Ratios of microbial biomass estimates to evaluate microbial ...

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Jul 12, 2005 - The Harjavalta Cu-Ni smelter is located in south-western. Finland (61°19′N, 22°9′E) and has been polluting the en- vironment with heavy ...
Biol Fertil Soils (2006) 42: 241–246 DOI 10.1007/s00374-005-0021-1

ORIGINA L PA PER

Oliver Dilly

Ratios of microbial biomass estimates to evaluate microbial physiology in soil

Received: 14 January 2005 / Revised: 30 May 2005 / Accepted: 6 June 2005 / Published online: 12 July 2005 # Springer-Verlag 2005

Abstract Soil microbial biomass data derived from fumigation–extraction (FE), substrate-induced respiration (SIR) and ATP estimations differed significantly and were significantly correlated, which agrees to previous studies. In a second step, the SIR/FE, ATP/FE and SIR/ATP ratios were calculated to evaluate the glucose-responsive and active component of the microbial (active and resting) biomass and the glucose-responsive component of the active microbiota. Soils were sampled along gradients within and between associated ecosystems in Northern Germany, Denmark and along a gradient of heavy metal pollution in Finland. The ratios indicated that the active portion and glucose-responsive component decreased with proceeding litter decomposition, higher degree of sustainable land management practices and higher degree of heavy metal contamination. Keywords ATP content . Fumigation–extraction . Gradient . Indicator . Microbial physiology . Pollution . Soil microbial biomass . Substrate-induced respiration

Introduction Soil microbial biomass is a fraction of soil organic matter that is sensitive to management practices and pollution (Powlson 1994). Four methods, fumigation–incubaThis work was presented at the workshop ‘Non-molecular manipulation of soil microbial communities’ at the University of Udine, Udine, Italy, 17–20 October 2004; convened by P.C. Brookes and M. De Nobili and supported by European Science Foundation. O. Dilly Ökologie-Zentrum, Universität Kiel, Schauenburgerstraße 112, 24118 Kiel, Germany O. Dilly (*) Lehrstuhl für Bodenschutz und Rekultivierung, Brandenburgische Technische Universität, Cottbus, Germany e-mail: [email protected]

tion (FI), substrate-induced respiration (SIR), fumigation– extraction (FE) and ATP content (Jenkinson and Powlson 1976; Anderson and Domsch 1978; Jenkinson and Ladd 1981; Vance et al. 1987), are widely used for the estimation of soil microbial biomass and are designed to quantify its C content (Elliott 1994). There exists an extensive bibliography concerning the correlation between the methods, calibration of the methods to obtain a realistic conversion factor for the estimation of soil microbial C and potentials and limitations of the methods (Alef 1993; Sparling and Ross 1993; Alef and Nannipieri 1995; Martens 1995; Dilly and Munch 1998; Burns 2004; Jenkinson et al. 2004). By considering the procedure involved in the four methods, the following basic considerations can be done: both FE and FI methods account for fumigation-sensitive organisms, e.g. organisms that are lysable in chloroform and are affected by the velocity of cell degradation and either extraction or mineralization efficiency, respectively. The SIR method estimates the microbial biomass that responds to easily degradable compounds (Vedder et al. 1996; Beck et al. 1997). The respiratory response to glucose was earlier referred to as the zymogenic response by Stout and Dutch (1968). ATP content represents a constituent that occurs in all living organisms (Nannipieri et al. 1990; Bentham et al. 1992) and is, therefore, controlled by the physiological state of cells. ATP measurements are sensitive to the extraction procedure (Martens 1995, 2001; Contin et al. 2002). Based on these basic considerations, the FE method detects both active or non-active (resting), zymogenous and autochthonous microorganisms (Winogradsky 1924). The SIR and ATP methods, as physiological estimates, refer to the active microbiota with regard to the substrateresponding and zymogenous component in the first and active, either zymogenous and autochthonous microorganisms in the latter case. Indeed, total microbial biomass was supposed to be determined by the FE method (Tessier et al. 1998), and the active microbial biomass (Hassink 1993, 1995; Kuperman and Carreiro 1997), glucose-responsive microbial biomass (Priha and Smolander 1994; Wardle and Ghani 1995a; Fritze et al. 1996; Lin and Brookes 1996)

