Abstract
Stabilized sewage sludge is applied to agricultural fields and farmland due to its high organic matter content. The aim of this study was to investigate the effects of two types of sludge stabilization, mesophilic anaerobic digestion (MAD) and thermophilic anaerobic digestion (TAD), on bacterial communities in sludge, including the presence of pathogenic microorganisms. Bacterial community structure and phylogenetic diversity were analyzed in four sewage sludge samples from the Czech Republic. Analysis of 16S ribosomal RNA (rRNA) genes showed that investigated sludge samples harbor diverse bacterial populations with only a few taxa present across all samples. Bacterial diversity was higher in sludge samples after MAD versus TAD treatment, and communities in MAD-treated sludge shared the highest genetic similarities. In all samples, the bacterial community was dominated by reads affiliated with Proteobacteria. The sludge after TAD treatment had considerably higher number of reads of thermotolerant/thermophilic taxa, such as the phyla Deinococcus-Thermus and Thermotogae or the genus Coprothermobacter. Only one operational taxonomic unit (OTU), which clustered with Rhodanobacter, was detected in all communities at a relative abundance >1 %. All of the communities were screened for the presence of 16S rRNA gene sequences of pathogenic bacteria using a database of 122 pathogenic species and ≥98 % identity threshold. The abundance of such sequences ranged between 0.23 and 1.57 % of the total community, with lower numbers present after the TAD treatment, indicating its higher hygienization efficiency. Sequences clustering with nontuberculous mycobacteria were present in all samples. Other detected sequences of pathogenic bacteria included Streptomyces somaliensis, Acinetobacter calcoaceticus, Alcaligenes faecalis, Gordonia spp., Legionella anisa, Bordetella bronchiseptica, Enterobacter aerogenes, Brucella melitensis, and Staphylococcus aureus.
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References
Arthurson V (2008) Proper sanitization of sewage sludge: a critical issue for a sustainable society. Appl Environ Microbiol 74:5267–5275. doi:10.1128/AEM.00438-08
Bibby K, Peccia J (2013) Identification of viral pathogen diversity in sewage sludge by metagenome analysis. Environ Sci Technol 47:1945–1951. doi:10.1021/es305181x
Bibby K, Viau E, Peccia J (2010) Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids. Water Res 44:4252–4260. doi:10.1016/j.watres.2010.05.039
Boonnorat J, Chiemchaisri C, Chiemchaisri W, Yamamoto K (2014) Microbial adaptation to biodegrade toxic organic micro-pollutants in membrane bioreactor using different sludge sources. Bioresour Technol 165:50–59. doi:10.1016/j.biortech.2014.04.024
Cai L, Zhang T (2013) Detecting human bacterial pathogens in wastewater treatment plants by a high-throughput shotgun sequencing technique. Environ Sci Technol 47:5433–5441. doi:10.1021/es400275r
Cai L, Ju F, Zhang T (2014) Tracking human sewage microbiome in a municipal wastewater treatment plant. Appl Microbiol Biotechnol 98:3317–3326. doi:10.1007/s00253-013-5402-z
Cardenas E, Tiedje JM (2008) New tools for discovering and characterizing microbial diversity. Curr Opin Biotechnol 19:544–549. doi:10.1016/j.copbio.2008.10.010
Choi JY, Kim Y, Ko EA, Park YK, Jheong WH, Ko G, Ko KS (2012) Acinetobacter species isolates from a range of environments: species survey and observations of antimicrobial resistance. Diagn Microbiol Infect Dis 74:177–180. doi:10.1016/j.diagmicrobio.2012.06.023
Cincinelli A, Martellini T, Misuri L, Lanciotti E, Sweetman A, Laschi S, Palchetti I (2012) PBDEs in Italian sewage sludge and environmental risk of using sewage sludge for land application. Environ Pollut 161:229–234. doi:10.1016/j.envpol.2011.11.001
Cole JR, Tiedje JM (2014) History and impact of RDP: a legacy from Carl Woese to microbiology. RNA Biol 11:239–243. doi:10.4161/rna.28306
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145. doi:10.1093/nar/gkn879
Gentry TJ, Wang G, Rensing C, Pepper IL (2004) Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels. Microb Ecol 48:90–102. doi:10.1007/s00248-003-1048-1
Gou C, Yang Z, Huang J, Wang H, Xu H, Wang L (2014) Effects of temperature and organic loading rate on the performance and microbial community of anaerobic co-digestion of waste activated sludge and food waste. Chemosphere 105:146–151. doi:10.1016/j.chemosphere.2014.01.018
