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Community-level responses of metabolically-active soil microorganisms to the quantity and quality of substrate inputs
Soil Biology & Biochemistry 36 (2004) 841–848 www.elsevier.com/locate/soilbio
Community-level responses of metabolically-active soil microorganisms to the quantity and quality of substrate inputs T. Pennanen*, S. Caul, T.J. Daniell, B.S. Griffiths, K. Ritz1, R.E. Wheatley Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received 12 December 2002; received in revised form 8 October 2003; accepted 21 January 2004
Abstract The community fingerprints of both the prevalent and the metabolically active microbial community were related to a quantitative estimation of microbial biomass in an arable soil, revealed by substrate-induced-respiration (SIR). Two concentrations of glucose or L -asparagine, representing those used in the SIR measurement or equivalent to those released in root exudates, were studied. Respiration rates and changes in community structure fingerprints were followed for 48 h. Bacterial and fungal community fingerprints were obtained using both reverse transcribed 16S and 18S ribosomal RNA (rRNA) regions and the corresponding rDNA as a template in PCR. Samples were then analysed by denaturing gradient gel electrophoresis (DGGE). Low concentrations of substrate amendments resulted in minor changes in rRNA or rDNA-based bacterial and fungal banding patterns during the whole 48 h incubation. High concentrations of substrates, especially L -asparagine, increased respiration rates and induced significant changes in both 16S rRNA and rDNA-community fingerprints. The prominent rRNA and rDNA bacterial community sequence types were common to all treatments, but in general the bacterial rDNA fingerprints had fewer bands than the corresponding rRNA profiles that assess the active fraction of the community. In contrast, there was little difference between fungal 18S rRNA and rDNA patterns. The number of fungal ribosomal sequence types in DGGE fingerprints was lower than the number of bacterial types. This study indicated that there was a rapid respiration response by the whole microbial community during SIR estimates in soil, but that community structure did not change during the conventional incubation period. In an extended (8– 48 h) incubation with high amounts of L -asparagine increased respiration was associated with growth of the microbial community. q 2004 Elsevier Ltd. All rights reserved. Keywords: Substrate-induced respiration; Ribosomal RNA; Soil microbial community; Denaturing gradient gel electrophoresis
1. Introduction The substrate-induced respiration (SIR) method (Anderson and Domsch, 1978) is commonly used to estimate soil microbial biomass. In the SIR method it is assumed that the majority of the soil microbes respond rapidly to glucose amendment and that the respiration response reflects the total microbial biomass. However, it is not directly known which components of the soil microbial community are the primary contributors to the observed respiratory flush. Although approaches that study the structure of * Corresponding author. Present address: Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FIN-01301 Vantaa, Finland. Tel.: þ358-10-211-2404; fax: þ 358-10-211-2204. E-mail address: [email protected] (T. Pennanen). 1 Present address: National Soil Resources Institute, Cranfield University, Bedfordshire MK45 4DT, UK. 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.01.014
the communities such as phospholipid fatty acid (Zelles, 1999; Pennanen, 2001) and DNA-based fingerprints (van Elsas et al., 1998; Tiedje et al., 1999) have the potential to characterize soil microbial communities, the structure of communities is not necessarily reflected in their biological activity. Most soil microbes are thought to be inactive (Olsen and Bakken, 1987) and DNA is also known to persist in dead cells and as extracellular DNA in soil (Ogram et al., 1987). However, as the cellular content of ribosomal RNA (rRNA) is directly correlated to cellular activity and growth rate (Rosset et al., 1966; Wagner, 1994; Manefield et al., 2002), an examination of rRNA should provide a more representative indication of which members of the microbial community are active. Our aim was to assess the metabolically-active fraction of the prevalent microbial community that responded to substrate amendments. We added substrates of varying
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quality and quantity to soil to invoke various microbial responses. We were especially interested in investigating relationships between respiration flux in SIR assays and microbial community profiles derived from both the prevalent and the active members of the community. A method for the rapid simultaneous extraction of both RNA and DNA from soil then the reverse transcription of both the 16S and 18S ribosomal RNA (rRNA) regions was developed. PCR amplifications were then performed for both partial 16S and 18S rRNA regions. Thereafter we related the rRNA community fingerprint, at both the RNA and DNA level, using denaturing gradient gel electrophoresis (DGGE) to general microbial respiration activity.
