Dynamics of Microbial Activity Related to N Cycling in Cd-Contaminated Soil During Growth of Soybean"

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1 Pedosphere 17(3): , 2007 ISSN /CN 2007 Soil Science Society of China Published by Elsevier Limited and Science Press PEDOSPHERE www elsevier com/locate/pcdosphere Dynamics of Microbial Activity Related to N Cycling in Cd-Contaminated Soil During Growth of Soybean" YANG Yell2, CHEN Ying-Xu', TIAN Guang-Mingl and ZHANG Zi-Jianl 'Department of Environmental Engineering, Zhejiang University, Hangzhou (China). yeyangaacee. org.cn Appraisal Centev for Environment and Engineering, SEP, Beajing (China) (Received August 4, 2006; revised January 25, 2007) ABSTRACT The potential influences of cadmium (Cd) on the biochemical processes of the soil nitrogen (N) cycle, along with the dynamics of ammonification, nitrification, and denitrification processes in the rhizosphere and non-rhizosphere (bulk soil), respectively, were investigated in a Cd-stressed system during an entire soybean growing season. In terms of Cd pollution at the seedling stage, the ammonifying bacteria proved to be the most sensitive microorganisms, whereas the effect.s of Cd on denitrification were not obvious. Following the growth of soybeans, the influences of Cd on ammonification in the bulk soil were: toxic impacts at the seedling stage, stimulatory effects during the early flowering stage, and adaptation to the pollutant during the podding and ripening stages. Although nitrification and deqitrification in the bulk soil decreased throughout the entire growth cycle, positive adaptation to Cd stress was observed during the ripening stage. Moreover, during the ripening stage, denitrification in the bulk soil under high Cd treatment (20 mg kg-') was even higher than that in the control, indicating a probable change in the ecology of the denitrifying microbes in the Cd-stressed system. Changes in the activity of microbes in the rhizosphere following plant growth were similar to those in the non-rhizosphere in Cd treatments; however, the tendency of change in the rhizosphere seemed to be more moderate. This suggested that there was some mitigation of Cd stress in the rhizosphere. Key Words: cadmium, microbial activity, nitrogen cycle, rhizosphere Citation: Yang, Y., Chen, Y. X., Tian, G. M. and Zhang, 2. J Dynamics of microbial activity related to N cycling in Cd-contaminated soil during growth of soybean. Pedosphere. 17(3): INTRODUCTION In heavily industrialized areas, numerous soil sites are contaminated with prohibitively high concentrations of heavy metals, which can affect normal agricultural practices. For instance, cadmium (Cd) is a non-essential element that can be highly phytotoxic (Bbbth, 1989). Although the average levels of Cd can be quite low, its inhibitory or toxic impacts on the metabolic activity of living systems or, in particular, on the natural microflora and microfauna in the soil can have a deleterious effect on soil quality, and consequently, on nutrient cycling (BBbth, 1989; Tonner and Kunze, 1990; He, 1997). As part of nutrient cycling and because of shortage of fertilizers and escalation of costs, the soil N cycle has become increasingly important (Berg and Rosswall, 1987; Li et al., 2005). One aspect of the soil N cycle is N mineralization. The addition of heavy metals to an acidic silty loam (Chang and Broadbent, 1982) and very high metal concentrations in other soils (Babich and Stotzky, 1985; Long et al., 2004) has been shown to inhibit net N mineralization. Low or no N mineralization was observed in the soil when fields were irrigated with wastewater containing relatively high heavy metal concentrations (Ramirez-F'uentes et al., 2002). These fields then required an N fertilizer application to maintain the normal level of crop production. However, Munn et al. (1997) found no adverse effects on net mineralization of legumes and soil organic N when heavy metals accumulated in the soil, whereas *'Project supported by the National Natural Science Foundation of China (No ) and the National Key Basic Research Program (973 Program) of China (No. 2002CB410807).

