Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6409-4

ENVIRONMENTAL BIOTECHNOLOGY

Analysis of microbial community structure and composition in leachates from a young landfill by 454 pyrosequencing Thorsten Köchling & José Luis Sanz & Sávia Gavazza & Lourdinha Florencio

Received: 8 October 2014 / Revised: 13 January 2015 / Accepted: 15 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Microorganisms are responsible for degrading the raw leachate generated in sanitary landfills, extracting the soluble fraction of the landfill waste and biotransforming organic matter and toxic residues. To increase our understanding of these highly contaminated ecosystems, we analyzed the microbial communities in the leachate produced by three landfill cells of different ages. Using high-throughput 454 pyrosequencing of the 16S rRNA gene, we describe the structure of the leachate communities and present their compositional characteristics. All three communities exhibited a high level of abundance but were undersampled, as indicated by the results of the rarefaction analysis. The distribution of the taxonomic operational units (OTUs) was highly skewed, suggesting a community structure with a few dominant members that are key for the degradation process and numerous rare microorganisms, which could act as a resilient microorganism seeder Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6409-4) contains supplementary material, which is available to authorized users. T. Köchling (*) : L. Florencio Laboratory of Environmental Sanitation, Department of Civil Engineering, Federal University of Pernambuco, Av. Acadêmico Hélio Ramos, s/n. Cidade Universitária, 50.740-530 Recife, PE, Brazil e-mail: [email protected] L. Florencio e-mail: [email protected] J. L. Sanz Department of Molecular Biology, Autonomous University of Madrid, 28049 Cantoblanco, Spain e-mail: [email protected] S. Gavazza Laboratory of Environmental Engineering, Federal University of Pernambuco, Academic Center of the Agreste, Rodovia BR-104, Km 62, Nova Caruaru, 55.002-970, PE Caruaru, Brazil e-mail: [email protected]

pool. Members of the phylum Firmicutes were dominant in all of the samples, accounting for up to 62 % of the bacterial sequences, and their proportion increased with increasing landfill age. Other abundant phyla included Bacteroidetes, Proteobacteria, and Spirochaetes, which together with Firmicutes comprised 90 % of the sequences. The data illustrate a microbial community that degrades organic matter in raw leachate in the early stages, before the methanogenic phase takes place. The genera found fit well into the classical pathways of anaerobic digestion processes. Keywords Landfill leachate . 16S rRNA gene . 454 pyrosequencing . Microbial community structure

Introduction The disposal of solid waste represents a global environmental problem that continues to grow as the world population and urban and industrial development in many countries increase. Currently, sanitary landfilling is the most common method of municipal solid waste (MSW) disposal; this method is superior to open dumping in terms of ecological impact and sustainability because the waste is contained in landfilling cells and the often highly toxic leachate is collected and treated onsite instead of seeping into the groundwater. Landfill leachate is a complex mixture of highly concentrated organic compounds with a chemical oxygen demand (COD) in the range from 140 to 152,000 mg/l (Kjeldsen et al. 2002) that is produced when the rainwater and the moisture content of the waste percolate through the trash, acting as a solvent for the contaminants that are released as the waste is digested. Raw landfill leachate constitutes a hazard to the environment and to public health because toxic organic and metal

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compounds as well as bacterial pathogens are released into the groundwater in instances of leakage. The inclusion of new compounds in daily care and pharmacological products widens the spectrum of xenobiotic and hard-to-degrade toxic compounds that enter landfills via municipal solid waste disposal and accumulate in the trash and leachate (Baun et al. 2004; Eggen et al. 2010). Given the finite impermeability of even the most suitable geomembrane materials and the long periods of time in which sealed landfills produce leachate after the landfilling phase is complete (Allen 2001), the high probability of hazardous leachate eventually entering the ecosystem surrounding the landfill should be considered. The microbial communities resident in landfill leachate are responsible for the conversion of organic matter into less complex and less toxic compounds. Knowledge of the structure and composition of these microbial populations is necessary to optimize the operational parameters of the leachate-treating bioreactor systems and to efficiently assess the public health risk from the release of pathogenic bacteria from leachate seeping into the soil and water table near the landfill site. Currently, relatively few published studies have characterized the microbial populations of landfill leachate, although this issue demands attention given the previously mentioned environmental and economic concerns. Several studies have focused on related ecosystems, such as leachate-contaminated sites (Röling et al. 2001; Liu et al. 2011) or leachate-treating bioreactors (Calli et al. 2006; Xie et al. 2012). The existing surveys of raw leachate communities rely on culture-dependent (Hata et al. 2004) and culture-independent techniques, such as denaturant gradient gel electrophoresis (DGGE), cloning, and Sanger sequencing of the microbial 16S rRNA gene (Huang et al. 2002, 2003, 2004, 2005; Laloui-Carpentier et al. 2006; Zhang et al. 2011a). The principal findings of these studies agree that landfill leachate harbors complex microbial communities in which members of the bacterial Firmicutes, Proteobacteria, and Bacteroidetes phyla are the most abundant taxonomic groups, whereas archaeal populations typically consist of methanogenic species. Although these studies have provided insight into the composition of resident microbial populations, the assessments remain incomplete and fail to provide information on community structure because the methodologies afford limited coverage of the overall population. Microbial communities usually contain large numbers of very rare taxa, which possibly act as a nearly inexhaustible reservoir of species (Sogin et al. 2006) and a buffer for the stabilization of the communities (Caron and Countway 2009) that often face important changes in nutritional and physicochemical conditions. This is especially the case in immature and developing ecosystems like raw landfill leachate in the transition phase from anoxic to methanogenic conditions. Therefore, the detection

of the low-abundance microbial species might reveal further valuable information about the metabolic capabilities and adaptability of bacterial assemblages. Here, we present the first study of leachate microbial communities using next-generation sequencing (NGS), which provides several orders of magnitude more sequence reads, enabling a more complete picture of the microbial populations that inhabit landfill leachate. The objectives of this survey were to characterize the biological diversity as well as the composition and structure of the microbial communities in raw landfill leachate from an MSW receiving sanitary landfill without leachate recirculation and to compare these parameters among the leachate samples derived from landfilled waste of increasing age (1, 2, and 3 years) that represent different time spans of environmental adaptation of the microorganisms.

