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Structure and function of the soil microbial community in a long-term fertilizer experiment
Soil Biology & Biochemistry 35 (2003) 453–461 www.elsevier.com/locate/soilbio
Structure and function of the soil microbial community in a long-term fertilizer experiment Petra Marschnera,*, Ellen Kandelerb, Bernd Marschnerc a Department of Soil and Water, University of Adelaide, PMB 1 Waite Campus, Glen Osmond, SA 5064, Australia Institut fu¨r Bodenkunde and Standortslehre, Fachgebiet Bodenbiologie, Universitat Hohenheim, D-70599 Stuttgart, Germany c Ruhr-Universita¨t Bochum, Geographisches Institut, Bodenkunde/Bodeno¨kologie, Universita¨tsstr., 150, D-44780 Bochum, Germany b
Received 12 March 2002; received in revised form 24 September 2002; accepted 3 December 2002
Abstract The effect of organic and inorganic fertiliser amendments is often studied shortly after addition of a single dose to the soil but less is known about the long-term effects of amendments. We conducted a study to determine the effects of long-term addition of organic and inorganic fertiliser amendments at low rates on soil chemical and biological properties. Surface soil samples were taken from an experimental field site near Cologne, Germany in summer 2000. At this site, five different treatments were established in 1969: mineral fertiliser (NPK), crop residues removed (mineral only); mineral fertiliser with crop residues; manure 5.2 t ha21 yr21; sewage sludge 7.6 t ha21 yr21 or straw 4.0 t ha21 yr21 with 10 kg N as CaCN2 t straw21. The organic amendments increased the Corg content of the soil but had no significant effect on the dissolved organic C (DOC) content. The C/N ratio was highest in the straw treatment and lowest in the mineral only treatment. Of the enzymes studied, only protease activity was affected by the different amendments. It was highest after sewage amendment and lowest in the mineral only treatment. The ratios of Gram þ to Gram 2 bacteria and of bacteria to fungi, as determined by signature phospholipid fatty acids, were higher in the organic treatments than in the inorganic treatments. The community structure of bacteria and eukaryotic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) and redundancy discriminate analyses of the DGGE banding patterns. While the bacterial community structure was affected by the treatments this was not the case for the eukaryotes. Bacterial and eukaryotic community structures were significantly affected by Corg content and C/N ratio. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Organic amendment; Sewage sludge; Mineral fertilizer; Microbial community structure; Soil enzyme activity; Denaturing gradient gel electrophoresis; Phospholipid fatty acid
1. Introduction Organic and inorganic fertiliser amendments are used primarily to increase nutrient availability to plants, but they can also affect soil microorganisms. Changes in microbial activity and composition can in turn influence plant growth, for example by increased nutrient turnover, or by increasing disease incidence or disease suppression. Amendments usually increase the amount of soil organic C (Corg) and the concentration of other nutrients such as N (Madejon et al., 2001; Crecchio et al., 2001). Organic matter can be added either directly by * Corresponding author. Tel.: þ 1-61-8-8303-7379; fax: þ 1-61-8-83036511. E-mail address: [email protected] (P. Marschner).
incorporating manure, straw, sewage sludge or municipal waste or indirectly via increased plant growth. Generally, microbial biomass increases with increasing Corg content of the soil (Dhillion, 1997). In a number of short-term studies it has been shown that organic amendments increase microbial biomass (Peacock et al., 2001; Hu et al., 1999; Pascual et al., 2000). Organic matter addition often leads to a rapid increase of soil CO2 evolution (Ajwa and Tabatabai, 1994) and increases the activity of various enzymes (Crecchio et al., 2001; Madejon et al., 2001; Kandeler et al., 1999b). An increase in enzyme activity was also observed after addition of either NPK fertiliser or farmyard manure in a long-term study (Kandeler et al., 1999a). Amendments can change amount and quality of dissolved organic matter (DOM) present in the soil solution (Marschner
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0038-0717(02)00297-3
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and Noble, 2000). DOM is an easily available substrate for soil microorganisms (Metting, 1993) and its importance for microbial activity has been shown by studies, in which soil respiration was closely related to DOM concentrations (Marschner and Noble, 2000). Changes in microbial community composition are often observed after addition of organic or inorganic amendments. Compared to NO3, NH4 fertilisation decreases take-all severity in wheat (Sarniguet et al., 1992a) and suppresses Thielaviopsis basicola (Harrison and Shaw, 2001). Soil fungi differ in their response to NPK fertilisers (Donnison et al., 2000) and N fertilisation changes the abundance of fungal species (Sarathchandra et al., 2001). Different types of amendments may differ in organic matter composition (e.g. C/N ratio), and this in turn, affects the decomposition rate and can change the microbial community structure. For example, presence of easily degradable plant material favours organisms that grow rapidly in nutrient-rich environments (copiotrophs) compared to organisms adapted to nutrientpoor conditions (oligotrophs) (Hu et al., 1999), while straw incorporation favours the competitive ability of cellulolytic microorganisms compared to those that can not degrade cellulose (Jensen and Nybroe, 1999). The increase in catabolic diversity that is observed in soils after addition of easily degradable organic matter (Degens et al., 2000) also indicates changes in microbial community structure. However, in a short-term study, Crecchio et al. (2001) found no changes in bacterial community structure after addition of municipal solid waste compost. In another study, Buyer and Kaufman (1996) found similar culturable bacterial and fungal species composition in low input and conventional agriculture. In most of the above mentioned studies the responses of soil microorganisms to organic or inorganic amendments were measured within a few years after a single application. Far less is known about the effects of longterm amendments at a low rate. The effect of these may differ substantially from those of a single high dose. In a field experiment with low rates of organic and inorganic amendments over 40 years we assessed soil chemical and microbiological properties using a range of approaches. We determined both total Corg and N and amount and aromaticity of DOC. The activities of enzymes involved in C, N, P and S cycling were studied, some of which have been previously shown to respond to organic amendments (Kandeler et al., 1999b). Phospholipid fatty acid (PLFA) patterns were used to estimate the relative abundance of bacteria and fungi. The molecular approach, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), provided an analysis of bacterial and eukaryotic community structure. The relationships between soil chemical properties and microbial indices were tested by regression and multivariate analyses.
