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Research article
Characterization of the Bacterial Community Associated with Methane and Odor in a Pilot-Scale Landfill Biocover under Moderately Thermophilic Conditions
1Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
2Green Environmental Complex Center, Suncheon 57992, Republic of Korea
J. Microbiol. Biotechnol. 2021; 31(6): 803-814
Published June 28, 2021 https://doi.org/10.4014/jmb.2103.03005
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Methane is a significant greenhouse gas that has a 28–36 times stronger effect on global warming than carbon dioxide [1]. Landfills are a major source of methane due to the anaerobic digestion of organic matter [2], during which odor compounds, including hydrogen sulfide (H2S), methanethiol, dimethyl sulfide (DMS), and volatile organic compounds, are also produced as byproducts [3-5]. Odor compounds are not only a nuisance but also pose a potential hazard to human health at high concentrations, with long-term exposure often leading to emotional stress and physical symptoms such as anxiety, headaches, vomiting, eye irritation, and respiratory problems [6-9]. Therefore, the mitigation of both methane and odor is necessary for anaerobic digestion processes.
Methane and odor compounds emitted by anaerobic digestion can be mitigated using biological, chemical, or physical treatment [3, 10-12]. Biological treatment is more environmentally friendly and safer than chemical and physical approaches because it does not require the use of chemicals and can be conducted at normal temperatures and pressures [3, 10, 11]. Biological treatment also offers high treatment efficiency, simple systems, and low treatment costs [3, 12]. Therefore, biological systems such as biocovers and biowindows have attracted attention as promising technologies for the control of the methane and odor compounds emitted from landfills [10, 11].
Microbial activity, which determines the performance of biological treatment systems [13], is primarily influenced by environmental factors such as temperature, pH, and moisture content [3, 14]. For example, if the temperature of the biological treatment system is outside the range that most microorganisms can tolerate, the microbial activity is dramatically lower [14]. Most biological systems designed to mitigate the methane and odor compounds emitted by landfills employ mesophilic microorganisms [15-17]. Because the activity of mesophilic microbes decreases rapidly at low temperatures, it could be expected that the performance of biological treatment systems would deteriorate during winter. However, this is not the case because the internal temperature of these systems is maintained at around 8–18°C due to the heat generated by the biodegradation of methane and odor compounds and the effect of insulating materials [10, 11, 18].
Mesophilic microorganisms are generally less active under moderately thermophilic conditions of 40°C or more. At midday during summer, the temperature inside biological treatment systems may rise to over 40°C due to the effects of solar radiation. Information on the composition and dynamics of the microbial community under moderately thermophilic conditions is thus essential when designing and setting the operating parameters for biological treatment systems during the hot season or in subtropical countries, but there is little information available on this. Therefore, in this study, the methane and odor removal performance of a pilot-scale biocover during the summer and non-summer seasons was compared. In particular, the structure of the bacterial community during summer was characterized and compared with the non-summer seasons. In addition, the degradation of methane and odor compounds by the packing material sampled from the biocover during summer was evaluated under moderately thermophilic conditions. Thermotolerant or thermophilic methane- and odor-degrading bacteria were also isolated and their roles in the degradation of methane and odor compounds investigated.
Materials and Methods
Packing Material for the Biocover
A pilot-scale biocover was constructed at the Gwangyang Sanitary Landfill, located at latitude 34°58’0’’ and longitude 127°38’35’’. This sanitary landfill began operation in 1996 and has a total disposal capacity of 3,145,291 m3 [10, 19]. The packing material for the biocover was a mixture of soil, perlite (Kyungdong One Co. Ltd., Korea), food waste compost, and earthworm cast (Kumhosilup, Korea). The physiochemical characteristics of these materials were described previously by Lee
Construction of the Pilot-Scale Biocover and Packing Material
A schematic diagram of the biocover (2.5 m wide × 2.5 m long × 1.2 m deep) is presented in Fig. S1. For thermal insulation, polystyrene foam boards (5 cm thick) were placed along the inside of the walls. The solid waste was first covered with a 0.23-m thick layer of gravel (particle diameter of 2–5 cm) and then a polypropylene non-woven textile sheet (Kyungdong One Co. Ltd.,). A 0.9-m-thick soil layer was placed on top of the textile cover. A perforated pipeline was installed at the bottom of the biocover and connected to polyvinyl chloride (PVC) pipes for biocover inlet gas sampling. An acrylic chamber (2.5 m wide × 2.5 m long × 0.3 m high) was installed on the surface of the biocover for gas sampling from the biocover surface (
Ambient Temperature, Precipitation, and Physicochemical Properties of the Packing Material
Ambient temperature and precipitation measurements during the experimental period were obtained from the Gwangyang Automatic Weather Station operated by the Korea Meteorological Administration. The packing material in the biocover was sampled between 11 and 12 o’clock on days 0, 40, 68, 99, 133, 163, 198, 238, and 252 at 0–15 cm (upper layer), 15–30 cm (middle layer), and 30–50 cm (bottom layer) from the surface of the biocover. Immediately before taking the sample, the temperature of the packing material in each layer was measured using a portable digital thermometer (SDT200, Summit Co. Ltd., Korea).
After being passed through a 2-mm sieve, the samples were stored at 4°C for the assessment of their physicochemical properties (pH, moisture content, and organic matter content) and at –20°C for the analysis of the bacterial community. The moisture and organic matter content of the samples were measured based on the Korean Standard Soil Analysis Method [10] and the Korean Standard Waste Analysis Method [10], respectively. To measure the pH of the samples, 3 g of each sample was mixed with 20 mL of distilled water, and the supernatant was collected after allowing the particles to settle for 5 min. The pH of the resulting supernatant was measured with a pH meter (Thermo Orion 535A, USA).
Gas Analysis
Gas samples from the inlet port and the surface of the biocover were collected on days 12, 39, 63, 98, 124, 165, 182, 223, and 253. The gas sampling was conducted using the same method described in a previous study [10]. The methane concentration in the gas samples was measured using a gas chromatograph equipped with a flame ionization detector [10]. The methane levels were also measured in the field using a biogas check analyzer (Geotechnical Instruments, UK) [10]. The concentrations of 22 odor compounds designated as key offensive odors by the Korean Odor Prevention Law were analyzed using the same methods described in a previous study [10]. Complex odor compounds were analyzed using the odor dilution ratio (ODR) [20]. Details on the calculation of the ODR are available in Lee
Bacterial Community Analysis Using Illumina MiSeq Sequencing
The packing material sampled at 15–30 cm on days 0–252 was used to characterize the bacterial community dynamics in the biocover. For DNA extraction, 0.5 g of each sample was transferred to a microtube from the stored bottle, and the DNA was extracted using a NucleoSpin Soil Kit (Macherey-Nagel GmbH, Germany) and a Mini-BeadBeater-8 system (BioSpec, USA). DNA extraction was performed following the manufacturer’s instructions. The extracted DNA samples were eluted with 50 μl of an elution buffer and stored at –20°C before analysis. The extracted DNA was used as a PCR template to analyze the bacterial community with an Illumina MiSeq sequencing platform (Macrogen Inc., Korea) using the same method described in our previous paper [11]. Each composite primer was designed based on 515f and 806r primers [21]. Sequences shorter or longer than the target sequence were cut using CD-HIT-OTU [22], and chimera and noise were eliminated. The sequences with over 97% similarity were classified into operational taxonomic units (OTUs). Using the UCLUST algorithm [23], the taxonomy for each OTU was assigned based on the 16S rRNA RDP database. The Chao1 richness estimator and the Shannon index were also calculated [24]. The obtained sequence data were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/) under accession number SRP185598. Bacterial community dynamics were analyzed using principal component analysis (PCA) with UniFrac [25] and CANOCO 4.5 software (Microcomputer Power, USA).
