Effect of Fe (III) addition on ammonium loss and associated microbial gene expression in soils

Abstract

The combined effects of temperature, Fe (III) contents, NH₄⁺-N contents, and soil-liquid ratio were evaluated relative to the loss of NH₄⁺-N in soils using a response surface methodology (RSM). The microbial mechanisms were explored for nitrogen transformation by quantifying functional genes related to nitrification and denitrification. According to parameter optimization analysis for prediction equation, the maximum NH₄⁺-N loss was 86.1% under the conditions of 17.0 °C, 0.772 g·kg^-1 Fe (III), 21.9 mg·kg⁻¹ NH₄⁺-N, and soil: liquid ratio of 1:1. The prediction result was similar to experimental data in the current study, which the NH₄⁺-N loss was 83.2% under the condition of 25 °C, 0.723 g·kg^-1 Fe (III), 20 mg·kg⁻¹ NH₄⁺-N, and soil-liquid ratio of 1:1. While the N₂O flux reached its minimum value of 8.35 μg·m⁻²·h^–¹ under the experimental conditions, correlating with gene copy numbers for ammonia-oxidizing bacteria ammonia monooxygenase subunit A gene (AOB-amoA), and nitrite reductase genes (nirK) were maximum values of 4.5×10⁵ and 4.8×10⁵ copies·g^-1, respectively. NH₄⁺-N loss resulted from multiple interacting processes beyond ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) mediated oxidation. The research findings can provide insights for reducing nitrogen application to avoid NH₄⁺ toxicity and increasing soil planting suitability.

Full Article

Effect of Fe (III) Addition on Ammonium Loss and Associated Microbial Gene Expression in Soils

Liyu Cheng,^a Hongjie Zhu,^b Weihao Xing,^a and Benjun Zhou  ,^a,*

The combined effects of temperature, Fe (III) contents, NH₄⁺-N contents, and soil-liquid ratio were evaluated relative to the loss of NH₄⁺-N in soils using a response surface methodology (RSM). The microbial mechanisms were explored for nitrogen transformation by quantifying functional genes related to nitrification and denitrification. According to parameter optimization analysis for prediction equation, the maximum NH₄⁺-N loss was 86.1% under the conditions of 17.0 °C, 0.772 g·kg^-1 Fe (III), 21.9 mg·kg⁻¹ NH₄⁺-N, and soil: liquid ratio of 1:1. The prediction result was similar to experimental data in the current study, which the NH₄⁺-N loss was 83.2% under the condition of 25 °C, 0.723 g·kg^-1 Fe (III), 20 mg·kg⁻¹ NH₄⁺-N, and soil-liquid ratio of 1:1. While the N₂O flux reached its minimum value of 8.35 μg·m⁻²·h^–¹ under the experimental conditions, correlating with gene copy numbers for ammonia-oxidizing bacteria ammonia monooxygenase subunit A gene (AOB-amoA), and nitrite reductase genes (nirK) were maximum values of 4.5×10⁵ and 4.8×10⁵ copies·g^-1, respectively. NH₄⁺-N loss resulted from multiple interacting processes beyond ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) mediated oxidation. The research findings can provide insights for reducing nitrogen application to avoid NH₄⁺ toxicity and increasing soil planting suitability.

DOI: 10.15376/biores.20.4.8713-8724

Keywords: Nitrogen fertilizers; Ammonium; Fe (III); Gene copy numbers

Contact information: a: School of Resources and Environmental Engineering, Hefei University of Technology, Hefei, 230009, China; b: Institute of Agricultural Engineering, Anhui Academy of Agricultural Sciences, Hefei 230001, China; *Corresponding author: benjamin8374@sina.com

INTRODUCTION

The use of chemical fertilizers is one of the key strategies to improve soil fertility and crop yield. Nitrate (NO₃^–)- and ammonium (NH₄⁺)-based nitrogen fertilizers are commonly employed to pursue high yields in modern intensive agriculture (Li et al. 2017), NH₄⁺-N is a preferred nitrogen source for most plant species in a certain concentration range, whereas at high concentrations, NH₄⁺-N has been found to inhibit the growth of most plants due to NH₄⁺ toxicity (Britto and Kronzucker 2002). Characteristics of NH₄⁺ toxicity are chlorosis of leaves, root suppression, stunted shoot growth, and so on (Li et al. 2014), which can be influenced by a combination of physical conditions, chemical conditions, and microorganisms of the soils, and depend on plant species and varieties (Cruz et al. 2011).

