Skip to main content
SpringerLink
Log in

Comprehensive analyses of functional bacteria and genes in a denitrifying EGSB reactor under Cd(II) stress

  • Environmental biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A bench-scale expanded granular sludge bioreactor (EGSB) was continuously operated to treat synthesized high-nitrate industrial wastewater with increasing bivalent cadmium (Cd(II)) stress. The bioreactor showed nearly complete nitrate removal regardless of Cd(II) loadings, while nitrite accumulated in the effluent when influent Cd(II) loading was over 64 mg/L. Mi-seq sequencing of 16S rRNA gene amplicons elucidated that denitrifiers had decreasing abundances while biodiversity showed increasing trend as the Cd(II) loading increased. In the bioreactor, genera Halomonas, Thauera, Pseudomonas, and Zoogloea played major roles in the denitrification under lower Cd(II) loadings (< 32 mg/L), while Halomonas sp. KM-1 and Halomonas sp. BC04 acted as the crucial Cd-resistant denitrifiers under 128 mg/L Cd(II) loading. Metagenomic analyses and real-time quantitative PCR consistently indicated that napA encoding nitrate reductase was the predominant denitrifying gene, that could be mainly functioning on the efficient nitrate removal. Statistical analyses revealed the significantly positive correlation between Halomonas and nirS gene, both of which were functionally responsible for nitrite reduction. The obtained results may be practically useful for regulation and optimization of the biological processes to treat industrial wastewater containing high levels of nitrate and Cd(II).

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

  • Almeida CM, Mucha AP, da Silva MN, Monteiro M, Salgado P, Necrasov T, Magalhaes C (2014) Salt marsh plants as key mediators on the level of cadmium impact on microbial denitrification. Environ Sci Pollut Res Int 21(17):10270–10278

    Article  CAS  PubMed  Google Scholar 

  • Amoozegar MA, Ghazanfari N, Didari M (2012) Lead and cadmium bioremoval by Halomonas sp., an exopolysaccharide-producing halophilic bacterium. Prog Biol Sci 2(1):1–11

    Google Scholar 

  • Aryal M, Liakopoulou-Kyriakides M (2015) Bioremoval of heavy metals by bacterial biomass. Environ Monit Assess 187(1):4173

    Article  CAS  PubMed  Google Scholar 

  • Bianucci E, Fullana C, Furlan A, Castro S (2013) Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium. Enzym Microb Technol 53(5):345–350

    Article  CAS  Google Scholar 

  • Chen C, Ren N, Wang A, Yu Z, Lee D-J (2008) Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor. Appl Microbiol Biotechnol 78(6):1057–1063

    Article  CAS  PubMed  Google Scholar 

  • Chen H-B, Wang D-B, Li X-M, Yang Q, Luo K, Zeng G-M, Tang M-M (2014) Effects of Cd(II) on wastewater biological nitrogen and phosphorus removal. Chemosphere 117:27–32

    Article  CAS  PubMed  Google Scholar 

  • Colussi I, Cortesi A, Della Vedova L, Gallo V, Robles FC (2009) Start-up procedures and analysis of heavy metals inhibition on methanogenic activity in EGSB reactor. Bioresour Technol 100(24):6290–6294

    Article  CAS  PubMed  Google Scholar 

  • Fajardo C, Gil-Diaz M, Costa G, Alonso J, Guerrero AM, Nande M, Lobo MC, Martin M (2015) Residual impact of aged nZVI on heavy metal-polluted soils. Sci Total Environ 535:79–84

    Article  CAS  PubMed  Google Scholar 

  • Fernandez-Nava Y, Maranon E, Soons J, Castrillon L (2008) Denitrification of wastewater containing high nitrate and calcium concentrations. Bioresour Technol 99(17):7976–7981

    Article  CAS  PubMed  Google Scholar 

  • Geets J, De Cooman M, Wittebolle L, Heylen K, Vanparys B, De Vos P, Verstraete W, Boon N (2007) Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Appl Microbiol Biotechnol 75(1):211–221

