Transformation of ammonium nitrogen and response characteristics of nitrifying functional genes in tannery sludge contaminated soil

Xiang-ke Kong ,Zi-xuan Zhang ,Ping Wang ,Yan-yan Wang ,Zhao-ji Zhang ,Zhan-tao Han ,Li-sha Ma*

1 Institute of Hydrogeology and Environmental Geology, Chinese Academy of Geological Sciences, Shijiazhuang 050061, China.

2 Key Laboratory of Groundwater Remediation of Hebei Province and China Geological Survey, Shijiazhuang 050061, China.

3 School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing 100083, China.

4 Technical Centre for Soil, Agriculture and Rural Ecology and Environment, Ministry of Ecology and Environment, Beijing 100012, China.

Abstract: High concentrations of ammonium nitrogen released from tannery sludge during storage in open air may cause nitrogen pollution to soil and groundwater.To study the transformation mechanism of NH4+-N by nitrifying functional bacteria in tannery sludge contaminated soils,a series of contaminated soil culture experiments were conducted in this study.The contents of ammonium nitrogen (as NH4+-N),nitrite nitrogen (as NO2--N) and nitrate nitrogen (as NO3--N) were analyzed during the culture period under different conditions of pollution load,soil particle and redox environment.Sigmodial equation was used to interpret the change of NO3--N with time in contaminated soils.The abundance variations of nitrifying functional genes (amoA and nxrA) were also detected using the real-time quantitative fluorescence PCR method.The results show that the nitrification of NH4+-N was aggravated in the contaminated silt soil and fine sand under the condition of lower pollution load,finer particle size and more oxidizing environment.The sigmodial equation well fitted the dynamic accumulation curve of the NO3--N content in the tannery sludge contaminated soils.The Cr(III) content increased with increasing pollution load,which inhibited the reproduction and activity of nitrifying bacteria in the soils,especially in coarse-grained soil.The accumulation of NO2--N contents became more obvious with the increase of pollution load in the fine sand,and only 41.5% of the NH4+-N was transformed to NO3--N.The redox environment was the main factor affecting nitrification process in the soil.Compared to the aerobic soil environment,the transformation of NH4+-N was significantly inhibited under anaerobic incubation condition,and the NO3--N contents decreased by 37.2%,61.9% and 91.9% under low,medium and high pollution loads,respectively.Nitrification was stronger in the silt soil since its copy number of amoA and nxrA genes was two times larger than that of fine sand.Moreover,the copy numbers of amoA and nxrA genes in the silt soil under the aerobic environment were 2.7 times and 2.2 times larger than those in the anaerobic environment.The abundance changes of the amoA and nxrA functional genes have a positive correlation with the nitrification intensity in the tannery sludge-contaminated soil.

Keywords: Tannery sludge;Transformation of ammonium nitrogen;Cr(III) aging;Fluorescence quantitative PCR;Functional gene

China is a major country in the world’s leather industry,and the leather industry occupies an important position in the national economy (Ma et al.2017).A tannery process usually produces large amount of wastewater and tannery sludge (Zeng et al.2016).Often,the tannery sludge contains high level of ammonium nitrogen (as NH4+-N) (often up to tens of thousands of mg/kg) (Kong et al.2020),during the process of deliming with ammonium salts and protein hydrolysis.In addition,the use of chromate as a tanning agent leads to a small amount of chromium (as Cr(III)) remained in the tannery sludge (Zheng and Zhou,2011).Up to now,due to the large volume and high treatment cost for the waste sludge,some tannery enterprises commonly dispose the sludge as ordinary garbage or solid waste,or directly use it fertilizer in farmland (Zou et al.2013).Although this type of waste disposal may increase the soil fertility(Martines et al.2010;Nakatani et al.2011),the high concentration NH4+-N in the sludge can make the soil quickly reach a saturated state that often causes the migration of extra NH4+-N to the ambient environments.Furthermore,the transformed NO3--N by nitrification is easier to migrate in soils,causing the nitrogen pollution to deep soil and even groundwater (Han et al.2014).Previous investigation of informal tannery sludge storage sites has confirmed that Cr(III) existed in the soils with both reducible,oxidizable and residual states,where the pollution depth of the Cr(III) is less than 20 cm deep,whilst the NH4+-N and NO3--N can reach the deep soil and shallow groundwater (M Barajas-Aceves et al.2014;Kong et al.2017).At present,there are many reports about chromium pollution in tannery sludge soils,but little attention has been paid to the pollution risk caused by high concentration of NH4+-N.The transformation of NH4+-N to NO2--N and NO3--N under the action of nitrifying bacteria is yet unclear in the tannery sludge soils,especially where presenting Cr(III)(Ma et al.2017;Araujo et al.2020;Guo et al.2021).

