ISSN 00262617, Microbiology, 2013, Vol. 82, No. 6, pp. 847–855. © Pleiades Publishing, Ltd., 2013. Original Russian Text © A.Yu. Kallistova, L. Montonen, G. Jurgens, U. Münster, M.V. Kevbrina, A.N. Nozhevnikova, 2014, published in Mikrobiologiya, 2014, Vol. 83, No. 1, pp. 109–118.
EXPERIMENTAL ARTICLES
Culturable Psychrotolerant Methanotrophic Bacteria in Landfill Cover Soil A. Yu. Kallistovaa, 1, L. Montonenb, G. Jurgensc, U. Münsterd, M. V. Kevbrinae, and A. N. Nozhevnikovaa a
Winogradsky Institute of Microbiology, Russian Academy of Sciences, pr. 60letiya Oktyabrya 7, k. 2, Moscow, 117312 Russia b Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Biocenter 1A, Viikinkaari 9, Helsinki, FIN00014 Finland c Department of Biotechnology and Chemical Technology, Aalto University School of Chemical Technology, P.O. Box 16100, Aalto, Kemistintie 1, Espoo, FIN00076 Finland d Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, P.O. Box 541, Korkeakoulunkatu 8, Tampere, FIN33101 Finland e Engineering and Technology Center OJSC Mosvodokanal, Pervyi Kur’yanovskii proezd 15, Moscow, 109235 Russia Received December 20, 2012
Abstract—Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10°C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20°C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40°C from a sample collected in May (the temperature of the cover soil was 11.5–12.5°C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundri paludum (98% sequence identity of 16S rRNA genes with the type strain SV96T) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15°C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripalu dum SV96. Keywords: aerobic methanotrophic bacteria (methanotrophs), psychrotolerant methanotrophs, landfills for disposal of municipal solid waste (MSW), denaturing gradient gel electrophoresis (DGGE) DOI: 10.1134/S0026261714010044
Aerobic methanotrophic bacteria (methanotrophs) use methane as a single carbon and energy source and play a key role in the global methane cycle by oxidizing this greenhouse gas and preventing its emission into the atmosphere. Mesophilic, moderately thermo philic, psychrotolerant, psychrophilic, alkaliphilic, halophilic, acidophilic, and thermoacidophilic me thanotrophs have been isolated from natural and anthropogenic ecosystems and classified based on 16S rRNA gene analysis [1–6]. The landfills for the disposal of municipal solid waste (MSW) are ecologically hazardous and repre sent anthropogenic sources of methane. Dense popu lations of methanotrophs, which spontaneously colo nize the upper aerobic layer of the anthropogenic soil, normally develop within the MSW landfills, including 1
Corresponding author; email:
[email protected]
the largest nearMoscow landfill Khmet’evo studied by us [7–10]. At the Khmet’evo landfill, methane emission was shown to be highly inhomogeneous, reaching 10 g C m–2 h–1 at some sites [7]. Methane oxidizing activity of the cover soil increased in the beginning of spring, reaching its maximum during the warm season (May–September). Conversely, methane emission decreased in summer and increased drasti cally in autumn (October–November) [8]. The num ber of methanotrophs (according to CARDFISH data) was maximal during the warm season, and posi tively correlated with methaneoxidizing activity of the cover soil. The high enough number of metha notrophs in the cold seasons indicated the presence of psychrotolerant methanotrophs in the cover soil [9]. By indirect immunofluorescence technique, members of the genera Methylomonas, Methylobacter, Methylo
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cystis, Methylosinus, and Methylocapsa were identified in the cover soil [10]. In the last years, the number of studies that ana lyzed the composition of methanotrophic populations at MSW landfills by molecular ecology methods increased significantly. Irrespective of the geographi cal disposition of the landfill (Germany, Great Britain, Ireland, United States, Canada, China) and of the variations in physicochemical characteristics of the soil and waste, methanotrophs of the genera Methylo bacter and Methylocystis prevailed in the cover soil. Members of the genera Methylosinus, Methylocella, Methylocapsa, Methylomonas, Methylomicrobium, Methylosarcina, Methylococcus, and Methylocaldum and methanotrophs closely related to acidophilic rep resentatives of the phylum Verrucomicrobia were also identified in the landfill cover soils [11–20]. The com position of the methanotrophic population of the cover soil has been studied as dependent on the vege tation cover [11], soil moisture [18, 20], soil tempera ture [21], oxygen [20] and nitrogen [13, 18, 20] con centrations, C/N ratio in organic soil substance [17], presence of the volatile