Materials and methods Landfill site and leachate sampling The Candeias sanitary landfill complex is located near Recife, the capital of the state of Pernambuco, which has a metropolitan population of approximately 3.7 million and is situated in Northeast Brazil (geographic coordinates: latitude −08° 09′ 57.46, longitude −34° 58′ 44.65). The installation was commissioned for operation in October 2007 and consists of the landfill, a treatment plant for the leachate effluent and a biogas recovery system for use as a renewable energy source. The landfill receives residues classified as nonhazardous (ABNT 2004) from the city of Recife and the surrounding communities. The landfill covers an effective surface of 70 ha (700,000 m2) and was designed as a three-dimensional grid of waste compartments with a total capacity of 10.56 million tons, a daily load capacity of 2100 t, and an expected operation period from 2007 to 2027. The generated leachate is not recirculated but is diverted via a drainage pipe system into collection ponds and treated at the aerobic on-site treatment plant (capacity 7–14 m3/h). One liter of raw leachate was sampled from the outlet of the drainage pipe of cells that were landfilled in 2009, 2010/2011, or 2011/2012. The samples were stored in airtight bottles and transferred immediately to the laboratory for analysis. Sample designations L1yo: leachate from waste that was landfilled in 2011/2012 (youngest cell). L2yo: leachate from waste that was landfilled in 2010/2011 (intermediate cell). L3yo: leachate from waste that was landfilled in 2009 (oldest cell).

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Physicochemical analysis

Phylogenetic and microbial community analysis

Sample temperature, pH, dissolved oxygen, and electrical conductivity were measured on-site with a Hach HQ40d multiprobe-meter using the PHC101, LDO101, and CDC401 probes (Hach, Loveland, CO, USA). The 5-day biochemical oxygen demand (BOD5) was determined with the pressure-based OxiTop system (WTW, Weilheim, Germany). The total organic carbon (TOC) content was measured using a Shimadzu TOC-V CSH analyzer (Shimadzu, Kyoto, Japan). Ion concentrations were determined with a Dionex ICS-1100 (cations) or a ICS-2100 (anions) system (Thermo Scientific, Sunnyvale, CA, USA).

The 454 Tools (454/Roche) and QIIME software packages (Caporaso et al. 2010) were used to demultiplex the raw data and perform the OTU picking, sequence alignment, taxonomy assignments, and the OTU table generation. For initial quality filtering of the sequences, only high-quality reads with a minimum length of 350 base pairs (bp), without ambiguous positions, and with a maximum homopolymer length of 6 bp were allowed; uncorrectable barcodes were discarded. The sequencer signal was enhanced by denoising the data (Reeder and Knight 2010), and chimeric sequences were removed (Haas et al. 2011). To assign the taxonomy, the sequence tags were compared to the SILVA 104 database, which contains both bacterial and archaeal 16S rDNA sequences (Quast et al. 2013), using the BLAST algorithm (Altschul et al. 1990). The phylogenetic affiliations of the 40 most abundant OTUs were manually cross-checked with the RDP classifier (Wang et al. 2007), with the SINA algorithm in the SILVA database (Quast et al. 2013), and with BLAST against the GenBank 16S rDNA reference set. Rarefaction curves and the Good, jackknife, Chao-1, and abundance-based coverage estimators were calculated to evaluate the completeness of the sampling effort. The biological diversity was assessed by computing the Hill numbers for each sample and converting them to effective number of OTUs (Jost 2006), which represent the equivalent number of OTUs in a community with maximum evenness. The R software environment (R Development Core Team 2008), including the vegan library (Oksanen et al. 2013), was used to calculate the abundance data and diversity metrics.

16S rDNA amplification and pyrosequencing Fifty milliliters of a homogenized 1-l leachate sample was centrifuged at 10,700g for 20 min, and the resulting pellet was resuspended in 250-μl PCR-grade water. Total community DNA was then extracted from the samples with the MO BIO PowerSoil kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol. Unidirectional 454 pyrosequencing of the 16S rRNA gene was performed with amplicons spanning the regions V6-V8 using the universal primer set 926 F (5′-AAAC TYAAAKGAATTGACGG-3′) (Lane 1991)/1392R (5′ACGGGCGGTGTGTRC-3′) (Lane et al. 1985), containing additional adapter, key and barcode sequences necessary for multiplexing and sequencing. Each PCR consisted of 5 ng of genomic template DNA, 1.25 U of Invitrogen Platinum Taq DNA polymerase (cat# 11304, Life Technologies, São Paulo, SP, Brazil), 1× reaction buffer, 2 mM Mg2+, 0.2 mM dNTPs (New England Biolabs, Ipswich, MA, USA), and 0.5 μM of each primer in a total volume of 50 μl. The cycling conditions were as follows: 3 min at 95 °C (initial denaturation) followed by 30 cycles of 30 s at 95 °C, 45 s at 55 °C (annealing), and 90 s at 68 °C (template extension). The program ended with an additional extension step of 10 min at 68 °C. Six replicate PCR products were pooled, concentrated, cleaned with the Invitrogen PureLink system (Life Technologies, São Paulo, SP, Brazil), and quantified on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purified barcoded amplicons were pooled for pyrosequencing in equimolar amounts. The multiplexed sample was sequenced by Macrogen (Seoul, South Korea) on a GS-FLX system (454 Life Sciences/Roche, Branford, CT, USA). All sequence data are available from the sequence read archive (SRA) of the National Center for Biotechnology Information (NCBI) under BioProject PRJNA241345 (SAMN02678185, SAMN02678186, SAMN02678187).

Results Physicochemical analysis The basic environmental characteristics of the Candeias landfill leachate are summarized in Table S1, and the ion concentrations measured by chromatography are provided in Table S2 (supporting material). The TOC, an indicator of COD as well as the NH4+ values, varied but did not show a gradual change that correlated with landfill age. The electrical conductivity was constant between the leachate samples (in the range of 15 to 17 mS/cm), which together with the elevated concentrations of sodium, potassium, and chloride ions indicated a high ionic strength of the leachate. None of the measured ion concentrations tended to correlate with increasing landfill age. The pH was slightly basic, with values between 7.2 and 7.7. The initially high BOD5 decreased sharply with increasing landfill cell age. Similarly, short-chain volatile fatty acids (VFAs) were present at a high concentration in the youngest

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sample and then nearly disappeared in the two subsequent samples. Microbial community structure After quality filtering, flowgram denoising and removing the chimeric sequences, a total of 620,982 high-quality reads were obtained: 205006 for L1yo, 205238 for L2yo, and 210738 for L3yo. The sequences clustered into 7448 distinct OTUs when applying a 97 % similarity threshold for inclusion. The comparative analysis using the SILVA database suggested that only 155 of the sequence tags (0.03 %) were affiliated with the domain Archaea. Given their extremely low abundance, these sequences were excluded from the community structure analysis. An amplification bias in the PCR step is unlikely to be the cause for the low number of archaea reads though; methanogenic OTUs have been found in dominant proportions in other studies using the same primer set (Callbeck et al. 2011; Berdugo-Clavijo and Gieg 2014). The rarefaction analysis of the three leachate samples at an OTU cutoff value of 97 % indicated that a full census had not been achieved for any of the samples (Fig. 1). All of the curves were far from reaching a plateau which did not allow for calculating the total species richness, a basic descriptor of biological diversity, solely based on the total number of identified OTUs. Therefore, a series of coverage estimators was Fig. 1 Rarefaction curves for the three leachate samples at an OTU threshold of 97 % sequence similarity

computed (Table 1) that take into account the underlying species abundance distribution (SAD) of the microbial populations to a different degree. Good’s estimator indicated nearly complete coverage. Several coverage estimators that take into account extremely rare species (OTUs), i.e., doubletons/singletons (Chao, jackknife), and rare species (ni ≤10, ACE) predicted that the real number of OTUs exceeded the number of detected OTUs to a varying degree. All of the estimates assigned the highest richness value to the L3yo leachate, which correlates with the total number of observed OTUs in the samples, being the highest for L3yo, while the number of high-quality sequences was uniform between samples. The log-abundance and Whittaker rank-abundance plots (Fig. 2) illustrated the presence of a high number of OTUs with few members, mainly singletons (OTUs with only one representative sequence) and doubletons (OTUs with two sequences), with a few OTUs represented by large sequence sets. This distribution is reflected in the pronounced parabolic shape of the log-abundance plot. Representing the distribution as a histogram using exponential bins suggested the presence of a subset of sequences affiliated with OTUs of intermediate abundance. A higher proportion of rare OTUs were detected in L3yo, which was most obvious on the Preston histogram. The mode of the SAD for all the samples, however, was unity (singleton OTUs).

Appl Microbiol Biotechnol Table 1

Coverage estimates for the bacterial leachate communities

Sample

Number of reads

OTU richness

Chao-1

ACE

ACE-1

Good’s coverage

1st order jackknife

2nd order jackknife

L1yo L2yo L3yo

204757 204959 210592

2994 2532 3541

13673 9523 18195

14410 10173 21041

30663 20455 47627

0.990 0.992 0.988

4971 4114 6086

6765 5517 8410

ACE abundance-based coverage estimator

The Wiener (H) and Simpson (1-D) indices of diversity were high, with almost no variation between samples (data not shown). However, when these metrics were converted to effective number of species/OTUs, the Shannon index (e H ) was highest in L3yo, whereas the transformed Simpson indices (1/D) and the Berger-Parker indices (1/d), which give more importance to the dominant members of the population, increased steadily from L1yo to L2yo to L3yo (Table 2). This behavior indicated that the biodiversity increased with progressing landfill age. To compare the community structure of the entire population to the subset of extremely rare species (singleton OTUs), the Shannon diversity was also computed at the phylum level for the singleton subset and compared to the diversity of the whole bacterial community (Table S3, supplementary material). The transformed indices were twice as high for the singleton subpopulation as for the whole community, Fig. 2 a–c Log-abundance plots of leachate samples L1yo, L2yo, and L3yo. d Semi-logarithmic rank-abundance plots of the three samples illustrating the size distribution of the detected OTUs

indicating that, in comparison, the subpopulation of very rare OTUs possessed a higher degree of biodiversity.

Community composition and function In total, 519 sequence reads in the entire dataset, grouped into 134 OTUs, could not be identified at the domain level using the BLAST algorithm for sequence comparison with the SILVA 104 database and were therefore excluded from further analysis. Only 155 sequences (0.03 % of the dataset) were affiliated with the domain Archaea, though the number of sequences increased with landfill age (L1yo, 24 reads; L2yo, 44; L3yo, 87). Seventeen of the 18 detected OTUs belonged to the phylum Euryarchaeota; the other sequence belonged to Crenarchaeota. All of the sequence tags identified on a genus level belonged to members of the methanogenic archaea

Appl Microbiol Biotechnol Table 2 The Hill numbers for the leachate samples representing the effective number of OTUs and their relation to the most commonly used diversity indices in community ecology

ENS effective number of species

Hill number

Related diversity index

N0

Richness (S)

as is (S)

