Abstract
The biological wastewater treatment systems are mostly based on the activated sludge method and endangered by exploitation problems connected to sludge bulking and/or foaming. Chloroflexi bacteria often cause bulking episodes and should be investigated in that matter, especially regarding the unusual metabolic activities. The aim of the study was to evaluate if the activity of microbial populations measured by the Biolog system is changing in reaction to diluent and homogenisation. The thesis was it that it differs for activated sludge samples with high and low abundance of Chloroflexi. The systematic review was performed to identify and fill a research gap concerning the best approach to study metabolism of activated sludge of various origins. We tested samples of municipal and industrial sludge with differing abundance of Chloroflexi and showed that activity of distinct types of sludge is influenced incongruously by preparation. The homogenisation unified the composition of activated sludge and influenced the microbial communities responsible for the hydrolysis of complex sugars. The homogenised sludge showed activity for the same substrates as the non-homogenised one, but the carbohydrate uptake was higher for the second one. Dilution with supernatant also influenced the metabolism. Substrate uptake was lower for 60% of the substrate groups for the less diluted sludge. On the other hand, the higher dilution of activated sludge inoculum resulted in a bigger spectrum of the substrates assimilated by bacteria on EcoPlate. Interestingly the type of diluent and time of homogenisation do not significantly affect the results within the same wastewater treatment plant.
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source utilisation for different substrate groups. A&A: amines and amides, AA: amino acids; C&AA: carboxylic and acetic acids, Carb: carbohydrates; Poly: polymers
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Data availability
All data gathered and analysed during this study are included in the article and its supplementary information files.
Abbreviations
- AA:
-
Amino acids
- A&A:
-
Amines and amides
- ANOSIM:
-
Analysis of similarities
- BOD:
-
Biological oxygen demand
- C&AA:
-
Carboxylic and acetic acids
- Carb:
-
Carbohydrates
- COD:
-
Chemical oxygen demand
- FISH:
-
Fluorescence in situ hybridisation
- MLSS:
-
Mixed liquor suspended solids
- N:
-
Nitrogen
- NMDS:
-
Nonparametric multidimensional scaling
- OD:
-
Optical density
- P:
-
Phosphorus
- PE:
-
Population equivalent
- PIX:
-
Iron-based coagulants
- Poly:
-
Polymers
- SIMPER:
-
Similarity percentage breakdown
- WWTP:
-
Wastewater treatment plant
References
Ahmar Siddiqui M, Biswal BK, Heynderickx PM, Kim J, Khanal SK, Chen G, Wu D (2022) Dynamic anaerobic membrane bioreactor coupled with sulfate reduction (SrDMBR) for saline wastewater treatment. Biores Technol. https://doi.org/10.1016/j.biortech.2021.126447
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56(6):1919–1925. https://doi.org/10.1128/aem.56.6.1919-1925.1990
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–169. https://doi.org/10.1128/mmbr.59.1.143-169.1995
Biggs CA, Olaleye OI, Jeanmeure LFC, Deines P, Jensen HS, Tait SJ, Wright PC (2011) Effect of temperature on the substrate utilization profiles of microbial communities in different sewer sediments. Environ Technol 32(2):133–144. https://doi.org/10.1080/09593330.2010.490852
Björnsson L, Hugenholtz P, Tyson GW, Blackall LL (2002) Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal. Microbiology 148(8):2309–2318. https://doi.org/10.1099/00221287-148-8-2309
Burger W, Krysiak-Baltyn K, Scales PJ, Martin GJO, Stickland AD, Gras SL (2017) The influence of protruding filamentous bacteria on floc stability and solid-liquid separation in the activated sludge process. Water Res. https://doi.org/10.1016/j.watres.2017.06.063
Cadoret A, Conrad A, Block JC (2002) Availability of low and high molecular weight substrates to extracellular enzymes in whole and dispersed activated sludges. Enzyme Microb Technol. https://doi.org/10.1016/S0141-0229(02)00097-2
Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Aust J Ecol 18(1):117. https://doi.org/10.1111/j.1442-9993.1993.tb00438.x