Nitrifying bacteria are the main functional groups affecting NH4+-N transformation in soil(Wendeborn,2020),and the ammonia oxidation and nitrite oxidation are two main stages controlling the nitrification (Mohamad et al.2021).The structure and function of nitrifying bacteria in soil are sensitive to exogenous chromium,even to the bacteria with the same functional genes.Previous studies have shown that chromium has obvious toxic effects on microorganisms in soil,resulting in a significant decrease in microbial abundance and activity of bacteria (Pantazopoulou and Zouboulis,2017;Yu et al.2021).Bacteria containing the genes encode key enzymes of ammonia monooxygenase (amoA) and nitrite oxidoreductase (nxrA) play an important role in ammonia and nitrite oxidation processes (Daims et al.2015;He et al.2012).Thus,monitoring the abundance of these two nitrifying functional genes can accurately determine the characteristics of nitrifying bacteria activity (Xu and Mao,2019).Based on the analysis of the abundances of functional genes that control the key processes of nitrogen cycling,and the transformation process of different forms of nitrogen content in soil,the transformability of NH4+-N to NO3--N in tannery sludge contaminated soil can be accurately explained (Wang,2018;An et al.2021).

Focusing on the pollution characteristics of typical tannery sludge storage sites,this study carried out the tannery sludge contaminated soil culture experiments under different pollution loads,soil particle sizes and redox conditions.The content changes of NH4+-N,NO2--N and NO3--N were analyzed during the culture period,and the sigmodial equation was used to fit the content change of NO3--N in contaminated soils.The abundance variations of nitrifying functional genes(amoAandnxrA) were also detected using the realtime quantitative fluorescence PCR method.The correlation between the abundance changes of nitrifying bacteria in soil and the conversion efficiency of NH4+-N were identified.The conclusion of this study provided the theoretical support for the prevention and control of NH4+-N pollution in tannery sludge contaminated soil.

1 Materials and methods

1.1 Tannery sludge and soil

The tannery sludge used in the experiments was obtained from an illegal tannery sludge disposal site in Hebei province of China,and the experimental soil was taken from uncontaminated silt soil and fine sand soils around the site.The proportions of clay,silt,and sand in the silt soil are 10.52%,74.39% and 15.09%,respectively.The tannery sludge and the soil were air-dried,crushed,homogenized,and screened with 20 mesh nylon sieves for subsequent culture experiments.The content of Cr(III) in the tannery sludge was 30 800 mg/kg,and the proportions of different forms of the Cr(III) were: Water soluble fraction (0.53%),weak acid extraction fraction (0.79%),reducible fraction (75.28%),oxidizable fraction (13.39%),and residual fraction (10.01%).The NH4+-N content was 16 080 mg/kg.The physicochemical characteristics of the tannery sludge and soil were shown in Table 1.

1.2 Soil culture experiment

Tannery sludge was well mixed with silt soil orfine sand in a certain proportion to prepare contaminated soil before conducting the soil culture experiment.The mass ratios of the silt soil or fine sand to tannery sludge for in the mixed soil samples were 255:45,210:90,150:150,respectively,which were stirred evenly to make the samples containing the mass content of 15% (mild),30% (moderate) and 50% (severe) tannery sludge(Table 2).By adding deionized water,the moisture content of different contaminated soil was maintained at field capacity (25 wt%),and the culture box was placed in a constant temperature incubator(JWINS,DRX-680E) at 25°C to avoid lighting.The incubator was kept in an aerobic condition,while the anaerobic environment was created by sealing and vacuuming the culture box in the soil culture process.The transformation process of NH4+-N and changes in the abundance of functional genesamoAandnxrAin the contaminated soils under different pollution loads,soil particle size and redox conditions were monitored.All the experiments were carried out in three parallel.

