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Plant and Soil 230: 145–160, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 145 Seasonal fluctuations in the population of denitrifying and N2 -fixing bacteria in an acid soil of a Norway spruce forest Alexander Mergel, Karin Kloos & Hermann Bothe1 Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, D-50923 Köln, Germany. 1 Corresponding author∗ Received 3 April 2000. Accepted in revised form 16 November 2000 Key words: denitrification, nitrogen fixation, seasonal fluctuations of soil bacteria, soil bacterial community, soil DNA isolation Abstract The seasonal fluctuations in the concentration of cultured denitrifying and N2 -fixing bacteria were followed in an ammonium fertilised and a control soil of a Norway spruce forest near Villingen/Black Forest from December 1994 to August 1998. The horizontal distribution of bacteria in three layers was determined by the MPN-method and by molecular probing (colony hybridisation) using specific 0.4–0.7 kb DNA probes for denitrification steps (narG, nirS, nirK and nosZ) and for N2 -fixation (nifH). The data showed that highest bacterial counts and higher numbers of denitrifying and N2 -fixing bacteria were generally detectable in the upper (= 5 cm) soil layer and that their amount decreased with soil depth. The concentration of these cultured bacteria showed seasonal fluctuations with highest numbers in autumn/winter/early spring and with low counts in summer. Denitrifying and N2 -fixing bacteria amounted to less than 10% of the total number of cultured bacteria determined by the MPN-method. Fertilisation with ammonium did not cause a shift in the population of these bacteria. These findings were corroborated by hybridisation experiments with genomic DNA isolated from the different layers. Strongest DNA–DNA hybridisation band intensities were obtained in the upper soil layer and their intensities decreased with soil depth. Soil samples from Villingen assayed in the laboratory produced N2 O (in dependence of nitrate and C2 H2 added to the vessels) and utilised this gas with higher activities in the assays with the fertilised soil. It is concluded that molecular techniques can successfully be applied for assessing seasonal fluctuations of bacterial populations in soil. Relative abundance of denitrifying and N2 -fixing bacteria can be determined from experiments with DNA isolated from soils. Attempts to transform these results to the total population of soil bacteria on a single cell basis are faced with many uncertainties. Introduction The bacterial populations in soils are said to be complex (Kruske et al., 1997). It was stated that soils contain 103-104 different genomes/g soil dry weight (Borneman et al., 1996; Sandaa et al., 1999; Torsvik et al., 1990) and only a very limited number of these can be cultivated (Amann et al., 1995; Felske et al., 1999). Investigations are further complicated by the fact that the bacterial populations are seemingly variable within short soil distances and may undergo seasonal fluctuations. These aspects, however, have not ∗ Fax No: 221-470-5181. Tel No: 221-470-2760. E-mail: [email protected] yet been thoroughly investigated. Information about seasonal variations of bacterial populations in other habitats, e.g. in aqueous biofilms or sediments (Joergensen, 1989; MacFarlane and Herbert, 1984), are also sparse. Whereas determinations were formerly restricted to activity measurements in soil cores both in situ and under laboratory conditions, molecular biological techniques based on DNA gene probing now offer new avenues for assessing the distribution of bacteria with special traits like denitrification and N2 -fixation in soils. In denitrification, nitrate is reduced via nitrite, nitric oxide and nitrous oxide finally to molecular nitrogen. The ability to denitrify is distributed 146 in phylogenetically diverse groups of unrelated bacteria (Knowles, 1982; Zumft, 1997). Any attempt to quantify denitrifying bacteria has to take into account that the reduction of nitrite to nitric oxide is catalysed by two totally different enzymes, either the cytochrome cd1 or the Cu-containing nitrite reductase. Denitrifying bacteria contain either one or the other enzyme (Coyne et al., 1989). In the present study, gene probes for all steps of denitrification, with the exception of NO-reductase, have been employed for monitoring the distribution of denitrifying bacteria in a selected soil site. Dinitrogen fixation is catalysed by nitrogenase which is encoded by the structural genes nifHDK. In spite of phylogenetic differences, nifH contains highly conserved DNA sequences among the N2 -fixing micro-organisms and has, therefore, been employed for developing probes for assessing the occurrence of nitrogenase in bacteria by molecular probing (Linne von Berg and Bothe, 1992; Widmer et al., 1999). In the present study, the seasonal fluctuations of the denitrifying and N2 -fixing bacterial population were monitored in the period from December 1994 to August 1998 in an acid Norway spruce forest in the Black Forest/Southern Germany. The location was selected because of low industrial activity in the area, therefore any N-input to the soil from air pollution is low. Two different sites in this forest were chosen for the present investigation. One plot had been fertilised with 150 kg of ammonium sulfate ha−1 in May 1994, and the other served as the non-treated control. The study aimed at determining the seasonal fluctuations by different microbiological methods. The initial studies were restricted to DNA hybridisation studies with cultured bacteria and to activity measurements with soil samples. Recently, we obtained soil DNA amenable to hybridisation with DNA-probes