Simultaneous Removal of Methane and Dimethyl Sulfide by the Packing Materials under Moderately Thermophilic Conditions
The simultaneous removal of methane and odor compounds was evaluated at 40°C and 50°C for three packing material samples taken on day 252 at 0–15 cm, 15–30 cm, and 30–50 cm from the surface of the biocover. In order to compare the removal efficiency under moderately thermophilic and medium temperature conditions, the same experiment was carried out at 30°Cs. DMS was selected as a representative odor compound. Five grams of the wet samples and 20 ml of nitrated mineral salt (NMS) medium were added to 600-ml serum bottles. The NMS medium contained 1.0 g/l MgSO4·7H2O, 0.2 g/l CaCl2·6H2O, 1.0 g/l KNO3, 0.272 g/l KH2PO4, and 0.717 g/l Na2HPO4·12H2O. The serum bottles were sealed with a butyl rubber stopper, and then methane gas from a cylinder (99%; Dong-A Gases, Korea) was injected into the bottles to a final concentration of 50,000 ppm [13]. In addition, DMS solution (99%; Acros Organics, Belgium) was injected into the bottles to a final concentration of 5,000 ppm. The serum bottles were incubated at 30°C, 40°C, and 50°C and 180 rpm in a shaking incubator. In order to prevent the stopper from falling out due to the expansion of the gas inside the serum bottle at 40°C and 50°C, 60 ml of the gas inside the serum bottle was removed using a syringe before putting the serum bottle into the incubator. The gas in the headspace of each serum bottle was periodically sampled using gas-tight syringes to measure the concentration of methane and DMS with a gas chromatography system (GC 7890, Agilent Technologies, USA) equipped with a 30 m × 320 μm × 1.8 μm capillary column (J&W Scientific, Inc., ISA) and a flame ionization detector (Agilent Technologies). The operating temperature of the oven, injector, and detector was 100°C, 230°C, and 230°C, respectively. The degradation rates for the methane and DMS were calculated as the reduction in the methane and DMS from their initial concentration to a concentration below 2,000 ppm and 500 ppm, respectively, divided by the incubation time per unit dry weight of the sample.
Isolation and Identification of Methane- and DMS-Degrading Bacteria under Moderately Thermophilic Conditions
To isolate methane- and DMS-degrading bacteria at 40°C and 50°C, enrichment cultures were developed. Packing material samples taken at 0–15 cm, 15–30 cm, and 30–50 cm from the surface of the biocover on day 252 were mixed in equal amounts with each other. Five grams of the mixed sample was placed in each of two 600-ml serum bottles containing 20 ml of NMS medium. After sealing the bottles with butyl rubber stoppers, methane gas and DMS solution were injected to final concentrations of 50,000 ppm and 5,000 ppm, respectively. One bottle was incubated at 40°C, and the other was incubated at 50°C in a shaking incubator (180 rpm). When the concentrations of methane and DMS in the headspace of each bottle decreased below the detection limit, methane gas and DMS solution were re-injected and the bottle was re-incubated at 40°C or 50°C. After repeating this process five times, 10 ml of the 1st enriched culture was transferred into 10 ml of fresh NMS medium in a 600 ml-serum bottle, and methane and DMS were injected into the bottle. Each bottle was incubated at 40°C or 50°C, and methane and DMS were re-supplied when their concentration fell below the detection limit before re-incubation. After repeating this process four times, 10 ml of the 2nd enriched culture was transferred into 10 ml of fresh NMS medium, methane and DMS were supplied, and the bottle was incubated at 40°C or 50°C.
After repeating this replenishment four times, the 3rd enriched culture was diluted with NMS medium, and the diluted culture was spread on NMS-agar (20 g/l) plates. The inoculated plates were incubated at 40°C or 50°C in a 5-L reactor, which was connected to a 3-L Tedlar bag containing 50,000 ppm of methane and 5,000 ppm of DMS. After incubation for 1–2 months, distinguishable colonies on the plates were carefully transferred to fresh NMS-agar plates and incubated in the same manner as described above. This process was repeated several times to produce four pure strains (HJ1, HJ2, HJ3, and HJ4) from the 40°C-enriched cultures and two pure strains (HJ5 and HJ6) from the 50°C-enriched cultures.
The simultaneous degradation of methane and DMS by the isolates in the serum bottles was evaluated at 40°C or 50°C using the same method as described in Section 2.6. To identify the isolates, genomic DNA samples were extracted using NucleoSpin Soil Kits (Macherey-Nagel GmbH Düren, Germany), and amplified with PCR using the primer set 340F (5’-TCCTACGGGAGGCAGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’)[26]. The sequence data were deposited in the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/) under the accession number MK577721 for the strain HY1, MK577720 for HY2, MK577719 for HY3, MK577718 for HY4, MK639430 for HY5, and MK577717 for HY6.
Results and Discussion
Variation in the Environmental Parameters
Environmental factors such as temperature, precipitation, moisture content, organic matter content, and pH affect biocover performance [10, 11, 27-32]. Fig. 1 presents the ambient temperature, precipitation, internal temperature, and physical characteristics of the packing material at different depths in the biocover. The average ambient temperature during winter (from December to February), spring (from March to May), and summer (from June to August) was –2.9–13.1ºC, 1.1–26.3ºC, and 18.0–31.5ºC, respectively (Fig 1A). Monthly precipitation was the highest in August (Fig. 1B). During winter and spring, the internal temperature tended to increase with greater distance from the surface of the biocover, with a range of 20–30ºC at midday (Fig. 1C), while that during summer was 41–49 ºC, even though the maximum ambient temperature was 21.4–37.0 ºC (Figs. 1A and 1C). There were four main reasons for this higher internal temperature: (1) the heat generated by biodegradation in the waste layer at the bottom of the biocover, (2) heat from the biodegradation of methane and odor compounds in the biocover, (3) heat from intense sunlight during the middle of the day, and (4) thermal insulation due to the polystyrene foam boards within the biocover (Fig. S1). Jung
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Fig. 1. Time profiles of the ambient temperature (A), precipitation (B), internal temperature (C), moisture content (D), organic matter content (E), and pH (F) of the packing material in the biocover.
The moisture content, organic matter content, and pH of the packing material in the biocover are presented in Figs. 1D-1F. The moisture content in the biocover mostly remained at around 20–25% at all sample depths, though it did increase to 30% on day 124 due to heavy precipitation (Fig. 1D). Cho and Ryu [3] reported that the optimal moisture content for biocover performance was 25–50%. The moisture content of the biocover in the present study thus fell within a suitable range for the simultaneous degradation of methane and odor compounds. During summer, despite the high evaporation rate, the moisture content in the biocover was maintained at a suitable level due to the frequent precipitation (Figs. 1A, 1B, and 1D). The successful removal of methane and odor compounds within a biocover with a low organic matter content of 5–10% has been reported [10, 11, 18]. In addition, most methane- and/or odor-degrading bacteria exhibit optimal activity at a neutral pH [3, 34, 35]. Considering these previous results, the moisture content and pH of the biocover were favorable for methane- and odor-degrading bacteria.