2. Materials and methods The study site is a reclaimed loess soil (calcareous Regosol) in the Rhineland lignite mining area near Cologne, Germany. After the end of the mining activities in 1968, the mine spoil was levelled and covered with a 90 cm layer of calcareous loess material with very low Corg content (0.1%). In 1969 an experimental field site was established with the following treatments: NPK mineral fertilizer, all plant residues removed (mineral only); mineral fertilizer, with plant residues incorporated into the soil; manure 5.2 t ha21 yr21; sewage sludge 7.6 t ha21 yr21; straw 4.0 t ha21 yr21 with 10 kg N as CaCN2 t straw21. Each treatment was replicated four times with plot sizes of 4 £ 9 m2 randomly distributed in the field. A crop rotation typical for the region consisting of sugar beet, wheat, rye, potato, wheat and barley was grown since the establishment of the site (for further details see Delschen (1999)). The treatments were sampled in June 2000 with winter barley as cover crop. In each replicate of the five treatments, eight soil samples were collected from the top 20 cm with an auger and mixed to give a bulk sample which was immediately stored in a cooler at 4 8C. In the laboratory, the stone-free samples were thoroughly homogenized and divided into several sub-samples, some of which were stored at 2 18 8C for microbial and enzyme analyses. 2.1. Soil chemical analyses For the extraction of soluble organic matter, about 80 g DW of field moist soil samples were packed into round steel cylinders (46 mm ID, 48 mm height) to give a bulk density similar to field conditions (approx. 1.1 g cm23). The cylinders were placed on porous sintered glass plates (pores , 16 mm) that were connected to a glass collection bottle pressurized to 2 60 hPa. The surface of the samples was covered with 1 mm glass beads and then drip-irrigated through a rainfall simulator with 25 capillaries of 0.2 mm diameter. A total of 250 ml 1 mM CaCl2 solution was applied within 6 h, equivalent to a rainfall intensity of about 25 mm h21. This mode of extraction, under unsaturated flow conditions, removes the mobile fraction of soluble organic matter and closely resembles field conditions where mainly medium-sized and larger pores (. 50 mm) participate in transport processes. Following 0.45 mm membrane filtration, acidification and gas-purging of the solutions to remove inorganic C, DOC was determined with a TOC-Analyser (Shimadzu 5050). In the filtrates, absorbance at 254 nm was measured and normalized to the DOC-concentration to give the specific UV-absorbance (SUVA254) as an estimate for the content of aromatic structures in the DOC (Chin et al., 1997). Total contents of Corg and N were determined in dried and ground samples with an elemental analyzer (ANA 1500, Carlo Erba, Milano, Italy)
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2.2. Microbial biomass and soil enzyme activity Ninhydrin-reactive N was measured according to a modification of the method of Amato and Ladd (1988). Briefly, 0.3 – 0.5 g moist soil was fumigated with 0.1 ml chloroform for 24 h at 25 8C. After removal of the chloroform, samples and unfumigated controls were extracted with 5.0 ml 2 M KCl solution for 60 min on a shaker. Two millilitre of the filtrates were mixed with 0.5 ml 0.4 M sodium citrate solution. Ninhydrin-reactive N was determined colorimetrically (Schinner et al., 1996). For the determination of urease activity, 0.3– 0.4 g moist soil were incubated with 1.5 ml 80 mM urea solution for 2 h at 37 8C. Released ammonium was extracted with 13.5 ml 2 M KCl solution, and determined colorimetrically by a modified Berthelot reaction (Kandeler and Gerber, 1988). Xylanase activity was determined by incubating 0.5 – 1.0 g soil with 5.0 ml substrate solution (1.7% w v21 xylan from oat spelts suspended in 2 M acetate buffer, pH 5.5) and 5.0 ml 2 M acetate buffer (pH 5.5) for 24 h at 50 8C. Reducing sugars released during the incubation period reduced potassium hexacyanoferrate (III) in an alkaline solution. Potassium hexacyanoferrate (II) was measured colorimetrically according to the Prussian blue reaction (Schinner et al., 1996). Protease activity was measured based on Ladd and Butler (1972), with the following modifications: Only 0.2 g soil were incubated for 2 h in 5 ml buffered casein solution (pH 8.1) and 5 ml TRIS buffer (0.05 M, pH 8.1) at 50 8C. The aromatic amino acids released were extracted with trichloroacetic acid (0.92 M) and measured colorimetrically using Folin –Ciocalteu reagent. Alkaline phosphomonoesterase (alkaline phosphatase) activity was assayed using a modified disodium phenylphosphate method: 0.3 –0.4 g soil was incubated in 2.0 ml 0.2 M borate buffer (pH 10.0) and 1.0 ml buffered phenylphosphate solution at 37 8C for 3 h; released phenol were determined colorimetrically at 20nm (Hoffmann, 1968). For the determination of arylsulfatase, 0.3 –0.4 g soil were incubated with 1 ml p-nitrophenylsulfate and 4 ml acetate buffer (0.05 M, pH 5.8) for 1 h at 37 8C (Tabatabai and Bremner, 1970). Released nitrophenol was determined colorimetrically at 420 nm. 2.3. PLFA extraction and analyses Lipids were extracted from soil, fractionated and quantified using the procedure described by Bardgett et al. (1996) (based on Blight and Dyer (1959) as modified by White et al. (1979)). Separated fatty acid methyl esters were identified by chromatographic retention time and mass spectral comparison using a standard qualitative bacterial methyl ester mix (Supelco) that ranged from C11 to C20. For each sample, the abundance of individual fatty acids is expressed on a dry weight of soil basis. Fatty acid
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nomenclature was used as described by Frostega˚rd et al. (1993). The fatty acids i15:0, a15:0, 15:0, i16:0, 17:0, i17:0, cy17:0 and cy19:0 were chosen to represent bacterial PLFAs with cy17:0 and iso and anteiso PLFAs characteristic for gram-negative bacteria and gram-positive bacteria, respectively (Frostega˚rd et al. 1993). The fatty acid 18:2v6 was used as an indicator of fungal biomass (Federle, 1986). 2.4. DNA extraction and DGGE DNA was isolated using the method of Borneman (personal communication) as described in Marschner et al. (2001). Bacterial 16S rDNA was amplified by using the eubacterial primer pair F984 and R1378 (Heuer et al., 1997). DNA of eukaryotic microorganisms (fungi, protozoa, red and green algae) was amplified using the universal primer pair NS1 and NS2 (White et al., 1990). In each case a GC-rich tail was attached to the forward primer to prevent complete separation of the strands in the DGGE (Heuer et al., 1997). For PCR, 2 ml of the 10fold diluted DNA extract were added to 23 ml PCR reaction mix composed of 0.2 ml Taq DNA polymerase (5 U ml21, Qbiogene; Illkirch, France), 2 ml dNTPs (2 mmol l21 each, Boehringer Mannheim, Germany), 2.5 ml 10 £ PCR-buffer (Qbiogene), 0.4 ml of each primer (0.5 mmol l21) and 17.5 ml ultra pure water. DNA was amplified in a Thermocycler (TRIO-Thermoblocke with TRIO Heated Lid, Biometra, Go¨ttingen, Germany). Bacterial DNA was amplified in 35 cycles of 1 min denaturation at 94 8C, 1 min at 55 8C for primer annealing and 2 min at 72 8C for primer extension. In the first cycle the denaturation phase was extended to 5 min at 94 8C to prevent annealing of the primers to non-target DNA. The 35 cycles were followed by a final step of 10 min at 72 8C and cooling at 10 8C (Heuer et al., 1997). Eukaryotic DNA was amplified by the following program: 5 min at 94 8C, followed by 10 min at 80 8C and 35 cycles of 30 s at 94 8C, 45 s at 45 8C and 90 s at 72 8C followed by a final step of 10 min at 72 8C and cooling at 10 8C. Successful amplification was verified by electrophoresis in 1.8% (w/v) agarose gels with SYBR green I nucleic acid staining (FMC Bio Products, Rockland, USA). DGGE polyacrylamide gels (8%) were used with the denaturing gradients 35 –55% with 100% of denaturant corresponding to 7 mol l21 urea and 40% (v/v) formamide. DGGE was performed using 20 ml of the PCR product in 1 £ TAE buffer at 60 8C, at a constant voltage of 150 V for 5 h (BIO-RAD Dcodee systems, Mu¨nchen, Germany). Gels were analysed as described previously (Marschner et al., 2001). 2.5. Statistical analysis Soil chemical properties, PLFA concentrations and ratios of different microbial groups were compared by a one-way
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Table 1 Soil organic carbon (Corg), total soil N (Nt), C/N ratio, water extractable organic C (DOC) and specific UV absorbance of DOC (SUVA254) in different longterm amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05) Amendment
Corg (%)
Nt (%)
C/N
DOC (mg kg21)
SUVA254 ( l mg21 m21)
Mineral only Mineral þ residues Manure Sewage Straw
0.67 ^ 0.04 a 0.70 ^ 0.01 a 0.80 ^ 0.03 b 0.82 ^ 0.05 b 0.83 ^ 0.05 b
0.099 ^ 0.002 ab 0.096 ^ 0.003 a 0.111 ^ 0.003 b 0.109 ^ 0.005 ab 0.103 ^ 0.004 ab
6.7 ^ 0.3 a 7.3 ^ 0.1 ab 7.2 ^ 0.2 ab 7.5 ^ 0.2 ab 8.0 ^ 0.3 b
100 ^ 12 a 116 ^ 12 a 123 ^ 13 a 108 ^ 5 a 113 ^ 3 a
2.1 ^ 0.1 2.1 ^ 0.1 2.5 ^ 0.1 2.2 ^ 0.1 2.5 ^ 0.1
ANOVA. Significant treatment effects were determined using the Tukey test in SigmaStat (SPSS, Chicago, USA). DGGE band patterns, PLFA patterns and enzyme activities were compared using redundancy discriminate analysis (RDA) with Monte Carlo permutation test (CANOCO, for Windows version 4, Microcomputer Power, Ithaca, USA). The Monte Carlo tests were based on 199 random permutations of the data. Soil chemical factors potentially affecting community structure as well as enzyme activities were used as environmental data.