for denitrification and N2 -fixation. Data obtained by these different approaches will be presented. Materials and methods area far remote from significant industrial activity. Two plots were selected for the investigations. One (at Glasergrenze, see topographic map no. 7916 of the Landesvermessungsamt Baden-Württemberg from 1994) had been fertilised with the fairly high load of 150 kg ha−1 ammonium sulfate in May 1994, whereas the other one (at Schlößlebühl, same map) served as the non-fertilised control. The surface of this fairly acid soil (pH ∼ 4.0) was covered with bilberry = whortleberry (Vaccinium myrtillus L.), cowberry (V. vitis-idea L.), diverse ferns, such as male fern (Dryopteris felix-mas (L.) Schott), mosses and lichens. The tree canopy contained predominantly Picea excelsior, some Scots pine (Pinus sylvestris L.) and, remarkably, considerable amounts silver firs (Abies alba Miller) of healthy appearance. This latter species is easily affected and indicative of air pollution at other stands. Other details of the Villingen spruce forest and its soil are given in Feger and Raspe (1992) and Stoermer et al. (1997). Soil sampling from the Norway spruce stand of Villingen Samples were taken from the following three layers: (a) 5 cm depth, thus from the upper part of the Ahorizon, just below the O-layer, consisting of accumulated decomposed organic matter, colour black. (b) 10 cm depths, from the middle of the A-horizon, brown-blackish decomposed organic matter (c) 25 cm, from the B-horizon, with an accumulation of brown-yellowish silicate clay, containing also roots, twigs and smaller stones which were discarded. All soil samples from Villingen were transported in plastic bags, kept at ∼ 10 ◦ C, to the Cologne laboratory and used the following day for activity measurements, DNA extraction or bacterial colony growth. Sampling dates were December 1994, March, May, July and September 1995, October 1996, April 1997, April 1998 and August 1998 (see Figures 1 and 2). Site investigated Gene probes used The soil of a dystric Cambisol (Stoermer et al., 1997) in a 100 year-old Norway spruce stand near Villingen, Black Forest (altitude about 780 m) was analysed for its bacterial community. The N-reserves of this soil have been estimated at 8.000 kg N ha−1 (Feger and Raspe, 1992) and are thus not low. The atmospheric N–input is regarded as being small in this The 0.4–0.7 kb DNA probes for denitrification, N2 fixation and for 16S-rRNA were essentially the same as in preceding publications (Fesefeldt et al., 1998; Kloos et al., 1995, 1998). They were generated by PCR using oligonucleotide primers of regions of the genes and the DNA of the target bacteria, followed 147 Figure 1. The seasonal fluctuation in the cell numbers and in water content in the two plots of the Villingen forest. Samples were taken from the 5, 10 and 25 cm layers of the nonfertilised (A) and the ammonium fertilised soil (B), diluted and determined by the MPN-method after growth in LB. The sampling dates are given in the Figure. The bars indicate the counts for the layers: black = 5 cm, grey = 10 cm and white, with small points = 25 cm. The water content of the soil samples (C) was determined as the difference between wet and dry weight of the soil samples. Straight line: nonfertilised soil. Dashed line: fertilised soil. Samples taken from 5 cm layer, N 10 cm layer, 25 cm layer. 148 Figure 2a–b. Hybridisation of the DNA from cultured bacteria with the gene probes for denitrification and for nitrogen fixation (dot blot analysis). Bacteria were isolated from the nonfertilised and fertilised soil of the Villingen spruce forest at the different sampling dates and grown on plates with either LB or YEM (both for 1 d) and heterotrophic mineral medium (5 d). Colonies were transferred to the filters which were used for the hybridisation with the gene probes. The percentage of positive isolates (giving a distinct signal with the probe) was referred to the absolute number of isolates determined by the MPN- method. Hybridisation with the following gene probes, in each case coding for part of the apoprotein of: (a) narG: apoprotein of dissimilatory nitrate reductase. (b) nirS: cytochrome cd1 containing nitrite reductase. (c) nirK: Cu-containing nitrite reductase. (d) nosZ: nitrous oxide reductase. (e) nifH: nitrogenase reductase. Bars represent: Samples from the upper 5 cm layer (in black), from the 10 cm depth (in darker grey) and for the 25 cm depth (in light grey). 149 Figure 2. c–d by cloning and sequencing of the amplificates. Their labeling with digoxigenin had been described earlier (Kloos et al., 1995). Previous studies (Kloos et al., 1995, 1998) had shown that these probes recognized the target DNA isolated from a wide range of reference organisms of all groups of proteobacteria plus some Gram positive and also several cyanobacteria. Probes for denitrification Dissimilatory nitrate reductase (narG) Template DNA from E. coli, size of the amplificate: 414 bp, homology to the published sequence (McPherson et al., 1984): 100%, both on the DNA and amino 150 Figure 2. e acid level, position of the probe within the DNA sequence: N 28–N 441. Cytochrome cd1 containing nitrite reductase (nirS) DNA from Pseudomonas aeruginosa, size of the amplificate: 596 bp, homology to the published sequence from Pseudomonas aeruginosa (Silvestrini et al., 1989): 100%, on the amino acid level, position of the probe within the DNA sequence: N 199 to N 795. Cu-containing nitrite reductase (nirK) DNA from Alcaligenes xylosoxidans NCIMIB 11015, size of the amplificate: 576 bp, homology to the published sequence from Alcaligenes faecalis (Nishiyama et al., 1993): 67%, on the amino acid level, position of the probe within the DNA