2 384 Y. YANG et al. more available N occurred in soils treated with metal-contaminated sewage sludge (Munn et al., 2000). In addition, previous studies have shown that microbes in a metal-stressed system play an important role in the soil N cycle (Wilson, 1977; Broos et al., 2004); however, information relating to physiological groups of bacteria involved in the soil nitrogen cycle is still limited. The objectives of this article were to investigate the potential influences of Cd on the biochemical processes of the soil N cycle and to characterize the dynamics of ammonification, nitrification, and denitrification processes in both the rhizosphere and non-rhizosphere (bulk soil) during the growth of soybeans. MATERIALS AND METHODS Selected soils The red soil (Ferralsol) used in the study was collected from the surface layer (0-20 cm) of a cultivated field in Lanxi of southwestern Zhejiang Province in southern China. The characteristics of the soil are listed in Table I. TABLE I Selected properties of the red soil collected from Lanxi in southwestern Zhejiang Province Soil type OM Clay (< 1 mm) Free Fez03 MnO ph (1:2.5) CEC Total Cd g kg-l cmol(+) kg-l mg kg-l Ferralsol Procedures First, the soil was air-dried, ground, and sieved to pass through a 1-mm sieve. Then, basal fertilizers with application rates of 0.15 g P2O5 kg-' dry soil from Ca(H2P04)~ and 0.15 g K20 kg-i dry soil from K2S04 were added. Next, to obtain four different Cd treatment levels of 2, 5, 10, and 20 mg kg-' soil, a solution of 3CdS04.8H20 was mixed with the soil. After each step, the soil and solution were thoroughly mixed with the soils, which were air-dried and ground to pass through a 1-mm sieve to ensure the thorough mixing of fertilizer and heavy metals with the soil. The soils were then transferred to 42 plastic rhizoboxes. Each rhizobox had two compartments, which were separated by a 300-mesh nylon screen. The inner compartment, designed as a growth chamber, held 0.5 kg soil where three germinated soybean seeds were sown, and the outside compartment contained 1.5 kg soil, with no crops being planted. The soil surfaces on either side of the nylon screen were maintained at the same level. The experiment was carried out in the greenhouse, and after four-week growth, the soils in fifteen rhizoboxes of five Cd treatment levels of 0, 2, 5, 10, and 20 mg kg-' soil, with each treatment in triplication, were sampled at the seedling stage for determining microbial activities and Cd concentration. Nine rhizoboxes of three Cd treatment levels of 0, 2, and 20 mg kg-' soil were dismantled at each stage of soybean growth, including the early flowering, podding, and ripening stages, and the soil in each compartment was separately sampled for analysis of the dynamic of ammonification, nitrification, and denitrification related to the N cycle during soybean growth. Additionally, the addition of 2 mg Cd kg-' soil was considered a slightly metal-contaminated condition, whereas 20 mg Cd kg-' soil was a heavily metal-contaminated condition. Measurement of microbial activities related to the N cycle Ammonification, nitrification, and denitrification of soil samples were determined using the methods proposed by Li et al. (1995). Ammonifying activity was estimated by measuring the ammonia released during the incubation period, expressed as mg NHi-N per kg soil. In brief, 20 g fresh soil and 2 ml of

3 MICROBIAL ACTIVITY AND N CYCLE 385 ammoniating bacteria culture were added to 500-mL conical flasks and incubated at 28 "C for 10 days. The ammonifying bacteria culture medium consisted of 5 g K2HP04, 2.5 g MgS04.7Hz0, 2.5 g CaClz, 0.05 g Fez(SO4)3, and 0.05 g MnS04 in one liter distilled water at ph 7.2, adjusted using KOH. The nitrifying activity was determined based on the disappearance of nitrite (%) during incubation. For determination of nitrifying activity, a 10 g soil sample was thoroughly mixed with 90 ml of distilled water, after which 1 ml of 10% soil solution was transferred to a 150-mL conical flask that contained 30 ml of nitrifying bacteria culture. This was then incubated for 15 days at 28 "C. The