2. Materials and methods 2.1. Soil The soil used was from the Carbrook association, an imperfectly drained soil with a clay loam texture, a pH (H2O) of 6.2, total C content of 2.33% (w:w), a total N content of 0.22% (w:w) and water-holding-capacity (WHC) of 0.47 g water g21 dry soil. Soil samples taken from a field in cultivation on a wheat– barley –potatoes intensive rotation were bulked and sieved to , 7 mm and then to , 2 mm. The sieved soil was then stabilized for a week at þ 4 8C. 2.2. Experimental design and sample preparation Soil samples (3 g fw) were weighed into 30 ml glass bottles and stabilized at 18– 20 8C over night (Anderson and Domsch, 1978). Soil moisture content was adjusted to 2 £ WHC by adding water only (control treatment), or in addition to glucose or L -asparagine solutions as substrates (Ritz and Wheatley, 1989). Solutions of glucose and L -asparagine were added to give final concentrations of 20 mg or 2000 mg C g21 dw. For each of these five treatments, 24 replicate bottles were prepared, sealed and incubated on a roller bed at 20 8C (Wheatley et al., 1989). Three replicates were removed and sampled immediately after sealing and subsequently after 0.5, 1, 2, 4, 8, 24 and 48 h. Headspace samples for CO2 determinations were taken by gas-tight syringes and 0.8 ml of the slurry was transferred to a sterile 2 ml plastic screw cap tube containing 0.1 g of diethyl pyrocarbonate (DEPC) treated glass beads (dia 1 mm). Tubes were snap frozen in liquid N2 and stored at 2 80 8C until nucleic acid extraction. 2.3. CO2 measurement The concentrations of CO2 in the headspace of the incubation bottles were measured by gas chromatography, using a Hewlett Packard 5890A g.c. fitted with a thermal conductivity detector; 1.8 m, 2 mm i.d., column packed with
Hayesep-Q; oven temperature 90 8C; carrier gas He, 20 ml min21. As the pH of the aqueous phase was , 6.5, the effective gas headspace was assumed to be that volume of the bottle not occupied by soil and liquid. 2.4. Nucleic acid extraction All glassware and water used for extraction was treated with 0.1% (v/v) DEPC to remove RNase activity. Samples were kept on ice during the whole procedure and all solutions used were ice-cold. Replicates were treated as separate samples. To each tube containing thawing soil samples, 500 ml of DEPC-treated phosphate buffer (0.1 M, pH 8.0) and 500 ml phenol (pH 4.5) were added. Nucleic acids were released by beating at 5000 rev min21 for 3 £ 20 s (tubes were kept on ice between pulses) and then centrifuging in a microfuge for 5 min at maximum speed. The aqueous phase was transferred to a clean 2 ml tube and 500 ml of phenol/ chloroform/isoamyl alcohol (25:24:1) was added. Following vortexing (30 s) and centrifugation (full speed 3 min) the aqueous phase was washed with 500 ml of chloroform/ isoamyl alcohol (24:1), vortexed and re-centrifugated. Then 200 ml of the aqueous phase was subjected to separate polyvinylpolypyrrolidone (PVPP, Sigma) and Sephadex G50 (Sigma) column purifications (modified from Cullen and Hirsch, 1998). PVPP powder was packed into MicroSpin chromatography columns (Bio-Rad) and columns were then stabilized three times with 250 ml of DEPC-treated water (centrifugation between water additions at 1300 g for 3 min) prior to sample application. Samples were eluted from the columns at 1300 g for 3 min. Subsamples (100 ml) of the product were further purified using freshly prepared Sephadex G50 -columns (Cullen and Hirsch, 1998). Nucleic acids were eluted from the columns at 100 g for 3 min. A 16 ml aliquot of purified nucleic acid from each sample was immediately digested by DNase (GibcoBRL, see manufacturer’s instructions) and subjected to reverse transription. 2.5. Reverse transcription (RT) of rRNA and PCR RNA (1 ml) was used as template for cDNA production using M-MLV reverse transcriptase (GibcoBRL) following the manufacturer’s instructions. Bacterial SSU rRNA was reverse-transcribed using a primer 1405R (50 -CGG GCG GTG TGT ACA AG) modified from the universal 16S rDNA primer 1401R for eubacteria (Nu¨bel et al., 1996). The FR1 primer was used to generate cDNA from fungal templates (Vainio and Hantula, 2000). A 100-fold dilution of cDNA and undiluted genomic DNA and RNA were used as a template for PCR using universal bacterial primers F968GC (Nu¨bel et al., 1996) þ R1405 or fungal FR1GC þ FF390 primers (Vainio and Hantula, 2000). Expand High Fidelity PCR System was used as a polymerase according to the manufacturer’s instructions (Roche Diagnostics GmbH). PCR conditions were as in Nu¨bel et al. (1996) and Vainio
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and Hantula (2000) with the exception of an annealing temperature of 60 8C for the 16S rDNA primers. PCR products were purified by phenol/chloroform (1:1) extraction and concentration normalised prior to DGGE analysis.
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variation between gels. Similarity matrices were then computed between sample profiles based on simple matching and the resultant matrices subjected to principal coordinate analysis (PCO) using Genstat (Payne et al., 2001). PCO scores were analysed by one-way ANOVA.