N1 N2 NInf

Shannon-Wiener (H) Simpson (D) Berger-Parker (d)

eH 1/D 1/d

Methanosaeta, Methanosarcina, Methanobacterium, and Methanoculleus. The sequences affiliated with the bacterial domain included members of 24 officially recognized phyla and six candidate divisions that do not yet contain any culturable representatives (Table S4). The proportion of reads assigned to bacterial candidate divisions was twice as high in the singleton population, indicating a greater presence of uncultured species within the rare members of the communities. The vast majority of the classified reads were affiliated with the phylum Firmicutes; the representation increased in relative abundance from 53 % in L1yo to 58 % in L2yo and to 62 % in L3yo (Fig. 3). Other dominant phyla included the Proteobacteria, Bacteroidetes, and Spirochaetes. The reads affiliated with these four major phylogenetic divisions accounted for up to 93 % of the complete set of bacterial sequences, leaving only a small portion of the bacterial richness of the leachate samples to the remaining 20 phyla and the candidate divisions. The next most abundant phyla were the Synergistetes, Tenericutes, and Lentisphaerae, Fig. 3 OTU abundance across the main bacterial phyla detected in the three leachate samples

Conversion to ENS

Effective number of OTUs/ENS

Sensitivity to OTUs

L1yo

L2yo

L3yo

2994

2532

3541

very rare

196 56 11

177 62 17

216 81 21

rare balanced abundant

after which other phyla contributed less than 0.5 % to the bacterial sequence set of each sample. Whereas the relative dominance of the Firmicutes increased with progressing landfill age, the Proteobacteria, initially the second most abundant taxonomic group, experienced a sharp decline from L1yo to L2yo and L3yo, mainly due to the decrease in abundance of members of the Beta- and Gammaproteobacteria. While the latter together accounted for 17 % of all bacterial sequence tags in L1yo but for only 1 % in L3yo, the abundance of Deltaproteobacteria-affiliated reads was below 2 % in all three samples. The third phylum that exhibited a gradual change in abundance based on landfill age was the Spirochaetes, which increased twofold, although their abundance was an order of magnitude lower than that of the Firmicutes. The Bacteroidetes showed no apparent linear trend in abundance over time. Within the dominating Firmicutes, the majority of the reads pertained to the class Clostridia; only 0.4 to 3.8 % of the Firmicutes sequence tags were affiliated with the other major subdivision, Bacilli. At the

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order level, the Clostridiales were the most abundant taxonomic subgroup (90 % of the Firmicutes-associated sequences), with high proportions from the Ruminococcaceae, Lachnospiraceae, and Syntrophomonadaceae families as well as the uncultured OP12 clade. The Bacteroidetes were highly abundant in all three samples, principally due to the presence of members of t he orders Bacteroi dales and Sphingobacteriales, most of which were related to WCHB169, a sequence retrieved from a hydrocarbon-contaminated aquifer (Dojka et al. 1998). With the objectives of evaluating the reliability of taxonomic low-level assignment in this pyrosequencing study and identifying possible key metabolic players in the leachate ecosystems, the QIIME classification of the 40 most abundant OTUs, which accounted for 54 to 58 % of the entire sequence set, was cross-checked with three different classifier/database methods for taxonomic assignment (RDP, BLAST/GenBank, and SINA/SILVA). Phylogenetic affiliations were consistent at the phylum level between the method used in this study to generate the OTU abundance table in QIIME (BLAST/ SILVA) and the three manual approaches, but on the class level, we observed three contradictory assignments using the different methods (Tables S5 and S6). The most abundant OTU in sample L1yo was misassigned by the method implemented in the QIIME pipeline and was identified on the genus level as Azospira, a member of Betaproteobacteria, whereas BLAST/GenBank assigned this sequence set to Pseudomonas aeruginosa, and the SINA classifier against the SILVA database assigned it to the genus Pseudomonas, both with a similarity score of 99 %. Two Spirochaetes-related sequence tags were classified as Deltaproteobacteria by BLAST/GenBank, although they had low similarity scores and could not be identified beyond the domain level using the RDP classifier. For the remaining 37 most abundant OTUs from the three samples, differences between the four approaches were primarily restricted to the maximum depth of taxonomic affiliation. Despite their compositional similarities at high phylogenetic levels, the three leachate communities had only a small subset of OTUs (7 %) in common. The majority of these most abundant OTUs could be identified on the genus level as Syntrophomonas, Sedimentibacter, Pelotomaculum, Ureibacillus, Anaerobranca, Desulfitibacter, Paenibacillus ( F i r m i c u t e s ) , P ro t e i n i p h i l u m , a n d P e t r i m o n a s (Bacteroidetes), reflecting the predominance of the Firmicutes and Bacteroidetes, which had also been observed for the whole community.

Discussion The values for absolute OTU richness varied considerably between the different estimators. By far, the lowest richness was predicted by Good’s estimator (99 % coverage by our

sampling effort), which, however, is considered prone to underestimation (Chao and Lee 1992) because it assumes that all species are present in equal proportions, which is virtually never the case. In this study, the 20 most abundant OTUs comprised 43 % of the entire sequence pool, and the 2202 singleton and doubleton OTUs accounted for only 1 % of the reads. The abundance distributions of the leachate populations appeared to be highly skewed toward extremely rare species, even for microbial communities. The ACE and Chao1 metrics take the inherent heterogeneity of abundance distributions into account and are therefore more adequate for application with our data. Their values were very similar in this study, and the Chao1 estimator seems to be particularly suited as it represents a robust but cautious assessment of the minimum richness of ecological communities (Shen et al. 2003). Thus, applying this conservative richness estimation, the microbial leachate communities contained at least between 10,000 and 18,000 different OTUs, indicating a considerably high degree of diversity for a severely contaminated ecosystem like raw landfill leachate. While the diversity indices that take into consideration mainly the high-abundance species (Simpson and BergerParker indices) increased linearly over the course of 3 years, metrics like the Shannon-Wiener index, which consider mainly the contribution of the rare OTUs, showed no clear tendency over time. This subpopulation of extremely rare microorganisms was probably shifting in composition and diversity during the process of adaptation of the microbial communities to the immature and variable leachate environment. This may have caused the seemingly random variability of the diversity indices that are sensitive to low-abundance taxa. A sampling interval of 1 year between samples was probably too short to allow for the observation of linear trends in this subpopulation. While the values for absolute richness were high, the majority of OTUs were present at low abundances, and all three communities were dominated by members of a single bacterial phylum, the Firmicutes. This phylogenetic group is most probably providing the majority of key players in the degradation process of organic matter and xenobiotics in the leachate, given their dominance and known capacity to break down a wide variety of often recalcitrant organic compounds (Winderl et al. 2008). The observed decline in BOD5 and VFA concentration coincided with an increasing biological diversity of the resident microbial communities, best reflected by the BergerParker index (1/d), a simple descriptor equal to the inverse of the proportional abundance of the most dominant OTU (d=nmax/N) in a community. The corresponding values increased over time, indicating a decrease in the level of dominance in the microbial communities and an increase in diversity. The Berger-Parker index has been described as appropriate for detecting disturbances in biodiversity due to

