1 s2.0 S004313542101054X Main
1 s2.0 S004313542101054X Main
1 s2.0 S004313542101054X Main
Water Research
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A R T I C L E I N F O A B S T R A C T
Keywords: The present nitrogen fixation industry is usually energy-intensive and environmentally detrimental. Therefore, it
Green nitrogen fixation is appealing to find alternatives. Here, we achieved both a synchronized biological nitrogen fixation and electric
Extracellular electron transfer energy production by using Geobacter sulfurreducens in a microbial electrochemical system. The results showed
Exoelectrogenic diazotroph
that G. sulfurreducens was able to fix nitrogen depending on anode respiration, producing a maximum current
Bioelectrochemical system
density of 0.17 ± 0.015 mA cm− 2 and a nitrogen-fixing activity of ca. 0.78 μmol C2H4 mg protein− 1 h− 1, thereby
N-deficient wastewater treatment
achieving a net total nitrogen-fixing rate of ca. 5.6 mg L− 1 day− 1. Specifically, nitrogen fixation did not impair
coulombic efficiency. Transcriptomic and metabolic analyses demonstrated that anode respiration provided
sufficient energy to drive nitrogen fixation, and in turn nitrogen fixation promoted anode respiration of the cell
by increasing acetate catabolism but reducing acetate anabolism. Furthermore, we showed that G. sulfurreducens
could be supplied in a bioelectrochemical system for N-deficient wastewater treatment to relieve N-deficiency
stress contributing to the formation of an electroactive biofilm, thereby simultaneously achieving nitrogen fix
ation, current generation and dissoluble organic carbon removal. Our study revealed a synergistic effect between
biological nitrogen fixation and current generation by G. sulfurreducens, providing a green nitrogen fixation
alternative through shifting the nitrogen fixation field from energy consumption to energy production and having
implications for N-deficient wastewater treatment.
1. Introduction diazotrophs (Hoffman et al., 2014; Kuypers et al., 2018). During that
process, cellular catabolism and respiration have been shown to provide
Nitrogen fixation, which involves converting inert N2 to reactive reduction power and energy to drive nitrogen fixation (Dixon and Kahn,
NH3, has been essential for the advancement of modern agriculture and 2004). Therefore, BNF is sustainable and environmentally benign and is
human society. The ever-increasing development of human civilization considered the best choice. Actually, BNF is widely carried out in natural
has aggravated the need for ammonium. Various endeavors have been ecosystems, contributing to over 50% of global nitrogen fixation
carried out to meet the demand (Cherkasov et al., 2015; Mus et al., (Fowler et al., 2013).
2018). Among them, the Haber–Bosch process has been widely applied, Various efforts have been devoted to harvesting BNF. For example,
contributing to approximately 90% of global artificial NH3 production Ortiz-Marquez et al. (2012) applied different mutagenesis strategies by
(Fowler et al., 2013). However, the process is both carbon- and point mutation of the glutamine synthase gene or deletion of the
energy-intensive, running at elevated temperatures (~x223C 500 ◦ C) nitrogenase transcription antiactivator nifL gene, and increased the
and pressures (150–300 bar), accounting for 1.4% of global carbon di ammonium secretion of azotobacter; Liu et al. (2017) sought an
oxide emissions and consuming 1% of the world’s total energy pro inorganic-biological hybrid combining inorganic catalysts to split water
duction (Kandemir et al., 2013; Milton et al., 2017). Therefore, it is for H2 generation and the H2-oxidizing diazotroph to fix N2 and achieved
appealing to find alternatives (Vicente and Dean, 2017). Biological ni the synthesis of NH3 under ambient conditions; and Ospina-Betancourth
trogen fixation (BNF) is a process catalyzed by nitrogenase in et al. (2020) employed a culture enrichment procedure and selected
* Corresponding authors.
E-mail addresses: [email protected] (X. Liu), [email protected] (S. Zhou).
https://doi.org/10.1016/j.watres.2021.117860
Received 18 May 2021; Received in revised form 4 October 2021; Accepted 8 November 2021
Available online 11 November 2021
0043-1354/© 2021 Elsevier Ltd. All rights reserved.
X. Jing et al. Water Research 208 (2022) 117860
superior N2-fixing microbial consortia, achieving a nitrogen-fixing effi supplied with 12 mM sodium acetate. To perform the BNF test, NH4Cl
ciency comparable to that of existing nitrogen-fixing technologies. was eliminated from the anolyte. The reactor was autoclaved at 121 ◦ C
Specifically, Ortiz-Medina et al. (2019) used anaerobic microbial elec for 30 min and then purged with sterile N2–CO2 (80:20) for at least 30
trolysis cells (MECs) inoculated with mixed microbial species not only to min to remove oxygen and to saturate N2. Geobacter sulfurreducens strain
achieve BNF but also generate electric energy. Here, the MECs selected KN400 was initially cultured in N-deficient NBAF medium (without
an anode consortium which was predominated by Geobacter species and NH4Cl) as previously reported (Liu et al., 2019). To start the process, 5
generated a maximum nitrogen fixation activity comparable to that of mL of bacterial cells in logarithmic phase was inoculated into the anode
model aerobic diazotrophs. Geobacter species are strictly anaerobic chamber, in which the anode was polarized at 0.3 V (vs. SCE) with a
heterotrophic microorganisms and generate energy by performing multichannel electrochemical workstation (CHI 1000 C, Shanghai,
extracellular electron transfer to respire extracellular electron acceptors China). The current was recorded continuously and normalized to the
(Lovley et al., 2011). They are the most efficient exoelectrogens known maximum projected area of the anode. All experiments were conducted
and usually dominate in the anode biofilms of MECs (Commault et al., in triplicate at 30 ◦ C, and the mean values were reported. All test groups
2013; Zakaria et al., 2018). In addition, nearly all Geobacter species have were listed in supplementary Table S1.
been shown to contain nitrogenase-encoding genes, and were able to fix
nitrogen coupled with the respiration of Fe(III) oxides (Ueki and Lovley, 2.2. Quantification of the total nitrogen, protein and dissolved organic
2010; Rӧling, 2014). Therefore, it is suspected that Geobacter species carbon
play a key part in both current generation and nitrogen fixation in that
MECs (Ortiz-Medina et al., 2019). Moreover, in another study, electro The nitrogen fixation rate was evaluated by calculating the increase
chemically active diazotrophs from Clostridium species were abundant in in total nitrogen (N total), including dissolved nitrogen in the anolyte and
the anode biofilm involved in current generation and BNF (Wong et al., biomass on the anode. To measure these parameters, destructive sam
2014). Obviously, these two studies suggested the possibility of using pling was carried out. Briefly, the whole anode biofilm was scraped and
electrochemically active diazotrophs to achieve a green nitrogen fixa suspended in the anolyte. The cells were lysed with an ultrasonic ho
tion that performs simultaneous electric energy generation and BNF. mogenizer (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ningbo,
However, this had previously not been corroborated. Furthermore, the China) at a frequency of 30 kHz and a potential of 180 W for 30 min. The
relationship between current generation and nitrogen fixation appears concentration of total nitrogen was determined directly using the per
mysterious, considering the following two issues. (i) For one biological sulfate digestion method (Hach kits 2672245, Beijing, China) (Liu et al.,
nitrogen gas fixation reaction to be completed, eight electrons must be 2017). To measure the protein concentration, the lysate was analyzed
consumed, and current generation also consumes electrons. Therefore, using the microBCA protein assay (Micro BCA protein assay kit, Thermo
these two processes are competitive. However, (ii) current generation Fisher Scientific, Rockford, USA) (Chen et al., 2017). The dissolved
drives the formation of a transmembrane proton gradient required for organic carbon (DOC) was measured using a multi 3100 analyzer (multi
energy generation (ATP), which supports nitrogen fixation. Therefore, N/C 3100, Analytik Jena, Germany).
in turn, these two processes are cooperative in that BNF should depend
on current generation.
To clarify the possibility and mechanism of simultaneous BNF and 2.3. NAD(P)H/NAD(P)+ and ATP quantification
current generation of electrochemically active diazotrophs, in this study,
a representative Geobacter species, Geobacter sulfurreducens strain Cells on the anode were collected after the current reached the
KN400, was selected since it generated the highest current in compari maximum. To determine the concentrations of ATP, NADH/NAD+ and
son to all other bacterial species and had been shown to fix nitrogen NADPH/NADP+, cells were first washed three times with 50 mM PBS
efficiently (Methé et al., 2005; Yi et al., 2009). G. sulfurreducens strain (pH 7.0) and then resuspended in PBS. The concentration of intracellular
KN400 was inoculated into the anode chamber of an N-deficient mi ATP was measured using the Enhanced ATP Assay Kit (Beyotime
crobial fuel cell, with the anode acting as the electron acceptor to induce Biotechnology, China) following the manufacturer’s instructions. The
current generation. The results showed that G. sulfurreducens fixed ni concentrations of intracellular NADH/NAD+and NADPH/NADP+were
trogen via anode reduction suggesting an anode respiration-dependent determined using an NAD(H) Quantification Kit (MAK037, Sigma-
BNF, and in return, BNF enhanced the anode respiration (energy Aldrich) and NADP(H) assay kit (BC1105, Solarbio, Beijing, China),
metabolism) of G. sulfurreducens cells. In addition, G. sulfurreducens respectively, as previously reported (Leonardo et al., 1996).
could be supplied as an additive in microbial electrochemical system for
N-deficient wastewater treatment to achieve not only dissoluble organic 2.4. Biological nitrogen fixation characterization
carbon removal but also BNF and current generation. Our study provides
the first direct evidence of synchronizing nitrogen fixation with electric Nitrogenase activity was determined following the acetylene
energy output and has implications for green nitrogen fixation and reduction assay by measuring the consumption of ethylene using gas
N-deficient wastewater treatment. chromatography (Shimadzu, GC-14, Kyoto, Japan) (Chen et al., 2021).
In detail, when the current reached the maximum, the anolyte was
2. Material and methods replaced with anaerobic fresh sterile anolyte and purged with Ar-CO2
(80: 20) for at least 30 min. Thereafter, C2H2 was injected into the anode
2.1. Reactor construction and operation chamber at a final concentration of 10% (v/v gas phase). The concen
tration of C2H4 was monitored by regularly applying 300 μL of head
Three electrodes of two-chambered H-shaped cells with a liquid space sample to the gas chromatograph.
volume of 60 mL and a headspace volume of 90 mL in each chamber The isotope labeling experiment was performed as previously
were assembled as previously described (Jing et al., 2019). The anode described (Chen et al., 2021). Briefly, the microbial fuel cell was purged
and cathode were made of graphite plates (2 × 3 × 0.3 cm) (JingLong with Ar2–CO2 (80: 20) firstly and then the headspace was purged with
15
Special Carbon, Beijing, China), which were polished using sandpaper N2–CO2 (80: 20). After a mature biofilm was formed, cells were
(grit type 400), sonicated for 30 mins, soaked in 1 M HCl overnight and collected by scraping and lyophilized. The 15N (m/z = 29)/14N (m/z =
then rinsed at least five times with Milli-Q water. A saturated calomel 28) ratio was measured by using the Thermo Scientific MAT 253 Plus gas
electrode (SCE) was used as the reference electrode. The electrolyte was bench isotope mass spectrometer (Thermo Fisher Scientific). The N2
freshwater medium (FWNN medium) buffered with carbonate as pre content in the headspace was determined by a robotized sampling and
viously reported (Nevin et al., 2009), except that the anolyte was analysis system (Molstad et al., 2007).
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X. Jing et al. Water Research 208 (2022) 117860
2.5. Biofilm morphology characterization source, G. sulfurreducens was still able to use the anode as an electron
acceptor, producing a current density with a maximum of approximately
The morphology of the anode biofilm was characterized when the 0.17 ± 0.015 mA cm− 2 (Fig. 1A). Accordingly, a rough anode biofilm
bioelectrochemical system produced the maximum current density. with a thickness of ca. 30 µm was formed (Fig. 1B), indicating that
Biofilms were rinsed briefly with sterile 0.9% NaCl, stained using the G. sulfurreducens was able to use nitrogen gas to grow. The nitrogen
LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, USA) for balance was further calculated by measuring the nitrogen consumption
15 min in the dark and then rinsed briefly with sterile 0.9% NaCl to and Ntotal. As indicated in Fig. 1C, the Ntotal increased by 107.79 ± 11.25
remove excess dyes. The 3-D structure of the biofilm was depicted using μmol which is consistent with the amount of nitrogen gas consumed
a confocal laser scanning microscope (CLSM) (Carl Zeiss LSM 880, (56.29 ± 6.98 μmol). These results indicated that G. sulfurreducens was
Germany) (Jing et al., 2019). able to perform BNF on the anode. An 15N isotope labeling experiment
was further performed by supplying 15N2 in the gaseous phase. Peak
2.6. Bacterial community analysis height values of 15N14N and 14N14N were measured and 15N/14N atom
ratio was calculated. Here, a higher 15N/14N ratio represents more 15N
Cells were collected when the current reached the maximum. The being fixed. A natural 15N abundance is usually around 0.366 atom%
total DNA was extracted according to the instructions of the EZNA™ (Shinoda et al., 2019). In contrast, as shown in Fig. 2A, the abundance of
15
Mag-Bind Soil DNA Kit (Omega Biotek, Inc., Norcross, GA, USA). The N increased to 6.574 atom% in the system. Furthermore, as shown in
primers Nobar_341F (CCTACGGGNGGCWGCAG) and Nobar_805R Fig. 2B, the nitrogenase activity of G. sulfurreducens in the system was
(GACTACHVGGGTATCTAATCC) were used to amplify the 16SV3-V4 calculated to be approximately 0.78 ± 0.037 μmol C2H4 mg− 1 protein
region of the 16S rRNA gene for pyrosequencing using a MiSeq h− 1. These results provided direct evidence of simultaneous current
sequencer by Sangon Biotech Co., Ltd., Shanghai, China. The sequencing generation and biological nitrogen fixation on the anode by
data were processed according to a previous study (Dong et al., 2017). G. sulfurreducens. Notably, strain KN400 also generated a current density
with a maximum at approximately 0.17 ± 0.013 mA cm− 2 (Fig. 1A)
when growing in N-sufficient electrolyte, but formed a compact and
2.7. Transcriptome sequencing and analysis
smooth biofilm with a thickness of ca. 28 µM (Fig. 1B). At this point, the
strain neither fixed nitrogen (Figs. 1C and 2A) nor showed nitrogenase
Cells on the anode were collected at the mid-log phase, and were
activity (Fig. 2B). Therefore, it appeared that BNF did not affect current
flash frozen in liquid nitrogen then stored at − 80 ◦ C until needed. The
generation of the G. sulfurreducens fuel cell.