a a b ab b
the mineral only treatment (Table 2). However, only protease activity was significantly higher in the sewage treatment than in the mineral fertilizer only treatment. Microbial biomass N was lower in the mineral fertilizer only and the straw amended soil than in the other three treatments. There were no significant differences in the total concentration of PLFAs between the treatments (data not shown). The concentrations of most PLFAs were similar across all treatments, except i15:0, a15:0 and 16.1v9, which were significantly higher after sewage sludge amendment than after mineral fertilization (Fig. 1). Signature PLFAs (see Section 2) were used to calculate the ratios between different microbial groups. The concentration of total bacterial and gram-positive bacterial PLFAs was highest after sewage sludge amendment and lowest in the mineral treatments (Table 3). The treatments did not differ in gramnegative or the fungal PLFAs. The ratios of gram-positive to gram-negative bacteria and of bacteria to fungi were higher in the organic amendments than in the mineral fertilizer treatments. These results indicate that the long-term fertilization treatments changed the microbial community structure in the soil. The sum of bacterial PLFAs (see Section 2), as an indicator for bacterial biomass, was weakly correlated with the activities of alkaline phosphatase, arylsulfatase, protease and invertase as well as with Corg concentration, total N and C/N ratio. Fungal biomass was not correlated with enzyme activities or with any of the soil chemical properties except for a weak correlation with pH. To study the effect on the microbial community structure in more detail, the community structure of bacteria and eukaryotic microorganisms was determined
3. Results The long-term addition of organic and inorganic amendments caused significant changes in soil chemical properties (Table 1). The amount of organic carbon was lower in the treatments with addition of mineral fertilizers (without or with plant residues) than in the soil with the organic amendments. Total N content was highest in the manure treatment and lowest in the mineral fertilizer þ residues treatment. The C/N ratio was highest after straw amendment and lowest in the treatment with mineral fertilizer only. The treatments did not differ significantly in the amount of dissolved organic carbon (DOC). However, the composition of the DOC was influenced by the different treatments; the aromaticity of the DOC (estimated by SUVA254) was lower in the mineral fertilizer treatments than in the manure and straw amendments. The activity of all enzymes tended to be higher in the treatments manure, sewage and straw compared to
Table 2 Biomass and enzyme activities in different long-term amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05)
Mineral only Mineral þ residue Manure Sewage Straw
Microbial biomass N (mg N g soil21)
Alkaline phosphatase (mg p-nitrophenol (g soil h)21)
Arylsulfatase (mg p-nitrophenol (g soil h)21)
Xylanase (mg GLC (g soil 24 h)21)
Invertase (mg GLC (g soil 3 h)21)
Protease (mg TYR (g soil 2 h)21)
Urease (mg N (g soil 2 h)21)
1.8 ^ 0.8 a 4.3 ^ 0.9 b 5.0 ^ 0.6 b 4.5 ^ 0.9 b 2.0 ^ 0.5 a
430 ^ 25 a 424 ^ 7 a 456 ^ 15 a 512 ^ 28 a 469 ^ 20 a
33 ^ 3 35 ^ 1 40 ^ 3 44 ^ 5 45 ^ 2
956 ^ 103 a 938 ^ 65 a 1037 ^ 96 a 1079 ^ 52 a 1144 ^ 149 a
2120 ^ 75 a 2259 ^ 65 a 2237 ^ 84 a 2372 ^ 79 a 2433 ^ 85 a
76 ^ 9 a 87 ^ 3 ab 94 ^ 6 ab 119 ^ 8 b 101 ^ 8 ab
25 ^ 2 a 28 ^ 3 a 28 ^ 2 a 25 ^ 2 a 30 ^ 2 a
a a a a a
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Fig. 1. Concentration of fatty acids in soil (nmol g soil21) after long-term application of inorganic or organic amendments. (A) mineral fertiliser only, ( ) mineral fertiliser þ residues, ( ) manure, ( ) sewage sludge, ( ) straw.
by PCR-DGGE. A total of 60 and 90 different bands were distinguished in the bacterial and eukaryotic communities, respectively. RDA of the DGGE patterns were carried using the soil chemical properties as environmental data (Fig. 2). The bacterial communities of the manure and straw treatments on the one hand and of the sewage sludge treatment on the other hand formed two distinct groups. The two mineral fertilizer treatments lay between these two groups. The variation in eukaryotic microbial community structure of a given treatment was large and no differences between the treatments were observed. Moreover, RDA showed that PLFA patterns (Fig. 2) and enzyme activities (data not shown) did not differ between the treatments. Multivariate analyses such as RDA in combination with Monte Carlo Permutation tests can be used to determine which environmental variables are significantly correlated with community structure as well as the relative importance
of these variables. The RDA showed that the bacterial community structure was significantly correlated with (listed in order of decreasing importance) aromaticity of the DOC (SUVA254), Corg concentration, DOC concentration and C/N ratio (Table 4). The bacterial community structure was also significantly correlated with the activities of arylsulfatase, alkaline phosphatase and protease. The structure of the eukaryotic microbial communities was significantly correlated with C/N ratio and Corg concentration whereas DOC and aromaticity of the DOC had no significant effect (Table 4). The eukaryotic community structure was also correlated with the amount of biomass N and invertase activity. The PFLA patterns were significantly correlated with DOC aromaticity, Corg and DOC concentration. There were no significant correlations between the PLFAs and enzyme activities or biomass N (Table 4). Enzyme activities were not significantly correlated to any of the soil chemical parameters measured.
Table 3 Concentration of PLFAs in soil characteristic for all bacteria, Gram 2 bacteria, Gram þ bacteria or fungi (nmol g soil-1) and ratios of Gram þ To gram 2 bacteria and bacteria to fungi in different long-term amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05)
Mineral only Mineral þ residues Manure Sewage sludge Straw
Bacteria
Gram 2 bacteria
Gram þ bacteria
Fungi
92.8 ^ 19.5 a 97.0 ^ 9.8 a 119.9 ^ 13.0 ab 159.0 ^ 18.9 b 133.1 ^ 8.5 ab
15.6 ^ 2.4 a 19.8 ^ 3.4 a 16.2 ^ 1.5 a 21.2 ^ 2.9 a 17.4 ^ 0.9 a
74.2 ^ 16.8 a 73.3 ^ 5.4 a 100.9 ^ 11.0 ab 132.0 ^ 13.5 b 111.6 ^ 7.2 ab
18.4 ^ 2.7 28.8 ^ 8.9 15.4 ^ 2.8 17.4 ^ 2.3 15.2 ^ 1.1
a a a a a
Gram þ /gram 2
Bacteria/fungi
4.6 ^ 0.4 3.9 ^ 0.3 6.2 ^ 0.2 6.3 ^ 0.2 3.4 ^ 0.2
5.2 ^ 0.8 a 4.1 ^ 0.8 a 8.4 ^ 1.2 b 9.2 ^ 0.1 b 8.8 ^ 0.3 b
a a b b b
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Fig. 2. Redundancy discriminate analysis plots of bacterial and eukaryotic DGGE patterns and PLFA patterns after long-term application of inorganic or organic amendments, using soil chemical data as environmental factors.
structure and biomass. The treatments differed in bacterial community structure and biomass but not in eukaryotic community structure or fungal biomass. Both bacterial and eukaryotic community structures were correlated with soil
4. Discussion This study showed that long-term inorganic or organic amendment at a low rate may affect microbial community
Table 4 Soil chemical properties (soil chemistry) and enzyme activities including CFE N (enzymes) with significant correlation (P # 0.10) to bacterial and eukaryotic DGGE patterns and PLFA patterns determined by Monte Carlo permutation tests. Listed in order of decreasing importance with P values in parentheses Bacteria
Eukaryotes
Soil chemistry SUVA254 Corg DOC C/N a
Enzymes (0.01) (0.03) (0.03) (0.01)
Arylsulfatase Phosphatase Protease
(0.01) (0.03) (0.03)
PLFA
Soil chemistry
Enzymes
C/N Corg
CFE N Invertase
(0.01) (0.10)
Soil chemistry (0.06) (0.03)
SUVA254 Corg DOC
None of the patterns were significantly correlated with Ct, Cinorg, Nt, biodegradability, St, C/S, urease or xylanase activity. No significant correlation with any of the enzyme activities.