Methane and Odor Removal during Summer
Fig. 2A presents a time profile of the methane concentration and removal efficiency. The methane concentration at the biocover inlet was below 32% during winter and spring but ranged from 20–38% during summer. It is believed that the inlet methane concentration was higher during summer because the biodegradation of landfill waste was higher under thermophilic conditions. During the 254-day experimental period, the methane removal efficiency was over 98% despite the fluctuation in the inlet concentration (Fig. 2A). The average inlet methane concentration during winter, spring, and summer was 22.0%, 16.3%, and 31.3%, respectively, and the outlet concentration was 0.1%, 0.1%, and 0.2%, respectively (Fig. 2B). The methane removal by the biocover was compared with that of the soil cover at a site adjacent to the biocover [19]. The methane concentration at the surface of the biocover (
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Fig. 2. Removal of methane within the biocover. (A) Time profile for the methane concentration and removal efficiency. (B) Seasonal changes in the methane concentration at the biocover inlet and outlet. (C) Comparison of the methane concentration at the biocover inlet, the biocover outlet, and the landfill soil cover. The landfill soil cover was monitored for 240 days (Dec. 2016 to Aug. 2017) at the soil surface at the same landfill (Yun
et al ., 2017). In the box plot, the boxes represent the 25th, 50th (median), and 75th percentiles, and the error bars indicate the 10th and 90th percentiles. Different letters indicate a significant difference within each plot (p < 0.05).
The concentration and removal efficiency for 22 odor compounds in the biocover are summarized in Table S1. Previous studies have reported that the key compounds that contribute to the complex odor intensity at the Gwangyang landfill are sulfur-containing compounds [10, 11, 18, 19, 36]. Similar to previous studies, sulfur-containing compounds were the primary contributors to the complex odor intensity in the present study (Table S1). The odor removal performance of the biocover is presented in Fig. 3. The average inlet ODR during summer (4735) was slightly higher than that during winter (1622) and spring (2046) (Figs. 3A and 3B). During summer, the average outlet ODR ranged from 30 to 100, with an average removal efficiency of 98%. The average odor removal efficiency was 96.6% during winter and 99.6% during spring. The ODR was 3–250 at the surface of the biocover across the entire sampling period but ranged from 7 to 10,000 at the surface of the landfill soil with no biocover (Fig. 3C).
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Fig. 3. Removal of odor within the biocover. (A) Time profile for complex odor compounds (ODR) and the removal efficiency. (B) Seasonal changes in the ODR at the biocover inlet and outlet. (C) Comparison of the ODR at the biocover inlet, the biocover outlet, and the landfill soil cover. The landfill soil cover was monitored for 240 days (Dec. 2016 to Aug. 2017) at the soil surface at the same landfill (Yun
et al ., 2017). In the box plot, the boxes represent the 25th, 50th (median), and 75th percentiles, and the error bars indicate the 10th and 90th percentiles. Different letters indicate a significant difference within each plot (p < 0.05).
In the pilot-scale biocover containing a mixture of soil, perlite, earthworm cast, and compost at a ratio of 6:2:1:1 (v/v), the methane and odor removal efficiency from the landfill gas was 85–96% and 93–98% during the spring and summer seasons, respectively [10]. A previous study reported that, across all seasons, odor removal by biocovers installed at a sanitary landfill ranged from 81 to 98% [18], which is similar to the biocover performance in the present study. In addition, there was no observed deterioration in methane or odor removal performance when the internal temperature of the biocover increased to more than 40ºC at midday during the summer season.
Bacterial Community Structure during Summer
Table 1 summarizes the bacterial community in the biocover. The number of OTUs increased from 545 to 1060 between day 0 and day 113 and then settled down to 441–686. The Shannon index, which is used to evaluate the diversity of a bacterial community, fluctuated between 4.703 and 5.587. The Shannon index during summer was 4.703–5.470, while that during winter and spring was 4.839–5.587, showing that the diversity of the bacterial community did not decrease significantly during summer. Principal component analysis (PCA) was also conducted to compare the structure of the bacterial community by season (Fig. 4), leading to the creation of three clear groups. The composition of the community during summer (days 198–252) differed from that during the winter and spring seasons (days 40–163).
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Table 1 . Bacterial community analysis results for the biocover.
Sampling time (d) No. of OTUs a Chao1b Shannonc Good coveraged 0 545 648 5.445 0.998 40 708 873 5.227 0.996 68 874 983 4.839 0.999 99 996 1160 5.411 0.997 133 1060 1193 5.587 0.998 163 650 801 4.890 0.989 198 441 603 5.373 0.985 238 686 818 4.703 0.998 252 673 885 5.470 0.989 aOperational taxonomic units
bChao1 is used to evaluate bacterial community richness.
cThe Shannon index is used to evaluate the diversity within a bacterial community.
dGood coverage is calculated as C=1-(s/n), where s is the number of unique OTUs and n is the number of individuals in the sample. This index provides a relative measure of how well the sample represents the wider community.
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Fig. 4. Principal component analysis (PCA) of the structure of the bacterial community in the biocover. The community structure was analyzed in duplicate.
Table 2 presents the relative abundance of the bacterial genera in the biocover at each sampling point. The correlation between the bacterial community and environmental parameters is shown in Table S2. The generation of a thermophilic environment in the biocover during summer promoted the growth of thermophilic heterotrophs, including
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Table 2 . Comparison of the relative abundance of bacterial genera in the biocover.
Genus Sampling time (d) 0 40 68 99 133 163 198 238 252 Non-Methanotroph Acinetobacter 15.70 -* - - - 1.10 - - - Actinomadura - 0.90 - - - - - 1.13 - Actinophytocola - - - - - - - 1.16 - Advenella 1.20 3.00 1.90 0.90 - - - - - Arthrobacter 8.00 - - 1.20 - - - - - Bacillus 1.00 - - - - 2.20 1.10 8.53 1.00 Bellilinea - - - - - - 4.00 - 1.90 Brevibacterium - - - - - - - 1.88 - Cellulosimicrobium - - - 1.50 - - - - - Chryseolinea - - - - - 1.10 - - - Desertibacter - - - - - - - - 1.70 Dokdonella - - - - 1.60 - - - - Homoserinibacter 1.10 - 1.20 1.30 1.20 - - - - Hydrogenophaga - - 1.30 - - - - - - Hyphomicrobium 0.44 0.46 0.35 0.49 0.52 1.10 1.20 6.42 0.94 Ignavibacterium - - - - - - - - 1.80 Lascolabacillus - - 1.20 - - - - - - Luteimonas - 7.50 1.60 1.70 1.20 2.00 - 4.80 - Microbispora 1.50 - - - - - - - - Nonomuraea - 4.90 2.00 1.80 1.20 0.90 - - - Ohtaekwangia - 1.70 0.90 1.70 1.70 4.10 1.50 7.28 5.90 Ornatilinea - - - - - - 2.20 - 2.70 Pedobacter 8.10 - - - - - - - - Planococcus 8.30 - - 1.20 - - - - - Porticoccus 2.90 - - - - - - - - Pseudomonas 3.80 1.10 - - - - - - - Pseudoxanthomonas - 1.00 - - - - - - - Rhodothermus - - - - - - 4.60 - 6.10 Rummeliibacillus 1.20 - - - - 1.70 - - - Serpens 15.70 27.20 13.90 13.40 12.40 7.30 18.10 - 2.00 Streptomyces 0.70 1.70 0.90 1.40 0.52 0.64 0.28 0.91 0.57 Thermanaerothrix 0.80 1.40 - 1.00 1.50 - 4.80 0.83 12.30 Thermomarinilinea - - - - - - 13.90 3.55 17.90 Methanotroph Methylocaldum 1.30 2.40 0.90 1.60 2.40 5.80 9.00 24.86 23.40 Methylococcus - - - 0.40 0.80 0.80 0.20 3.46 - Methylobacter 0.80 15.90 41.90 37.80 37.30 37.70 13.00 6.60 6.60 Methylomicrobium - 0.10 - 0.40 0.10 0.10 - - - Methylosarcina - - 0.10 0.20 0.50 0.60 - - - Methylomonas - - - - 0.20 - - - - Methylocystis - - - 0.10 0.20 0.10 0.10 - - Others 27.46 30.73 31.85 31.91 36.66 32.76 26.02 28.59 15.19 Total 100 100 100 100 100 100 100 100 100 *-, Less than 0.1%
The relative abundance of the genus
The pattern for the genus
The relative abundance of
The most dominant methanotrophs were
Simultaneous Removal of Methane and DMS by the Packing Material under Moderately Thermophilic Conditions
Fig. 5 presents time profiles for methane and DMS degradation by the packing material sampled during summer at 30°C, 40°C, and 50°C. All of the packing material samples from the top (0–15 cm), middle (15–30 cm), and bottom (30–50 cm) layers of the biocover simultaneously degraded methane and DMS under moderately thermophilic conditions (40–50°C) and mesophilic conditions (30°C). Table 3 presents the degradation rates for methane and DMS at different temperatures. The degradation rate for both methane and DMS at 50°C was significantly higher than at 30°C and 40°C and increased with a greater sampling depth, with the degradation rate for methane and DMS highest at 30–50 cm (3.059 ± 0.074 and 0.256 ± 0.005 μmol·g-dry sample-1·h-1, respectively). Because the internal temperature during summer increased to 41–49ºC at midday during summer (Fig. 1C), the methane and DMS degradation was higher under moderately thermophilic conditions than under mesophilic conditions. Degradation was also assumed to be highest at 30–50 cm because the internal temperatures tended to rise as the depth of the biocover increased (Fig. 1C).