nitrifying bacteria culture medium consisted of 1 g NaNO2, 0.75 g KzHP04, 0.25 g NaHzPO4.7Hz0, 1.0 g NazCO3, 0.03 g MgS04.7H20, 0.01 g MnS04, and 3.0 g CaC03 in 1 L of distilled water. Denitrifying activity, expressed as a percent, was measured by the decrease in nitrate. During the experimental period, 15 g of soil and 10 ml of 50 mg KN03 L-l were added to a 50-mL bottle, which was then anaerobically incubated for three days at 28 "C. Analysis Microbial activities based on the levels of ammonification, nitrification, and denitrification at different Cd concentrations were analyzed for soils sampled at the seedling stage of soybeans and then for the entire soybean growing season. The data on microbial activities were subjected to statistical analysis. Mean separation was performed at the 5% and 1% probability levels using a LSD test. RESULTS AND DISCUSSION Microbial activity related to the N cycle of Cd-stressed seedlings Microbial activities and Cd concentration of soils sampled at the seedling stage after four-week growth are listed in Table 11. It was observed that the ammonifying and nitrifying activities in the bulk soils (non-rhizosphere) sharply decreased with an increase in Cd concentration, with the ammonifying bacteria being more sensitive to Cd pollution than the nitrifying bacteria. For example, in non-rhizosphere with 20 mg Cd kg-' soil, the relative inhibition on nitrification was 41.4%, whereas a 50.2% inhibition was found on ammonification. However, Hu et al. (1990) reported that the toxic impacts of heavy metals on nitrification were stronger than those on ammonification. The soil ph might have been an important factor that led to the differences between Hu et al.'s study and the results obtained in this study. The soil ph in this experiment was about 4.3 (Table I), which was much lower than the optimum ph condition for nitrifying bacteria (ph ). Consequently, the sensitivity of nitrifying bacteria to heavy metals could be expected to decrease. However, at the investigated levels it seemed that the impact of Cd on denitrification in the bulk soil was not obvious, as, with the exception of the 5 and 20 mg Cd kg-' soil, no obvious inhibitory effect was found (Table 11). TABLE I1 Microbial activities in the rhizosphere (R) and non-rhizosphere (NR) of Cd-stressed soybeans at the seedling stage Cd concentration Ammonifying activity Nitrifying activity Denitrifying activity NR R NR R NR R mg Cd kg-' soil - mg NHZ-N kg-l soil - % ** * * * 57.7** 50.5* ** ** 45.4** ** 84.55** 38.3** 36.0** 39.7** 38.1 *, **Significant at P = 0.05 and P = 0.01 levels, respectively. The effects of plant roots on the soil's biological, chemical, and physical properties are considered

4 386 Y. YANG et al. to be of agronomic and ecological interest (Rovira, 1979; Youssef et al., 1988). As the diffusible watersoluble root exudates and insoluble root debris formed the primary nutrient sources for most of the microbes in the rhizosphere, improvement of ammonifying activities was found in the rhizosphere. The enhancement of ammonification in the rhizosphere reached a maximum at 10 mg Cd kg- soil, and then declined at 20 mg Cd kg- soil (Table 11) owing to a critical inhibition on root growth. However, the denitrifying activities in the rhizosphere were lower than those in the non-rhizosphere, and similar nitrification levels were found between the rhizosphere and non-rhizosphere (Table 11). Dynamics of microbial activities related to the N cycle during the soybean growth Toxic effects of Cd were found in most of the microbes related to the N cycle at the seedling stage (Table 11). Does this result also apply to the long-term situation? Would microbe activities in the rhizosphere arise as the plants grew? To further understand the microbial process of soil N cycling in a Cd-stressed agricultural system, the processes of ammonification, nitrification, and denitrification were characterized for an entire soybean-growing season. At the seedling stage, Cd inhibited ammonification in the bulk soil (Fig. la). For the bulk soil at the early flowering stage, a maximum ammonifying activity