Daims H, Brühl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22(3):434. https://doi.org/10.1016/S0723-2020(99)80053-8
Dunkel T, de León Gallegos EL, Bock C, Lange A, Hoffmann D, Boenigk J, Denecke M (2018) Illumina sequencing for the identification of filamentous bulking and foaming bacteria in industrial activated sludge plants. Int J Environ Sci Technol 15(6):1139. https://doi.org/10.1007/s13762-017-1484-y
Eikelboom DH (2000) Process control of activated sludge plants by microscopic investigation. IWA Publishing, London
Gich F, Garcia-Gil J, Overmann J (2001) Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes. Arch Microbiol 177(1):1–10. https://doi.org/10.1007/s00203-001-0354-6
Gruber W, Niederdorfer R, Ringwald J, Morgenroth E, Bürgmann H, Joss A (2021) Linking seasonal N2O emissions and nitrification failures to microbial dynamics in a SBR wastewater treatment plant. Water Res X. https://doi.org/10.1016/j.wroa.2021.100098
Hammer Ø, Harper DAT, Ryan PD (2001) Past: Paleontological statistics software package for education and data analysis. Palaeontol Electron 4(1):9
Huang X, Xue D, Xue L (2015) Changes in soil microbial functional diversity and biochemical characteristics of tree peony with amendment of sewage sludge compost. Environ Sci Pollut Res 22(15):11617. https://doi.org/10.1007/s11356-015-4407-9
Jatowiecki Ł, Chojniak JM, Dorgeloh E, Hegedusova B, Ejhed H, Magnér J, Płaza GA (2016) Microbial community profiles in wastewaters from onsite wastewater treatment systems technology. PLoS ONE. https://doi.org/10.1371/journal.pone.0147725
Justyna M, Danuta W, Izabela G (2016) Investigation of functional diversity and activated sludge condition using biolog® system. Archit Civ Eng Environ 9(1):119–126. https://doi.org/10.21307/acee-2016-013
Kiersztyn B, Chróst R, Kaliński T, Siuda W, Bukowska A, Kowalczyk G, Grabowska K (2019) Structural and functional microbial diversity along a eutrophication gradient of interconnected lakes undergoing anthropopressure. Sci Rep. https://doi.org/10.1038/s41598-019-47577-8
Kragelund C, Remesova Z, Nielsen JL, Thomsen TR, Eales K, Seviour R, Nielsen PH (2007) Ecophysiology of mycolic acid-containing Actinobacteria (Mycolata) in activated sludge foams. FEMS Microbiol Ecol 61(1):174. https://doi.org/10.1111/j.1574-6941.2007.00324.x
Kragelund C, Thomsen TR, Mielczarek AT, Nielsen PH (2011) Eikelboom’s morphotype 0803 in activated sludge belongs to the genus Caldilinea in the phylum Chloroflexi. FEMS Microbiol Ecol 76(3):451. https://doi.org/10.1111/j.1574-6941.2011.01065.x
Li Y, Chróst RJ (2006a) Enzymatic activities in petroleum wastewater purification system by an activated sludge process. J Microbiol Biotechnol 16(2):200
Li Y, Chróst RJ (2006b) Microbial enzymatic activities in aerobic activated sludge model reactors. Enzym Microb Technol 39(4):568. https://doi.org/10.1016/j.enzmictec.2005.10.028
Li J, Jin Z, Yu B (2010) Changes in the structure and diversity of bacterial communities during the process of adaptation to organic wastewater. Can J Microbiol. https://doi.org/10.1139/w10-009
McIlroy SJ, Saunders AM, Albertsen M, Nierychlo M, McIlroy B, Hansen AA, Karst SM, Nielsen JL, Nielsen PH (2015) MiDAS: the field guide to the microbes of activated sludge. Database. https://doi.org/10.1093/database/bav062
Miłobędzka A, Muszyński A (2016) Selection of methods for activated sludge bulking control using a molecular biology technique combined with respirometric tests. Biotechnologia 97(3):187. https://doi.org/10.5114/bta.2016.62356
Miłobedzka A, Muszyński A (2017) Can DNA sequencing show differences between microbial communities in Polish and Danish wastewater treatment plants? Water Sci Technol 75(6):1447–1454. https://doi.org/10.2166/wst.2017.015
Muszyński A, Miłobędzka A (2015) Differences in populations of filamentous bacteria involved in foaming and bulking of activated sludge. Chall Mod Technol 6(3):30
Muszyński A, Tabernacka A, Miłobedzka A (2015) Long-term dynamics of the microbial community in a full-scale wastewater treatment plant. Int Biodeterior Biodegrad. https://doi.org/10.1016/j.ibiod.2015.02.008