In recent years there has been some progress in understanding the mechanisms of NH₄⁺ toxicity. Generally, factors such as protein glycosylation defects, inefficient NH₄⁺-N cycling, accumulation of reactive oxygen species (ROS), cytoplasm acidification, consumption of inorganic cations and organic acids, disruption of the photosystem, and interference with hormone signaling, may contribute to NH₄⁺ toxicity (Britto and Kronzucker 2002; Liu and Von Wirén 2017; Jia et al. 2020; Kong et al. 2022). In agricultural contexts, the input of a large amount, or even excessive nitrogen fertilizers for increasing yields, can lead to NH₄⁺ toxicity. Therefore, improving nitrogen use efficiency and alleviating NH₄⁺ loss is crucial for reducing nitrogen fertilizers input and increasing plant growth.

The nitrogen cycle in soils is mainly derived by the processes of nitrification and denitrification (Pajares and Bohannan 2016). Ammonia oxidation is a crucial step in the nitrification process (Bhambri et al. 2025), which correlates with soil microbial ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and different enzymes activity. Denitrification is a microbial process in which nitrate (NO₃^–) is sequentially reduced to nitrite (NO₂^–), nitric oxide (NO), nitrous oxide (N₂O), and finally dinitrogen gas (N₂). Steps in the process are catalyzed by different enzymes, including nitrous oxide reductase, and nitrite reductase, among others. Thus, the abundance of the nitrite reductase genes (nirK and nirS), nitrous oxide reductase gene (nosZ), ammonia-oxidizing archaeal ammonia monooxygenase subunit A gene (AOA-amoA), and ammonia-oxidizing bacteria ammonia monooxygenase subunit A gene (AOB-amoA) affect nitrogen cycling and nitrogen balance (Guo et al. 2017; Xu et al. 2022). Additionally, Fe (III) affects the diversity and abundance of microorganisms in the soil, specific concentrations of Fe (III) can even impact microbial metabolites and pathways, thereby affecting chemical reactions and material cycles in the soils (Chen et al. 2022). The addition of Fe³⁺ can effectively stimulate nitrogen cycling processes by Feammox (Fe (III) reduction coupled to anaerobic ammonium oxidation), where NH₄⁺-N can serves as an electron donor while Fe³⁺ can act as a terminal electron acceptor under anaerobic conditions (Wang et al. 2025). The addition of Fe (III) in agricultural soils to alter the content of soil NH₄⁺ and alleviate the toxicity of NH₄⁺ to plants is worthy of further exploration.

Therefore, this study aimed to systematically investigate the effect of Fe (III) addition on NH₄⁺-N loss under different conditions in agricultural soils, which will be helpful for the interpretation of microbial dynamics in iron and nitrogen coupling (Fe-N) cycle and provide reference for reducing the loss of NH₄⁺ and improving soil productivity.

EXPERIMENTAL

Materials

Soil sampling

Soil samples (0 to 20 cm depth) were collected from agricultural fields (30°59′4″, 119°13′14″) in Langxi County, Anhui Province, China. The collected soil samples were immediately placed in sterile plastic bags without aeration and transferred to the laboratory as soon as possible. The specimens were then dried at room temperature (25 ± 1 °C) and were subsequently sieved through a 0.15-mm sieve to remove crop residues, gravels, etc. The physical and chemical properties of soil samples include moisture content (MC), pH, total phosphorus (TP), soil organic carbon (SOC) content, NH₄⁺-N content, NO₃^–-N content, and Fe (III) and Fe (II) concentrations were measured.

Methods

After mixing deionized water with soil samples at a liquid-to-soil ratio of 2.5:1 (v/w; L/kg), soil pH was measured using a pH meter (ZXHD/Bante920, China). The MC was determined by heating 50 g soil samples to a constant value at 60 °C, SOC was measured using a potassium bichromate external heating method, total phosphorus determination by the Molybdenum-Antimony-Ascorbic acid colorimetric method (Bao 2005). Soil samples were extracted using a 2 mol·L^-1 KCl solution at a v/w ratio of 10:1, followed by a 30-minute extraction period at 25 °C. The extracted solution was used for NH₄⁺-N and NO₃^–-N determination. The NH₄⁺-N was determined using the hypochlorite-phenol method, while NO₃^–-N was measured using the dual-wavelength spectrophotometric method (Cawse 1967). The soil samples were extracted using a 0.5 mol·L^-1 HCl solution at liquid-to-soil ratio of 20:1 for a 30-minute extraction period, and the extracted solution was used for soil Fe (II) and Fe (III) determination. Meanwhile, the Fe (II) was measured using the ortho-phenanthroline colorimetric method. Total iron content was determined after reducing Fe (III) to Fe (II) by adding hydroxylamine hydrochloride and employing the same method. The content of Fe (III) was calculated by subtracting the Fe (II) content from the total iron content (Viollier et al. 2000). Headspace gas samples were collected from the culture boxes using a 10-mL polyethylene syringe twice each week for N₂O flux analysis. The boxes were sealed with parafilm for 4 h before collection. N₂O flux was measured using a gas chromatograph (GC-2014C, Shimadzu, Tokyo, Japan).