organic compounds [12] and earthworms [16], and the season [18, 19]. In the cover soil of a landfill in Great Britain, methanotrophs of the genus Methylocystis prevailed in the period from April to September; in April (soil temperature of 5.4⎯17.8°C) and September (9.5–19.2°C), metha notrophs of the genus Methylocaldum were detected, and in April and June (11–20.4°C), those of the genus Methylococcus [18]. In a Canadian MSW landfill, the methanotrophic population of the upper soil layer (0– 0.1 m) was more dependent on seasonal variations. In all soil samples, methanotrophs of the genera Methy localdum and Methylobacter prevailed. Maximal spe cies diversity, as well as appearance of methanotrophs of the genera Methylomicrobium, Methylomonas, and Methylosarcina, was registered in the period from October to December, at the soil temperature of 3.4– 11°C [19]. The aim of the present work was to obtain enrich ment cultures of psychrotolerant methanotrophic bac teria from the Khmet’evo landfill cover soil sampled in the cold (April, October, and November) and warm (May–September) seasons, to identify metha notrophs in these enrichment cultures, and to investi gate the effect produced by introduction of the bio mass of psychrotolerant methanotrophs on the meth ane emission from the landfill surface in the cold sea son (autumn). MATERIALS AND METHODS Samples of cover soil collected at the Khmet’evo MSW landfill (Moscow oblast, Solnechnogorskii region) were used in this study. Detailed characteriza tion of this landfill has been provided previously [7– 10]. The temperature of the cover soil was measured using temperature sensors (Dallas Semiconductor,
United States) [9]. The samples of the cover soil were taken by the Izmail’skii’s borer from the depths of 0– 20 and 20–40 cm during snowfree period (from the beginning of April until the end of November). In win ter (December–March), soil samples were not taken since the soil layer was covered with snow and was fro zen, and the places of thawing were impassable. Methaneoxidizing activity was determined as described previously [10] in water suspensions of soil samples taken in May from the depth of 20–40 cm. The soil suspensions were incubated at 2, 5, 10, 20, 25, 30, 40, 50, and 60°C. Methane concentration was measured every 24 h on a GC Chrom5 (Czech Republic) using a thermal conductivity detector and AG3 activated carbon as the sorbent. All experiments were performed in triplicate. The initial methaneoxi dizing activity was calculated from the decrease in methane content over the first 1–3 days of incubation and was expressed in µmol of CH4 per g of absolutely dry mass (ADM) of soil per day. ADM of soil was determined after the samples were dried at 105°C until reaching constant mass. Enrichment cultures of methanotrophs were obtained from cover soil sampled at experimental sites where high rate of methane oxidation was recorded [10]. The soil was sampled from the upper layer (0– 20 cm) in the warm (July, September) and cold (April, October, November) seasons (Table 1). Parallel enrichment cultures from the same soil samples were incubated at 10 and 20°C in the P liquid mineral medium [22] under a gas phase containing 10 vol % methane. Enrichment cultures were also obtained at 5 and 40°C from the soil suspensions in which the abovedescribed measurement of methaneoxidizing activity was performed in May. The soil sampled in October from experimental site 6 was used to obtain enrichment cultures of methanotrophs at 10 and 20°C, at methane concentration of 5, 10, and 50 vol %. The site 6 was characterized by high me thane concentration (35–60 vol %) in the pore gas at the depth of 20–60 cm and by its dramatic decrease to zero in the nearsurface layer, as well as by high me thane emission in April, May, and November and by absence of methane emission or even by methane con sumption from the nearsoil air in the period from June to October [7]. To obtain enrichment cultures, soil suspensions were prepared from freshly taken cover soil samples (pH 6.8–7.3) [10]. The suspension was inoculated at a dose of 10% into 60mL vials containing 10 mL of sterile P medium and a required methane concentra tion in the gas phase (5, 10 or 50 vol %). The vials were incubated statically in the dark at 5, 10, 20, and 40°C. Bacterial growth was estimated qualitatively from the appearance of turbidity and films in the medium. The growth of methanotrophs was assessed from the con sumption of the preintroduced methane from the gas phase; methane concentration was measured using GC Chrom5 (Czech Republic). Enrichment cultures MICROBIOLOGY
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Table 1. Conditions of obtaining enrichment cultures of methanotrophic bacteria Soil sample no.