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than 5 and 2 mm were used for the analysis of the Ohf horizon and topsoils, respectively. The Danish soils were taken from the Euroflux site ‘Sorø’ (55°29′N, 11°38′E) and were sampled in June 1999. The forest L, Of1, Of2 and Ah and arable Ap horizon were quantitatively taken below continuous flow boxes (area=200 cm2) after finishing soil respiration measurements. Sample preparation and FE, SIR and ATP determination were done as previously described, except that SIR was carried out in closed jars (Alef 1995). Three independent replicates were analysed from each soil horizon. Materials and methods The Harjavalta Cu-Ni smelter is located in south-western The first series of soil samples was collected from ara- Finland (61°19′N, 22°9′E) and has been polluting the enble and forest soils of a landscape in Northern Germany vironment with heavy metals for nearly 50 years. An 8-km(Bornhöved Lake district), the second from a Danish forest long transect in Scots pine stands has been established, and an adjacent arable soil and the third set from three starting from the point emission source, and fertilisation humus horizons in Scots pine stands along a gradient of experiments were performed in spring 1992. Here, the data heavy metal pollution in Finland. The original data of published by Fritze et al. (1996) from the sampling in June the Finnish Scots pine stands were already published by 1993 (four replicates) were used. The FE and SIR measurements of Germany and Danish Fritze et al. (1996). Soil properties are displayed in Table 1. Soils from two arable sites, a grassland and a beech soils were carried out by the methods of Vance et al. (1987) forest, were sampled from the ‘Bornhöved Lake district’ in and Anderson and Domsch (1978), respectively, as deNorthern Germany (54°06′N, 10°14′E). Bulk samples con- scribed in detail by Dilly and Munch (1995) with one sisting of multiple cores were taken from the topsoils (A modification. Whereas the topsoils were extracted with horizons) of all sites. Material from the L and Ohf horizons the usual soil-extractant ratio of 1:4, the L or Ohf horizons in the beech forest was sampled at four locations each of of the forest soil was extracted with a ratio of 1:16 to gain about 40×40 cm. The samples were homogenised and sufficient extract for analysis (dry weight/volume). The sieved. Material from the L horizon consisted mainly of ATP content was measured in soils that have been staleaf litter and could not be sieved. It was cut in pieces bilised for 4 weeks at 4°C (Jenkinson and Oades 1979). smaller than 5 mm immediately before the FE and ATP Soil samples were extracted with the trichloracetic-acid/ determinations, but not before SIR measurements, to phosphate (without paraquat) solution and sonificated on avoid damage to microbiota, particularly to the fungal ice for 2 min using the power of about 150 W. To increase mycelium (Maraun and Scheu 1995). The fractions smaller the amount of the extractant, 2.00 and 0.40 g material were or the microbial biomass decomposing plant residues (Pfender et al. 1996), by the SIR method. Thus, the SIR/ FE, ATP/FE and SIR/ATP ratios were calculated to assemble for glucose-responsive and active component in the total microbial biomass and the glucose-responsive component in the active microbiota, respectively. Because these ratios are based on at least one physiological method, it is hypothesised that they can be used to evaluate the physiology of the soil microflora.

Table 1 Soil characteristics in the landscape in Northern Germany and in Denmark and along a gradient of heavy metal pollution in Finland

a pH values in soils of the landscape in Northern Germany and Denmark were measured in 0.01 M CaCl2, those in the humus horizons of Scots pine stands growing along a gradient of heavy metal pollution in Finland were measured in 0.1 M BaCl2 b Estimated by fumigation–extraction uS Silty sand, lS loamy sand, ND not determined

Horizon Depth (cm) Landscape in Northern Germany Field crop rotation Ap Field maize monoculture Ap Grassland Ah Beech forest L Ohf Ah Landscape in Denmark Beech forest L Of1 Of2 A(e)h Crop rotation field Ap Gradient in Finland (controls) 0.5 (high pollution) F/H 4 F/H 8 (low pollution) F/H

pHa

Corg (mg C/N Texture Microbial biomassb g−1 soil) (μg Cmic g−1 soil)

0–20 0–20 0–10 4–3 3–0 0–5

5.2 4.6 5.1 4.5 3.6 3.5

15 12 18 412 308 29

5–3.6 3.6–2 2–0 0–3 0–20

ND 4.8 5.0 4.5 6.9

440 289 200 64 22

3–0 3–0 3–0

3.9 3.7 3.7

322 499 452

9 10 10 20 19 15 ND ND ND ND ND 30 37 38

uS uS uS ND ND lS

249 136 330 3,802 3,205 355

ND ND ND ND ND

17,390 4,982 3,883 1,112 762

ND ND ND

1,584 4,194 5,623

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used for topsoils and L and Ohf horizon, respectively. Results were corrected for recovery of an ATP ‘spike’ to the soil samples that was 2.5 times higher than the previously measured ATP content. Each analysis has been carried out with three replicates. Soil was stored no longer than 3 weeks at 4°C before analysis. Microbial biomass of Finnish soils was determined in the Finnish laboratory similarly as for the German and Danish locations.

Results and discussion

Microbial biomass values are given in Table 1 and significant correlations with coefficients exceeding 0.8 are presented in Table 2. Although soils under contrasting land use systems from the Bornhöved Lake District produced lower correlation coefficients between the FE and SIR and also between SIR and ATP, they showed the highest coefficients between FE and ATP. German soils of the Bornhöved Lake district The highest SIR/FE ratio was observed in the fresh litter (L horizon) of the beech forest (Table 3). This ratio decreased with increasing soil depth. In mineral topsoils, the SIR/FE ratio was highest in grassland and lowest in beech forest. The highest ATP/FE ratio was detected in the L and Ohf horizon of the forest soil, whereas there was no significant difference in the ATP/FE ratio values of monoculture, grassland and forest topsoils. In contrast, the ratio was reduced in the field crop rotation. The ratio between SIR and ATP was the highest in the L horizon of the beech forest (Table 3). These results confirm findings by Priha and Smolander (1997) who observed higher SIR than FE microbial biomass values in the litter horizons but not in the respective underlying topsoils. The decline in SIR/FE and

Table 2 Spearman rank order correlation coefficients (r) between FE, SIR and ATP in soils in the landscape in Northern Germany and in Denmark and along a gradient of heavy metal pollution in Finland FE

Landscape in Northern Germany SIR 6 ATP 6 Landscape in Denmark SIR 15 ATP 14 Gradient in Finland SIR 12 ATP 12

Site

Horizon SIR/FE ATP/FE SIR/ATP

Field crop rotation Field maize monoculture Grassland Beech forest

Ap Ap Ah L Ohf Ah

1.3bc 1.3bc 1.8b 10.6a 2.1b 0.8c

0.43c 0.46c 0.44c 1.87a 1.06b 0.50c

2.94a 2.23b 4.00a 5.69a 1.89c 1.66c

Different letters indicate significant differences when applying the Student–Newman–Keuls method (p