Enzymes (0.04) (0.06) (0.06)
nsa
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chemistry and enzyme activity. The changes in community structure were, however, not accompanied by significant changes in functional diversity. The increase in soil Corg after addition of organic material (Table 1) is in agreement with other studies (Crecchio et al., 2001) and often leads to increased microbial biomass and activity (Dhillion, 1997; Kandeler et al., 1999b). Easily degradable compounds such as organic acids in the insoluble or soluble organic matter can be utilised by a wide range of organisms. Presence of these compounds can lead to a rapid increase in biomass and activity (Ku¨sel and Drake, 1999) and favour the growth of copiotrophic compared to oligotrophic organisms (Hu et al., 1999). Once the easily degradable compounds have been metabolised, only the more recalcitrant material remains. The decomposition of this material requires enzymes that are produced by a limited number of microbial species (Moorhead and Sinsabaugh, 2000) and may increase the competitive ability of cellulolytic microorganisms (Jensen and Nybroe, 1999). Changes in organic matter composition can lead to a succession of microorganisms such as that observed by Ponge (1991) for fungi during the decomposition of pine needles. The temporal dynamics in decomposition depend on the chemical composition of the organic matter that is added to the soil (Lettau and Kuzyakov, 1999; Northup et al., 1998). Differences in organic matter composition and thus substrate availability are likely to be the reason for the differences in microbial community structure observed in the present study such as the increased bacteria/fungi ratio and the increased gram-positive/gramnegative ratio (Table 3). RDA showed that both bacteria and eukaryotic community structures were correlated with Corg concentration but only bacterial community structure was influenced by DOC concentration as well as aromaticity of the DOC (indicated by the SUVA254) (Table 4). The DOC aromaticity was highest in the straw and manure treatments. This can be attributed to the high lignin content of straw and thus also manure, which contains substantial amounts of straw, and the release of soluble lignin degradation products (Guggenberger and Zech, 1993). Aromatic compounds are generally less degradable (Boissier and Fontvieille, 1993) and can be toxic and thus decrease litter turnover (Northup et al., 1998). The readily decomposable compounds in DOC such as organic acids and carbohydrates (Ku¨sel and Drake, 1999; Guggenberger and Zech, 1993) are probably mainly utilised by soil bacteria while fungi decompose the more recalcitrant and insoluble materials. This may explain why DOC amount and composition had no significant effect on the eukaryotic community structure (Table 4). Nitrogen is often a key limiting factor for soil organisms and addition of N can change microbial biomass, activity and species composition (Sarathchandra et al., 2001). In the present study, manure amendment increased total N concentration compared to the other treatments (Table 1).
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Interestingly, soil total N concentration had no significant effect on microbial community composition (Table 4) but was weakly correlated with bacterial biomass. It should be noted, that the total N concentration in the present study is low compared to that in other studies (Crecchio et al., 2001; Kandeler et al., 1999b). The highest C/N ratio was found after addition of straw (Table 1) although mineral N had been added with the straw. Organic matter with a high C/N ratio is only slowly degraded by microorganisms (Wagner and Wolf, 1999). The present study showed that the C/N ratio also affects microbial community structure as both bacterial and eukaryotic community structures were significantly correlated with the soil C/N ratio (Table 4). The treatments manure, sewage sludge and mineral fertilizer þ residues increased microbial biomass N compared to straw and mineral only (Table 2). In the straw treatment the low microbial biomass N is probably due to the high C/N ratio of the soil. In the mineral only treatment it may be explained by low Corg content. However, despite of a low Corg content in the mineral þ residues treatment, the microbial biomass N in this treatment was as high as in the manure or sewage amendments. It may be speculated that the plant residues were rapidly decomposed leading to a low soil Corg and a high microbial biomass. The concentration of bacterial PLFAs was lower in both mineral treatments as compared to organic amendments. This indicates that the active bacterial biomass may be declining in response to the low Corg content. The activities of most enzymes were not significantly affected by the long-term treatments in the present study (Table 2). In the short-term, addition of organic matter often leads to increased enzyme activities (Kandeler et al., 1999b; Crecchio et al., 2001; Madejon et al., 2001) and long-term organic amendments can also stimulate enzyme activities and microbial biomass (Kandeler et al., 1999a). The lack of stimulation in the present study may be due to the relatively small amounts of organic amendments added per year which were only about half as much as in the studies by Kandeler et al. (1999b) or Crecchio et al. (2001). This is also reflected by the lower Corg and total N content in this study compared to Kandeler et al. (1999b) and Crecchio et al. (2001). Thus, despite changes in microbial community structure, enzyme activities were generally not affected by the treatments and showed no correlation with any of the soil chemical parameters measured. Due to the functional redundancy of soil microorganisms (i.e. one function can be carried out by a range of different microorganisms), changes in microbial community structure do not necessarily lead to changes in enzyme activities. This is particularly true for the breakdown of sugars, proteins and organic P but may be less so for the decomposition of recalcitrant compounds. Two points regarding enzyme activities should be noted (Nannipieri et al., 2002). Firstly, any short-term changes in enzyme activity may be masked by enzymes that are adsorbed to soil particles and which can retain their activity for many years. Secondly, enzyme
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activity tests measure the potential activity of a given enzyme in soil, not the actual activity. The long-term addition of sewage sludge did not differ in its effect on bacterial and fungal biomass, PLFA patterns or eukaryotic community structure from that of the other two organic amendments (Tables 2 and 3, Figs. 1 and 2). However, the bacterial community structure of the sewage sludge treatment differed from that of the straw and manure treatments. This effect on bacterial community structure can be explained by the higher concentration of aromatic compounds in the DOC of the straw and manure treatments. Sewage sludge may contain high concentrations of heavy metals. However, in the present study, although the concentration of Zn and Cu in the sewage sludge amended soil were elevated in comparison to the other treatments they reached only 15 and 89 mg kg21, respectively (Delschen, 1999), which is well below levels where toxic effects can be expected (Chaudri et al., 2000). In other studies no negative effects of heavy metals added with municipal waste, dump material or sewage sludge on soil biomass or activity were found (Chander et al., 2001; Madejon et al., 2001). It should be noted that bacteria added with the sewage sludge may also have contributed to the observed change in bacterial community structure compared to the other organic treatments. The soil that had received mineral fertilizer without residues was characterised by a very low Corg content, a low C/N ratio as well as low enzyme activities and bacterial biomass. However, the microbial community structure did not differ from that of the other treatments. It may be speculated that a further decrease in soil Corg content will have a strong negative impact on microbial activity in the soil. The incorporation of harvest residues did not offset the decline in soil Corg but increased the microbial biomass. The results of the present study show that long-term addition of organic amendments at a low rate may increase bacterial biomass while having no effect on fungal biomass. They also demonstrate the fundamental importance of organic C for soil microorganisms. Different amendments affected the bacterial and eukaryotic community structure through their effect on Corg and C/N ratio of the soil. The bacterial community structure was also influenced by changes in DOC concentration and composition. Moreover, the study revealed that changes in community composition are not necessarily accompanied by changes in enzyme activities.
Acknowledgements We would like to thank Karen Baumann and Sabine Rudolph for excellent technical assistance and Peter Leinweber for the elemental analyses.
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P. Marschner et al. / Soil Biology & Biochemistry 35 (2003) 453–461 Hoffmann, G., 1968. Eine photometrische Methode zur Bestimmung der Phosphataseaktivita¨t in Bo¨den. Zeitschrift fu¨r Pflanzenerna¨hrung und Bodenkunde 118, 161 –172. Hu, S.J., Van Bruggen, A.H.C., Gru¨nwald, N.J., 1999. Dynamics of bacterial populations in relation to carbon availability in a residueamended soil. Applied Soil Ecology 13, 21–30. Jensen, L.E., Nybroe, O., 1999. Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere. Applied Environmental Microbiology 65, 4320–4328. Kandeler, E., Gerber, H., 1988. Short-term assay of soil urease activity using colorimetric determination of ammonium. Biology and Fertility of Soils 6, 68–72. Kandeler, E., Luxhøi, J., Tscherko, D., Magid, J., 1999a. Xylanase, invertase and protease at the soil-litter interface of a loamy sand. Soil Biology & Biochemistry 31, 1171–1179. Kandeler, E., Stemmer, M., Klimanek, E.-M., 1999b. Response of soil microbial biomass, urease and xylanase within particle size fractions to long-term soil management. Soil Biology & Biochemistry 31, 261–273. Ku¨sel, K., Drake, H.L., 1999. Microbial turnover of low molecular weight organic acids during leaf litter decomposition. Soil Biology & Biochemistry 31, 107 –118. Ladd, J.N., Butler, J.H.A., 1972. Short-term assays of soil proteolytic enzyme activities using proteins and dipeptide derivatives as substrates. Soil Biology & Biochemistry 4, 19– 30. Lettau, T., Kuzyakov, Y., 1999. Verwertung organischer Substanzen durch mikrobielle Bodenbiomasse als eine Funktion chemisch—thermodynamischer Parameter. Journal of Plant Nutrition and Soil Science 162, 171–177. Madejon, E., Burgos, P., Lopez, R., Cabrera, F., 2001. Soil enzymatic response to addition of heavy metals with organic residues. Biology and Fertility of Soils 34, 144– 150. Marschner, B., Noble, A., 2000. Chemical and biological processes leading to the neutralisation of soil acidity after incubation with different litter materials. Soil Biology & Biochemistry 32, 805 –813. Marschner, P., Yang, C.H., Lieberei, R., Crowley, D.E., 2001. Soil and plant specific effects on bacterial community composition in the rhizosphere. Soil Biology & Biochemistry 33, 1437–1445. Metting, F.B., 1993. Structure and physiological ecology of soil microbial communities. In: Metting, F.B., (Ed.), Soil Microbial Ecology— Application in Agricultural and Environmental Management, Marcel Dekker, New York, pp. 3–24.