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Table 3 . Comparison of the methane and DMS degradation rate by the packing material at 30, 40, and 50°C.
Temp. (℃) Sampling layer (cm) Degradation rate for the packing material (μmol·g-dry sample-1·h-1) CH4 DMS 30℃ 0–15 1.502 ± 0.060 C, D* 0.136 ± 0.001 D, E 15–30 1.614 ± 0.060 C 0.148 ± 0.004 D 30–50 1.682 ± 0.074 C 0.154 ± 0.005 D 40℃ 0–15 1.388 ± 0.060 C, D 0.124 ± 0.004 E, F 15–30 1.335 ± 0.060 D 0.125 ± 0.004 E, F 30–50 1.407 ± 0.060 C, D 0.110 ± 0.004 F 50℃ 0–15 2.052 ± 0.060 B 0.208 ± 0.004 C 15–30 2.101 ± 0.060 B 0.228 ± 0.004 B 30–50 3.059 ± 0.074 A 0.256 ± 0.005 A *Different letters denote a significant difference (
n = 3,p < 0.05).
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Fig. 5. Time profiles for methane (A–C) and DMS (D–F) degradation by the packing material sampled at 0– 15, 15–30, and 30–50 cm from the surface of the biocover. Incubation temperatures: (A) and (D) 30°C, (B) and (D) 40°C, and (C) and (F) 50°C.
Isolation and Identification of Thermophilic Methanotrophs and Simultaneous Removal of Methane and DMS
Four methane- and DMS-degrading bacterial species –
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Fig. 6. Time profiles for methane (A and C) and DMS (B and D) degradation by the isolates from the biocover. Incubation temperatures: (A) and (B) 40°C and (C) and (D) at 50°C.
The bacterial community results reveal that the abundance of
Although three strains belonging to
Except for
It took around 8 to 10 days for the packing material to completely degrade the methane and DMS at different depths in the biocover (Fig. 5). However, it took more than 40 days for methane and DMS to be completely decomposed by pure bacteria isolated from the enrichment culture at 40°C (Figs. 6A and 6B). In addition, the degradation rates for
In this study, the simultaneous removal of methane and odor in the biocover was compared between the summer and non-summer seasons. The biocover performance for the methane and odor compounds did not deteriorate even when the internal temperature of the biocover increased to more than 40°C at midday during summer. The packing material sampled from the biocover in summer was able to degrade methane and DMS at 40–50°C, while the isolated bacteria simultaneously degraded methane and DMS at 40°C and/or 50°C. The diversity of the bacterial community in the biocover also remained constant regardless of the season, though the relative abundance of thermotolerant and thermophilic bacteria increased during summer. The major methane-oxidizing bacterial group shifted from
Supplemental Materials
Acknowledgments
This research was supported by the Korean Ministry of Environment as a Converging Technology Project (201500164003).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(6): 803-814
Published online June 28, 2021 https://doi.org/10.4014/jmb.2103.03005
Copyright © The Korean Society for Microbiology and Biotechnology.
Characterization of the Bacterial Community Associated with Methane and Odor in a Pilot-Scale Landfill Biocover under Moderately Thermophilic Conditions
Hyoju Yang1, Hyekyeng Jung1, Kyungcheol Oh2, Jun-Min Jeon2, and Kyung-Suk Cho1*
1Department of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
2Green Environmental Complex Center, Suncheon 57992, Republic of Korea
Correspondence to:Kyung Suk Cho, kscho@ewha.ac.kr
Abstract
A pilot-scale biocover was constructed at a sanitary landfill and the mitigation of methane and odor compounds was compared between the summer and non-summer seasons. The average inlet methane concentrations were 22.0%, 16.3%, and 31.3%, and the outlet concentrations were 0.1%, 0.1%, and 0.2% during winter, spring, and summer, respectively. The odor removal efficiency was 98.0% during summer, compared to 96.6% and 99.6% during winter and spring, respectively. No deterioration in methane and odor removal performance was observed even when the internal temperature of the biocover increased to more than 40°C at midday during summer. During summer, the packing material simultaneously degraded methane and dimethyl sulfide (DMS) under both moderately thermophilic (40–50°C) and mesophilic conditions (30°C). Hyphomicrobium and Brevibacillus, which can degrade methane and DMS at 40°C and 50°C, were isolated. The diversity of the bacterial community in the biocover during summer did not decrease significantly compared to other seasons. The thermophilic environment of the biocover during summer promoted the growth of thermotolerant and thermophilic bacterial populations. In particular, the major methane-oxidizing species were Methylocaldum spp. during summer and Methylobacter spp. during the nonsummer seasons. The performance of the biocover remained stable under moderately thermophilic conditions due to the replacement of the main species and the maintenance of bacterial diversity. The information obtained in this study could be used to design biological processes for methane and odor removal during summer and/or in subtropical countries.
Keywords: Methane, odor, thermophile, thermotolerance, bacterial community, biological system
Introduction
Methane is a significant greenhouse gas that has a 28–36 times stronger effect on global warming than carbon dioxide [1]. Landfills are a major source of methane due to the anaerobic digestion of organic matter [2], during which odor compounds, including hydrogen sulfide (H2S), methanethiol, dimethyl sulfide (DMS), and volatile organic compounds, are also produced as byproducts [3-5]. Odor compounds are not only a nuisance but also pose a potential hazard to human health at high concentrations, with long-term exposure often leading to emotional stress and physical symptoms such as anxiety, headaches, vomiting, eye irritation, and respiratory problems [6-9]. Therefore, the mitigation of both methane and odor is necessary for anaerobic digestion processes.
Methane and odor compounds emitted by anaerobic digestion can be mitigated using biological, chemical, or physical treatment [3, 10-12]. Biological treatment is more environmentally friendly and safer than chemical and physical approaches because it does not require the use of chemicals and can be conducted at normal temperatures and pressures [3, 10, 11]. Biological treatment also offers high treatment efficiency, simple systems, and low treatment costs [3, 12]. Therefore, biological systems such as biocovers and biowindows have attracted attention as promising technologies for the control of the methane and odor compounds emitted from landfills [10, 11].