Green SJ, Prakash O, Jasrotia P, Overholt WA, Cardenas E, Hubbard D, Tiedje JM, Watson DB, Schadt CW, Brooks SC, Kostka JE (2012) Denitrifying bacteria from the genus Rhodanobacter dominate bacterial communities in the highly contaminated subsurface of a nuclear legacy waste site. Appl Environ Microbiol 78:1039–1047. doi:10.1128/aem.06435-11
Guzman C, Jofre J, Montemayor M, Lucena F (2007) Occurrence and levels of indicators and selected pathogens in different sludges and biosolids. J Appl Microbiol 103:2420–2429. doi:10.1111/j.1365-2672.2007.03487.x
Harder R, Heimersson S, Svanström M, Peters GM (2014) Including pathogen risk in life cycle assessment of wastewater management. 1. Estimating the burden of disease associated with pathogens. Environ Sci Technol 48:9438–9445. doi:10.1021/es501480q
Heimersson S, Harder R, Peters GM, Svanström M (2014) Including pathogen risk in life cycle assessment of wastewater management. 2. Quantitative comparison of pathogen risk to other impacts on human health. Environ Sci Technol 48:9446–9453. doi:10.1021/es501481m
Horackova S (2008) Vodovody, kanalizace a vodní toky v roce 2008. Czech Statistical Office. http://csugeo.i-server.cz/csu/2009edicniplan.nsf/publ/2003-09-v_roce_2008. Accessed February 2015
Huse SM, Welch DM, Morrison HG, Sogin ML (2010) Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environ Microbiol 12:1889–1898. doi:10.1111/j.1462-2920.2010.02193.x
Jost L (2006) Entropy and diversity. Oikos 113:363–375. doi:10.1111/j.2006.0030-1299.14714.x
Kirby R, Sangal V, Tucker NP, Zakrzewska-Czerwińska J, Wierzbicka K, Herron PR, Chu CJ, Chandra G, Fahal AH, Goodfellow M, Hoskisson PA (2012) Draft genome sequence of the human pathogen Streptomyces somaliensis, a significant cause of actinomycetoma. J Bacteriol 194:3544–3545. doi:10.1128/JB.00534-12
Kitajima M, Haramoto E, Iker BC, Gerba CP (2014) Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona. Sci Total Environ 484:129–136. doi:10.1016/j.scitotenv.2014.03.036
Kristensen P (2014) Urban waste water treatment (CSI 024/WAT 005) - Assessment published Jan 2013. European Environment Agency. http://www.eea.europa.eu/data-and-maps/indicators/urban-waste-water-treatment/urban-waste-water-treatment-assessment-3. Accessed February 2015
Lahiri A, Kneisel J, Kloster I, Kamal E, Lewin A (2014) Abundance of Mycobacterium avium ssp. hominissuis in soil and dust in Germany—implications for the infection route. Lett Appl Microbiol 59:65–70. doi:10.1111/lam.12243
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82:6955–6959
Levantesi C, Beimfohr C, Blanch AR, Carducci A, Gianico A, Lucena F, Tomei MC, Mininni G (2014) Hygienization performances of innovative sludge treatment solutions to assure safe land spreading. Environ Sci Pollut Res. doi:10.1007/s11356-014-3572-6
Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235. doi:10.1128/aem.71.12.8228-8235.2005
Lozupone CA, Hamady M, Kelley ST, Knight R (2007) Quantitative and qualitative β diversity measures lead to different insights into factors that structure microbial communities. Appl Environ Microbiol 73:1576–1585. doi:10.1128/aem.01996-06
Luo W, D’Angelo EM, Coyne MS (2008) Organic carbon effects on aerobic polychlorinated biphenyl removal and bacterial community composition in soils and sediments. Chemosphere 70:364–373. doi:10.1016/j.chemosphere.2007.07.022
Martín-González L, Castro R, Pereira MA, Alves MM, Font X, Vicent T (2011) Thermophilic co-digestion of organic fraction of municipal solid wastes with FOG wastes from a sewage treatment plant: reactor performance and microbial community monitoring. Bioresour Technol 102:4734–4741. doi:10.1016/j.biortech.2011.01.060
Meyer F, Paarmann D, D’Souza M, Olson R, Glass E, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J, Edwards R (2008) The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinf 9:386. doi:10.1186/1471-2105-9-386
Nakano M, Niwa M, Nishimura N (2013) Specific and sensitive detection of Alcaligenes species from an agricultural environment. Microbiol Immunol 57:240–245. doi:10.1111/1348-0421.12026
Nalin R, Simonet P, Vogel TM, Normand P (1999) Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium. Int J Syst Evol Microbiol 49(Pt 1):19–23