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Moorhead, D.L., Sinsabaugh, R.L., 2000. Simulated patterns of litter-decay predict patterns of extracellular enzyme activities. Applied Soil Ecology 14, 71 –79. Nannipieri, P., Kandeler, E., Ruggiero, P., 2002. Enzyme activities and microbiological and biochemical processes in soil. In: Burns, R., Dick, R. (Eds.), Enzymes in the Environment: Activity, Ecology, and Application, Marcel Dekker, New York, pp. 1–34. Northup, R.R., Dahlgren, R.A., McColl, J.G., 1998. Polyphenols as regulators of plant-litter-soil interactions in northern Californian pygmy forest—a positive feedback. Biogeochemistry 42, 189 –220. Pascual, J.A., Garcia, C., Hernandez, T., Moreno, J.L., Ros, M., 2000. Soil microbial activity as a biomarker of degradation and remediation processes. Soil Biology & Biochemistry 32, 1877–1877. Peacock, A.D., Mullen, M.D., Ringelberg, D.B., Tyler, D.D., Hedrick, D.B., Gale, P.M., White, D.C., 2001. Soil microbial community responses to dairy manure or ammonium nitrate applications. Soil Biology and Biochemistry 33, 1011–1019. Ponge, J.F., 1991. Succession of fungi and fauna during decomposition of needles in a small area of Scots pine litter. Plant and Soil 138, 99– 113. Sarathchandra, S.U., Ghani, A., Yeates, G.W., Burch, G., Cox, N.R., 2001. Effect of nitrogen and phosphate fertilizers on microbial and nematode diversity in pasture soils. Soil Biology & Biochemistry 33, 953–964. Sarniguet, A., Lucas, P., Lucas, M., 1992. Relationships between take-all, soil conduciveness to the disease, populatios of fluorescent pseudomonads and nitrogen fertilizers. Plant and Soil 145, 17 –27. ¨ hlinger, R., Kandeler, E., Margesin, R., 1996. Methods in Schinner, F., O Soil Biology, Springer, Berlin. Tabatabai, M.A., Bremner, J.M., 1970. Arylsulfatase activity in soils. Soil Science Society of America Proceedings 34, 225 –229. Wagner, G.H., Wolf, D.C., 1999. Carbon transformations and soil organic matter formation. In: Sylvia, D.M., Fuhrmann, J.J., Hartel, P.G., Zuberer, D.A. (Eds.), Principles and Applications of Soil Microbiology, Prentice Hall, New Jersey, pp. 218–258. White, D.C., Davis, W.M., Nickels, J.S., King, J.C., Bobbie, R.J., 1979. Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40, 51 –62. White, T.J., Bruns, S., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. A Guide to Methods and Applications, Academic Press, San Diego, pp. 315– 322.
Structure and function of the soil microbial community in a long-term fertilizer experiment Petra Marschnera,*, Ellen Kandelerb, Bernd Marschnerc a Department of Soil and Water, University of Adelaide, PMB 1 Waite Campus, Glen Osmond, SA 5064, Australia Institut fu¨r Bodenkunde and Standortslehre, Fachgebiet Bodenbiologie, Universitat Hohenheim, D-70599 Stuttgart, Germany c Ruhr-Universita¨t Bochum, Geographisches Institut, Bodenkunde/Bodeno¨kologie, Universita¨tsstr., 150, D-44780 Bochum, Germany b
Received 12 March 2002; received in revised form 24 September 2002; accepted 3 December 2002
Abstract The effect of organic and inorganic fertiliser amendments is often studied shortly after addition of a single dose to the soil but less is known about the long-term effects of amendments. We conducted a study to determine the effects of long-term addition of organic and inorganic fertiliser amendments at low rates on soil chemical and biological properties. Surface soil samples were taken from an experimental field site near Cologne, Germany in summer 2000. At this site, five different treatments were established in 1969: mineral fertiliser (NPK), crop residues removed (mineral only); mineral fertiliser with crop residues; manure 5.2 t ha21 yr21; sewage sludge 7.6 t ha21 yr21 or straw 4.0 t ha21 yr21 with 10 kg N as CaCN2 t straw21. The organic amendments increased the Corg content of the soil but had no significant effect on the dissolved organic C (DOC) content. The C/N ratio was highest in the straw treatment and lowest in the mineral only treatment. Of the enzymes studied, only protease activity was affected by the different amendments. It was highest after sewage amendment and lowest in the mineral only treatment. The ratios of Gram þ to Gram 2 bacteria and of bacteria to fungi, as determined by signature phospholipid fatty acids, were higher in the organic treatments than in the inorganic treatments. The community structure of bacteria and eukaryotic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) and redundancy discriminate analyses of the DGGE banding patterns. While the bacterial community structure was affected by the treatments this was not the case for the eukaryotes. Bacterial and eukaryotic community structures were significantly affected by Corg content and C/N ratio. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Organic amendment; Sewage sludge; Mineral fertilizer; Microbial community structure; Soil enzyme activity; Denaturing gradient gel electrophoresis; Phospholipid fatty acid
1. Introduction Organic and inorganic fertiliser amendments are used primarily to increase nutrient availability to plants, but they can also affect soil microorganisms. Changes in microbial activity and composition can in turn influence plant growth, for example by increased nutrient turnover, or by increasing disease incidence or disease suppression. Amendments usually increase the amount of soil organic C (Corg) and the concentration of other nutrients such as N (Madejon et al., 2001; Crecchio et al., 2001). Organic matter can be added either directly by * Corresponding author. Tel.: þ 1-61-8-8303-7379; fax: þ 1-61-8-83036511. E-mail address: [email protected] (P. Marschner).
incorporating manure, straw, sewage sludge or municipal waste or indirectly via increased plant growth. Generally, microbial biomass increases with increasing Corg content of the soil (Dhillion, 1997). In a number of short-term studies it has been shown that organic amendments increase microbial biomass (Peacock et al., 2001; Hu et al., 1999; Pascual et al., 2000). Organic matter addition often leads to a rapid increase of soil CO2 evolution (Ajwa and Tabatabai, 1994) and increases the activity of various enzymes (Crecchio et al., 2001; Madejon et al., 2001; Kandeler et al., 1999b). An increase in enzyme activity was also observed after addition of either NPK fertiliser or farmyard manure in a long-term study (Kandeler et al., 1999a). Amendments can change amount and quality of dissolved organic matter (DOM) present in the soil solution (Marschner
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0038-0717(02)00297-3
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and Noble, 2000). DOM is an easily available substrate for soil microorganisms (Metting, 1993) and its importance for microbial activity has been shown by studies, in which soil respiration was closely related to DOM concentrations (Marschner and Noble, 2000). Changes in microbial community composition are often observed after addition of organic or inorganic amendments. Compared to NO3, NH4 fertilisation decreases take-all severity in wheat (Sarniguet et al., 1992a) and suppresses Thielaviopsis basicola (Harrison and Shaw, 2001). Soil fungi differ in their response to NPK fertilisers (Donnison et al., 2000) and N fertilisation changes the abundance of fungal species (Sarathchandra et al., 2001). Different types of amendments may differ in organic matter composition (e.g. C/N ratio), and this in turn, affects the decomposition rate and can change the microbial community structure. For example, presence of easily degradable plant material favours organisms that grow rapidly in nutrient-rich environments (copiotrophs) compared to organisms adapted to nutrientpoor conditions (oligotrophs) (Hu et al., 1999), while straw incorporation favours the competitive ability of cellulolytic microorganisms compared to those that can not degrade cellulose (Jensen and Nybroe, 1999). The increase in catabolic diversity that is observed in soils after addition of easily degradable organic matter (Degens et al., 2000) also indicates changes in microbial community structure. However, in a short-term study, Crecchio et al. (2001) found no changes in bacterial community structure after addition of municipal solid waste compost. In another study, Buyer and Kaufman (1996) found similar culturable bacterial and fungal species composition in low input and conventional agriculture. In most of the above mentioned studies the responses of soil microorganisms to organic or inorganic amendments were measured within a few years after a single application. Far less is known about the effects of longterm amendments at a low rate. The effect of these may differ substantially from those of a single high dose. In a field experiment with low rates of organic and inorganic amendments over 40 years we assessed soil chemical and microbiological properties using a range of approaches. We determined both total Corg and N and amount and aromaticity of DOC. The activities of enzymes involved in C, N, P and S cycling were studied, some of which have been previously shown to respond to organic amendments (Kandeler et al., 1999b). Phospholipid fatty acid (PLFA) patterns were used to estimate the relative abundance of bacteria and fungi. The molecular approach, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), provided an analysis of bacterial and eukaryotic community structure. The relationships between soil chemical properties and microbial indices were tested by regression and multivariate analyses.