sequence: N 526–N 1101. N2 O-reductase (nosZ) DNA from Pseudomonas stutzeri ZoBell, size of the amplificate: 598 bp, homology to the published sequence from the same bacterium (Viebrock and Zumft, 1988): 88%, on the DNA and 91% on the amino acid level, position of the probe within the DNA sequence: N 630–N 1227. Probe for N2 -fixation Nitrogenase reductase (nifH) DNA from Azospirillum brasilense Sp7, size of the amplificate: 435 bp, homology to the published sequence from the same bacterium (De Zamaroczy et al., 1989): 100%, both on the DNA an amino acid level, position of the probe within the DNA sequence: N 19–N 453. A general probe for recognising bacterial DNA 16S-rRNA DNA from Azospirillum brasilense Sp7, size of the amplificate: 693 bp, homology to the published sequence from E. coli (Brosius et al., 1981): 85%, on the DNA level, position of the probe within the DNA sequence: N 8–N 701. Isolation of bacteria and colony hybridisations The method is similar as described previously (Kloos et al., 1998). Ten g of soil were suspended in 10 ml H2 O and stirred for 1 h, and larger particles were removed with a sieve of 0.5 µm pore size. The filtrate was diluted (102–104 fold) and streaked out onto agar plates containing either LB, YEM or heterotrophic mineral medium (for the exact composition see Kloos 151 et al., 1998). All media contained 250 µg ml−1 cycloheximide to suppress fungal growth. Plates were incubated at 30 ◦ C for 1 d (in the case of LB and YEM) or 5 d (for heterotrophic mineral medium). Colonies grown were transferred to sterile microtiter plates which contained 250 µl liquids of either LB, YEM or heterotrophic mineral medium. Samples were analysed from each soil layer and medium. On the average, 250–288 randomly selected colonies for each trial were grown up to an optical density > 0.1 (620 nm) and subsequently analysed by colony hybridisation (Grunstein and Hogness, 1975) using the above mentioned gene probes. Bacteria in 50 µl aliquots were transferred to nylon+ -membranes (Qiagen, Hilden) under vacuum blotting. Filters were treated with denaturating solution, neutralising solution, and then with UV-light for 2 min. The treatment with proteinase K and PMSF was also exactly the same as described (Kloos et al., 1998). Hybridisation was done overnight at 68 ◦ C with 200–300 ng labelled probe/100 cm2 membrane in the mixture 5 × SSC, 0.5% blocking reagent, 0.1% Na-lauroylsarcosine, 0.02% SDS. Filters were rinsed twice with 2 × SSC, 0.1% SDS (10 min, 68 ◦ C), and signals were detected immunologically following the standard protocol of Roche-Boehringer, Mannheim. Extraction of DNA from the soil samples and quantifications Acid soils in particular are contaminated by humic acids, tannins, carbohydrates, free DNA and other interfering substances. The following protocol was developed for the isolation of DNA which was essentially freed from such compounds and which was amenable to digestion by the restriction enzymes (BamHI, SmaI, SacI and EcoRI) and to hybridisation with the gene probes for denitrification and N2 fixation. Soil samples (5 g) were stirred into 10 ml 0.1% Na4 P2 O7 /10 mm Tris–HCl/ 1 mM EDTA (=TE buffer). After an incubation (10 min, RT) and centrifugation (10 min, 6000 × g), the pellet was washed with 10 ml TE buffer and centrifuged (10 min, 6000 × g). The pellet was suspended in 5 ml TE/25 mg lysozyme and incubated (1 h, 37 ◦ C, under shaking), followed by the addition of 1 ml of 10% SDS (= sodium dodecyl sulphate), incubation (1 h, 65 ◦ C, under shaking) and was centrifuged (10 min, 6000 × g). Supernatants were then subjected to phenol extraction (using equal vol of phenol) and centrifuged (15 min, 12 000 × g). The aqueous phase was removed and cleaned from residual phenol by treatment with 5 ml chloroform/isoamyl alcohol (24/1,v/v). The DNA from the upper phase (∼ 5 ml) was then precipitated with potassium acetate (300 µl) and isopropanol (3 ml) for 16 h at 4 ◦ C. The sample was centrifuged (10 min, 10 000 × g), washed with 300 µl 70% ethanol, centrifuged once more, dried in a vacuum centrifuge and suspended with 1 ml 1.2 M NaCl. After storage for 2 h at RT, the sample was supplemented with 1/10 vol 10% CTAB (= hexadecyl-trimethylammonium bromide) dissolved in 1.2 M NaCl, stored (10 min, 65 ◦ C), supplemented with chloroform (1 ml) and spun down in an Eppendorf centrifuge at maximal speed. After repeating this washing step, the DNA in the aqueous phase was precipitated with 50 µl 3 m K+ -acetate/0.6 ml isopropanol, stored (16 h, 4 ◦ C), centrifuged (20 min, 18 000 × g), washed once more with 70% ethanol, dried in the SpeedVac SC 100 (Savant) vacuum centrifuge and suspended in 20 µl TE buffer. The DNA obtained was then electrophoresed on 0.6% PeqGold Low MeltAgaraose (from Peqlab, D-Erlangen) at 40–50 V for 1–2 h. The DNA bands which were discernible by UV-light were cut out and transferred to 1.5 ml Eppendorf tubes, supplemented with 3 vol of TE, incubated at 65 ◦ C to melt the agarose (5–10 min) and extracted with choroform/ isoamylalcohol (24/1). The DNA was precipitated with 1/10 vol of 3 m K+ -acetate/0.6 ml isopropanol (RT, 1 h), centrifuged (15 min, 18 000 × g), washed with 70% ethanol, dried in the SpeedVac and finally suspended in 20 µl TE buffer. This somewhat laborious preparation gave DNA of high purity (O.D. ratio 260 nm /280 nm ∼ 1.7). Seeding experiments with Azospirillum brasilense Sp7, Alcaligenes eutrophus H16 or E. coli K12 gave recoveries between 47 and 69% (not documented). Signal intensities of the genomic DNA isolated from the soil samples or of the DNA–DNA hybridisation bands were determined densitometrically using the NIH Image 1.61 picture analyser program for MacIntosh. Activity measurements in the soil cores Samples from the Villingen forest were assayed in aqueous extract for pH using a PHM 61 laboratory pHmeter electrode (Radiometer, Copenhavn) and for nitrate content by the salicylic acid method (Cataldo et al., 1975). The soil moisture was calculated from the dry weight of the soil. MPN-determinations after diluting the soil samples 103 – 107 fold and incubating in LB for 4 weeks were performed as described by Al- 152 exander (1982). Formation or utilization of gases were assayed with 2 g soil samples in ∼ 7.0 ml Fernbach flasks covered with gas-tight suba seals. For N2 Oformations, the flasks were supplemented with 2 ml 1 mM NaNO3 , evacuated and refilled with argon, and 0.5 ml C2 H2 was added by a syringe. For determining N2 O-uptake rates, 1 ml of this gas was injected into the anaerobic Fernbach flasks. Incubation was generally performed in a shaking water bath (30 ◦ C, 1 d). Gases (N2 O, CO2 and O2 ) were determined by standard gas chromatography as described earlier (Kloos et al., 1995). Results The seasonal fluctuations in the total number of cultured bacteria and in the percentages of denitrifying and N2 -fixing isolates Any investigation with soil samples is faced with the problem that soils can show large fluctuations in their chemical and physical composition within short distances. In the case of the Villingen soil, horizons had a homogenous appearance judging from looking into different holes dug within a few m of one another on the two plots. In the fertilised soil, the upper 10–15 cm in depth contained black soil of decomposed plant litter material, and then from this depth till 80 cm the soil was homogeneously brown-yellowish containing a few small stones. The nonfertilised soil had similar eye-visible appearance, but the black cover spread from the top to approximately 20 cm, probably due to the fact that this stand was more extensively covered with small woody plants like Vaccinium myrtillus, V. vitis-idaea and Calluna vulgaris. This black upper cover was more thoroughly interspersed with plant roots than in the fertilised plot. In October 1996, samples from five places separated from each other by few meters were collected from the upper soil layer (5 cm depth) of both the N-fertilised and the nonfertilised soil, and five independent determinations of nitrate content and cfus after growth on LB for 1d were performed for each of the two plots. The nitrate content was 4.4±1.8 µmol g−1 dry weight of soil for the fertilised plot and 2.4±1.8 for the control. Likewise, the data for the colony forming units (cfu) after growth on LB for 1 d were variable: 6.7±6.1 × 10 5 cfu g−1 dry weight of soil in the fertilised plot and 6.2 ± 5.1 × 10 5 cfu g−1 dry weight (n = 5, always) in the case of the control. Variations in these counts by a factor of maximally 10 had to be encountered when cfus grown on LB were determined from soil samples taken within short distances. When the cells had been grown on plates with yeast extract/mannitol (YEM) for 1 d or on heterotrophic mineral medium supplemented with malate, variations in cfu counts were only two- to threefold. The pH value of the samples taken from the upper 5 cm of the two plots varied between 3.8 and 4.1 (Table 1). The seasonal fluctuations of cultured bacteria (determined by the MPN-method after growth in LB for 4 weeks) and of cultured denitrifying and N2 -fixing microorganisms hybridising with the specific gene probes was followed during the period from December 1994 till August 1998. Due to the large amount of data compiled from the different samples (two plots, of each three horizons, growth of the bacteria in three different media on the plates in most cases, then hybridisation with 5 gene probes), only duplicate determinations for each trial were feasible. The total cell number showed fluctuations with peak values in autumn – winter – early spring and with low values in late spring – summer (Figure 1a, b). In April 1997, scores were fairly high, whereas they were low in April 1998. The forest soil was unusually dry in all three soil layers (5, 10, 25 cm) in April 98 (Figure 1c) which could account for the low MPN at that date. The soil at other dates was, however, not drier in summer than in autumn–winter–spring. Thus, there was no obvious correlation between water content and bacterial cell counts. The maximal MPN-values did not exceed 3 × 107 cells g−1 dry weight of soil even in the upper (5 cm) zone. Highest counts were often, particularly in the case of the fertilised plot, in the upper layer and lowest counts were at 25 cm (Figure 1a, b). Hybridisations with the different gene probes for denitrification (nirS, nirK, nosZ and narG, experiments with narG could be performed only from March 1995 on) and for nifH gave a similar pattern in the seasonal fluctuations of the counts in the soil layers (Figure 2a–e) as obtained in the case of the MPN (Figure 1). Scores were generally higher in autumn-winter-early spring and lower in summer. Highest positive hybridisations were often obtained with the isolates from the 5 cm or the upper two (5 cm and 10 cm) layers. Sometimes, however, apparently after prolonged rainfalls (e.g. in March 1995), high scores were also measured with the isolates from the 25 cm zone. It should be noted that the nitrate content decreased with the depth of the soil and was 153 Table 1. Parameters of soil samples taken from the two plots of the Villingen forest Soil Soil depths [cm] Non-fertilised 5 10 pH [H2 O] Water content [%] Nitrate content [µmol g−1 dry weight × h−1 ] N2 O-formation [nmol g−1 dry weight × h−1 ] N2 O-utilization [nmol g−1 dry weight × h−1 ] CO2 -formation in air [µmol g−1 dry weight × h−1 ] O2 -uptake in air [µmol g−1 dry weight × h−1 ] CO2 -form., under Ar [µmol g−1 dry weight × h−1 ] Cell number [MPN, 105 g−1 dry weight] cfu × 105 g−1 dry weight] grown on LB cfu × 105 g−1 dry weight] grown on YEM cfu × 105 g−1 dry weight] grown on heter. MIN Total cfu × 105 g−1 dry weight grown on the three media 3.8 72.7 2.4 3.8 23.4 0.4 4.1 16.6 0.0 4.1 64.9 4.4 4.2 21.7 0.1 4.5 19.3 0.0 3.2 0.5 0.6 15.9 2.2 0.4 167.1 150.3 108.9 447.9 128.7 162.5 1.1 0.25 0.1 0.4 0.15 0.05 1.3 0.4 0.15 1.1 0.25 0.05 0.3 0.1 0.05 0.25 0.1 0.05 48.1 5.1 1.8 41.3 9.1 6.5 6.2 0.2 0.1 6.7 0.7 0.03 8.9 1.2 1.7 5.2 3.7 2.9 6.1 0.7 0.8 4.4 2.0 1.4 21.2 2.1 2.6 16.3 6.4 4.4 25 Fertilised 5 10 25 Non-standard abbreviations: wt = weight, cfu = colony forming units, MPN = most probable number, LB = Luria-Bertani, YEM = yeast extract-mannitol, heterMIN = heterotrophic mineral medium. The determinations of the parameters are described under ‘Materials and methods’. The formation of N2 O (in the presence of 1 mM nitrate) and the utilisation of this gas was performed with the soil samples under laboratory conditions at 20 ◦ C. The total cfu data represent the sum of the colonies grown on all three media, thus including those isolates which grow on more than one medium. Data are from October 1996. low (< 0.1 µmol/g dry weight) at 25 cm (Table 1). The total number of isolates hybridising with the gene probes for denitrification and for nifH were low, generally amounting to only 2–5% and rarely exceeding 10 % of the total amounts of cultured bacteria (Figure 2 a–e). Isolates hybridizing with all three probes for denitrification: narG, nirS or nirK, and nosZ were under 1% (not documented). DNA of none of the isolates hybridised with both nirS and nirK (not documented). Scores were considerably higher with nirK than with nirS (Figure 2b, 2c). Experiments performed with genomic DNA isolated from the Villingen soil Genomic DNA was extracted from the three soil layers of the two Villingen plots in August 1998. The DNA preparation obtained had molecular weights between 15 and 25 kb with no smear above and below these sizes indicating that the portion of sheared DNA was negligible (Porteous et al., 1994). The yields were calculated from a standard curve with E. coli DNA and gave 10.4/10.5 µg DNA g−1 dry weight of soil for the upper 5 cm zone of the nonfertilised/fertilised Villingen soil, respectively. The corresponding values for the 10 cm and 25 layers were 5.0/3.7 and 1.4/1.6 µg DNA g−1 dry weight of soil, respectively (see also Figure 3). The DNA preparation obtained was suitable for hybridisation with the DNA-probe for the 16S-rRNA (a general probe recognising only bacterial DNA) and with the other segments representing parts of the denitrification and nifH genes. DNA–DNA hybridisation signal intensities can only be compared 154 filterwise because hybridisation conditions cannot be standardised to such an extent as to get reproducible signals from one filter to the next. Therefore, the DNA on the filter was first hybridised with the physiological gene probe, then the filter was stripped and hybridisation was subsequently performed with the 16S-rRNA probe (Figure 3). As documented for the hybridisation with nirK, nirS and nifH (Figure 3), signal intensities were highest in the upper, 5 cm zone. For quantitative data, the signals were scanned and the maximal values, always obtained in the hybridisations with the DNA from the 5 cm layer, were arbitrarily set to 100% (Table 2). The data indicate that the signal intensities decreased parallel with the soil depth in the case of all five physiological gene probes available and also in parallel with the signal intensity obtained with the 16S-rRNA probe. Thus, there was no selective enrichment of denitrifying and N2 -fixing bacteria in lower soil layer (= 25 cm) of the Villingen soil. At the same sampling date (August 1998), the total number of cfu was determined for comparison. Total cfu after growth on LB and YEM for 1–2 days were in the range of 104 – 105 g−1 dry weight of soil (Table 2). The cfu values were always 1–2 orders of magnitudes lower than the counts determined by the MPN-methods (Table 1). Bacteria grown on the LB and YEM containing plates in August 1998 were then used for colony hybridisations (Table 2). Positive scores with the physiological gene probes were in the range of 102 – 103 (Table 2) and thus amounted to grossly 5% of the bacterial counts. When the percentages of isolates hybridising with one of the gene probes were referred to the total number of cultured bacteria obtained by the MPN-method, a clearer picture about the distribution of bacteria in horizontal layers emerged. (Figure 2). However, it is not clear whether the percentage of bacteria hybridising with the probes is the same on growth in suspensions (for the MPN-method) as on plates (for the determination of the cfu). Determination of the potential physiological activities Soil from Villingen was tested for several physiological activities (Table 1). The rates of CO2 -evolution and of O2 -uptake were approximately the same when the soil samples were assayed in air. The CO2 formation activity under anaerobic conditions was about fourfold lower than in air. The samples utilised N2 O (Table 1), and this activity was totally inhibited by approximately 10% C2 H2 in the gas phase (data not shown). The samples also produced N2 O (Table 1), however, only in strict dependence on the addition of nitrate and C2 H2 in the case of the Villingen soil. In the 5 cm soil layer, samples from the fertilised soil formed and utilised N2 O with significantly higher activities than those from the non-fertilised soil in these experiments performed in October 1996. The temperature dependence of the N2 O-formation activity was tested with soil from the 5 cm layer at 9 ◦ , 20 ◦ and 30 ◦ C. Figure 4 shows a typical experiment where the assay vessels were supplemented with different concentrations of nitrate. Activities were readily measurable after 24 h in the 20 ◦ and 30 ◦ C assays, and unambiguous values were obtained after 48 h in the 9 ◦ C assay. The reaction, however, proceeded for at least 60 h. The apparent Vmax –values at 30 ◦ C were 29.5 for samples from the fertilised plot and 14.5 nmol N2 O formed h−1 × mg−1 dry weight of soil for those from the control area. Corresponding data were 19.0/8.2 for 20 ◦ C and 0.5/0.3 nmol N2 O formed h−1 × mg−1 dry weight of soil for 9 ◦ C for samples from the fertilised/ nonfertilised soil, respectively. Biological reactions generally show an approximately twofold increase when the assay temperature is raised by 10 ◦ C. This was, indeed, the case for the data for the fertilised soil obtained at 20 and at 30 ◦ C, whereas the data at 9 ◦ C were 5–6 fold lower than at 20 ◦ C. Remarkably, the samples from the fertilised plot consistently showed higher activities than those from the control in this specific test. In these N2 O-evolutions, the S[0.5V ] -values (= apparent Michaelis constants) for nitrate using the samples from the fertilised plot were roughly 1/2 of those obtained with the control soil. Discussion Both denitrification and N2 -fixation are widely distributed among diverse bacteria of totally unrelated systematic affinities. Therefore, general DNA probes based on 16S-rRNA oligonucleotide sequences cannot be developed for recognising bacteria with these physiological traits (Braker et al., 1998). The 0.4–0.7 kb probes developed in our laboratory specifically for the different steps of denitrification and for N2 -fixation recognize these genes in a wide range of bacteria by heterologous hybridisations (Kloos et al., 1998) and have successfully been employed for analysing the populations of Hyphomicrobium isolates from aqueous systems (Fesefeldt et al., 1998; Kloos et al., 155 Figure 3. Hybridisation of genomic DNA isolated from the three different soil layers with the gene probes. DNA was isolated as described under ‘Materials and methods’ and blotted onto the nylon+ -filters. Hybridisation on each filter was first performed with the physiological gene probe (nirK, nirS or nifH). Then the filters were stripped and hybridisations were performed with the general 16S-rRNA probe recognising bacterial DNA. DNA isolated from 0.5 g soil (fresh weight) was blotted to the filters for the 5 cm and 10 cm samples and from 1.2 g for the sample from the 25 cm depth. Band intensities were quantified densitometrically for the data of Table 2. 156 Table 2. Hybridisation of the total genomic DNA isolated from the different layers of the two Villingen plots and of genomic DNA from cultured bacteria with the gene probes for denitrification and nitrogen fixation Fertilised Soil depth Non-fertilised 5 cm 10 cm 25 cm 5 cm 10 cm 25 cm Total cfu after growth on LB (number × 104 × g soil) YEM (number × 104 × g soil) 8.8±3.9 8.2±1.7 9.5±1.3 3.0±0.4 1.5±0.5 4.9±0.2 13.4±2.8 18.5±9.4 6.0±1.2 17.7±5.5 0.8±0.3 6.7±5.9 100 100 70.2±9.9 79.8±7.8 13.5±0.5 16.9±3.6 100 100 39.9±4.6 48.2±7.0 14.9±1.4 17.8±0.2 6.4 35.8 32.3 14.0 3.6 22.8 34.4 23.0 27.8 30.6 1.2 25.9 100 100 33.0±6.9 56.6±2.8 25.3±1.7 16.3±0.5 100 100 29.1±7.1 20.3±1.0 19.4±7.3 11.3±1.2 6.4 37.3 16.7 4.7 11.7 7.0 159.4 55.6 29.1 46.4 1.1 0.8 100 61.2±6.7 25.0±3.7 100 26.2±2.4 17.6±0.5 100 68.8±18.1 17.5±6.4 100 40.9±3.2 19.1±4.7 24.8 0 49.1 11.4 15.8 29.5 183.7 119.5 17.2 0.0 2.3 0.0 100 100 58.9±3.3 47.5±0.2 18.6±9.1 9.2±0.3 100 100 29.3±1.7 29.2±3.7 15.1±4.1 13.8±3.2 6.3 91.3 41.1 14.4 2.0 63.9 16.5 152.7 37.8 0.0 0.0 0.0 100 100 44.0±3.9 40.3±0.1 21.0±4.1 11.0±3.2 100 100 50.6±2.9 46.1±4.7 20.9±2.7 18.6±3.2 12.7 62.3 67.8 60.4 0.0 0.0 0.0 87.1 0.0 0.0 1.8 1.9 Hybridisation of soil DNA with NifH 16S-rRNA Hybridising isolates from medium LB (number × 102 × g soil) YEM (number × 102 × g soil) Hybridisation of soil DNA with NarG 16S-rRNA Hybridising isolates from medium LB (number × 102 × g soil) YEM (number × 102 × g soil) Hybridisation of soil DNA with NirS 16S-rRNA Hybridising isolates from medium LB (number × 102 × g soil) YEM (number × 102 × g soil) Hybridisation of soil DNA with NirK 16S-rRNA Hybridising isolates from medium LB (number × 102 × g soil) YEM (number × 102 × g soil) Hybridisation of soil DNA with NosZ 16S-rRNA Hybridising isolates from medium LB (number × 102 × g soil) YEM (number × 102 × g soil) Soil samples from the 5, 10 and 25 cm layer was diluted and plated onto agar containing either LB or YEM. After 1 d, the number of colonies grown (cfu) were counted, and 250–288 colonies were used for hybidization with the gene probes in the dot- blot analyis. The percentage of positive counts was referred to the total number of cfu. For the other part of the data of this table, genomic DNA was isolated from the 5, 10 and 25 cm layers of the two Villingen plots and then hybridised with the gene probes for denitrification or nifH. The signal intensities were quantified densitometrically and DNA–DNA hybridisation bands were then stripped off the filter, and the same filter was subsequently used for the hybridisations with the DNA probe coding for the bacterial 16S-rRNA. The signal intensities obtained with the DNA from the 5 cm soil layers were always set to 100%. All data refer in% to this intensity. The soil was sampled in August 1998. 