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The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs
Journal Pre-proof The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs Yutian Hu, Nan Chen, Tong Liu, Chuanping Feng, Linlin Ma, Si Chen, Miao Li
PII:
S0304-3894(20)30423-4
DOI:
https://doi.org/10.1016/j.jhazmat.2020.122434
Reference:
HAZMAT 122434
To appear in:
Journal of Hazardous Materials
Received Date:
3 February 2020
Revised Date:
28 February 2020
Accepted Date:
28 February 2020
Please cite this article as: Hu Y, Chen N, Liu T, Feng C, Ma L, Chen S, Li M, The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122434
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The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs
Yutian Hua, Nan Chena* [email protected], Tong Liua, Chuanping Fenga, Linlin Maa, Si Chena, Miao Lib a
School of Water Resources and Environment, MOE Key Laboratory of Groundwater
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Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing, 100083, P.R. China b
School of Environment, Tsinghua University, Beijing 100084, P.R. China
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* Corresponding Author:
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(N. Chen), Phone: +86-10-82322281, Fax: +86-10-82321081
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Graphicalabstrct
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Highlights
Both Cr(VI) and nitrate were completely reduced at initial pH 10.0 and 11.0.
The primary genes for reducing nitrate and Cr(VI) were narG and azoR, respectively.
The dominant genus under alkaline environments was Pannonibacter.
The relative abundances of metabolic pathways were higher at pH 10.0 and 11.0.
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Potential interspecies cooperation was found to benefit removal of co-contaminant.
Abstract 2
To date, comparatively little research is known about the role of pH conditions in bioremediation of Cr(VI) contaminated aquifers. This study explored microbial Cr(VI) reduction and denitrification under different initial pHs. The underlying mechanism was also investigated. When testing 50 mg/L-N nitrate and 10 mg/L Cr(VI), complete contaminants removal was observed at initial pH 10.0 and 11.0, and only 10%-30% of removal achieved under other conditions, which might be ascribe to the significant up-
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regulation of functional genes narG (8.31 and 10.46 folds) and azoR (24.90 and 15.96
folds) at initial pH 10.0 and 11.0. Metagenomic sequencing showed that alkali tolerant bacteria played major roles in the NO3--Cr(VI) reduction (i.e. Pannonibacter increased
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by 13.08% and 25.24% at initial pH 10.0 and 11.0), and metabolic pathways of
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Degradation and Energy were found of increased abundant. Furthermore, a significative study suggested that potential interspecies cooperation existed at initial pH 11.0 to
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facilitating the simultaneous removal of contaminants, and Pannonibacter indicus might
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be an important participant in the degradation of contaminants. The results of this study will fully understand the metabolic patterns of bacteria under alkaline conditions, expand
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the range of available functional bacteria, and enhance the practical aspects of cocontaminants remediation.
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Keywords: pH; Nitrate-Cr(VI) bio-reduction; Metabolic pathway; Functional genes; Potential interspecies cooperation. 1. Introduction The residual nitrate in soil and groundwater initiated a cascade of large-scale environmental concerns with the widespread use of nitrogen fertilizer (Nieder et al., 3
2018). Excessive nitrate and nitrite (intermediate of nitrate reduction) loadings lead to severe water deterioration (such as eutrophication), and bring toxic effects on humans once using the contaminated water for drinking(Zhao et al., 2018). The U.S. Environment Protection Agency (USEPA) has set the maximum contaminant levels (MCL) of nitrate and nitrite at 10 and 1.0 mg L−1 (measured as N) in drinking water, respectively. Based on it, biological denitrification has attracted widespread attention
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(Fan et al., 2018), which consecutively reduce nitrate to nitrite, nitric oxide, nitrous oxide and finally nitrogen gas with the participation of nitrate reductase, nitrite
reductase, nitric oxide reductase, nitrous oxide reductase (Diaz and Rosenberg, 2008).
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At the same time, the combined pollution of nitrate and Cr(VI), occurs in
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intensive mining and smelting activities especially, poses a serious threat to the geological environment (Hausladen et al., 2018). Emissions from geological
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weathering and industrial processes arise the chromium flux in groundwater, primarily
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as Cr(III) and Cr(VI) (Jobby et al., 2018). Cr(VI) (0.1 mg L−1 of MCL in drinking water proposed by USEPA) is recognized as carcinogens and toxins that can easily
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penetrate cells and inhibit biological processes (Sun et al., 2019), which hampers the denitrification process through decreasing the rate of conversion nitrate to (Sun et al.,
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2019). Compared with the Cr(VI), Cr(III) is of lower mobility and bioavailable at neutral pH, and can be removed easily from water (Bhattacharya et al., 2019). Therefore, bio-reduction of Cr(VI) to Cr(III) is one promising approach to remove chromium from contaminated groundwater (Chen et al., 2019). The pH of the nitrate and Cr (VI) contaminated area is of highly complexity. For 4
example, numerous chromium pollution sites in California have pH values varied from 3.0 to 12.0 (Hausladen et al., 2018). pH, one of the most important environmental index, governs the biogeochemical activities of microorganisms (Han et al., 2019). The optimum pH for most environmental strains of denitrifying bacteria has been reported to be between 7.0 and 8.0 (Gan et al., 2019). However, some researchers found that the aerobic granular denitrifying sludge was more adaptable to acidic rather conditions
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alkaline conditions (Jiang et al., 2019). In addition, Cr(VI) reduction by some chromate
reduction bacteria (such as Geobacter sulfurreducens) was observed performed better
under neutral conditions (He et al., 2019), while another chromate reduction bacteria,
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Pannonibacter phragmitetus LSSE-09, preformed best in alkaline wastewaters (Xu et
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al., 2011). In summary, pH affects the metabolism of microorganisms which utilize the substrates in wastewater, resulting in variations in the sludge stability and pollutant
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removal performance. However, to date, the behaviors of microorganisms under stress
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from both Cr(VI) and nitrate at different initial pH values have not yet been reported, especially in terms of the energy supply, functional genes levels, and metabolism
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pathway. Therefore, it is important to investigate the response to the pH shock of the simultaneous Cr(VI) and nitrate removal in the mixed system by microorganisms, and it
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will help to have a deeper understanding of the biogeochemical behavior of nitrogen and chromium.