Table 1 Basic physical and chemical properties of the tannery sludge and soil

The soil samples were collected and mixed well by five subsample points at the time intervals of 0,3,8,15,30,45 and 60 days,and then parts of the soil samples were saved in the sterilized centrifuge tubes for contaminant content and microbial function gene test,respectively.Samples for microbial functional gene testing were stored in -70°C refrigerator before sending to the Shanghai Meiji Biomedical Technology Co.,Ltd.for detection.The water contents of the contaminated soils were retained at close to 25 wt% by periodic addition of distilled water.

1.3 Analytical methods

The NH4+-N,NO2--N and NO3--N were analyzed using the UV-2 550 spectrophotometer at λmax630 nm and 543 nm by the spectrophotometric methods(Kong et al.2019).The absolute contents ofamoAgene of ammonia oxidizing bacteria andnxrAgene of nitrosobacteria in the soil samples were detected by quantitative fluorescence PCR,using the Bori 9600plus fluorescence quantitative PCR apparatus.The Quantitative PCR reaction system was 20 μL,including: 1 μL DNA template,10 μL 2X Taq Plus Master Mix,0.8 μL (5μm) upstream and downstream primers,7.4 μL ultrapure sterile water.The amplification primers and reaction procedures for quantitative PCR were shown in Table 3.

Table 2 Culture conditions of tannery sludge contaminated soils

Table 3 Quantitative PCR primer and reaction procedure

1.4 Data analysis

According to the ‘S’ curve model proposed by Sabey and Frederick (1959),the sigmodial equation was used to analyse the NO3--N content change with time in the contaminated soils.The delay period,maximum rate period and stagnation period of soil nitrification process were quantitatively described by the model,and the maximum nitrification rate,delay time and maximum accumulation of nitrate in the nitrification process were calculated according to the formula (Rovita and Killorn,2008;Yuan et al.2007).The equation was described as follows:

Where: △(NO3-) (mg·kg-) is the accumulationof NO3--N;A1andA2represent the minimum and maximum conversion of NO3--N (mg·kg-1),respectively;t0(d) is maximum increase time of NO3--N conversion;(A2-A1)/(4dt) is the maximum increase in NO3--N conversion (mg·kg-1).The SPSS 20.0 was used for statistical analysis of the experimental data.The obtained copy numbers ofamoAgene andnxrAgene were analyzed by the single-factor variance method and the minimum significant difference method for significant analysis and multiple comparison (α=0.05).

2 Results and discussion

2.1 Effect of pollution load on ammonium nitrogen transformation and functional nitrification gene abundance

As shown in Fig.1,the inorganic nitrogen in the contaminated soils are mainly NH4+-N in the initial culturing stage (0-15 d),and there are almost no NO2--N and NO3--N.During this stage,great amount of NH4+-N is formed and accumulated,due to the hydrolysis of organic nitrogen (protein) in the tannery sludge and microbial ammonification.With the increase of incubation time (15-60 d),the NH4+-N content decreases and NO3--N content both increases in the silt soil and fine sand under different pollution loads,indicating that the nitrification intensity in soil increases gradually.With the increase of pollution load,the conversion efficiency of the NH4+-N in the soils at the initial culturing stage gradually decreases.In addition,the difference between nitrification rate of NH4+-N and the final conversion of NO3--N is limited in the condition of low pollution load.The NO3--N content appears after 30 days,and more than 90%of NH4+-N is converted to NO3--N after 60 days of incubation.Under the condition of high pollution load,the nitrification rate of the NH4+-N and the final conversion of the NO3--N in the fine sand decreases significantly.As a result,the NO2--N content is accumulated after 30 days incubation.

Fig.1 Transformation of ammonium nitrogen in contaminated silt soil and fine sand under different pollution loads

The abundance changes ofamoAandnxrAgenes in the silt soil under different pollution loads are shown in Fig.2.In the beginning of culturing,the ammonia-oxidizing bacteria and nitrite-oxidizing bacteria,sensitive to environmental conditions,are in the growth adaptation period (Smolders et al.2001).The abundance ofamoAandnxrAgenes is relatively low at this stage (gene copy order of 105),and the nitrification intensity is relatively weak prior to 30 days of incubation.After that,as the NH4+-N in contaminated soil is used as a nitrogen source,the nitrifying bacteria grow significantly (e.g.,theamoAandnxrAgene copies increase to the order of magnitude of 107and 106,respectively).There is a good correlation between the numbers ofamoAandnxrAgene copy and NO3--N accumulation in the soil at different incubation stages.