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anthropogenic pollution in soil ecosystems (Caruso et al. 2008). Applying this interpretation, the diversity and stability of the communities increased over time as the leachate became less contaminated. As it is reasonable to assume that the more dominant bacterial species are ecologically and functionally the most important microorganisms in the leachate ecosystems, the metrics that are more sensitive to dominant species should be considered for the evaluation of diversity in terms of functional stability. The Berger-Parker index most prominently reflected the increase in diversity over time. While biodiversity increased over time in the leachate microbial communities, the Firmicutes clearly represented the dominant phylum in all three samples. Members of the Firmicutes have been observed to provide an important proportion of the microbial communities in comparable ecosystems. Together with the Bacteroidetes, Spirochaetes, as well as the Proteobacteria, they predominated the resident communities in studies of samples from other landfills or leachatecontaminated environments (Röling et al. 2001; Liu et al. 2011; Huang et al. 2004; Huang et al. 2005; Zhang et al. 2011a). Members of the phylum Actinobacteria have also been detected in other surveys (Röling et al. 2001; Huang et al. 2004), but the Actinobacteria were represented by very few sequence counts in the Candeias leachates. As discussed above, the great majority of the detected OTUs in this study occurred at extremely low abundances in the data set. These members of the microbial communities represent the tail of the rank-abundance curve, and while they probably do not contribute to the metabolic function of the community, they could most likely act as a microorganism seeder pool (Wittebolle et al. 2008), of which the most adapted members are selected under certain environmental conditions to give rise to a resilient and trophically adapted population. The comparison of diversity measures at the phylum level between the whole sequence set and the singleton subpopulations supports this hypothesis. Higher diversity values for the singleton subpopulations than those for the entire communities (Table S3) suggest the existence of a more diverse subset of species from which the most suited rise to dominance in response to the environmental conditions. This phenomenon has been observed in the literature in controlled reactor experiments, where significant differences were observed in the microbial populations in several reactors seeded with the same inoculum (anaerobic granular sludge) after 3 months of feeding with different substrates (Diaz et al. 2003). The analysis of the phylogenetic affiliation of the sequence reads suggested the nearly complete absence of sequences within the domain Archaea. The methanogenic population in the leachate is most likely in its initial state of development. As the Candeias landfill is still adapting to the high organic input from the waste deposition, it may be too young to provide the environment for these microorganisms to grow optimally. Previous studies on the microbial community

composition of leachate ecosystems detected the presence of archaea in samples from landfills that were at least 10 years old (Röling et al. 2001; Liu et al. 2011; Huang et al. 2002; Huang et al. 2003), which explains the marked differences in community composition at the domain level. The large number of high-quality reads and taxonomic assignments obtained in this survey allowed us to carry out an analysis of the metabolic network formed by the members of the microbial communities present in the leachate. For this functional assessment, only the OTUs with a relative abundance of at least 0.5 % (number of sequences in the OTU over the total number of sequences recovered in a cell) were considered. Due to the high number of singletons, doubletons, and other very rare OTUs, the coverage of these OTUs ranged from 54 to 58 % in the three cells. Considering the entire sequence set of the three samples, most of the identified OTUs were fermentative anaerobes affiliated with the phyla Firmicutes and Bacteroidetes; the order Clostridiales in the phylum Firmicutes is one of the most common fermentative acetogenic microbial groups isolated from ecosystems. Whereas Proteobacteria were less abundant in L2yo and L3yo, the Firmicutes increased in number from L1yo to L3yo. A similar progression has been described in anaerobic granular sludge (Diaz et al. 2006): Young granules were predominantly composed of Proteobacteria (52 %), whereas 55 % of mature granules contained Gram-positive bacteria (Firmicutes+Actinobacteria) and only 12 % included Proteobacteria. An increase in the proportion of Firmicutes was also observed during the maturation of an anaerobic biofilm (Fernandez et al. 2008). The majority of the detected Firmicutes belonged to the class Clostridia and was affiliated to genera such as Syntrophomonas, Sedimentibacter, Clostridium, and Pelotomaculum, all of which were present in the three leachate samples. Sedimentibacter growth is supported by the fermentation of pyruvate or aminoacids in a Stickland-type reaction, whereas Clostridium utilizes carbohydrates and/or proteins, depending on the species. The end-products of fermentation are VFAs (mainly acetate, propionate, and butyrate) and, in the case of Clostridium, also short-chain alcohols and hydrogen. Sedimentibacter has been observed to participate in the dehalogenation/dechlorination of organic compounds (Maphosa et al. 2012) and has been found in contaminated anaerobic sediments, where it could possibly be involved in surfactant biodegradation (Lara-Martín et al. 2007). Syntrophomonas was one of the most abundant genera in a leachate study on a landfill in China (Huang et al. 2004) and has been encountered in biogas reactor systems (Krause et al. 2008). It is usually found together with methanogenic archaea, with which it syntrophically degrades fatty acids. Interestingly, there was a universal presence of OTUs remotely related to the genera Anaerobranca (92 % similarity), Desulfitibacter (93 %), Eubacterium (91 %), Ureibacillus