2.6. Denaturing gradient gel electrophoresis The 18S rDNA primer pair and the unmodified version of the 16S rDNA primers have been shown to give reproducible DGGE or temperature gradient gel electrophoresis (TGGE) fingerprints that reflect the fungal and bacterial sequence diversity of the microbial community (Felske and Akkermans, 1998; Pennanen et al., 2001). PCR products were separated by DGGE using 8% acrylamide/bisacrylamide gels according to the manufacturer’s instructions (DCode Universal Mutation Detection System, Bio-Rad). Equal amounts of the PCR products (visual estimation from the agarose gels) were loaded. The denaturing gradient was 46 –60% for both the bacterial and fungal amplification products. Samples were run at 60 8C, 65 V for 16 h. Bands were visualized by silver staining (McCaig et al., 2001). 2.7. Data analysis Digital images of the gels were made and banding profiles normalized to marker lanes (composed of reference environmental samples) using Gel Compar software (Applied Maths. Kortrijk, Belgium). Bands were then automatically identified by thresholding, and their relative positions logged by image analysis. The resultant banding patterns were scored as binary presence or absence matrices. Comparisons were confined to lanes within gels: Inter-gel comparisons were not deemed appropriate due to significant
3. Results 3.1. Substrate induced respiration C mineralization rates showed characteristic patterns for the water controls and both the different substrates and amendment concentrations (Fig. 1). CO2 evolution increased in all samples over the first hour, but was significantly greater in samples amended with C than the unamended controls. Thereafter the respiration rate of the low substrate amendments (20 mg C g21 dw) decreased to the rates of the water controls although they remained significantly above the control rates for 8 h. High concentrations of both glucose and L -asparagine concentrations (2000 mg C g21 dw) supported significantly ðP , 0:001Þ higher respiration rates than the lower concentrations from 4 –48 h after amendment. In the high L -asparagine treatment the respiration rate started to increase again after 8 h. 3.2. Microbial community structure PCR of the RT controls, i.e. samples subjected to DNase digestion but not to RT, produced no amplification products indicating that no contaminating DNA was present in the 16S or 18S cDNA template (data not shown). All bacterial and fungal fingerprints contained a large number of
Fig. 1. Respiration rates from triplicate samples following glucose (a) and L -asparagine (b) amendments, over the 48 h incubation. Broken line indicates control treatment, filled circles indicate high concentrations of substrate amendment (2000 mg C g21 dw) and open circles low concentrations (20 mg C g21 dw). Error bars show standard deviation.
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sequence types (bands), although fungal analysis revealed fewer types compared to bacterial. Bacterial DGGE profiles derived from corresponding rDNA and rRNA templates were different although the prominent bands were common. There seemed to be higher number of metabolically-active bacterial sequence types compared to the number of dominant sequence types seen in the rDNA profiles (Fig. 2a,b). In contrast, the fungal rDNA and rRNA profiles were very similar (Fig. 2c,d). The very high number of bands in the bacterial gels may have reduced the detection resolution of the rRNA-DGGE-profiles and made isolation of the individual bands for sequencing impossible. However, the aim of this study was to show the response of the bacterial and fungal communities to the substrates additions without any deliberate selection by specific primers in the PCR amplifications. All control treatment DGGE profiles, in which only water had been added to the soil remained relatively unchanged during the incubation. Thus PCO analyses demonstrated
hardly any alterations in community profiles (data not shown). Low concentrations of substrate amendments caused only few alterations in both the rRNA or rDNAbased bacterial and fungal (Figs. 2c,d and 3 as an example) banding patterns during the first 48 h of the incubation. The first two components of the PCO analyses of the bacterial and fungal banding patterns accounted for 21 –37% of the variation, but in most cases variations were small and not related to the temporal changes. For example the L -asparagine amended bacterial community showed some separation along PCO1 after 24 and 48 h (Fig. 3a) but this was caused by the appearance of only two sequence types. High concentrations of substrate addition resulted in clear temporal patterns in banding profiles from bacteria (Figs. 4 and 5). The effect was particularly apparent in high asparagine treatments (Fig. 5) where separation of the late time points is pronounced. The trends were due to several changes in band profiles with new dominant bands
Fig. 2. DGGE of (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry and (c) 18S rDNA and (d) 18S rRNA fragments from L -asparagine (20 mg C g21 dw) amended slurry. Numbers on the top of the lines refer to the hours of incubation before sampling.
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Fig. 4. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from glucose (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3. Fig. 3. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (20 mg C g21 dw) amended slurry. Numbers within symbols refer to the hours of incubation before sampling. Points show means ðn ¼ 3Þ; bars show standard errors.
appearing in the bacterial profiles, weak bands becoming prominent and loss of bands (see Figs. 2a,b). PCO plots showed that the changes in the active bacterial community were apparent earlier in the time course with separation between 4 and 8 h time points (Figs. 4b and 5b) whereas the rDNA-based fingerprint did not show any clear incubation time-related separation until after 24 h (Figs. 4a and 5a). The fungal community showed less of a response to the high concentrations of substrate addition than the bacteria (Fig. 6 as an example). High substrate amendments induced a change in only one sequence type in the fungal profiles, which shows as a separation of the late sampling occasions in Fig. 6a.