157 Figure 4. N2 O-formation activity with soil samples from the fertilised and nonfertilised soil. Soil samples from Villingen were brought to the Cologne laboratory and assayed for N2 O-formation in the presence of varying amounts of nitrate and at different temperatures. The Fernbach flasks contained 10% C2 H2 to block N2 O-reductase activity. The samples assayed were from the upper (= 5 cm) soil layer. Dashed lines: samples from the fertilised plot. Straight lines: samples from the nonfertilised control. experiment performed at 30 ◦ C N experiment performed at 20 ◦ C experiment performed at 9 ◦ C. 1995). Hybridisations with these probes also indicated seasonal fluctuations in the population of cultured denitrifying and N2 -fixing bacteria in both plots of the Villingen forest, and these seasonal variations grossly matched with the fluctuations of the total number of bacteria determined by the MPN-method. The high variability of the soil constituents within short distances is a general problem in soil microbiology. The question arises whether this variability is larger than any difference in the data obtained or which definitive conclusions can be drawn from such studies. First of all, it can be stated that the total bacterial counts in such rather acid soil are – not unexpectedly – considerably lower than in soils with higher pH values. For example, bacterial numbers in the soil of the Chorbusch forest (a gleysol, pH around 5) or in chalk meadows in the vicinity of Cologne are 1–2 orders of magnitude higher than in the Villingen soil (Kloos et al., 1998; this study). Secondly, bacterial life and activity are highest in the upper zone and decrease with the depth of the soil. This was shown for all soils investigated so far (Kloos et al., 1998) and is also seen in the Villingen plots, although not so distinctly as in the other soils, possibly because of the low bacterial numbers in the soil profiles of this spruce forest. Data are clear-cut for the activity measurements (highest gas formations or utilisations always in the upper layer) and for the experiments performed with the isolated DNA (band intensity also highest in upper 5 cm). In the MPN– determinations and in the colony hybridisations with the cultured bacteria, the method allowed to conclude that seasonal fluctuations occurred with peak activities in autumn/winter and early spring and with low scores in summer. The refinement of the data was, however, not sufficient for more generalisations mainly because of the low number of cultured bacteria and because it was not feasible to accumulate more data, e.g. by monthly determinations. Such fluctuations with peak activities in winter/early spring during freezing/thawing conditions have also been reported for N2 O-formations for grassland and 158 other soils (Bremner et al., 1980; Sommerfeld et al., 1993). In combination with chemical processes, biological reactions, in particular those performed by denitrifying bacteria, appear to be responsible for this large fluxes of the gas (Müller et al., 1997). The population of cultured denitrifying bacteria assessed by molecular probing are likely too small to account for these rates in the Villingen soil. Denitrifying bacteria amounted to less than 10% of the cultured bacteria and did not exceed 106 cells g−1 soil dry weight (Figure 2). With soil samples assayed, rates (in the presence of nitrate and C2 H2 ) amounted to maximally 15–20 nmol N2 O g−1 soil sample × h−1 at 20 ◦ C in the 5 cm layer (Figure 4). For comparison, a culture of Alcaligenes eutrophus in the exponential growth phase produced about 0.2 nmol N2 O h−1 per 10 6 cells at 20 ◦ C, and the same cell number of Bacillus cereus formed even less under the same assay conditions (B. Schmitz and H. Bothe, unpublished data). Since there is a difference of two orders of magnitude, other bacteria or chemical processes than the cultured, ‘classical’ denitrifying bacteria must have been major contributors to the 15–20 nmol h−1 detected in the soil samples. This consideration is in line with the statement that only 1% or less of the soil bacteria have been cultured (Amann et al., 1995). Thus a concentration of at least 108 cells g−1 soil dry weight is to be expected in order to produce the rates of 15–20 nmol N2 O h−1 × g−1 soil sample. Which are the organisms (factors) responsible for this high activity in soil samples? Besides noncultured bacteria, fungi and heterotrophic nitrifiers could be major contributors to the N2 O-production. Some fungi have been reported to perform denitrification (Shoun et al., 1992) but the relative contributions of fungi to the N2 O-release from soils have not yet been examined. Heterotrophic nitrifiers may also produce N2 O, although the major gas formed by them appears to be nitric oxide (Daum et al., 1998). Such strongly acid soils have been reported to contain heterotrophic nitrifiers in the extremely and thus surprisingly high numbers of 108 to 1011 cells/g soil (Papen and Von Berg, 1998). A gene probe developed for nitrification (from the amoA gene of Nitrosomonas europaea) recognised the corresponding gene in the heterotrophic nitrifier Pseudomonas putida (Daum et al., 1998) but could not be tested for the occurrence of this gene in a wide range of heterotrophic nitrifiers because only few of them can be cultured nowadays. Therefore, the amoA probe (Kloos et al., 1998) was not used in the present study. The data of the present study indicated that the characterization of cultured bacteria provided some information about the seasonal fluctuations in such soils with low bacterial counts. The major conclusion is that cultured denitrifying and N2 -fixing bacteria occur in the upper soil layer in higher numbers than in depth which poses the question of their advantage in having retained these genes for life in such a habitat. The results also showed that cultured bacteria possessing the Cu-nitrite reductase (nirK) occur more abundantly at such a location than those with the cytochrome cd1 nitrite reductase (nirS), whereas it was suggested that bacteria with