The Impact of Silver Nanoparticles on the Composting of Municipal

Oct 21, 2013 - School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, Cincinnati, Ohio. ‡ Pegasus Technical ...
0 downloads 0 Views 3MB Size
Article pubs.acs.org/est

The Impact of Silver Nanoparticles on the Composting of Municipal Solid Waste Alireza Gitipour,† Amro El Badawy,† Mahendranath Arambewela,‡ Bradley Miller,§ Kirk Scheckel,§ Michael Elk,§ Hodon Ryu,§ Vicente Gomez-Alvarez,§ Jorge Santo Domingo,§ Stephen Thiel,† and Thabet Tolaymat*,§ †

School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, Cincinnati, Ohio Pegasus Technical Services Inc., Cincinnati, Ohio § U.S. Environmental Protection Agency, Office of Research and Development, Cincinnati, Ohio ‡

S Supporting Information *

ABSTRACT: The study evaluates the impact of polyvinylpyrrolidone (PVP) coated silver nanoparticles (PVP-AgNPs) on the composting of municipal solid waste. The results suggest that there was no statistically significant difference in the leachate, gas, and solid quality parameters and overall composting performance between the treatments containing the AgNPs, Ag + , and negative control. Nonetheless, taxonomical analyses of 25 Illumina 16S rDNA barcoded libraries containing 2 393 504 sequences indicated that the bacterial communities in composted samples were highly diverse and primarily dominated by Clostridia (48.5%), Bacilli (27.9%), and beta-Proteobacteria (13.4%). Bacterial diversity studies showed that the overall bacterial community structure in the composters changed in response to the Ag-based treatments. However, the data suggest that functional performance was not significantly affected due to potential bacterial functional redundancy within the compost samples. The data also indicate that while the surface transformation of AgNPs to AgCl and Ag2S can reduce the toxicity, complexation with organic matter may also play a major role. The results of this study further suggest that at relatively low concentrations, the organically rich waste management systems’ functionality may not be influenced by the presence of AgNPs.

1. INTRODUCTION Silver has been recognized for its antimicrobial properties for over 2000 years.1 Recently, silver was engineered into nanoparticles, structures having at least one dimension ranging from 1 to 100 nm in size, with unique physiochemical properties that differentiate them from their bulk counterparts. The relatively high surface area to volume ratio of nanoparticles leads to more reactivity and sometimes-higher toxicity than the bulk material.2 Silver nanoparticles (AgNPs) are receiving much attention and are currently employed in a broad range of applications.2 The strong antibacterial properties of AgNPs have encouraged their use as antibacterial/antifungal agents in various types of consumer products.3 The increasing rate of applications has caused AgNPs to become one of the most widely commercialized nanomaterials today.2 While a number of studies attempted to explain the mechanisms by which AgNPs exert their antimicrobial activity, the proposed mechanisms are still not fully understood. Proposed mechanisms include a combination of a release of silver ions by AgNP dissolution under aerobic conditions and specific AgNP properties such as nanoparticle transport by a Trojan-horse type mechanism followed by generation of © XXXX American Chemical Society

reactive oxygen species (ROS) leading to cell membrane damage.4−7 Several studies have demonstrated AgNPs to be toxic in both aerobic and anaerobic environments.8,9 Like many other nanoparticles, AgNPs have the potential for release into the environment throughout their life cycle. At the end of their useful life, products containing nanoparticles are often disposed of with the municipal solid waste stream. In a disposal scenario, nanoparticles (e.g., AgNPs) may leach from products into the solid waste. Research by Benn and Westerhoff suggested that AgNPs could be released from AgNP-coated socks. Nanoparticles could also enter the solid waste stream with biosolids from wastewater treatment plants.10 While landfilling is still the dominant method for solid waste management in the United States (U.S.),11 composting is gaining momentum. Of the approximately 250 million tons of solid waste generated in the U.S in 2010, the recycled and composted fraction of this waste exceeded 85 million tons.11 Received: June 5, 2013 Revised: August 28, 2013 Accepted: October 21, 2013

A

dx.doi.org/10.1021/es402510a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

Figure 1. Schematic of the experimental setup of the compost reactors.

The study also examines relatively low concentrations that may be encountered in a real world scenario and how such exposure may impact the function and composition of microbial communities associated with compost samples. More recently, studies measuring the impact of AgNPs on mixed microbial communities have used molecular profiling techniques such as T-RFLP23 and DGGE analysis of 16S-rRNA gene sequences.23,24 For example, Colman et al.23 showed that AgNPs can have an effect on the microbial composition of sediments as determined by T-RFLP. It should be noted that these community profiling methods can only provide an idea of how treatments can impact the most abundant populations and therefore generate an incomplete picture for the rest of the bacterial community members. Moreover, such profiling methods do not identify the members of the community. Alternatively, next generation sequencing methods utilized in the current study can identify a large number of the bacterial populations within samples exposed to AgNPs, including relatively low abundant members, and provide relevant information on the population dynamics within compost samples as a result of exposure to AgNP treatments.

Composting is recognized as a low cost, environmentally sound process and has been recommended as an alternative to landfilling of food and green wastes.12 There are concerns associated with the growing use of AgNPs and their potential impact on waste management systems that largely depend on microbial decomposition for waste stabilization. Modeling results indicate that up to 15% of the total silver in the forms of Ag+ and/or AgNPs could be released from biocidal plastics and textiles.13,14 Other studies have shown between 34% and 80% of the Ag released from commercially available functional (nano) textiles are in the form of AgNPs (nanocomposites, elemental AgNPs, and nanoAgCl particles).15 The properties of the released AgNPs (e.g., stabilization mechanism and the chemistry of the capping agent) and the surrounding environmental conditions (e.g., the pH, ionic strength, electrolyte type, and the presence of natural organic matter (NOM)) govern their environmental fate and toxicity.16,17 Ho et al. reported that aggregated nanoparticles posed lower toxicity relative to stable nanoparticles.18 In addition to aggregation, AgNPs may undergo surface transformations that will influence their behavior. For example, in a system that has high concentrations of chlorides and sulfides (e.g., wastewater, composter leachate, and landfill leachate), the released AgNPs may transform to silver chlorides (AgCl) and silver sulfides (Ag2S), which are less toxic relative to the metallic AgNPs.19,20 The current study aims at investigating the impacts of PVPAgNPs on the composting process of the biodegradable organic fraction of municipal solid waste. This research represents one of the few studies that evaluate end-of-life management concerns with regard to the increasing use of nanomaterials in everyday life. PVP-AgNPs are used since sterically stabilized NPs showed minimal aggregation in high ionic strength solutions with different background electrolyte valences (up to 1 M Na+ and Ca2+).21 The conductivity of compost leachate has been reported to range between 750 and 9000 uS cm−1,22 which translates to approximately 0.01−0.2 M ionic strength. Therefore, PVP-AgNPs are not expected to aggregate under the experimental ionic strength.

2. EXPERIMENTAL SECTION 2.1. Nanoparticles, Selection, Synthesis, and Purification. The toxicity of nanomaterials including AgNPs is impacted by the size of the nanoparticles, and thus, it is critical to utilize a nanoparticle that could resist size change resulting from aggregation. Aggregation may also lead to the nanoparticles settling out of solution and as a result a lower level of interaction between the AgNPs and the microorganisms in the compost media, which may further confound the results of the experiment and increase the uncertainty and accuracy of the conclusions on the toxicological impacts of nanomaterials. Therefore, the highly stable PVP-AgNPs were utilized in the current study. It has been reported that PVP-AgNPs resist aggregation in high ionic strength solutions with high valence background electrolytes25 which are typical conditions in a compost system. Additionally, research showed that the most commonly used capping agents for silver nanoparticles are B

dx.doi.org/10.1021/es402510a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

citrate and polyvinylpyrrolidone (PVP).25 This makes PVPAgNPs a suitable nanoparticle to be utilized for investigating the impacts of AgNPs in complex environmental settings. The polyvinylpyrrolidone (PVP) coated AgNPs (PVP-AgNPs) were prepared and purified according to a method described previously by El Badawy et al.26 (more details are presented in Supporting Information (SI)). The hydrodynamic diameter (HDD) and zeta potential of the AgNPs were measured using a Zetasizer Nanoseries (Malvern Instruments). Transmission electron microscopy (TEM) was used to verify nanoparticles’ characteristics (size and shape). TEM samples were prepared by depositing a drop of the sample suspension on a carbon coated copper grid. Samples were air-dried at room temperature overnight in a dust-free box. Images were captured using a JEOL-1200 EX TEM (JEOL Inc.) operated at 120 kV. Total Ag concentrations were measured using a PerkinElmer AAnalyst 800 atomic absorption spectrometer after microwave acid digestion following EPA method 3015A. 2.2. The Compost Reactors. Nine composters (Figure 1) each with a volume of 130 L were used in the study. To maintain the uniformity of food waste composition, a synthetic food waste mixture (Figure S1, SI) was prepared in a manner to obtain an initial carbon-to-nitrogen (C/N) ratio of 34 ± 3, which is in the range 18−40 considered ideal for the composting process, and as composting proceeds, C/N gradually decreases until reaching levels below 12, indicating compost maturity.27 Each composter received 27 kg size reduced waste because solid waste systems are rather heterogeneous and to minimize the heterogeneity in the waste composition among the compost reactors, the waste components were size reduced by cutting each individual component into approximately 2 cm × 2 cm × 2 cm pieces. The nine reactors were divided into three batches (three reactors each); the first was used as a negative control (no treatment); the second was treated with Ag+ (using AgNO3 salt), and the third was treated with PVP-AgNPs. The total silver concentration in both treatments was 2 mg kg−1 of compost. The composters were operated at a temperature of 50 °C (optimum for the composting process) and an initial moisture content of 65% (w/w) to be in the average range reported for compost moisture content.28 Warm humidified air was added to each reactor at a rate of 2.5 L min−1 for the duration of the study. The composters were mixed daily and monitored for 60 days, during which most of the compost activity occurs.29 2.3. Composting Sampling and Analysis. To evaluate the performance of the composters, gas, leachate, and solid samples were monitored. Gas composition was evaluated at 8-h intervals for the initial two weeks and 12-h intervals for the remainder of the experiment. The gas samples were collected using a 50 mL airtight syringe and immediately analyzed for the concentration of CO2, O2, CH4, H2, and N2O using a gas chromatograph (GC, Shimadzu) equipped with a thermal conductivity detector (TCD) and a flame ionization detector (FID). Prior to gas sampling, air introduction was stopped for 15 min to allow headspace gas to equilibrate. Leachate samples were collected twice per week and analyzed for pH, conductivity, chemical oxygen demand (COD; HACH, DR890 Colorimeter), total organic carbon (SHIMADZU, TOC-V,EPA Method 9060A), ammonia-nitrogen (HACH, DR890 Colorimeter), and chloride (DIONEX ICS-2000 Ion Chromatograph). The leachate samples were also analyzed for

total silver content (GF-AA) after digestion using EPA method 3015. Solid samples were analyzed for carbon to nitrogen ratio (C/ N) using a Carlo Erba NC2500 elemental analyzer (more details are presented in the SI). The surface transformations of AgNPs were determined using X-ray Photoelectron Spectroscopy (XPS; Surface Science Laboratories SSX-100 XPS) and Xray absorption spectroscopy (XAS; details available in the SI). The total silver concentrations of the solid samples were determined using (GF-AA) after acid digestion method 3051. The solid sample collection was performed after rotating and lifting the compost reactors in order to obtain representative samples. The leachate and gas data were analyzed using an ANOVA test (SigmaPlot 12.0, Systat Software Inc.) where statistical significance was calculated using untreated samples (control) and treated samples (AgNO3 and AgNPs), and those with a p value < 0.05 are considered significant. 2.4. DNA Sequencing and Bacterial Composition and Diversity Analyses. Approximately, 0.25 g (wet weight) of each compost sample was used to extract total DNA using the UltraClean Soil DNA kit following the manufacturer’s instructions (MoBio Laboratories Inc., Solana Beach, CA). An aliquot of the DNA extracts (2 μL) was used to partially amplify the 16S rRNA gene using barcoded primers 515F and 806R,30 which allows the generation of overlapping forward and reverse reads for each sequence. The amplification step was performed using a BioRad Tetrad and using the following thermal cycling conditions: (1) 95 °C for 3 min; (2) 35 cycles of 45 s at 95 °C, 60s at 50 °C, and 72C; (3) 72 °C for 10 min. Illumina MiSeq coupled with pair end 250 bp kits were used to generate sequencing data. Prior to analyses, primer sequences were removed resulting in average sequence reads of 251 bp. The sequences were processed and analyzed using the software MOTHUR v1.30.1.31 Briefly, fastq files for forward and reverse reads were used to form contigs which were first screened for sequence length (no greater than 255 bp). Sequences with ambiguous bases (N’s), containing homopolymers greater than seven bases, and classified as chloroplasts and mitochondria were removed. To reduce computational time, unique sequences were used to identify chimeras and to generate distance matrix and cluster analyses. MOTHUR was used to align and sort sequences with >97% similarity into operational taxonomic units (OTUs). Chimera-free sequences were classified using the tool Classifier in Ribosomal Database Project II release 10.28.32 Barcoded 16S rRNA gene libraries were developed for 25 out of the 27 compost samples available from weeks one, two, and four. After removing undesirable sequences (i.e., chimeras, and contigs containing ambiguous bases, homopolymers, or outside of the sequence length range), a total of 2 393 504 reads were used in downstream analyses. The total number of contigs containing unique sequences was 178 085. The latter data set was composed of 120 699 unique sequences (OTUs) at the 99% sequence identity, which were then used in diversity analyses to further reduce computational needs. The latter sequences were used to determine the bacterial composition of the samples and the impact of Ag-based treatment on community structure and bacterial diversity. Sequences from rare members were eliminated from analyses that focused on the most abundant OTUs. 2.5. Species Richness, Diversity, and Statistical Analysis of Microbial Communities. Prior to analysis, read libraries (i.e., samples) were normalized by randomization C

dx.doi.org/10.1021/es402510a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

matrices such as composting. Furthermore, purification of the suspension ensured that any observed effects were associated with nanoparticles and not impurities carried over from the synthesis process. 3.2. Gas Quantity and Quality. Microbial respiration is a commonly used indicator when evaluating the extent of biological activity present in the composting process.40,41 Therefore, potential impacts of AgNPs on composting could be indirectly evaluated using gaseous emissions. Gas samples were analyzed for CO2, O2, CH4, H2, and N2O concentration. Regardless of the treatment, CH4 and H2 concentrations were below the method detection limits (0.3% for CH4 and 0.04% for H2). As expected, the average O2 concentrations (Figure 3a)

to the smallest data set (i.e., 35 913 reads). Species richness (S), Shannon−Wiener diversity (H), and evenness (EH) were calculated for each sample according to methods described elsewhere.33 Subsampling and diversity indices were calculated with MOTHUR.34 In within-sample diversity (i.e., Shannon− Wiener) and taxonomic distribution (e.g., % of phylum Clostridia) for each sampling time were calculated using the coefficient of variation (CV). The CV is described in Shade et al. and is defined by the equation CV = σ/μ, where σ is the standard deviation and μ is the mean.35 Nonmetric multidimensional scaling (nMDS) analysis based on the Bray−Curtis similarity coefficient of the transformed data (log[x + 1]) was used to describe the relationships among microbial communities based on the relative distribution of OTU groups. A twoway crossed analysis of similarities (ANOSIM) was used to identify with significant differences (p < 0.05) the community assemblages among treatments and between sampling times. R values near 0 indicate no difference between groups, whereas those greater than 0 indicate dissimilarities between groups.36 Similarity Percentage (SIMPER) analysis was used to determine which species were most responsible for the differences observed between communities of different treatments and sampling times.34 All statistical analyses were performed with the software PAST v2.17c.37

3. RESULTS AND DISCUSSION 3.1. AgNPs Characterization. The PVP-AgNPs investigated, suspenstion pH of 7.0, had a zeta potential (ζ) of −10.2 mV and a HDD of 12.3 ± 0.5 nm. The weight of evidence from the literature suggests that engineered nanoparticles are likely to be of concern owing to their unique properties when they have diameters of 30 nm or less31.38 The AgNPs average size obtained from TEM was 25.8 (Figure 2), which is different than

Figure 2. TEM image of PVP-coated AgNPs. Figure 3. Average gas levels in composters’ headspace (a) O2, (b) CO2, and (c) N2O.

