Skip to main content

Advertisement

SpringerLink
Log in

Use of functional gene expression and respirometry to study wastewater nitrification activity after exposure to low doses of copper

  • Research Article
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript

Abstract

Autotrophic nitrification in biological nitrogen removal systems has been shown to be sensitive to the presence of heavy metals in wastewater treatment plants. Using transcriptase-quantitative polymerase chain reaction (RT-qPCR) data, we examined the effect of copper on the relative expression of functional genes (i.e., amoA, hao, nirK, and norB) involved in redox nitrogen transformation in batch enrichment cultures obtained from a nitrifying bioreactor operated as a continuous reactor (24-h hydraulic retention time). 16S ribosomal RNA (rRNA) gene next-generation sequencing showed that Nitrosomonas-like populations represented 60–70 % of the bacterial community, while other nitrifiers represented <5 %. We observed a strong correspondence between the relative expression of amoA and hao and ammonia removal in the bioreactor. There were no considerable changes in the transcript levels of amoA, hao, nirK, and norB for nitrifying samples exposed to copper dosages ranging from 0.01 to 10 mg/L for a period of 12 h. Similar results were obtained when ammonia oxidation activity was measured via specific oxygen uptake rate (sOUR). The lack of nitrification inhibition by copper at doses lower than 10 mg/L may be attributed to the role of copper as cofactor for ammonia monooxygenase or to the sub-inhibitory concentrations of copper used in this study. Overall, these results demonstrate the use of molecular methods combined with conventional respirometry assays to better understand the response of wastewater nitrifying systems to the presence of copper.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Ahn JH, Kwan T, Chandran K (2011) Comparison of partial and full nitrification processes applied for treating high-strength nitrogen wastewaters: microbial ecology through nitrous oxide production. Environ Sci Technol 45:2734–2740

    Article  CAS  Google Scholar 

  • Ahn JH, Yu R, Chandran K (2008) Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor. Biotechnol Bioeng 100:1078–1087

    Article  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Barber WP, Stuckey DC (2000) Nitrogen removal in a modified anaerobic baffled reactor (ABR): 2, nitrification. Water Res 34:2423–2432

    Article  CAS  Google Scholar 

  • Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108:4516–4522

    Article  CAS  Google Scholar 

  • Çeçen F, Semerci N, Geyik AG (2010) Inhibition of respiration and distribution of Cd, Pb, Hg, Ag and Cr species in a nitrifying sludge. J Hazard Mater 178:619–627

    Article  Google Scholar 

  • Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M, Hauser L, Hooper A, Klotz M, Norton J (2003) Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J Bacteriol 185:2759–2773

    Article  CAS  Google Scholar 

  • Chandran K, Love NG (2008) Physiological state, growth mode, and oxidative stress play a role in Cd (II)-mediated inhibition of Nitrosomonas europaea 19718. Appl Environ Microbiol 74:2447–2453

    Article  CAS  Google Scholar 

  • Chandran K, Smets BF (2000) Applicability of two-step models in estimating nitrification kinetics from batch respirograms under different relative dynamics of ammonia and nitrite oxidation. Biotechnol Bioeng 70:54–64

    Article  CAS  Google Scholar 

  • Chipasa KB (2003) Accumulation and fate of selected heavy metals in a biological wastewater treatment system. Waste Manag 23:135–143

    Article  CAS  Google Scholar 

  • Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen A, McGarrell DM, Marsh T, Garrity GM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145

    Article  CAS  Google Scholar 

  • Elnabarawy MT, Robideau RR, Beach SA (1988) Comparison of three rapid toxicity test procedures: Microtox,® polytox,® and activated sludge respiration inhibition. Toxicity Assessment 3:361–370

    Article  CAS  Google Scholar 

  • Ensign SA, Hyman MR, Arp DJ (1993) In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper. J Bacteriol 175:1971–1980

    CAS  Google Scholar 

  • Francis CA, Beman JM, Kuypers MM (2007) New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME J 1:19–27