of mg NH,f-N kg- was found in the 20 mg Cd kg- treatment, followed by mg NH,f-N kg- in the 2 mg Cd kg- treatment (Fig. la). This suggested a stimulatory effect of Cd on ammonification at the early flowering stage. However, a decrease of ammonifying activities in all the treatments was observed in the bulk soil from the early flowering stage to the podding stage. Nevertheless, during the ripening stage, no distinct differences were found among the three treatments. >- 160 [a Control [ 2 rng Cd kg 120 mg Cd kg Non-rhizosphere Rhizosphere S E P R S E P R S E P R S E P R S E P R S E P R S E P R S E P R S E P R Growth stage Fig. 1 Arnmonifying (a), nitrifying (b), and denitrifying (c) activities in the rhizosphere and non-rhizosphere (bulk soil) under different Cd stress conditions during the growth of soybeans. S = seedling stage; E = early flowering stage; P = podding stage; and R = ripening stage. Vertical bars indicate standard errors of the means (n = 3). Stronger ammonifying activities were observed throughout all the stages of soybean growth in the rhizosphere of the control treatment compared with non-rhizosphere. Li et al. (1993) indicated that the enhancement of enzyme activities in the rhizosphere could reach the maximum level during the peak periods of plant growth. It was shown that the improvement of ammonification in the rhizosphere increased during the early flowering stage and then declined during the podding and ripening stages. The decline in ammonification during the podding stage might be attributed to the strong N uptake

5 MICROBIAL ACTIVITY AND N CYCLE 387 during the early flowering stage, which led to a relative deficit of N in the rhizospheric soil and then weakened ammonification. In the Cd treatments, the changes of ammonification with soybean growth in the rhizosphere were similar to those in the bulk soil. However, the changes in the rhizosphere seemed to be less extreme compared to those in the bulk soil, suggesting there was some mitigation of Cd stress in the rhizosphere. Fig. lb showed that nitrification in the bulk soil decreased throughout the entire soybean-growing season. Furthermore, the relative inhibitory effects of Cd on nitrification were weakened with soybean growth, and no distinctive toxic effects of Cd were found during the podding and ripening stages. The changes of nitrification in the rhizosphere were similar to those in the bulk soil (Fig. lb). No obvious differences were observed between the rhizosphere and the bulk soil in the control or Cd treatments. The denitrification changes in the rhizosphere and bulk soil are shown in Fig. lc. In the bulk soil, the denitrifying activities consistently decreased as soybean growth progressed; however, denitrification in the control and low Cd treatment sharply decreased during the entire soybean growth period, whereas the decline observed in the high Cd treatment was more moderate. In addition, the denitrifying activity in the 20 ing Cd kg-i soil treatment at the ripening stage was even higher than that in the control. It is difficult to determine the factor that might induce this change. Nevertheless, it has been reported that the presence of heavy metals could significantly affect microbial populations and bacterial communities in the soil, thereby potentially altering the soil ecology (Wilson, 1977; BQBth, 1989; Tonner and Kunze, 1990). Therefore, the most likely explanation could be the change in ecology for denitrifying microbes, which induced a positive adaptation to high Cd contamination. In terms of the effects resulting from plant roots, the enhancement of denitrification in the rhizosphere in all treatments reached the maximum during the podding stage, with decline to a certain extent during the ripening stage (Fig. lc). Overall, for all treatments, the denitrification changes in the rhizosphere were similar to those in the bulk soil, with some mitigation being observed in the rhizosphere (Fig. Ic). This showed that changes in the rhizosphere were more gradual than those in the bulk soil. CONCLUSIONS The present study demonstrated a positive metal-adaptation process of