the average size obtained by the DLS (12.3 nm). It has been reported in the literature that the TEM size of nanoparticles may be different from the DLS size for various reasons, including the potential increase in size as a result of the drying process during the preparation of the samples for TEM analysis.25 As previously mentioned, the PVP stabilized AgNPs were used in this experiment because they are known to resist aggregation under various environmentally relevant conditions.25 Kvitek et al. determined that PVP is the most effective polymer for stabilizing AgNPs,39 and thus a good candidate for investigating the toxicological impacts of AgNPs under complex

correlated with CO2 concentrations (Figure 3b) as a result of aerobic microbial respiration. No statistically significant differences were observed for the average daily levels of CO2 and NO2 (Figure 3b and c; p > 0.05; the averages with the standard deviations of the gas measurements are presented in Figure S2, SI). As will be discussed later in the manuscript, most of the silver in the current study is bound to the organic matter present and may have caused a reduction in the potential toxicity of silver. D

dx.doi.org/10.1021/es402510a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

3.3. Leachate Quality. The pH of the compost leachate ranged between 6.7 and 8.8, which is well within the range commonly reported for the process42 (Figure S3, SI). The conductivity values (10−60 uS cm−1) for the compost leachate (Figure S4, SI) were a result of the high concentration of the water-soluble salts within the food waste.43 The TOC (4000− 25 000 mg·L−1) and COD levels (25 000−100 000 mg·L−1), presented in Figures S5 and S6 (SI), indicate that the compost materials were initially very high in organic matter, and as the composting process proceeded, the organic matter was mineralized by microbial activity, resulting in lower concentrations. In all treatments, the ammonia-nitrogen level peaked between days 8 and 12 and, by day 25, had decreased to relatively stable levels (Figure S7, SI). Overall, no statistically significant differences (p > 0.05) were observed between the various treatments. Low concentrations of total silver (Figure S8, SI) were detected in the leachate of the composters treated with silver nitrate and PVP-AgNPs, which may indicate a precipitation of silver as silver chlorides and adsorption to the waste materials. This is supported by the high concentrations of chlorides detected in the leachate samples as presented in (Figure S9, SI). In order to verify the transformations of AgNO3 and AgNPs to AgCl and other species, XPS and XAS analyses were performed on leachate samples. Silver was not detected, which can be attributed to the low concentration of silver present in the leachate (Table S1, SI). In order to overcome the detection problem, leachate samples were collected from the positive control (Ag+) and from the composters treated with PVP-AgNPs after one month. These samples were then spiked with 200 mg L−1 of Ag+ and PVPAgNPs, respectively. The spiked samples were analyzed using the XAS technique after one week of preparation. In the case of the positive control samples (Ag+), a strong silver signal was obtained, and the results confirmed the transformation of silver ions to AgCl, Ag cystine, and Ag humic phases (Figure 4 and Table S2; more detail about the identification of these species is