Pervin HM, Batstone DJ, Bond PL (2013a) Previously unclassified bacteria dominate during thermophilic and mesophilic anaerobic pre-treatment of primary sludge. Syst Appl Microbiol 36:281–290. doi:10.1016/j.syapm.2013.03.003
Pervin HM, Dennis PG, Lim HJ, Tyson GW, Batstone DJ, Bond PL (2013b) Drivers of microbial community composition in mesophilic and thermophilic temperature-phased anaerobic digestion pre-treatment reactors. Water Res 47:7098–7108. doi:10.1016/j.watres.2013.07.053
Pilloni G, Granitsiotis MS, Engel M, Lueders T (2012) Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes. PLoS ONE 7, e40467. doi:10.1371/journal.pone.0040467
Prakash O, Green SJ, Jasrotia P, Overholt WA, Canion A, Watson DB, Brooks SC, Kostka JE (2012) Rhodanobacter denitrificans sp. nov., isolated from nitrate-rich zones of a contaminated aquifer. Int J Syst Evol Microbiol 62:2457–2462. doi:10.1099/ijs.0.035840-0
Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ (2011) Removing noise from pyrosequenced amplicons. BMC Bioinf 12:38. doi:10.1186/1471-2105-12-38
Reeder J, Knight R (2010) Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat Methods 7:668–669. doi:10.1038/nmeth0910-668b
Schloss PD (2013) Secondary structure improves OTU assignments of 16S rRNA gene sequences. ISME J 7:457–460. doi:10.1038/ismej.2012.102
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. doi:10.1128/AEM.01541-09
Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS ONE 6, e27310. doi:10.1371/journal.pone.0027310
Seipke RF, Kaltenpoth M, Hutchings MI (2012) Streptomyces as symbionts: an emerging and widespread theme? FEMS Microbiol Rev 36:862–876. doi:10.1111/j.1574-6976.2011.00313.x
Stiborová H, Vrkoslavová J, Lovecká P, Pulkrabová J, Hrádková P, Hajšlová J, Demnerová K (2015). Aerobic biodegradation of selected polybrominated diphenyl ethers (PBDEs) in wastewater sewage sludge. Chemosphere 118: 315-321. doi:10.1016/j.chemosphere.2014.09.048
Tandishabo K, Nakamura K, Umetsu K, Takamizawa K (2012) Distribution and role of Coprothermobacter spp. in anaerobic digesters. J Biosci Bioeng 114:518–520. doi:10.1016/j.jbiosc.2012.05.023
Uhlík O, Wald J, Strejček M, Musilová L, Rídl J, Hroudová M, Vlček Č, Cardenas E, Macková M, Macek T (2012) Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil. PLoS ONE 7, e40653. doi:10.1371/journal.pone.0040653
Uhlík O, Musilová L, Rídl J, Hroudová M, Vlček C, Koubek J, Holečková M, Macková M, Macek T (2013) Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil. Appl Microbiol Biotechnol 97:9245–9256. doi:10.1007/s00253-012-4627-6
United States Environmental Protection Agency (2009) 2009 Hazardous Waste Report. http://www.epa.gov/osw/inforesources/data/br09/br2009rpt.pdf. Accessed February 2015
van Ingen J, Boeree MJ, Dekhuijzen PNR, van Soolingen D (2009) Environmental sources of rapid growing nontuberculous mycobacteria causing disease in humans. Clin Microbiol Infect 15:888–893. doi:10.1111/j.1469-0691.2009.03013.x
Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267. doi:10.1128/Aem.00062-07
Ye L, Zhang T (2011) Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing. Environ Sci Technol 45:7173–7179. doi:10.1021/Es201045e
Yu D, Kurola JM, Lähde K, Kymäläinen M, Sinkkonen A, Romantschuk M (2014) Biogas production and methanogenic archaeal community in mesophilic and thermophilic anaerobic co-digestion processes. J Environ Manag 143:5460. doi:10.1016/j.jenvman.2014.04.025
Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. doi:10.1089/10665270050081478
Acknowledgments
We acknowledge Miluše Hroudová and Jakub Rídl of the Institute of Molecular Genetics AS CR for having performed the pyrosequencing run. We thank too Dr. Monika Stavělová for providing the samples. Dr. Mary-Cathrine Leewis is acknowledged for her comments on the manuscript. Financial support was provided by a grant from the Czech Ministry of Education, Youth and Sports (no. LH14004).
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Stiborova, H., Wolfram, J., Demnerova, K. et al. Bacterial community structure in treated sewage sludge with mesophilic and thermophilic anaerobic digestion. Folia Microbiol 60, 531–539 (2015). https://doi.org/10.1007/s12223-015-0396-9
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DOI: https://doi.org/10.1007/s12223-015-0396-9