the ammonification, nitrification, and denitrification in a Cd-stressed agricultural system. Induced by soil microbes and plant roots, the long-time effects of heavy metals on soil N-cycling differed from those at the seedling stage of plants. As far as the effects of plant roots were concerned, the microbe activity changes with the soybean growth in the rhizosphere were similar to those in the bulk soil in the Cd treatments, but the tendency of change in the rhizosphere seemed to be more moderate. This suggested that there was some mitigation of Cd stress in the rhizosphere. Further studies on the biological and the physicochemical conditions in the rhizosphere should be conducted to enhance the capacity to alleviate heavy metal contamination in a metal-stressed agricultural system. ACKNOWLEDGEMENTS The authors express their sincere thanks to Miss Q. Lin from Zhengjiang University for her advice on microbial activity assays and Mr. S. Zhu from Zhengjiang University for his assistance with the construction of the rhizoboxes. We also thank Mrs. J. Gellatly from Zhengjiang University of Technology for her helpful comments on this article. REFERENCES BBBth, E Effects of heavy metals in soil on microbial processes and population (a review). Water, Air, and Soil Pollution. 47(3/4): Babich, H. and Stotzky, G Heavy metal toxicity to microbe-mediated ecologic processes: A review and potential application to regulatory policies. Environ. Res. 36:

6 388 Y. YANG et al. Berg, P. and Rosswall, T Seasonal variations in abundance and activity of nitrifiers in four arable cropping systems. Microbial Ecol. 13: Broos, K., Uyttebroek, M., Mertens, J. and Smolders, E A survey of symbiotic nitrogen fixation by white clover grown on metal contaminated soils. Soil Biology and Biochemistry. 36: Chang, F. H. and Broadbent, F. E Influence of trace metals on some soil nitrogen transformations. J. Environ. Qual. 11: 1-4. He, 2. L Soil microbe biomass and significance innutrient cycling and quality assessment. Soils (in Chinese). 29: Hu, G. R., Li, Y. L. and Peng, P. Q Study on microbial effects of cadmium and lead in soil. Agro-Environ. Protection (in Chinese). 9(4): 6-9. Li, F. L., Yu, Z. N. and He, S. J Research Methods for Microorganisms in Agriculture (in Chinese). CGna Agriculture Publishing House, Beijing. pp Li, Z. G., Pan, Y. H. and Li, L. M Dynamics of bacteria and their enzymes activity in the rhizosphere of wheat of different genotypes. Acta Pedologica Sinica (in Chinese). 30( 1): 1-8. Li, Z. P., Zhang, T. L., Han, F. X. and Felix-henningsen, P Changes in soil C and N contents and mineralization across a cultivation chronosequence of paddy fields in subtropical China. Pedosphere. 15( 5): Long, J., Huang, C. Y., Teng, Y. and Yao, H. Y The microbial biomass and enzyme activities of reclaimed minesoils in the heavy metal pollution area. Chinese Journal of Eco-Agriculture (in Chinese). 12(3): Munn, K. J., Evans, J. and Chalk, P. M Mineralization of soil and legume nitrogen in soils treated with metalcontaminated sewage sludge. Soil Biology and Biochemistry. 32: Munn, K. J., Evans, J., Chalk, P. M., Morris, S. G. and Whatmuff, M Symbiotic effectiveness of rhizobium trifolii and mineralisation of legume nitrogen in response to past amendment of a soil with sewage sludge. J. Sustain. Agric. 11: Ramirez-Fuentes, E., Lucho-Constantino, C., Escamilla-Silva, E. and Dendooven, L Characteristics, and carbon and nitrogen dynamics in soil irrigated with wastewater for different lengths of time. Btoresource Technology. 85: Rovira, A. D Biology of soil-root interface. In Harley, J. L. and Russell, R. S. (eds.) The Soil-Root Interface. Academic Press, New York. pp Tonner, G. and Kunze, C The influence of heavy metals on nitrogen mineralization with regard to the relative ptoportions of fungi and bacteria. Angewandte Botanik. 64(3/4): Wilson, D Nitrification in soil treated with domestic and industrial sewage sludge. Environ. Pollut. 12: Youssef, R. A., Kanazawa, S. and Chino, M Distribution of microbial biomass across the rhizosphere of barley (Hondeum vulgare L.). Biology and Fertility of soils. 7: