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Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions Article in Aerobiologia · January 2013 DOI: 10.1007/s10453-013-9287-1
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Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions Tina Šantl-Temkiv, Kai Finster, Bjarne Munk Hansen, Lejla Pašić & Ulrich Gosewinkel Karlson Aerobiologia International Journal of Aerobiology including the online journal `Physical Aerobiology' ISSN 0393-5965 Aerobiologia DOI 10.1007/s10453-013-9287-1
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Author's personal copy Aerobiologia DOI 10.1007/s10453-013-9287-1
ORIGINAL PAPER
Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions Tina Sˇantl-Temkiv • Kai Finster • Bjarne Munk Hansen • Lejla Pasˇic´ Ulrich Gosewinkel Karlson
•
Received: 7 September 2012 / Accepted: 7 January 2013 Ó Springer Science+Business Media Dordrecht 2013
T. Sˇantl-Temkiv K. Finster Department of Physics and Astronomy, Stellar Astrophysics Centre, Aarhus University, Ny Munkegade 120, 8000 Aarhus, Denmark
their presence and activity in the atmosphere has not been investigated so far. We enriched airborne methanotrophs from air and rainwater and showed that they oxidized methane at atmospheric concentration. The majority of seven OTUs, detected using pmoA gene clone libraries, were affiliated to the type II methanotrophic genera Methylocystis and Methylosinus. Furthermore, 16S rRNA gene clone libraries revealed the presence of OTUs affiliated with the genera Hyphomicrobium and Variovorax, members of which can stimulate methane oxidation by yet unidentified mechanisms. Simulating cloud-like conditions revealed that although both low pH and the presence of common cloud-borne organics negatively affected methane oxidation, airborne methanotrophs were able to degrade atmospheric methane in most cases. We demonstrate here for the first time that viable methanotrophic bacteria are present in air and rain and thus expand our knowledge on the global distribution of methanotrophs to include the atmosphere. The fact that they can degrade methane to below atmospheric concentrations when inoculated into artificial cloud water leads to an important possible effect of these organisms: the atmosphere may not only function as a medium for microbial dissemination, but also as a site of active microbial methane turnover.
L. Pasˇic´ Section for Molecular Genetics and Biology of Microorganisms, Department of Biology, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
Keywords Air microbiology Methane Methanotrophs Methylocystis Aerial dispersal Airborne bacteria
Abstract Atmospheric methane is degraded by both photooxidation and, in topsoils, by methanotrophic bacteria, but this may not totally account for the global sink of this greenhouse gas. Topsoils are a prominent source of airborne bacteria, which can degrade some organic atmospheric compounds at rates similar to photooxidation. Although airborne methanotrophs would have direct access to atmospheric methane,
Electronic supplementary material The online version of this article (doi:10.1007/s10453-013-9287-1) contains supplementary material, which is available to authorized users. T. Sˇantl-Temkiv B. M. Hansen U. G. Karlson (&) Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark e-mail: uka@dmu.dk T. Sˇantl-Temkiv K. Finster Microbiology Section, Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus, Denmark
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1 Introduction Methane is among the major greenhouse gasses due to its high capacity to absorb infrared radiation, 25 times higher than the capacity of CO2 (Kvenvolden and Rogers 2005). Human activity has caused the atmospheric concentration of methane to more than double over the past 300 years (Isaksen et al. 2009), making it the most abundant organic compound in air. Aerobic methanotrophic bacteria, which use methane as their sole source of carbon and energy, act as a major methane sink. Methanotrophs consume over half of the methane produced, before it enters the atmosphere (Kvenvolden and Rogers 2005). In the atmosphere, photooxidation by hydroxyl radicals is the dominant sink for atmospheric methane, accounting for 90 % of its degradation (Wuebbles and Hayhoe 2002). However, recent studies have indicated that the role of hydroxyl radicals might have been overestimated (Wang et al. 2008), which leaves a yet unidentified sink for atmospheric methane. Most of the methanotrophic bacteria are found in areas with high methane fluxes from anaerobic methanogenic environments. Nevertheless, it has been demonstrated repeatedly that methanotrophs residing in top layers of different soil types oxidize methane at atmospheric concentrations (*1.8 ppm, v/v) (Bull et al. 2000; Knief et al. 2003), thus acting as a sink for about 5 % of the methane emitted to the atmosphere (Wuebbles and Hayhoe 2002). Methanotrophs that are responsible for the oxidation of atmospheric methane display a high affinity toward methane. Cultivated methanotrophic bacteria have been classified as type I, affiliating with c-Proteobacteria, and type II methanotrophs, affiliating with a-Proteobacteria. Cultures of type II methanotrophs of the Methylocystis/Methylosinus group were shown to have a high affinity to methane (Dunfield et al. 1999). Unlike type I methanotrophs, which did not exhibit high affinity toward methane, type II methanotrophs could utilize methane at atmospheric concentrations for cell maintenance, although they could not sustain growth at these concentrations (Knief and Dunfield 2005). It was suggested that the growth of methanotrophs in environments, exposed only to atmospheric methane, is stimulated either by periodically elevated methane concentrations or
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by the presence of other C-1 compounds, such as methanol or formate (Jensen et al. 1998; Knief and Dunfield 2005). The key enzyme involved in methane oxidation in most methanotrophs is the particulate methane monooxygenase (pmo), which catalyzes the conversion of methane into methanol. Among cultivated type II methanotrophs a pmo-like gene cluster ‘‘pmo2’’ of low sequence similarity to the pmo1 cluster was commonly identified in addition to the pmo1 cluster (Yimga et al. 2003). Recently, the high-affinity methane oxidation in a Methylocystis strain SC2 has been associated with the isoenzymes, encoded by the pmo2 cluster (Baani and Liesack 2007). While pmo1 was expressed only at high methane concentrations, pmo2 was constitutively expressed and was proposed to provide Methylocystis SC2 with a selective advantage in environments exposed to methane at atmospheric concentrations. As soil is among the most prominent sources of airborne bacteria (Burrows et al. 2009), methanotrophic bacteria of topsoil layers may get aerosolized and could even be metabolically active when getting scavenged by cloud droplets. Cloud droplets play a potentially important role as sites where atmospheric organic compounds are processed (Blando and Turpin 2000). In several studies, it has been shown that cloud-borne heterotrophic bacteria can degrade common cloud-borne organics, such as aldehydes, alcohols, mono- and dicarboxylic acids, at similar rates as photooxidation (Ariya et al. 2002; Amato et al. 2005; Vaı¨tilingom et al. 2010; Temkiv et al. 2012), and on the basis of these observations, it has been hypothesized that cloud-borne bacteria have an important influence on atmospheric chemistry. This raises the question, whether airborne methanotrophic bacteria likewise could impact the turnover of methane in the atmosphere. Although cloud-borne bacteria have easy access to atmospheric methane as well as metabolic intermediates of methane oxidation (Marinoni et al. 2004), it remains to be investigated firstly whether methanotrophic bacteria are present in air and clouds at all and secondly whether they would be able to utilize methane at atmospheric concentrations. Here we report for the first time on the presence and activity of rain- and airborne methanotrophs.
Author's personal copy Aerobiologia Fig. 1 Phylogenetic tree depicting the relationship of translated pmoA sequences, identified in air and rain. The most likely topology shown here was obtained by using the JTT model of sequence evolution along with a C distribution of rate heterogeneity with a proportion of invariant sites. Names in bold correspond to enrichment culture clones obtained from air sample Lpl or rain sample R1. The scale represents expected number of substitutions per site
Table 1 The coverage (no. of sequences) of pmoA gene (N = 158) and 16S rRNA gene (N = 219) clone libraries for all enrichment replicates of samples Lpl and R1 Sample
Lpl
Enrichment nr.