It is worth noting that the numbers ofamoAandnxrAgene copy in the silt soil with high pollution load increase slowly at the early culturing stage(sample FW3,Fig.2).For example,the numbers ofamoAgene copy in the FW3 is 1.35×105copies/g at 30 d,which is two orders of magnitude less than that in low pollution load silt soil as 4.75×107copies/g in the FW1.Due to the presence of high content Cr (III) in the FW3,it may toxicate and reduce activity of the nitrifying bacteria in soil(Pantazopoulou and Zouboulis,2017;Ramírez-Díaz et al.2008).In addition,the competition for oxygen between heterotrophic bacteria and nitrifying bacteria during the degradation of organic matter in contaminated soil have also inhibited the activity and reproductively of the aerobic nitrifying bacteria in the early stage,thereby decelerating the NH4+-N transformation to NO3--N (Liu et al.2016;Ke et al.2015).After 60 days of incubation,the activity of the nitrifying bacteria increases with the decrease of Cr (III) content in the soil.As a result,the number ofamoAgene under different pollution loads increases significantly from 105to 107-108in the orders of magnitude,and the copy number ofnxrAgene accordingly increases from 105to 106-107.The nitrification rate of NH4+-N in soil was significantly improved after 60 d incubation.

Fig.2 Richness characteristics of amoA and nxrA genes in silt soil under different pollution loads

2.2 Effect of soil particle size on ammonia nitrogen transformation and nitrification functional gene abundance

The abundance variation characteristics ofamoAandnxrAgenes in low pollution load silt soil(FW1) and fine sand (SW1) are further analyzed,with the results shown in Fig.3,after 30 days of culturing,the copies ofamoAandnxrAgenes in the FW1 reached to 1.84×107copies/g and 2.86×105copies/g,respectively,which are significantly higher than 2.36×106copies/g and 1.48×105copies/g in the SW1.This is basically consistent with their conversion efficiency of NH4+-N to NO2--N and NO3--N (Fig.1).

Fig.3 Richness characteristics of amoA and nxrA genes in silt soil and fine sand under low pollution load

With the increase of pollution load,the conversion efficiency of NH4+-N in the fine sand decreases significantly,and the occurrence of NO2--N and NO3--N in the soil delays in about 15 days.After 60 days,41.5% of NH4+-N has been converted to NO3--N in fine sand,with about 27.5% of NO2--N accumulated.Since the iron oxides and organic matter are the main factors which enhance the aging process of the Cr(III) and reduce its bioavailability content in the soil (Kong et al.2019),the contents of iron oxides and organic matter in silt soil are about twice of that in the fine sand (Table 1).The content of bioavailable Cr(III)in the fine sand is relatively higher than that in the silt soil,which hence has restrained the activity of nitrifying bacteria from fast culturing in the early stage (Ramírez-Díaz et al.2008).In addition,the nitrite oxidizing bacteria may be more susceptible to the Cr(III) and other pollution in the sludge,showing that the number ofnxrAgenes increases slowly.Although the number ofamoAgenes that control the transformation of NH4+-N to NO2--N in fine sand increases rapidly (an order of magnitude increase after 30 days of incubation),the subsequent transformation of NO2--N to NO3--N is inhibited due to the slow increase ofnxrAgenes,resulting in the accumulation of large amount of NO2--N generated in the first step of ammonia oxidation.

2.3 Effects of redox conditions on ammonium nitrogen transformation and abundance of nitrification functional genes

The transformation process of NH4+-N in the contaminated silt soil in an anaerobic condition can be seen from Fig.4,where the nitrification rate of the NH4+-N and final conversion amount of NO3--N are significantly lower than those in an aerobic condition (Fig.1A,C and E).This figure also shows that,after 60 days of incubation,the NH4+-N content no longer decreases,and the transformation of NO3--N does either not work well in the soil with a high pollution load.Since nitrification is a reaction process taking place under an aerobic environment,the activity of nitrifying bacteria is hence fairly low in the anaerobic condition,which inhibits the generation of NO3--N (Pettridge et al.2013).In the case of low,medium and high pollution loads,respectively,the conversion rates of the NO3--N in the silt soil decrease by 37.2%,61.9% and 91.9%.

