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Article

Efficiency Determination and Mechanism Investigation of Autotrophic Denitrification Strain F1 to Promote Low-Carbon Development

1
Shaanxi Key Lab of Environmental Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China
2
XAUAT UniSA An De College, Xi’an University of Architecture and Technology, Xi’an 710055, China
3
Department of Environmental Engineering, College of Environmental & Resource Science, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Water 2022, 14(21), 3353; https://doi.org/10.3390/w14213353
Received: 23 August 2022 / Revised: 1 October 2022 / Accepted: 18 October 2022 / Published: 22 October 2022

Abstract

:
Shewanella sp. strain F1, isolated from a lab-scale Fe(II)−dependent anaerobic denitrifying reactor, could reduce nitrate by oxidizing Fe(II). Its nitrate reduction rate and Fe(II) oxidation rate were 0.48 mg/(L·h) and 5.05 mg/(L·h) at OD600 of 0.4786 with a five-fold diluent. Shewanella sp. was popular in Fe(III) reduction. Fewer studies about its ability for Fe(II) oxidation are available. The low pH was determined to be the switch for Shewanella sp. strain F1 to perform Fe(III) reduction or Fe(II) oxidation. Even under a low pH, the produced Fe(III) precipitated around cells from iron encrustation. By observation of the morphologies of strain F1, two corresponding microbial mechanisms were proposed. One was named Cyc 2−based Fe(II)-dependent denitrification, in which Fe(II) was oxidized by Cyc 2 in the outer cell membrane, and the produced Fe(III) precipitated on the cell wall surface to form tiled iron encrustation. The other was named Cyc 1−based Fe(II)−dependent denitrification, in which Fe(II) was oxidized on the existing iron precipitation on the cell wall surface to form towery iron encrustation, and the electron was transported to Cyc 1 in the periplasm. The efficiency determination and mechanism investigation of strain F1 will promote the development of autotrophic denitrification technology and meet the requirement of a low−carbon policy.

1. Introduction

Nitrate, as one of the most common pollutants in underground and surface water, may cause serious environmental problems and great public health risks [1]. It is necessary to remove nitrate from water bodies [2]. In the traditional denitrification process, organics are necessary as electron donors, thus increasing the cost and meanwhile generating CO2, which goes against the aims of the low−carbon economy. Recently, an autotrophic denitrification process with Fe(II) as an electron donor to reduce nitrate in low C/N ratio wastewater has attracted much attention, because (1) it is economical as ferrous salt is much cheaper than organics, e.g., methanol and acetate, (2) it is environmentally friendly because its product is ferric salt rather than greenhouse gas, and (3) its product could be used as a precipitant to remove phosphate and arsenate from wastewater [3,4,5]. As a new and valuable bioprocess, Fe(II)−dependent autotrophic denitrification is a significant discovery for environmental engineering.
Fe(II)−dependent autotrophic denitrification had been carried out in lab-scale reactors and the volumetric removal rate reached 0.70 kg-N/(m3∙d) [6,7,8]. To improve the efficiency and reveal the autotrophic denitrification mechanism, pure culture should be isolated and characterized. As was reported, Fe(II)−dependent autotrophic denitrification strains were distributed widely in Acidovorax, Dechloromonas, Paracoccus, and Pseudogulbenkiania [9,10,11]. However, no information about the autotrophic denitrification mechanism is available.
Therefore, several strains were isolated from the Fe(II)−dependent autotrophic denitrification reactor in our pre-experiments. Among them, strain F1 was chosen for further study as it showed remarkable denitrification activity with Fe(II) as the electron donor. Strain F1 was identified as Shewanella sp. However, to our knowledge, Shewanella sp. was popular for its ability to reduce Fe(III) and its contribution to the Fe cycle [12,13,14]. Few reports of their ability to oxidize Fe(II) are available.
Based on the above, first of all, the phenomenon of strain F1 to oxidize Fe(II) and reduce nitrate should be re−verified. The Fe(II) oxidation and nitrate reduction rates should be calculated by Haldane modeling. Then morphological observations on strain F1 cells should be performed to reveal the mechanisms. All results obtained will support the development of Fe(II)−dependent autotrophic denitrification technology, and achieve carbon zero during the denitrification process.

