Extraction of soil nitrogen by chloroform fumigation – A new index for the evaluation of soil nitrogen supply

Soil Biology & Biochemistry 43 (2011) 2423e2426

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Extraction of soil nitrogen by chloroform fumigation e A new index for the evaluation of soil nitrogen supply Wantai Yu a, Philip C. Brookes b, *, Qiang Ma a, Hua Zhou a, Yonggang Xu a, Shanmin Shen a a b

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, PR China Sustainable Soils and Grassland Systems Department, Rothamsted Research, Harpenden, Herts. AL5 2JQ, UK1

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2010 Received in revised form 11 July 2011 Accepted 28 July 2011 Available online 29 August 2011

The N extracted after chloroform (CHCl3) fumigation was determined as a possible index of soil N supply to plants. The relationships between extractable N following fumigation and reference indices such as total N, alkali-hydrolyzable N, N released by the Stanford short-term incubation method, and the N extracted by KCl and by CaCl2, were measured in nine soils of differing soil N supply capacity. A highly significant correlation was achieved between the extractable N released by fumigation and the N released by the Stanford method, i.e. a short-term aerobic incubation (r ¼ 0.87). Similarly, the correlation between extractable N by fumigation and the N uptake by ryegrass was highly statistically significant (r ¼ 0.93). Using the N extracted following fumigation has the advantage that laboratory results are available in two days and are both reproducible and of high precision. Therefore, the N extracted following fumigation is a valid, timesaving and precise index of soil N supply capacity. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: N supply N extracted by chloroform fumigation N uptake

Nitrogen (N) is an essential plant nutrient and many researchers have tried to measure soil N supply. With the rapid development of the N fertilizer industry and the increasing application of N fertilizers to soil since the 1980s, its excessive use has threatened both human health, e.g. by encouraging the development of toxic Cyanobacterial blooms (Prommana et al., 2006) and otherwise damaging the environment (Longo and York, 2008). Therefore, research has focused not only on soil N and soil N supply and fertilizer application, but also on assessing the environmental effects of N and the protection of the environment. Soil N supply refers to the N supplying ability of soil to crops or other plants. Indicators of soil N supply may be both chemical and biological. The chemical indices include total N (Smith et al., 1977), alkali-hydrolyzable N (Cornfield, 1960), KCl extractable N (Bremner, 1965a,b; McTaggart and Smith, 1993), and CaCl2 extractable N (Stanford and Legg, 1968). These methods provide simple and rapid estimates of available N. However, they have several disadvantages. For example, they take no account of the biological process of N mineralizationeimmobilization in soil. The biological indices include N assessment by a short-term (14 days) aerobic incubation of Stanford (1982) (temperature: 35  1  C, soil moisture: 50% of

* Corresponding author. Tel.: þ44 1582 763133; fax: þ44 1582 760981. E-mail address: [email protected] (P.C. Brookes). 1 Current address. 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.07.023

WHC); one of 7 days by Thicke and Russelle (1993) and a short-term anaerobic incubation of 7 days by Keeney and Bremner (1966). These indices are obtained by more or less complex procedures involving incubations under strictly defined incubation conditions. However, they have sound scientific rationales, especially the Stanford aerobic incubation method, which has become a standard method to determine the soil N supply capacity (Zhao et al., 2009). Nevertheless, chemical procedures still receive considerable attention. Many researchers have focused on trying to develop chemical methods that have wide application, are easy to determine and are related to mineralizationeimmobilization of soil N. The increase in nitrogen extracted following fumigation is a common method to measure soil microbial biomass N (Brookes et al., 1985). Biomass N plays an important role in soil N supply, being both an important sink and source of available soil N (Lethbridge and Davidson, 1983). Unfortunately, it only comprises a small fraction of the total N mineralized in soil, which has resulted in it being ignored as an index of soil N availability. We investigated the extractable N released by chloroform fumigation as an index of soil N supply. Other indices, such as total N, alkali-hydrolyzable N, the N extracted by KCl, the N extracted by CaCl2, the N extracted after shortterm aerobic incubation and microbial biomass N were also compared as indices of N supply by correlating them with ryegrass N uptake. The experiment was conducted at the Shenyang Experimental Station (SES) of the Institute of Applied Ecology, CAS (41320 N, 123 230 E). The mean annual air temperature is 7e8  C, the mean