2. Materials and methods The study site is a reclaimed loess soil (calcareous Regosol) in the Rhineland lignite mining area near Cologne, Germany. After the end of the mining activities in 1968, the mine spoil was levelled and covered with a 90 cm layer of calcareous loess material with very low Corg content (0.1%). In 1969 an experimental field site was established with the following treatments: NPK mineral fertilizer, all plant residues removed (mineral only); mineral fertilizer, with plant residues incorporated into the soil; manure 5.2 t ha21 yr21; sewage sludge 7.6 t ha21 yr21; straw 4.0 t ha21 yr21 with 10 kg N as CaCN2 t straw21. Each treatment was replicated four times with plot sizes of 4 £ 9 m2 randomly distributed in the field. A crop rotation typical for the region consisting of sugar beet, wheat, rye, potato, wheat and barley was grown since the establishment of the site (for further details see Delschen (1999)). The treatments were sampled in June 2000 with winter barley as cover crop. In each replicate of the five treatments, eight soil samples were collected from the top 20 cm with an auger and mixed to give a bulk sample which was immediately stored in a cooler at 4 8C. In the laboratory, the stone-free samples were thoroughly homogenized and divided into several sub-samples, some of which were stored at 2 18 8C for microbial and enzyme analyses. 2.1. Soil chemical analyses For the extraction of soluble organic matter, about 80 g DW of field moist soil samples were packed into round steel cylinders (46 mm ID, 48 mm height) to give a bulk density similar to field conditions (approx. 1.1 g cm23). The cylinders were placed on porous sintered glass plates (pores , 16 mm) that were connected to a glass collection bottle pressurized to 2 60 hPa. The surface of the samples was covered with 1 mm glass beads and then drip-irrigated through a rainfall simulator with 25 capillaries of 0.2 mm diameter. A total of 250 ml 1 mM CaCl2 solution was applied within 6 h, equivalent to a rainfall intensity of about 25 mm h21. This mode of extraction, under unsaturated flow conditions, removes the mobile fraction of soluble organic matter and closely resembles field conditions where mainly medium-sized and larger pores (. 50 mm) participate in transport processes. Following 0.45 mm membrane filtration, acidification and gas-purging of the solutions to remove inorganic C, DOC was determined with a TOC-Analyser (Shimadzu 5050). In the filtrates, absorbance at 254 nm was measured and normalized to the DOC-concentration to give the specific UV-absorbance (SUVA254) as an estimate for the content of aromatic structures in the DOC (Chin et al., 1997). Total contents of Corg and N were determined in dried and ground samples with an elemental analyzer (ANA 1500, Carlo Erba, Milano, Italy)
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2.2. Microbial biomass and soil enzyme activity Ninhydrin-reactive N was measured according to a modification of the method of Amato and Ladd (1988). Briefly, 0.3 – 0.5 g moist soil was fumigated with 0.1 ml chloroform for 24 h at 25 8C. After removal of the chloroform, samples and unfumigated controls were extracted with 5.0 ml 2 M KCl solution for 60 min on a shaker. Two millilitre of the filtrates were mixed with 0.5 ml 0.4 M sodium citrate solution. Ninhydrin-reactive N was determined colorimetrically (Schinner et al., 1996). For the determination of urease activity, 0.3– 0.4 g moist soil were incubated with 1.5 ml 80 mM urea solution for 2 h at 37 8C. Released ammonium was extracted with 13.5 ml 2 M KCl solution, and determined colorimetrically by a modified Berthelot reaction (Kandeler and Gerber, 1988). Xylanase activity was determined by incubating 0.5 – 1.0 g soil with 5.0 ml substrate solution (1.7% w v21 xylan from oat spelts suspended in 2 M acetate buffer, pH 5.5) and 5.0 ml 2 M acetate buffer (pH 5.5) for 24 h at 50 8C. Reducing sugars released during the incubation period reduced potassium hexacyanoferrate (III) in an alkaline solution. Potassium hexacyanoferrate (II) was measured colorimetrically according to the Prussian blue reaction (Schinner et al., 1996). Protease activity was measured based on Ladd and Butler (1972), with the following modifications: Only 0.2 g soil were incubated for 2 h in 5 ml buffered casein solution (pH 8.1) and 5 ml TRIS buffer (0.05 M, pH 8.1) at 50 8C. The aromatic amino acids released were extracted with trichloroacetic acid (0.92 M) and measured colorimetrically using Folin –Ciocalteu reagent. Alkaline phosphomonoesterase (alkaline phosphatase) activity was assayed using a modified disodium phenylphosphate method: 0.3 –0.4 g soil was incubated in 2.0 ml 0.2 M borate buffer (pH 10.0) and 1.0 ml buffered phenylphosphate solution at 37 8C for 3 h; released phenol were determined colorimetrically at 20nm (Hoffmann, 1968). For the determination of arylsulfatase, 0.3 –0.4 g soil were incubated with 1 ml p-nitrophenylsulfate and 4 ml acetate buffer (0.05 M, pH 5.8) for 1 h at 37 8C (Tabatabai and Bremner, 1970). Released nitrophenol was determined colorimetrically at 420 nm. 2.3. PLFA extraction and analyses Lipids were extracted from soil, fractionated and quantified using the procedure described by Bardgett et al. (1996) (based on Blight and Dyer (1959) as modified by White et al. (1979)). Separated fatty acid methyl esters were identified by chromatographic retention time and mass spectral comparison using a standard qualitative bacterial methyl ester mix (Supelco) that ranged from C11 to C20. For each sample, the abundance of individual fatty acids is expressed on a dry weight of soil basis. Fatty acid
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nomenclature was used as described by Frostega˚rd et al. (1993). The fatty acids i15:0, a15:0, 15:0, i16:0, 17:0, i17:0, cy17:0 and cy19:0 were chosen to represent bacterial PLFAs with cy17:0 and iso and anteiso PLFAs characteristic for gram-negative bacteria and gram-positive bacteria, respectively (Frostega˚rd et al. 1993). The fatty acid 18:2v6 was used as an indicator of fungal biomass (Federle, 1986). 2.4. DNA extraction and DGGE DNA was isolated using the method of Borneman (personal communication) as described in Marschner et al. (2001). Bacterial 16S rDNA was amplified by using the eubacterial primer pair F984 and R1378 (Heuer et al., 1997). DNA of eukaryotic microorganisms (fungi, protozoa, red and green algae) was amplified using the universal primer pair NS1 and NS2 (White et al., 1990). In each case a GC-rich tail was attached to the forward primer to prevent complete separation of the strands in the DGGE (Heuer et al., 1997). For PCR, 2 ml of the 10fold diluted DNA extract were added to 23 ml PCR reaction mix composed of 0.2 ml Taq DNA polymerase (5 U ml21, Qbiogene; Illkirch, France), 2 ml dNTPs (2 mmol l21 each, Boehringer Mannheim, Germany), 2.5 ml 10 £ PCR-buffer (Qbiogene), 0.4 ml of each primer (0.5 mmol l21) and 17.5 ml ultra pure water. DNA was amplified in a Thermocycler (TRIO-Thermoblocke with TRIO Heated Lid, Biometra, Go¨ttingen, Germany). Bacterial DNA was amplified in 35 cycles of 1 min denaturation at 94 8C, 1 min at 55 8C for primer annealing and 2 min at 72 8C for primer extension. In the first cycle the denaturation phase was extended to 5 min at 94 8C to prevent annealing of the primers to non-target DNA. The 35 cycles were followed by a final step of 10 min at 72 8C and cooling at 10 8C (Heuer et al., 1997). Eukaryotic DNA was amplified by the following program: 5 min at 94 8C, followed by 10 min at 80 8C and 35 cycles of 30 s at 94 8C, 45 s at 45 8C and 90 s at 72 8C followed by a final step of 10 min at 72 8C and cooling at 10 8C. Successful amplification was verified by electrophoresis in 1.8% (w/v) agarose gels with SYBR green I nucleic acid staining (FMC Bio Products, Rockland, USA). DGGE polyacrylamide gels (8%) were used with the denaturing gradients 35 –55% with 100% of denaturant corresponding to 7 mol l21 urea and 40% (v/v) formamide. DGGE was performed using 20 ml of the PCR product in 1 £ TAE buffer at 60 8C, at a constant voltage of 150 V for 5 h (BIO-RAD Dcodee systems, Mu¨nchen, Germany). Gels were analysed as described previously (Marschner et al., 2001). 2.5. Statistical analysis Soil chemical properties, PLFA concentrations and ratios of different microbial groups were compared by a one-way
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Table 1 Soil organic carbon (Corg), total soil N (Nt), C/N ratio, water extractable organic C (DOC) and specific UV absorbance of DOC (SUVA254) in different longterm amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05) Amendment
Corg (%)
Nt (%)
C/N
DOC (mg kg21)
SUVA254 ( l mg21 m21)
Mineral only Mineral þ residues Manure Sewage Straw
0.67 ^ 0.04 a 0.70 ^ 0.01 a 0.80 ^ 0.03 b 0.82 ^ 0.05 b 0.83 ^ 0.05 b
0.099 ^ 0.002 ab 0.096 ^ 0.003 a 0.111 ^ 0.003 b 0.109 ^ 0.005 ab 0.103 ^ 0.004 ab
6.7 ^ 0.3 a 7.3 ^ 0.1 ab 7.2 ^ 0.2 ab 7.5 ^ 0.2 ab 8.0 ^ 0.3 b
100 ^ 12 a 116 ^ 12 a 123 ^ 13 a 108 ^ 5 a 113 ^ 3 a
2.1 ^ 0.1 2.1 ^ 0.1 2.5 ^ 0.1 2.2 ^ 0.1 2.5 ^ 0.1
ANOVA. Significant treatment effects were determined using the Tukey test in SigmaStat (SPSS, Chicago, USA). DGGE band patterns, PLFA patterns and enzyme activities were compared using redundancy discriminate analysis (RDA) with Monte Carlo permutation test (CANOCO, for Windows version 4, Microcomputer Power, Ithaca, USA). The Monte Carlo tests were based on 199 random permutations of the data. Soil chemical factors potentially affecting community structure as well as enzyme activities were used as environmental data.
a a b ab b
the mineral only treatment (Table 2). However, only protease activity was significantly higher in the sewage treatment than in the mineral fertilizer only treatment. Microbial biomass N was lower in the mineral fertilizer only and the straw amended soil than in the other three treatments. There were no significant differences in the total concentration of PLFAs between the treatments (data not shown). The concentrations of most PLFAs were similar across all treatments, except i15:0, a15:0 and 16.1v9, which were significantly higher after sewage sludge amendment than after mineral fertilization (Fig. 1). Signature PLFAs (see Section 2) were used to calculate the ratios between different microbial groups. The concentration of total bacterial and gram-positive bacterial PLFAs was highest after sewage sludge amendment and lowest in the mineral treatments (Table 3). The treatments did not differ in gramnegative or the fungal PLFAs. The ratios of gram-positive to gram-negative bacteria and of bacteria to fungi were higher in the organic amendments than in the mineral fertilizer treatments. These results indicate that the long-term fertilization treatments changed the microbial community structure in the soil. The sum of bacterial PLFAs (see Section 2), as an indicator for bacterial biomass, was weakly correlated with the activities of alkaline phosphatase, arylsulfatase, protease and invertase as well as with Corg concentration, total N and C/N ratio. Fungal biomass was not correlated with enzyme activities or with any of the soil chemical properties except for a weak correlation with pH. To study the effect on the microbial community structure in more detail, the community structure of bacteria and eukaryotic microorganisms was determined
3. Results The long-term addition of organic and inorganic amendments caused significant changes in soil chemical properties (Table 1). The amount of organic carbon was lower in the treatments with addition of mineral fertilizers (without or with plant residues) than in the soil with the organic amendments. Total N content was highest in the manure treatment and lowest in the mineral fertilizer þ residues treatment. The C/N ratio was highest after straw amendment and lowest in the treatment with mineral fertilizer only. The treatments did not differ significantly in the amount of dissolved organic carbon (DOC). However, the composition of the DOC was influenced by the different treatments; the aromaticity of the DOC (estimated by SUVA254) was lower in the mineral fertilizer treatments than in the manure and straw amendments. The activity of all enzymes tended to be higher in the treatments manure, sewage and straw compared to
Table 2 Biomass and enzyme activities in different long-term amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05)
Mineral only Mineral þ residue Manure Sewage Straw
Microbial biomass N (mg N g soil21)
Alkaline phosphatase (mg p-nitrophenol (g soil h)21)
Arylsulfatase (mg p-nitrophenol (g soil h)21)
Xylanase (mg GLC (g soil 24 h)21)
Invertase (mg GLC (g soil 3 h)21)
Protease (mg TYR (g soil 2 h)21)
Urease (mg N (g soil 2 h)21)
1.8 ^ 0.8 a 4.3 ^ 0.9 b 5.0 ^ 0.6 b 4.5 ^ 0.9 b 2.0 ^ 0.5 a
430 ^ 25 a 424 ^ 7 a 456 ^ 15 a 512 ^ 28 a 469 ^ 20 a
33 ^ 3 35 ^ 1 40 ^ 3 44 ^ 5 45 ^ 2
956 ^ 103 a 938 ^ 65 a 1037 ^ 96 a 1079 ^ 52 a 1144 ^ 149 a
2120 ^ 75 a 2259 ^ 65 a 2237 ^ 84 a 2372 ^ 79 a 2433 ^ 85 a
76 ^ 9 a 87 ^ 3 ab 94 ^ 6 ab 119 ^ 8 b 101 ^ 8 ab
25 ^ 2 a 28 ^ 3 a 28 ^ 2 a 25 ^ 2 a 30 ^ 2 a
a a a a a
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Fig. 1. Concentration of fatty acids in soil (nmol g soil21) after long-term application of inorganic or organic amendments. (A) mineral fertiliser only, ( ) mineral fertiliser þ residues, ( ) manure, ( ) sewage sludge, ( ) straw.
by PCR-DGGE. A total of 60 and 90 different bands were distinguished in the bacterial and eukaryotic communities, respectively. RDA of the DGGE patterns were carried using the soil chemical properties as environmental data (Fig. 2). The bacterial communities of the manure and straw treatments on the one hand and of the sewage sludge treatment on the other hand formed two distinct groups. The two mineral fertilizer treatments lay between these two groups. The variation in eukaryotic microbial community structure of a given treatment was large and no differences between the treatments were observed. Moreover, RDA showed that PLFA patterns (Fig. 2) and enzyme activities (data not shown) did not differ between the treatments. Multivariate analyses such as RDA in combination with Monte Carlo Permutation tests can be used to determine which environmental variables are significantly correlated with community structure as well as the relative importance
of these variables. The RDA showed that the bacterial community structure was significantly correlated with (listed in order of decreasing importance) aromaticity of the DOC (SUVA254), Corg concentration, DOC concentration and C/N ratio (Table 4). The bacterial community structure was also significantly correlated with the activities of arylsulfatase, alkaline phosphatase and protease. The structure of the eukaryotic microbial communities was significantly correlated with C/N ratio and Corg concentration whereas DOC and aromaticity of the DOC had no significant effect (Table 4). The eukaryotic community structure was also correlated with the amount of biomass N and invertase activity. The PFLA patterns were significantly correlated with DOC aromaticity, Corg and DOC concentration. There were no significant correlations between the PLFAs and enzyme activities or biomass N (Table 4). Enzyme activities were not significantly correlated to any of the soil chemical parameters measured.
Table 3 Concentration of PLFAs in soil characteristic for all bacteria, Gram 2 bacteria, Gram þ bacteria or fungi (nmol g soil-1) and ratios of Gram þ To gram 2 bacteria and bacteria to fungi in different long-term amendments. Means of four replicates ^ standard error. Different letters in the same column indicate significant differences (P , 0.05)
Mineral only Mineral þ residues Manure Sewage sludge Straw
Bacteria
Gram 2 bacteria
Gram þ bacteria
Fungi
92.8 ^ 19.5 a 97.0 ^ 9.8 a 119.9 ^ 13.0 ab 159.0 ^ 18.9 b 133.1 ^ 8.5 ab
15.6 ^ 2.4 a 19.8 ^ 3.4 a 16.2 ^ 1.5 a 21.2 ^ 2.9 a 17.4 ^ 0.9 a
74.2 ^ 16.8 a 73.3 ^ 5.4 a 100.9 ^ 11.0 ab 132.0 ^ 13.5 b 111.6 ^ 7.2 ab
18.4 ^ 2.7 28.8 ^ 8.9 15.4 ^ 2.8 17.4 ^ 2.3 15.2 ^ 1.1
a a a a a
Gram þ /gram 2
Bacteria/fungi
4.6 ^ 0.4 3.9 ^ 0.3 6.2 ^ 0.2 6.3 ^ 0.2 3.4 ^ 0.2
5.2 ^ 0.8 a 4.1 ^ 0.8 a 8.4 ^ 1.2 b 9.2 ^ 0.1 b 8.8 ^ 0.3 b
a a b b b
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Fig. 2. Redundancy discriminate analysis plots of bacterial and eukaryotic DGGE patterns and PLFA patterns after long-term application of inorganic or organic amendments, using soil chemical data as environmental factors.
