School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, UK1
Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK2
Author for correspondence: Alan J. McCarthy. Tel: +44 151 794 4413. Fax: +44 151 794 4401. e-mail: aj55m{at}liverpool.ac.uk
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ABSTRACT |
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Keywords: sulfate-reducing bacteria, 16S rDNA, landfill, PCR primers, oligonucleotide probes
Abbreviations: DIG, digoxigenin; RDP, ribosomal database project; SRB, sulfate-reducing bacteria
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INTRODUCTION |
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Landfill sites are essentially bioreactors in which anaerobic bacterial communities mediate the mineralization and stabilization of organic matter (Barlaz, 1997 ). They have long been overlooked as important habitats for SRB due to the fact that methanogenesis predominates as the key terminal process of carbon mineralization in the absence of significant concentrations of sulfate. Our knowledge of the occurrence and distribution of SRB in landfill is therefore extremely limited. The SRB are a diverse group of anaerobic bacteria that have the ability to use sulfate as a terminal electron acceptor in the consumption of organic matter, with the concomitant production of H2S. They are ubiquitous in the environment and have pivotal roles in the biogeochemical cycling of carbon and sulfur. Sulfate reduction could be responsible for up to 50% of organic matter degradation in high-sulfate environments such as estuarine and marine sediments (Jorgensen, 1982
); however, active sulfate reduction has also been reported in low-sulfate environments such as soils and freshwater sediments (Postgate, 1984
; Jones & Simon, 1984
; Bak & Pfennig, 1991a
, b
). In landfill sites, the breakdown of waste material ultimately to methane is a complex process involving a series of microbially driven transformations that harness the co-ordinated activity of several trophic groups of bacteria. While the key terminal process is methanogenesis, SRB can compete with methanogenic bacteria for available electron donors such as acetate and H2, and have the potential to inhibit the methanogenic decomposition of waste organic matter, resulting in the increased production of H2S and the phenomenon of souring (Gurijala & Suflita, 1993
; Harvey et al., 1997
). Conventional wisdom suggests that the low availability of sulfate outside the marine environment will limit sulfate reduction and therefore SRB populations, but this may not be true of landfill sites. Exogenous sources of sulfate, e.g. gypsum from construction and demolition debris, have been thought to be responsible for sulfate levels as high as 80 mmol per kg dry weight waste material in particular landfill sites (Suflita et al., 1992
; Gurijala & Suflita, 1993
). Cellulosic material can account for over 40% of the volume of a landfill site and act as a reservoir of sulfate that originates from other waste fractions (Suflita et al., 1992
; Gurijala & Suflita, 1993
). Consequently sulfate may be present in landfills in significant amounts.
Inhibition of methanogenesis by sulfate has been observed in a range of environments (Oremland & Polcin, 1982 ; Beeman & Suflita, 1987
; Raskin et al., 1996
) and so could clearly occur in landfill (Gurijala & Suflita, 1993
). The SRB are therefore one of a number of important functional bacterial groups whose structure and activity in landfill sites needs to be directly addressed. Data on their occurrence and distribution should ultimately enable the development of detection protocols that can be used to monitor the microbiology of landfill sites in order to provide information for site management. For example, molecular biological methods could give SRB population profiles that provide an early warning of interference with methanogenesis by sulfate reduction.
Phylogenetic analysis based on 16S rRNA sequence comparisons has classified the major SRB genera into a number of distinct lineages (Devereux et al., 1989 ) and this was used as the starting point for the study reported here. Oligonucleotide probes designed by Devereux et al. (1992)
target a number of these groups; however, the suite of probes currently available in the literature does not encompass all of the main groups of SRB, nor have specific PCR amplification primers for SRB detection in environmental samples been described or applied. In this paper, we describe and evaluate combinations of PCR primers and oligonucleotide probes for the six major phylogenetic groups of SRB, and apply these to DNA extracted from samples of landfill leachate to provide baseline information on the occurrence and distribution of SRB taxa.
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METHODS |
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The leachate samples were processed immediately upon receipt. Each 1 litre sample was concentrated by centrifugation (27000 g, 40 min) and the pellet resuspended in 20 ml 0·1 M K2HPO4. Aliquots (1·5 ml) of this concentrated sample were centrifuged (22000 g, 5 min) and the pellets stored at -80 °C until required.