1
pmoA 16S rRNA
R1 2
3
4
5
F
Sum
1
2
8
14
20
16
18
16
19
20
19
19
16
92
12
13
9
20
113
18
19
19
2 Results 2.1 Diversity of methanotrophs in enrichment cultures The air sample that was collected over the landfill, and the first rain sample contained cultivable methanotrophs, which became enriched on 2 % (v/v) methane. From the corresponding 12 replicate enrichments, a clone library of 158 pmoA sequences was obtained (Table 1), from which 7 OTUs were constructed at an evolutionary distance of 7 % (Fig. 1). The same OTUs were in most cases present in several replicate enrichments of the same sample. However, distinct OTUs were present in air or rain enrichments. Most of the sequences from both air and rain enrichments belonged to type II methanotrophs within the a-Proteobacteria subphylum. Sequences affiliated to the genera Methylocystis and Methylosinus accounted for 77.2 % of all 158 sequences, forming 5 OTUs. One of these OTUs
3
4
5
F
Sum
3
13
16
66
12
18
20
106
was closely affiliated to the pmo2 gene cluster of Methylocystis sp. SC2. Type I methanotrophs of the cProteobacteria subphylum were only found in 3 replicates of air enrichment cultures and accounted for 22.8 % of all 158 sequences. They clustered with members of the Methylococcus-group and were closely related to strains within the genus Methylocaldum. 2.2 Members of rain and air enrichment cultures A clone library of 219 16S rRNA gene sequences was obtained from the 12 replicate enrichments (Table 1). We constructed 42 OTUs at an evolutionary distance of 3 %. The phylogenetic position of OTU representatives that belong to Proteobacteria is presented in SI-Fig. 1. OTUs affiliated with methanotrophs belonged to three genera, which were already detected in pmoA gene clone libraries. The dominant OTU (20.9 % of all 215 sequences) was present in 5 rain enrichment cultures and was affiliated with the genus Methylocystis.
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Author's personal copy Aerobiologia Table 2 Genera enriched from air or rain, based on 16S rRNA sequences Group
Genus
No. of OTUs
No. of sequences
No. of replicate enrichments, in which genus was detected Enrichment cultures from rain sample, R1
Enrichment cultures from air sample, Lpl
a-Proteobacteria
Afipia
1
4
3
0
a-Proteobacteria
Brevundimonas
2
13
2
2
b-Proteobacteria
Ramlibacter
1
1
0
1
a-Proteobacteria
Devosia
1
1
2
0
b-Proteobacteria
Hydrogenophaga
1
25
0
6
a-Proteobacteria c-Proteobacteria
Hyphomicrobium Lysobacter
5 1
17 2
4 0
3 1
a-Proteobacteria
Mesorhizobium
1
1
0
1
a-Proteobacteria
Woodsholensia
2
4
1
2
a-Proteobacteria
Reyranella
1
1
0
1
a-Proteobacteria
Methylobacterium
2
6
2
1
c-Proteobacteria
Methylocaldum
2
10
0
2 (3)a a
0 (2)a
a-Proteobacteria
Methylocystis
1
45
5 (4)
a-Proteobacteria
Methylosinus
1
10
0
6 (6)a 1
Actinobacteria
Mycobacterium
1
1
0
d-Proteobacteria
Nannocystis
1
1
0
1
Bacteroidetes
Pedobacter
1
4
0
4
a-Proteobacteria
Phenylobacterium
1
1
0
1
c-Proteobacteria
Pseudomonas
4
11
1
4
c-Proteobacteria
Pseudoxanthomonas
1
6
4
0
a-Proteobacteria
Rhizobium
1
1
0
1
Bacteroidetes a-Proteobacteria
Roseivirga Sphingopyxis
1 1
1 1
0 0
1 1
c-Proteobacteria
Stenotrophomonas
1
4
0
3
b-Proteobacteria
Variovorax
2
12
5
1
Other
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Italic type indicates known methanotrophic genera a
Indicates the number of replicate enrichment cultures, in which methanotrophic genera were detected by pmoA clone libraries
The sequences related to Methylosinus (1 OTU) and Methylocaldum (2 OTUs) represented 4.6 % each of the analyzed 215 sequences and were detected only in air enrichments. Besides these three genera of methanotrophs, some other characteristic community members were found. A few genera were prevalent in enrichments from both rain and air (Table 2), as for example Hyphomicrobium (7.9 % of all 215 sequences), Variovorax (5.6 %), and Pseudomonas (5.1 %), while others were common in enrichments from only one source, such as Hydrogenophaga (11.6 %), Pedobacter (1.9 %), Stenotrophomonas (1.9 %) and Pseudoxanthomonas (2.8 %).