Herein, the feasibility and mechanism of simultaneous bio-reduction of nitrate and Cr(VI) were investigated. To illustrate the bacterial diversity, succession and metabolic mechanisms in the process of microorganism degrading co-contaminants 5
under different initial pH values, the adaptive mechanism of functional flora acclimating to both nitrate and Cr(VI) stress was studied using metagenomic analysis. In addition, metagenome binning was utilized to find the main and potential players of the microbial communities in NO3--Cr(VI) degrading under alkaline condition. This study is likely to shed new light on the attenuation mechanism of nitrate and chromium-contaminated aquifers, and provides further understanding of the
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biogeochemical cycle of nitrogen and chromium. 2. Materials and methods 2.1. Batch experiments
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A series of batch experiments were conducted in 100 mL serum bottles to explore
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the effect of the initial pHs on contemporaneous nitrate and Cr(VI) reduction in triplicate. Then, approximately 3 mL anaerobic sludge (the best removal performance according to
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the results of different sludge dosage in Fig. S1) washed by 0.9% NaCl was re-inoculated
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into a 100 mL serum bottle containing 100 mL of synthetic wastewater (0.4217 g CH3COONa, 0.0561 g K2HPO4 and 0.5 g yeast extract per liter) (Chen et al., 2019), in
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which 10 mg of Cr(VI) L-1 in the form of K2Cr2O7 and 50 mg L-1 of NO3--N in the form of NaNO3 were also involved in the synthetic wastewater, which were monitored using
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a spectrophotometer (DR6000, Hach, USA). The initial pH values were adjusted to 5.0, 7.0, 10.0, 11.0, and 12.0 through the manual addition of 1.0 M HCl or 1.0 M NaOH (Qi et al., 2020), which determined from the analysis of pH in the contaminated site such as California (the range of pH was from 5 to 12) (Dhal et al., 2013). Then gentle mixing was provided by a shaker at 150 rpm. Other reactors inoculated with autoclaved 6
anaerobic sludge and without anaerobic sludge were also carried out under the same experimental conditions for comparison. At the same time, the experiments with actual water diluted 10 times (the initial Cr(VI) and TOC concentration were 120.5 ± 0.6 and 10.25 ± 0.64 mg L-1, respectively) were also carried out, adding 50 mg L-1 NO3--N and 0.4217 g CH3COONa, the adjusted pH was 11.0 (the results was showed in Fig. S2). Of which, each serum bottle was purged with nitrogen for 20 minutes (Du et al., 2020). And
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mixed liquor samples were taken for nitrate, nitrite, ammonium, Cr(VI), total chromium,
electron transport system activity (ETSA), and cytochrome C (Cyt C) analysis, and the pH values were also monitored during the experiment. An environmental scanning
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electron microscope (ESEM, Quanta 200, Netherlands) and a fluorescence microscope
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(BX53, Olympus, Japan) with an ultraviolet blue light-excited filter plate for microorganisms were used after batch experiments at initial pH 5.0, 7.0, and 11.0 to
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explore the differences in morphology (Duan et al., 2019, 2020). These differences were
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distinguished with an AO/EB Staining Kit (Sangon, Shanghai, China), in which uniformly green, and orange represented live cells and necrotic cells, respectively.
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To characterize the reduction capacity of bacteria for Cr(VI) and nitrate, the effects of the initial Cr(VI) concentration (2, 5, 10, 20, or 50 mg L-1) and initial NO3--N
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concentration (10, 20, 50, 70, or 100 mg L-1) were also investigated. The other experimental conditions were similar with those described above. The chemical methods and microorganismal analysis used in this study, including the cultivation of microorganisms, cell lysis, and cell separation methods, are detailed in Supporting Information (SI). 7
2.2. Molecular analysis Four biofilm samples at the fastest reduction rate were collected, marked with its pH values (5.0, 7.0, 10.0, and 11.0), and another sample (named as control) was collected from initial sludge. Total genomic DNA was extracted and amplified with the PCR primers
338F
(5’-ACTCCTACGGGAGGCAGCAG-3’)
and
806R
(5’-
GGACTACHVGGGTWTCTAAT-3’). Amplicon sequencing was then performed using
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the MiSeq platform (Illumina, U.S.) at Majorbio Technology Ltd. (Shanghai, China). Raw sequences were submitted to the NCBI SRA database under the accession number
of SUB6207731. Variations in functional genes (narG, napA, nirS, nirK, norB, nosZ,
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chrA, chrR, nfsA, nemA, and azoR) were further quantified with quantitative real-time
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PCR (q-PCR) using a StepOne Plus™ Real-Time PCR System (Thermo, U.S.) while the total RNA was extracted with a FastRNA Pro Soil-Indirect Kit (MP Bio, U.S.), and the
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cDNA was inversely transcribed with Revert Premium Reverse Transcriptase (Beyotime,
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Shanghai, China). The q-PCR primers (Table S1) were synthesized by Sangon Biotech (Shanghai, China). Detailed methods about metagenome sequencing, assembling,
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annotating, and binning are provided in the SI Appendix. 3. Results and discussion
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3.1 Performance of co-contaminants at different pH values pH is an important environmental factor influencing biogeochemical processes
significantly (Mary Mangaiyarkarasi et al., 2011). In this study, the nitrate and Cr(VI) concentrations decreased progressively over time in the experimental treatments with varied pH values (Fig. 1a, b, Figure S1). The average removal rates were 0.52 and 0.83 8
mg h-1 for nitrate and 0.12 and 0.14 mg h-1 for Cr(VI), with k of 0.0256 and 0.0892 h-1 for nitrate and 0.0202 and 0.0296 h-1 for Cr(VI), respectively (Table S2). Compared with the removal efficiencies of about 67% for nitrate (Vilardi et al., 2020) and 75% for Cr(VI) (Vilardi et al., 2019) under the additions of biomass and nZVI particles, they were all up to 99.8% at initial pH of 10.0 and 11.0 with the participation of microorganisms. For treatments with other initial pH, the removal efficiencies for nitrate and Cr(VI) were
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only 10%-20% and 20%-30%, respectively, and the removal effects were not as well as
chemical method at pH 5.0-9.0 (Vilardi et al., 2018; Vilardi, 2019). It was also observed that the concentration of total Cr was below to 2 mg L-1 at initial pH of 10.0 and 11.0
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(Fig. S3c), implying that Cr(Ⅲ) was the main form of remaining chromium, and
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precipitates as hydroxides.
At initial pH of 10.0 and 11.0, the accumulation of nitrite was less than 6 mg L-1,
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and complete nitrite reduction was observed within 144 and 72 h, respectively (Fig. 1c),
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indicating that the potential NO2- reduction rate was at least as high as that of the NO3-. In addition, the complete reduction of nitrite was not observed at other pH values, which
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might be due to the significant effect of the initial pH on nitrite reduction pathway by affecting the activity of nitrite reductases (Kim et al., 2017). Furthermore, the pH was
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also a factor in determining the NO3- fate for microorganisms (Šimek et al., 2002), and the concentration of NH4+ remained below the detection limit throughout the experiment under all pH conditions (data not shown), which can be speculated that NH4+ might be used by microorganisms as a nitrogen source. It was found that the carbon source utilization varied at different initial pH 9
conditions from Fig. 1d, and the utilization under pH of 10.0 and 11.0 were higher than that under other initial pHs. Approximately 232.5 ± 5 mg L-1 CH3COONa is required to reduce 10 mg L-1 Cr(VI) and 50 mg L-1 NO3--N, suggesting that microorganisms need to use carbon sources as nutrients to survive and reduce nitrate and Cr(VI). Differences in the pollutions reduction kinetic constant by microorganism might be due to differences in the energy supply, and they were measured by the value of ETSA and Cyt
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C (Fig. 1e and f) which reflecting bacterial activities (Zimmermann et al., 1978). The ETSA value increased with the increasing initial pH (5.0-11.0). Additionally,
microorganism obtained electrons from Cyt C to reduce Cr(VI) and nitrate (Lovley, 1995;
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Zeng et al., 2018). Compared with the initial activity of bacteria after 36 h (the maximum
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reduction time for nitrate and Cr(VI)), the Cyt C concentration increased by 1.2-fold and 1.6-fold at initial pH of 10.0 and 11.0, respectively, while it decreased under other initial
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pH values. This finding might be due to the bacteriocin production, which had the ability
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to cause cell death and was favored under stressful growth conditions, suggesting that the initial pH had direct and indirect impacts on the growth and metabolic activity of
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microorganisms (Han et al., 2019). The analysis of the performance of simultaneous microbial nitrate and Cr(VI) reduction at initial pH 11.0 was shown in SI. In addition,
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the microbial analysis and co-contaminant reduction location were also in SI 2.1 and 2.2. Cr(VI) and nitrate in mixed systems affect each other's reduction, the presence of Cr(VI) had a negative effect on denitrification, while the removal of Cr(VI) accelerated with the increasing of nitrate concentration. Furthermore, membrane-associated enzymes had a dominant effect on the microbial mediated reduction of Cr(VI) and nitrate. 10
3.2 Microbial community analysis Fig. 2 showed the different relative abundances of the dominant bacteria in all reactors. Proteobacteria is a typical phylum in the reactor with initial pH 10.0 and 11.0 as well as the initial microorganisms, but it only accounted for 16.01% and 9.68% of the total bacterial community structure in the reactors with pH 5.0 and 7.0. And Fimicutes dominated the two reactors, resulting in the acceleration or suppression of denitrification
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and the removal of Cr(VI).