Nucleic acid extraction and purification.
Pellets of concentrated leachate stored at -80 °C were thawed on ice and resuspended in 200 µl sterile distilled H2O to give a final 375-fold concentration of the leachate solids. DNA was extracted and purified from this concentrated leachate using the FastDNA SPIN kit (Bio 101) and a Hybaid Ribolyser according to the manufacturers instructions. DNA was extracted from control strains by resuspending freeze-dried cultures in 200 µl sterile distilled H2O and applying the Bio 101 kit and Hybaid Ribolyser protocol described above. DNA recovery, purity and yield were evaluated by agarose gel electrophoresis as described below.
Design of 16S rDNA-targeted PCR primers and internal 16S rRNA-targeted oligonucleotide probes.
A phylogenetic tree showing the lineage of the six main groups of SRB was constructed from aligned 16S rRNA sequences obtained from the GenBank, EMBL and RDP (Maidak et al., 1997 ) databases using the neighbour-joining method of Jukes & Cantor (1969)
and produced by the TREEVIEW program (PHYLIP 3.4) (Felsenstein, 1993
). Bootstrap analysis consisting of 100 resamplings of the data was performed using SEQBOOT (PHYLIP 3.4) and a consensus phenogram was generated using CONSENSE (PHYLIP 3.4). 16S rDNA-targeted PCR primers and internal 16S rRNA-targeted oligonucleotide probes were designed from a collection of 60 SRB 16S rRNA sequences obtained from the databases. Escherichia coli and Bacillus subtilis 16S rRNA sequences were used as reference points for the alignment of the SRB sequences. Regions of variability between sequences representing each SRB group and the reference sequences were located by eye. Potential candidates for PCR primers and internal oligonucleotide probes were compared to the aligned SSU_rRNA database of the RDP using the CHECK_PROBE utility.
PCR amplification of 16S rDNA with SRB group-specific primers.
Direct PCR amplification with the group-specific PCR primers (Table 1) was attempted on the DNA extracted from each landfill site. Reactions were carried out as follows: 95 °C for 1 min, annealing for 1 min and 72 °C for 1 min for 30 cycles. Each reaction tube (100 µl) contained: 2 µl each primer (10 pmol µl-1), 2 µl dNTP (10 mM each), 85 µl distilled H2O, 10 µl 10xPCR buffer (HT Biotech), 0·2 µl 10% (w/v) BSA, 1 U SuperTaq polymerase (HT Biotech) and DNA template (approx. 100150 ng). The amplifications were carried out using a hot-start PCR protocol whereby each reaction, without Taq polymerase, was heated at 95 °C for 5 min to fully denature the DNA template. The tubes were then cooled to 80 °C and maintained at this temperature while the enzyme was added. Each reaction was then overlaid with mineral oil prior to cycling. In addition to the direct amplification of landfill DNA, nested amplification was also applied. DNA extracted from landfill leachate was first amplified with the eubacterial primers pA and pH' (Edwards et al., 1989
) at low stringency (annealing temperature 45 °C), then aliquots of these eubacterial PCR amplification products were diluted 100-fold into fresh reaction mixtures containing a pair of SRB group-specific primers.
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Oligonucleotide probing.
For the optimization of hybridization conditions, DNA extracted from each control strain was diluted in an equal volume of denaturing solution (1 M NaOH, 3 M NaCl) and transferred to positively charged nylon membrane (Boehringer Mannheim) using a dot-blot apparatus (Minifold, Schleicher and Schuell). DNA was then fixed to membranes by UV cross-linking. For the environmental samples, PCR amplification products were transferred and fixed to positively charged nylon membrane (Boehringer Mannheim) by Southern blotting and UV cross-linking. Membranes were first incubated in standard prehybridization solution [5xSSC, 0·1% (w/v) N-lauroyl sarcosine, 0·02% (w/v) SDS, 1% (w/v) blocking reagent [Boehringer Mannheim)] at the appropriate hybridization temperature for 1 h to prevent non-specific binding of the probe.