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annual precipitation is about 700 mm and the frost-free period is 147e164 days. The soils (0e20 cm depth) were collected from different treatments of three long-term experiments of SES and Shenyang Agricultural University (SAU). There were nine soils (given as IeIX) in the pot experiment with four replicates per treatment. The soil samples of I and II were collected from the Nil (CK) and NP þ M (farmyard manure) treatments of a long-term experiment in SAU (Wang et al., 2008). Soil samples III, IV, V and VI were collected from the CK, M, NPK, and NPK þ M treatments of a long-term experiment at SES (Yu et al., 2009). Samples VII and VIII were obtained from the plantation and fallow long-term experiment, respectively, in SES (Yu et al., 1995, 2007). Samples IX was obtained from a nonexperimental grassland field in SES. The soils were Clay-loam mixed Aquic Mesic Dystrochrepts and Clay-loam mixed Udic Mesic Umbraqualfs (USDA, 1992). Although only two soil types were adopted in the present study, they are widespread in the North China Plain and are from different long-term experiments which have been running for almost 20 years. The soil total N was 0.9 g kg1 to 1.8 g kg1 (Table 1), covering a range including almost all of the cultivated surface soils in China (Zhu, 1998).

Standard techniques were used to measure pH (1:2.5 soil-water suspension) (Anon, 1986), extractable P (0.5 M NaHCO3 at pH 8.5) (Olsen et al., 1954), soil-exchangeable K (1 M NH4OAc at pH 7) (Jones, 1973), and total N and C by automated combustion analysis. Soil available N was measured by an alkaline hydrolysis method (Cornfield, 1960). Nitrogen mineralized over a short-term aerobic incubation of 14 days was extracted by 2 M KCl. The NHþ 4 eN and NO 3 eN were distilled after the addition of MgO and Devardas alloy (Keeney and Nelson, 1982). Total N extracted by 1 M KCl and by 0.01 M CaCl2 were measured by a semi-micro Kjeldahl digestion method (Table S1). Total N after fumigation was extracted by 0.5 M K2SO4 and determined by semi-micro Kjeldahl digestion, followed by steam distillation, as described by Brookes et al. (1985), and  NHþ 4 eN and NO3 eN were measured by the same method as above. The organic N was determined from the difference between the total N and the NHþ 4 eN. Total N in plants was determined by the method described by Bremner (1965a,b). Each moist soil was passed through a 5-mm sieve and divided into two parts. One part was air-dried and ground to pass a 2-mm sieve and used to determine soil chemical properties. The other part was adjusted with distilled water to 50% WHC after measure-

Table 1 Soil properties. Soil numbera

I

II

III

IV

V

VI

VII

VIII

IX

Mean

ST. DEV.

C.V.

Total N (g kg1) Organic C (g kg1) Available P (mg kg1) Exchangeable K (mg kg1) pH

0.91 10.31 11.50 88.59 6.45

1.77 17.86 16.71 106.78 6.22

0.94 10.45 3.22 69.74 6.44

1.17 12.04 3.43 67.75 6.44

1.05 11.55 17.11 84.34 6.03

1.36 13.54 21.14 101.48 6.09

1.43 15.85 11.42 161.34 6.83

0.89 11.66 7.73 72.05 5.98

1.66 18.8 8.02 171.92 6.47

1.24 13.56 11.14 102.67 6.33

0.33 3.19 6.23 38.78 0.27

0.2655 0.2352 0.5590 0.3777 0.0429

a

For soil details see text.

Table 2 The biomass of ryegrass and N uptake by ryegrass in various treatments. Soil No.a

Ryegrass shoot Biomass (g pot1)

I II III IV V VI VII VIII IX Mean

10.05 16.85 10.02 11.78 13.31 15.71 12.53 16.98 16.08 13.70

        

0.61c 0.83a 0.48c 0.72bc 0.84b 0.77ab 1.07bc 0.98a 1.11a

Ryegrass root N uptake (mg pot1) 168.84 394.29 142.28 190.84 199.65 278.07 206.75 258.10 287.83 236.29

        

10.48cd 29.73a 10.12d 8.50bc 10.99bc 12.91b 9.68bc 18.81b 20.39b

Biomass (g pot1) 2.54 2.76 2.88 2.64 2.46 2.36 1.88 3.65 2.40 2.62

        

0.11ab 0.15ab 0.14ab 0.20ab 0.17ab 0.10ab 0.08b 0.27a 0.13ab

Total biomass (g pot1)

N uptake (mg pot1) 42.16 52.99 39.46 39.60 41.33 40.59 28.01 54.39 36.00 41.61

        

3.12b 3.63a 1.95b 2.36b 1.73b 2.36b 1.48c 3.74a 1.86b

12.59 19.61 12.90 14.42 15.77 18.07 14.41 20.63 18.48 16.32

        

1.02d 1.68ab 0.96d 1.24cd 0.88c 1.30b 0.73cd 1.63a 1.52ab

Total N uptake (mg pot1) 211.00 447.28 181.74 230.44 240.98 318.66 234.76 312.48 323.83 277.90

        

16.61cd 30.40a 12.19d 19.15c 18.06c 25.99b 13.75c 25.62b 26.24b

Letter indicates the significant difference in one column at the 5% level of probability. a See text for soil details.

Table 3 The concentrations of mineral N following Stanford short-term aerobic incubation (mg kg1). Total Mineral Na

Soil No.

After incubation

Before incubation

NHþ 4 eN

NO 3 eN

NHþ 4 eN

NO 3 eN

I II III IV V VI VII VIII IX Mean

5.93  0.39c 8.05  0.41a 8.05  0.45a 7.63  0.61ab 7.04  0.30b 7.04  0.33b 4.24  0.37d 3.39  0.24e 8.05  0.48a 6.60

34.76  2.41c 69.27  4.56a 26.20  1.60d 36.46  2.25c 45.53  2.58b 59.94  3.99ab 48.33  3.43b 44.09  3.42b 59.95  3.27ab 47.17

5.98  0.37c 6.36  0.43bc 6.82  0.31b 4.12  0.18d 7.18  0.52b 9.74  0.49a 6.48  0.39bc 3.46  0.17d 3.64  0.20d 5.98

20.30  1.23bc 38.06  2.18a 15.76  0.96c 21.32  1.17bc 23.35  1.30b 31.97  1.82a 19.26  0.79bc 23.94  1.15b 33.67  1.66a 25.29

14.41  0.74c 32.90  2.65a 11.67  0.78c 18.65  1.37bc 22.04  0.81b 25.27  1.36ab 26.83  1.13ab 20.08  1.08bc 30.70  1.47a 22.51

Letter indicates the significant difference at 5% level of probability for the same index within Stanford short-term aerobic incubation and the extractable N concentrations, respectively. a  þ  Total mineral N ¼ (NHþ 4 eN þ NO3 eN) after incubation  (NH4 eN þ NO3 eN) before incubation.

21.25  48.77  19.06  25.44  29.37  35.12  25.31  33.61  43.78  31.30  0.79cd  1.73a  0.66d  1.03cd  1.56bc  2.13ab  0.91c  1.38b  1.70a 17.59 39.42 14.33 18.78 23.12 31.24 20.81 28.25 38.57 25.79  0.04d  0.10c  0.15b  0.21bc  0.27ab  0.12c  0.02e  0.21a  0.18a 0.93 2.50 3.13 2.75 3.55 2.54 0.51 4.06 3.99 2.66 50.17  4.57cd 102.43  7.22a 40.05  2.41d 56.83  3.16c 57.57  2.87c 80.04  5.20b 54.12  3.11c 66.84  4.69bc 101.43  6.74a 67.72 1.36c 3.12a 0.89d 1.60c 1.57c 2.44b 1.68c 2.79bc 3.04a 31.15  58.46  19.88  32.76  31.41  44.76  29.89  35.82  56.84  37.89  1.59c  4.11a  1.07d  3.09bc  1.76c  2.60b  1.46c  2.89bc  4.15a 32.80 63.07 24.70 37.71 34.56 48.99 33.88 38.93 62.46 41.90  0.78c  1.67a  0.70c  1.03c  2.04bc  2.53ab  1.21bc  1.34b  1.59a 17.37 39.36 15.35 19.12 23.01 31.05 20.24 27.91 38.97 25.82  0.06e  0.19b  0.26ab  0.31ab  0.14d  0.33bc  0.19c  0.12d  0.26a 1.65 4.61 4.82 4.95 3.15 4.23 3.99 3.11 5.62 4.01 I II III IV V VI VII VIII IX Mean