It was observed that the dominant genera were Enterococcus, Bacillus, and Pannonibacter under acidic, neutral, and alkaline conditions, respectively. This result
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indicated that bacteria were sensitive to the changes of pH values, and bacteria gradually
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adapted to the changes in the environment through self-adjustment. Once the initial pH value changed, different operating conditions might select specific bacterial populations
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to adapt to these conditions. Interestingly, Enterococcus (a metal-reducing bacteria)
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reportedly could reduce chromate (Sayel et al., 2012), but energy analysis (Fig. 1 e and f) implied that it might have lost the ability to reduce Cr(VI) owing to the lack of energy.
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A typical anaerobic bacteria -- Bacillus (61.87% with pH 7.0), belonging to Fimicutes, was associated with dissimilatory metal reduction and denitrification (Lovley, 1993; Ito
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et al., 2019; Ma et al., 2019). Yet the kinetic constant of denitrification in system was only 0.0016 h-1, because the action of Cr(VI) converting to Cr(III) led to more generation of ROS and lower abundance of antioxidant genes including lexR and mdh at pH 7.0 (Fig. S9), thus denitrification would be inhibited through exerting deleterious effects on proteins and DNA in cells at initial pH 7.0 (Lyon and Alvarez, 2008). Compared to the 11
initial microorganisms, the abundance of Pannonibacter (facultative anaerobic bacteria) increased by 13.08% and 25.24% in reactors at initial pH of 10.0 and pH 11.0, respectively. This result associated with both chromate bio-reduction and denitrification (Bai et al., 2019; Chai et al., 2019), providing evidence for higher nitrate and Cr(Ⅵ) reduction kinetic constant at same conditions. In addition, it was speculated that Chryseobacterium (25.46%) and Anaerobacillus (20.76%) (potential candidates for the
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wastewater treatment) had ability to reduce pollutant under alkaline environments
according to high percentage in reactors of pH 10.0 and 11.0. In summary, these findings
indicated that the pH had a significant effect on the bacterial community of the reactor,
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and the results confirmed that pH 10.0 and 11.0 might be more beneficial to bacterial
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metabolism than other pH values. Additionally, the microorganism morphology, primary reduction location under different pH values were also explored in SI 2.3, consistent with
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the results that organism evolution, microbes built self-protecting structures against pH
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or toxic pollutant shocks through forming different morphology. And the reduction of contaminants occurred primarily in the membrane region.
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3.3 Metabolic pathways analysis
It was observed that initial pH affected the microbial metabolic function (Fig. 3).
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The relative abundance of Degradation metabolic pathways including amine and polyamine degradation, fatty acid degradation, and carbohydrates and carboxylates degradation under alkaline conditions was higher than those under acidic and neutral conditions (Fig. 3a). These metabolic pathways played important roles in supporting microorganism survival through the degrading of amino acids, fatty acid, and 12
carbohydrates. The results suggested that microorganisms could utilize the sodium acetate to serve as sources of nutrients and energy, which was consistent with the degradation of carbon source at different initial pHs (Fig. 1d). Meanwhile, the relative abundance of other metabolic pathways including Energy supply (Fig. 3b), Response to Stimulus (Fig. 3c), and Cellular Processes (Fig. 3d) were consistent with Degradation, indicating that microorganisms could provide more energy to survive. Compared to other
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pHs, the microorganisms were more resistant to environmental changes through
metabolism such as TCA cycle, proteins involved in response to pH/DNA damage, and Quorum Sensing under alkaline conditions. Therefore, varied pH values could change
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the metabolic pathways by changing the gene abundance, benefitting the attenuation
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(acidic and natural pH) or propagation (alkaline pH) to improve the reduction ability of nitrate and chromate.
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The relative expression of denitrifying functional genes might provide evidence for the extent or level of active denitrification, including napA, narG, nirS, nirK, norB, and
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nosZ, which played major roles in the step of NO3-→NO2-, NO2-→NO, NO→N2O, N2O→
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N2, respectively (Henderson et al., 2010). As shown in Fig. 3e, pH exerted direct control on the reduction efficiency by affecting the relative expression of denitrification genes
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(Liu et al., 2010). The relative expression levels of all the genes were significantly upregulated at initial pH of 10.0 and 11.0, compared with other pH conditions. The narG gene (8.31 and 10.46 folds) had a significant enhancement compared to napA gene (2.80 and 4.46 folds) at initial pH of 10.0 and 11.0, probably because the anaerobic denitrifying bacteria primarily expressed membrane-bound nitrate reductase to catalyze the 13
denitrification process. During the nitrite reduction process, nirK gene was the major coding gene of nitrite reductase according to the results of qPCR. NorB upregulated 24.88 and 33.12 folds, while nosZ showed no obvious fluctuations at different pH values, suggesting that the pH affected the sequence reduction during the denitrification process (Wang et al., 2018). It was reported that genes including nemA, nfsA, azoR, chrA, and chrR were
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associated with the reduction and efflux of chromate (Thatoi et al., 2014). It was observed that the expression of nemA and nfsA did not differ significantly before and
after exposure to contaminants, while azoR provided a significant enhancement (24.90
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and 15.96 folds with pH 10.0 and 11.0 respectively) (Fig. 3e), which had an important
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role in microorganismal resistance to oxidative stress (Misal and Gawai, 2018). The gene of azoR also showed a similar Cr(VI) reductases characteristic (He et al., 2010), In
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addition, the genes of chrA and chrR had slightly increment, and they were responsible
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for chromate efflux (Alvarez et al., 2000) and reducing ROS production (Cheung and Gu, 2007), respectively. This result indicated that the cells had higher resistance to Cr(VI)
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toxicity under alkaline conditions.
3.4 Metabolic mechanism of bacterial cooperation
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3.4.1 Metabolic potential of MAGs The qualities of the MAGs were shown in Table S5 and the phylogenomic analyses
of 74 genomic bins also showed in SI. A metagenomic sequence analysis could provide definitive evidence for the positive selection of functional genes under nitrate and Cr(VI) stress. As shown in Fig. S9, Enterococcus (metal reducing bacteria) only carried the 14
napA gene, and this series of evidence supported the low kinetic constant for denitrification at pH 5.0 and 7.0. The Halomonas genus (M5.13 and M7.1) (a typical denitrification bacteria) had a complete denitrification pathway, and yet its denitrification capacity was weak under both acidic and neutral conditions, illustrating why denitrification efficiencies were lower. It was found that Thauera sp000310145 (a typical denitrification bacteria) (M5.4, M7.10, M10.14, and M11.12) encoded the key
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enzymes for complete denitrification. However, it was not conducive to nitrogen
removal according to the analysis of energy (Fig. 1) and qPCR (Fig. 3e). Rhodocyclaceae (Nitrate reduction bacteria) had no ability to use nitrite as an electron donor, causing a
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lack of nitrite reduction pathway, though it was extremely resistant to the pH (5.0-10.0).