total RNA was extracted using TRIzol reagent (Invitrogen, California,
USA) as previously described (Liu et al., 2021). Transcriptome
sequencing was performed at Novogene Bioinformatics Technology Co., 3.2. Biological nitrogen fixation enhances anode respiration
Ltd., (Tianjin, China). Briefly, mRNA was purified from total RNA by
removing 16S and 23S rRNA and sequenced using the Illumina Novaseq Nitrogen fixation is a reduction process that consumes eight elec
platform. Raw data were quality checked and filtered, and then were trons per reduction of one molecule of nitrogen gas (N2). Therefore, the
processed through in-house perl scripts and then used to map against the two processes of current generation and nitrogen fixation compete for
published reference genome of G. sulfurreducens KN400 the electrons generated from acetate oxidation. The results demon
(NZ_CP002031.1). HTSeq v0.6.1 was used to count the reads numbers strating that BNF did not affect current generation suggested that
mapped to each gene. Fragments per kilobase per million mapped reads G. sulfurreducens used a mechanism to dissipate electrons in a distinct
(FPKM) of each gene was calculated based on the length of the gene and ratio, which balanced energy production and BNF. The electron dissi
reads count mapped to this gene. And then the DESeq R package (1.18.0) pations under N-deficient and N-sufficient conditions were analyzed by
was used to analyze the differential expression. The P values were calculating the coulombic efficiency which represents the ratio of elec
adjusted using the TMM (trimmed mean of M-values) method. trons used for current generation versus the total electrons released by
electron donor oxidation (Liu et al., 2015). As indicated in Table 1,
3. Results strain KN400 catabolized a comparable amount of acetate to generate a
comparable amount of current either under N-deficient or N-sufficient
3.1. Simultaneous biological nitrogen fixation and current generation in conditions in the same time period. Therefore, similar coulombic effi
Geobacter sulfurreducens ciencies of 40.05 ± 9.65% under N-deficient conditions and 38.97 ±
8.52% under N-sufficient conditions were calculated. This result is
A three-electrode two-chambered H-shaped cell was constructed and surprising, considering that nitrogen fixation should lead to additional
inoculated with G. sulfurreducens strain KN400. The anode was poised at electron consumption that would decrease the coulombic efficiency
0.3 V to act as an infinite electron acceptor. As expected, in the absence when the same amount of acetate is oxidized. Notably, the biomass
of ammonium and with nitrogen gas being the only candidate nitrogen formed in N-deficient fuel cells (56.2 ± 1.24 mg L− 1) was significantly
Fig. 1. Current generation and biofilm formation. (A) The average current densities, (B) the morphology of biofilms, and (C) total nitrogen (Ntotal) accumulation and
nitrogen gas consumption of the microbial fuel cells with or without ammonium in the electrolyte. The current densities were calculated from five replicates, and the
dashed area represents the standard deviation. The biofilms were stained by LIVE/DAED staining. The total nitrogen includes dissolved nitrogen in the electrolyte and
nitrogen in the biomass. Shown are representatives from at least five images.
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X. Jing et al. Water Research 208 (2022) 117860
Fig. 2. Biological nitrogen fixation on the anode. (A) elemental analyzer-isotopic ratio mass spectrometry of the 15N14N and 14N14N, and (B) the normalized
nitrogenase activity (***p< 0.001) of G. sulfurreducens in microbial fuel cells with or without ammonium in the electrolyte. The error bars represent the standard
deviation (n= 3).
Table 1
Acetate consumption, electron recovery, and growth of Geobacter sulfurreducens.
Acetate consumed Acetate for current generation Cell mass (mg Acetate assimilated Coulombic efficiency Acetate for N2 fixation
(mM) (mM) dw/L) (mM) (%) (mM)
lower than the biomass (68.2 ± 1.17 mg L− 1) formed in N-sufficient fuel will enter the anabolic pathway of the cell during acetate metabolism.
cells (Table 1). In addition to being an electron donor for anode Under N-deficient conditions, less biomass was formed, suggesting that
reduction, acetate also provides a carbon source to support biomass acetate in part probably had flown into the catabolic cycle for energy
formation (Mollaei et al., 2021). Therefore, part of the supplied acetate and reducing power generation to meet the needs of BNF but not
Fig. 3. Analysis of the metabolism of cells from the microbial fuel cells with or without ammonium in the anolyte. (A) The modular acetate flux. (B) Normalized
acetate consumption and current generation by the G. sulfurreducens cell (**p< 0.01). (C) The ratios of NADH/NAD+ and NADPH/NADP+ and (D) the concentration
of ATP (***p< 0.001). The error bars represent the standard deviation (n = 3).
