Abstract
The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at −20°C with and without glycerol as cryoprotectant and at 4 and 20°C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4°C with and without nitrate and at 20°C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4°C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.
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References
Ahn YH (2006) Sustainable nitrogen elimination biotechnologies: a review. Process Biochem 41:1709–1721
Amann RI, Krumholz L, Stahl DA (1990a) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990b) Combination of 16s ribosomal-RNA-targeted oligonucleotide probes with flow-cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
Boon N, Goris J, De Vos P, Verstraete W, Top EM (2000) Bioaugmentation of activated sludge by an indigenous 3-chloroaniline-degrading Comamonas testosteroni strain, I2gfp. Appl Environ Microbiol 66:2906–2913
Braker G, Fesefeldt A, Witzel KP (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol 64:3769–3775
Casciotti KL, Ward BB (2001) Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing bacteria. Appl Environ Microbiol 67:2213–2221
Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M, Hauser L, Hooper A, Klotz M, Norton J, Sayavedra-Soto L, Arciero D, Hommes N, Whittaker M, Arp D (2003) Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J Bacteriol 185:2759–2773
Daims H, Bruhl 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 22:434–444
deGraaf AAV, deBruijn P, Robertson LA, Jetten MSM, Kuenen JG (1996) Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology-UK 142:2187–2196
Egli K, Fanger U, Alvarez PJJ, Siegrist H, van der Meer JR, Zehnder AJB (2001) Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch Microbiol 175:198–207
Geets J, Boon N, Verstraete W (2006) Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations. FEMS Microbiol Ecol 58:1–13
Greenberg AE, Clesceri LS, Eaton AD (1992) Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC
Guven D, Dapena A, Kartal B, Schmid MC, Maas B, van de Pas-Schoonen K, Sozen S, Mendez R, Op den Camp HJM, Jetten MSM, Strous M, Schmidt I (2005) Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria. Appl Environ Microbiol 71:1066–1071
Hippen A, Rosenwinkel KH, Baumgarten G, Seyfried CF (1997) Aerobic deammonification: a new experience in the treatment of wastewaters. Water Sci Technol 35:111–120
Jetten MSM, Wagner M, Fuerst J, van Loosdrecht M, Kuenen G, Strous M (2001) Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr Opin Biotechnol 12:283–288
Kindaichi T, Ito T, Okabe S (2004) Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization. Appl Environ Microbiol 70:1641–1650
Kloos K, Mergel A, Rosch C, Bothe H (2001) Denitrification within the genus Azospirillum and other associative bacteria. Aust J Plant Physiol 28:991–998
Kowalchuk GA, Stephen JR, DeBoer W, Prosser JI, Embley TM, Woldendorp JW (1997) Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63:1489–1497
Kuai LP, Verstraete W (1998) Ammonium removal by the oxygen-limited autotrophic nitrification–denitrification system. Appl Environ Microbiol 64:4500–4506
Laurin V, Labbe V, Juteau P, Parent S, Villemur R (2006) Long-term storage conditions for carriers with denitrifying biomass of the fluidized, methanol-fed denitrification reactor of the Montreal Biodome, and the impact on denitrifying activity and bacterial population. Water Res 40:1836–1840
Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA (1996) Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol 62:2156–2162
Moraghan JT, Buresh R (1977) Correction for dissolved nitrous oxide in nitrogen studies. Soil Sci Soc Am J 41:1201–1202
Neef A, Amann R, Schlesner H, Schleifer KH (1998) Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology-UK 144:3257–3266
Norton JM, Alzerreca JJ, Suwa Y, Klotz MG (2002) Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria. Arch Microbiol 177:139–149