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Contrary to the previous studies (Sotres et al., 2016; Tu et al., 2019; Vijay et al., 2019), Burkholderiaceae (a metal reducing bacteria) (Bins M5.11, M7.6, and M10.8) and
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Acholeplasmataceae (Bins M5.2, M5.5, M5.12, M7.3, M7.7, M7.12, and M7.16) had no
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genes for N metabolism at pH 5.0, 7.0, or 10.0, which might be due to the fact that the pH might lead to bacterial gene selection (Lund et al., 2014).
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It was found that 20.34% and 23.73% of the MAGs in the 59 MAGs did not carry the chromium reduction genes and the chromium transport genes under different pH
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values, indicating that the reduction of chromate in a mixed microbial system was not the function of a single microbe, but it depended on multiple microbial collaboration. It was found that the azoR gene (52.54%) played a major role in the reduction of chromate, compared to the nemA (32.20%) and chrR genes (32.20%), confirmed by genes expression analysis. NfsA (Erysipelotrichaceae (M5.3)) and nfsB (Burkholderiaceae 15
(M5.11, M7.6, M10.8), Dysgonomonadaceae (M10.3), Flavobacteriaceae (M10.1), and Pseudomonadaceae (M7.13)) were the major oxygen-insensitive nitroreductases, and Cr(VI) reduction gave rise to the product of reactive oxygen species (ROS) and Cr(V) (Ackerley et al., 2006; Shang et al., 2019). Thus, the presence of the ROS response enzymes (recA, recG, and ruvB) was essential for bacteria to protect themselves from the ROS attacks. In addition, the Chryseobacterium (potential candidates for the
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wastewater treatment) genus encoded enzyme about degrading Cr(VI) without
denitrification at pH 10.0, indicating that it had potential to reduce the chromate under alkaline conditions, consistent with Romaniuk et al. (2018). Moreover, the strain
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Pannonibacter indicus was isolated under alkaline conditions and had not been reported
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for denitrification or Cr(VI) reduction, but it had complete denitrification genes and chromium reduction transport genes. Hence it was inferred that Pannonibacter indicus
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had great potential for simultaneously reduce nitrate and chromium. Notably, the
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Pannonibacter (facultative anaerobic bacteria) genus could reduce nitrate (Bai et al., 2019), heavy metal (e.g. Cr(VI) (Chai et al., 2019) and As(V) (Bandyopadhyay et al.,
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2013)).
3.4.2 Bacterial metabolic pathways reconstruction at initial pH 11.0
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The cooperation between different bacteria and various functional genes could
further reveal the common adaptations of bacterial communities under alkaline stress and joint nitrate and chromium stress (Fig. 4). The discrepant nitrogen metabolism pathways in varied MAGs provided insight into possible interbacterial transfer of inorganic nitrogen compounds (Hug et al., 2015). We found that most microorganisms 16
that used inorganic N were specialists, harboring only one or two pathways (Fig. 4). The decoupling of modular pathways with more than one enzymatic step was common (Fig. 4). For denitrification, 85% of the microbes had only a partial pathway (i.e., only one step was present for the transformation of NO2− to NO, N2O, and N2), rather than the whole pathway (NO3− to N2). For example, some bins (e.g. M11.1, M11.2 et al) were able to conduct dissimilative nitrate reduction to nitrite with narGHI and napAB encoded
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enzymes, while M11.10 and M11.12 could only convert nitrate into nitrite assimilative by narB and nasAB encoded enzymes. M11.4 and M11.11 might use nitric oxide as
electron donors, while M11.9 converted nitrous oxide into nitrogen and released it into
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the atmosphere. Meanwhile, M11.14 had ability to convert nitrogen to ammonia nitrogen
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(nifDKH) as a nutrient for its own use through fixing nitrogen. Notably, only the strains of Pannonibacter indicus and Thauera (denitrifying bacteria) had complete
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denitrification pathways. Thauera lacked energy supply due to the absence of most genes
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including idh and acnB that played an important role in TCA cycle, so its weak denitrification could be conjectured, demonstrating that Pannonibacter indicus played a
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key role in this reduce co-contaminants process. In summary, denitrification was the result of a variety of microbial cooperation actions, while quorum sensing, flagellar
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assembly, and the ABC transport system provided a possible microbial collaborative reduction of nitrates (Van Alst et al., 2007). The presence of different main chromate-reducing genes (chrR, frp, nemA, azoR, nfsA, nfsB, and kefF) indicated that different species had different chromium reductases, including flavin reductases, nitrate reductases, and flavin proteins. Notably, bins carrying 17
the chrR, nemA and azoR genes accounted for 43%, 36%, and 64% of the total bins assembled at pH 11.0, respectively, suggesting that they might be the primary genes for chromium reduction, which was consistent with the qPCR results. M11.6, M11.4, M11.13 and M11.14 only carried a chromium-reducing gene such as nemA or azoR but not contain chromium transporter genes (chrA and srpC), which might not be involved in the reduction of chromate. In addition, genes related to DNA protection
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pathway (recAG, ruvAB, and lexR) were found in lots of MAGs, which protected cells from ROS attack.
In summary, our results suggest that NO3- and Cr(VI) can be reduced efficiently by
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the co-metabolism of bacterial communities, and the coordination between different
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bacteria is essential for complete reduction of nitrate and chromium simultaneously. 4. Conclusions
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This study clarified the mechanisms of simultaneous nitrate and chromate reduction
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with bacteria, which probed the biogeochemical cycle of nitrogen and chromium in aquifers at different initial pH conditions with the participation of microorganisms. The
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survival of microorganisms depended on initial pH conditions. Different initial pH values made the shape and dominant species of the microorganisms different, which
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caused the microorganisms to show large differences in behavior under different initial pHs with 0.0014, 0.0016, 0.0045, 0.0256, 0.0892, and 0.0015 h-1 for nitrate reduction kinetic constant and 0.0015, 0.0018, 0.002, 0.0202, 0.0296, and 0.0013 h-1 for Cr(VI) reduction kinetic constant. Significant increments in metabolic pathways relative abundance (up to 40 folds) and relative expression level of functional genes (up to 10 18
folds) were observed in samples from pH 10.0 and 11.0 conditions, compared to the represent values from other pH conditions. To sum up, the above differences explained clearly why the removal efficiency was as high as 99.8% at initial pH of 10.0 and 11.0, but it was below 30% at other pHs. At the same time, the analysis of bins showed that potential inter-species cooperation under alkaline conditions had great potential to
(such as nitrate and chromate) in alkaline groundwater.
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Author contribution statement
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remove complex pollutants, which provided a reference for remediation of co-pollutants
Yutian Hu and Nan Chen conceived the idea and designed the research; Yutian Hu, Tong
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Liu, Linlin Ma, and Chen Si performed the experiment; Yutian Hu and Tong Liu
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analyzed data; Yutian Hu draft the manuscript; Yutian Hu, Nan Chen, Chuanping Feng, and Miao Li consulting the results, and revised the manuscript.
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Conflict of interest
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We declare that we do not have any commercial or associative interest that represents a
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conflict of interest in connection with the work submitted.
Declarations of interest: none Declaration of Competing Interest There is no competing interest to declare.
Acknowledgments 19
The authors acknowledge financial support from the Major Science and Technology Program for Water Pollution Control and Treatment (No.2017ZX07202002) and the
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National Natural Science Foundation of China (NSFC) (No. 21876159).
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Figure 1. Effect of the initial pH on the synchronous nitrate and Cr(VI) removal (a) the
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Figure 2. Microbial community compositions and functional species revealed by sequencing the microbial samples, before and after the experiment by (a) phylum-level
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Figure 3. The different metabolic pathway relative abundance including Degradation (a),
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Figure 4. A co-metabolic model of a nitrate-Cr(VI)-reducing bacterial community based on genomic bins generated from metagenomic data at initial pH 11.0.