Fig.4 Transformation of ammonia nitrogen in contaminated silt soil under anaerobic condition

The abundance variation characteristics ofamoAandnxrAgenes in silt soil with a low pollution load under different redox conditions are further analyzed with results shown in Fig.5.The figure shows that,after 60 days of incubation,the copies ofamoAandnxrAgenes in the aerobic soil are 4.04×107copies/g and 1.47×106copies/g,respectively,while the values in the anaerobic soil are 1.52×107copies/g and 6.69×105copies/g.The copy numbers ofamoAandnxrAgenes increase by more than 2.7 and 2.2 times in comparison with those under an anaerobic condition,respectively,indicating that the anaerobic environment significantly impacts on the reproduction and growth of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria (Liu and Wang,2013),thereby affecting conversion efficiency of NH4+-N to NO2--N and NO3--N in the soil.

Fig.5 Genes richness characteristics of amoA and nxrA under different redox condition

2.4 Accumulation characteristics of nitrate nitrogen

The S-growth curve has been used to model the accumulation characteristics of NO3--N in the contaminated soil (Fig.6 and Table 4).Fig.6 shows that the sigmodial equation well explains the curve fitting (R2＞ 0.97) of the NO3--N contents in tannery sludge contaminated soil cultured in 60 days.The range values (A2-A1) of the accumulated NO3--N are as follows (Fig.6A): FW3 ＞FW2 ＞ FW1.Although the cumulative conversion value of NO3--N in the soil under a high pollution load is low at the early stage,a large amount of NH4+-N can be transformed into NO3--N after 60 days,owing to the increase of abundance and activity of nitrifying bacteria.Under the same pollution load,the cumulative transformation of NO3--N in the silt soil is greater than that in fine sand,as shown in Fig.6B where the cumulative NO3--N contents are: FW2 ＞ SW2,indicating the transformation of NH4+-N in the soil with finer particles is more efficient.The cumulative transformation amount of NO3--N in silt soil under aerobic conditions is greater than that under anaerobic conditions (FW2 ＞ OFW2) (Fig.6C),indicating that the nitrification efficiency is higher under aerobic conditions.The maximum increase point(t0) of NO3--N accumulation in soil under different environmental conditions is shown in Table 4:FW1 ＜ FW2 ＜ FW3,FW2 ＜ SW2,FW2 ＜ OFW2.Above all,For the tannery sludge contaminated soil,a lower contamination load,finer particle size and more oxidizing environment could reduce the start time of nitrification and enhance the transformation of NH4+-N to NO3--N.

Table 4 Sigmodial fitting parameters of nitrate accumulation process under different environment conditions

Fig.6 S-growth fitting curve of nitrate accumulation process under different environmental condition

3 Conclusions

The nitrification process in tannery sludge contaminated soil can start at a early stage and has a complete reaction in the conditions of low pollution load,fine soil particle size and oxidizing environment.The sigmodial equation can well fit the cumulative variation characteristics of NO3--N content in the contaminated soil.High Cr (III)content and an anaerobic environment in the tannery sludge contaminated soil greatly inhibits the reproduction and activity of nitrifying bacteria,resulting in a decrease in the conversion efficiency of NH4+-N to NO3--N.The transformation of NH4+-N to NO2--N and NO3--N in the soils studied is interpreted on the basis of the abundance variation of functional genesamoAandnxrA.The copy numbers of theamoAandnxrAgenes in silt soil is twice higher than those in fine sand soil.And the numbers ofamoAandnxrAgenes in aerobic conditions are 2.7 and 2.2 times as many as those in anaerobic conditions.The abundance ofamoAandnxrAgenes is positively correlated with nitrification intensity in the soils.

Acknowledgements

This study was financially supported jointly by Natural Science Foundation of Hebei Province(D2020504003),and National Key Research and Development Program of China (No.2019YFC18 05300).