ASV data were imported into R using phyloseq (McMurdie and Holmes, 2013MCMURDIE P. J.; HOLMES, S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE, v. 8, p. e61217, 2013. https://dx.doi.org/10.1371/journal.pone.0061217
https://dx.doi.org/10.1371/journal.pone....
). Unassigned taxa and any residual ASVs identified as chloroplast, mitochondria, or eukaryote were excluded from the analysis. The remaining sequences were analyzed as described by Heinz et al. (2017)HEINZ, K. G. H.; ZANONI, P. R. S.; OLIVEIRA, R. R.; MEDINA-SILVA, R.; SIMÃO, T. L. L.; TRINDADE, F. J. et al. Recycled paper sludge microbial community as a potential source of cellulase and xylanase enzymes. Waste Biomass Valorization, v. 8, p. 1907-1917, 2017. https://dx.doi.org/10.1007/s12649-016-9792-x
https://dx.doi.org/10.1007/s12649-016-97...
. Sequencing results were deposited in the National Center for Biotechnology Information (NCBI) under BioProject ID PRJNA471748.
Canonical Correspondence Analysis (CCA) was used to evaluate linkages between microbial communities (ten most-abundant phyla) and chemical parameters (TOC, COD, DO, TSS, SS, SD, and TKN) using Past3 software (Hammer et al., 2001HAMMER, O.; HARPER, D. A. T.; RYAN, P. D. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontology Electronica, v. 4, n. 1, p. 1-9, 2001.).
3. RESULTS AND DISCUSSION
After removing the low-quality sequences, the amplicon sequencing from the four samples collected seasonally from the petrochemical industry active sludge yielded a total of 241,859 16S rRNA gene sequences samples, representing an average of 60,465 sequences per sample. The average sequence length was 273 bp.
The microbiota was classified within 31 phyla, 65 classes, 146 orders, 167 families and 185 genera or respective taxa. The domain Bacteria had the highest number of classified microorganisms (94.9% of the total sequences). The occurrence of four archaeal phyla was observed: Crenarchaeota, Halobacterota, Nanoarchaeota, Aenigmarchaeota. The phylum Aenigmarchaeota was present only in sequences from sample C3, comprising 0.10% of the total sequences in sample C3.
The classified bacterial community was composed of thirteen phyla with an abundance higher than 1% of the total sequences (Figure 2). Proteobacteria was the most abundant phylum in samples C1, C2, and C4, representing up to 37% of the total sequences in C2, followed by the phylum Bacteroidota present in samples C1, C2 and C4 (16.22%, 15.36% and 17.59% of the total sequences, respectively). In sample C3, the most abundant phylum was Armatimonadota and Proteobacteria; they represented 49.16% and 21.09% of the total sequences, respectively (Figure 2). Armatimonadota was the second-most abundant phylum in C1, after Proteobacteria, accounting for 11.74% of the total sequences. Unclassified sequences at the phylum level presented an average of 0.01% of the total sequences in the samples.
Classification of the most abundant phyla (≥ 1% of the total sequences in at least one sample) of microorganism present in activated sludge samples over a year (samples C1 to C4). “Others” represents the phyla whose abundances are lower than 1% of the total sequences. * Archaea phyla.
From the 336 detected taxa, 33 presented a relative abundance higher than 1% in at least one sample (Table 2) and were considered the predominant microbiota. From that, seventeen microorganisms were classified at the genus level. Hyphomicrobium was the most abundant genus in samples C1, C2 and, C4, accounting for 13.98%, 12.72% and 13.07% of the total sequences, respectively. The most abundant microorganism of sample C3 was a taxa belonging to the family Fimbriimonadaceae (phylum Armatimonadota), representing 48.96% of the total sequences in that sample. The majority of the 336 detected taxa were considered rare microbiota for presenting a relative abundance below 1% of the total sequences. From that, 185 microorganisms were classified at the genus level (Supplementary Table 1).
Canonical Correspondence Analysis (CCA) showed that the values of the physicochemical parameters of SS, SD and TSS are correlated, as well as ammoniacal nitrogen (Figure 3). According to the analyzed chemical parameters (Table 1), C3 presents the highest COD, AN and solids (SS, SD and TSS) compared to the other samples. These microbiological and chemical characteristics found in sample C3 make it different from C1, C2, and C4 (Figure 4). The highest COD, AN, and solids values are correlated to the high frequency of the phylum Armatimonadota.
Our study provided 16S rRNA gene sequence analyses of the microbial community present in activated sludge from the petrochemical industry. Our findings are in accordance with previous studies of activated sludge, with the predominance of Proteobacteria (Xia et al., 2010XIA, S.; DUAN, L.; SONG, Y.; LI, J.; PICENO, Y. M.; ANDERSEN, G. L.; COHEN, A. L. et al. Bacterial community structure in geographically distributed biological wastewater treatment reactors. Environmental Science and Technology, v. 44, p. 7391-7396, 2010. https://doi.org/10.1021/es101554m
https://doi.org/10.1021/es101554m...
). Sidhu et al. (2017)SIDHU, C.; VIKRAM, S.; PINNAKA, A. K. Unraveling the microbial interactions and metabolic potentials in pre- and post-treated sludge from a wastewater treatment plant using metagenomic studies. Frontiers in Microbiology, v. 8, p. 1382, 2017. https://dx.doi.org/10.3389/fmicb.2017.01382
https://dx.doi.org/10.3389/fmicb.2017.01...
characterized and dissected the phylogenetic and functional structures from the sludge community at the phylum level and found the dominance of Proteobacteria in raw and dried sludge samples, representing 97.9% and 92.6%, respectively.