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PII:
S0304-3894(20)30423-4
DOI:
https://doi.org/10.1016/j.jhazmat.2020.122434
Reference:
HAZMAT 122434
To appear in:
Journal of Hazardous Materials
Received Date:
3 February 2020
Revised Date:
28 February 2020
Accepted Date:
28 February 2020
Please cite this article as: Hu Y, Chen N, Liu T, Feng C, Ma L, Chen S, Li M, The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122434
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The mechanism of nitrate-Cr(VI) reduction mediated by microbial under different initial pHs
Yutian Hua, Nan Chena* [email protected], Tong Liua, Chuanping Fenga, Linlin Maa, Si Chena, Miao Lib a
School of Water Resources and Environment, MOE Key Laboratory of Groundwater
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Circulation and Environmental Evolution, China University of Geosciences (Beijing), Beijing, 100083, P.R. China b
School of Environment, Tsinghua University, Beijing 100084, P.R. China
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* Corresponding Author:
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(N. Chen), Phone: +86-10-82322281, Fax: +86-10-82321081
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Graphicalabstrct
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Highlights
Both Cr(VI) and nitrate were completely reduced at initial pH 10.0 and 11.0.
The primary genes for reducing nitrate and Cr(VI) were narG and azoR, respectively.
The dominant genus under alkaline environments was Pannonibacter.
The relative abundances of metabolic pathways were higher at pH 10.0 and 11.0.
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Potential interspecies cooperation was found to benefit removal of co-contaminant.
Abstract 2
To date, comparatively little research is known about the role of pH conditions in bioremediation of Cr(VI) contaminated aquifers. This study explored microbial Cr(VI) reduction and denitrification under different initial pHs. The underlying mechanism was also investigated. When testing 50 mg/L-N nitrate and 10 mg/L Cr(VI), complete contaminants removal was observed at initial pH 10.0 and 11.0, and only 10%-30% of removal achieved under other conditions, which might be ascribe to the significant up-
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regulation of functional genes narG (8.31 and 10.46 folds) and azoR (24.90 and 15.96
folds) at initial pH 10.0 and 11.0. Metagenomic sequencing showed that alkali tolerant bacteria played major roles in the NO3--Cr(VI) reduction (i.e. Pannonibacter increased
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by 13.08% and 25.24% at initial pH 10.0 and 11.0), and metabolic pathways of
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Degradation and Energy were found of increased abundant. Furthermore, a significative study suggested that potential interspecies cooperation existed at initial pH 11.0 to
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facilitating the simultaneous removal of contaminants, and Pannonibacter indicus might
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be an important participant in the degradation of contaminants. The results of this study will fully understand the metabolic patterns of bacteria under alkaline conditions, expand
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the range of available functional bacteria, and enhance the practical aspects of cocontaminants remediation.
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Keywords: pH; Nitrate-Cr(VI) bio-reduction; Metabolic pathway; Functional genes; Potential interspecies cooperation. 1. Introduction The residual nitrate in soil and groundwater initiated a cascade of large-scale environmental concerns with the widespread use of nitrogen fertilizer (Nieder et al., 3
2018). Excessive nitrate and nitrite (intermediate of nitrate reduction) loadings lead to severe water deterioration (such as eutrophication), and bring toxic effects on humans once using the contaminated water for drinking(Zhao et al., 2018). The U.S. Environment Protection Agency (USEPA) has set the maximum contaminant levels (MCL) of nitrate and nitrite at 10 and 1.0 mg L−1 (measured as N) in drinking water, respectively. Based on it, biological denitrification has attracted widespread attention
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(Fan et al., 2018), which consecutively reduce nitrate to nitrite, nitric oxide, nitrous oxide and finally nitrogen gas with the participation of nitrate reductase, nitrite
reductase, nitric oxide reductase, nitrous oxide reductase (Diaz and Rosenberg, 2008).
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At the same time, the combined pollution of nitrate and Cr(VI), occurs in
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intensive mining and smelting activities especially, poses a serious threat to the geological environment (Hausladen et al., 2018). Emissions from geological
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weathering and industrial processes arise the chromium flux in groundwater, primarily
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as Cr(III) and Cr(VI) (Jobby et al., 2018). Cr(VI) (0.1 mg L−1 of MCL in drinking water proposed by USEPA) is recognized as carcinogens and toxins that can easily
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penetrate cells and inhibit biological processes (Sun et al., 2019), which hampers the denitrification process through decreasing the rate of conversion nitrate to (Sun et al.,
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2019). Compared with the Cr(VI), Cr(III) is of lower mobility and bioavailable at neutral pH, and can be removed easily from water (Bhattacharya et al., 2019). Therefore, bio-reduction of Cr(VI) to Cr(III) is one promising approach to remove chromium from contaminated groundwater (Chen et al., 2019). The pH of the nitrate and Cr (VI) contaminated area is of highly complexity. For 4
example, numerous chromium pollution sites in California have pH values varied from 3.0 to 12.0 (Hausladen et al., 2018). pH, one of the most important environmental index, governs the biogeochemical activities of microorganisms (Han et al., 2019). The optimum pH for most environmental strains of denitrifying bacteria has been reported to be between 7.0 and 8.0 (Gan et al., 2019). However, some researchers found that the aerobic granular denitrifying sludge was more adaptable to acidic rather conditions
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alkaline conditions (Jiang et al., 2019). In addition, Cr(VI) reduction by some chromate
reduction bacteria (such as Geobacter sulfurreducens) was observed performed better
under neutral conditions (He et al., 2019), while another chromate reduction bacteria,
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Pannonibacter phragmitetus LSSE-09, preformed best in alkaline wastewaters (Xu et
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al., 2011). In summary, pH affects the metabolism of microorganisms which utilize the substrates in wastewater, resulting in variations in the sludge stability and pollutant
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removal performance. However, to date, the behaviors of microorganisms under stress
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from both Cr(VI) and nitrate at different initial pH values have not yet been reported, especially in terms of the energy supply, functional genes levels, and metabolism
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pathway. Therefore, it is important to investigate the response to the pH shock of the simultaneous Cr(VI) and nitrate removal in the mixed system by microorganisms, and it
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will help to have a deeper understanding of the biogeochemical behavior of nitrogen and chromium.