2. Materials and Methods

2.1. Shewanella sp. Strain F1

In total, 7 strains were isolated from a lab−scale Fe(II)−dependent autotrophic denitrification reactor by streak plate method. Among them, strain F1 showed the highest denitrification activity with Fe(II) as an electron donor (Figure S1); thus, the following experiments were carried out by strain F1. By 16s rRNA sequencing analysis, strain F1 was classified into Shewanella sp. The GenBank accession number of Shewanella sp. strain F1 was KT932999 (Figure S2, noting that the strain was named strain NF−1 in GenBank). Figure S3 showed the detailed morphology characteristics of strain F1. Its colonies presented milk white, spheroidal with a diameter of about 0.7 mm, smooth, convex, and with irregular edges after cultivating for 5 days. Gram test showed that strain F1 was Gram−negative.
For all experiments, a cell suspension of strain F1 was used to make sure equal biomass was added for parallel tests. Cells on the solid medium were washed with sterile water (three times) and collected into a centrifuge tube to be stored at 4 °C as the cell suspension.

2.2. Chemicals and Media

To cultivate the Shewanella sp. strain F1, the electron donor was supplied in the form of FeSO4∙7H2O, while the electron acceptor was supplied in the form of NaNO3. The chemicals in this experiment were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China.
The basal medium was shown in Table 1. The pH of the medium was regulated to 6.2 ± 0.2 by adding NaOH (1 M) and HCl (1 M). The cultivation of strain F1 both on the solid medium and in the liquid medium was performed in the anaerobic incubator statically at 30 °C.

2.3. Morphology Observation

The morphology of the bacterial colony on Petri dishes was observed by a stereoscope (Carl Zeiss AG, Berlin, Germany). A Gram staining test was performed under the aseptic condition and examined under an optical microscope (Leica, Berlin, Germany). The surface morphology of a single cell was observed by a field emission scanning electron microscope (FESEM) (Hitachi, Tokyo, Japan), and the profile morphology of a single cell was observed by a transmission electron microscope (TEM) (Gtontorn, NJ, USA). Moreover, in view of the specificity of iron−oxidizing bacteria, an energy−dispersive spectrometer (EDS) (Arun, Oxford, UK) was used to determine the extracellular substance.
All bio-samples for morphology observations were collected and stored in glutaric dialdehyde solution at 4 °C. Further treatment and observation were delegated to the Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University.

2.4. Determination of the Denitrification Activity

To determine the Fe(II)−dependent autotrophic denitrification activity of Shewanella sp. strain F1, batch experiments were performed in serum bottles. All bottles with the medium were aerated with argon gas for 10 min and sterilized to get rid of oxygen as well as microbes. The dilution ratio was 1:20. All operations were sterile, and bottles were cultivated in an Electrotek anaerobic workstation at 32 °C. Dividing the removed nitrate (and Fe(II)) by the reaction time, the nitrate reduction (and Fe(II) oxidation) rate was calculated.
Further, the effects of the substrate concentration on the activity of strain F1 were studied. The gradient nitrate–nitrogen concentration was 21, 42, 63, 84, 105, 147, or 210 mg/L, while the gradient Fe(II) concentration was 420, 840, 1260, 1680, 2100, 2940, or 4200 mg/L. For each concentration, a parallel group was set, and the concentrations of NO3−N (and Fe(II)) were tested after 24 h. The NO3−N reduction rate and Fe(II) oxidation rate were calculated as described above. The Haldane model was used to fit the data to see the effects of substrate concentration on nitrate reduction rate and Fe(II) oxidation rate [15] (Equation (1)):
r i = r m a x 1 + K m [ S ] + [ S ] K i                    
where ri is the removal rate (mg/(L·h)), rmax is the maximum removal rate (mg/(L·h)), S is the substrate concentration (mg/L), Km is the half−rate constant (mg/L), and Ki is the half-inhibition constant (mg/L).