Microbial activity, which determines the performance of biological treatment systems [13], is primarily influenced by environmental factors such as temperature, pH, and moisture content [3, 14]. For example, if the temperature of the biological treatment system is outside the range that most microorganisms can tolerate, the microbial activity is dramatically lower [14]. Most biological systems designed to mitigate the methane and odor compounds emitted by landfills employ mesophilic microorganisms [15-17]. Because the activity of mesophilic microbes decreases rapidly at low temperatures, it could be expected that the performance of biological treatment systems would deteriorate during winter. However, this is not the case because the internal temperature of these systems is maintained at around 8–18°C due to the heat generated by the biodegradation of methane and odor compounds and the effect of insulating materials [10, 11, 18].
Mesophilic microorganisms are generally less active under moderately thermophilic conditions of 40°C or more. At midday during summer, the temperature inside biological treatment systems may rise to over 40°C due to the effects of solar radiation. Information on the composition and dynamics of the microbial community under moderately thermophilic conditions is thus essential when designing and setting the operating parameters for biological treatment systems during the hot season or in subtropical countries, but there is little information available on this. Therefore, in this study, the methane and odor removal performance of a pilot-scale biocover during the summer and non-summer seasons was compared. In particular, the structure of the bacterial community during summer was characterized and compared with the non-summer seasons. In addition, the degradation of methane and odor compounds by the packing material sampled from the biocover during summer was evaluated under moderately thermophilic conditions. Thermotolerant or thermophilic methane- and odor-degrading bacteria were also isolated and their roles in the degradation of methane and odor compounds investigated.
Materials and Methods
Packing Material for the Biocover
A pilot-scale biocover was constructed at the Gwangyang Sanitary Landfill, located at latitude 34°58’0’’ and longitude 127°38’35’’. This sanitary landfill began operation in 1996 and has a total disposal capacity of 3,145,291 m3 [10, 19]. The packing material for the biocover was a mixture of soil, perlite (Kyungdong One Co. Ltd., Korea), food waste compost, and earthworm cast (Kumhosilup, Korea). The physiochemical characteristics of these materials were described previously by Lee
Construction of the Pilot-Scale Biocover and Packing Material
A schematic diagram of the biocover (2.5 m wide × 2.5 m long × 1.2 m deep) is presented in Fig. S1. For thermal insulation, polystyrene foam boards (5 cm thick) were placed along the inside of the walls. The solid waste was first covered with a 0.23-m thick layer of gravel (particle diameter of 2–5 cm) and then a polypropylene non-woven textile sheet (Kyungdong One Co. Ltd.,). A 0.9-m-thick soil layer was placed on top of the textile cover. A perforated pipeline was installed at the bottom of the biocover and connected to polyvinyl chloride (PVC) pipes for biocover inlet gas sampling. An acrylic chamber (2.5 m wide × 2.5 m long × 0.3 m high) was installed on the surface of the biocover for gas sampling from the biocover surface (
Ambient Temperature, Precipitation, and Physicochemical Properties of the Packing Material
Ambient temperature and precipitation measurements during the experimental period were obtained from the Gwangyang Automatic Weather Station operated by the Korea Meteorological Administration. The packing material in the biocover was sampled between 11 and 12 o’clock on days 0, 40, 68, 99, 133, 163, 198, 238, and 252 at 0–15 cm (upper layer), 15–30 cm (middle layer), and 30–50 cm (bottom layer) from the surface of the biocover. Immediately before taking the sample, the temperature of the packing material in each layer was measured using a portable digital thermometer (SDT200, Summit Co. Ltd., Korea).
After being passed through a 2-mm sieve, the samples were stored at 4°C for the assessment of their physicochemical properties (pH, moisture content, and organic matter content) and at –20°C for the analysis of the bacterial community. The moisture and organic matter content of the samples were measured based on the Korean Standard Soil Analysis Method [10] and the Korean Standard Waste Analysis Method [10], respectively. To measure the pH of the samples, 3 g of each sample was mixed with 20 mL of distilled water, and the supernatant was collected after allowing the particles to settle for 5 min. The pH of the resulting supernatant was measured with a pH meter (Thermo Orion 535A, USA).
Gas Analysis
Gas samples from the inlet port and the surface of the biocover were collected on days 12, 39, 63, 98, 124, 165, 182, 223, and 253. The gas sampling was conducted using the same method described in a previous study [10]. The methane concentration in the gas samples was measured using a gas chromatograph equipped with a flame ionization detector [10]. The methane levels were also measured in the field using a biogas check analyzer (Geotechnical Instruments, UK) [10]. The concentrations of 22 odor compounds designated as key offensive odors by the Korean Odor Prevention Law were analyzed using the same methods described in a previous study [10]. Complex odor compounds were analyzed using the odor dilution ratio (ODR) [20]. Details on the calculation of the ODR are available in Lee
Bacterial Community Analysis Using Illumina MiSeq Sequencing
The packing material sampled at 15–30 cm on days 0–252 was used to characterize the bacterial community dynamics in the biocover. For DNA extraction, 0.5 g of each sample was transferred to a microtube from the stored bottle, and the DNA was extracted using a NucleoSpin Soil Kit (Macherey-Nagel GmbH, Germany) and a Mini-BeadBeater-8 system (BioSpec, USA). DNA extraction was performed following the manufacturer’s instructions. The extracted DNA samples were eluted with 50 μl of an elution buffer and stored at –20°C before analysis. The extracted DNA was used as a PCR template to analyze the bacterial community with an Illumina MiSeq sequencing platform (Macrogen Inc., Korea) using the same method described in our previous paper [11]. Each composite primer was designed based on 515f and 806r primers [21]. Sequences shorter or longer than the target sequence were cut using CD-HIT-OTU [22], and chimera and noise were eliminated. The sequences with over 97% similarity were classified into operational taxonomic units (OTUs). Using the UCLUST algorithm [23], the taxonomy for each OTU was assigned based on the 16S rRNA RDP database. The Chao1 richness estimator and the Shannon index were also calculated [24]. The obtained sequence data were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/) under accession number SRP185598. Bacterial community dynamics were analyzed using principal component analysis (PCA) with UniFrac [25] and CANOCO 4.5 software (Microcomputer Power, USA).
Simultaneous Removal of Methane and Dimethyl Sulfide by the Packing Materials under Moderately Thermophilic Conditions
The simultaneous removal of methane and odor compounds was evaluated at 40°C and 50°C for three packing material samples taken on day 252 at 0–15 cm, 15–30 cm, and 30–50 cm from the surface of the biocover. In order to compare the removal efficiency under moderately thermophilic and medium temperature conditions, the same experiment was carried out at 30°Cs. DMS was selected as a representative odor compound. Five grams of the wet samples and 20 ml of nitrated mineral salt (NMS) medium were added to 600-ml serum bottles. The NMS medium contained 1.0 g/l MgSO4·7H2O, 0.2 g/l CaCl2·6H2O, 1.0 g/l KNO3, 0.272 g/l KH2PO4, and 0.717 g/l Na2HPO4·12H2O. The serum bottles were sealed with a butyl rubber stopper, and then methane gas from a cylinder (99%; Dong-A Gases, Korea) was injected into the bottles to a final concentration of 50,000 ppm [13]. In addition, DMS solution (99%; Acros Organics, Belgium) was injected into the bottles to a final concentration of 5,000 ppm. The serum bottles were incubated at 30°C, 40°C, and 50°C and 180 rpm in a shaking incubator. In order to prevent the stopper from falling out due to the expansion of the gas inside the serum bottle at 40°C and 50°C, 60 ml of the gas inside the serum bottle was removed using a syringe before putting the serum bottle into the incubator. The gas in the headspace of each serum bottle was periodically sampled using gas-tight syringes to measure the concentration of methane and DMS with a gas chromatography system (GC 7890, Agilent Technologies, USA) equipped with a 30 m × 320 μm × 1.8 μm capillary column (J&W Scientific, Inc., ISA) and a flame ionization detector (Agilent Technologies). The operating temperature of the oven, injector, and detector was 100°C, 230°C, and 230°C, respectively. The degradation rates for the methane and DMS were calculated as the reduction in the methane and DMS from their initial concentration to a concentration below 2,000 ppm and 500 ppm, respectively, divided by the incubation time per unit dry weight of the sample.