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X. Jing et al. Water Research 208 (2022) 117860
affecting the coulombic efficiency. To examine this hypothesis, the conditions were obtained (Table S3).
amount of acetate consumption that was used as carbon source for In accordance with a higher catabolism and a less efficient anabolism
biomass synthesis was calculated according to the assimilation Eq. (1) during BNF, the energy-yielding tricarboxylic acid cycle (TCA cycle) was
(Galushko and Schink, 2000) after measuring the biomass amount upregulated while the acetate to biomass conversion pathways therefore
which could be displayed as the amount of total protein: assimilation, were downregulated (Fig. 4). Here, the TCA cycle gener
ated reducing power in the form of NADH and NADPH which will either
17CH3COO− + 11H2O = 8(C4H7O3) + 2HCO3− + 15OH− ((1)) be oxidized for energy production via anode respiration or provide
As indicated in Table 1 and Fig. 3A, the ratio of acetate to biomass electrons for BNF. Reduced flavodoxin or ferredoxin are direct electron
conversion accounts for 13.9% and 16.8% of consumed acetate under N- donors to nitrogenase. Previous study suggested that the activity of
deficient and N-sufficient condition, respectively. The amount of acetate pyruvate-flavodoxin oxidoreductase (PFOR) contributed to the genera
consumed for nitrogen fixation was calculated based on the assumption tion of flavodoxin in G. sulfurreducens (Poudel et al., 2018). However,
that the oxidization of 1 acetate could release 8 electrons eligible for 2 the expression of PFOR was downregulated during BNF (Fig. S4A).
NH3 generation (Eq. (2)) and the carbon-to-nitrogen ratio was shown to [Ni-Fe]-hydrogenase also could catalyze the reduction of ferredoxin but
be approximately 5:1 in bacteria (Persson, 1989). As indicated in was also downregulated (Fig. S4A). In contrast, the expression of the
Fig. 3A, 1.4% of equivalent acetate would enter the BNF pathway. The electron-transferring flavoprotein (Etf) complex (etfA, etfB and
extra acetate flux toward a catabolic pathway requires the cell boosting KN400_2732) and NADH-dependent ferredoxin: NADP reductase
catabolism. (NfnAB) were upregulated (Fig. S4A). Particularly, the Eft complex is
homologous to the Fix complex (encoded by fixABCX) and is able to
N2 + 8H− + 8e− + 16 AT→2NH3 + H2 + 16ADP + 16Pi (2) catalyze the oxidation of two NADH to generate a reduced quinone and a
The metabolism of single G. sulfurreducens cell was calculated further reduced ferredoxin (Ledbetter et al., 2017). NfnAB was also shown to
(Fig. S1). As expected (Fig. 3B), the cell displayed a higher acetate catalyze an electron-bifurcating process via oxidation of two NADPH for
metabolism rate (1.84 ± 0.11 mM mg− 1 protein) and current generation the generation of reduced ferredoxin and NADH (Buckel and Thauer,
(26.30 ± 1.39 μA mg− 1 protein) when performing BNF than when 2018). Therefore, the activity of Etf complex and NfnAB should
growing under N-sufficient conditions, with an acetate metabolism of contribute to the generation of reduced ferredoxin in G. sulfurreducens.
1.52 ± 0.10 mM mg− 1 protein and current generation of 21.02 ± 0.99 The expression of nitrogenase and its regulatory cascade were also
μA mg− 1 protein. The results indicated that the cells boosted their en upregulated in N-deficient electrolyte (Fig. S4B) which is consistent with
ergy metabolism to meet energy requirements when performing nitro the high nitrogenase activity, also indicating a process of BNF in
gen fixation. Therefore, ATP generation was significantly higher in G. sulfurreducens.
nitrogen-fixing cells (328.83 ± 19.19 nmol mg− 1 protein), reaching
2.15 times higher than the ATP generation in nonnitrogen-fixing cells 3.4. Inoculating G. sulfurreducens promotes N-deficient wastewater
(152.70 ± 13.39 nmol mg− 1 protein) (Fig. 3C). Furthermore, both the treatment in microbial fuel cells
NADH/NAD+ and NADPH/NADP+ ratios were also higher in nitrogen-
fixing cells, at 0.043 ± 0.003 and 0.873 ± 0.036, respectively, than in Microbial fuel cells (MFCs) are an effective device for facilitating
nonnitrogen-fixing cells, which yielded 0.020 ± 0.005 and 0.647 ± wastewater treatment due to it enhancing the anaerobic respiration
0.023, respectively (Fig. 3D). This is also in accordance with the (catabolism) of the microbial community (Liu et al., 2013; Zhang et al.,
calculation (Fig. 3A). BNF is an energy- and electron-consuming process 2011; Zhang et al., 2013). It has been reported that MFCs could also be
that requires 16 ATP and 8 electrons for the fixation of 1 molecule of used for synthetic N-deficient wastewater, which had the characteristics