    Article  CAS  PubMed  Google Scholar 

  • Giblin A, Tobias C, Song B, Weston N, Banta G, Rivera-Monroy V (2013) The importance of dissimilatory nitrate reduction to ammonium (DNRA) in the nitrogen cycle of coastal ecosystems. Oceanography 26(3):124–131

    Article  Google Scholar 

  • González-Domenech CM, Martínez-Checa F, Béjar V, Quesada E (2010) Denitrification as an important taxonomic marker within the genus Halomonas. Syst Appl Microbiol 33(2):85–93

    Article  PubMed  CAS  Google Scholar 

  • Gouia H, Habib Ghorbal M, Meyer C (2000) Effects of cadmium on activity of nitrate reductase and on other enzymes of the nitrate assimilation pathway in bean. Plant Physiol Biochem 38(7):629–638

    Article  CAS  Google Scholar 

  • Gumaelius L, Smith EH, Dalhammar G (1996) Potential biomarker for denitrification of wastewaters: effects of process variables and cadmium toxicity. Water Res 30(12):3025–3031

    Article  CAS  Google Scholar 

  • Guo G-X, Deng H, Qiao M, Yao H-Y, Zhu Y-G (2013) Effect of long-term wastewater irrigation on potential denitrification and denitrifying communities in soils at the watershed scale. Environ Sci Technol 47(7):3105–3113

    Article  CAS  PubMed  Google Scholar 

  • Guo XC, Miao Y, Wu B, Ye L, Yu HY, Liu S, Zhang XX (2015) Correlation between microbial community structure and biofouling as determined by analysis of microbial community dynamics. Bioresour Technol 197:99–105

    Article  CAS  PubMed  Google Scholar 

  • Hawari AH, Mulligan CN (2006) Biosorption of lead(II), cadmium(II), copper(II) and nickel(II) by anaerobic granular biomass. Bioresour Technol 97(4):692–700

    Article  CAS  PubMed  Google Scholar 

  • Hinojosa MB, Carreira JA, García-Ruíz R, Dick RP (2004) Soil moisture pre-treatment effects on enzyme activities as indicators of heavy metal-contaminated and reclaimed soils. Soil Biol Biochem 36(10):1559–1568

    Article  CAS  Google Scholar 

  • Holtan-Hartwig L, Bechmann M, Risnes Høyås T, Linjordet R, Reier Bakken L (2002) Heavy metals tolerance of soil denitrifying communities: N2O dynamics. Soil Biol Biochem 34(8):1181–1190

    Article  CAS  Google Scholar 

  • Jia S, Wang Z, Zhang XX, Liu B, Li WX, Cheng SP (2013) Metagenomic analysis of cadmium and copper resistance genes in activated sludge of a tannery wastewater treatment plant. J Environ Biol 34(2):375–380

    PubMed  Google Scholar 

  • Jia S, Shi P, Hu Q, Li B, Zhang T, Zhang XX (2015) Bacterial community shift drives antibiotic resistance promotion during drinking water chlorination. Environ Sci Technol 49(20):12271–12279

    Article  CAS  PubMed  Google Scholar 

  • Jia S, Zhang XX, Miao Y, Zhao YT, Ye L, Li B, Zhang T (2017) Fate of antibiotic resistance genes and their associations with bacterial community in livestock breeding wastewater and its receiving river water. Water Res 124:259–268

    Article  CAS  PubMed  Google Scholar 

  • Ju F, Lau F, Zhang T (2017) Linking microbial community, environmental variables, and methanogenesis in anaerobic biogas digesters of chemically enhanced primary treatment sludge. Environ Sci Technol 51(7):3982–3992

    Article  CAS  PubMed  Google Scholar 

  • Kawata Y, Kawasaki K, Shigeri Y (2012) Draft genome sequence of Halomonas sp. strain KM-1, a moderately halophilic bacterium that produces the bioplastic poly (3-hydroxybutyrate). J Bacteriol 194(10):2738–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kristiansson E, Fick J, Janzon A, Grabic R, Rutgersson C, Weijdegard B, Soderstrom H, Larsson DGJ (2011) Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS One 6(2):e17038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li W, Zheng P, Wang L, Zhang M, Lu H, Xing Y, Zhang J, Wang R, Song J, Ghulam A (2013) Physical characteristics and formation mechanism of denitrifying granular sludge in high-load reactor. Bioresour Technol 142:683–687

    Article  CAS  PubMed  Google Scholar 

  • Li S, Xu Q, Ma B, Guo L, She Z, Zhao Y, Gao M, Jin C, Dong J, Wan Y (2018) Performance evaluation and microbial community of a sequencing batch reactor under divalent cadmium (Cd (II)) stress. Chem Eng J 336:325–333

    Article  CAS  Google Scholar 

  • Liao R, Shen K, Li A-M, Shi P, Li Y, Shi Q, Wang Z (2013) High-nitrate wastewater treatment in an expanded granular sludge bed reactor and microbial diversity using 454 pyrosequencing analysis. Bioresour Technol 134:190–197

    Article  CAS  PubMed  Google Scholar 

  • Ma L, Xia Y, Li B, Yang Y, Li L-G, Tiedje JM, Zhang T (2015) Metagenomic assembly reveals hosts of antibiotic resistance genes and the shared resistome in pig, chicken, and human feces. Environ Sci Technol 50(1):420–427

    Article  CAS  PubMed  Google Scholar 

  • Magalhães C, Costa J, Teixeira C, Bordalo AA (2007) Impact of trace metals on denitrification in estuarine sediments of the Douro River estuary, Portugal. Mar Chem 107(3):332–341

    Article  CAS  Google Scholar 

  • Manasi Rajesh V, Krishna Kumar AS, Rajesh N (2014) Biosorption of cadmium using a novel bacterium isolated from an electronic industry effluent. Chem Eng J 235:176–185

    Article  CAS  Google Scholar 

  • Miao Y, Liao R, Zhang XX, Wang Y, Wang Z, Shi P, Liu B, Li A-M (2015) Metagenomic insights into Cr (VI) effect on microbial communities and functional genes of an expanded granular sludge bed reactor treating high-nitrate wastewater. Water Res 76:43–52

    Article  CAS  PubMed  Google Scholar 

  • Mudhoo A, Kumar S (2013) Effects of heavy metals as stress factors on anaerobic digestion processes and biogas production from biomass. Int J Environ Sci Technol 10(6):1383–1398

    Article  CAS  Google Scholar 

  • Osaka T, Shirotani K, Yoshie S, Tsuneda S (2008) Effects of carbon source on denitrification efficiency and microbial community structure in a saline wastewater treatment process. Water Res 42(14):3709–3718

    Article  CAS  PubMed  Google Scholar 

  • Penton CR, Johnson TA, Quensen IIIJF, Iwai S, Cole JR, Tiedje JM (2013) Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter. Front Microbiol 4(279):1–17

    Google Scholar 

  • 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(23):7537–7541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shapleigh JP (2013) The prokaryotes. Springer, Berlin, pp 405–425

    Book  Google Scholar 

  • Smith CJ, Nedwell DB, Dong LF, Osborn AM (2007) Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments. Appl Environ Microbiol 73(11):3612–3622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sobolev D, Begonia M (2008) Effects of heavy metal contamination upon soil microbes: lead-induced changes in general and denitrifying microbial communities as evidenced by molecular markers. Int J Environ Res Public Health 5(5):450–456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Srinandan C, Shah M, Patel B, Nerurkar AS (2011) Assessment of denitrifying bacterial composition in activated sludge. Bioresour Technol 102(20):9481–9489

    Article  CAS  PubMed  Google Scholar 

  • Tsai Y-P, You S-J, Pai T-Y, Chen K-W (2006) Effect of Cd(II) on different bacterial species present in a single sludge activated sludge process for carbon and nutrient removal. J Environ Eng 132(2):173–180

    Article  CAS  Google Scholar 

  • Vig K (2003) Bioavailability and toxicity of cadmium to microorganisms and their activities in soil: a review. Adv Environ Res 8(1):121–135

    Article  CAS  Google Scholar 

  • Waldron PJ, Wu L, Nostrand JDV, Schadt CW, He Z, Watson DB, Jardine PM, Palumbo AV, Hazen TC, Zhou J (2009) Functional gene array-based analysis of microbial community structure in groundwaters with a gradient of contaminant levels. Environ Sci Technol 43(10):3529–3534

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Zhang XX, Lu X, Liu B, Li Y, Long C, Li A (2014) Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing. PLoS One 9(11):e113603

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yang Y, Yu K, Xia Y, Lau FTK, Tang DTW, Fung WC, Fang HHP, Zhang T (2014) Metagenomic analysis of sludge from full-scale anaerobic digesters operated in municipal wastewater treatment plants. Appl Microbiol Biotechnol 98(12):5709–5718

    Article  CAS  PubMed  Google Scholar 

  • Ye L, Shao MF, Zhang T, Tong AH, Lok S (2011) Analysis of the bacterial community in a laboratory-scale nitrification reactor and a wastewater treatment plant by 454-pyrosequencing. Water Res 45(15):4390–4398

    Article  CAS  PubMed  Google Scholar 

  • Yu K, Zhang T (2013) Construction of customized sub-databases from NCBI-nr database for rapid annotation of huge metagenomic datasets using a combined BLAST and MEGAN approach. PLoS One 8(4):e59831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang T, Shao M-F, Ye L (2011) 454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. ISME J 6(6):1137–1147

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhou Z, Zheng Y, Shen J, Zhang L, Liu Y, He J (2012) Responses of activities, abundances and community structures of soil denitrifiers to short-term mercury stress. J Environ Sci 24(3):369–375

    Article  CAS  Google Scholar 

  • Zou G, Ylinen A, Di Capua F, Papirio S, Lakaniemi AM, Puhakka J (2013) Impact of heavy metals on denitrification of simulated mining wastewaters. Adv Mater Res 825:500–503

    Article  CAS  Google Scholar 

  • Zou G, Papirio S, Ylinen A, Di Capua F, Lakaniemi AM, Puhakka JA (2014) Fluidized-bed denitrification for mine waters. Part II: effects of Ni and Co. Biodegradation 25(3):417–423

    CAS  PubMed  Google Scholar 

  • Zou G, Papirio S, van Hullebusch ED, Puhakka JA (2015) Fluidized-bed denitrification of mining water tolerates high nickel concentrations. Bioresour Technol 179:284–290

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the High Performance Computing Center (HPCC) of Nanjing University for the help of computation.

Funding

This study was financially supported by the National Key Technology Support Program of China (2014BAC08B04), the Jiangsu Science Foundation for Distinguished Young Scholars (BK20150019), and the Six Talent Peaks Project in Jiangsu Province (2015-JNHB-001).

Author information

Authors and Affiliations

  1. State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 163 Xianlin Road, Nanjing, 210023, China

    Yu Miao, Xu-Xiang Zhang, Shuyu Jia, Runhua Liao & Aimin Li

  2. School of Materials Science and Engineering, Jingdezhen Ceramic Institute, Jingdezhen, 333403, China

    Runhua Liao

Authors
  1. Yu Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xu-Xiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuyu Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Runhua Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aimin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xu-Xiang Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(PDF 3588 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Miao, Y., Zhang, XX., Jia, S. et al. Comprehensive analyses of functional bacteria and genes in a denitrifying EGSB reactor under Cd(II) stress. Appl Microbiol Biotechnol 102, 8551–8560 (2018). https://doi.org/10.1007/s00253-018-9228-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-018-9228-6

Keywords

Search

Navigation