Isolation and Identification of Methane- and DMS-Degrading Bacteria under Moderately Thermophilic Conditions
To isolate methane- and DMS-degrading bacteria at 40°C and 50°C, enrichment cultures were developed. Packing material samples taken at 0–15 cm, 15–30 cm, and 30–50 cm from the surface of the biocover on day 252 were mixed in equal amounts with each other. Five grams of the mixed sample was placed in each of two 600-ml serum bottles containing 20 ml of NMS medium. After sealing the bottles with butyl rubber stoppers, methane gas and DMS solution were injected to final concentrations of 50,000 ppm and 5,000 ppm, respectively. One bottle was incubated at 40°C, and the other was incubated at 50°C in a shaking incubator (180 rpm). When the concentrations of methane and DMS in the headspace of each bottle decreased below the detection limit, methane gas and DMS solution were re-injected and the bottle was re-incubated at 40°C or 50°C. After repeating this process five times, 10 ml of the 1st enriched culture was transferred into 10 ml of fresh NMS medium in a 600 ml-serum bottle, and methane and DMS were injected into the bottle. Each bottle was incubated at 40°C or 50°C, and methane and DMS were re-supplied when their concentration fell below the detection limit before re-incubation. After repeating this process four times, 10 ml of the 2nd enriched culture was transferred into 10 ml of fresh NMS medium, methane and DMS were supplied, and the bottle was incubated at 40°C or 50°C.
After repeating this replenishment four times, the 3rd enriched culture was diluted with NMS medium, and the diluted culture was spread on NMS-agar (20 g/l) plates. The inoculated plates were incubated at 40°C or 50°C in a 5-L reactor, which was connected to a 3-L Tedlar bag containing 50,000 ppm of methane and 5,000 ppm of DMS. After incubation for 1–2 months, distinguishable colonies on the plates were carefully transferred to fresh NMS-agar plates and incubated in the same manner as described above. This process was repeated several times to produce four pure strains (HJ1, HJ2, HJ3, and HJ4) from the 40°C-enriched cultures and two pure strains (HJ5 and HJ6) from the 50°C-enriched cultures.
The simultaneous degradation of methane and DMS by the isolates in the serum bottles was evaluated at 40°C or 50°C using the same method as described in Section 2.6. To identify the isolates, genomic DNA samples were extracted using NucleoSpin Soil Kits (Macherey-Nagel GmbH Düren, Germany), and amplified with PCR using the primer set 340F (5’-TCCTACGGGAGGCAGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’)[26]. The sequence data were deposited in the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/) under the accession number MK577721 for the strain HY1, MK577720 for HY2, MK577719 for HY3, MK577718 for HY4, MK639430 for HY5, and MK577717 for HY6.
Results and Discussion
Variation in the Environmental Parameters
Environmental factors such as temperature, precipitation, moisture content, organic matter content, and pH affect biocover performance [10, 11, 27-32]. Fig. 1 presents the ambient temperature, precipitation, internal temperature, and physical characteristics of the packing material at different depths in the biocover. The average ambient temperature during winter (from December to February), spring (from March to May), and summer (from June to August) was –2.9–13.1ºC, 1.1–26.3ºC, and 18.0–31.5ºC, respectively (Fig 1A). Monthly precipitation was the highest in August (Fig. 1B). During winter and spring, the internal temperature tended to increase with greater distance from the surface of the biocover, with a range of 20–30ºC at midday (Fig. 1C), while that during summer was 41–49 ºC, even though the maximum ambient temperature was 21.4–37.0 ºC (Figs. 1A and 1C). There were four main reasons for this higher internal temperature: (1) the heat generated by biodegradation in the waste layer at the bottom of the biocover, (2) heat from the biodegradation of methane and odor compounds in the biocover, (3) heat from intense sunlight during the middle of the day, and (4) thermal insulation due to the polystyrene foam boards within the biocover (Fig. S1). Jung
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Figure 1. Time profiles of the ambient temperature (A), precipitation (B), internal temperature (C), moisture content (D), organic matter content (E), and pH (F) of the packing material in the biocover.
The moisture content, organic matter content, and pH of the packing material in the biocover are presented in Figs. 1D-1F. The moisture content in the biocover mostly remained at around 20–25% at all sample depths, though it did increase to 30% on day 124 due to heavy precipitation (Fig. 1D). Cho and Ryu [3] reported that the optimal moisture content for biocover performance was 25–50%. The moisture content of the biocover in the present study thus fell within a suitable range for the simultaneous degradation of methane and odor compounds. During summer, despite the high evaporation rate, the moisture content in the biocover was maintained at a suitable level due to the frequent precipitation (Figs. 1A, 1B, and 1D). The successful removal of methane and odor compounds within a biocover with a low organic matter content of 5–10% has been reported [10, 11, 18]. In addition, most methane- and/or odor-degrading bacteria exhibit optimal activity at a neutral pH [3, 34, 35]. Considering these previous results, the moisture content and pH of the biocover were favorable for methane- and odor-degrading bacteria.
Methane and Odor Removal during Summer
Fig. 2A presents a time profile of the methane concentration and removal efficiency. The methane concentration at the biocover inlet was below 32% during winter and spring but ranged from 20–38% during summer. It is believed that the inlet methane concentration was higher during summer because the biodegradation of landfill waste was higher under thermophilic conditions. During the 254-day experimental period, the methane removal efficiency was over 98% despite the fluctuation in the inlet concentration (Fig. 2A). The average inlet methane concentration during winter, spring, and summer was 22.0%, 16.3%, and 31.3%, respectively, and the outlet concentration was 0.1%, 0.1%, and 0.2%, respectively (Fig. 2B). The methane removal by the biocover was compared with that of the soil cover at a site adjacent to the biocover [19]. The methane concentration at the surface of the biocover (
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Figure 2. Removal of methane within the biocover. (A) Time profile for the methane concentration and removal efficiency. (B) Seasonal changes in the methane concentration at the biocover inlet and outlet. (C) Comparison of the methane concentration at the biocover inlet, the biocover outlet, and the landfill soil cover. The landfill soil cover was monitored for 240 days (Dec. 2016 to Aug. 2017) at the soil surface at the same landfill (Yun
et al ., 2017). In the box plot, the boxes represent the 25th, 50th (median), and 75th percentiles, and the error bars indicate the 10th and 90th percentiles. Different letters indicate a significant difference within each plot (p < 0.05).
The concentration and removal efficiency for 22 odor compounds in the biocover are summarized in Table S1. Previous studies have reported that the key compounds that contribute to the complex odor intensity at the Gwangyang landfill are sulfur-containing compounds [10, 11, 18, 19, 36]. Similar to previous studies, sulfur-containing compounds were the primary contributors to the complex odor intensity in the present study (Table S1). The odor removal performance of the biocover is presented in Fig. 3. The average inlet ODR during summer (4735) was slightly higher than that during winter (1622) and spring (2046) (Figs. 3A and 3B). During summer, the average outlet ODR ranged from 30 to 100, with an average removal efficiency of 98%. The average odor removal efficiency was 96.6% during winter and 99.6% during spring. The ODR was 3–250 at the surface of the biocover across the entire sampling period but ranged from 7 to 10,000 at the surface of the landfill soil with no biocover (Fig. 3C).
