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Keywords:

  • municipal solid waste;
  • nitrous oxide;
  • ammonia-oxidizing bacteria;
  • denitrifying bacteria;
  • amoA;
  • nosZ

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

High emissions of nitrous oxide (N2O) have recently been documented at municipal solid waste (MSW) landfills. However, the biodiversity of the bacterial populations involved remains unexplored. In this study, we investigated communities of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria associated with the leachates from three MSW disposal sites by examining the diversity of the ammonia monooxygenase structural gene amoA and the nitrous oxide reductase gene nosZ, respectively. Cloning and phylogenetic analysis of the functional genes revealed novel and similar groups of prokaryotes involved in nitrogen cycling in the leachates with different chemical compositions. All amoA sequences recovered grouped within the Nitrosomonas europaea cluster in the Betaproteobacteria, with the vast majority showed only relatively moderate sequence similarities to known AOB but were exclusively most similar to environmental clones previously retrieved from wastewater treatment plants. All nosZ sequences retrieved did not cluster with any hitherto reported nosZ genes and were only remotely related to recognized denitrifiers from the Gammaproteobacteria and thus could not be affiliated. Significant overlap was found for the three denitrifying nosZ leachate communities. Our study suggests a significant selection of the novel N-cycling groups by the unique environment at these MSW disposal sites.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Nitrous oxide (N2O) is a potent greenhouse gas, accounting for 5–6% of observed greenhouse warming (Barnes & Owens, 1998). Its 100-year global warming potential is about 320 times as strong as that of carbon dioxide (CO2), and it has a lifetime of c. 120 years (IPCC, 1996). In addition, some N2O react with atomic oxygen in the stratosphere, and the resultant nitric oxide (NO) induces the destruction of the stratospheric ozone (Bliefert, 1994; Upstill-Goddard et al., 1999).

Human interference in the nitrogen cycle in the past century has intensified global N2O production during microbial nitrification and denitrification; in particular, the increasing inputs of anthropogenic nitrogen into agricultural systems dominate the emissions of anthropogenic N2O (Mosier et al., 1998; Barton & Atwater, 2002). The accumulation of nitrogen in wastewater and solid wastes may be a significant fate of much anthropogenic nitrogen, and N2O could be emitted during each process of waste management (Barton & Atwater, 2002).

Landfilling is still the most common form of disposal of municipal solid waste (MSW) worldwide, and nearby municipal dumps represent a large part of the waste disposal system in developing countries. In fact, a considerable portion of the MSW is disposed in open dumps or poorly managed landfills even in industrialized countries (Christensen, 1989). MSW-deposited sites are potentially high emitters of N2O as the environmental conditions in these systems are favorable for the microbial production of this important atmospheric trace gas, as the deposited organic waste is rich in nitrogen, and alternating aerobic and anaerobic sites are expected in the waste volume (Rinne et al., 2005). In spite of this, measurements of N2O emissions from MSW disposal sites are scare, and discrepancies in the results concerning N2O emissions exist among the few conducted studies (Borjesson & Svensson, 1997; Lee et al., 2002; McBain et al., 2005; Rinne et al., 2005), indicating that N2O productions may vary greatly among different systems. Although high emissions from several landfills have recently been reported (Lee et al., 2002; Rinne et al., 2005), the diversity and structure of the bacterial populations involved remain unexplored. In the past 8 years, functional gene-based molecular techniques have been proven to be effective for examining the genetic diversity of nitrifiers and denitrifiers in the environment (Rotthauwe et al., 1997; Scala & Kerkhof, 1998). Here, we report on two groups of microorganisms, ammonia oxidizers and denitrifiers, involved in nitrogen cycling associated with the effluent leachates from two MSW landfills operated under different criteria and one municipal open dump site. Two functional genes, the ammonia monooxygenase structural gene amoA and nitrous oxide reductase gene nosZ, served as molecular markers for ammonia-oxidizing bacteria (AOB) and denitrifying bacteria, respectively. High emission of N2O was documented previously for one of the two landfills under study (Lee et al., 2002), and comparative rRNA gene sequencing surveys have been conducted to identify members of the domain Bacteria inhabiting the leachates of the two landfills (Huang et al., 2004, 2005). Our study suggests that novel and similar ammonia-oxidizing and denitrifying bacterial communities harbored these important MSW systems.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

