Enrichment of anodophilic nitrogen fixing bacteria in a bioelectrochemical system

doi:10.1016/j.watres.2014.06.046

Water Research

Volume 64, 1 November 2014, Pages 73-81

https://doi.org/10.1016/j.watres.2014.06.046 Get rights and content

Highlights

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Nitrogen deficient wastewater was treated using a bioelectrochemical system.
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Good carbon removal efficiency was achieved.
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Anode-depended nitrogen fixation was proven using acetylene reduction assay.
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Microbial analysis confirmed the presence of nitrogen fixing bacterial genera.

Abstract

We demonstrated the ability of a bio-anode to fix dinitrogen (N₂), and confirmed that diazotrophs can be used to treat N-deficient wastewater in a bioelectrochemical system (BES). A two-compartment BES was fed with an N-deficient medium containing glucose for >200 days. The average glucose and COD removal at an anodic potential of +200 mV vs. Ag/AgCl was 100% and 76%, respectively. Glucose removal occurred via fermentation under open circuit (OC), with acetate as the key byproduct. Closing circuit remarkably reduced acetate accumulation, suggesting the biofilm could oxidise acetate under N-deficient conditions. Nitrogen fixation required an anode and glucose; removing either reduced N₂ fixation significantly. This suggests that diazotroph utilised glucose directly at the anode or indirectly through syntrophic interaction of an N₂-fixing fermenter and an anodophile. The enriched biofilm was dominated (68%) by the genus Clostridium, members of which are known to be electrochemically active and capable of fixing N₂.

Graphical abstract

Introduction

Industrial wastewaters, including wastewater produced from pulp and paper industries, are carbon (C) rich but nitrogen (N) deficient (Pratt et al., 2007, Pokhrel and Viraraghavan, 2004). To enable efficient biological treatment, a C:N ratio of 100:5 in the raw influent is usually recommended (Peng et al., 2003, Slade et al., 2011). Hence, external supplementation of N (as ammonium or nitrate) is needed to treat N-deficient wastewater (Dennis et al., 2004). N supplementation incurs costs, and intense monitoring is required to prevent discharge of excess N to the environment (Gauthier et al., 2000).

As an alternative to supplementing N, the use of diazotrophic (N₂-fixing) bacteria has been proposed as a method for treating N-deficient wastewater in activated sludge systems (Pratt et al., 2007, Gauthier et al., 2000). N₂-fixing bacteria are capable of converting atmospheric nitrogen (N₂) to ammonia (NH₃) as a means of supplementing N requirements for growth (Nair, 2010). Biological N₂ fixation is catalysed by the nitrogenase enzyme complex, and the reduction of N₂ to NH₃ takes place according to reaction (1) (Nair, 2010):N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16Pi

Although N₂-fixing bacteria could be used in activated sludge processes to oxidise carbon in N-deficient wastewater, the widespread use of this approach has not been possible because nitrogenase is irreversibly inhibited by oxygen (O₂), and conventional activated sludge processes require aeration to facilitate oxidation of organic carbon (Nair, 2010). To prevent O₂ inhibition of nitrogenase, diazotrophs often secrete extracellular polymeric substances (EPS; also known as slime) to limit O₂ diffusion into cells (Nair, 2010). Excessive EPS production can cause sludge bulking, resulting in poor solid/liquid separation and reduced effluent quality (Peng et al., 2003). Hence, the use of N₂-fixing activated sludge processes to oxidise carbon in N-deficient wastewater is problematic.

One approach to eliminating the negative impact of O₂ on N₂ fixation is to combine N₂-fixing microorganisms with bioelectrochemical systems (BESs). A typical BES consists of an anode and a cathode chamber (Logan et al., 2008). The anodic chamber facilitates the growth of microorganisms (anode respiring bacteria; ARB) under anaerobic conditions, using the electrode (anode) as the sole electron acceptor. If the ARB are diazotrophs, oxidation of N-deficient wastewater in the absence of O₂ becomes feasible because a solid electrode (not O₂) is the final electron acceptor. The anode potential regulates the thermodynamics of bacterial metabolism. Therefore, N₂ fixation in diazotrophic ARB is likely to be regulated by the anodic potential. Consequently, inhibitory effects of O₂ on N₂ fixation may be eliminated because of the maintenance of anaerobic conditions in the anode chamber. The electrons donated by the ARB flow to the cathode via an external circuit, where they combine with protons and O₂ to form water (Logan et al., 2008).