    Article  CAS  Google Scholar 

  • Gernaey K, Verschuere L, Luyten L, Verstraete W (1997) Fast and sensitive acute toxicity detection with an enrichment nitrifying culture. Water Environ Res 69:1163–1169

    Article  CAS  Google Scholar 

  • Grüttner H, Winther-Nielsen M, Jørgensen L, Bøgebjerg P, Sinkjaer O (1994) Inhibition of the nitrification process in municipal wastewater treatment plants by industrial discharges. Water Sci Technol 29:69–77

    Google Scholar 

  • Harms G, Layton AC, Dionisi HM, Gregory IR, Garrett VM, Hawkins SA, Robinson KG, Sayler GS (2003) Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant. Environ Sci Technol 37:343–351

    Article  CAS  Google Scholar 

  • Hermansson A, Lindgren P-E (2001) Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR. Appl Environ Microbiol 67:972–976

    Article  CAS  Google Scholar 

  • Hooper AB, Terry KR (1973) Specific inhibitors of ammonia oxidation in Nitrosomonas. J Bacteriol 115:480–485

    CAS  Google Scholar 

  • Hu Z, Chandran K, Grasso D, Smets BF (2002) Effect of nickel and cadmium speciation on nitrification inhibition. Environ Sci Technol 36:3074–3078

    Article  CAS  Google Scholar 

  • Hu Z, Chandran K, Grasso D, Smets BF (2003) Impact of metal sorption and internalization on nitrification inhibition. Environ Sci Technol 37:728–734

    Article  CAS  Google Scholar 

  • Hu Z, Chandran K, Grasso D, Smets BF (2004) Comparison of nitrification inhibition by metals in batch and continuous flow reactors. Water Res 38:3949–3959

    Article  CAS  Google Scholar 

  • Jönsson K, Grunditz C, Dalhammar G, La Cour JJ (2000) Occurrence of nitrification inhibition in Swedish municipal wastewaters. Water Res 34:2455–2462

    Article  Google Scholar 

  • Juliastuti S, Baeyens J, Creemers C, Bixio D, Lodewyckx E (2003) The inhibitory effects of heavy metals and organic compounds on the net maximum specific growth rate of the autotrophic biomass in activated sludge. J Hazard Mater 100:271–283

    Article  CAS  Google Scholar 

  • Kapoor V, Pitkänen T, Ryu H, Elk M, Wendell D, Santo Domingo JW (2015) Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA-and DNA-based quantitative PCR assays. Appl Environ Microbiol 81:91–99

    Article  Google Scholar 

  • Karvelas M, Katsoyiannis A, Samara C (2003) Occurrence and fate of heavy metals in the wastewater treatment process. Chemosphere 53:1201–1210

    Article  CAS  Google Scholar 

  • Kim YM, Lee DS, Park C, Park D, Park JM (2011) Effects of free cyanide on microbial communities and biological carbon and nitrogen removal performance in the industrial activated sludge process. Water Res 45:1267–1279

    Article  CAS  Google Scholar 

  • Kim YM, Park D, Lee DS, Park JM (2008) Inhibitory effects of toxic compounds on nitrification process for cokes wastewater treatment. J Hazard Mater 152:915–921

    Article  CAS  Google Scholar 

  • Kuo DH-W, Robinson KG, Layton AC, Meyers AJ, Sayler GS (2010) Transcription levels (amoA mRNA-based) and population dominance (amoA gene-based) of ammonia-oxidizing bacteria. J Ind Microbiol Biotechnol 37:751–757

    Article  CAS  Google Scholar 

  • Madoni P, Davoli D, Guglielmi L (1999) Response of SOUR and AUR to heavy metal contamination in activated sludge. Water Res 33:2459–2464

    Article  CAS  Google Scholar 

  • Miller DJ, Nicholas D (1985) Characterization of a soluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea. J Gen Microbiol 131:2851–2854

    CAS  Google Scholar 

  • Milner M, Curtis T, Davenport R (2008) Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant. Water Res 42:2863–2872