Canonical correlation analysis (CCA) associating the sample collection and chemical parameters to the activated sludge sampling point.
Canonical correlation analysis (CCA) associating the most abundant bacterial phyla and chemical parameters to the activated sludge sampling point.
Analysis of the microbial community revealed key groups for degradation of recalcitrant compounds present in the industrial effluent. Proteobacteria prevail in WWTPs treating pharmaceutical, oil refinery, and biological reactors (Xia et al., 2010XIA, S.; DUAN, L.; SONG, Y.; LI, J.; PICENO, Y. M.; ANDERSEN, G. L.; COHEN, A. L. et al. Bacterial community structure in geographically distributed biological wastewater treatment reactors. Environmental Science and Technology, v. 44, p. 7391-7396, 2010. https://doi.org/10.1021/es101554m
https://doi.org/10.1021/es101554m...
; Ibarbalz et al., 2013IBARBALZ, F. M.; FIGUEROLA, E. L. M.; ERIJMAN, L. Industrial activated sludge exhibit unique bacterial community composition at high taxonomic ranks. Water research, v. 47, p. 3854-3864, 2013. https://doi.org/10.1016/j.watres.2013.04.010
https://doi.org/10.1016/j.watres.2013.04...
; Kwiatkowska and Zielinska, 2016KWIATKOWSKA, A. C.; ZIELINSKA, M. Bacterial communities in full-scale wastewater treatment systems. World Journal Microbiology Biotechnology, v. 32, p. 66, 2016. https://dx.doi.org/10.1007/s11274-016-2012-9
https://dx.doi.org/10.1007/s11274-016-20...
). Alphaproteobacteria and Gammaproteobacteria were the most dominant class in Proteobacteria. The filamentous Alphaproteobacteria are versatile consumers of various organic substrates (Kragelund et al., 2006KRAGELUND, C.; KONG, Y.; VAN DER, W. J.; THELEN, K.; EIKELBOOM, D.; TANDOI, V. et al. Ecophysiology of different filamentous Alphaproteobacteria species from industrial wastewater treatment plants. Microbiology, v. 152, p.3003-3012, 2006. https://doi.org/10.1099/mic.0.29249-0
https://doi.org/10.1099/mic.0.29249-0...
). Most species are aerobic or facultatively anaerobic; many are oligotrophic, preferring to grow in environments with low nutrient concentration (Madigan et al., 2016MADIGAN, M. T.; MARTINKO, J. M.; BENDER, K. S.; BUCKLEY, D. H.; STAHL, D. A. Microbiologia de Brock. Porto Alegre: Artmed, 2016. 1032 p.).
Activated sludge has a very diverse microbial community structure depending on both wastewater composition and operational conditions in the treatment plant. However, in several studies of microbial community structure, it has been found that the composition of activated sludge from different plants is quite similar in terms of overall dominating bacterial phylogenetic groups. In nutrient removal of activated sludge, the dominating group frequently found is Alphaproteobacteria, Gammaproteobacteria and Betaproteobacteria (Klausen et al., 2004KLAUSEN, M. M.; THOMSEN, T. R.; NIELSEN, J. L.; MIKKELSEN, L. H.; NIELSEN, P. H. Variations in microcolony strength of probe-defined bacteria in activated sludge flocs. FEMS Microbiology Ecology, v. 50, p.123-132, 2004. https://doi.org/10.1016/j.femsec.2004.06.005
https://doi.org/10.1016/j.femsec.2004.06...
; Lee et al. 2002LEE, N.; LA COUR JANSSEN, J.; ASPEGREN, H.; HENZE, M. N. P. H.; WAGNER, M. Population dynamics in wastewater treatment plants with enhanced biological phosphorus removal operated without nitrogen removal. Water Science Technology, v. 46, p.163-170, 2002. https://doi.org/10.2166/wst.2002.0472
https://doi.org/10.2166/wst.2002.0472...
; Schmid et al., 2003SCHMID, M.; THILL, A.; PURKHOLD, U.; WALCHER, M.; BOTTERO, J. Y.; GINESTET, P. et al. Characterization of activated sludge flocs by confocal laser scanning microscopy and image analysis. Water Research, v. 37, p. 2043-2052, 2003. https://doi.org/10.1016/S0043-1354(02)00616-4
https://doi.org/10.1016/S0043-1354(02)00...
; Wagner and Loy, 2002WAGNER, M.; LOY, A. Bacterial community composition and function in sewage treatment systems. Current Opinion Biotechnology. v. 13, p. 218-227, 2002. https://doi.org/10.1016/S0958-1669(02)00315-4
https://doi.org/10.1016/S0958-1669(02)00...
). Studies in WWTPs suggested a higher diversity of active denitrifiers, including uncharacterized Alphaproteobacteria, Gammaproteobacteria and Actinobacteria (Osaka et al. 2006OSAKA, T.; YOSHIE, S.; TSUNEDA, S.; HIRATA, A.; IWAMI, N.; INAMORI, Y. Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microbiology Ecology, v. 52, p. 253-266, 2006. https://doi.org/10.1007/s00248-006-9071-7
https://doi.org/10.1007/s00248-006-9071-...