Nielsen PH (2009) FISH handbook for biological wastewater treatment. Water Intell Online. https://doi.org/10.2166/9781780401775
Nielsen JL, Kragelund C, Nielsen PH (2010a) Ecophysiological analysis of microorganisms in complex microbial systems by combination of fluorescence in situ hybridization with extracellular staining techniques. In: Cummings SP (ed) Bioremediation. Humana Press, Totowa, NJ, pp 117–128. https://doi.org/10.1007/978-1-60761-439-5_8
Nielsen PH, Mielczarek AT, Kragelund C, Nielsen JL, Saunders AM, Kong Y, Vollertsen J (2010b) A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants. Water Res 44(17):5070. https://doi.org/10.1016/j.watres.2010.07.036
Nierychlo M, Miłobȩdzka A, Petriglieri F, McIlroy B, Nielsen PH, McIlroy SJ (2019) The morphology and metabolic potential of the Chloroflexi in full-scale activated sludge wastewater treatment plants. FEMS Microbiol Ecol. https://doi.org/10.1093/femsec/fiy228
Nierychlo M, Andersen KS, Xu Y, Green N, Jiang C, Albertsen M, Dueholm MS, Nielsen PH (2020a) MiDAS 3: an ecosystem-specific reference database, taxonomy and knowledge platform for activated sludge and anaerobic digesters reveals species-level microbiome composition of activated sludge. Water Res. https://doi.org/10.1016/j.watres.2020.115955
Nierychlo M, McIlroy SJ, Kucheryavskiy S, Jiang C, Ziegler AS, Kondrotaite Z, Stokholm-Bjerregaard MS, Nielsen PH (2020b) Candidatus Amarolinea and Candidatus Microthrix are mainly responsible for filamentous bulking in danish municipal wastewater treatment plants. Front Microbiol. https://doi.org/10.3389/fmicb.2020.01214
Speirs L, Nittami T, McIlroy S, Schroeder S, Seviour RJ (2009) Filamentous bacterium Eikelboom Type 0092 in activated sludge plants in Australia is a member of the phylum chloroflexi. Appl Environ Microbiol 75(8):2446. https://doi.org/10.1128/AEM.02310-08
Speirs LBM, Rice DTF, Petrovski S, Seviour RJ (2019) The phylogeny, biodiversity, and ecology of the Chloroflexi in activated sludge. Front Microbiol. https://doi.org/10.3389/fmicb.2019.02015
Tsigkou K, Terpou A, Treu L, Kougias PG, Kornaros M (2022) Thermophilic anaerobic digestion of olive mill wastewater in an upflow packed bed reactor: evaluation of 16S rRNA amplicon sequencing for microbial analysis. J Environ Manag. https://doi.org/10.1016/j.jenvman.2021.113853
Weber KP, Legge RL (2009) One-dimensional metric for tracking bacterial community divergence using sole carbon source utilization patterns. J Microbiol Methods 79(1):55. https://doi.org/10.1016/j.mimet.2009.07.020
West-Roberts JA, Matheus-Carnevali PB, Schoelmerich MC, Al-Shayeb B, Thomas AD, Sharrar A, He C, Chen L-X, Lavy A, Keren R, Amano Y, Banfield JF (2021) The Chloroflexi supergroup is metabolically diverse and representatives have novel genes for non-photosynthesis based CO2 fixation. BioRxiv
Wingender J, Jaeger KE, Flemming HC (1999) Interaction between extracellular polysacccharides and enzymes. In microbial extracellular polymeric substances: characterization, structure and function. Springer, Berlin, pp 231–251
Zak JC, Willig MR, Moorhead DL, Wildman HG (1994) Functional diversity of microbial communities: a quantitative approach. Soil Biol Biochem 26(9):1101. https://doi.org/10.1016/0038-0717(94)90131-7
Acknowledgements
We thank Dr. Marta Nierychło and professor Per Nielsen for hosting our FISH procedures at the Department of Chemistry and Bioscience, Center for Microbial Communities, Aalborg University, Aalborg, Denmark
Funding
This work has received funding from the National Science Centre, Poland [Grant Number 2015/17/B/ NZ9/01552].
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BK contributed to formal analysis, visualisation, writing—review and editing. RC contributed to conceptualisation and funding acquisition. AM contributed to conceptualisation, formal analysis, writing—original draft, and writing—review and editing.
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Kiersztyn, B., Chróst, R. & Miłobędzka, A. Homogenisation and dilution in metabolic evaluation of activated sludge rich in Chloroflexi. Int. J. Environ. Sci. Technol. 20, 3295–3308 (2023). https://doi.org/10.1007/s13762-022-04191-y
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DOI: https://doi.org/10.1007/s13762-022-04191-y