Low-Dissolved-Oxygen Nitrifying Systems Exploit Ammonia-Oxidizing Bacteria with Unusually High Yields

ABSTRACT In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of aeration to avoid nitrification failures. This approach contributes significantly to operational costs and the carbon footprint of nitrifying wastewater treatment processes. In this study, we tested the effect of aeration rate on nitrification by correlating ammonia oxidation rates with the structure of the ammonia-oxidizing bacterial (AOB) community and AOB abundance in four parallel continuous-flow reactors operated for 43 days. Two of the reactors were supplied with a constant airflow rate of 0.1 liter/min, while in the other two units the airflow rate was fixed at 4 liters/min. Complete nitrification was achieved in all configurations, though the dissolved oxygen (DO) concentration was only 0.5 ± 0.3 mg/liter in the low-aeration units. The data suggest that efficient performance in the low-DO units resulted from elevated AOB levels in the reactors and/or putative development of a mixotrophic AOB community. Denaturing gel electrophoresis and cloning of AOB 16S rRNA gene fragments followed by sequencing revealed that the AOB community in the low-DO systems was a subset of the community in the high-DO systems. However, in both configurations the dominant species belonged to the Nitrosomonas oligotropha lineage. Overall, the results demonstrated that complete nitrification can be achieved at low aeration in lab-scale reactors. If these findings could be extended to full-scale plants, it would be possible to minimize the operational costs and greenhouse gas emissions without risk of nitrification failure.

[1]  C. Criddle,et al.  Occurrence of Ammonia-Oxidizing Archaea in Wastewater Treatment Plant Bioreactors , 2006, Applied and Environmental Microbiology.

[2]  Francis L. de los Reyes,et al.  Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors , 2005, Applied and Environmental Microbiology.

[3]  L. Raskin,et al.  Diversity and dynamics of microbial communities in engineered environments and their implications for process stability. , 2003, Current opinion in biotechnology.

[4]  James R. Cole,et al.  rrndb: the Ribosomal RNA Operon Copy Number Database , 2001, Nucleic Acids Res..

[5]  J. M. González,et al.  A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. , 2002, Environmental microbiology.

[6]  Daniel B. Oerther,et al.  Bacterial Competition in Activated Sludge: Theoretical Analysis of Varying Solids Retention Times on Diversity , 2004, Microbial Ecology.

[7]  J. Prosser.  Autotrophic nitrification in bacteria. , 1989, Advances in microbial physiology.

[8]  M. Spérandio,et al.  Modelling an aerobic submerged membrane bioreactor with ASM models on a large range of sludge retention time , 2008 .

[9]  P. Reich,et al.  Diversity and Productivity in a Long-Term Grassland Experiment , 2001, Science.

[10]  T. Curtis,et al.  Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant. , 2008, Water research.

[11]  E. Bock,et al.  Anaerobic ammonia oxidation by cell-free extracts of Nitrosomonas eutropha , 1998, Antonie van Leeuwenhoek.

[12]  M. Hermansson,et al.  Effects of environmental conditions on the nitrifying population dynamics in a pilot wastewater treatment plant. , 2007, Environmental microbiology.

[13]  Awwa,et al.  Standard Methods for the examination of water and wastewater , 1999 .

[14]  K. Chandran,et al.  Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes , 2011, Biotechnology and bioengineering.

[15]  A. Boehm,et al.  Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary. , 2008, Environmental microbiology.

[16]  J. G. Kuenen,et al.  A mathematical description of the behaviour of mixed chemostat cultures of an autotrophic nitrifier and a heterotrophic nitrifier/aerobic denitrifier; a comparison with experimental data , 1993 .

[17]  G. Kowalchuk,et al.  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 , 1997, Applied and environmental microbiology.

[18]  James R. Cole,et al.  A new version of the RDP (Ribosomal Database Project) , 1999, Nucleic Acids Res..

[19]  M. V. van Loosdrecht,et al.  Nitrous oxide emission during wastewater treatment. , 2009, Water research.

[20]  J. Waterbury,et al.  Nitrospira marina gen. nov. sp. nov.: a chemolithotrophic nitrite-oxidizing bacterium , 1986, Archives of Microbiology.

[21]  M. Nei,et al.  A Simple Method for Estimating and Testing Minimum-Evolution Trees , 1992 .

[22]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[23]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[24]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[25]  D. Tilman.  Resource competition and community structure. , 1983, Monographs in population biology.

[26]  J. O. O N H O A H N, † S U N G P Y O K I M, †, § H O N G.  N2O Emissions from Activated Sludge Processes, 2008-2009: Results of a National Monitoring Survey in the United States , 2010 .