-
Figure 3. Removal of odor within the biocover. (A) Time profile for complex odor compounds (ODR) and the removal efficiency. (B) Seasonal changes in the ODR at the biocover inlet and outlet. (C) Comparison of the ODR at the biocover inlet, the biocover outlet, and the landfill soil cover. The landfill soil cover was monitored for 240 days (Dec. 2016 to Aug. 2017) at the soil surface at the same landfill (Yun
et al ., 2017). In the box plot, the boxes represent the 25th, 50th (median), and 75th percentiles, and the error bars indicate the 10th and 90th percentiles. Different letters indicate a significant difference within each plot (p < 0.05).
In the pilot-scale biocover containing a mixture of soil, perlite, earthworm cast, and compost at a ratio of 6:2:1:1 (v/v), the methane and odor removal efficiency from the landfill gas was 85–96% and 93–98% during the spring and summer seasons, respectively [10]. A previous study reported that, across all seasons, odor removal by biocovers installed at a sanitary landfill ranged from 81 to 98% [18], which is similar to the biocover performance in the present study. In addition, there was no observed deterioration in methane or odor removal performance when the internal temperature of the biocover increased to more than 40ºC at midday during the summer season.
Bacterial Community Structure during Summer
Table 1 summarizes the bacterial community in the biocover. The number of OTUs increased from 545 to 1060 between day 0 and day 113 and then settled down to 441–686. The Shannon index, which is used to evaluate the diversity of a bacterial community, fluctuated between 4.703 and 5.587. The Shannon index during summer was 4.703–5.470, while that during winter and spring was 4.839–5.587, showing that the diversity of the bacterial community did not decrease significantly during summer. Principal component analysis (PCA) was also conducted to compare the structure of the bacterial community by season (Fig. 4), leading to the creation of three clear groups. The composition of the community during summer (days 198–252) differed from that during the winter and spring seasons (days 40–163).
-
Table 1 . Bacterial community analysis results for the biocover..
Sampling time (d) No. of OTUs a Chao1b Shannonc Good coveraged 0 545 648 5.445 0.998 40 708 873 5.227 0.996 68 874 983 4.839 0.999 99 996 1160 5.411 0.997 133 1060 1193 5.587 0.998 163 650 801 4.890 0.989 198 441 603 5.373 0.985 238 686 818 4.703 0.998 252 673 885 5.470 0.989 aOperational taxonomic units.
bChao1 is used to evaluate bacterial community richness..
cThe Shannon index is used to evaluate the diversity within a bacterial community..
dGood coverage is calculated as C=1-(s/n), where s is the number of unique OTUs and n is the number of individuals in the sample. This index provides a relative measure of how well the sample represents the wider community..
-
Figure 4. Principal component analysis (PCA) of the structure of the bacterial community in the biocover. The community structure was analyzed in duplicate.
Table 2 presents the relative abundance of the bacterial genera in the biocover at each sampling point. The correlation between the bacterial community and environmental parameters is shown in Table S2. The generation of a thermophilic environment in the biocover during summer promoted the growth of thermophilic heterotrophs, including
-
Table 2 . Comparison of the relative abundance of bacterial genera in the biocover..
Genus Sampling time (d) 0 40 68 99 133 163 198 238 252 Non-Methanotroph Acinetobacter 15.70 -* - - - 1.10 - - - Actinomadura - 0.90 - - - - - 1.13 - Actinophytocola - - - - - - - 1.16 - Advenella 1.20 3.00 1.90 0.90 - - - - - Arthrobacter 8.00 - - 1.20 - - - - - Bacillus 1.00 - - - - 2.20 1.10 8.53 1.00 Bellilinea - - - - - - 4.00 - 1.90 Brevibacterium - - - - - - - 1.88 - Cellulosimicrobium - - - 1.50 - - - - - Chryseolinea - - - - - 1.10 - - - Desertibacter - - - - - - - - 1.70 Dokdonella - - - - 1.60 - - - - Homoserinibacter 1.10 - 1.20 1.30 1.20 - - - - Hydrogenophaga - - 1.30 - - - - - - Hyphomicrobium 0.44 0.46 0.35 0.49 0.52 1.10 1.20 6.42 0.94 Ignavibacterium - - - - - - - - 1.80 Lascolabacillus - - 1.20 - - - - - - Luteimonas - 7.50 1.60 1.70 1.20 2.00 - 4.80 - Microbispora 1.50 - - - - - - - - Nonomuraea - 4.90 2.00 1.80 1.20 0.90 - - - Ohtaekwangia - 1.70 0.90 1.70 1.70 4.10 1.50 7.28 5.90 Ornatilinea - - - - - - 2.20 - 2.70 Pedobacter 8.10 - - - - - - - - Planococcus 8.30 - - 1.20 - - - - - Porticoccus 2.90 - - - - - - - - Pseudomonas 3.80 1.10 - - - - - - - Pseudoxanthomonas - 1.00 - - - - - - - Rhodothermus - - - - - - 4.60 - 6.10 Rummeliibacillus 1.20 - - - - 1.70 - - - Serpens 15.70 27.20 13.90 13.40 12.40 7.30 18.10 - 2.00 Streptomyces 0.70 1.70 0.90 1.40 0.52 0.64 0.28 0.91 0.57 Thermanaerothrix 0.80 1.40 - 1.00 1.50 - 4.80 0.83 12.30 Thermomarinilinea - - - - - - 13.90 3.55 17.90 Methanotroph Methylocaldum 1.30 2.40 0.90 1.60 2.40 5.80 9.00 24.86 23.40 Methylococcus - - - 0.40 0.80 0.80 0.20 3.46 - Methylobacter 0.80 15.90 41.90 37.80 37.30 37.70 13.00 6.60 6.60 Methylomicrobium - 0.10 - 0.40 0.10 0.10 - - - Methylosarcina - - 0.10 0.20 0.50 0.60 - - - Methylomonas - - - - 0.20 - - - - Methylocystis - - - 0.10 0.20 0.10 0.10 - - Others 27.46 30.73 31.85 31.91 36.66 32.76 26.02 28.59 15.19 Total 100 100 100 100 100 100 100 100 100 *-, Less than 0.1%.
The relative abundance of the genus
The pattern for the genus
The relative abundance of
The most dominant methanotrophs were
Simultaneous Removal of Methane and DMS by the Packing Material under Moderately Thermophilic Conditions
Fig. 5 presents time profiles for methane and DMS degradation by the packing material sampled during summer at 30°C, 40°C, and 50°C. All of the packing material samples from the top (0–15 cm), middle (15–30 cm), and bottom (30–50 cm) layers of the biocover simultaneously degraded methane and DMS under moderately thermophilic conditions (40–50°C) and mesophilic conditions (30°C). Table 3 presents the degradation rates for methane and DMS at different temperatures. The degradation rate for both methane and DMS at 50°C was significantly higher than at 30°C and 40°C and increased with a greater sampling depth, with the degradation rate for methane and DMS highest at 30–50 cm (3.059 ± 0.074 and 0.256 ± 0.005 μmol·g-dry sample-1·h-1, respectively). Because the internal temperature during summer increased to 41–49ºC at midday during summer (Fig. 1C), the methane and DMS degradation was higher under moderately thermophilic conditions than under mesophilic conditions. Degradation was also assumed to be highest at 30–50 cm because the internal temperatures tended to rise as the depth of the biocover increased (Fig. 1C).
-
Table 3 . Comparison of the methane and DMS degradation rate by the packing material at 30, 40, and 50°C..