Okabe S, Kindaichi T, Ito T (2005) Fate of C-14-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms. Appl Environ Microbiol 71:3987–3994
Ovreas L, Forney L, Daae FL, Torsvik V (1997) Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol 63:3367–3373
Philippot L (2002) Denitrifying genes in bacterial and archaeal genomes. Biochim Biophys Acta N Gene Struct Expr 1577:355–376
Pynaert K, Smets BF, Wyffels S, Beheydt D, Siciliano SD, Verstraete W (2003) Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl Environ Microbiol 69:3626–3635
Rittmann BE, Regan JM, Stahl DA (1994) Nitrification as a source of soluble organic substrate in biological treatment. Water Sci Technol 30:1–8
Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63:4704–4712
Rysgaard S, Glud RN (2004) Anaerobic N-2 production in Arctic sea ice. Limnol Oceanogr 49:86–94
Strous M, vanGerven E, Kuenen JG, Jetten M (1997) Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (Anammox) sludge. Appl Environ Microbiol 63:2446–2448
Strous M, Heijnen JJ, Kuenen JG, Jetten MSM (1998) The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl Microbiol Biotechnol 50:589–596
Strous M, Kuenen JG, Jetten MSM (1999) Key physiology of anaerobic ammonium oxidation. Appl Environ Microbiol 65:3248–3250
Strous M, Pelletier E, Mangenot S, Rattei T, Lehner A, Taylor MW, Horn M, Daims H, Bartol-Mavel D, Wincker P, Barbe V, Fonknechten N, Vallenet D, Segurens B, Schenowitz-Truong C, Medigue C, Collingro A, Snel B, Dutilh BE, Op den Camp HJM, van der Drift C, Cirpus I, van de Pas-Schoonen KT, Harhangi HR, van Niftrik L, Schmid M, Keltjens J, van de Vossenberg J, Kartal B, Meier H, Frishman D, Huynen MA, Mewes HW, Weissenbach J, Jetten MSM, Wagner M, Le Paslier D (2006) Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440:790–794
Third KA, Sliekers AO, Kuenen JG, Jetten MSM (2001) The CANON system (completely autotrophic nitrogen removal over nitrite) under ammonium limitation: interaction and competition between three groups of bacteria. Syst Appl Microbiol 24:588–596
Throback IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401–417
Vandegraaf AA, Mulder A, Debruijn P, Jetten MSM, Robertson LA, Kuenen JG (1995) Anaerobic oxidation of ammonium is a biologically mediated process. Appl Environ Microbiol 61:1246–1251
Verstraete W, Philips S (1998) Nitrification–denitrification processes and technologies in new contexts. Environ Pollut 102:717–726
Vogelsang C, Gollembiewski K, Ostgaard K (1999) Effect of preservation techniques on the regeneration of gel entrapped nitrifying sludge. Water Res 33:164–168
Windey K, De Bo I, Verstraete W (2005) Oxygen-limited autotrophic nitrification–denitrification (OLAND) in a rotating biological contactor treating high-salinity wastewater. Water Res 39:4512–4520
Wyffels S, Pynaert K, Boeckx P, Verstraete W, Van Cleemput O (2003) Identification and quantification of nitrogen removal in a rotating biological contactor by N-15 tracer techniques. Water Res 37:1252–1259
Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616
Acknowledgment
This research was funded by a Ph.D. grant (aspirant) for Siegfried E. Vlaeminck from the Fund of Scientific Research-Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen). Part of the work also derives from project grant GOA 1205073 (2003–2008) of the Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek (Belgium).
The authors gratefully thank Greet Van de Velde for technical support, Pascal Boeckx for kind assistance with N2O analyses and Loïs Maignien, Kim Heylen, Peter Deschryver, Ilse Forrez, Lieven Wittebolle and the anonymous reviewers for critically reviewing this manuscript.
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Vlaeminck, S.E., Geets, J., Vervaeren, H. et al. Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage. Appl Microbiol Biotechnol 74, 1376–1384 (2007). https://doi.org/10.1007/s00253-006-0770-2
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DOI: https://doi.org/10.1007/s00253-006-0770-2