; Hagman et al., 2008HAGMAN, M.; NIELSEN, J. L.; NIELSEN, P. H.; JANSEN, J. Mixed carbon sources for nitrate reduction in activated sludge-identification of bacteria and process activity studies. Water Research, v. 42, p. 1539-1546, 2008. https://doi.org/10.1016/j.watres.2007.10.034
https://doi.org/10.1016/j.watres.2007.10...
; Morgan-Sagastume et al., 2008MORGAN-SAGASTUME, F.; NIELSEN, J. L.; NIELSEN, P. H. Substrate-dependent denitrification of abundant probe-defined denitrifying bacteria in activated sludge. FEMS Microbiology Ecology, v. 66, p. 447-461, 2008. https://doi.org/10.1111/j.1574-6941.2008.00571.x
https://doi.org/10.1111/j.1574-6941.2008...
). Filamentous Alphaproteobacteria have been shown as essential microorganisms in industrial WWTPs, often related to bulking incidents or deteriorating settling sludge properties (Levantesi et al., 2004LEVANTESI, C.; BEIMFOHR, C.; GEURKINK, B.; ROSSETTI, S.; THELEN, K.; KROONEMAN, J. et al. Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants. System Applied Microbiology, v. 27, p.716-727, 2004. https://doi.org/10.1078/0723202042369974
https://doi.org/10.1078/0723202042369974...
).
At the order level, it was found that the dominant populations in the activated sludge samples were Burkolderiales and Rhizobiales, which represented 8.03% and 7.44% of those populations. This low percentage indicates a great diversity of the bacterial populations present in the activated sludge.
Sample C3 presented the most different microbial composition of the four samples, mainly because of the dominance of the individuals from the phylum Armatimonadota (Lee et al., 2013LEE, K. C. Y.; HERBOLD, C. W.; DUNFIELD, P. F.; MORGAN, X. C.; MCDONALD, I. R.; STOTT, M. B. Phylogenetic delineation of the novel phylum Armatimonadetes (former candidate division OP10) and definition of two novel candidate divisions. Applied Environmental Microbiology, v. 79, p. 2484-2487, 2013. https://doi.org/10.2166/wst.2002.0472
https://doi.org/10.2166/wst.2002.0472...
). This phylum is found in a diverse array of environments, such as geothermal soils (Stott et al., 2008STOTT, M. B.; SAITO, J. A.; CROWE, M. A.; DUNFIELD, P. F.; HOU, S.; NAKASONE, E. et al. Culture-independent characterization of a novel microbial community at a hydrothermal vent at Brothers volcano, Kermadec arc, New Zealand. Journal of Geophysical Research: Solid Earth, v. 113, 2008. https://dx.doi.org/10.1029/2007JB005477
https://dx.doi.org/10.1029/2007JB005477...
), freshwater lakes and rivers (Crump and Hobbie, 2005CRUMP, B. C.; HOBBIE, J. E. Synchrony and seasonality in bacterioplankton communities of two temperate rivers. Limnology Oceanography, v. 50, p. 1718-1729, 2005. https://doi.org/10.4319/lo.2005.50.6.1718
https://doi.org/10.4319/lo.2005.50.6.171...
), the water discharged from manures (Simpsonet al., 2004SIMPSON, J. M.; DOMINGO, J. W.; REASONER, D. J. Assessment of equine fecal contamination: the search for alternative bacterial source-tracking targets. FEMS Microbiology Ecology, v. 47, p. 65-75, 2004. https://doi.org/10.1016/S0168-6496(03)00250-2
https://doi.org/10.1016/S0168-6496(03)00...
), and activated sludge (Dalevi et al., 2001DALEVI, D.; HUGENHOLTZ, P.; BLACKALL, L. L. A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data. International Journal of Systematic Evolutionary Microbiology, v. 51, p. 385-391, 2001. https://doi.org/10.1099/00207713-51-2-385
https://doi.org/10.1099/00207713-51-2-38...
). Portillo et al. (2009)PORTILLO, M. C.; GONZALEZ, J. M. Members of the Candidate Division OP10 are spread in a variety of environments. World Journal Microbiology Biotechnology, v. 25, p. 347-353, 2009. https://dx.doi.org/10.1007/s11274-008-9895-z
https://dx.doi.org/10.1007/s11274-008-98...
pointed out that this bacterial phylum could constitute an average of 5% among the total bacterial sequences recovered in hypersaline soils, geothermal springs, lake and river, bioreactors, and endolithic environments. Among the phylum Armatimonadetes, a more extensive geographical distribution was found in anaerobic niches (Harris et al., 2004HARRIS, J. K.; KELLEY, S. T.; PACE, N. R. New perspective on uncultured bacterial phylogenetic division OP11. Applied Environmental Microbiology, v. 70, p. 845-849, 2004. https://dx.doi.org/10.1128/AEM.70.2.845-849.2004
https://dx.doi.org/10.1128/AEM.70.2.845-...
; Stott et al., 2008STOTT, M. B.; SAITO, J. A.; CROWE, M. A.; DUNFIELD, P. F.; HOU, S.; NAKASONE, E. et al. Culture-independent characterization of a novel microbial community at a hydrothermal vent at Brothers volcano, Kermadec arc, New Zealand. Journal of Geophysical Research: Solid Earth, v. 113, 2008. https://dx.doi.org/10.1029/2007JB005477
https://dx.doi.org/10.1029/2007JB005477...