Experimental design

The experiments were conducted in April to December 2023 in a light incubator (GZX-80, China), where 200 g of soil samples were placed in in a series of 400-mL culture boxes. Box-Behnken design (BBD) design experiments was employed to investigate the effects of soil-liquid ratio (w/w), temperature (°C), Fe (III) content (g·kg^-1), and NH₄⁺-N (mg·kg^-1) content on NH₄⁺-N loss rate in the soils, and the design of the factors and levels are shown in Table 1. The loss rate of NH₄⁺-N (NO₃^–-N) was calculated as followed,

NH₄⁺-N (NO₃^–– N) loss rate (%)    (1)

where C₀ is the initial concentration of NH₄⁺-N or NO₃^–-N (mg·kg^-1), and C₁ is the concentration of NH₄⁺-N or NO₃^–-N at predetermined intervals (mg·kg^-1).

Table 1. Experimental Design of Variables Factors and Levels

DNA extraction and bacterial PCR gene amplification

Using a headless plastic syringe (50 mL, tip cut off), three replicate soil samples were collected from the culture boxes. Soil DNA extraction using a PowerSoilTM DNA extraction kit (MoBio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturers’ instructions, and quantitative polymerase chain reaction (PCR) targeting the denitrifying genes (nosZ, nirK) and nitrifying genes (AOA-amoA, AOB-amoA).

To amplify the target genes, AOA-amoA (635 bp) was amplified using the forward primer Arch-amoAF (STA ATG GTC TGG CTT AGA CG) and the reverse primer Arch-amoAR (GCG GCC ATC CAT CTG TAT GT) (Francis et al. 2005). The standard curve had a slope of -3.257, an R² of 0.9992, and a PCR efficiency of 102.8%. AOB-amoA (491bp) was amplified using forward primer amoA-1F (GGG GTT TCT ACT GGT GGT) and reverse primer amoA-2R (CCC CTC KGS AAA GCC TTC TTC) (Rotthauwe et al. 1997). The standard curve had a slope of -3.353, an R² of 0.9931, and a PCR efficiency of 98.76%. NirK (165bp) was amplified using forward primer nirK876F (ATY GGC GGG VAY GGC GA) and reverse primer nirK1040R (GCC TCG ATC AGR TTR TGG GTT) (Henry et al. 2004), the standard curve had a slope of -3.583, an R² of 0.9979, and a PCR efficiency of 90.2%. NosZ (267bp) was amplified using forward primer nosZ2FHenry (CGC RAC GGG CAA SAA GGT SMS SGT) and reverse primer nosZ2RHenry (CAK RTG CAK SGC RTGG CAG AA) (Henry et al. 2006). The standard curve had a slope of -4.012, an R² of 0.9971, and a PCR efficiency of 77.5%. DNA purity and concentration were measured using a Micro Drop spectrophotometer (SMA4000, Beijing Meilin Hengtong Technology Co., Ltd., Beijing, China).

Statistical analyses

All experiments were performed in triplicate, such that reported results represent the mean of the triplicates. Design-Expert (version 13, Stat-Ease Inc., Minneapolis, MO, USA) and Origin2022 (Inc., OriginLab, Northampton, MA, USA) software were used to assess the correlation between Fe (III) and soil physic-chemical properties, abundance of AOA-amoA, AOB-amoA, nosZ, and nirK. The significance level was set at P < 0.05.

RESULTS

Physico-chemical Characteristics of Agricultural Soils

The physico-chemical characteristics of the agricultural soil samples are shown in Table 2, including soil moisture, NH₄⁺-N, NO₃^–-N, TP, SOC, pH, Fe (III) content, and Fe (II) content.