presented in SI). On the other hand, a weak signal was obtained from the leachate samples spiked with the PVP-AgNPs (data not shown). The weak signal may have resulted from the attachment of the NPs on the solid organic fractions present in the leachate and the formation of bigger aggregates that subsequently settle, along with the attached, AgNPs out of solution and as a result were not detected. Nonetheless, under the current experimental conditions, phase transformations can occur and significantly affect the stability, bioavailability, and toxicity of Ag-NPs.19 Also, the Cl− ions present in the media react with the dissolved silver ions and form AgCl as confirmed by XAS analysis. Therefore, the surface transformation of AgNPs as well as Ag+ to AgCl may further explain the results observed in the current study. Choi et al.5 demonstrated that the toxicity of AgNPs to nitrifying bacteria was reduced in the presence of various anions such as chloride and sulfate. It is noted that previous studies have suggested that one of the toxicity mechanisms of AgNPs is through the dissolution to Ag+.7 With the presence of a relatively high concentration of Cl− in solution, most of the ionic silver transforms to AgCl.19 3.4. Solids Quality. One of the key indicators of compost decomposition is the carbon to nitrogen (C/N) ratio. A decreasing trend in the ratio of C/N, followed by the eventual stabilization of the organics, can generally be observed as composting proceeds. This is caused by the loss of carbon from the system as a result of the decomposition of organic substrates and the release of CO2.43,44 The C/N ratio of the compost materials used in the current study is presented in (Figure S10, SI). Initially, the C/N of the utilized composting materials was 34, and as the experiment progressed, the C/N ratio gradually decreased to 10 by the end of the study, indicating compost maturity. The relatively high initial value could be a result of the higher proportion of waste high in carbon (e.g., paper and cardboard) to waste high in nitrogen (e.g., grass; Figure S1, SI). No significant differences were found for C/N among the treatments (p > 0.05). The C/N ratio obtained herein was in agreement with ratios and trends reported in previous studies.45 The concentration of total silver in the final composted solid samples ranged between 3 and 6 mg kg−1 (Figure S11, SI), suggesting that a large fraction of the silver was adsorbed onto or complexed with the natural organic matter of the solid media.46 Even with the increase of silver in the solid phase, the silver concentrations in the samples were below the detection limit of the XPS as presented in Table S1 (SI). XAS analysis was also used to determine possible surface transformation of the silver nanoparticles in the compost solid samples. At time zero (the first possible time a sample was collected once the NPs were mixed into the system), the XAS spectra showed that the PVP-AgNPs attached to the compost material was mostly in the metallic form (Figure 4 and Table S2 (SI)). After one month, the silver was not detected using XAS analysis. It is noted that heterogeneity is rather high in the 27 kg compost reactor utilized in the current study. Even with the mixing, variability in AgNPs content in the collected samples is inevitable. With silver content around the detection limit for the XAS, it is not surprising that silver was not detected. Therefore, neither the XPS nor the XAS analyses confirm the surface transformations of AgNPs to AgCl in the solid compost samples. Nevertheless, this does not eliminate the possibility of this transformation occurring especially with the high amounts of chlorides present in the system. Gunawan et al. showed the influence of the oxidation state of Ag on its antimicrobial

Figure 4. Ag Kα XAS spectra of AgCl, Ag Cystine, and Ag Humic as pure phases and PVP-AgNPs reacted with the compost material and AgNO3 with the compost leachate. E

dx.doi.org/10.1021/es402510a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

action.47 AgNPs are not thermodynamically stable under most environmental conditions and will oxidize or react with organic ligands.48 As an example, silver is known to react strongly with sulfide, chloride, and organic matter in relevant environmental situations.19 Another possible explanation of the lack of overall impact of AgNPs on the composting process is the complexation with the compost products containing high amounts of natural organic matter (NOM), which can limit the AgNPs bioavailability.46 3.5. Bacterial Diversity Analyses. Differences in community structure profiles were observed between control samples and treated samples, although differences were not statistically significant between treatments (Table 1and Table

bacterial community structure (i.e., regardless of treatment). The variation could also depend on the level of dissolved ions as well as direct contact of NPs with microbial cells.49 For example, Xiu et al.50 proposed that under anaerobic conditions environmental parameters that stimulate Ag+ ion release play a larger role that particle size and physical contact. In contrast with community sequencing profiles, richness, evenness, and Shannon diversity indices were not significantly different between treatments and over time (Figure 6, Figures S15 and S16, SI) suggesting that, while the treatments were perturbing the microbial composition, they were not catastrophic. The data also suggest that highly diverse microbial communities can help to deal with perturbations associated with AgNP exposure, perhaps due to the presence of functional redundant bacterial groups. Relatively low impacts in microbial composition have been observed for other environmental matrices after AgNP exposure. For example, Sun et al.24 found that AgNPs did not change the community structure of unsettled activated sludge after 24 h treatments. Using pyrosequencing, Doolette et al.51 showed that the dominant populations in wastewater reactors were not significantly impacted by the addition of low doses of AgNPs. Microbial processes such as nitrification and methanogenesis were not impacted either. The presence of 120 699 OTUs suggests that these compost samples are very diverse. Even when rare members were removed (sequences present only once in all the samples, Figure S12, SI), more than 9368 OTUs were identified. To the authors’ knowledge, this is the highest number of OTUs reported for compost samples. The most represented bacterial taxa at the class level were Clostridia (48.5%), Bacilli (27.9%), and beta-Proteobacteria (13.4%) (Figure 5). Other classes represented were gamma- and alpha-Proteobacteria and Erysipelotrichia, but to a much lesser extent (