Herein, the feasibility and mechanism of simultaneous bio-reduction of nitrate and Cr(VI) were investigated. To illustrate the bacterial diversity, succession and metabolic mechanisms in the process of microorganism degrading co-contaminants 5
under different initial pH values, the adaptive mechanism of functional flora acclimating to both nitrate and Cr(VI) stress was studied using metagenomic analysis. In addition, metagenome binning was utilized to find the main and potential players of the microbial communities in NO3--Cr(VI) degrading under alkaline condition. This study is likely to shed new light on the attenuation mechanism of nitrate and chromium-contaminated aquifers, and provides further understanding of the
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biogeochemical cycle of nitrogen and chromium. 2. Materials and methods 2.1. Batch experiments
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A series of batch experiments were conducted in 100 mL serum bottles to explore
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the effect of the initial pHs on contemporaneous nitrate and Cr(VI) reduction in triplicate. Then, approximately 3 mL anaerobic sludge (the best removal performance according to
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the results of different sludge dosage in Fig. S1) washed by 0.9% NaCl was re-inoculated
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into a 100 mL serum bottle containing 100 mL of synthetic wastewater (0.4217 g CH3COONa, 0.0561 g K2HPO4 and 0.5 g yeast extract per liter) (Chen et al., 2019), in
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which 10 mg of Cr(VI) L-1 in the form of K2Cr2O7 and 50 mg L-1 of NO3--N in the form of NaNO3 were also involved in the synthetic wastewater, which were monitored using
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a spectrophotometer (DR6000, Hach, USA). The initial pH values were adjusted to 5.0, 7.0, 10.0, 11.0, and 12.0 through the manual addition of 1.0 M HCl or 1.0 M NaOH (Qi et al., 2020), which determined from the analysis of pH in the contaminated site such as California (the range of pH was from 5 to 12) (Dhal et al., 2013). Then gentle mixing was provided by a shaker at 150 rpm. Other reactors inoculated with autoclaved 6
anaerobic sludge and without anaerobic sludge were also carried out under the same experimental conditions for comparison. At the same time, the experiments with actual water diluted 10 times (the initial Cr(VI) and TOC concentration were 120.5 ± 0.6 and 10.25 ± 0.64 mg L-1, respectively) were also carried out, adding 50 mg L-1 NO3--N and 0.4217 g CH3COONa, the adjusted pH was 11.0 (the results was showed in Fig. S2). Of which, each serum bottle was purged with nitrogen for 20 minutes (Du et al., 2020). And
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mixed liquor samples were taken for nitrate, nitrite, ammonium, Cr(VI), total chromium,
electron transport system activity (ETSA), and cytochrome C (Cyt C) analysis, and the pH values were also monitored during the experiment. An environmental scanning
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electron microscope (ESEM, Quanta 200, Netherlands) and a fluorescence microscope
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(BX53, Olympus, Japan) with an ultraviolet blue light-excited filter plate for microorganisms were used after batch experiments at initial pH 5.0, 7.0, and 11.0 to
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explore the differences in morphology (Duan et al., 2019, 2020). These differences were
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distinguished with an AO/EB Staining Kit (Sangon, Shanghai, China), in which uniformly green, and orange represented live cells and necrotic cells, respectively.
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To characterize the reduction capacity of bacteria for Cr(VI) and nitrate, the effects of the initial Cr(VI) concentration (2, 5, 10, 20, or 50 mg L-1) and initial NO3--N
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concentration (10, 20, 50, 70, or 100 mg L-1) were also investigated. The other experimental conditions were similar with those described above. The chemical methods and microorganismal analysis used in this study, including the cultivation of microorganisms, cell lysis, and cell separation methods, are detailed in Supporting Information (SI). 7
2.2. Molecular analysis Four biofilm samples at the fastest reduction rate were collected, marked with its pH values (5.0, 7.0, 10.0, and 11.0), and another sample (named as control) was collected from initial sludge. Total genomic DNA was extracted and amplified with the PCR primers
338F
(5’-ACTCCTACGGGAGGCAGCAG-3’)
and
806R
(5’-
GGACTACHVGGGTWTCTAAT-3’). Amplicon sequencing was then performed using
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the MiSeq platform (Illumina, U.S.) at Majorbio Technology Ltd. (Shanghai, China). Raw sequences were submitted to the NCBI SRA database under the accession number
of SUB6207731. Variations in functional genes (narG, napA, nirS, nirK, norB, nosZ,
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chrA, chrR, nfsA, nemA, and azoR) were further quantified with quantitative real-time
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PCR (q-PCR) using a StepOne Plus™ Real-Time PCR System (Thermo, U.S.) while the total RNA was extracted with a FastRNA Pro Soil-Indirect Kit (MP Bio, U.S.), and the
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cDNA was inversely transcribed with Revert Premium Reverse Transcriptase (Beyotime,
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Shanghai, China). The q-PCR primers (Table S1) were synthesized by Sangon Biotech (Shanghai, China). Detailed methods about metagenome sequencing, assembling,
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annotating, and binning are provided in the SI Appendix. 3. Results and discussion
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3.1 Performance of co-contaminants at different pH values pH is an important environmental factor influencing biogeochemical processes
significantly (Mary Mangaiyarkarasi et al., 2011). In this study, the nitrate and Cr(VI) concentrations decreased progressively over time in the experimental treatments with varied pH values (Fig. 1a, b, Figure S1). The average removal rates were 0.52 and 0.83 8
mg h-1 for nitrate and 0.12 and 0.14 mg h-1 for Cr(VI), with k of 0.0256 and 0.0892 h-1 for nitrate and 0.0202 and 0.0296 h-1 for Cr(VI), respectively (Table S2). Compared with the removal efficiencies of about 67% for nitrate (Vilardi et al., 2020) and 75% for Cr(VI) (Vilardi et al., 2019) under the additions of biomass and nZVI particles, they were all up to 99.8% at initial pH of 10.0 and 11.0 with the participation of microorganisms. For treatments with other initial pH, the removal efficiencies for nitrate and Cr(VI) were
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only 10%-20% and 20%-30%, respectively, and the removal effects were not as well as
chemical method at pH 5.0-9.0 (Vilardi et al., 2018; Vilardi, 2019). It was also observed that the concentration of total Cr was below to 2 mg L-1 at initial pH of 10.0 and 11.0
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(Fig. S3c), implying that Cr(Ⅲ) was the main form of remaining chromium, and
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precipitates as hydroxides.
At initial pH of 10.0 and 11.0, the accumulation of nitrite was less than 6 mg L-1,
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and complete nitrite reduction was observed within 144 and 72 h, respectively (Fig. 1c),
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indicating that the potential NO2- reduction rate was at least as high as that of the NO3-. In addition, the complete reduction of nitrite was not observed at other pH values, which
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might be due to the significant effect of the initial pH on nitrite reduction pathway by affecting the activity of nitrite reductases (Kim et al., 2017). Furthermore, the pH was
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also a factor in determining the NO3- fate for microorganisms (Šimek et al., 2002), and the concentration of NH4+ remained below the detection limit throughout the experiment under all pH conditions (data not shown), which can be speculated that NH4+ might be used by microorganisms as a nitrogen source. It was found that the carbon source utilization varied at different initial pH 9
conditions from Fig. 1d, and the utilization under pH of 10.0 and 11.0 were higher than that under other initial pHs. Approximately 232.5 ± 5 mg L-1 CH3COONa is required to reduce 10 mg L-1 Cr(VI) and 50 mg L-1 NO3--N, suggesting that microorganisms need to use carbon sources as nutrients to survive and reduce nitrate and Cr(VI). Differences in the pollutions reduction kinetic constant by microorganism might be due to differences in the energy supply, and they were measured by the value of ETSA and Cyt
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C (Fig. 1e and f) which reflecting bacterial activities (Zimmermann et al., 1978). The ETSA value increased with the increasing initial pH (5.0-11.0). Additionally,
microorganism obtained electrons from Cyt C to reduce Cr(VI) and nitrate (Lovley, 1995;
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Zeng et al., 2018). Compared with the initial activity of bacteria after 36 h (the maximum
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reduction time for nitrate and Cr(VI)), the Cyt C concentration increased by 1.2-fold and 1.6-fold at initial pH of 10.0 and 11.0, respectively, while it decreased under other initial
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pH values. This finding might be due to the bacteriocin production, which had the ability
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to cause cell death and was favored under stressful growth conditions, suggesting that the initial pH had direct and indirect impacts on the growth and metabolic activity of
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microorganisms (Han et al., 2019). The analysis of the performance of simultaneous microbial nitrate and Cr(VI) reduction at initial pH 11.0 was shown in SI. In addition,
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the microbial analysis and co-contaminant reduction location were also in SI 2.1 and 2.2. Cr(VI) and nitrate in mixed systems affect each other's reduction, the presence of Cr(VI) had a negative effect on denitrification, while the removal of Cr(VI) accelerated with the increasing of nitrate concentration. Furthermore, membrane-associated enzymes had a dominant effect on the microbial mediated reduction of Cr(VI) and nitrate. 10
3.2 Microbial community analysis Fig. 2 showed the different relative abundances of the dominant bacteria in all reactors. Proteobacteria is a typical phylum in the reactor with initial pH 10.0 and 11.0 as well as the initial microorganisms, but it only accounted for 16.01% and 9.68% of the total bacterial community structure in the reactors with pH 5.0 and 7.0. And Fimicutes dominated the two reactors, resulting in the acceleration or suppression of denitrification
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and the removal of Cr(VI).