Incubation temperature, °C
Methane concentration, %
The obtained enrichment culture designation*
April
4
10, 20
10
4/10, 4/20
May
10 11
5 40
10 10
July
2
10, 20
10
2/10, 2/20
September
5
10, 20
10
5/10, 5/20
October
7 8 9
10, 20 10, 20 10, 20
5 10 50
7/10, 7/20 8/10, 8/20 9/10, 9/20
November
3
10, 20
10
3/10, 3/20
Time of soil sampling
10/5 11/40
* Culture designations include sample number and incubation temperature.
were reinoculated every 1–4 weeks (depending on the cultivation temperature) into the medium of the same composition. Isolation of total DNA from enrichment cultures, PCR amplification of the 16S rRNA genes, and DGGE were carried out as described in the paper by Pimenov et al. [23]. The primers used were 984F, GC984F, 1492R [24], and MethT1dF [25]. The PCR products obtained upon amplification with the MethT1dF–1492R primer pair were purified and sequenced. The PCR products obtained upon amplification with the GC984F–1492R primer pair were separated by DGGE [23]. Sequencing was carried out in two directions on a 310 Genetic Analyzer (ABI Prism, Applied Biosys tems, United States) using a Big Dye Terminator v. 3.0 Cycle Sequencing Ready Reaction DNA Sequencing kit (ABI Prism, Applied Biosystems) and 310 Data Collection Software Version 3.0, at the Department of Applied Chemistry and Microbiology, University of Helsinki (Finland). Chromatograms were inspected using Chromas Version 1.45 software package. The obtained nucleotide sequences were compared with those of representatives of the Bacteria domain using the BLAST software [http://www.ncbi.nlm.nih.gov/ blast]. Nucleotide sequences were aligned and the phylogenetic trees were constructed using ARB soft ware package. The backbone tree was constructed using the neighborjoining method with the Jukes– Cantor distance correction. Newly determined partial sequences (500 bp and shorter) were then added, using the maximum parsimony option of the ARB software package, to the backbone tree without changing the overall tree topology [26]. The statistical significance of the branching order was determined using bootstrap analysis of 1000 alternative trees. The newly deter MICROBIOLOGY
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mined nucleotide sequences were deposited in Gen Bank under accession nos. HF565143–HF565161. The inoculum of methanotrophic bacteria, which was introduced into the Khmet’evo landfill cover soil to test the feasibility of decreasing methane emission in the cold season (October, November) by increasing the density of the psychrotolerant methanotrophic population, was derived from the enrichment cultures that we obtained. Specifically, methanotrophic enrichment cultures obtained at 10 and 20°C from various samples of the cover soil were mixed, inocu lated at a dose of 15 vol % into fresh P medium, and grown at 15 ± 1°C under a gas phase with 10 vol % methane. The field experiment on the introduction of me thanotrophic inoculum into the cover soil was started in September. For this, experimental and control sites sized 1 ± 1.5 m were chosen. 5 L of inoculum was poured into 10cmdeep grooves that were dug in the cover soil across the whole surface of the experimental site. At a control site, the grooves were filled with 5 L of water instead of the inoculum. The grooves were covered with fresh soil, and the sites were evenly poured with five more liters of inoculum or water (in the control). At both sites, methane emission was measured by the static chamber method [7] before and after the introduction of the inoculum (water), and cover soil samples were taken. In the soil samples, the most probable number of culturable methanotrophs was determined by the serial tenfold dilutions method [9, 10]. Excessive inoculum not used for the introduc tion was stored at 4°C for several months. Before iden tification of methanotrophs by DGGE, the stored material was inoculated into fresh P medium under a gas phase with 10 vol % methane and incubated at 20°C (culture 6/20).