Organic Nb Total N NO 3 eN

Letter indicates the significant difference at 5% level of probability for the same index within Stanford short-term aerobic incubation and the extractable N concentrations, respectively. a Extractable N concentrations after fumigation ¼ (NO 3 eN þ Total N). b Organic N ¼ Total N  NHþ 4 eN.

20.32 46.27 15.93 22.69 25.82 32.58 24.80 29.55 39.79 28.64

 1.62cd  3.03a  0.95d  2.20cd  1.49c  2.16bc  0.94c  2.38c  3.02ab

38.84 88.19 33.39 44.22 52.49 66.36 46.12 61.86 82.35 57.09

 2.91de  3.42a  1.87e  3.21d  2.82c  3.19b  3.13d  4.29b  5.16a

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0.91d 2.64a 1.12d 1.76cd 1.51c 2.10b 1.97cd 1.73bc 3.13ab

Organic Nb Total N NHþ 4 eN

NO 3 eN

Non-Fumigated extracts

NHþ 4 eN

Extractable Na Fumigated extracts Soil No.

Table 4 The concentrations of extractable N with and without fumigation (mg kg1).

ment of soil moisture for microbial biomass measurements and 70% of field moisture capacity for pot experiments. The ryegrass was grown in 36 plastic pots, each with a diameter of 30 cm and a height of 25 cm. Each pot was filled with the equivalent of 5 kg dry soil and supplied with 50 mg P and 50 mg K kg1 soil. Thirty five ryegrass seeds were sown in each pot on April 25 2007, and the plants thinned to 30 seedlings per pot at 7 days after germination. The final harvest was on August 5, 2007. All visible roots were picked out, washed, then oven-dried. Because of the vigorous growth, the ryegrass was harvested once during the growing period in addition to the final harvest. The harvested ryegrass shoots and plant residues were then oven-dried and weighed. Differences between measurements were examined by analysis of variance (ANOVA) with SPSS 11.0. The least significant difference (LSD) was set at the 5% probability level. The biomass of ryegrass was significantly different between treatments (P < 0.05) and the average biomass dry matter yield was 16.32 g pot1 (Table 2). The proportion of the shoot biomass was 77.67e87.01% with an average of 83.95%, while the proportion of the root biomass averaged 16.05%. The biomass differences indicated that the various soils had markedly different fertilities. The soil N supply includes N uptake by crops and soil residual available N. The latter is usually negligible and only the N uptake by crops is considered when measuring the soil N supply capacity. The average N uptakes by ryegrass shoots and roots were 236.29 mg pot1 and 44.61 mg pot1 respectively, equivalent to 85.03% and 14.97% of total plant N uptakes, respectively. The N uptake by ryegrass was significantly different between treatments (P < 0.05) (Table 2). The discrepancy was attributed to the soil N supply capacity. The mineral N concentrations extracted before and after the short-term aerobic incubation in various treatments are given in Table 3. The NHþ 4 eN concentrations remained unchanged but the nitrate N concentrations were much larger after incubation. The mean NO 3 eN concentration in the various treatments was 25.29 mg kg1 before incubation and 47.17 mg kg1 after incubation (P < 0.05). The soil organic N after mineralization was mainly converted to nitrate; little NHþ 4 eN was detected (Zhang et al., 1997). The extractable N concentrations with and without fumigation in the various treatments are also shown in Table 4. The NHþ 4 eN concentrations were small without fumigation with an average of 2.66 mg kg1. Differences in NHþ 4 eN concentrations among the various treatments were not evident. However, the NHþ 4 eN concentrations increased significantly with fumigation. The mean, of 4.01 mg kg1, was about 50% larger than that extracted from nonfumigated soils (P < 0.05). The soil NO 3 eN concentrations hardly changed with and without fumigation in the various treat ments. In contrast to NHþ 4 eN and NO3 eN, the changes in organic N were considerably larger, with an average of 28.64 mg kg1 in nonfumigated soil. Following fumigation, the extracted organic N significantly increased in the various treatments. The mean concentration was 37.89 mg kg1, which increased 32% over that without fumigation. The organic N comprises a fraction of biomass N and is potentially plant available (Paul and Beauchamp, 1996; Xu et al., 2010). The correlations between the N extracted following fumigation with the other indices of N availability were statistically significant (P < 0.05) (Table 5). The strongest correlation was with the N mineralized by short-term aerobic incubations because both indices were relevant to the microbial processes which drive mineralizationeimmobilization. Part of the N measured in aerobic incubations was probably derived from the organic N extracted by K2SO4 after fumigation. The correlation coefficient between extractable N after fumigation and mineral N after short-term