2.5. Effects of Initial pH

Batch experiments were performed in serum bottles to determine the effects of initial pH on the Fe(II)−dependent autotrophic denitrification activity. Bottles with the medium were purged with argon gas for 10 min to maintain anaerobic conditions. The bacteria suspension was inoculated into bottles at a dosage of 5% (v/v) by a sterile operation. The gradient initial pH was set to 5.4, 5.8, 6.2, 6.6, or 7.0 using 1 M HCl and 1 M NaOH. The concentrations of nitrate and Fe(II) were tested after 24 h. The GaussAmp model (Equation (2)) was used to fit the data to determine the optimal initial pH [16]:
r = y 0 + r m a x e 2 ( p H p H m ) 2 w                  
where r is the determined nitrate/Fe(II)removal rate (µg/h), y0 is the basal activity value (µg/h), rmax is the maximum removal rate at different initial pH (µg/h), pH is the initial pH value, pHm is the optimal initial pH value, and w is the reaction coefficient.

2.6. Chemical Analysis

All the samples were analyzed immediately after sampling. The concentrations of nitrate–nitrogen and Fe(II) were determined according to standard methods [17]. The pH values were determined by an S20K pH meter (Mettler Toledo, Geneva, Switzerland). The OD600 value of cell suspension was determined by the spectrophotometer (Shimadzu, Tokyo, Japan) at 600 nm.

3. Results

3.1. The Specific Denitrification Activity of Shewanella sp. Strain F1

A batch test was performed to determine the Fe(II)−dependent autotrophic denitrification activity of Shewanella sp. strain F1. Both nitrate reduction and Fe(II) oxidation by Shewanella sp. strain F1 followed first-order kinetics (Figure 1A,B). When the OD600 was 0.4786 with a five−fold diluent, the nitrate reduction rate was 0.48 mg/(L·h) and the Fe(II) oxidation rate was 5.05 mg/(L·h). Fitted with the first-order kinetics, the rate constant of nitrate reduction and Fe(II) oxidation by Shewanella sp. strain F1 was 0.0089 h−1 and 0.0085 h−1, respectively. The Fe(II)−dependent autotrophic denitrification activity of Shewanella sp. strain F1 was much higher than that of reported strains, e.g., Pseudogulbenkiania strain 2002, Microbacterium strain W5, and Citrobacter freundii strain PXL1 [11,18,19]. This remarkable activity was probably because strain F1 was isolated from the high−rate lab-scale Fe(II)−dependent autotrophic denitrification reactor.
The Fe(II)−dependent autotrophic denitrification activity of Shewanella sp. strain F1 varied at different substrate concentrations (Figure 1C,D). When the initial nitrate concentration ranged from 21 to 147 mg N/L, the nitrate reduction rate went from 0.45 to 2.56 mg N/(L·h). When the initial nitrate concentration increased to 210 mg N/L, the nitrate reduction rate was inhibited and decreased to 2.31 mg N/(L·h). The Fe(II) oxidation rate shared the same trend as the nitrate reduction rate. When the initial Fe(II) concentration ranged from 420 mg to 2940 mg Fe/L, the Fe(II) oxidation rate went from 15.37 to 81.94 mg Fe/(L·h). When the initial Fe(II) concentration increased to 4200 mg Fe/L, the Fe(II) oxidation rate was inhibited and decreased to 77.40 mg Fe/(L·h). Fitted with the Haldane model (Equation (1)), the maximum removal rate (vmax), the half−rate constant (Km), and the half−inhibition constant (Ki) of nitrate were 2.64 mg N/(L·h), 61.25 mg N/L, and 349.78 mg N/L, respectively. Meanwhile, the vmax, Km, and Ki of Fe(II) were 81.94 mg Fe/(L·h), 942.93 mg Fe/L, and 7811.40 mg Fe/L, respectively (Figure 1). Shewanella sp. strain F1 has broad prospects to be applied as an optimal inoculum for autotrophic denitrification technology.

3.2. The Effects of pH on the Denitrification Activity of Shewanella sp. Strain F1

Figure 2A,B illustrate the autotrophic denitrification activity by Shewanella sp. strain F1 at different pH values. Data at pH 8.2 are not shown as Fe(II) precipitated as soon as it was added into the reaction system. Little nitrate reduction was observed at pH 7.2, but the decrease in Fe(II) was remarkable. The decrease in Fe(II) at pH 7.2 was caused by chemical precipitation rather than bio-oxidation. Shewanella sp. strain F1 had better activity at pH 6.2 than at pH 5.2, because the neutral pH condition benefited the denitrification process [20].
The pH affected Shewanella sp. strain F1’s ability to perform Fe−(II)dependent autotrophic denitrification. A range of pH values was set to determine the optimal pH for strain F1 to reduce nitrate and oxidize Fe(II). The results are shown in Figure 2C,D. In the range of tested pH values, both the nitrate reduction rate and Fe(II) oxidation rate showed peak values, and the peak values were obtained when the initial pH was 6.6. When the initial pH was lower than 6.6, both the nitrate reduction rate and Fe(II) oxidation rate increased to their peak values, then began to decrease when the initial pH exceeded 6.6. Fitted by the GaussAmp model (Equation (2)), the optimal pH for nitrate reduction and Fe(II) oxidation was 6.66 and 6.69, respectively (Figure 2C,D).

3.3. The Morphology Observation of Shewanella sp. Strain F1

Figure 3 shows the morphological characteristics of strain F1. When cultivating strain F1 with nitrate and ferrous salts at pH 6.6, some attachments were observed on the cell wall surface. They were tiled or towery. The tiled attachment formed a layer to cover the cell, while the towery attachment accumulated on the specific point on the cell wall surface.
The attachment on the cell wall surface was speculated to be ferric/ferrous oxyhydroxide [21,22,23,24]. To ascertain the composition of attachments, EDS was used and the results are shown in Figure 4. The major elements on the cell wall surface were carbon, oxygen, and iron, in descending order (Figure 4C–E). Carbon was distributed evenly in the view, while oxygen and iron were distributed only on the cell wall surface. Iron and oxygen accounted for only a small part compared with carbon, because the conductive adhesive that acted as the carrier for FESEM samples was made of carbon. From Figure 4, we concluded that the attachment on the cell wall surface mainly consisted of iron and oxygen. The product of Fe(II) oxidation, Fe(III), tended to precipitate on the cell wall surface as soon as it was produced because of the high pH (~6.2). The precipitated Fe(III) formed attachments to cells and will be referred to as iron encrustation hereafter.

4. Discussion

4.1. The Switch for Shewanella sp. Strain F1 to Perform Autotrophic Denitrification

In recent decades, Shewanella spp. have received attention for their role in the iron cycle [25,26,27]. However, Shewanella spp. were only deemed to reduce Fe(III) [28,29,30]. Few reports of their ability to oxidize Fe(II) are available. From the results in Section 3.1, we see that Shewanella sp. strain F1 possesses remarkable Fe(II)−dependent autotrophic denitrification activity. That indicates that the strain could oxidize Fe(II) and reduce nitrate effectively.
As reported, the Shewanella sp. prefer to live in alkaline or circumneutral pH environments, and only in these environments could they reduce Fe(III) [31,32]. In this study, Shewanella sp. strain F1 was tested in weakly acidic conditions (pH 6.2) and it oxidized Fe(II). As shown in Section 3.2, obvious Fe(II)−dependent autotrophic denitrification activity was only observed at pH 5.2 and 6.2. Thus, the pH was speculated to be the switch for Shewanella sp. strain F1 to oxidize Fe(II) rather than reduce Fe(III). The reason might be that the low pH inhibited the activity of iron reductase, and the microorganism had to exploit other ways to obtain energy. However, further studies need to be carried out to prove this hypothesis.