    Article  CAS  Google Scholar 

  • Ochoa-Herrera V, León G, Banihani Q, Field JA, Sierra-Alvarez R (2011) Toxicity of copper (II) ions to microorganisms in biological wastewater treatment systems. Sci Total Environ 412:380–385

    Article  Google Scholar 

  • Painter HA (1970) A review of literature on inorganic nitrogen metabolism in microorganisms. Water Res 4:393–450

    Article  CAS  Google Scholar 

  • Park S, Ely RL (2008) Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals. Appl Environ Microbiol 74:5475–5482

    Article  CAS  Google Scholar 

  • Radniecki T, Ely R (2008) Zinc chloride inhibition of Nitrosococcus mobilis. Biotechnol Bioeng 99:1085–1095

    Article  CAS  Google Scholar 

  • Radniecki TS, Semprini L, Dolan ME (2009) Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions. Biotechnol Bioeng 102:546–553

    Article  CAS  Google Scholar 

  • Rowan AK, Snape JR, Fearnside D, Barer MR, Curtis TP, Head IM (2003) Composition and diversity of ammonia‐oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater. FEMS Microbiol Ecol 43:195–206

    Article  CAS  Google Scholar 

  • Sakano Y, Pickering KD, Strom PF, Kerkhof LJ (2002) Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support. Appl Environ Microbiol 68:2285–2293

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541

    Article  CAS  Google Scholar 

  • Siripong S, Rittmann BE (2007) Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants. Water Res 41:1110–1120

    Article  CAS  Google Scholar 

  • Walker C, De La Torre J, Klotz M, Urakawa H, Pinel N, Arp D, Brochier-Armanet C, Chain P, Chan P, Gollabgir A (2010) Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci U S A 107:8818–8823

    Article  CAS  Google Scholar 

  • You S, Hsu C, Ouyang C (2002) Identification of the microbial diversity of wastewater nutrient removal processes using molecular biotechnology. Biotechnol Lett 24:1361–1366

    Article  CAS  Google Scholar 

  • You S-J, Tsai Y-P, Huang R-Y (2009) Effect of heavy metals on nitrification performance in different activated sludge processes. J Hazard Mater 165:987–994

    Article  CAS  Google Scholar 

  • Yu R, Chandran K (2010) Strategies of Nitrosomonas europaea 19718 to counter low dissolved oxygen and high nitrite concentrations. BMC Microbiol 10:70

    Article  Google Scholar 

  • Yu R, Lai B, Vogt S, Chandran K (2011) Elemental profiling of single bacterial cells as a function of copper exposure and growth phase. PLoS One 6:e21255

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Kit Daniels for building the nitrifying bioreactor and for technical assistance. VK and XL were supported by ORISE-EPA Research Fellowship. KC was supported by the Water Environment Research Foundation. The US Environmental Protection Agency, through its Office of Research and Development, funded and managed, or partially funded and collaborated in, the research described herein. This work has been subjected to the agency’s administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the agency; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Author information

Authors and Affiliations

  1. Oak Ridge Institute for Science and Education, Oak Ridge, TN, 37831, USA

    Vikram Kapoor & Xuan Li

  2. Office of Research and Development, US Environmental Protection Agency, Cincinnati, OH, 45268, USA

    Vikram Kapoor, Xuan Li, Christopher A. Impellitteri & Jorge W. Santo Domingo

  3. Department of Earth and Environmental Engineering, Columbia University, 500 West 120th Street, New York, NY, 10027, USA

    Kartik Chandran

Authors
  1. Vikram Kapoor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kartik Chandran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher A. Impellitteri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jorge W. Santo Domingo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jorge W. Santo Domingo.

Additional information

Responsible editor: Gerald Thouand

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kapoor, V., Li, X., Chandran, K. et al. Use of functional gene expression and respirometry to study wastewater nitrification activity after exposure to low doses of copper. Environ Sci Pollut Res 23, 6443–6450 (2016). https://doi.org/10.1007/s11356-015-5843-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11356-015-5843-2

Keywords

Search

Navigation