[27]  R. Amann,et al.  Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm , 2001, Applied and Environmental Microbiology.

[28]  Katie Bloor,et al.  Experimental demonstration of chaotic instability in biological nitrification , 2007, The ISME Journal.

[29]  M. Moo-young,et al.  Environmental Biotechnology: Principles and Applications , 2010 .

[30]  D. Noguera,et al.  Characterization of two ammonia‐oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations , 2007, Journal of applied microbiology.

[31]  E. Bock,et al.  Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha , 1997, Archives of Microbiology.

[32]  K. Schleifer,et al.  Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology , 1999, Applied and Environmental Microbiology.

[33]  G. Tallec,et al.  Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation. , 2008, Bioresource technology.

[34]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[35]  M. Wagner,et al.  Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors , 2003, Applied and Environmental Microbiology.

[36]  Thomas P. Curtis,et al.  Modeling Risk of Failure in Nitrification: Simple Model Incorporating Abundance and Diversity , 2009 .

[37]  Ying Huang,et al.  Taxonomic evaluation of the Streptomyces griseus clade using multilocus sequence analysis and DNA-DNA hybridization, with proposal to combine 29 species and three subspecies as 11 genomic species. , 2010, International journal of systematic and evolutionary microbiology.

[38]  M. Nei,et al.  Prospects for inferring very large phylogenies by using the neighbor-joining method. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  P. Lindgren,et al.  Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR , 2001, Applied and Environmental Microbiology.

[40]  J. Hollibaugh,et al.  Ammonia oxidation and ammonia-oxidizing bacteria and archaea from estuaries with differing histories of hypoxia , 2007, The ISME Journal.

[41]  Ø. Hammer,et al.  PAST: PALEONTOLOGICAL STATISTICAL SOFTWARE PACKAGE FOR EDUCATION AND DATA ANALYSIS , 2001 .

[42]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[43]  J. Beman,et al.  Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[44]  K. Schleifer,et al.  Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization , 2001, Applied and Environmental Microbiology.

[45]  L. Sayavedra-Soto,et al.  Chemolithoorganotrophic Growth of Nitrosomonas europaea on Fructose , 2003, Journal of bacteriology.

[46]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[47]  M. V. van Loosdrecht,et al.  Mechanisms and specific directionality of autotrophic nitrous oxide and nitric oxide generation during transient anoxia. , 2010, Environmental science & technology.

[48]  A. Oskooi.  Molecular Evolution and Phylogenetics , 2008 .

[49]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[50]  O Hammer-Muntz,et al.  PAST: paleontological statistics software package for education and data analysis version 2.09 , 2001 .

[51]  Thomas P. Curtis,et al.  Agreement between Theory and Measurement in Quantification of Ammonia-Oxidizing Bacteria , 2005, Applied and Environmental Microbiology.

[52]  T. Jin,et al.  Occurrence of ammonia‐oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants , 2009, Journal of applied microbiology.

[53]  Denny S. Parker,et al.  Process Design Manual for Nitrogen Control. , 1975 .

[54]  G. Kowalchuk,et al.  Shifts in the dominant populations of ammonia-oxidizing ß subclass Proteobacteria along the eutrophic Schelde estuary , 2001 .

[55]  H. D. Stensel,et al.  Design and retrofit of wastewater treatment plants for biological nutrient removal , 1992 .

[56]  Zhiguo Yuan,et al.  Nitrous oxide generation in full-scale biological nutrient removal wastewater treatment plants. , 2010, Water research.

[57]  D. Stahl,et al.  Molecular and modeling analyses of the structure and function of nitrifying activated sludge , 1999 .

[58]  C. Knapp,et al.  Nitrite-oxidizing bacteria guild ecology associated with nitrification failure in a continuous-flow reactor. , 2007, FEMS microbiology ecology.

[59]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[60]  I. Schmidt.  Chemoorganoheterotrophic Growth of Nitrosomonas europaea and Nitrosomonas eutropha , 2009, Current Microbiology.

[61]  A. Pollice,et al.  Influence of aeration and sludge retention time on ammonium oxidation to nitrite and nitrate. , 2002, Water research.

[62]  Tong Zhang,et al.  Ammonia‐oxidizing bacteria dominates over ammonia‐oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading , 2011, Biotechnology and bioengineering.

[63]  D. Stahl,et al.  Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria , 2009, Nature.

[64]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[65]  D. Noguera,et al.  Evaluating the effect of dissolved oxygen on ammonia-oxidizing bacterial communities in activated sludge. , 2004, Water research.

[66]  O. Ulloa,et al.  High diversity of ammonia-oxidizing archaea in permanent and seasonal oxygen-deficient waters of the eastern South Pacific. , 2010, Environmental microbiology.