It was observed that the dominant genera were Enterococcus, Bacillus, and Pannonibacter under acidic, neutral, and alkaline conditions, respectively. This result
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indicated that bacteria were sensitive to the changes of pH values, and bacteria gradually
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adapted to the changes in the environment through self-adjustment. Once the initial pH value changed, different operating conditions might select specific bacterial populations
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to adapt to these conditions. Interestingly, Enterococcus (a metal-reducing bacteria)
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reportedly could reduce chromate (Sayel et al., 2012), but energy analysis (Fig. 1 e and f) implied that it might have lost the ability to reduce Cr(VI) owing to the lack of energy.
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A typical anaerobic bacteria -- Bacillus (61.87% with pH 7.0), belonging to Fimicutes, was associated with dissimilatory metal reduction and denitrification (Lovley, 1993; Ito
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et al., 2019; Ma et al., 2019). Yet the kinetic constant of denitrification in system was only 0.0016 h-1, because the action of Cr(VI) converting to Cr(III) led to more generation of ROS and lower abundance of antioxidant genes including lexR and mdh at pH 7.0 (Fig. S9), thus denitrification would be inhibited through exerting deleterious effects on proteins and DNA in cells at initial pH 7.0 (Lyon and Alvarez, 2008). Compared to the 11
initial microorganisms, the abundance of Pannonibacter (facultative anaerobic bacteria) increased by 13.08% and 25.24% in reactors at initial pH of 10.0 and pH 11.0, respectively. This result associated with both chromate bio-reduction and denitrification (Bai et al., 2019; Chai et al., 2019), providing evidence for higher nitrate and Cr(Ⅵ) reduction kinetic constant at same conditions. In addition, it was speculated that Chryseobacterium (25.46%) and Anaerobacillus (20.76%) (potential candidates for the
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wastewater treatment) had ability to reduce pollutant under alkaline environments
according to high percentage in reactors of pH 10.0 and 11.0. In summary, these findings
indicated that the pH had a significant effect on the bacterial community of the reactor,
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and the results confirmed that pH 10.0 and 11.0 might be more beneficial to bacterial
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metabolism than other pH values. Additionally, the microorganism morphology, primary reduction location under different pH values were also explored in SI 2.3, consistent with
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the results that organism evolution, microbes built self-protecting structures against pH
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or toxic pollutant shocks through forming different morphology. And the reduction of contaminants occurred primarily in the membrane region.
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3.3 Metabolic pathways analysis
It was observed that initial pH affected the microbial metabolic function (Fig. 3).
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The relative abundance of Degradation metabolic pathways including amine and polyamine degradation, fatty acid degradation, and carbohydrates and carboxylates degradation under alkaline conditions was higher than those under acidic and neutral conditions (Fig. 3a). These metabolic pathways played important roles in supporting microorganism survival through the degrading of amino acids, fatty acid, and 12
carbohydrates. The results suggested that microorganisms could utilize the sodium acetate to serve as sources of nutrients and energy, which was consistent with the degradation of carbon source at different initial pHs (Fig. 1d). Meanwhile, the relative abundance of other metabolic pathways including Energy supply (Fig. 3b), Response to Stimulus (Fig. 3c), and Cellular Processes (Fig. 3d) were consistent with Degradation, indicating that microorganisms could provide more energy to survive. Compared to other
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pHs, the microorganisms were more resistant to environmental changes through
metabolism such as TCA cycle, proteins involved in response to pH/DNA damage, and Quorum Sensing under alkaline conditions. Therefore, varied pH values could change
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the metabolic pathways by changing the gene abundance, benefitting the attenuation
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(acidic and natural pH) or propagation (alkaline pH) to improve the reduction ability of nitrate and chromate.
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The relative expression of denitrifying functional genes might provide evidence for the extent or level of active denitrification, including napA, narG, nirS, nirK, norB, and
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nosZ, which played major roles in the step of NO3-→NO2-, NO2-→NO, NO→N2O, N2O→
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N2, respectively (Henderson et al., 2010). As shown in Fig. 3e, pH exerted direct control on the reduction efficiency by affecting the relative expression of denitrification genes
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(Liu et al., 2010). The relative expression levels of all the genes were significantly upregulated at initial pH of 10.0 and 11.0, compared with other pH conditions. The narG gene (8.31 and 10.46 folds) had a significant enhancement compared to napA gene (2.80 and 4.46 folds) at initial pH of 10.0 and 11.0, probably because the anaerobic denitrifying bacteria primarily expressed membrane-bound nitrate reductase to catalyze the 13
denitrification process. During the nitrite reduction process, nirK gene was the major coding gene of nitrite reductase according to the results of qPCR. NorB upregulated 24.88 and 33.12 folds, while nosZ showed no obvious fluctuations at different pH values, suggesting that the pH affected the sequence reduction during the denitrification process (Wang et al., 2018). It was reported that genes including nemA, nfsA, azoR, chrA, and chrR were
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associated with the reduction and efflux of chromate (Thatoi et al., 2014). It was observed that the expression of nemA and nfsA did not differ significantly before and
after exposure to contaminants, while azoR provided a significant enhancement (24.90
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and 15.96 folds with pH 10.0 and 11.0 respectively) (Fig. 3e), which had an important
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role in microorganismal resistance to oxidative stress (Misal and Gawai, 2018). The gene of azoR also showed a similar Cr(VI) reductases characteristic (He et al., 2010), In
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addition, the genes of chrA and chrR had slightly increment, and they were responsible
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for chromate efflux (Alvarez et al., 2000) and reducing ROS production (Cheung and Gu, 2007), respectively. This result indicated that the cells had higher resistance to Cr(VI)
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toxicity under alkaline conditions.
3.4 Metabolic mechanism of bacterial cooperation
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3.4.1 Metabolic potential of MAGs The qualities of the MAGs were shown in Table S5 and the phylogenomic analyses
of 74 genomic bins also showed in SI. A metagenomic sequence analysis could provide definitive evidence for the positive selection of functional genes under nitrate and Cr(VI) stress. As shown in Fig. S9, Enterococcus (metal reducing bacteria) only carried the 14
napA gene, and this series of evidence supported the low kinetic constant for denitrification at pH 5.0 and 7.0. The Halomonas genus (M5.13 and M7.1) (a typical denitrification bacteria) had a complete denitrification pathway, and yet its denitrification capacity was weak under both acidic and neutral conditions, illustrating why denitrification efficiencies were lower. It was found that Thauera sp000310145 (a typical denitrification bacteria) (M5.4, M7.10, M10.14, and M11.12) encoded the key
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enzymes for complete denitrification. However, it was not conducive to nitrogen
removal according to the analysis of energy (Fig. 1) and qPCR (Fig. 3e). Rhodocyclaceae (Nitrate reduction bacteria) had no ability to use nitrite as an electron donor, causing a
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lack of nitrite reduction pathway, though it was extremely resistant to the pH (5.0-10.0).