structure and biomass. The treatments differed in bacterial community structure and biomass but not in eukaryotic community structure or fungal biomass. Both bacterial and eukaryotic community structures were correlated with soil
4. Discussion This study showed that long-term inorganic or organic amendment at a low rate may affect microbial community
Table 4 Soil chemical properties (soil chemistry) and enzyme activities including CFE N (enzymes) with significant correlation (P # 0.10) to bacterial and eukaryotic DGGE patterns and PLFA patterns determined by Monte Carlo permutation tests. Listed in order of decreasing importance with P values in parentheses Bacteria
Eukaryotes
Soil chemistry SUVA254 Corg DOC C/N a
Enzymes (0.01) (0.03) (0.03) (0.01)
Arylsulfatase Phosphatase Protease
(0.01) (0.03) (0.03)
PLFA
Soil chemistry
Enzymes
C/N Corg
CFE N Invertase
(0.01) (0.10)
Soil chemistry (0.06) (0.03)
SUVA254 Corg DOC
None of the patterns were significantly correlated with Ct, Cinorg, Nt, biodegradability, St, C/S, urease or xylanase activity. No significant correlation with any of the enzyme activities.
Enzymes (0.04) (0.06) (0.06)
nsa
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chemistry and enzyme activity. The changes in community structure were, however, not accompanied by significant changes in functional diversity. The increase in soil Corg after addition of organic material (Table 1) is in agreement with other studies (Crecchio et al., 2001) and often leads to increased microbial biomass and activity (Dhillion, 1997; Kandeler et al., 1999b). Easily degradable compounds such as organic acids in the insoluble or soluble organic matter can be utilised by a wide range of organisms. Presence of these compounds can lead to a rapid increase in biomass and activity (Ku¨sel and Drake, 1999) and favour the growth of copiotrophic compared to oligotrophic organisms (Hu et al., 1999). Once the easily degradable compounds have been metabolised, only the more recalcitrant material remains. The decomposition of this material requires enzymes that are produced by a limited number of microbial species (Moorhead and Sinsabaugh, 2000) and may increase the competitive ability of cellulolytic microorganisms (Jensen and Nybroe, 1999). Changes in organic matter composition can lead to a succession of microorganisms such as that observed by Ponge (1991) for fungi during the decomposition of pine needles. The temporal dynamics in decomposition depend on the chemical composition of the organic matter that is added to the soil (Lettau and Kuzyakov, 1999; Northup et al., 1998). Differences in organic matter composition and thus substrate availability are likely to be the reason for the differences in microbial community structure observed in the present study such as the increased bacteria/fungi ratio and the increased gram-positive/gramnegative ratio (Table 3). RDA showed that both bacteria and eukaryotic community structures were correlated with Corg concentration but only bacterial community structure was influenced by DOC concentration as well as aromaticity of the DOC (indicated by the SUVA254) (Table 4). The DOC aromaticity was highest in the straw and manure treatments. This can be attributed to the high lignin content of straw and thus also manure, which contains substantial amounts of straw, and the release of soluble lignin degradation products (Guggenberger and Zech, 1993). Aromatic compounds are generally less degradable (Boissier and Fontvieille, 1993) and can be toxic and thus decrease litter turnover (Northup et al., 1998). The readily decomposable compounds in DOC such as organic acids and carbohydrates (Ku¨sel and Drake, 1999; Guggenberger and Zech, 1993) are probably mainly utilised by soil bacteria while fungi decompose the more recalcitrant and insoluble materials. This may explain why DOC amount and composition had no significant effect on the eukaryotic community structure (Table 4). Nitrogen is often a key limiting factor for soil organisms and addition of N can change microbial biomass, activity and species composition (Sarathchandra et al., 2001). In the present study, manure amendment increased total N concentration compared to the other treatments (Table 1).
459
Interestingly, soil total N concentration had no significant effect on microbial community composition (Table 4) but was weakly correlated with bacterial biomass. It should be noted, that the total N concentration in the present study is low compared to that in other studies (Crecchio et al., 2001; Kandeler et al., 1999b). The highest C/N ratio was found after addition of straw (Table 1) although mineral N had been added with the straw. Organic matter with a high C/N ratio is only slowly degraded by microorganisms (Wagner and Wolf, 1999). The present study showed that the C/N ratio also affects microbial community structure as both bacterial and eukaryotic community structures were significantly correlated with the soil C/N ratio (Table 4). The treatments manure, sewage sludge and mineral fertilizer þ residues increased microbial biomass N compared to straw and mineral only (Table 2). In the straw treatment the low microbial biomass N is probably due to the high C/N ratio of the soil. In the mineral only treatment it may be explained by low Corg content. However, despite of a low Corg content in the mineral þ residues treatment, the microbial biomass N in this treatment was as high as in the manure or sewage amendments. It may be speculated that the plant residues were rapidly decomposed leading to a low soil Corg and a high microbial biomass. The concentration of bacterial PLFAs was lower in both mineral treatments as compared to organic amendments. This indicates that the active bacterial biomass may be declining in response to the low Corg content. The activities of most enzymes were not significantly affected by the long-term treatments in the present study (Table 2). In the short-term, addition of organic matter often leads to increased enzyme activities (Kandeler et al., 1999b; Crecchio et al., 2001; Madejon et al., 2001) and long-term organic amendments can also stimulate enzyme activities and microbial biomass (Kandeler et al., 1999a). The lack of stimulation in the present study may be due to the relatively small amounts of organic amendments added per year which were only about half as much as in the studies by Kandeler et al. (1999b) or Crecchio et al. (2001). This is also reflected by the lower Corg and total N content in this study compared to Kandeler et al. (1999b) and Crecchio et al. (2001). Thus, despite changes in microbial community structure, enzyme activities were generally not affected by the treatments and showed no correlation with any of the soil chemical parameters measured. Due to the functional redundancy of soil microorganisms (i.e. one function can be carried out by a range of different microorganisms), changes in microbial community structure do not necessarily lead to changes in enzyme activities. This is particularly true for the breakdown of sugars, proteins and organic P but may be less so for the decomposition of recalcitrant compounds. Two points regarding enzyme activities should be noted (Nannipieri et al., 2002). Firstly, any short-term changes in enzyme activity may be masked by enzymes that are adsorbed to soil particles and which can retain their activity for many years. Secondly, enzyme
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activity tests measure the potential activity of a given enzyme in soil, not the actual activity. The long-term addition of sewage sludge did not differ in its effect on bacterial and fungal biomass, PLFA patterns or eukaryotic community structure from that of the other two organic amendments (Tables 2 and 3, Figs. 1 and 2). However, the bacterial community structure of the sewage sludge treatment differed from that of the straw and manure treatments. This effect on bacterial community structure can be explained by the higher concentration of aromatic compounds in the DOC of the straw and manure treatments. Sewage sludge may contain high concentrations of heavy metals. However, in the present study, although the concentration of Zn and Cu in the sewage sludge amended soil were elevated in comparison to the other treatments they reached only 15 and 89 mg kg21, respectively (Delschen, 1999), which is well below levels where toxic effects can be expected (Chaudri et al., 2000). In other studies no negative effects of heavy metals added with municipal waste, dump material or sewage sludge on soil biomass or activity were found (Chander et al., 2001; Madejon et al., 2001). It should be noted that bacteria added with the sewage sludge may also have contributed to the observed change in bacterial community structure compared to the other organic treatments. The soil that had received mineral fertilizer without residues was characterised by a very low Corg content, a low C/N ratio as well as low enzyme activities and bacterial biomass. However, the microbial community structure did not differ from that of the other treatments. It may be speculated that a further decrease in soil Corg content will have a strong negative impact on microbial activity in the soil. The incorporation of harvest residues did not offset the decline in soil Corg but increased the microbial biomass. The results of the present study show that long-term addition of organic amendments at a low rate may increase bacterial biomass while having no effect on fungal biomass. They also demonstrate the fundamental importance of organic C for soil microorganisms. Different amendments affected the bacterial and eukaryotic community structure through their effect on Corg and C/N ratio of the soil. The bacterial community structure was also influenced by changes in DOC concentration and composition. Moreover, the study revealed that changes in community composition are not necessarily accompanied by changes in enzyme activities.
Acknowledgements We would like to thank Karen Baumann and Sabine Rudolph for excellent technical assistance and Peter Leinweber for the elemental analyses.
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