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2.3 Methane oxidation at low methane concentrations Oxidation of methane at atmospheric concentrations was observed in all replicates of rain and air enrichment cultures after they had grown on 2 % (v/v) methane. A more detailed characterization of methane degradation conducted in two freshly diluted enrichments showed that 2 % methane was fully degraded within 5 (for the air enrichment Lpl.5) or 8 days (rain enrichment R1.1), while atmospheric methane (1.8 ppm (v/v)) was degraded to levels below the detection limit (below 0.2 ppm) within 7–16 h.
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Organic compounds contained in artificial cloud water medium also had a negative effect on methane oxidation (Fig. 3c, d). The effect increased with increasing concentration of DOM. Artificial cloud water medium with *40 lM DOM and *200 lM DOM did not affect the oxidation rate. Enrichments growing in the presence of *400 lM of DOM exhibited a reduced oxidation rate, and at *1,600 lM DOM, methane oxidation ceased completely. Even though the oxidation rate was reduced in the presence of *400 lM DOM, we observed methane oxidation at below 20 ppm headspace concentration. However, at the end of the experiment the methane concentration was still above atmospheric levels. As with the pH, the rain enrichment R1.1 was affected more strongly than the air enrichment Lpl.5. Fig. 2 Degradation of low concentrations of methane (\350 nM, corresponding to 5.6 lg/l) over 2.5 days in the headspace of serum bottles containing enrichment cultures from rain or air. Exponential models are presented by the full line (air enrichments, p \ 0.001) and the dashed line (rain enrichments, p [ 0.5). plus—rain enrichment culture; upright triangle—air enrichment culture
In actively growing enrichment cultures, the oxidation rates decreased from 35 to 45 nM/day at *15 ppm methane to 4–6 nM/day at *1.5 ppm. In general, the air enrichment expressed higher oxidation rates than the rain enrichment. The highaffinity oxidation rate was dependent upon methane concentration; that is, it followed first-order kinetics, with oxidation rate constants of 4.4 and 4.5 h-1 for rain and air enrichments, respectively (Fig. 2). 2.4 Methane oxidation under simulated cloud conditions Low pH values affected methane oxidation negatively (Fig. 3) and in two ways: firstly, there was a longer lag phase, during which the methane concentration remained constant, and secondly, the rate of methane consumption was reduced (Fig. 3a, b). The rain enrichment culture R1.1 was slightly more sensitive to low pH than the air enrichment Lpl.5. In R1.1 methane oxidation rates were reduced already at pH 5.0, whereas Lpl.5 only was affected at pH 4.0. Regardless of pH value, the enrichment cultures retained their methane oxidation capacity at low concentrations and eventually depleted methane to below atmospheric concentrations.