nitrogen gas (Eq. (2)) (Foster et al., 2018). In particular, NADH/NADPH of pulp and paper, and the sugar refining industries, treatment after
acts as the reducing power, thereby providing electron sources for inducing the enrichment of electrochemically active nitrogen-fixing
nitrogenase (Poudel et al., 2018). Therefore, the accumulation of bacteria (Wong et al., 2014). The abilities of G. sulfurreducens cell to
reducing power and ATP was necessary to accommodate conditions fix nitrogen by reducing the anode and to generate current more effi
necessary for nitrogen fixation by the cell. These data demonstrated that ciently in N-deficient electrolyte suggest that G. sulfurreducens could be
at the single-cell level, the cell accelerated the acetate catabolic pathway applied in N-deficient wastewater treatment. This possibility was tested
and then increased its energy yield to produce conditions favorable for in the three-electrode two-chambered H-shaped cell with the anode
efficient nitrogen fixation. Furthermore, electrochemical kinetic anal chamber directly supplied with the real pulp and paper mills wastewater
ysis also demonstrated that the nitrogen-fixing anode biofilm had a (collected from Nine Dragons Paper Limited, Dongguan, China) and the
lower electron transfer resistance (Fig. S3). All these factors contributed catholyte of FMNN medium. The wastewater had a pH of 7.56 and an
to an efficient extracellular electron transfer path that facilitated anode initial DOC of approximately 950 mg L− 1 and total nitrogen of 43.33 ±
reduction by G. sulfurreducens cells and finally served in nitrogen fixa 1.51 mg L− 1. As shown in Fig. 5A, the MFC slowly generated a current
tion. Additionally, the higher NAD(P)H/NAD(P)+ ratios but lower that achieved a maximum of approximately 0.012 mA cm− 2 in 5 days
biomass formed also corroborated the alteration of electron flow in and then remained stable when treated with crude N-deficient waste
G. sulfurreducens under N-deficient conditions (Fig. 3A). water. Meanwhile, the DOC of the wastewater decreased slowly at a rate
of 8.99 ± 2.02 mg L− 1 day− 1, and the net Ntotal of the MFC increased
steadily at a rate of 0.488 ± 0.14 mg L− 1 day− 1 (Fig. 5B). In contrast,
3.3. Anode respiration drives biological nitrogen fixation after inoculation of 5 mL of G. sulfurreducens culture in the anolyte, the
current increased continuously and arrived at a maximum of ca. 0.075
Geobacter species perform anaerobic extracellular respiration and mA cm− 2 at 10 days, thereafter decreasing due to the depletion of DOC
transfer electrons extracellularly to drive the formation of a trans (Fig. 5A). Here, the average DOC removal rate and Ntotal increasing rate
membrane proton gradient, thereby contributing to energy generation were 45.42 ± 4.02 mg L− 1 day− 1 and 1.44 ± 0.19 mg L− 1 day− 1,
(Lovley et al., 2011). Therefore, anode reduction and subsequent current respectively (Fig. 5B). Therefore, the inoculation of G. sulfurreducens
generation generated energy to support nitrogen fixation; otherwise, the increased the electrocatalytic activity and nitrogen fixation of the mi
cell could not respire the unpoised anode and then fix nitrogen (Fig. S2). crobial community. In particular, the nitrogenase activity at day 12 was
Thus, the process should be described as being an anode-respiration 1.7 ± 0.29 μmol C2H4 mg protein− 1 h− 1, which was much higher than
driven BNF. To further analyze the coupling between the nitrogenase activity without G. sulfurreducens inoculation (0.097 ±
anode-respiration and BNF, the global transcriptional profiles of 0.04 μmol C2H4 mg protein− 1h− 1) (Fig. 5C). This result demonstrated
G. sulfurreducens cells growing under N-deficient and N-sufficient that supplementation with G. sulfurreducens could accelerate N-deficient
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X. Jing et al. Water Research 208 (2022) 117860
Fig. 4. The mechanism of synchronizing current generation and biological nitrogen fixation in G. sulfurreducens. Inset heat maps show the differential expression
analysis of genes involved in nitrogen fixation, anode respiration, tricarboxylic acid cycle and acetate assimilation in G. sulfurreducens cell growing in N-deficient
anolyte versus in N-sufficient anolyte.
Fig. 5. Characterization of the effect with or without inoculation of G. sulfurreducens on the N-deficient wastewater treatment. (A) Current generation. (B) The
decrease in dissolved organic carbon (DOC) and the increase in total nitrogen (Ntotal). (C) Nitrogenase activity (***p< 0.001). (D) The relative abundances of
bacterial 16S rRNA genes at the genus level.