4. Discussion In general the availability of C, nutrients, O2 and water governs the activity of soil microbes (West and Sparling,
1986). In this experiment, O2 and water limitation were removed by using the roller bed incubation method (Ritz and Wheatley, 1989). In addition to the quality of the C and other nutrients, microbial community function can also be affected by the quantity of the substrate (Griffiths et al., 1999). Thus, two application concentrations of glucose and L -asparagine were selected to represent C concentrations giving maximum heterotrophic activity, i.e. suitable for the SIR measurement and C concentrations equivalent to those released in root exudates and difference in resource quality (Wheatley et al., 2001). CO2 evolution increased in all samples over the first hour, this was likely to be due to water amendment and physical disturbance during incubation on the roller bed. Immediately after addition the low amendment concentrations of glucose and L -asparagine applications produced similar respiration responses to the high amendment concentrations (Fig. 1) but started to slowly decrease after 1 h, probably due to substrate limitation. Addition of substrates at 2000 mg C g21 dw resulted in the highest respiration rates, and these remained high throughout the incubation. The relatively small difference in immediate respiration rates between the different substrate loads may reflect the wide range of optimal glucose concentrations for
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Fig. 5. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3.
Fig. 6. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on fungal-specific primers: (a) 18S rDNA and (b) 18S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3.
SIR (West and Sparling, 1986). On the other hand, Marstorp and Witter (1999) reported that even very low rates of glucose application resulted in microbial uptake of C without any increase in the amount of dsDNA. They concluded this from the appearance of a chloroform labile pool of C without any increase in the amount of dsDNA after the addition of a low concentration of glucose. Our observation, which showed few qualitative changes in the 16S or 18S rDNA fingerprints with low concentrations of substrate amendment during the 48 h incubation, corresponds well with their results. In addition, all the low rates of substrate amendments showed that a large number of metabolically-active bacterial and fungal sequence types were present at the start of the incubation. This is logical because in addition to the conditioning at þ 4 8C after sieving, the soil was stabilized for 16 h before the start of the SIR experiment. Thus the microbial community should have been relatively active and able to respond quickly to water amendment and to physical disturbance on the roller bed. The prominent bacterial sequence types were common to both the 16S rRNA and rDNA fingerprints but generally
the bacterial rDNA fingerprints showed a lower number of bands than the corresponding rRNA profiles. This may indicate that metabolic functions are carried out by microorganisms which are not dominant in the community at a DNA level. In contrast, 18S rRNA and rDNA templates gave a relatively consistent view of the diversity of the fungal community in the arable soil, suggesting that the dominant fungal sequence types were also metabolically active. There seemed to be fewer fungal ribosomal sequence types than bacterial. Borneman et al. (1996) reported that less than 1% of the sequences found in agricultural soil were of fungal origin. Using PLFA-analysis, Frostega˚rd and Ba˚ a˚ th (1996) reported fungal/bacterial ratios about five times lower in arable compared to forest soil. It has been reported that there is a low diversity of arbuscular mycorrhizal (AM) fungi in arable soils (Helgason et al., 1998; Daniell et al., 2001), which may at least partly result from the frequent physical disturbances associated with tillage. The high L -asparagine amendments resulted in both increased respiration rates and clear changes in both the 16S
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rRNA and rDNA-community fingerprints at the later sampling occasions. This implies that the final peak in CO2 production was a result of bacterial growth of specific types capable of either efficient L -asparagine utilization, or by the removal of N limitation. Similarly, the exponential increase in respiration rate peaking after 25 h incubation coincided with the exponential increase in dsDNA concentration (Marstorp and Witter, 1999). Tsai et al. (1997) observed a rapid increase in respiration after 12 –30 h, followed later by an increase in both biomass C and ATP. Anderson and Domsch (1978) showed that SIR estimates after 1 – 3 h best represented the size of the original microbial community. Our results support this as neither selective cell growth nor activity limited to certain microbial sequence types occurred during the first 8 h of incubation. In addition, SIR estimates assume that all components of the microbial biomass respond equally to the substrate, and that the initial substrate-induced maximal respiratory response is correlated to the actual size of the microbial population (Anderson and Domsch, 1978). The large diversity of metabolically-active sequence types observed in our bacterial and fungal rRNA fingerprints suggests that a wide range of organisms responded to the amendments and were involved in the early increase in respiration rate. SIR has been shown to correlate more or less well with other microbial biomass techniques based on different approaches, such as fumigation extraction and ATP measurements (West and Sparling, 1986; Wardle and Parkinson, 1990b; Martens, 1987; Hassink, 1993; Ba˚a˚th and Arnebrant, 1994; Wardle and Ghani, 1995). As emphasized in many of these studies, SIR is assumed to be a measure of all the active microbial biomass. This study using independent molecular methods supports this assumption.
Acknowledgements The work was financially supported by the Academy of Finland (T.P.). SCRI receives grant-in-aid form the Scottish Executive Environment and Rural Affairs Department.