3 Discussion 3.1 Diversity of methanotrophs in enrichment cultures The topsoils of the Skellingsted landfill have previously been characterized as exhibiting high methane oxidation activity (Christophersen et al. 2000) and as containing between 2.7 9 105 and 4.1 9 106 cultivable methanotrophs per gram of soil (dw) (Svenning et al. 2003) (SI-Table 1). Topsoils covering landfills are exposed to very high methane fluxes, which promote the development of large, active methanotrophic microbial communities (Hanson and Hanson 1996). Such soil surfaces could serve as important sources for the emission of methanotrophic bacteria into the atmosphere. Indeed, studying the presence of methanotrophic bacteria in the air over the Skellingsted landfill, we found 4 species of methanotrophs. Assuming that the average particle mass over the landfill was 45 lg/m3 of air (Hueglin et al. 2004), we estimated an upper limit of density of cultivable methanotrophs in air above the landfill. Airborne particles in the atmospheric boundary layer were likely derived from the landfill soil and the grass cover. Based on previously reported densities of cultivable methanotrophs in Skellingsted landfill topsoil (Svenning et al. 2003) and assuming all particles in air to be soil-derived (this assumption was made in order to retrieve the upper limit of density), the highest expected density of cultivable methanotrophs was
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Author's personal copy Aerobiologia Fig. 3 a Effect of pH on methane oxidation by an enrichment from rain. b Effect of pH on methane oxidation by an enrichment from air; Fig. 3a, b: diamond—pH 6.8; ex—pH 6.0; plus—pH 5.0; upright triangle—pH 4.0. c Effect of different concentrations of ACW medium on methane oxidation by an enrichment from rain. d Effect of different concentrations of ACW medium on methane oxidation by an enrichment from air. Fig. 3c, d: filled diamond—control; downright triangle—40 lM DOM; asterisk—200 lM DOM; diamond—400 lM DOM; filled squares— 1,600 lM DOM. Fig. 3a, b, c, d: The horizontal line marks the level of the atmospheric methane concentration, 1.8 ppm
from 10 to 200 cells/m3 of air (SI-Table 1). Hence, our air sample (513 m3) contained at a maximum between 5,000 and 100,000 methanotrophs. In the atmosphere, bacteria get rapidly desiccated and, during daytime, are exposed to UV radiation and oxidative stress, all of which compromises their survival. Thus, besides the presence of cultivable airborne methanotrophs close to their potential origin, we also investigated the wet deposition of methanotrophs during two rain events. We enriched 3 specieslevel types of methanotrophs from one of the rain samples, which indicates that (1) their presence is not limited to the near-surface air masses and that (2) they can get scavenged by cloud or rain droplets. The absence of viable methanotrophs from the remaining two samples, however, points to the fact that the presence of airborne methanotrophs is spatially and temporarily variable, depending on yet unidentified conditions. The methanotrophs enriched from air and rain were closely affiliated to known cultivated methanotrophs. Our type II sequences were closely related to
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sequences of methanotrophs, previously isolated from landfill soil (Wise et al. 1999). The same three genera (Methylocaldum spp, Methylocystis spp, and Methylosinus spp) were previously detected in many upland and hydromorphic soils (Knief et al. 2003). Comparing the response of different methanotrophic isolates to low methane concentrations, Knief and Dunfield (2005) found that some strains of Methylocystis were the most oligotrophic, utilizing methane at atmospheric concentrations for several months without declining in cell numbers. And while strains belonging to the genus Methylosinus were also able to utilize methane at low concentrations, strains belonging to the genus Methylocaldum were unable to do so. In addition to their ability to utilize atmospheric methane, which could be beneficial for active airborne cells, Methylocystis sp. and Methylosinus sp. can also form desiccation-resistant resting stages (i.e., cysts or exospores) (Staley et al. 2005), which may help them to survive the aerial transport. Cultivable type II methanotrophs were dominant in the atmosphere above Skellingsted landfill, as they
