Elsevier

Water Research

Volume 76, 1 June 2015, Pages 43-52
Water Research

Metagenomic insights into Cr(VI) effect on microbial communities and functional genes of an expanded granular sludge bed reactor treating high-nitrate wastewater

https://doi.org/10.1016/j.watres.2015.02.042Get rights and content

Highlights

  • Nearly complete nitrate removal by anaerobic bioreactors with influent Cr(VI) at 120 mg/L.

  • Application of high-throughput sequencing technologies to characterize microbial community structure and function.

  • Predominance of Thauera and Halomonas in the bioreactor fed with Cr(VI).

  • Different responses of Thauera and Halomonas to Cr(VI) stress.

Abstract

In this study, a lab-scale expanded granular sludge bed reactor was continuously operated to treat high-nitrate wastewater containing different concentrations of hexavalent chromium (Cr(VI)). Nearly complete nitrate removal was achieved even at 120 mg/L influent Cr(VI). Pyrosequencing of 16S rRNA gene showed that Cr(VI) decreased the biodiversity of the bacterial community and potential denitrifiers. Proteobacteria dominated in the bioreactor, and Betaproteobacteria had increased abundance after Cr(VI) feeding. Thauera and Halomonas were the two predominant genera in the bioreactor fed with Cr(VI), demonstrating opposite responses to the Cr(VI) stress. Metagenomic analysis indicated that Cr(VI) feeding posed no obvious effect on the overall function of the bacterial community, but altered the abundance of specific denitrifying genes, which was evidenced by quantitative real time PCR. This study revealed that Halomonas mainly contributed to the denitrification under no or low Cr(VI) stress, while Thauera played a more important role under high Cr(VI) stress.

Introduction

In some industrial processes, such as alloy and stainless steel production, leather tanning, textile dyeing and metal finishing, extensive application of chromium results in high concentration of both chromium and nitrate in the produced wastewater (Sahinkaya et al., 2013). Microbial denitrification is a reliable and efficient method for high-nitrate wastewater treatment, and chromium at low level can simulate microbial growth (Gikas and Romanos, 2006), but high concentration of Cr(VI) can inhibit microbial growth (Vaiopoulou and Gikas, 2012) by altering enzyme conformation and blocking essential functional groups (Zou et al., 2014), and affect their denitrifying capacity (Colussi et al., 2009).

As an anaerobic technology, expanded granular sludge bed (EGSB) reactor has higher settling capability of granules biomass, and is also capable of diluting the wastewater by recirculation to improve substrate diffusion on the liquid/granule interface (Liao et al., 2013). The bioreactor is often used to treat high concentration of organic wastewater containing toxic pollutants, e.g. Cr(VI) (Sheng et al., 2011).

Previous studies mainly focused on the performance of bioreactors on nitrate removal in existence of chromium (Cecen et al., 2010, Sahinkaya and Kilic, 2014), while limited information is available about the roles of the bacterial community in nitrogen removal by EGSB. Several conventional molecular methods have been widely used to determine bacterial species and functional genes responsible for the nitrogen removal, e.g. polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (Sahinkaya et al., 2013), terminal restriction fragment length polymorphism (Osaka et al., 2008) and DNA cloning (Ye et al., 2011). The methods are considered specific, but low-throughput, which cannot provide comprehensive insights into the structural and functional changes of the microbial communities. Recent studies have shown that high-throughput sequencing technologies including amplicon and shotgun sequencing have enough sequencing depth and high accuracy to cover complex bacterial communities (Zhang et al., 2012, Zhou et al., 2013a). Pyrosequencing of 16S rRNA gene amplicon has been widely used to characterize microbial structure (Zhou et al., 2011a), and annotation of shotgun reads generated by Illumina high-throughput sequencing of environmental genomes is considered reliable method for comprehensive exploration of functional genes in the environments (Hemme et al., 2010, Ye et al., 2012, Yu and Zhang, 2012).

Cr(VI) shock can alter microbial community structure and function, subsequently affecting denitrification performance of bioreactors, but how denitrifiers resist the Cr(VI) stress through community structure shift remains unknown. To test the hypothesis, this study investigated the effects of Cr(VI) shock on the denitrification capacity of an EGSB reactor treating high-nitrate wastewater, and attempted to uncover the underlying molecular ecological mechanisms by characterizing the alterations of functional bacteria and genes using high-throughput sequencing and metagenomic analysis. The results may help to extend our knowledge about the microbial denitrification processes in EGSB reactors under Cr(VI) stress, and might be technically useful to regulate and optimize treatment processes of the industrial wastewater containing high concentrations of nitrate and Cr(VI).

Section snippets

Bioreactor operation and chemical analysis

The column of the EGSB reactor had a working volume of 1.74 L with inter diameter of 60 mm and height of 91 cm. Table S1 shows the components of the synthetic raw wastewater of the bioreactor, which was prepared following Liao et al. (2013). During the start-up period, the reactor was operated with increasing influent NO3N concentration from 200 to 2000 mg/L, and each concentration phase lasted for about 7 d until the bioreactor reached stable state (Table S2). The liquid up-flow velocity of

Performance of the EGSB reactor

After start-up, the bioreactor showed nearly complete denitrification with influent nitrate concentration at 2000 mg/L, although influent Cr(VI) concentration increased to 40 mg/L (Fig. 1). However, when influent Cr(VI) concentration reached 80 mg/L, effluent nitrite concentration gradually increased (Fig. 1). During phase 5 with influent Cr(VI) at 120 mg/L, both denitrification and Cr(VI) reduction capacity were obviously inhibited since effluent nitrite and Cr(VI) concentrations increased to

Discussion

This study showed that nearly complete nitrate removal was achieved during the operational period of the EGSB reactor. Gikas and Romanos (2006) reported that Cr(VI) at <25 mg/L could simulate microbial growth, but Sahinkaya and Kilic (2014) have recently indicated that 50 mg/L Cr(VI) can inhibit heterotrophic and elemental-sulfur-based autotrophic denitrification. In this study, the bacterial cultivation and biomass accumulation in the EGSB reactor might reduce the adverse effect of the toxic

Conclusion

  • Joint use of metagenomic analyses and molecular methods can provide comprehensive insights into Cr(VI) effects on microbial community structure and function of the EGSB reactor treating high-nitrate wastewater.

  • Cr(VI) feeding can cause bacterial community shift and biodiversity reduction in the EGSB reactors, and alter the abundance of the specific denitrifying genes.

  • Thauera and Halomonas are the predominant genera in the denitrifying bioreactors fed with Cr(VI). Thauera may play a more

Acknowledgements

This study was financially supported by the Major Science and Technology Program of China for Water Pollution Control and Treatment (2012ZX07101-002-004 and 2012ZX07506-004-004), National Natural Science Foundation of China (51378252), Jiangsu Science and Technology Support Program of China (BE2013704) and Environmental Protection Research Foundation of Jiangsu Province (China) (2012045). We also would like to thank the High Performance Computing Center (HPCC) of Nanjing University for the help

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