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(92 %), and Paenibacillus (92 %) affiliated to the orders Clostridiales and Bacillales. These taxa comprise strictly or facultatively anaerobic microorganisms that primarily grow via the fermentation of carbohydrates or proteins and the production of VFAs. Desulfitibacter and Ureibacillus utilize respiratory metabolism: Desulfitibacter utilizes a limited number of substrates as electron donors (formate, lactate, methanol, and pyruvate) with elemental sulfur, sulfite, thiosulfate, nitrate, and nitrite as electron acceptors, whereas Ureibacillus is ureolytic. Most of these organisms are spore-forming, which might contribute to their persistence in the system. Although the carbon cycle is dominant in a landfill, microorganisms involved in other cycles were also detected. However, the nitrogen cycle is barely present in this environment. Nitrifying bacteria, strict aerobes, and N2-fixing microorganisms were not detected. Only a few of the retrieved sequences were affiliated with genera that perform respiratory metabolism and are able to grow via denitrification using nitrate/nitrite as an electron acceptor (the Firmicutes C. denitrificans, Paenibacillus and Bacillus, as well as Zobellella and certain strains of Pseudomonas, which are Gammaproteobacteria). With respect to the sulfur cycle, aerobic colorless sulfur bacteria were absent, as were members of the N-cycle. However, the reductive pathway of the cycle was well represented, mainly by members of the Firmicutes. Several OTUs were remotely related to the sulfate/sulfite-reducing genera Desulfitibacter, Desulfosporosinus, Desulfotomaculum, D e s u l f o v i rg u l a , D e s u l f o r u d i s ( F i r m i c u t e s ) , a n d Desulforegula (Deltaproteobacteria). Various OTUs exhibited only low sequence similarities to described species (levels below 93 %), indicating the presence of new and yet uncultured SRB taxa in this environment. The syntrophobacteria (phylum Firmicutes) deserve further consideration. As obligate hydrogen-producing acetogens, these microorganisms oxidize the fermentation end-products to acetate using the protons as electron acceptors. Several strains of the genus Syntrophomonas, including species such as S. wolfei and S. sapovorans, formed the dominant or codominant groups in the three cells with abundances of 9.9, 4.5, and 11.4 % in cells 1, 2, and 3, respectively. Syntrophomonas spp. beta-oxidize fatty acids in syntrophic association with hydrogenotrophic microorganisms, such as methanogens or sulfate-reducing bacteria (SRB). In coculture with hydrogenotrophs, Syntrophomonas spp. produce acetate and hydrogen. Moreover, Pelotomaculum (P. schinkii) was present in all the cells (relative abundance of 0.5 to 1.1 %) and syntrophically grows on propionate, alcohols, and lactate with methanogens. The low concentrations of C3 to C5 VFAs (propionic, butyric, and valeric acid) in the three cells (data not shown) support the significance of this group of bacteria. Some of the genera and included species are moderate thermophiles (Anaerobaculum, Anaerobranca, Syntrophothermus,

Ureibacillus, Desulfovirgula, C. straminisolvens, and H. hydrothermalis). This finding is not surprising because the degradation of organic matter releases heat into the environment. The heterogeneous inner structure of a sanitary landfill provides thermophilic niches that enhance the maturation of the raw organic matter. Although most of the identified OTUs were strictly anaerobic, the L1yo sample constituted an exception. The dominant L1yo population was OTUs affiliated with the genera Pseudomonas (11.4 %), Comamonas (0.7 %), and Zobellella (0.5 %), all of which are Proteobacteria chemoorganotrophs that undergo respiratory metabolism using oxygen as the terminal electron acceptor. Some strains are capable of using nitrate as an alternative (denitrifying strains). These OTUs were not recovered in L2yo and L3yo. Because of the young age of the sanitary landfill, aerobic niches were still present, enabling the aerobic degradation of organic matter to CO2, which is energetically more favorable than the corresponding anaerobic pathways. The most abundant OTU in sample L1yo (99 % similarity with Pseudomonas aeruginosa) was absent in the other two samples. The presence in this environment of P. aeruginosa, most likely the most prevalent of all bacterial species, is not surprising because strains exist that are involved in the biodegradation of various organic molecules, such as amino acids, sugars, alkanes, phenols, and polycyclic aromatic hydrocarbons (Zhang et al. 2011b). Pseudomonasaffiliated sequences and isolates have also been recovered in other studies of leachate communities or leachatecontaminated ecosystems (Huang et al. 2005; Tao et al. 2014). With respect to the Bacteroidetes, both of the identified genera affiliated with this phylum, Proteiniphilum, and Petrimonas were found in all three cells with relatively high abundances of 7.2, 8.9, and 5.7 %, respectively. Members of the Bacteroidetes are strict anaerobes with a hydrolytic and fermentative metabolism. Nevertheless, there is an important difference in the metabolism of both encountered genera: Proteiniphilum is exclusively proteolytic, and Petrimonas prefers carbohydrates. The major end-products of fermentation are acetate, propionate, H2, and CO2. Members of both genera have been identified in ecosystems where the degradation of concentrated organic matter occurs. Proteiniphilum was the predominant OTU found by 16S rDNA pyrosequencing of a full-scale anaerobic digester that was treating the organic fraction of domestic solid waste (J. Cardinale and J.L. Sanz, unpublished) and has been isolated from a brewery wastewatertreating UASB (Upflow Anaerobic Sludge Blanket) system (Chen and Dong 2005), while 16S rRNA sequences of Petrimonas sulfuriphila have been detected in clone libraries of anaerobic manure-treating bioreactors (Kampmann et al. 2012). Other OTUs affiliated with the phylum Bacteroidetes were also present in all three landfill cells (with relative abundances of 2.9, 5.4, and 3.7 %), but these OTUs were phylogenetically