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were present in all the replicate enrichments, while type I methanotrophs were only detected in 3 replicates (Table 2). It has previously been reported that type II methanotrophs were much more abundant in soils from the same landfill (Svenning et al. 2003). In rain exclusively type II methanotrophs were found. Four species-level methanotrophs present in our air and rain enrichments clustered with type II methanotrophic isolates (Fig. 1) that have been shown to be capable of high-affinity methane oxidation (Dunfield et al. 1999, 2002; Knief and Dunfield 2005). We also detected a pmoA sequence that affiliated with the pmo2 gene cluster, which was recently found in many type II methanotrophs (Yimga et al. 2003). In Methylocystis strain SC2, this cluster has been associated with the ability of this strain to utilize methane at low concentrations (Baani and Liesack 2007). We postulate that it is very likely that cells of methanotrophs belonging to the genus Methylocystis are responsible for the oxidation of methane at low concentrations by our enrichments. None of the pmoA sequences present in our enrichments fell within the still uncultivated clusters USC a, USC c or cluster I (Fig. 1), which have been proposed to be responsible for the oxidation of methane at atmospheric concentrations in topsoils (Knief et al. 2003; Ricke et al. 2005). In order to look for the presence of these clusters in atmospheric samples, direct molecular approaches, by which it is possible to avoid cultivation biases, should be employed in the future. 3.2 Members of rain and air enrichment cultures Based on their 16S rRNA gene sequences, we identified several other bacterial genera common to most air and rain enrichment replicates (SI-Fig. 1). Members of Pseudomonas spp., Hyphomicrobium spp. and Variovorax spp were previously isolated from soil enrichment cultures grown on low methane concentrations (\275 ppm) (Dunfield et al. 1999). Although none of these isolates were able to grow on methane, Dunfield et al. (1999) demonstrated that Variovorax spp. stimulated the growth of type II methanotrophs from the Methylocystis-Methylosinus group and a co-culture of these methanotrophs with Variovorax spp. showed high-affinity kinetics of methane oxidation. Interestingly, Hyphomicrobium sp., another common member of our enrichment
cultures, was also proposed to promote the growth of methanotrophs on methane by removing excess methanol that builds up in their environment (Wilkinson and Harrison 1973). By forming clumps or rosettes (Staley et al. 2005), which may be associated with cells of methanotrophs, Hyphomicrobium sp. could provide a beneficial microenvironment for methanotrophs by removing methanol as well as shielding them from UV radiation. It seems that some viable bacteria common in both the dry and the liquid phase of the atmosphere belong to genera that could promote the activity of methanotrophs. These bacteria may even get aerosolized together with methanotrophs, for example by being either attached to each other or to the same soil particle. However, this raises the question of whether there are microbial interactions in the atmosphere. 3.3 Methane oxidation under simulated cloud conditions For cloud water droplets, pH values between 3.9 and 7.0 have been reported (Marinoni et al. 2004). We performed microcosm experiments, in order to investigate the effect of acidic conditions on the activity of airborne methanotrophs. Although we observed decreased rates of methane oxidation at low pH values, the enrichment cultures were still able to degrade atmospheric methane even at pH 4.0. Cloud water organic compounds affected methane oxidation negatively. This is surprising as it has been reported for some of the organic compounds in ACW medium that they stimulated methane oxidation (West and Schmidt 1999), probably by serving as an additional carbon and energy source. In addition, ACW medium was shown to have a positive effect on the activity of cloud-borne bacteria, when oxidizing organic compounds that are common in cloud water (Vaı¨tilingom et al. 2010). In our study, none of the DOM concentrations, which were applied to simulate the concentration range of DOM in cloud water (100–1,300 lM) (Marinoni et al. 2004), had a positive influence on methane oxidation by our enrichments. The highest concentration of DOM even completely inhibited methane oxidation, despite the fact that we could observe bacterial growth in the enrichments. This reconfirms that in highly populated, nutrient-rich conditions methanotrophs are outcompeted by other microbiota (Svenning et al. 2003). However, natural
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cloud droplets, usually having much lower concentrations of DOM and being only sparsely populated by bacteria, could offer an appropriate environment for methanotrophic activity.