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30 2.5
5
1
3 2
4
Fig. 1. Temperature of the cover soil at the Khmet’evo landfill. The temperature was measured on April 10 (1), May 18 (2), July 8 (3), September 9 (4), and November 28 (5); the determination error was ~0.02–1.19°C.
RESULTS Dependence of the cover soil methaneoxidizing activity on temperature. In the regions with a tempe rate continental climate, the soil temperature changes with the season. At the old site of the Khmet’evo land fill, the cover soil layer (0–60 cm) was completely fro zen in winter; in the period from April to November the soil temperature varied from 0 to 28°C. In spring and summer, the soil was warmed from the surface, and its temperature decreased with depth. In contrast, in autumn the soil temperature was higher at the depths of 40–60 cm. Usually, more significant tem perature variations (by 2–9°C) occurred in the upper soil layer (0–20 cm), while the temperature of the middle layer (20–40 cm) varied insignificantly (in the range of 1°C) (Fig. 1). The methaneoxidizing activity was determined in the soil sample taken in May from a depth of 20–40 cm; the temperature of this layer was similar to the mean soil temperature in the vegetation season (11°C). Methane oxidation occurred both at 2 and at 60°C. Maximal rate of methane oxidation was registered at 40 and 50°C, being 1.5 to 2fold higher than that at 20°C (Fig. 2). The ability of the cover soil to oxidize methane in the range of temperatures from 2 to 60°C indicated the presence in the soil of methanotrophs with different temperature preferences. Thermotole rant methanotrophs could be only slightly active in the cover soil in situ but developed rapidly in laboratory conditions at 40 and 50°C. It should be noted that in the methanogenic zone of landfills the temperature can be 50°C and higher [27]. The heating of some of the aerated sites of the cover soil cannot be excluded either. Methanotrophs identified in enrichment cultures. Methanotrophs closely related to Methylosinus spo rium NCIMB 11126 (Y18946) were isolated from the culture enriched at 10°C under a gas phase with 5 vol % methane. Methanotrophs closely related to Methylobacter tundripaludum SV96 (AJ414655) were identified in cultures enriched at 10°C with 10 vol %
Methaneoxidizing activity, µmol CH4 (g soil ADM)–1
Depth, cm
–10 –20 –30 –40 –50 –60 –70
Temperature, °C 10 15 20
5
0
2.0 1.5 1.0
0.5
0
10
20 30 40 Temperature, °C
50
60
Fig. 2. Dependence of the methaneoxidizing activity of the cover soil on the incubation temperature. Methane oxidizing activity was determined in a soil sample taken in May from a depth of 20–40 cm.
methane in the gas phase. In the culture enriched at 10°C with 50 vol % methane in the gas phase, metha notrophs related to members of the genus Methylocella were found (Table 2, Fig. 3). In cultures enriched at 20°C at 10 vol % methane in the gas phase, methanotrophs closely related to Methylobacter tundripaludum SV96 (AJ414655), M. luteus NCIMB 11914 (AF304195), M. marinus A45 (AF304197), and Methylosinus trichosporium BF1 (AJ868424) were identified. In the enrichment culture obtained at 20°C with 50 vol % methane the gas phase, methanotrophs related to Methylosinus sporium F10/1b (AJ458489) were found (Table 2, Fig. 3). In cultures enriched at 40°C and 10 vol % methane in the gas phase, a methanotroph closely related to Methylocaldum gracile VKM14LT (U89298) was identified (Table 2, Fig. 3). Other bacteria identified in enrichment cultures. In addition to methanotrophs, in