nirS predominate in nature (Coyne et al., 1989). Moreover, the results indicated seasonal fluctuations of the total number of bacteria and the percentage of denitrifying and N2 -fixing microorganisms. More details could, however, not be gained, because the method with its high statistic error was too coarse. In the present study, 250–300 bacterial colonies were characterized for their hybridisation with the gene probes at each date and plot. For obtaining more accurate differences, an at least tenfold higher number of isolates needs to be investigated which is hardly feasible. Even then the problem would still remain that less than 1% of the bacteria detectable by fluorescence methods in soils (Torsvik et al., 1990) would have been cultured. Hybridisation with genomic DNA isolated from the different layers and with the gene probes indicated more convincingly that denitrifying and N2 -fixing bacteria mainly occur in the upper soil zone, and that their concentration decreases in parallel with the total number of bacteria assessed by the MPN-method or by the cfu-determination. If one assumes that the DNA of the bacteria from the different layers hybridises with the same concentration dependence, this approach gives clear-cut results on the relative abundance. A crucial factor in these assays is, however, the isolation of DNA in high yield and purity. The preparation obtained in the present study was amenable to digestion by restriction enzymes and to hybridisations with the gene probes. The method employed was timeconsuming but allowed the recovery of 50–70% of the DNA as shown in seeding experiments with Acaligenes eutrophus H16, Azospirillum brasilense Sp7 and Escherichia coli K12 (A. Mergel, unpublished data). A recent examination of the different protocols for the extraction of DNA from soils came to the conclusion that not more than 6% of the DNA of indigenous bacteria can be recovered from different soils and that the clay content strongly influences the retrieval of DNA 159 (Frostegard et al., 1999). An even more severe bias is that such relative signal intensities of the hybridisation band cannot be transformed to cell numbers or referred to activities based on single cells (Amann et al., 1997). The quantitative determination of individual genes present in single copy or low copy numbers in intact bacterial cells of soils by in situ hybridisation methods is not possible (Hodson et al., 1995), and quantitative in situ PCR-techniques for characterising bacterial populations in habitats are still in their infancy. Such methods are, however, required if one wants to correlate numbers in the bacterial populations to global fluxes of NOx or other gases. Well aware of these facts, we nevertheless venture to forward the following estimations: The genome size of E. coli is 4.7 × 106 bp or 3.1 × 109 dalton (standard table from Boehinger-Roche; see also Torsvik et al., 1990). A calibration curve with DNA isolated from E. coli indicated that the nonfertilised/fertilised plot contained 10.5/10.5 µg DNA g−1 dry weight of soil in the 5 cm layer and 5.0/3.7 in 10 cm and 1.4/1.6 in 25 cm. This is equivalent to a total number of E. coli genomes of 2.0/2.0 × 109 g−1 soil in 5 cm, 0.9/0.7 × 109 in 10 cm and 0.25/0.3 × 109 in 25 cm. If the average genome size of the bacteria from the Villingen soil is similar to that of E. coli, the comparison of these data with the MPN-determinations shows that 1– 5% of the bacteria of that soil have been cultured. The same can be deduced from the concentration of DNA in bacteria cells, varying between 2 and 15 fg DNA (Fuhrman et al., 1988; Paul and Myers, 1982). If the average value is 8.5 fg, then the nonfertilised/fertilised plot contains 1.4/1.4 × 109 cells in 5 cm, 0.65/ 0.5 × 109 in 10 cm and 0.2/0.2 × 109 in 25 cm. All these estimations, together with the activity measurements seem to indicate that such soils contain more than 109 cells g−1 dry weight of soils in the upper layer, of which grossly 107 to 108 appear to be potential N2 O-producing microorganisms. One plot had been fertilised with ammonium sulfate in May 1994, and the soil samples from this plot investigated in October 1996 showed higher N2 Oformations and utilizations than those from the control area. Other parameters (CO2 -evolution and O2 -uptake activities, MPN-counts, hybridisations with cultured bacteria, hybridisations with DNA isolated from the different horizons), however, did not show significant differences between the samples from the two plots. Fertilisation with nitrate may cause a shift in the population of denitrifying bacteria in other habitats (Nijburg and Laanbroek, 1997). The ammonium added to the Villingen soil may have been converted to nitrate by the action of nitrifying bacteria. As there was no increase in nitrate content in the horizons of the fertilised Villingen soil and as the increased denitrification activity in the laboratory experiments was only detected when nitrate was added to samples, this higher activity may be caused by the activation of ‘dormant’, noncultured denitrifying or nitrifying bacteria which may have selectively been enriched by the N-fertilisation. Acknowledgements The authors are indebted to Regina Hiltawsky, Karin Kaiser, Dr Ralf Schlichting and Valer Schulte-Fischedick for participating in some of the experiments, to Kerstin Nawrath and Barbara Schmitz for skilful technical assistance and to Dr Oliver Schmitz for helpful comments. This work was kindly supported by grants from the Deutsche Forschungsgemeinschaft within the priority program "Microbial Ecology" and from the BMBF within the program Spurenstoffkreisläufe (through the Fraunhofer Institute in GarmischPartenkirchen). 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