Temp. (℃) Sampling layer (cm) Degradation rate for the packing material (μmol·g-dry sample-1·h-1) CH4 DMS 30℃ 0–15 1.502 ± 0.060 C, D* 0.136 ± 0.001 D, E 15–30 1.614 ± 0.060 C 0.148 ± 0.004 D 30–50 1.682 ± 0.074 C 0.154 ± 0.005 D 40℃ 0–15 1.388 ± 0.060 C, D 0.124 ± 0.004 E, F 15–30 1.335 ± 0.060 D 0.125 ± 0.004 E, F 30–50 1.407 ± 0.060 C, D 0.110 ± 0.004 F 50℃ 0–15 2.052 ± 0.060 B 0.208 ± 0.004 C 15–30 2.101 ± 0.060 B 0.228 ± 0.004 B 30–50 3.059 ± 0.074 A 0.256 ± 0.005 A *Different letters denote a significant difference (
n = 3,p < 0.05)..
-
Figure 5. Time profiles for methane (A–C) and DMS (D–F) degradation by the packing material sampled at 0– 15, 15–30, and 30–50 cm from the surface of the biocover. Incubation temperatures: (A) and (D) 30°C, (B) and (D) 40°C, and (C) and (F) 50°C.
Isolation and Identification of Thermophilic Methanotrophs and Simultaneous Removal of Methane and DMS
Four methane- and DMS-degrading bacterial species –
-
Figure 6. Time profiles for methane (A and C) and DMS (B and D) degradation by the isolates from the biocover. Incubation temperatures: (A) and (B) 40°C and (C) and (D) at 50°C.
The bacterial community results reveal that the abundance of
Although three strains belonging to
Except for
It took around 8 to 10 days for the packing material to completely degrade the methane and DMS at different depths in the biocover (Fig. 5). However, it took more than 40 days for methane and DMS to be completely decomposed by pure bacteria isolated from the enrichment culture at 40°C (Figs. 6A and 6B). In addition, the degradation rates for
In this study, the simultaneous removal of methane and odor in the biocover was compared between the summer and non-summer seasons. The biocover performance for the methane and odor compounds did not deteriorate even when the internal temperature of the biocover increased to more than 40°C at midday during summer. The packing material sampled from the biocover in summer was able to degrade methane and DMS at 40–50°C, while the isolated bacteria simultaneously degraded methane and DMS at 40°C and/or 50°C. The diversity of the bacterial community in the biocover also remained constant regardless of the season, though the relative abundance of thermotolerant and thermophilic bacteria increased during summer. The major methane-oxidizing bacterial group shifted from
Supplemental Materials
Acknowledgments
This research was supported by the Korean Ministry of Environment as a Converging Technology Project (201500164003).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

Fig 6.

-
Table 1 . Bacterial community analysis results for the biocover..
Sampling time (d) No. of OTUs a Chao1b Shannonc Good coveraged 0 545 648 5.445 0.998 40 708 873 5.227 0.996 68 874 983 4.839 0.999 99 996 1160 5.411 0.997 133 1060 1193 5.587 0.998 163 650 801 4.890 0.989 198 441 603 5.373 0.985 238 686 818 4.703 0.998 252 673 885 5.470 0.989 aOperational taxonomic units.
bChao1 is used to evaluate bacterial community richness..
cThe Shannon index is used to evaluate the diversity within a bacterial community..
dGood coverage is calculated as C=1-(s/n), where s is the number of unique OTUs and n is the number of individuals in the sample. This index provides a relative measure of how well the sample represents the wider community..
-
Table 2 . Comparison of the relative abundance of bacterial genera in the biocover..
Genus Sampling time (d) 0 40 68 99 133 163 198 238 252 Non-Methanotroph Acinetobacter 15.70 -* - - - 1.10 - - - Actinomadura - 0.90 - - - - - 1.13 - Actinophytocola - - - - - - - 1.16 - Advenella 1.20 3.00 1.90 0.90 - - - - - Arthrobacter 8.00 - - 1.20 - - - - - Bacillus 1.00 - - - - 2.20 1.10 8.53 1.00 Bellilinea - - - - - - 4.00 - 1.90 Brevibacterium - - - - - - - 1.88 - Cellulosimicrobium - - - 1.50 - - - - - Chryseolinea - - - - - 1.10 - - - Desertibacter - - - - - - - - 1.70 Dokdonella - - - - 1.60 - - - - Homoserinibacter 1.10 - 1.20 1.30 1.20 - - - - Hydrogenophaga - - 1.30 - - - - - - Hyphomicrobium 0.44 0.46 0.35 0.49 0.52 1.10 1.20 6.42 0.94 Ignavibacterium - - - - - - - - 1.80 Lascolabacillus - - 1.20 - - - - - - Luteimonas - 7.50 1.60 1.70 1.20 2.00 - 4.80 - Microbispora 1.50 - - - - - - - - Nonomuraea - 4.90 2.00 1.80 1.20 0.90 - - - Ohtaekwangia - 1.70 0.90 1.70 1.70 4.10 1.50 7.28 5.90 Ornatilinea - - - - - - 2.20 - 2.70 Pedobacter 8.10 - - - - - - - - Planococcus 8.30 - - 1.20 - - - - - Porticoccus 2.90 - - - - - - - - Pseudomonas 3.80 1.10 - - - - - - - Pseudoxanthomonas - 1.00 - - - - - - - Rhodothermus - - - - - - 4.60 - 6.10 Rummeliibacillus 1.20 - - - - 1.70 - - - Serpens 15.70 27.20 13.90 13.40 12.40 7.30 18.10 - 2.00 Streptomyces 0.70 1.70 0.90 1.40 0.52 0.64 0.28 0.91 0.57 Thermanaerothrix 0.80 1.40 - 1.00 1.50 - 4.80 0.83 12.30 Thermomarinilinea - - - - - - 13.90 3.55 17.90 Methanotroph Methylocaldum 1.30 2.40 0.90 1.60 2.40 5.80 9.00 24.86 23.40 Methylococcus - - - 0.40 0.80 0.80 0.20 3.46 - Methylobacter 0.80 15.90 41.90 37.80 37.30 37.70 13.00 6.60 6.60 Methylomicrobium - 0.10 - 0.40 0.10 0.10 - - - Methylosarcina - - 0.10 0.20 0.50 0.60 - - - Methylomonas - - - - 0.20 - - - - Methylocystis - - - 0.10 0.20 0.10 0.10 - - Others 27.46 30.73 31.85 31.91 36.66 32.76 26.02 28.59 15.19 Total 100 100 100 100 100 100 100 100 100 *-, Less than 0.1%.
-
Table 3 . Comparison of the methane and DMS degradation rate by the packing material at 30, 40, and 50°C..
Temp. (℃) Sampling layer (cm) Degradation rate for the packing material (μmol·g-dry sample-1·h-1) CH4 DMS 30℃ 0–15 1.502 ± 0.060 C, D* 0.136 ± 0.001 D, E 15–30 1.614 ± 0.060 C 0.148 ± 0.004 D 30–50 1.682 ± 0.074 C 0.154 ± 0.005 D 40℃ 0–15 1.388 ± 0.060 C, D 0.124 ± 0.004 E, F 15–30 1.335 ± 0.060 D 0.125 ± 0.004 E, F 30–50 1.407 ± 0.060 C, D 0.110 ± 0.004 F 50℃ 0–15 2.052 ± 0.060 B 0.208 ± 0.004 C 15–30 2.101 ± 0.060 B 0.228 ± 0.004 B 30–50 3.059 ± 0.074 A 0.256 ± 0.005 A *Different letters denote a significant difference (
n = 3,p < 0.05)..
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