cultures enriched at 5 and 10°C we identified obligate methylobacteria closely related to Methylovorus glucosotrophus DSM 6874T (FR733702) and Methylotenera varsatilis 301 (CP002056), respectively (99% 16S rRNA gene sequence identity) (Table 2). In the culture enriched at 20°C and 5 vol % me thane in the gas phase, we found an organism (HF565155) closely related (99% 16S rRNA gene sequence identity) to the F061 (FM253612) and G121 (FM253626) clones of uncultured verrucomi crobia. The closest cultivated relatives of this organism (92% 16S rRNA gene sequence identity) were Verru comicrobium spinosum DSM 4136 (NR_026266) and Prosthecobacter vanneervenii DSM 12252 (AJ966883). MICROBIOLOGY
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Table 2. Methanotrophs and methylobacteria identified in enrichment cultures obtained from the Khmet’evo landfill cover soil Incubation temperature, °C
Methane concentration in the gas phase, %
5
10
10
5
Methylotenera versatilis 301 (99)
40
10/5 (HF565153) 7/10 (HF565152)
Methylovorus glucosotrophus DSM 6874T (99)
7/10 (HF565151)
Methylovorus glucosotrophus DSM 6874T (99)
8/10 (HF565151)
Methylobacter tundripaludum SV96T(99)
2/10, 3/10, 5/10 (HF565150)
Methylobacter marinus A45 (98), isolate 4/20 (100)
4/10 (HF565144)
50
Methylocella tundrae Y1 (99), Methylocella palustris H4 (99)
9/10 (HF565154)
10
Methylobacter marinus A45 (99), Methylobacter luteus NCIMB 11914 (99)
2/20, 3/20 (HF565145)
Methylobacter tundripaludum SV96T (98, 99)
4/20 (HF565143), 6/20 (HF565146)
Methylosinus trichosporium BF1 (100)
8/20 (HF565147)
50
Methylosinus sporium F10/1b (99)
9/20 (HF565148)
10
Methylocaldum gracile VKM14LT (99)
In the enrichment cultures we also identified bac teria closely related to Pseudomonas marginalis LMG 2238 (HE586396, 100% 16S rRNA gene sequence identity), Pseudoxanthomonas sacheonensis BDc54 (EF575564, 100%), Dokdonella koreensis NML 010233 (EF589679, 98%), D. koreensis DS140 (AY987369, 98%), Protochlamydia naegleriophila KNic (DQ339144, 98%), and other bacteria with lower 16S rRNA gene similarity with known species. Determination of the phylogenetic affiliation of the methanotrophic isolate 4/20 with the genus Methylo bacter. The methanotroph belonging to the genus Methylobacter was isolated in pure culture at 20°C from a cover soil sample taken in April. The DGGE profile of the culture 4/20 contained one band which did not divide upon repeated DGGE, thus indicating the presence of a single bacterial species. The DNA fragment of 484 bp obtained from this DGGE band demonstrated 98% sequence identity with the 16S rRNA genes of M. marinus A45 (AF304197, NR_025132). The DNA isolated from the culture 4/20 was also amplified using the MethT1dF–1492R primer pair. Sequencing of this amplificate yielded a 16S rRNA gene sequence (1314 bp) that showed 98% sequence identity with that from M. tundripaludum SV96 (AJ414655), 97% with that of M. psychrophilus Z0021 (AF152597, MICROBIOLOGY
Culture designation (GenBank accession)
Methylosinus sporium NCIMB 11126 (100)
10
20
Closely related species (% sequence identity of 16S rRNA genes)
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11/40(HF565149)