Table 2. Physico-chemical Characteristics of Sample Soil

Response Surface Design Results

The effects of Temperature (A), Fe (III) content (B), NH₄⁺-N content (C), and soil-liquid ratio (D) on the loss rate of NH₄⁺-N (Y) are shown in Table 3. The data were processed and regression analyzed using the software Design Expert 13.0, and the prediction equation for the loss rate of NH₄⁺-N was obtained as follows:

Y = 257.34272 – 1.83296A – 58.36061B – 2.51492C – 1.60374D + 0.447421AB + 0.008438AC + 0.007169AD – 0.029211BC – 0.16818BD – 0.003135 CD – 0.006934A² + 9.064717B² + 0.032288C² + 0.007769D²

Table 3. Experimental Design and Result for NH₄⁺-N Removal by RSM

Table 4. ANOVA of Model Equation for NH₄⁺ -N Removal

The results of the final model analysis of variance (ANOVA) are shown in Table 4. The model was highly significant (P = 0.0024), the effect of B, BD, B², C², and D² on Y are significant (P < 0.05), while other terms did not yield a significant effect on Y.

Analysis of interaction effects of impact factors

Figure 1(a) shows the combined effect of temperature and Fe (III) on NH₄⁺-N loss. NH₄⁺-N loss increased significantly with increasing Fe (III) content when the temperature was constant, but the relatively flat curvilinear shows that the NH₄⁺-N loss was not significantly affected by temperature when the Fe (III) content was constant.

Figure 1(b) shows the combined effect of temperature and NH₄⁺-N concentration on NH₄⁺-N loss. NH₄⁺-N loss initially decreased, followed by an increase with NH₄⁺-N concentration when the temperature was constant. The flat curvilinear shows NH₄⁺-N loss was not significantly affected by temperature when NH₄⁺-N concentration was constant.

Figure 1(c) shows the combined effect of temperature and soil-liquid ratio on NH₄⁺-N loss. As shown, the overall variation was small, indicating that the interaction of temperature and soil-liquid ratio did not have a significant effect on the NH₄⁺-N loss.

Figure 1(d) shows the combined effect of Fe (III) and NH₄⁺-N concentration on NH₄⁺-N loss. It can be seen that NH₄⁺-N loss increased with increasing Fe (III) content when the NH₄⁺-N was constant, but NH₄⁺-N loss showed a tendency of decreasing and then increasing with NH₄⁺-N concentration when the Fe (III) content was constant, and the maximum NH₄⁺-N loss rate was appeared when the Fe (III) content was 0.868 g·kg^-1 and NH₄⁺-N content was 20 mg·kg^-1

_. Figure 1(e) shows the combined effect of Fe (III) and soil-liquid ratio on NH₄⁺-N loss. The overall magnitude of the curves varied steeply, which means NH₄⁺-N loss was significantly affected by the interaction of Fe (III) and soil-liquid ratio, which was consistent with the results reflected in the statistical significance of individual interactions (Table 4). In all interaction combinations, the combined effect of Fe (III) and NH₄⁺-N concentration on NH₄⁺-N loss was significantly higher than other interaction combination (P<0.05).

Figure 1(f) shows the combined effect of NH₄⁺-N concentrations and soil-liquid ratio on NH₄⁺-N loss. It can be seen NH₄⁺-N loss showed a tendency of decreasing and then increasing with NH₄⁺-N concentration when soil-liquid ratio was constant. The minimum NH₄⁺-N loss rate was achieved when soil-liquid ratio was 0.6:1 and NH₄⁺-N content was 40 mg·kg^-1_.

Optimization analysis for NH₄⁺-N loss

According to parameter optimization analysis for prediction equation, the optimal NH₄⁺-N loss condition was obtained as: Temperature of 17.0 °C, 0.772 g·kg^-1 Fe (III), 21.9 mg·kg⁻¹ NH₄⁺-N, and soil-liquid ratio of 1:1. It was assumed that NH₄⁺-N loss was 86.1% under these conditions. The prediction result was similar to the current study, where the NH₄⁺-N loss was 83.2% under the condition of 25 °C, 0.723 g·kg^-1 Fe (III), 20 mg·kg⁻¹ NH₄⁺-N, and soil-liquid ratio of 1:1.