wastewater treatment by increasing anaerobic respiration and nitrogen inoculation of G. sufurreducens affected the structure of the bacterial
fixation of the anode microbial community. community. In detail, genera belonging to Tepidiphilus, Pseudomonas and
The microbial community architectures in the two different treat Thermicanus dominated in crude wastewater. After being treated by
ments were analyzed by examining the 16S rDNA profiles after per MFC, genera belonging to Tepidiphilus, Bacteroides, Pseudomonas,
forming Illumina MiSeq sequencing. The overall phylogenetic Desulfovibrio, Ideonella, and unclassified Pseudomonadaceae dominated
characteristics at the genus level are plotted in Fig. 5D. As illustrated, the in the anode biofilm. Here, species from Pseudomonas and Ideonella
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X. Jing et al. Water Research 208 (2022) 117860
contain diazotrophs that can perform anaerobic growth and fix nitrogen regulate the carbon metabolism. This is consistent with previous studies
(Noar and Buckley, 2009; Yan et al., 2010), thereby providing nitrogen showing that G. sulfurreducens possessed a mechanism to upregulate
sources to other species, and species from Bacteroides, Pseudomonas and acetate catabolism for energy production and reduce acetate anabolism
Desulfovibrio can perform extracellular electron transfer (Ha et al., 2012; under conditions of electron acceptor limitation (Esteve-Núñez et al.,
Ouyang et al., 2021; Yu et al., 2018), which can contribute to anodic 2005; Mahadevan et al., 2006). This type of response was suggested to
respiration for current generation. Furthermore, species from Bacter be universal in providing cells with enough ATP to adapt to fluctuating
oides, Pseudomonas and Ideonella are predicted to also contribute to the subsurface environment (Esteve-Núñez et al., 2005), thereafter satis
decomposition of DOC, including lignin, hemicelluloses, benzoic acid, fying the extensive energy demand during nitrogen fixation. It was re
phenylacetic acid, hexadecenoic acid, etc., in wastewater (Ismail and ported that the intracellular metabolite level and the redox state are two
Hossain, 2015; Thorell et al., 2003), and could provide simple carbon major factors determining the global regulation of cellular activities
and energy sources for other species (Wong et al., 2014). The diversity of (Kouzuma et al., 2015). Considering the accumulation of NAD(P)H in
the microbial community was further increased after inoculation with cells under both nitrogen fixing and electron acceptor limiting condi
G. sulfurreducens. For example, the community of diazotrophs expanded tions, a more reducing intracellular environment might trigger a similar
to include species from Azospirillum (Steenhoudt and Vanderleyden, cellular response in shifting metabolism toward energy generation.
2000), Geobacter, Ideonella and Clostridiales (Chen, 2004), and the Actually, the transcriptome data have shown a different expression of
community of exoelectrogens included Bacteroides, Desulfovibrio, Pseu signal transduction pathways (Table S3), such as second messenger
domonas and Geobacter species. In particular, Geobacter species domi system and two-component system, in G. sulfurreducens under different
nated the anode microbial community, accounting for 4.51%, and are nitrogen conditions, indicating an active metabolic regulation.
predicted to be the predominant contributors to current generation and
nitrogen fixation, considering that they are the most efficient exoelec 4.3. Using electrogenic diazotroph for N-deficient wastewater treatment
trogens and are able to synchronize biological nitrogen fixation and
current generation. Using Geobacter species as additives to facilitate N-deficient waste
water treatment in a bioelectrochemical system (BES) is intriguing and
4. Discussion represents a novel technique. Traditionally, N-deficient wastewater
treatment strategies include adding ammonium, supplying nitrogen-
4.1. Using electrogenic diazotroph for green nitrogen fixation fixing microorganisms, and treating with activated sludge (Ismail and
Hossain 2015; Vashi et al., 2018). However, corresponding problems
Geobacter species are the most efficient exoelectrogens and contain arise, including the risk of nitrogen contamination, the
genes encoding nitrogen-fixing functions (Lovley et al., 2011; Methé energy-consuming process of aeration and the concomitant oxygen
et al., 2005). Applying Geobacter species as an electrogenic diazotroph toxicity to nitrogenase (Vashi et al., 2018). In contrast, combining
and an anode as an electron acceptor showed promise for realizing Geobacter species and BES for N-deficient wastewater treatment elimi
simultaneous current generation and biological nitrogen fixation, nates these problems since the BES is an anaerobic system, and the
especially considering that performing electron-intensive nitrogen fix anode can act as an infinite electron acceptor to support the nitrogen
ation was not shown to affect current generation or columbic efficiency fixation of Geobacter species, thereby generating electrical energy.
of microbial fuel cells and that the current nitrogen-fixing industry is Notably, the specific function of Geobacter species in this process has not
energy-intensive. Moreover, previous progresses in increasing the been identified. Electroactive diazotrophs have been proposed to
loading of exoelectrogens on the anode or the current generation of the directly oxidize organic waste to reduce the anode during N-deficient
anode provides possibilities to further increase nitrogen fixation (Liu wastewater treatment (Wong et al., 2014). However, Geobacter species
et al., 2019; Yaqoob et al., 2020). For example, upon shifting the were shown to only be able to grow on a few small organic acids,
operation of this G. sulfurreducens fuel cell from fed-batch mode to including the fermentation products of other species (Lovley et al.,
continuous mode, the maximum current increases to 0.3 ± 0.02 mA 2011). Therefore, it is possible that they can form symbiosis with other
cm− 2, and the nitrogen fixation rate reaches approximately 8.6 mg L− 1 fermenting bacteria and establish a syntrophic community, thereby
day− 1 (Fig. S5). This is approaching the highest rate of microbial ni accelerating DOC removal. Notably, Geobacter species can express
trogen fixation as recently reported (Ospina-Betancourth et al., 2020). conductive extracellular matrices (Liu et al., 2020a, 2020b, 2019) or
Notably, even though the combination of a bioelectrochemical system form direct electric contacts with other species (Liu et al., 2018; Rotaru
with nitrogen-fixing bacteria for nitrogen fixation has been reported et al., 2014), thereby contributing to the extracellular respiration of
(Chen et al., 2021; Knoche et al., 2017), in these systems electric energy other species. The simultaneous abundance of the other diazotrophs and
was consumed to drive nitrogen fixation. exoelectrogens strengthens the possibility of Geobacter species contrib
Our results showed that nitrogen fixation affected the biofilm for uting to or facilitating the anodic respiration of those species. Further
mation of G. sulfurreducens inducing the formation of a rough biofilm. studies are warranted. Furthermore, the finding that biological nitrogen
Transcriptome analysis demonstrated that the genes related to chemo fixation consumes DOC in wastewater and generates electric energy is
taxis and flagella expression were down-regulated during nitrogen fix not only beneficial for the environment but also exemplifies green ni
ation (Table S2). We also showed that a rough anode biofilm was formed trogen fixation.