4.2. The Conceptual Mechanism of Shewanella sp. Strain F1 to Perform Autotrophic Denitrification

From Section 3.3, two kinds of iron encrustation were observed around cells, in tiled or towery shapes (Figure 3). From our previous publications and literature by other research groups [19,20,21,22,33,34], the iron encrustations were mainly ferric compounds with a small part of ferrous compounds. From Figure 3C,D, for most of the strain F1 cells, both tiled or towery iron encrustation existed. The iron encrustation affected the nutrient transportation and decreased the activity of strain F1 cells. However, there were still large areas of exposure for each cell, signifying the nutrient transportation was not cut off. Based on the formation of iron encrustation and the electron transport chain of denitrification, two conceptual mechanisms of autotrophic denitrification by Shewanella sp. strain F1 are shown in Figure 5. In Figure 5A, which shows the formation of the tiled iron encrustation, the oxidation of Fe(II) to Fe(III) is carried out by a C−type cytochrome (Cyc 2) present in the outer cell membrane [35]. This is called Cyc 2−based autotrophic denitrification. Electrons are transferred to the cytoplasmic membrane through a terminal ‘aa3 cytochrome oxidase’, and again to the electron transport chain that supports the downstream denitrification. Molecules that shuttle electrons are Cyc 1 and rusticyanin [36,37]. Figure 5B shows the formation of the towery iron encrustation. Different from the formation of tiled iron encrustation, to form the towery iron encrustation, Fe(II) gives the electron to the iron precipitate and becomes Fe(III) [38,39]. Fe(III) continues precipitating on the iron precipitate. The electron travels to Cyc 1 and participates in the electron transport chain, which supports the denitrification process. This is called Cyc 1−based autotrophic denitrification. Both mechanisms were observed in this study.

5. Conclusions

Shewanella sp. strain F1 showed remarkable autotrophic denitrification activity and could be the optimal seeding sludge for Fe(II)−dependent autotrophic denitrification processes. Differing from the conventional understanding of Shewanella sp. being the Fe(III) reducer, Shewanella sp. strain F1 showed Fe(II) oxidizing activity with nitrate as the electron acceptor. That may ‘clue’ new Fe cycles in the lithosphere and biosphere. The pH was determined to be the trigger for Shewanella sp. strain F1 to perform Fe(III) oxidation from Fe(II) reduction under batch tests. Further observation and more proving tests should be carried out. Combining the electron transport chain of denitrification and the observed results, Cyc 2−based and Cyc 1−based autotrophic denitrification mechanisms were proposed. Even though the formation of iron encrustation was inevitable, there were still exposed areas for strain F1 cells to transport nutrients, maintaining the activity of nitrate reduction and Fe(II) oxidation. The isolation and efficiency determination of Shewanella sp. strain F1, and the corresponding mechanism investigation will promote the development of Fe(II)−dependent autotrophic denitrification technology that meets the requirement of a low−carbon policy.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w14213353/s1, Figure S1: The nitrate and ferrous ion removal efficiency of seven isolated strains from the lab-scale reactor; Figure S2: Phylogenetic tree of strain F1. Numbers in parentheses represent the sequences’ accession number in GenBank. The number at each branch points is the percentage supported by bootstrap (1000 re-samplings). Bar: 0.5% sequence divergence; Figure S3: Morphological characteristics of strain F1. A: colony morphology by the stereoscope; B: Gram stain observed by the optical microscope.