Current knowledge of diazotrophic ARB (DARB) is limited. Although potential diazotrophs including Azoarcus, Clostridium and Geobacter have been reported in association with anodes of microbial fuel cells (MFCs) under N supplemented conditions (Phung et al., 2004, Kim et al., 2004), it is unclear whether these bacteria met their N requirements via fixation of atmospheric N₂. Belleville et al. (2011) and Clauwaert et al. (2007) operated BESs to treat N-deficient wastewater, but did not provide direct experimental evidence of N₂ fixation, although they assumed that this was how the N requirements of the ARB were met, and did not investigate the bacterial diversity in their systems.

The objectives of this study were to: (1) investigate the efficiency of anodic oxidation of an N-deficient wastewater by an enriched microbial biofilm community; (2) elucidate the possible routes of glucose metabolism by the anodic biofilm; (3) assess the influence of anodic current production on N₂ fixation; and (4) characterise the enriched anodic bacterial community using 454 sequencing.

Section snippets

Composition of the N-deficient medium

The synthetic N-deficient medium used in this study contained glucose as the sole source of carbon and energy, and represents wastewater characteristic of pulp and paper, and sugar refining industries. The medium contained (per litre of DI water): MgSO₄·7H₂O (25 mg), CaCl₂·2H₂O (25 mg), glucose monohydrate (374−1684 mg), KH₂PO₄ (2300 mg), K₂HPO₄ (5750 mg) and 0.40 mL of trace element solution. The trace element solution contained (per litre): nitrilotriacetic acid (5000 mg), H₃BO₃ (310 mg), FeSO

Establishment of the N₂-fixing anodophilic biofilm

The BES performance during 200 days of operation is summarised in Fig. 2. Measurable current recorded after approximately 20 days of operation indicated the enrichment of an N₂-fixing anodophilic biofilm. Establishment of the active anodophilic biofilm led to a current increase, and the cathodic potential decreased (Fig. 2). At an organic loading rate (as COD) of approximately 4 mg L⁻¹ h⁻¹, a current of approximately 100 mA was recorded.

The removal of glucose from the anodic compartment

Conclusions

We confirmed that N-deficient wastewater could be treated using a BES anode, provided the first experimental evidence of nitrogen fixation, and characterised biofilm ARB in a BES treating N-deficient wastewater. Our results suggest that:

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Good carbon removal efficiencies were achieved under anaerobic conditions
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The N₂-fixing activity was anode- and glucose-dependent (removing either reduced N₂-fixing activity).
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The presence of N₂-fixing bacterial genera was demonstrated using 454 pyrosequencing.
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Acknowledgements

This project was funded by the CSIRO Water for a Healthy Country Flagship. We acknowledge the Australian Commonwealth Government for an Australian Postgraduate Award provided through the University of Western Australia to the first author. We are grateful to Dr. Trevor Bastow and Ms. Yasuko Geste (CSIRO, Land and Water) for their assistance with the acetylene measurement. We also thank Ms. Annachiara Codello for her assistance with the DNA extraction, and Dr. Joy Vadhanabhuti and Assistant

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Enrichment of anodophilic nitrogen fixing bacteria in a bioelectrochemical system

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Composition of the N-deficient medium

Establishment of the N2-fixing anodophilic biofilm

Conclusions

Acknowledgements

Bioresour. Technol.

Curr. Opin. Biotechnol.

Trends Microbiol.

Anaerobe

FEMS Microbiol. Lett.

Sci. Total Environ.

Process Biochem.

Standard Methods for the Examination of Water and Wastewater

Cellular Energy Metabolism and its Regulation

Environmentally Friendly Production of Pulp and Paper

Influence of nitrogen limitation on performance of a microbial fuel cell

Water Sci. Technol.

PyNAST: a flexible tool for aligning sequences to a template alignment

Bioinformatics

Ano-cathodophilic biofilm catalyzes both anodic carbon oxidation and cathodic denitrification

Environ. Sci. Technol.

Affinity of microbial fuel cell biofilm for the anodic potential

Environ. Sci. Technol.

Open air biocathode enables effective electricity generation with microbial fuel cells

Environ. Sci. Technol.

The performance of a nitrogen-fixing SBR

Water Sci. Technol.

Establishment of the N₂-fixing anodophilic biofilm