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Community-level responses of metabolically-active soil microorganisms to the quantity and quality of substrate inputs T. Pennanen*, S. Caul, T.J. Daniell, B.S. Griffiths, K. Ritz1, R.E. Wheatley Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received 12 December 2002; received in revised form 8 October 2003; accepted 21 January 2004
Abstract The community fingerprints of both the prevalent and the metabolically active microbial community were related to a quantitative estimation of microbial biomass in an arable soil, revealed by substrate-induced-respiration (SIR). Two concentrations of glucose or L -asparagine, representing those used in the SIR measurement or equivalent to those released in root exudates, were studied. Respiration rates and changes in community structure fingerprints were followed for 48 h. Bacterial and fungal community fingerprints were obtained using both reverse transcribed 16S and 18S ribosomal RNA (rRNA) regions and the corresponding rDNA as a template in PCR. Samples were then analysed by denaturing gradient gel electrophoresis (DGGE). Low concentrations of substrate amendments resulted in minor changes in rRNA or rDNA-based bacterial and fungal banding patterns during the whole 48 h incubation. High concentrations of substrates, especially L -asparagine, increased respiration rates and induced significant changes in both 16S rRNA and rDNA-community fingerprints. The prominent rRNA and rDNA bacterial community sequence types were common to all treatments, but in general the bacterial rDNA fingerprints had fewer bands than the corresponding rRNA profiles that assess the active fraction of the community. In contrast, there was little difference between fungal 18S rRNA and rDNA patterns. The number of fungal ribosomal sequence types in DGGE fingerprints was lower than the number of bacterial types. This study indicated that there was a rapid respiration response by the whole microbial community during SIR estimates in soil, but that community structure did not change during the conventional incubation period. In an extended (8– 48 h) incubation with high amounts of L -asparagine increased respiration was associated with growth of the microbial community. q 2004 Elsevier Ltd. All rights reserved. Keywords: Substrate-induced respiration; Ribosomal RNA; Soil microbial community; Denaturing gradient gel electrophoresis
1. Introduction The substrate-induced respiration (SIR) method (Anderson and Domsch, 1978) is commonly used to estimate soil microbial biomass. In the SIR method it is assumed that the majority of the soil microbes respond rapidly to glucose amendment and that the respiration response reflects the total microbial biomass. However, it is not directly known which components of the soil microbial community are the primary contributors to the observed respiratory flush. Although approaches that study the structure of * Corresponding author. Present address: Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FIN-01301 Vantaa, Finland. Tel.: þ358-10-211-2404; fax: þ 358-10-211-2204. E-mail address: [email protected] (T. Pennanen). 1 Present address: National Soil Resources Institute, Cranfield University, Bedfordshire MK45 4DT, UK. 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.01.014
the communities such as phospholipid fatty acid (Zelles, 1999; Pennanen, 2001) and DNA-based fingerprints (van Elsas et al., 1998; Tiedje et al., 1999) have the potential to characterize soil microbial communities, the structure of communities is not necessarily reflected in their biological activity. Most soil microbes are thought to be inactive (Olsen and Bakken, 1987) and DNA is also known to persist in dead cells and as extracellular DNA in soil (Ogram et al., 1987). However, as the cellular content of ribosomal RNA (rRNA) is directly correlated to cellular activity and growth rate (Rosset et al., 1966; Wagner, 1994; Manefield et al., 2002), an examination of rRNA should provide a more representative indication of which members of the microbial community are active. Our aim was to assess the metabolically-active fraction of the prevalent microbial community that responded to substrate amendments. We added substrates of varying
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quality and quantity to soil to invoke various microbial responses. We were especially interested in investigating relationships between respiration flux in SIR assays and microbial community profiles derived from both the prevalent and the active members of the community. A method for the rapid simultaneous extraction of both RNA and DNA from soil then the reverse transcription of both the 16S and 18S ribosomal RNA (rRNA) regions was developed. PCR amplifications were then performed for both partial 16S and 18S rRNA regions. Thereafter we related the rRNA community fingerprint, at both the RNA and DNA level, using denaturing gradient gel electrophoresis (DGGE) to general microbial respiration activity.