MSW disposal sites, sample collection and physico-chemical analysis

Two MSW landfills [Likeng Landfill (LK) and Gouzikeng Landfill (GZK)] and one municipal open dump (Guoqiaowo, GQW) were included in the present study. The LK and GZK landfills were operated under two different modes and were described in detail elsewhere (Huang et al., 2002, 2003). Very briefly, effluent leachate from the neighboring on-going site (accepting newly placed waste) was recirculated through the LK landfill body that was closed several years ago, while no leachate recirculation was used at the GZK landfill. The GQW open dump began operations in 1994 and major disposal activities lasted for c. 10 years. It has a total capacity of 350 000 m3, with a typical daily loading rate of 330 t of MSW. No daily soil covers and final topsoil cover were applied during its operation and after its closure. Triplicate samples of leachate oozing from the bottom of each site were collected in 1-L screw-cap bottles and immediately placed on ice for transport to the laboratory. Physico-chemical analyses were performed according to the Standard Methods (Greenberg et al., 1992) as described previously (Huang et al., 2002). Ammoniacal N was determined by the Indophenol-blue method (Allen, 1989), while NO3-N and NO2-N were determined with ion-selective probes (Models 9307BN and C9346BN, respectively, Orion, Beverly, MA).

DNA extraction and clone library construction

For effective comparison, we extracted total community DNA from the leachate samples using the same procedures, which comprise a lysozyme/proteinase K/sodium dodecyl sulfate (SDS) treatment, followed by standard phenol/chloroform extractions (Bond et al., 1995). DNA extracts from triplicate leachate samples were pooled. The same bulk DNA samples used in the previous studies of LK and GZK landfill prokaryotes (Huang et al., 2002, 2003, 2004, 2005) were used in the present study.

The amoA gene fragments (c. 490 bp) were amplified from bulk DNA samples using the PCR primers (amoA-1F and amoA-2R) and cycling parameters of Rotthauwe et al. (1997). nosZ gene fragments (c. 1110 bp) were amplified with the specific primer set (661F and 1773R) and PCR conditions previously described by Scala & Kerkhof (1999). Amplified products were concentrated and purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and the appropriately sized fragments were then excised from 1% agarose gels and eluted with a QIAquick gel extraction kit (Qiagen). The purified amplicons were ligated into the T-vector (Takara), and the ligation products were used to transform into Escherichia coli DH-5α competent cells.

Rrestriction fragment length polymorphism (RFLP), DNA sequencing and phylogenetic analysis

For RFLP analysis, nosZ inserts from randomly selected recombinant clones were reamplified by PCR with primers 661F and 1773R. The amplicons were subjected to enzymatic digestion with the endonuclease MspI (Takara), and the corresponding clones were grouped by manually comparing the cleavage patterns generated as described elsewhere (Huang et al., 2002). One to three representative clones from each unique RFLP type were selected for sequencing. Sequencing was performed on an ABI 377 sequencer using the Dye-Terminator Cycle Sequencing Ready Reaction FS Kit with M13/pUC universal sequencing primers P47 and P48 as described by the manufacturer (PE Applied Biosystems). Without RFLP screening, recombinant amoA clones were randomly selected for sequencing with the primer P47. The resulting sequences were compared with those available in GenBank using the blast network service to determine their approximate phylogenetic affiliation. Neighbor-joining phylogenetic trees were generated using the paup software package (version 4.0 b8). One thousand bootstraps were performed to assign confidence levels to the nodes in the trees. Sequences were checked for potential chimeric origin by comparing the distance trees generated with different regions of the sequence.

Nucleotide sequence accession numbers

The amoA and nosZ gene sequences obtained in this study have been deposited in the EMBL database under accession numbers AM293384AM293545.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results of the physico-chemical analyses are given in Table 1. Compared with the LK and GQW leachates, the GZK landfill leachates were relatively weak in terms of levels of total solids (TS), chemical oxygen demand (COD) and ammoniacal N (NH4+-N). In general, all effluent leachates from the three MSW sites were slightly alkaline, with pH values ranging from 8.2 to 8.6. The high NH4+-N contents are mainly due to the fact that after NH4+-N is formed by ammonification, it is not further degraded under the anaerobic conditions prevalent in these systems.

Table 1.   Physico-chemical characteristics of effluent leachates from the three MSW disposal sites* (mean concentrations in mg L−1)
 pHBicarb. Alkalinity (as CaCO3)Total solids (TS)Chemical oxygen demand (COD)NH4+-NNO3-NNO2-N
  • *

    Some characteristics of the LK and GZK leachates, including pH, alkalinity, TS and COD, have been published previously (Huang et al., 2002, 2003).

  • TOC, instead of COD, was determined for the GQW leachates.

  • ND, Not determined.

LK8.610128425445620511781.4
GQW8.411018387274512774.9ND
GZK8.245936601086751280.6

Evaluations of the natural diversity of amoA and nosZ genes were conducted for the three MSW leachates using a standard procedure involving selectively PCR amplification, cloning and comparative sequence analysis. amoA gene clone libraries were constructed only for the LK and GZK landfill leachates due to failure of PCR amplification of amoA fragments with the DNA extracts from the GQW open dump. The levels of similarity between different pairs of retrieved amoA genes ranged from 74.1% to 100%. Phylogenetic analysis revealed that all these landfill sequences retrieved belonged to the Nitrosomonas europaea-like cluster and comprised three major subgroups (Fig. 1). Approximately half of the clones (22 out of the 48 GZK clones and 16 out of the 27 LK clones) fell into Subgroup 1 and clustered with environmental sequences previously recovered from a nitrifying wastewater treatment plant (WWTP) (Purkhold et al., 2000), but they were distinct from any other reported amoA sequences and not closely related to any cultivated AOB (similarity <87%). Twenty-two GZK clones and 10 LK clones, accounting for 45% and 37% of their respective amoA libraries, formed a common branch (Subgroup 2) with S12, an amoA sequence previously retrieved from a nitrifying WWTP (Purkhold et al., 2000). Again, these clones were not closely related to any other environmentally retrieved amoA sequences and showed only low sequence similarities (<85%) to those from known AOB. Additionally, four GZK and one LK clones, along with their closely related counterparts (WWTP clones ST-C-gene-1 and SBBR1-8, 92.2–99.1% similarities), grouped with sequences from well-described representatives in the N. europaea-like cluster.

image

Figure 1.  Phylogenetic tree of AOB based on amoA gene fragments recovered from two landfill leachates (GZK and LK, shown in bold). The tree was constructed using the neighbor-joining distance method. The reference sequence of Nitrosococcus oceani (AF047705) was used as an outgroup. Bootstrap values (n=1000 replicates) of ≥50% are reported as percentages. The scale bar represents 10% sequence divergence. To simplify tree illustration, closely related amoA sequences originating from the same site are represented by a single clone. If ≥2, the number of clones is indicated at the end of the corresponding representative in parentheses.

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One nosZ gene library was generated for each of the three MSW disposal sites. Restriction enzyme analyses were performed on the nosZ clones randomly selected from each library. Digestion with the MspI endonuclease revealed 34, 24 and 8 unique RFLP types among the 76 GQW, 67 GZK and 10 LK clones screened. Only 10 nosZ clones were processed for the LK library due to poor PCR amplification with the primer set 661F/1773R. One to three representative clones in each unique RFLP type in each library were fully sequenced. Comparative analysis showed that the similarity of the nosZ genes from the GQW, GZK and LK libraries was in the range of 70.2–100%, 70.3–100% and 85.7–100%, respectively. Comparisons with the GenBank database using the blastn search confirmed that all the retrieved sequences showed homology to known nosZ sequences. The levels of similarity between these MSW sequences and the nosZ genes previously deposited in the GenBank only ranged from 70.6% to 85.9%, and no closely related cultured denitrifiers were found for these novel sequences (Fig. 2), suggesting that they likely represent denitrifying bacteria that constitute new taxa. Phylogenetic analysis revealed that all the MSW nosZ sequences did not form common branches along with their closest relatives in the GenBank, including sequences of cultivated species from the genus Pseudomonas, and clones from cultivated field soil (Stres et al., 2004), earthworm gut (Horn et al., 2006) and marine sediment (Scala & Kerkhof, 1998, 1999); instead, they were individually (and deeply in the case of Subgroup 2) branched in the phylogenetic tree and formed three major unaffiliated subgroups without any pure-culture sequences (Fig. 2). Subgroup 1 encompassed a considerable diversity of nosZ sequences and constituted the majority of the GQW and GZK libraries (73.7% and 92.2%, respectively) and all of the 10 clones from the LK library. Separating from Subgroup 1 were two minor branches, one of which obviously formed a GQW-specific group while the other comprised exclusively GQW and GZK clones.

image

Figure 2.  Phylogenetic tree of denitrifier based on nosZ gene fragments recovered from leachates of three MSW disposal sites (GQW, GZK and LK, shown in bold). The tree was generated using the neighbor-joining distance method. The reference sequence of Ralstonia solanacearum (AL646084) was used as an outgroup. Bootstrap values (n=1000 replicates) of ≥50% are reported as percentages. The scale bar represents 10% sequence divergence. To simplify tree illustration, cloned nosZ sequences with similarities of ≥97% that originated from the same site are represented by a single clone. If ≥2, the number of clones is indicated at the end of the corresponding representative in parentheses.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Recent investigations have revealed significant emission of N2O from MSW-deposited sites (Lee et al., 2002; Rinne et al., 2005). We investigated the bacterial groups involved in nitrogen cycling from three MSW disposal sites operated under different criteria. N2O is formed as a side product during nitrification and occurs as an intermediate during denitrification (Conrad, 1996). The nitrate produced by nitrification serves as an electron acceptor in denitrification and is reduced to N2 via nitrite, NO and N2O. The first step of aerobic nitrification is performed by AOB, while the capacity for denitrification is found in a wide variety of taxonomic groups, mostly in the Proteobacteria species (Zumft, 1999). In addition, AOB with denitrifying ability may also produce N2O via the so-called nitrifier denitrification pathway (Wrage et al., 2001; Shaw et al., 2006).

Comparative sequence analysis of amoA clones obtained from the LK and GZK leachate samples demonstrated that nitrosomonads exclusively from the N. europaea-like cluster are responsible for ammonia oxidation at these landfill sites. Significantly different and more diverse AOB communities have been reported in a variety of other habitats, including soil, marine, estuarine and freshwater systems (Speksnijder et al., 1998; Phillips et al., 1999; Avrahami et al., 2002; Cebron et al., 2003). It is somewhat surprising to detect this similar and low scale of species richness of AOB at the two landfill sites as previous studies have indicated that the LK and GZK landfill leachates harbored diverse and significantly different bacterial communities (Huang et al., 2004, 2005). The low diversity of the AOB diversity might render the nitrification in situ more susceptible to environmental perturbation. All of the landfill amoA sequences recovered from the present study are most similar to those N. europaea-like sequences retrieved from various wastewater treatment plants; however, unlike the landfill systems, wastewater treatment plants generally harbor a relatively high AOB diversity with amoA sequences related to most of the recognized Nitrosomonas species, and different wastewater treatment plants differ significantly with regard to species of AOB (Purkhold et al., 2000). The difference in ammonium concentration did not affect the structure of the amoA communities in the LK and GZK landfill leachates. Similarly, Avrahami et al. (2002) did not observe any clustering of amoA genes with respect to ammonium treatment when studying the effect of ammonium addition on soil microbial communities. However, previous studies have found that both increased N2O emission rates and a greater contribution of ammonia-oxidation to total N2O production were in correlation with elevated ammonium concentrations in soil (Muller et al., 1998; Avrahami et al., 2002), and the response of the AOB activity was mainly due to a physiological shift instead of a major change in the ammonia-oxidizing population (Avrahami et al., 2002). It remains unclear why amoA genes were not detected in the samples from the GQW open dump while they were readily amplified from the GZK landfill leachate, which had a much lower ammonium concentration.

Examination of the 153 environmental nosZ clones revealed that there is substantial and novel diversity of this gene within the MSW-deposited systems. Previously unknown nosZ gene sequences have been frequently detected in various environments (Scala & Kerkhof, 1999; Stres et al., 2004). Tree organization suggested that the three denitrifying nosZ leachate communities are related and with significant overlap. The majority of the nosZ clones did not form separate phylogenetic groups according to disposal form and/or site (Fig. 2). Similar results have been reported in previous studies comparing different habitats or treatments (Philippot et al., 2002; Prieme et al., 2002; Rich et al., 2003). On the other hand, specific nosZ gene clusters corresponding to sampling location have been detected in some other studies (Scala & Kerkhof, 1999). A few GQW and GZK clones did form specific groups in the phylogenetic tree (Fig. 2). Without doubt, practical operations have significant influences on the microbial diversity in the MSW-deposited environment; however, further examination of additional sites for each disposal mode will be necessary to elucidate whether disposal mode-specific denitrifier assemblages have evolved and, in particular, what is the role of these specific groups in differentiating the denitrification processes at their respective sites. Although high N2O emission rates (113 mg m−2 h−1) were recorded from the effluent leachate pond at the LK recirculating landfill (Lee et al., 2002), PCR attempts with the DNA extract from aqueous samples collected from the same leachate pond only resulted in very weak nosZ-specific (and relatively weak amoA-specific) banding. It is unlikely that this was directly due to the unsuitability of the applied PCR conditions or impurity of the DNA template, as the same amplification conditions worked well with the GZK and GQW leachate DNA, and archaeal and bacterial 16S rRNA gene fragments were readily amplified from the same bulk LK DNA sample (Huang et al., 2002, 2004). This finding suggests that a very small subfraction of the total community with efficient N-transforming capacity might be responsible for the high N2O emission from this landfill site, which was receiving high-strength leachate from the newly placed waste matrix. In addition, MspI restriction analysis of the 10 LK nosZ clones resulted in eight unique RFLP types, indicating that the nosZ community is diverse and its actual diversity was only partially revealed.

Overall, our results suggest that representatives from unknown taxa are likely responsible for the N2O emissions from the MSW disposal sites. Differences in waste disposal mode did not cause dramatic changes in the nitrifying and denitrifying communities. As numerous environmental factors can vary the amount of N2O produced during the nitrification and denitrification pathways, further research should be oriented towards exploring the abundance and dynamics of the different amoA and nosZ genes at these potential high-N2O emitting sites by quantitative molecular approaches. Additional knowledge from these studies would aid in developing strategies of effective control of N2O emission from these important and complex systems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported financially by the National Natural Science Foundation of China (40401057) and the Natural Science Foundation of Guangdong Province, PR China (05003330). We kindly thank the two anonymous reviewers for their constructive comments on the manuscript. We are indebted to Yong-Qiang Zhang for excellent field assistance in collecting the GQW open dump leachate samples.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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