NR_025016), and 97% with that of M. marinus A45 (AF304197, NR_025132) (Fig. 4). The 4/20 isolate grew well at 10 and 20°C, pH 7.0. The cells in the cul ture were roundish rods measuring 0.6–0.7 ± 1.1 µm. Decrease in methane emission during the cold sea son after the introduction of psychrotolerant metha notrophs into the cover soil. In the field experiment at the Khmet’evo landfill, an inoculum derived from enrichment cultures of methanotrophs active at low temperatures (15 ± 1°C) was introduced into the cover soil. The total number of microbial cells in the inocu lum, determined by DAPI staining, was 13 × 107 cells mL–1. During the first months after introduction of the inoculum (October, November), methane emission from the experimental site was lower than that from the control site (Table 3), and the most probable num ber of culturable methanotrophs was by an order of magnitude higher. DGGE analysis of the inoculum revealed methanotrophs closely related to M. tundri paludum SV96 (99% 16S rRNA gene identity). DISCUSSION Psychrotolerant, mesophilic, thermotolerant, and moderately acidophilic methanotrophs have been found in enrichment cultures obtained from the cover soil of the Khmet’evo MSW landfill. In cultures
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KALLISTOVA et al. Methylobacter bovis, L20839 Methylobacter vinelandii, L20841 Methylobacter capsulatus, L20843 Methylobacter luteus, AF304195 2/20a; 3/20a 59 Methylobacter marinus, AF304197 Methylobacter sp. 5FB, AJ868427 6/20a,b 53 T 81 Methylobacter tundripaludum strain SV96 , AJ414655 Methylobacter psychrophilus, AF152597 Methylobacter sp. T20, AF131868 99 4/10a,c 2/10a, c; 3/10c; 5/10a,b,c Methylobacter whittenburyi, X72773 91 Methylobacter sp. HG1, AF495887 60 Methylobacter sp. LW12, AY007295 Methylobacter alcaliphilus, AF096091 Methylomonas sp. strain KSWIII, AB015602 96 97 Methylomonas sp. LW13, AF150792 86 Methylomonas methanica, AF15 Methylomonas sp. LW16, AF150796 99 Methylomonas rubra, AF304194 Methylomonas scandinavica, AJ131369 96 Methylocaldum gracile, U89298 11/40c 67 Methylocaldum sp. E10a, AJ868426 96 Methylocaldum tepidum, U89297 Methylocaldum sp. 5FB, AJ868425 Methylocaldum szegediense, AJ627387 63 Methylocaldum sp. O12, DQ496233 74 Methylococcus capsulatus, AJ563935 Methylococcus thermophilus, X73819 Methylosinus sporium, AJ458486 7/10c 82 Methylosinus sporium, AJ458488 9/20b Methylocystis echinoides, AJ458473 88 Methylocystis parvus, AF150805 Methylocystis aldrichii, DQ364433 58 Methylosinus trichosporium, AJ458477 8/20f Methylocella silvestris, AJ49184 66 Methylocella tundrae, AJ563928 55 Methylocella palustris, AJ563927 98 9/10b Methyloccapsa acidiphila, AJ563926 Methylobacillus flagellatus KT, CP000284 7/10a; 8/10a Methylobacillus flagellatus, M95651 Methylobacillus pratensis, AY298905
0.10
97
50
Fig. 3. Methanotrophs and obligate methylobacteria identified in enrichment cultures obtained from the Khmet’evo landfill cover soil. Scale bar corresponds to 10% sequence difference between the 16S rRNA gene sequences. In the designations of the 16S rRNA gene sequences found in enrichment cultures, the “number/number” portion is the designation of the enrichment culture (sample number/incubation temperature), and the letters a–f designate bands in the gel from which the DNA was extracted and sequenced.
Methylobacter vinelandii, L20841 Methylobacter capsulatus, L20843 Methylobacter bovis, L20839
91 52 61
62 94
Methylobacter luteus, AF304195 Methylobacter marinus, AF304197 Methylobacter sp. 5FB, AJ868427 76 Methylobacter tundripaludum strain SV96T, AJ414655 99 Methylobacter psychrophilus, AF152597 Methylobacter sp. T20, AF131868 4/20 Methylobacter whittenburyi, X72773 Methylobacter sp. LW12, AY007295 Methylobacter alcaliphilus, AF096091 0.10
Fig. 4. Affiliation of the methanotrophic isolate 4/20 with the genus Methylobacter. Scale bar corresponds to 10% sequence dif ference between the 16S rRNA genes.