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distant from any described species, making it difficult to extrapolate their metabolic role. Members of the Firmicutes and Bacteroidetes form part of the core microbial population in the sanitary landfill that plays an important role in transforming organic matter into small intermediate degradation compounds. The Spirochaetes, a group of mostly anaerobic chemoheterotrophs, were the fourth most abundant phylum detected in all three leachate samples. Leptospira and Treponema, both known for their potential pathogenicity, were encountered while species of Treponema have also been associated with ecological functions as they are capable of degrading cellulose and other often resistant plant-derived polysaccharides (Dröge et al. 2006). The relatively high abundances of Spirochaetesaffiliated bacteria in the leachate suggest their important role in the transformation of plant matter, which is one of the main components of municipal solid waste (McDonald et al. 2012).

Fig. 4 Proposed metabolic network of the leachate communities: 1 only in cell 1, 2 nitrate can be used as an electron acceptor, 3 most of the H2 and CO2 is released into the atmosphere with the biogas, and 4 methanogens represent only 0.03 % of the retrieved sequences

Some Spirochaetes, like members of the genus Spirochaeta, which were also detected in the leachate samples, appear to enhance cellulose biodegradation in symbiosis with Clostridia species (Pohlschroeder et al. 1994). The presence of both genera in the landfill leachate probably indicates such a relationship, showing how microbial networks form, conditioned by the physicochemical characteristics of their environment and make the biotransformation of often recalcitrant compounds more efficient. Anaerobaculum was the most abundant member of the phylum Synergistetes in all three samples, with OTUs in cells 2 and 3 exhibiting 98 % sequence similarity to A. thermoterrenum. This thermophilic strict anaerobe can ferment proteins, organic acids, and a limited number of carbohydrates and has previously been identified in at least one microbial landfill leachate community study (Uchida et al. 2009).

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Considering the 40 most abundant OTUs identified in this study, a diagram of organic matter degradation in the studied sanitary landfill is presented in Fig. 4; only OTUs with a similarity percentage higher than 90 % to described genera have been included. The Candeias site is a landfill in its pre-methanogenic stage. Even at this early point in community development, we observed a shift toward a more diverse and stable population dominated by members of the order Clostridiales. We detected a high level of microdiversity within the set of lowabundance OTUs, which probably serve as a species pool that permits the functional adaptation of the microbial communities to the environmental conditions. To obtain a picture of the entire transformation process toward a methanogenic ecosystem, the Candeias landfill should be revisited, and the structure and composition of the resident microbial communities should be analyzed again in the future. Acknowledgments This work was supported by grant APQ-10653.07/10 from the FACEPE foundation (Fundação de Amparo à Ciência e Tecnologia de Pernambuco) and grants 560389/2010-8 and 481357/ 2011-4 from the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). We thank the Sanitary Landfill CTR Candeias for the samples and for their cooperation and CENAPAD/ Dept. of Mechanical Engineering, UFPE–Recife for providing the parallel computing resources.

References ABNT- Brazilian National Standards Organization (2004) MBR 10004, Solid Waste Classification Allen A (2001) Containment landfills: the myth of sustainability. Eng Geol 60:3–19 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Baun A, Ledin A, Reitzel L, Bjerg P, Christensen T (2004) Xenobiotic organic compounds in leachates from ten Danish MSW landfills— chemical analysis and toxicity tests. Water Res 38:3845–3858 Berdugo-Clavijo C, Gieg LM (2014) Conversion of crude oil to methane by a microbial consortium enriched from oil reservoir production waters. Front Microbiol 5. doi:10.3389/fmicb.2014.00197 Callbeck CM, Dong X, Chatterjee I, Agrawal A, Caffrey SM, Sensen CW, Voordouw G (2011) Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection. Appl Environ Microbiol 91:799–810 Calli B, Mertoglu B, Roest K, Inanc B (2006) Comparison of long-term performances and final microbial compositions of anaerobic reactors treating landfill leachate. Bioresour Technol 97:641–647 Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336 Caron DA, Countway PD (2009) Hypotheses on the role of the protistan rare biosphere in a changing world. Aquat Microb Ecol 57:227–238 Caruso T, Pigino G, Bernini F, Bargagli R, Migliorini M (2008) The Berger–Parker index as an effective tool for monitoring the