Oligonucleotides specific for each of the six main groups of SRB (Table 2) were 3'-end labelled with non-radioactive DIG-11-ddUTP (1 mM) using terminal transferase (Boehringer Mannheim) according to the manufacturers instructions. The concentrated labelled probes were diluted in prehybridization solution and membranes incubated overnight at a temperature appropriate to the melting temperature (Tm) of the probe used (Table 2
). After hybridization, two 15 min high-stringency washes were performed at the hybridization temperature. DIG-labelled DNA was then detected using the standard DIG luminescent detection procedure (Boehringer Mannheim) and membranes were exposed to X-ray film at room temperature.
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RESULTS |
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16S rDNA-targeted PCR primers were designed from this collection of 60 SRB 16S rRNA sequences and potential candidates for PCR primers were compared to the aligned SSU_rRNA database of the RDP using the CHECK_PROBE utility. Dr Mark Munson (University of Essex, Colchester, UK) kindly provided five potentially specific PCR primer sequences, one of which was derived from an oligonucleotide probe designed by Devereux et al. (1992) (see Table 1
). The results of this comprehensive cross-specificity check on the complete RDP database enabled the final selection of six 16S rDNA-targeted PCR primer pairs theoretically specific for each of the six main groups of SRB (Table 1
). In some cases, one or both of the primers were not completely specific, but no single non-target organism matched both primers and, in any case, Southern hybridization was always used for verification. The theoretical cross-specificity of the primers and probes with SRB as predicted by the RDP database is presented in Table 3
. The specificity of the primers was confirmed by screening the current release of the RDP database.
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Desulfotomaculum-like (group 1) amplification prod ucts were obtained from three of the seven landfill sites (P, S and C) and shown to contain the target 16S rDNA by hybridization against probe DFM228 (Fig. 4 and not shown).
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These results are summarized in Table 4(a).
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Desulfotomaculum-like (group 1) amplification products were obtained from all seven landfill sites, confirmed by hybridization against probe DFM228 (data not shown).
Desulfobulbus-like (group 2) amplification products were obtained from four landfill sites (P, B, R and W) and these hybridized with probe DBB660 (Fig. 6 and not shown).
Desulfobacter-like (group 4) amplification products were obtained from four landfill sites (P, B, S and W) with hybridization against probe DSB623 (data not shown).
Desulfococcus Desulfonema Desulfosarcina- like (group 5) and Desulfovibrio-like (group 6) amplification products were obtained from six of the seven landfill sites (all except site S), confirmed by hybridization against probes DCC868 and DSV687 respectively (data not shown).
Desulfobacterium-like (group 3) amplification products were never obtained from any of the landfill site leachate samples using this nested PCR approach. Although the direct PCR amplification of 16S rDNA demonstrated that SRB were present in five of the seven landfill sites sampled, application of nested PCR was able to show that SRB 16S rDNA could be detected in all seven leachate samples.
These data are summarized in Table 4(b).
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DISCUSSION |
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Theoretical cross-specificity analysis of the primers and probes designed in this study indicated that primerprobe combinations would provide highly specific molecular tools for unequivocal detection of each of the six SRB groups in environmental samples. This was confirmed experimentally, providing further confidence in the data on SRB group detection in the landfill leachate samples. The data showed that populations of SRB were detectable in landfill leachate by PCR amplification and probing, and that their occurrence would appear to be widespread. SRB 16S rDNA was successfully amplified from five out of seven landfill sites using the direct PCR approach and from all seven sites sampled using nested PCR.
The results obtained using the direct PCR amplification approach suggest that there are one or two dominant groups of SRB in each of the landfill sites: Desulfotomaculum (group 1) in landfills S and C; Desulfotomaculum (group 1) and DesulfococcusDesulfonemaDesulfosarcina (group 5) in landfill P; Desulfobacter (group 4) in landfill B; DesulfococcusDesulfonemaDesulfosarcina (group 5) in landfill W. Only in two landfill sites (R and H) were no SRB detected using this direct PCR approach.