Fig. 1. Response surface plot and contour plot showing the effect of (a) A and B, (b) A and C, (c) A and D, (d) B and C, (e) B and D, and (f) C and D on the loss of NH₄⁺-N

DISCUSSION

Iron (Fe) is a key factor in the regulation of soil nitrification, denitrification processes, and nitrogen turnover. The nitrogen and the Fe cycle are coupled in soil environments (Kampschreur et al. 2011). Changes of nitrogen and Fe (III) concentrations can lead to shifts in soil microbial communities (Chen et al. 2022), which will impact nitrification and denitrification processes, thereby affecting nitrogen distribution and transformation in soils (Davidson et al. 2003; Heckman et al. 2013; Park and Novak 2013; Cai et al. 2014). In this study, the maximum NH₄⁺-N loss of 84.6% occurred when the addition of Fe (III) was 0.868 g·kg^-1 in soils (Table 5). It was found that gene copy numbers for AOB-amoA, nirK, and nosZ under that condition increased 44%, 87.6%, and 0.48% compared with control, respectively. However, the gene copy numbers for AOA-amoA dropped 46.5%, simultaneously. The decrease in AOA-amoA abundance alongside the increase in AOB-amoA might result from AOB could inhibit AOA through rapid growth under resource-limited condition (Guo et al. 2021). The gene copy numbers for nirK and nosZ, which have been categorized as denitrifying genes, have higher population abundance than nitrifying genes in the soils. Table 5 further indicated that N₂O flux from control and test 2 were significantly higher than those in the other tests but were not correlated to population abundance of denitrifying genes (nirK + nosZ). A possible explanation is that the loss of NH₄⁺-N in this system is not only caused by AOA and AOB interactions, but also by other reaction processes, such as anaerobic ammonium oxidation, the oxidation of NH₄⁺ coupled with Fe (III) reduction (Van der Star et al. 2007), etc. Feammox is a promising technology that can proceed without oxygen and minimize N₂O production. Previous studies have also shown that Fe (III)-mediated Feammox was positively correlated with NH₄⁺-N loss (Zhu et al. 2021; Hu et al. 2022).

In addition, it has been widely reported that pH is also an important factor influencing the distribution and transformation of N in soils (Clement et al. 2005; Jiang et al. 2022). The current results are consistent with this, compared with control, the pH increased from 5.5 to 6.0 as the loss of NH₄⁺-N increased from 25% to 84.6% after 14 d incubation experiments. A possible explanation is the loss of NH₄⁺-N was related with the decrease of pH due to the ammonia oxidation process, but the increase of NO₃^– caused by nitrification might enhance the denitrification (Chen et al. 2016), thereby increasing the pH with higher loss rate of NH₄⁺-N.

The authors also found that the loss of NH₄⁺-N was increased from 79.0% to 83.2% with the soil-liquid ratio increased from 0.4:1 to 1:1 under the same condition of temperature, Fe (III) addition, and NH₄⁺-N content, while the gene copy numbers for AOB-amoA and nirK were increased 68.5% and 45%, the gene copy numbers for AOA-amoA and nosZ were decreased 32.4% and 3.35% (Table 5), respectively.

Table 5. Changes in Abundance of Functional Genes and N Turnover in Soils

This means that NH₄⁺-N loss was not significantly affected by the soil-liquid ratio. A possible explanation is the decrease soil-liquid ratio leads to an increase in aerobic conditions in the soil, thereby promoting nitrification reactions. However, it is difficult to explore the specific mechanisms in depth because the various processes can be mixed and interact with each other (Maag and Vinther 1999; Xu et al. 2003; Hu et al. 2010). Future research should focus on investigating the interrelationships between microbial interactions and Fe–N cycle, to promote its application in broader soil types.

CONCLUSIONS

1. In this study, the authors systematically analyzed the combined effects of temperature, Fe (III) contents, NH₄⁺-N contents, and soil-liquid ratio on the magnitude of loss of NH₄⁺-N in soils using response surface methodology (RSM). The results showed that Fe (III) addition affected NH₄⁺-N loss by various processes that interact with each other, which could contribute significantly to nitrogen management and soil productivity enhancement.
2. The N₂O flux reached its minimum value, correlating with gene copy numbers for AOB-amoA and nirK were maximum values, respectively. Nitrogen turnover was not correlated with corresponding functional gene abundance.
3. Iron-nitrogen interaction in soils play a fundamental role in plant growth by influencing nitrogen turnover, water retention, and microbial activity. Further research is needed for NH₄⁺-N loss mechanisms with Fe (III) addition.

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Article submitted: March 3, 2025; Peer review completed: July 25, 2025; Revised version received and accepted: Aug. 6, 2025; Published: August 13, 2025.

DOI: 10.15376/biores.20.4. 8713-8724