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Contrary to the previous studies (Sotres et al., 2016; Tu et al., 2019; Vijay et al., 2019), Burkholderiaceae (a metal reducing bacteria) (Bins M5.11, M7.6, and M10.8) and
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Acholeplasmataceae (Bins M5.2, M5.5, M5.12, M7.3, M7.7, M7.12, and M7.16) had no
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genes for N metabolism at pH 5.0, 7.0, or 10.0, which might be due to the fact that the pH might lead to bacterial gene selection (Lund et al., 2014).
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It was found that 20.34% and 23.73% of the MAGs in the 59 MAGs did not carry the chromium reduction genes and the chromium transport genes under different pH
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values, indicating that the reduction of chromate in a mixed microbial system was not the function of a single microbe, but it depended on multiple microbial collaboration. It was found that the azoR gene (52.54%) played a major role in the reduction of chromate, compared to the nemA (32.20%) and chrR genes (32.20%), confirmed by genes expression analysis. NfsA (Erysipelotrichaceae (M5.3)) and nfsB (Burkholderiaceae 15
(M5.11, M7.6, M10.8), Dysgonomonadaceae (M10.3), Flavobacteriaceae (M10.1), and Pseudomonadaceae (M7.13)) were the major oxygen-insensitive nitroreductases, and Cr(VI) reduction gave rise to the product of reactive oxygen species (ROS) and Cr(V) (Ackerley et al., 2006; Shang et al., 2019). Thus, the presence of the ROS response enzymes (recA, recG, and ruvB) was essential for bacteria to protect themselves from the ROS attacks. In addition, the Chryseobacterium (potential candidates for the
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wastewater treatment) genus encoded enzyme about degrading Cr(VI) without
denitrification at pH 10.0, indicating that it had potential to reduce the chromate under alkaline conditions, consistent with Romaniuk et al. (2018). Moreover, the strain
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Pannonibacter indicus was isolated under alkaline conditions and had not been reported
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for denitrification or Cr(VI) reduction, but it had complete denitrification genes and chromium reduction transport genes. Hence it was inferred that Pannonibacter indicus
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had great potential for simultaneously reduce nitrate and chromium. Notably, the
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Pannonibacter (facultative anaerobic bacteria) genus could reduce nitrate (Bai et al., 2019), heavy metal (e.g. Cr(VI) (Chai et al., 2019) and As(V) (Bandyopadhyay et al.,
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2013)).
3.4.2 Bacterial metabolic pathways reconstruction at initial pH 11.0
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The cooperation between different bacteria and various functional genes could
further reveal the common adaptations of bacterial communities under alkaline stress and joint nitrate and chromium stress (Fig. 4). The discrepant nitrogen metabolism pathways in varied MAGs provided insight into possible interbacterial transfer of inorganic nitrogen compounds (Hug et al., 2015). We found that most microorganisms 16
that used inorganic N were specialists, harboring only one or two pathways (Fig. 4). The decoupling of modular pathways with more than one enzymatic step was common (Fig. 4). For denitrification, 85% of the microbes had only a partial pathway (i.e., only one step was present for the transformation of NO2− to NO, N2O, and N2), rather than the whole pathway (NO3− to N2). For example, some bins (e.g. M11.1, M11.2 et al) were able to conduct dissimilative nitrate reduction to nitrite with narGHI and napAB encoded
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enzymes, while M11.10 and M11.12 could only convert nitrate into nitrite assimilative by narB and nasAB encoded enzymes. M11.4 and M11.11 might use nitric oxide as
electron donors, while M11.9 converted nitrous oxide into nitrogen and released it into
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the atmosphere. Meanwhile, M11.14 had ability to convert nitrogen to ammonia nitrogen
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(nifDKH) as a nutrient for its own use through fixing nitrogen. Notably, only the strains of Pannonibacter indicus and Thauera (denitrifying bacteria) had complete
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denitrification pathways. Thauera lacked energy supply due to the absence of most genes
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including idh and acnB that played an important role in TCA cycle, so its weak denitrification could be conjectured, demonstrating that Pannonibacter indicus played a
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key role in this reduce co-contaminants process. In summary, denitrification was the result of a variety of microbial cooperation actions, while quorum sensing, flagellar
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assembly, and the ABC transport system provided a possible microbial collaborative reduction of nitrates (Van Alst et al., 2007). The presence of different main chromate-reducing genes (chrR, frp, nemA, azoR, nfsA, nfsB, and kefF) indicated that different species had different chromium reductases, including flavin reductases, nitrate reductases, and flavin proteins. Notably, bins carrying 17
the chrR, nemA and azoR genes accounted for 43%, 36%, and 64% of the total bins assembled at pH 11.0, respectively, suggesting that they might be the primary genes for chromium reduction, which was consistent with the qPCR results. M11.6, M11.4, M11.13 and M11.14 only carried a chromium-reducing gene such as nemA or azoR but not contain chromium transporter genes (chrA and srpC), which might not be involved in the reduction of chromate. In addition, genes related to DNA protection
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pathway (recAG, ruvAB, and lexR) were found in lots of MAGs, which protected cells from ROS attack.
In summary, our results suggest that NO3- and Cr(VI) can be reduced efficiently by
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the co-metabolism of bacterial communities, and the coordination between different
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bacteria is essential for complete reduction of nitrate and chromium simultaneously. 4. Conclusions
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This study clarified the mechanisms of simultaneous nitrate and chromate reduction
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with bacteria, which probed the biogeochemical cycle of nitrogen and chromium in aquifers at different initial pH conditions with the participation of microorganisms. The
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survival of microorganisms depended on initial pH conditions. Different initial pH values made the shape and dominant species of the microorganisms different, which
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caused the microorganisms to show large differences in behavior under different initial pHs with 0.0014, 0.0016, 0.0045, 0.0256, 0.0892, and 0.0015 h-1 for nitrate reduction kinetic constant and 0.0015, 0.0018, 0.002, 0.0202, 0.0296, and 0.0013 h-1 for Cr(VI) reduction kinetic constant. Significant increments in metabolic pathways relative abundance (up to 40 folds) and relative expression level of functional genes (up to 10 18
folds) were observed in samples from pH 10.0 and 11.0 conditions, compared to the represent values from other pH conditions. To sum up, the above differences explained clearly why the removal efficiency was as high as 99.8% at initial pH of 10.0 and 11.0, but it was below 30% at other pHs. At the same time, the analysis of bins showed that potential inter-species cooperation under alkaline conditions had great potential to
(such as nitrate and chromate) in alkaline groundwater.
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Author contribution statement
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remove complex pollutants, which provided a reference for remediation of co-pollutants
Yutian Hu and Nan Chen conceived the idea and designed the research; Yutian Hu, Tong
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Liu, Linlin Ma, and Chen Si performed the experiment; Yutian Hu and Tong Liu
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analyzed data; Yutian Hu draft the manuscript; Yutian Hu, Nan Chen, Chuanping Feng, and Miao Li consulting the results, and revised the manuscript.
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Conflict of interest
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We declare that we do not have any commercial or associative interest that represents a
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conflict of interest in connection with the work submitted.
Declarations of interest: none Declaration of Competing Interest There is no competing interest to declare.
Acknowledgments 19
The authors acknowledge financial support from the Major Science and Technology Program for Water Pollution Control and Treatment (No.2017ZX07202002) and the
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ro of
National Natural Science Foundation of China (NSFC) (No. 21876159).
20
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Figure 1. Effect of the initial pH on the synchronous nitrate and Cr(VI) removal (a) the
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Figure 2. Microbial community compositions and functional species revealed by sequencing the microbial samples, before and after the experiment by (a) phylum-level
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Figure 3. The different metabolic pathway relative abundance including Degradation (a),
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Figure 4. A co-metabolic model of a nitrate-Cr(VI)-reducing bacterial community based on genomic bins generated from metagenomic data at initial pH 11.0.
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