). Chemical parameters influenced the bacterial community of C3. The canonical correlation analysis (CCA) shows that the phylum Armatimonadota presented a positive correlation with the increasing COD, TOC and total dissolved and suspended solids of the C3 sample. This sample showed the highest COD and the second-highest TOC and Solids (TSS, SS, and SD) quantification; these parameters contribute to the formation of an environment with low oxygen concentrations, which may have favored the occurrence of the phylum Armatimonadota. Also, sample C3 showed bacterial diversity differences between the other collections of activated sludge, such the phyla Aenigmarchaeota, Caldisericota, Cloacimonadota, MBNT15 and Sva0485, which were only detected in C3 (Supplementary Table 1).
CCA analysis also showed the correlation of Actinobacteriota with the presence of dissolved oxygen (DO). Most genera from this phylum are aerobic (Goodfellow and Williams, 1983GOODFELLOW, M.; WILLIAMS, S. T. Ecology of Actinomycetes. Annual Review of Microbiology, v. 37, n. 1, p. 189-216, 1983. https://doi.org/10.1146/annurev.mi.37.100183.001201
https://doi.org/10.1146/annurev.mi.37.10...
) and this phylum presented significant quantification in sample C2 (2 mg per liter).
Nitrospirae shows a correlation with the presence of NTK. The ability to perform nitrite reduction was a physiological characteristic observed in Nitrospirae (Sidhu et al., 2017SIDHU, C.; VIKRAM, S.; PINNAKA, A. K. Unraveling the microbial interactions and metabolic potentials in pre- and post-treated sludge from a wastewater treatment plant using metagenomic studies. Frontiers in Microbiology, v. 8, p. 1382, 2017. https://dx.doi.org/10.3389/fmicb.2017.01382
https://dx.doi.org/10.3389/fmicb.2017.01...
). According to Ward et al. (2009)WARD, N. L.; CHALLACOMBE, J. F.; JANSSEN, P. H.; HENRISSAT, B.; COUTINHO, P. M.; WU, M. et al. Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Applied Environmental Microbiology, v. 75, p. 2046-56, 2009. https://dx.doi.org/10.1128/AEM.02294-08
https://dx.doi.org/10.1128/AEM.02294-08...
, genomic evidence suggested that the role of acidobacteria in nitrogen cycling in soils and sediments is the reduction of nitrate, nitrite, and possibly nitric oxide due to assimilatory nitrate reductase gene sequences. The presence of nif genes related to conventional nitrogenase was found in a study by Inoue et al. (2015)INOUE, J.; OSHIMA, K.; SUDA, W.; SAKAMOTO, M.; IINO, T.; NODA, S.; OHKUMA, M. Distribution and Evolution of Nitrogen Fixation Genes in the Phylum Bacteroidetes. Microbes Environmental, v. 30, n. 1, p. 44-50, 2015. http://doi.org/10.1264/jsme2.ME14142
http://doi.org/10.1264/jsme2.ME14142...
, suggesting nitrogen fixation ability in some Bacteroidetes species.
Acidobacteriota shows a correlation with the presence of AN, SS, SD and TSS. Bacteria belonging to the phylum Acidobacteria have also been observed in a wide variety of environments, including extreme (Hobel et al., 2005HOBEL, C. F. V.; MARTEINSSON, V. T.; HREGGVIDSSON, G. O.; KRISTJÁNSSON, J. K. Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16 S rRNA and chitinase genes. Applied Environmental Microbiology, v. 71, p. 2771-2776, 2005. https://dx.doi.org/10.1128/aem.71.5.2771-2776.2005
https://dx.doi.org/10.1128/aem.71.5.2771...
), polluted (Bobbink et al., 2010BOBBINK, R.; HICKS, K.; GALLOWAY, J.; SPRANGER, T.; ALKEMADE, R.; ASHMORE, M. et al. Global assessment of nitrogen deposition effects on terrestrial plant diversity: a synthesis. Ecological Applications, v. 20, p. 30-59, 2010. http://dx.doi.org/10.1890/08-1140.1
http://dx.doi.org/10.1890/08-1140.1...
), and effluent wastewater environments (LaPara et al., 2000LAPARA, T. M.; NAKATSU, C. H.; PANTEA, L.; ALLEMAN, J. E. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Applied Environmental Microbiology, v. 66, p. 3951-3959, 2000. https://dx.doi.org/10.1128/aem.66.9.3951-3959.2000
https://dx.doi.org/10.1128/aem.66.9.3951...
). Ward et al. (2009)WARD, N. L.; CHALLACOMBE, J. F.; JANSSEN, P. H.; HENRISSAT, B.; COUTINHO, P. M.; WU, M. et al. Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Applied Environmental Microbiology, v. 75, p. 2046-56, 2009. https://dx.doi.org/10.1128/AEM.02294-08
https://dx.doi.org/10.1128/AEM.02294-08...
found that Acidobacteria were involved in nitrogen cycling, promoting the conversion of nitrate and nitrite.
All the sequences were classified at the phylum level, and up to 55% were associated with a bacterial genus. Among the most abundant microorganisms, Hyphomicrobium and Fimbriimonadaceae were described in the literature as potential denitrifiers and degradators. The genus Hyphomicrobium is a denitrifier and can degrade C-1 compounds such as methanol (Rissanen et al., 2017RISSANEN, A. J.; OJALA, A.; FRED, T.; TOIVONEN, J.; TIIROLA, M. Methylophilaceae and Hyphomicrobium as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants. Journal of Industrial Microbiology Biotechnology, v. 44, p. 35-47, 2017. https://doi.org/10.1007/s10295-016-1860-5
https://doi.org/10.1007/s10295-016-1860-...
). Sequences representing the phylum Armatimonadetes have been isolated by culture-independent methods from various environments, including aerobic and anaerobic wastewater treatment processes, the rhizosphere, hypersaline microbial mats and subsurface geothermal water streams (Portillo and Gonzalez, 2009PORTILLO, M. C.; GONZALEZ, J. M. Members of the Candidate Division OP10 are spread in a variety of environments. World Journal Microbiology Biotechnology, v. 25, p. 347-353, 2009. https://dx.doi.org/10.1007/s11274-008-9895-z
https://dx.doi.org/10.1007/s11274-008-98...
; Lee et al., 2013LEE, K. C. Y.; HERBOLD, C. W.; DUNFIELD, P. F.; MORGAN, X. C.; MCDONALD, I. R.; STOTT, M. B. Phylogenetic delineation of the novel phylum Armatimonadetes (former candidate division OP10) and definition of two novel candidate divisions. Applied Environmental Microbiology, v. 79, p. 2484-2487, 2013. https://doi.org/10.2166/wst.2002.0472
https://doi.org/10.2166/wst.2002.0472...
; Tamaki et al., 2011TAMAKI, H.; TANAKA, Y.; MATSUZAWA, H.; MURAMATSU, M.; MENG, X.Y.; HANADA, S. et al. Armatimonas rosea gen. nov., sp nov., of a novel bacterial phylum, Armatimonadetes phyl. nov., formally called the candidate phylum OP10. International Journal Systematic and Evolutionary Microbiology. v.61, p.1442-1447, 2011. https://doi.org/10.1099/ijs.0.025643-0
https://doi.org/10.1099/ijs.0.025643-0...
). Fimbriimonadaceae belonging to Armatimonadetes was detected in an anammox consortia where ammonium was removed without nitrite and oxygen (Liang et al., 2014LIANG, Y.; LI, D.; ZHANG, X.; ZENG, H.; YANG, Z.; ZHANG, J. Microbial characteristics and nitrogen removal of simultaneous partial nitrification, anammox and denitrification (SNAD) process treating low C/N ratio sewage. Bioresource Technology, v.169, p.103-109, 2014. https://doi.org/10.1016/j.biortech.2014.06.064
https://doi.org/10.1016/j.biortech.2014....
).
4. CONCLUSION
Even with the advances brought about by the new generation sequencing, there are still challenges regarding the classification of the microorganisms in environmental samples. The classification of sequences at a lower taxonomic level, such as family or genus, is essential to understanding a WWTP as a whole and the real participation of each microorganism in the different stages of treatment. The present study contributed to the characterization of the microbial communities involved in the sewage treatment of the petrochemical industry. Identifying the microorganisms has the broader impact of contributing to the knowledge of biological wastewater treatment.
5. ACKNOWLEDGMENTS
We would like to thank Sistema Integrado de Tratamento de Efluentes Líquidos do Polo Petroquímico (SITEL-CORSAN) for authorizing the sample collection. We thank High Performance Computing Lab - LAD/PUCRS for allowing access to run the high-throughput sequences analyses. Luiz Gustavo A. Borges thanks PEGA/PUCRS. We also thank CNPq and CAPES for their financial support.
6. REFERENCES
- ABE, T.; USHIKI, N.; FUJITANI, H.; TSUNEDA, S. A rapid collection of yet unknown ammonia oxidizers in pure culture from activated sludge. Water Research, v. 108, p. 169-178, 2017. https://doi.org/10.1016/j.watres.2016.10.070
» https://doi.org/10.1016/j.watres.2016.10.070 - ANTUNES, T. C.; BALLARINI, A. E.; VAN DER SAND, S. Temporal variation of bacterial population and response to physical and chemical parameters along a petrochemical industry wastewater treatment plant. Annals of the Brazilian Academy of Sciences, v. 91, n. 2, 2018. https://doi.org/10.1590/0001-3765201920180394
» https://doi.org/10.1590/0001-3765201920180394 - APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p.
- BATES, S. T.; BERG-LYONS, D.; CAPORASO, W. W. A; KNIGHT, R.; FIERER, N. Examining the global distribution of dominant archaeal populations in soil. The ISME Journal, v. 5, p. 908-17, 2011. https://doi.org/10.1038/ismej.2010.171
» https://doi.org/10.1038/ismej.2010.171 - BOBBINK, R.; HICKS, K.; GALLOWAY, J.; SPRANGER, T.; ALKEMADE, R.; ASHMORE, M. et al Global assessment of nitrogen deposition effects on terrestrial plant diversity: a synthesis. Ecological Applications, v. 20, p. 30-59, 2010. http://dx.doi.org/10.1890/08-1140.1
» http://dx.doi.org/10.1890/08-1140.1 - CALLAHAN, B. J.; MCMURDIE, P. J.; ROSEN, M. J.; HAN, A. W.; JOHNSON, A. J.; HOLMES, S. P. DADA2: High-resolution sample inference from Illumina amplicon data. Nature Methods, v. 13, p. 581-583, 2016. https://dx.doi.org/10.1038/nmeth.3869
» https://dx.doi.org/10.1038/nmeth.3869 - CLAESSON, M. J.; WANG, Q.; O'SULLIVAN, O.; GREENE-DINIZ, R.; COLE J. R.; ROSS, R. P.; O'TOOLE, P. W. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Research, v. 38, p. e200, 2010. https://doi.org/10.1093/nar/gkq873
» https://doi.org/10.1093/nar/gkq873 - CRUMP, B. C.; HOBBIE, J. E. Synchrony and seasonality in bacterioplankton communities of two temperate rivers. Limnology Oceanography, v. 50, p. 1718-1729, 2005. https://doi.org/10.4319/lo.2005.50.6.1718
» https://doi.org/10.4319/lo.2005.50.6.1718 - DALEVI, D.; HUGENHOLTZ, P.; BLACKALL, L. L. A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data. International Journal of Systematic Evolutionary Microbiology, v. 51, p. 385-391, 2001. https://doi.org/10.1099/00207713-51-2-385
» https://doi.org/10.1099/00207713-51-2-385 - FIGUEROLA, E. L.; ERIJMAN, L. Bacterial taxa abundance pattern in an industrial wastewater treatment system determined by the full rRNA cycle approach. Environmental Microbiology, v.9, p.1780-1789, 2007. https://doi.org/10.1111/j.1462-2920.2007.01298.x
» https://doi.org/10.1111/j.1462-2920.2007.01298.x - GOODFELLOW, M.; WILLIAMS, S. T. Ecology of Actinomycetes. Annual Review of Microbiology, v. 37, n. 1, p. 189-216, 1983. https://doi.org/10.1146/annurev.mi.37.100183.001201
» https://doi.org/10.1146/annurev.mi.37.100183.001201 - GWIN, C. A.; LEFEVRE, E.; ALITO, C. L.; GUNSCH, C. K. Microbial community response to silver nanoparticles and Ag+ in nitrifying activated sludge revealed by ion semiconductor sequencing. The Science of the Total Environmental, v. 616-617, p. 1014-1021, 2018. https://doi.org/10.1016/j.scitotenv.2017.10.217
» https://doi.org/10.1016/j.scitotenv.2017.10.217 - HAGMAN, M.; NIELSEN, J. L.; NIELSEN, P. H.; JANSEN, J. Mixed carbon sources for nitrate reduction in activated sludge-identification of bacteria and process activity studies. Water Research, v. 42, p. 1539-1546, 2008. https://doi.org/10.1016/j.watres.2007.10.034
» https://doi.org/10.1016/j.watres.2007.10.034 - HAMMER, O.; HARPER, D. A. T.; RYAN, P. D. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontology Electronica, v. 4, n. 1, p. 1-9, 2001.
- HARRIS, J. K.; KELLEY, S. T.; PACE, N. R. New perspective on uncultured bacterial phylogenetic division OP11. Applied Environmental Microbiology, v. 70, p. 845-849, 2004. https://dx.doi.org/10.1128/AEM.70.2.845-849.2004
» https://dx.doi.org/10.1128/AEM.70.2.845-849.2004 - HEIDENWAG, I.; LANGHEINRICH, U.; LÜDERITZ, V. Self Purification in upland and lowland streams. Acta Hydrochimica at Hydrobiologica, v. 29, n. 1, p. 22-33, 2001.
- HEINZ, K. G. H.; ZANONI, P. R. S.; OLIVEIRA, R. R.; MEDINA-SILVA, R.; SIMÃO, T. L. L.; TRINDADE, F. J. et al Recycled paper sludge microbial community as a potential source of cellulase and xylanase enzymes. Waste Biomass Valorization, v. 8, p. 1907-1917, 2017. https://dx.doi.org/10.1007/s12649-016-9792-x
» https://dx.doi.org/10.1007/s12649-016-9792-x - HOBEL, C. F. V.; MARTEINSSON, V. T.; HREGGVIDSSON, G. O.; KRISTJÁNSSON, J. K. Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16 S rRNA and chitinase genes. Applied Environmental Microbiology, v. 71, p. 2771-2776, 2005. https://dx.doi.org/10.1128/aem.71.5.2771-2776.2005
» https://dx.doi.org/10.1128/aem.71.5.2771-2776.2005 - IBARBALZ, F. M.; FIGUEROLA, E. L. M.; ERIJMAN, L. Industrial activated sludge exhibit unique bacterial community composition at high taxonomic ranks. Water research, v. 47, p. 3854-3864, 2013. https://doi.org/10.1016/j.watres.2013.04.010
» https://doi.org/10.1016/j.watres.2013.04.010 - INOUE, J.; OSHIMA, K.; SUDA, W.; SAKAMOTO, M.; IINO, T.; NODA, S.; OHKUMA, M. Distribution and Evolution of Nitrogen Fixation Genes in the Phylum Bacteroidetes. Microbes Environmental, v. 30, n. 1, p. 44-50, 2015. http://doi.org/10.1264/jsme2.ME14142
» http://doi.org/10.1264/jsme2.ME14142 - KLAUSEN, M. M.; THOMSEN, T. R.; NIELSEN, J. L.; MIKKELSEN, L. H.; NIELSEN, P. H. Variations in microcolony strength of probe-defined bacteria in activated sludge flocs. FEMS Microbiology Ecology, v. 50, p.123-132, 2004. https://doi.org/10.1016/j.femsec.2004.06.005
» https://doi.org/10.1016/j.femsec.2004.06.005 - KRAGELUND, C.; KONG, Y.; VAN DER, W. J.; THELEN, K.; EIKELBOOM, D.; TANDOI, V. et al Ecophysiology of different filamentous Alphaproteobacteria species from industrial wastewater treatment plants. Microbiology, v. 152, p.3003-3012, 2006. https://doi.org/10.1099/mic.0.29249-0
» https://doi.org/10.1099/mic.0.29249-0 - KRISHNAN, M.; SUGANYA, T.; PANDIARAJAN, J. Bacterial community exploration through Ion Torrent sequencing from different treatment stages of CETP for tannery. Expert Opinion Environmental Biology Journal, v. 5, p. 3, 2016. https://dx.doi.org/10.4172/2325-9655.1000136
» https://dx.doi.org/10.4172/2325-9655.1000136 - KWIATKOWSKA, A. C.; ZIELINSKA, M. Bacterial communities in full-scale wastewater treatment systems. World Journal Microbiology Biotechnology, v. 32, p. 66, 2016. https://dx.doi.org/10.1007/s11274-016-2012-9
» https://dx.doi.org/10.1007/s11274-016-2012-9 - LAPARA, T. M.; NAKATSU, C. H.; PANTEA, L.; ALLEMAN, J. E. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Applied Environmental Microbiology, v. 66, p. 3951-3959, 2000. https://dx.doi.org/10.1128/aem.66.9.3951-3959.2000
» https://dx.doi.org/10.1128/aem.66.9.3951-3959.2000 - LEE, N.; LA COUR JANSSEN, J.; ASPEGREN, H.; HENZE, M. N. P. H.; WAGNER, M. Population dynamics in wastewater treatment plants with enhanced biological phosphorus removal operated without nitrogen removal. Water Science Technology, v. 46, p.163-170, 2002. https://doi.org/10.2166/wst.2002.0472
» https://doi.org/10.2166/wst.2002.0472 - LEE, K. C. Y.; HERBOLD, C. W.; DUNFIELD, P. F.; MORGAN, X. C.; MCDONALD, I. R.; STOTT, M. B. Phylogenetic delineation of the novel phylum Armatimonadetes (former candidate division OP10) and definition of two novel candidate divisions. Applied Environmental Microbiology, v. 79, p. 2484-2487, 2013. https://doi.org/10.2166/wst.2002.0472
» https://doi.org/10.2166/wst.2002.0472 - LEVANTESI, C.; BEIMFOHR, C.; GEURKINK, B.; ROSSETTI, S.; THELEN, K.; KROONEMAN, J. et al Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants. System Applied Microbiology, v. 27, p.716-727, 2004. https://doi.org/10.1078/0723202042369974
» https://doi.org/10.1078/0723202042369974 - LIANG, Y.; LI, D.; ZHANG, X.; ZENG, H.; YANG, Z.; ZHANG, J. Microbial characteristics and nitrogen removal of simultaneous partial nitrification, anammox and denitrification (SNAD) process treating low C/N ratio sewage. Bioresource Technology, v.169, p.103-109, 2014. https://doi.org/10.1016/j.biortech.2014.06.064
» https://doi.org/10.1016/j.biortech.2014.06.064 - MADIGAN, M. T.; MARTINKO, J. M.; BENDER, K. S.; BUCKLEY, D. H.; STAHL, D. A. Microbiologia de Brock. Porto Alegre: Artmed, 2016. 1032 p.
- MCMURDIE P. J.; HOLMES, S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE, v. 8, p. e61217, 2013. https://dx.doi.org/10.1371/journal.pone.0061217
» https://dx.doi.org/10.1371/journal.pone.0061217 - MORGAN-SAGASTUME, F.; NIELSEN, J. L.; NIELSEN, P. H. Substrate-dependent denitrification of abundant probe-defined denitrifying bacteria in activated sludge. FEMS Microbiology Ecology, v. 66, p. 447-461, 2008. https://doi.org/10.1111/j.1574-6941.2008.00571.x
» https://doi.org/10.1111/j.1574-6941.2008.00571.x - OSAKA, T.; YOSHIE, S.; TSUNEDA, S.; HIRATA, A.; IWAMI, N.; INAMORI, Y. Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microbiology Ecology, v. 52, p. 253-266, 2006. https://doi.org/10.1007/s00248-006-9071-7
» https://doi.org/10.1007/s00248-006-9071-7 - PORTILLO, M. C.; GONZALEZ, J. M. Members of the Candidate Division OP10 are spread in a variety of environments. World Journal Microbiology Biotechnology, v. 25, p. 347-353, 2009. https://dx.doi.org/10.1007/s11274-008-9895-z
» https://dx.doi.org/10.1007/s11274-008-9895-z - QUAST, C.; PRUESSE, E.; YILMAZ, P.; GERKEN, J.; SCHWEER, T.; YARZA, P. et al The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research, v. 41, p. D590-D596, 2013. https://dx.doi.org/10.1093/nar/gks1219
» https://dx.doi.org/10.1093/nar/gks1219 - R CORE TEAM. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2019.
- RISSANEN, A. J.; OJALA, A.; FRED, T.; TOIVONEN, J.; TIIROLA, M. Methylophilaceae and Hyphomicrobium as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants. Journal of Industrial Microbiology Biotechnology, v. 44, p. 35-47, 2017. https://doi.org/10.1007/s10295-016-1860-5
» https://doi.org/10.1007/s10295-016-1860-5 - SÁNCHEZ, O.; FERRERA, I.; GONZÁLEZ, J.M.; MAS, J. Assessing bacterial diversity in a seawater-processing wastewater treatment plant by 454-pyrosequencing of the 16S rRNA and amoA genes. Microbial Biotechnology, v. 6, n. 4, p. 435-442, 2013. https://dx.doi.org/10.1111/1751-7915.12052
» https://dx.doi.org/10.1111/1751-7915.12052 - SCHMID, M.; THILL, A.; PURKHOLD, U.; WALCHER, M.; BOTTERO, J. Y.; GINESTET, P. et al Characterization of activated sludge flocs by confocal laser scanning microscopy and image analysis. Water Research, v. 37, p. 2043-2052, 2003. https://doi.org/10.1016/S0043-1354(02)00616-4
» https://doi.org/10.1016/S0043-1354(02)00616-4 - SIDHU, C.; VIKRAM, S.; PINNAKA, A. K. Unraveling the microbial interactions and metabolic potentials in pre- and post-treated sludge from a wastewater treatment plant using metagenomic studies. Frontiers in Microbiology, v. 8, p. 1382, 2017. https://dx.doi.org/10.3389/fmicb.2017.01382
» https://dx.doi.org/10.3389/fmicb.2017.01382 - SIMPSON, J. M.; DOMINGO, J. W.; REASONER, D. J. Assessment of equine fecal contamination: the search for alternative bacterial source-tracking targets. FEMS Microbiology Ecology, v. 47, p. 65-75, 2004. https://doi.org/10.1016/S0168-6496(03)00250-2
» https://doi.org/10.1016/S0168-6496(03)00250-2 - STOTT, M. B.; SAITO, J. A.; CROWE, M. A.; DUNFIELD, P. F.; HOU, S.; NAKASONE, E. et al Culture-independent characterization of a novel microbial community at a hydrothermal vent at Brothers volcano, Kermadec arc, New Zealand. Journal of Geophysical Research: Solid Earth, v. 113, 2008. https://dx.doi.org/10.1029/2007JB005477
» https://dx.doi.org/10.1029/2007JB005477 - TAMAKI, H.; TANAKA, Y.; MATSUZAWA, H.; MURAMATSU, M.; MENG, X.Y.; HANADA, S. et al Armatimonas rosea gen. nov., sp nov., of a novel bacterial phylum, Armatimonadetes phyl. nov., formally called the candidate phylum OP10. International Journal Systematic and Evolutionary Microbiology. v.61, p.1442-1447, 2011. https://doi.org/10.1099/ijs.0.025643-0
» https://doi.org/10.1099/ijs.0.025643-0 - WAGNER, M.; LOY, A. Bacterial community composition and function in sewage treatment systems. Current Opinion Biotechnology. v. 13, p. 218-227, 2002. https://doi.org/10.1016/S0958-1669(02)00315-4
» https://doi.org/10.1016/S0958-1669(02)00315-4 - WARD, N. L.; CHALLACOMBE, J. F.; JANSSEN, P. H.; HENRISSAT, B.; COUTINHO, P. M.; WU, M. et al Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Applied Environmental Microbiology, v. 75, p. 2046-56, 2009. https://dx.doi.org/10.1128/AEM.02294-08
» https://dx.doi.org/10.1128/AEM.02294-08 - XIA, S.; DUAN, L.; SONG, Y.; LI, J.; PICENO, Y. M.; ANDERSEN, G. L.; COHEN, A. L. et al Bacterial community structure in geographically distributed biological wastewater treatment reactors. Environmental Science and Technology, v. 44, p. 7391-7396, 2010. https://doi.org/10.1021/es101554m
» https://doi.org/10.1021/es101554m - YANG, Y.; YU, K.; XIA, Y.; LAU, F. T.; TANG, D. T.; FUNG, W. C.; FANG, H. H. Metagenomic analysis of sludge from full-scale anaerobic digesters operated in municipal wastewater treatment plants. Applied Microbiology Biotechnology, v. 98, p. 5709, 2014. https://doi.org/10.1007/s00253-014-5648-0
» https://doi.org/10.1007/s00253-014-5648-0 - YE, L.; ZHANG, T. Bacterial communities in different sections of a municipal wastewater treatment plant revealed by 16S rDNA 454 pyrosequencing. Applied Microbiology Biotechnology, v. 97, p. 2681, 2013. https://doi.org/10.1007/s00253-012-4082-4
» https://doi.org/10.1007/s00253-012-4082-4 - ZHANG, T.; SHAO, M. F.; YE, L. 454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. The ISME Journal, v. 6, p. 1137-1147, 2011. https://doi.org/10.1038/ismej.2011.188
» https://doi.org/10.1038/ismej.2011.188
Supplementary Table 1.
Publication Dates
-
Publication in this collection
25 June 2021 -
Date of issue
2021
History
-
Received
02 Oct 2020 -
Accepted
03 May 2021