in N-sufficient electrolyte when the expression of the flagellum was
inhibited (Fig. S6). So, it can be speculated that a deficiency of flagellum 5. Conclusions
expression during nitrogen fixation contributed to the formation of a
rough biofilm, highlighting the structural function of flagella in In this study, we demonstrated simultaneous biological nitrogen
G. sulfurreducens biofilm formation (Liu et al., 2019). Inhibiting the fixation and electric energy production by G. sulfurreducens. Specifically,
expression of flagella might be a mechanism to save energy to meet the the biological nitrogen fixation of G. sulfurreducens was driven by anode
energy demand needed for nitrogen fixation. respiration after coupling the extracellular electron transfer for energy
production with nitrogen fixation, and in return, biological nitrogen
4.2. Balancing act between catabolism and anabolism fixation could enhance anode respiration of the G. sulfurreducens cell by
increasing acetate catabolism but decreasing acetate anabolism and
The finding showing that G. sulfurreducens cell increased acetate increasing extracellular electron transfer. Furthermore, we revealed that
respiration but decreased acetate to cell carbon conversion during ni biological nitrogen fixation did not impair the current generation or
trogen fixation indicated that G. sulfurreducens harbors a mechanism to coulombic efficiency of the G. sulfurreducens fuel cell. Finally, we
7
X. Jing et al. Water Research 208 (2022) 117860
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findings show great promise to further green nitrogen fixation and could Miller, J., Plunkett, M.H., Hoben, J.P., Barney, B.M., Carlson, R.P., Miller, A.F.,
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Declaration of Competing Interest Escherichia coli. J. Bacteriol. 178 (20), 6013–6018.
Liu, C., Sakimoto, K.K., Colon, B.C., Silver, P.A., Nocera, D.G., 2017. Ambient nitrogen
reduction cycle using a hybrid inorganic-biological system. Proc. Natl. Acad. Sci. U.
The authors declare no conflict of interest. S. A. 114 (25), 6450–6455.
Liu, W., Cai, W., Ma, A., Ren, G., Li, Z., Zhuang, G., Wang, A., 2015. Improvement of
bioelectrochemical property and energy recovery by acylhomoserine lactones
Funding (AHLs) in microbial electrolysis cells (MECs). J. Power Sources 284, 56–59.
Liu, X., Huang, L., Rensing, C., Ye, J., Nealson, K.H., Zhou, S., 2021. Syntrophic
This research was supported by the National Science Fund for interspecies electron transfer drives carbon fixation and growth by
Rhodopseudomonas palustris under dark, anoxic conditions. Sci. Adv. 7 (27),
Distinguished Young Scholars of China, grants no. 41925028, National
eabh1852.
Natural Science Foundation of China, Grants no. 42077218, and the Liu, X., Jing, X., Ye, Y., Zhan, J., Ye, J., Zhou, S., 2020a. Bacterial vesicles mediate
Scientific Research Foundation of Graduated School of Fujian Agricul extracellular electron transfer. Environ. Sci. Technol. Lett. 7 (1), 27–34.
Liu, X., Ye, Y., Xiao, K., Rensing, C., Zhou, S., 2020b. Molecular evidence for the adaptive
ture and Forestry University (324–1122yb072).
evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in
natural environments. Mol. Microbiol. 113 (4), 783–793.
Supplementary materials Liu, X., Zhuo, S., Jing, X., Yuan, Y., Rensing, C., Zhou, S., 2019. Flagella act as Geobacter
biofilm scaffolds to stabilize biofilm and facilitate extracellular electron transfer.
Biosens. Bioelectron. 146, 111748.
Supplementary material associated with this article can be found, in Liu, X., Zhuo, S., Rensing, C., Zhou, S., 2018. Syntrophic growth with direct interspecies
the online version, at doi:10.1016/j.watres.2021.117860. electron transfer between pili-free Geobacter species. ISME J. 12 (9), 2142–2151.
Liu, Y., Liu, H., Wang, C., Hou, S.X., Yang, N., 2013. Sustainable energy recovery in
wastewater treatment by microbial fuel cells: stable power generation with nitrogen-
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