2. Materials and methods 2.1. Soil The soil used was from the Carbrook association, an imperfectly drained soil with a clay loam texture, a pH (H2O) of 6.2, total C content of 2.33% (w:w), a total N content of 0.22% (w:w) and water-holding-capacity (WHC) of 0.47 g water g21 dry soil. Soil samples taken from a field in cultivation on a wheat– barley –potatoes intensive rotation were bulked and sieved to , 7 mm and then to , 2 mm. The sieved soil was then stabilized for a week at þ 4 8C. 2.2. Experimental design and sample preparation Soil samples (3 g fw) were weighed into 30 ml glass bottles and stabilized at 18– 20 8C over night (Anderson and Domsch, 1978). Soil moisture content was adjusted to 2 £ WHC by adding water only (control treatment), or in addition to glucose or L -asparagine solutions as substrates (Ritz and Wheatley, 1989). Solutions of glucose and L -asparagine were added to give final concentrations of 20 mg or 2000 mg C g21 dw. For each of these five treatments, 24 replicate bottles were prepared, sealed and incubated on a roller bed at 20 8C (Wheatley et al., 1989). Three replicates were removed and sampled immediately after sealing and subsequently after 0.5, 1, 2, 4, 8, 24 and 48 h. Headspace samples for CO2 determinations were taken by gas-tight syringes and 0.8 ml of the slurry was transferred to a sterile 2 ml plastic screw cap tube containing 0.1 g of diethyl pyrocarbonate (DEPC) treated glass beads (dia 1 mm). Tubes were snap frozen in liquid N2 and stored at 2 80 8C until nucleic acid extraction. 2.3. CO2 measurement The concentrations of CO2 in the headspace of the incubation bottles were measured by gas chromatography, using a Hewlett Packard 5890A g.c. fitted with a thermal conductivity detector; 1.8 m, 2 mm i.d., column packed with
Hayesep-Q; oven temperature 90 8C; carrier gas He, 20 ml min21. As the pH of the aqueous phase was , 6.5, the effective gas headspace was assumed to be that volume of the bottle not occupied by soil and liquid. 2.4. Nucleic acid extraction All glassware and water used for extraction was treated with 0.1% (v/v) DEPC to remove RNase activity. Samples were kept on ice during the whole procedure and all solutions used were ice-cold. Replicates were treated as separate samples. To each tube containing thawing soil samples, 500 ml of DEPC-treated phosphate buffer (0.1 M, pH 8.0) and 500 ml phenol (pH 4.5) were added. Nucleic acids were released by beating at 5000 rev min21 for 3 £ 20 s (tubes were kept on ice between pulses) and then centrifuging in a microfuge for 5 min at maximum speed. The aqueous phase was transferred to a clean 2 ml tube and 500 ml of phenol/ chloroform/isoamyl alcohol (25:24:1) was added. Following vortexing (30 s) and centrifugation (full speed 3 min) the aqueous phase was washed with 500 ml of chloroform/ isoamyl alcohol (24:1), vortexed and re-centrifugated. Then 200 ml of the aqueous phase was subjected to separate polyvinylpolypyrrolidone (PVPP, Sigma) and Sephadex G50 (Sigma) column purifications (modified from Cullen and Hirsch, 1998). PVPP powder was packed into MicroSpin chromatography columns (Bio-Rad) and columns were then stabilized three times with 250 ml of DEPC-treated water (centrifugation between water additions at 1300 g for 3 min) prior to sample application. Samples were eluted from the columns at 1300 g for 3 min. Subsamples (100 ml) of the product were further purified using freshly prepared Sephadex G50 -columns (Cullen and Hirsch, 1998). Nucleic acids were eluted from the columns at 100 g for 3 min. A 16 ml aliquot of purified nucleic acid from each sample was immediately digested by DNase (GibcoBRL, see manufacturer’s instructions) and subjected to reverse transription. 2.5. Reverse transcription (RT) of rRNA and PCR RNA (1 ml) was used as template for cDNA production using M-MLV reverse transcriptase (GibcoBRL) following the manufacturer’s instructions. Bacterial SSU rRNA was reverse-transcribed using a primer 1405R (50 -CGG GCG GTG TGT ACA AG) modified from the universal 16S rDNA primer 1401R for eubacteria (Nu¨bel et al., 1996). The FR1 primer was used to generate cDNA from fungal templates (Vainio and Hantula, 2000). A 100-fold dilution of cDNA and undiluted genomic DNA and RNA were used as a template for PCR using universal bacterial primers F968GC (Nu¨bel et al., 1996) þ R1405 or fungal FR1GC þ FF390 primers (Vainio and Hantula, 2000). Expand High Fidelity PCR System was used as a polymerase according to the manufacturer’s instructions (Roche Diagnostics GmbH). PCR conditions were as in Nu¨bel et al. (1996) and Vainio
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and Hantula (2000) with the exception of an annealing temperature of 60 8C for the 16S rDNA primers. PCR products were purified by phenol/chloroform (1:1) extraction and concentration normalised prior to DGGE analysis.
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variation between gels. Similarity matrices were then computed between sample profiles based on simple matching and the resultant matrices subjected to principal coordinate analysis (PCO) using Genstat (Payne et al., 2001). PCO scores were analysed by one-way ANOVA.