biodiversity of disturbed soils: a case study on Mediterranean oribatid (Acari: Oribatida) assemblages. In: Hawksworth D, Bull A (eds) Biodiversity and Conservation in Europe. Springer, Netherlands, pp 35–43 Chao A, Lee SM (1992) Estimating the number of classes via sample coverage. J Am Stat Assoc 87:210–217 Chen S, Dong X (2005) Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater. Int J Syst Evol Microbiol 55:2257–2261 R Development Core Team (2008) R: a language and environment for statistical computing. http://www.R-project.org. Diaz EE, Amils R, Sanz JL (2003) Molecular ecology of anaerobic granular sludge grown at different conditions. Water Sci Technol 48:57– 64 Diaz EE, Stams F, Amils R, Sanz JL (2006) Phenotypic properties and microbial diversity of methanogenic granules from a full-scale UASB reactor treating brewery wastewater. Appl Environ Microbiol 72:4942–4949 Dojka MA, Hugenholtz P, Haack SK, Pace NR (1998) Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl Environ Microbiol 64: 3869–3877 Dröge S, Fröhlich J, Radek R, König H (2006) Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite, Neotermes castaneus. Appl Environ Microbiol 72:392–397 Eggen T, Moeder M, Arukwe A (2010) Municipal landfill leachates: a significant source for new and emerging pollutants. Sci Total Environ 408:5147–5157 Fernandez N, Diaz EE, Amils R, Sanz JL (2008) Analysis of microbial community during biofilm development in an anaerobic wastewater reactor. Microb Ecol 56:121–132 Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methe B, DeSantis TZ, Petrosino JF, Knight R, Birren BW (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454pyrosequenced PCR amplicons. Genome Res 21:494–504 Hata J, Miyata N, Kim E-S, Takamizawa K, Iwahori K (2004) Anaerobic degradation of cis-1,2-dichloroethylene and vinyl chloride by Clostridium sp. strain DC1 isolated from landfill leachate sediment. J Biosci Bioeng 97:196–201 Huang L-N, Zhou H, Chen Y-Q, Luo S, Lan C-Y, Qu L-H (2002) Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval. FEMS Microbiol Lett 214:235–240 Huang L-N, Chen Y-Q, Zhou H, Luo S, Lan C-Y, Qu L-H (2003) Characterization of methanogenic Archaea in the leachate of a closed municipal solid waste landfill. FEMS Microbiol Ecol 46: 171–177 Huang L-N, Zhou H, Zhu S, Qu L-H (2004) Phylogenetic diversity of bacteria in the leachate of a full-scale recirculating landfill. FEMS Microbiol Ecol 50:175–183 Huang L-N, Zhu S, Zhou H, Qu L-H (2005) Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill. FEMS Microbiol Lett 242:297–303 Jost L (2006) Entropy and diversity. Oikos 113:363–375 Kampmann K, Ratering S, Baumann R, Schmidt M, Zerr W, Schnell S (2012) Hydrogenotrophic methanogens dominate in biogas reactors fed with defined substrates. Syst Appl Microbiol 35:404–413 Kjeldsen P, Barlaz MA, Rooker AP, Baun A, Ledin A, Christensen TH (2002) Present and long-term composition of MSW landfill leachate: a review. Crit Rev Env Sci Tech 32:297–336 Krause L, Diaz NN, Edwards RA, Gartemann K-H, Krömeke H, Neuweger H, Pühler A, Runte K, Schlüter A, Stoye J, Szczepanowski R, Tauch A, Goesmann A (2008) Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor. J Biotechnol 136:91–101

Appl Microbiol Biotechnol Laloui-Carpentier W, Li T, Vigneron V, Mazéas L, Bouchez T (2006) Methanogenic diversity and activity in municipal solid waste landfill leachates. Anton Leeuw 89:423–434 Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics, Stackebrandt, E Goodfellow, M. Wiley, New York, pp 115–175 Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82:6955–6959 Lara-Martín PA, Gómez-Parra A, Köchling T, Sanz JL, Amils R, González-Mazo E (2007) Anaerobic degradation of linear alkylbenzene sulfonates in coastal marine sediments. Environ Sci Technol 41:3573–3579 Liu J, Wu W, Chen C, Sun F, Chen Y (2011) Prokaryotic diversity, composition structure, and phylogenetic analysis of microbial communities in leachate sediment ecosystems. Appl Microbiol Biotechnol 91:1659–1675 Maphosa F, van Passel MW, de Vos WM, Smidt H (2012) Metagenome analysis reveals yet unexplored reductive dechlorinating potential of Dehalobacter sp. E1 growing in co-culture with Sedimentibacter sp. Environ Microbial Rep 6:604–616 McDonald JE, Houghton JN, Rooks DJ, Allison HE, McCarthy AJ (2012) The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters. Environ Microbiol 14:1077–1087 Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Wagner H (2013) vegan: community ecology package. http://CRAN.R-project.org/package= vegan. Pohlschroeder M, Leschine SB, Canale-Parola E (1994) Spirochaeta caldaria sp.nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum. Arch Microbiol 161:17–24 Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596 Reeder J, Knight R (2010) Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat Methods 7: 668–669

Röling WFM, van Breukelen BM, Braster M, Lin B, van Versefeld HW (2001) Relationships between microbial community structure and hydrochemistry in a landfill leachate-polluted aquifer. Appl Environ Microbiol 67:4619–4629 Shen T, Chao A, Lin C (2003) Predicting the number of new species in further taxonomic sampling. Ecology 84:798–804 Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored Brare biosphere^. Proc Natl Acad Sci U S A 103:12115–12120 Tao Y, Zhou Y, He X, Hu X, Li D (2014) Pseudomonas chengduensis sp. nov., isolated from landfill leachate. Int J Syst Evol Microbiol 64: 95–100 Uchida M, Hatayoshi H, Aoi S, Shimoyama T, Nakayama T, Okuwaki A, Nishino T, Hemmi H (2009) Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of microbial community in landfill leachate. J Hazard Mater 164:1503–1508 Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267 Winderl C, Anneser B, Griebler C, Meckenstock RU, Lueders T (2008) Depth-resolved quantification of anaerobic toluene degraders and aquifer microbial community patterns in distinct redox zones of a tar oil contaminant plume. Appl Environ Microbiol 74:792–801 Wittebolle L, Vervaeren H, Verstraete W, Boon N (2008) Quantifying community dynamics of nitrifiers in functionally stable reactors. Appl Environ Microbiol 74:286–293 Xie B, Xiong S, Liang S, Hu C, Zhang X, Lu J (2012) Performance and bacterial compositions of aged refuse reactors treating mature landfill leachate. Bioresour Technol 103:71–77 Zhang W, Yue B, Wang Q, Huang Z, Huang Q, Zhang Z (2011a) Bacterial community composition and abundance in leachate of semi-aerobic and anaerobic landfills. J Environ Sci 23:1770–1777 Zhang Z, Hou Z, Yang C, Ma C, Tao F, Xu P (2011b) Degradation of nalkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8. Bioresour Technol 102:4111–4116

Analysis of microbial community structure and composition in leachates from a young landfill by 454 pyrosequencing.

Microorganisms are responsible for degrading the raw leachate generated in sanitary landfills, extracting the soluble fraction of the landfill waste a...
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