enriched at 10°C with 10 vol % methane in the gas phase, methanotrophs closely related to members of Methylobacter tundripaludum were identified. The Arctic wetland soil isolate M. tundripaludum is a psy
chrotolerant organism growing well in the range of temperatures from 10 to 27°C with the optimum at 23°C [28]. In the culture enriched at 10°C and 50 vol % methane in the gas phase, psychrotolerant MICROBIOLOGY
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Table 3. Methane emission from the surfaces of experimental and control sites of the Khmet’evo landfill Methane emission, mg C m–2 h–1 Month of measurement experiment September
control
1031.10 ± 23.32
871.59 ± 30.40
October
–4.78 ± 1.83
300.85 ± 16.42
November
777.31 ± 9.17
7620.10 ± 609.40
methanotrophs related to members of the genus Me thylocella were found. These moderately acidophilic organisms, isolated from acidic sphagnum bogs, grow at a low temperature (5–10°C) and are facultative methanotrophs. Members of Methylocella are wide spread both in acidic and in neutral natural and anthropogenic habitats [29]. Methanotrophs closely related to M. tundripaludum, M. psychrophilus, and Methylocella spp. were also identified in the cover soil of an Irish MSW landfill, composed of weakly acidic peat with pH 6.2 [14, 15]. In the cover soil of a landfill in Sweden, a selective effect of temperature on the composition of metha notrophic population was revealed: at low tempera tures (3–10°C), only type I methanotrophs devel oped, whereas both types of methanotrophs developed at 20°C [21]. In the present study, we identified in a culture enriched at 10°C methanotrophs closely related to members of the mesophilic species Methy losinus sporium (type II), thus confirming our earlier data on the ability of some mesophilic type II metha notrophs to grow at 10–15°C [30]. Unlike other authors who studied the composition of metha notrophic populations at MSW landfills [12–18], we did not find members of the genus Methylocystis in our enrichment cultures. All of the type II methanotrophs that we revealed belonged to the genera Methylosinus and Methylocella. A methanotroph closely related to the thermoto lerant species Methylocaldum gracile, which grows in the range of temperatures from 20 to 47°C with the optimum at 42°C [31], was isolated from our enrich ment culture obtained at 40°C from a sample of the soil with a temperature of 11.7–12.5°C. Members of the genus Methylocaldum frequently occur at MSW landfills [13, 16–19]. Methylocaldum spp. have been shown to prevail over other methanotrophs in soil samples with temperatures of no more than 20°C. Thus, Methylocaldum spp. prevailed in laboratory landfill lysimeters with a mean soil temperature of 14– 19.8°C [11], in landfill cover soil in April (soil tempe rature of 5.4–17.8°C) and September (9.5–19.2°C) [18], and also in the landfill soil in the period from October to December (3.4–11°C) [19]. MICROBIOLOGY
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In addition to methanotrophs, we also identified in enrichment cultures obligate methylobacteria that uti lize methanol and methylamines (but not methane) as growth substrates and employ the RuMP pathway of formaldehyde assimilation [32]. High proportion of methylotrophs was also found in the microbial popu lation of the cover soil of an Irish MSW landfill [15]. Studies of various MSW landfills in Germany de monstrated that, irrespective of the landfill location, depth of the soil horizon, and soil parameters, all of the landfills were dominated by methanotrophs of the genera Methylocystis, Methylobacter, and Methylococ cus. The authors assume that the high methane con centration is the most important factor that controls the composition of the methanotrophic populations at MSW landfills [13]. In our opinion, in regions with a temperate continental climate, an important factor that affects the composition of the active metha notrophic population of the cover soil is its tempera ture. During the warm seasons (summer), mesophilic and thermotolerant methanotrophs are active in the cover soil. This follows from the fact that just these methanotrophs were present in most of the enrich ment cultures initiated from cover soil samples over the whole year and incubated at 20°C, whereas they were not found in enrichment cultures grown from the same soil samples at an incubation temperature of 10°C. On the contrary, during the cold seasons (spring and late autumn), the decreased soil temperature resulted in the activation of psychrotolerant metha notrophs in the soil. Therefore, the introduction of the inoculum into the cover soil, causing an increase in the number of psychrotolerant methanotrophs, promoted a decrease in the methane emission during the cold season (autumn). Inoculation of the cover soil with methanotrophs may be a promising technique for decreasing methane emission within old sites of land fills, where the cover soil was locally damaged during recultivation, and also in the sites with a thin layer of cover soil. ACKNOWLEDGMENTS This work was supported by the Russian Academy of Sciences (project no. 3 of Russian−Finnish scien
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tific exchange) and by the Finnish Academy of Scien ces (project no. 214719). 12.
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