2.6. Denaturing gradient gel electrophoresis The 18S rDNA primer pair and the unmodified version of the 16S rDNA primers have been shown to give reproducible DGGE or temperature gradient gel electrophoresis (TGGE) fingerprints that reflect the fungal and bacterial sequence diversity of the microbial community (Felske and Akkermans, 1998; Pennanen et al., 2001). PCR products were separated by DGGE using 8% acrylamide/bisacrylamide gels according to the manufacturer’s instructions (DCode Universal Mutation Detection System, Bio-Rad). Equal amounts of the PCR products (visual estimation from the agarose gels) were loaded. The denaturing gradient was 46 –60% for both the bacterial and fungal amplification products. Samples were run at 60 8C, 65 V for 16 h. Bands were visualized by silver staining (McCaig et al., 2001). 2.7. Data analysis Digital images of the gels were made and banding profiles normalized to marker lanes (composed of reference environmental samples) using Gel Compar software (Applied Maths. Kortrijk, Belgium). Bands were then automatically identified by thresholding, and their relative positions logged by image analysis. The resultant banding patterns were scored as binary presence or absence matrices. Comparisons were confined to lanes within gels: Inter-gel comparisons were not deemed appropriate due to significant
3. Results 3.1. Substrate induced respiration C mineralization rates showed characteristic patterns for the water controls and both the different substrates and amendment concentrations (Fig. 1). CO2 evolution increased in all samples over the first hour, but was significantly greater in samples amended with C than the unamended controls. Thereafter the respiration rate of the low substrate amendments (20 mg C g21 dw) decreased to the rates of the water controls although they remained significantly above the control rates for 8 h. High concentrations of both glucose and L -asparagine concentrations (2000 mg C g21 dw) supported significantly ðP , 0:001Þ higher respiration rates than the lower concentrations from 4 –48 h after amendment. In the high L -asparagine treatment the respiration rate started to increase again after 8 h. 3.2. Microbial community structure PCR of the RT controls, i.e. samples subjected to DNase digestion but not to RT, produced no amplification products indicating that no contaminating DNA was present in the 16S or 18S cDNA template (data not shown). All bacterial and fungal fingerprints contained a large number of
Fig. 1. Respiration rates from triplicate samples following glucose (a) and L -asparagine (b) amendments, over the 48 h incubation. Broken line indicates control treatment, filled circles indicate high concentrations of substrate amendment (2000 mg C g21 dw) and open circles low concentrations (20 mg C g21 dw). Error bars show standard deviation.
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sequence types (bands), although fungal analysis revealed fewer types compared to bacterial. Bacterial DGGE profiles derived from corresponding rDNA and rRNA templates were different although the prominent bands were common. There seemed to be higher number of metabolically-active bacterial sequence types compared to the number of dominant sequence types seen in the rDNA profiles (Fig. 2a,b). In contrast, the fungal rDNA and rRNA profiles were very similar (Fig. 2c,d). The very high number of bands in the bacterial gels may have reduced the detection resolution of the rRNA-DGGE-profiles and made isolation of the individual bands for sequencing impossible. However, the aim of this study was to show the response of the bacterial and fungal communities to the substrates additions without any deliberate selection by specific primers in the PCR amplifications. All control treatment DGGE profiles, in which only water had been added to the soil remained relatively unchanged during the incubation. Thus PCO analyses demonstrated
hardly any alterations in community profiles (data not shown). Low concentrations of substrate amendments caused only few alterations in both the rRNA or rDNAbased bacterial and fungal (Figs. 2c,d and 3 as an example) banding patterns during the first 48 h of the incubation. The first two components of the PCO analyses of the bacterial and fungal banding patterns accounted for 21 –37% of the variation, but in most cases variations were small and not related to the temporal changes. For example the L -asparagine amended bacterial community showed some separation along PCO1 after 24 and 48 h (Fig. 3a) but this was caused by the appearance of only two sequence types. High concentrations of substrate addition resulted in clear temporal patterns in banding profiles from bacteria (Figs. 4 and 5). The effect was particularly apparent in high asparagine treatments (Fig. 5) where separation of the late time points is pronounced. The trends were due to several changes in band profiles with new dominant bands
Fig. 2. DGGE of (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry and (c) 18S rDNA and (d) 18S rRNA fragments from L -asparagine (20 mg C g21 dw) amended slurry. Numbers on the top of the lines refer to the hours of incubation before sampling.
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Fig. 4. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from glucose (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3. Fig. 3. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (20 mg C g21 dw) amended slurry. Numbers within symbols refer to the hours of incubation before sampling. Points show means ðn ¼ 3Þ; bars show standard errors.
appearing in the bacterial profiles, weak bands becoming prominent and loss of bands (see Figs. 2a,b). PCO plots showed that the changes in the active bacterial community were apparent earlier in the time course with separation between 4 and 8 h time points (Figs. 4b and 5b) whereas the rDNA-based fingerprint did not show any clear incubation time-related separation until after 24 h (Figs. 4a and 5a). The fungal community showed less of a response to the high concentrations of substrate addition than the bacteria (Fig. 6 as an example). High substrate amendments induced a change in only one sequence type in the fungal profiles, which shows as a separation of the late sampling occasions in Fig. 6a.