However, nested PCR amplification revealed the presence of other groups not detected by the direct PCR: Desulfotomaculum (group 1) in landfills B, R, H and W; Desulfobulbus (group 2) in landfills P, B, R and W; Desulfobacter (group 4) in landfills S and W; DesulfococcusDesulfonemaDesulfosarcina (group 5) in landfills R and H; Desulfovibrio (group 6) in all except landfill S.
It is presumed that SRB groups that can only be detected in landfill leachates when a second round of amplification is employed (nested PCR) are present in lower numbers than members of the dominant groups detectable by direct PCR. Therefore, the dual application of direct and nested PCR can permit a rapid estimate of the relative predominance of SRB groups in landfill leachate. However, this is only a qualitative estimation of relative numbers based on detection through one round of PCR (direct) compared to two rounds of PCR (nested) and bears no statistical significance.
It is possible that the requirement for nested PCR to detect members of group 2 (DBB) and group 6 (DSV-DMB) in any leachate sample could be a feature of the PCR efficiency of these specific primers, rather than reflection of a relatively small population size. However, PCR amplifications of DNA extracted from pure cultures using all six group-specific primer sets yielded approximately equivalent amounts of PCR product, i.e. no significant differences in performance of the primer pairs were noted. Desulfobacterium-like (group 3) amplification products were never obtained from any of the landfill sites by either direct or nested PCR; this would appear to correlate with the association of most of the known species of the genus Desulfobacterium with the marine environment (Postgate, 1984 ; Fauque, 1995
).
Nevertheless, the results obtained from the nested PCR (Table 4) suggest that there is a high level of SRB diversity in landfill, as a distribution of the other five main groups was observed. This correlates with investigations of SRB occurrence and distribution in other environments in which most of the main groups have been detected by oligonucleotide probing (Kane et al., 1993
; Ramsing et al., 1993
; Risatti et al., 1994
; Devereux et al. 1996a
, b
; Raskin et al., 1996
; Purdy et al., 1997
; Trimmer et al., 1997
; Rooney-Varga et al., 1997
; Manz et al., 1998
; Sahm et al., 1999
). This is, to our knowledge, the first investigation of SRB occurrence in landfill using molecular biological techniques, and the only study of SRB molecular ecology described to date in which DNA extracts have been amplified by specific PCR prior to confirmation by oligonucleotide hybridization.
This apparent diversity of SRB, at least at the generic/suprageneric level, in landfill sites is not unexpected. The extremely high and varied organic carbon load together with long retention times encourages large and active populations of fermentative micro-organisms, which in turn produce various volatile fatty acids that serve as substrates for SRB. The scale of landfill sites and their extreme heterogeneity would promote microbial diversity. Also, as leachate results from the percolation of water through the site, high diversity would be expected even though SRB distribution is undoubtedly non-uniform throughout the site. While it would be of interest to study SRB populations in solid landfill material, leachate is going to be the only practical sample material for routine analysis and SRB monitoring. Thus, the argument that SRB populations in leachate may be a poor representation of SRB population size and distribution in the landfill does not preclude their use as a practical source of useful information on the landfill site as a whole.
It is now well established that SRB and methanogens compete for fermentation products such as acetate and H2 and that, in the presence of non-limiting levels of sulfate, SRB generally outcompete methanogenic bacteria (Oremland & Polcin, 1982 ; Beeman & Suflita, 1987
; Raskin et al., 1996
), with sulfate reduction being the key process of carbon mineralization in these environments. However, in landfill it is usually methanogenic bacteria that dominate, with methanogenesis, not sulfate reduction, as the key terminal process of carbon mineralization. This suggests that SRB populations in landfill are limited by the availability of sulfate, thereby allowing methanogenesis to dominate. However, the detection of SRB in these landfill sites suggests that the potential for sulfate reduction and the possible inhibition of methane production is present (Suflita et al., 1992
; Gurijala & Suflita, 1993
). It is important to be able to monitor SRB populations in landfill sites because their proliferation can potentially affect site performance via the inhibition of methanogenesis.
This study provides 16S rRNA-based methods for detecting SRB in landfill and also provides the first insight into SRB diversity in landfill sites.
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ACKNOWLEDGEMENTS |
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Received 4 November 1999;
revised 20 March 2000;
accepted 27 March 2000.