Author Contributions

Conceptualization, P.Z. and R.W.; methodology, R.W.; software, S.-Z.W.; validation, F.-K.Q. and B.-Y.L.; formal analysis, F.-K.Q. and B.-Y.L.; investigation, S.-Z.W.; resources, R.W. and P.Z.; data curation, R.W.; writing—original draft preparation, F.-K.Q.; writing—review and editing, R.W.; visualization, R.W.; supervision, R.W.; project administration, R.W.; funding acquisition, R.W. and P.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant no. 51808433).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Nitrate reduction and Fe(II) oxidation activities of Shewanella sp. strain F1. (A) The decrease of nitrate and Fe(II) concentration along with cultivation time. (B) The first−order kinetics fitting of nitrate reduction and Fe(II) oxidation rate by strain F1. (C) The nitrate reduction rate by strain F1 at different nitrate concentrations. (D) The Fe(II) oxidation rate by strain F1 at different Fe(II) concentrations.
Figure 1. Nitrate reduction and Fe(II) oxidation activities of Shewanella sp. strain F1. (A) The decrease of nitrate and Fe(II) concentration along with cultivation time. (B) The first−order kinetics fitting of nitrate reduction and Fe(II) oxidation rate by strain F1. (C) The nitrate reduction rate by strain F1 at different nitrate concentrations. (D) The Fe(II) oxidation rate by strain F1 at different Fe(II) concentrations.
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Figure 2. Effects of initial pH on the activity of Shewanella sp. strain F1. (A) The nitrate reduction activity along with cultivation time. (B) The Fe(II) oxidation activity along with time. (C) The removal rate of nitrate−nitrogen under different initial pHs. (D) The removal rate of Fe(II) under different initial pHs.
Figure 2. Effects of initial pH on the activity of Shewanella sp. strain F1. (A) The nitrate reduction activity along with cultivation time. (B) The Fe(II) oxidation activity along with time. (C) The removal rate of nitrate−nitrogen under different initial pHs. (D) The removal rate of Fe(II) under different initial pHs.
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Figure 3. Morphological characteristics of strain F1. (A,B) Surface morphology of cell wall by FETEM. (C,D) Profile morphology of the cell wall by TEM. Arrows refer to the iron encrustation.
Figure 3. Morphological characteristics of strain F1. (A,B) Surface morphology of cell wall by FETEM. (C,D) Profile morphology of the cell wall by TEM. Arrows refer to the iron encrustation.
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Figure 4. FESEM images of strain F1 and the surface contents analysis by EDS. (A) The view of the targeted cell and the composition of main elements. (B) Overlay chart of the distribution of carbon, oxygen, and iron. (C) Distribution of carbon in the view. (D) Distribution of oxygen in the view. (E) Distribution of iron in the view.
Figure 4. FESEM images of strain F1 and the surface contents analysis by EDS. (A) The view of the targeted cell and the composition of main elements. (B) Overlay chart of the distribution of carbon, oxygen, and iron. (C) Distribution of carbon in the view. (D) Distribution of oxygen in the view. (E) Distribution of iron in the view.
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Figure 5. The conceptual autotrophic denitrification mechanisms by Shewanella sp. strain F1. The tiled iron encrustation was formed as shown in (A), while the towery iron encrustation was formed as shown in (B).
Figure 5. The conceptual autotrophic denitrification mechanisms by Shewanella sp. strain F1. The tiled iron encrustation was formed as shown in (A), while the towery iron encrustation was formed as shown in (B).
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Table 1. Composition of the basal medium.
Table 1. Composition of the basal medium.
ComponentConcentrationComponentConcentration
NaNO30.3 g/LFeSO4∙7H2O5 g/L
NaHCO32.5 g/LMgSO4∙7H2O0.5 g/L
CaCl2∙2H2O0.01 g/L(NH4)2SO40.28 g/L
KH2PO40.25 g/LEDTA3.00 mg/L
Na2MoO4∙2H2O0.036 mg/LH3BO30.010 mg/L
CoCl2∙6H2O0.19 mg/LNiCl3∙6H2O0.024 mg/L
MnCl2∙2H2O0.50 mg/LCuCl2∙2H2O0.010 mg/L
ZnCl20.07 mg/L
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Qin, F.-K.; Wan, S.-Z.; Liu, B.-Y.; Wang, R.; Zheng, P. Efficiency Determination and Mechanism Investigation of Autotrophic Denitrification Strain F1 to Promote Low-Carbon Development. Water 2022, 14, 3353. https://doi.org/10.3390/w14213353

AMA Style

Qin F-K, Wan S-Z, Liu B-Y, Wang R, Zheng P. Efficiency Determination and Mechanism Investigation of Autotrophic Denitrification Strain F1 to Promote Low-Carbon Development. Water. 2022; 14(21):3353. https://doi.org/10.3390/w14213353

Chicago/Turabian Style

Qin, Fang-Kai, Si-Zhuo Wan, Bing-Yin Liu, Ru Wang, and Ping Zheng. 2022. "Efficiency Determination and Mechanism Investigation of Autotrophic Denitrification Strain F1 to Promote Low-Carbon Development" Water 14, no. 21: 3353. https://doi.org/10.3390/w14213353

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