4. Discussion In general the availability of C, nutrients, O2 and water governs the activity of soil microbes (West and Sparling,
1986). In this experiment, O2 and water limitation were removed by using the roller bed incubation method (Ritz and Wheatley, 1989). In addition to the quality of the C and other nutrients, microbial community function can also be affected by the quantity of the substrate (Griffiths et al., 1999). Thus, two application concentrations of glucose and L -asparagine were selected to represent C concentrations giving maximum heterotrophic activity, i.e. suitable for the SIR measurement and C concentrations equivalent to those released in root exudates and difference in resource quality (Wheatley et al., 2001). CO2 evolution increased in all samples over the first hour, this was likely to be due to water amendment and physical disturbance during incubation on the roller bed. Immediately after addition the low amendment concentrations of glucose and L -asparagine applications produced similar respiration responses to the high amendment concentrations (Fig. 1) but started to slowly decrease after 1 h, probably due to substrate limitation. Addition of substrates at 2000 mg C g21 dw resulted in the highest respiration rates, and these remained high throughout the incubation. The relatively small difference in immediate respiration rates between the different substrate loads may reflect the wide range of optimal glucose concentrations for
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Fig. 5. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on eubacterial-specific primers: (a) 16S rDNA and (b) 16S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3.
Fig. 6. Principal co-ordinate scores of DGGE profiles derived from PCR reactions of soil community nucleic acids based on fungal-specific primers: (a) 18S rDNA and (b) 18S rRNA fragments from L -asparagine (2000 mg C g21 dw) amended slurry. Symbols and error bars as in Fig. 3.
SIR (West and Sparling, 1986). On the other hand, Marstorp and Witter (1999) reported that even very low rates of glucose application resulted in microbial uptake of C without any increase in the amount of dsDNA. They concluded this from the appearance of a chloroform labile pool of C without any increase in the amount of dsDNA after the addition of a low concentration of glucose. Our observation, which showed few qualitative changes in the 16S or 18S rDNA fingerprints with low concentrations of substrate amendment during the 48 h incubation, corresponds well with their results. In addition, all the low rates of substrate amendments showed that a large number of metabolically-active bacterial and fungal sequence types were present at the start of the incubation. This is logical because in addition to the conditioning at þ 4 8C after sieving, the soil was stabilized for 16 h before the start of the SIR experiment. Thus the microbial community should have been relatively active and able to respond quickly to water amendment and to physical disturbance on the roller bed. The prominent bacterial sequence types were common to both the 16S rRNA and rDNA fingerprints but generally
the bacterial rDNA fingerprints showed a lower number of bands than the corresponding rRNA profiles. This may indicate that metabolic functions are carried out by microorganisms which are not dominant in the community at a DNA level. In contrast, 18S rRNA and rDNA templates gave a relatively consistent view of the diversity of the fungal community in the arable soil, suggesting that the dominant fungal sequence types were also metabolically active. There seemed to be fewer fungal ribosomal sequence types than bacterial. Borneman et al. (1996) reported that less than 1% of the sequences found in agricultural soil were of fungal origin. Using PLFA-analysis, Frostega˚rd and Ba˚ a˚ th (1996) reported fungal/bacterial ratios about five times lower in arable compared to forest soil. It has been reported that there is a low diversity of arbuscular mycorrhizal (AM) fungi in arable soils (Helgason et al., 1998; Daniell et al., 2001), which may at least partly result from the frequent physical disturbances associated with tillage. The high L -asparagine amendments resulted in both increased respiration rates and clear changes in both the 16S
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rRNA and rDNA-community fingerprints at the later sampling occasions. This implies that the final peak in CO2 production was a result of bacterial growth of specific types capable of either efficient L -asparagine utilization, or by the removal of N limitation. Similarly, the exponential increase in respiration rate peaking after 25 h incubation coincided with the exponential increase in dsDNA concentration (Marstorp and Witter, 1999). Tsai et al. (1997) observed a rapid increase in respiration after 12 –30 h, followed later by an increase in both biomass C and ATP. Anderson and Domsch (1978) showed that SIR estimates after 1 – 3 h best represented the size of the original microbial community. Our results support this as neither selective cell growth nor activity limited to certain microbial sequence types occurred during the first 8 h of incubation. In addition, SIR estimates assume that all components of the microbial biomass respond equally to the substrate, and that the initial substrate-induced maximal respiratory response is correlated to the actual size of the microbial population (Anderson and Domsch, 1978). The large diversity of metabolically-active sequence types observed in our bacterial and fungal rRNA fingerprints suggests that a wide range of organisms responded to the amendments and were involved in the early increase in respiration rate. SIR has been shown to correlate more or less well with other microbial biomass techniques based on different approaches, such as fumigation extraction and ATP measurements (West and Sparling, 1986; Wardle and Parkinson, 1990b; Martens, 1987; Hassink, 1993; Ba˚a˚th and Arnebrant, 1994; Wardle and Ghani, 1995). As emphasized in many of these studies, SIR is assumed to be a measure of all the active microbial biomass. This study using independent molecular methods supports this assumption.
Acknowledgements The work was financially supported by the Academy of Finland (T.P.). SCRI receives grant-in-aid form the Scottish Executive Environment and Rural Affairs Department.
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