Extractable Na

W. Yu et al. / Soil Biology & Biochemistry 43 (2011) 2423e2426

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W. Yu et al. / Soil Biology & Biochemistry 43 (2011) 2423e2426

Table 5 The correlation of the indices of N supply capacity to the Fumigated extracted N, N uptake by ryegrass and by ryegrass shoot, respectively (n ¼ 9). Fumigated extracted N Extractable N by Fumigated P value

Total N

Alkaline N

Short-term incubation N

KCl Extracted N

CaCl2 Extracted N

0.794* 0.011

0.846** 0.004

0.869** 0.002

0.825** 0.006

0.885** 0.002

N uptake by ryegrass P value

0.929*** 0.000

0.726* 0.027

0.652 0.057

0.813** 0.008

0.855** 0.005

0.876** 0.002

N uptake by ryegrass shoot P value

0.912*** 0.001

0.777* 0.014

0.703* 0.035

0.856** 0.003

0.822** 0.007

0.868** 0.002

Significance levels are: *** for P < 0.001; ** for P < 0.01; * for P < 0.05.

aerobic incubations was 0.87 (P < 0.01) which was higher than that of other correlation coefficients (Table 5). The correlations between the total N uptake (i.e. shoot plus root) and the N extracted after fumigation, and other reference indices except alkaline N (Table 5), were statistically significant (P < 0.05). The correlation between the total N uptake by ryegrass (i.e. root plus shoot) and the N extracted after fumigation (r ¼ 0.93) was very similar to, indeed actually numerically larger than, the correlation between the N uptake by ryegrass and the N extracted following short-term aerobic incubation (r ¼ 0.81). Therefore, the N extracted following fumigation can provide a valid indicator of the soil N supply capacity. The N released by aerobic incubation is generally considered to be the standard method of assessing soil N supply capacity (Zhao et al., 2009). The advantages of this method include widespread application, stable and accurate data, and consideration of the soil mineralizationeimmobilization process. Its main limitation is that the measurement time is too long. While the new index has as a close correlation, or even a stronger one, with the N uptake by ryegrass or ryegrass shoots as the Stanford method, the procedure only take two days. Another advantage is that both chemical and biological procedures are both taken into account in the new approach, and the results are consistent and of high precision. However, it must be noted that the soils were all clay soils of similar pH, and it will be necessary to extend the soil types tested in order to be confident of the wider application of this new approach. In summary, the N extracted following fumigation is a valid, time saving and precise index of soil N supply capacity, at least in the soils so far tested, giving results comparable to the N mineralized by the short-term Stanford aerobic incubation. Acknowledgements This work was financially supported by the Key Innovational Project in Environment and Resources Fields from CAS, China (No. KZCX2-YW-405), National Natural Science Foundation of China (Nos. 31070547, 41171242). Rothamsted Research is a BBSRC funded Institute. Appendix. Supplementary information Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.soilbio.2011.07.023. References Anon, 1986. Analysis of Agricultural Materials, third ed. HMSO, London. Reference Book 427. Bremner, J.M., 1965a. Nitrogen availability indexes. In: Black, C.A. (Ed.), Methods of Soil Analysis. Part 2, vol. 9. American Society of Agronomy, Madison. WI, pp. 1324e1345. Agronomy.

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