4 Conclusions To date, methanotrophic bacteria have been studied in soils, deserts, landfills, tundra, wetlands, rice paddies, sediments, lakes and marine environments (Hanson and Hanson 1996) but not in the atmosphere. We report for the first time that viable methanotrophic bacteria are present in both the dry and the wet phase of the atmosphere, indicating that their presence is not just sporadic. However, due to the limited number of samples, future studies possibly employing cultivationindependent methods, for example, Q-PCR, should focus on enumerating methanotrophs in the atmosphere. We also showed that airborne methanotrophs were able to oxidize methane at atmospheric concentrations even at the low pH characteristic for cloud droplets. These bacteria seem to be more competitive in non-crowded, nutrient poor environments; therefore, cloud droplets may provide an appropriate environmental niche for their activity and growth. We hypothesize that the atmosphere serves both as a medium for dissemination of viable airborne methanotrophs from sources with dense and active populations to more remote or newly established environments and as an environment where significant methane oxidation could take place. Studies in cloud chambers, designed to investigate the activity of airborne methanotrophs in simulated in situ conditions are needed to obtain conclusive insights into biological methane oxidation in the atmosphere. We may thus be able to elucidate whether viable methane degrading bacteria in the liquid phase of the atmosphere may contribute to the missing sink of atmospheric methane.
5 Experimental procedures 5.1 Sampling procedure Two rain samples were collected into a sterile stainless steel funnel from a platform of a water tower at 30 m above ground level (N55°410 29.5100 /E12°60 17.5200 ).
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The first rain sample (R1, 152 ml) was collected on October 28, 2009, and the second between November 1 and 2 (R2, 60 ml), 2009. Prior to collection, the funnel was rinsed with a mixture of 1 % benzalkonium chloride and 62 % ethanol in deionized water and then rinsed 5 times with sterile deionized water to remove the remaining sterilization mixture. Afterward, the equipment was autoclaved for 1 h. Sterile deionized water, poured over the sampling equipment on the site of collection and treated in the same way as the samples, was used as a negative control. Two air samples of 513 m3 each were collected 1 m above ground using the Ka¨rcher DS 5600 commercial vacuum cleaner (Alfred Ka¨rcher GmbH & Co. KG, Germany), whose use as a microbial air sampler has been reported previously (Zweifel et al. 2012). Its inner water holding vortex chamber is designed to form a water vortex causing the particles carried by the air stream to be captured in the liquid that is contained in the vortex chamber. The air flow through the vortex chamber was maintained at 1.8 m3 min-1, measured immediately after sampling by fitting a wind speed meter with a rotating vane sensor (Kimo LVB, Marnela-Valle´e, France) to the inlet pipe of the vacuum cleaner. Prior to use, the vortex chamber and all ducts and materials in contact with airstream and sample were washed three times in 1 M HCl, rinsed in filtered (0.2 lm poresize, Supor-filter, all Life Sciences, St Petersburg, FL) deionized water, and thereafter sterilized in 70 % ethanol, which was left to evaporate under sterile conditions. Then the assembled collection system was sealed and stored in sterile polycarbonate bags. Immediately before sampling, 500 ml of freshly 0.2-lm-filter-sterilized 1 9 PBS (containing 0.137 M NaCl, 2.7 mM KCl, 4.29 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2) was used to rinse the vortex chamber. The rinse was processed as negative control along with the samples. The vortex chamber was immediately refilled with 3 L of freshly filtered 1 9 PBS that served as collection buffer. During sampling for 285 min, approx. 300 ml of buffer was lost due to evaporation, which was within the design limits of the vortex chamber. After sampling, the remaining buffer was further processed as described below. The first air sample (A1) was collected above a lawn (N55°410 44.1200 /E12°60 4.7500 ) near the tower on October 27, 2009, and the second sample (Lpl) was collected above the grass-covered Skellingsted landfill (N55°350 31.2300 /E11°260 39.1500 ) on October 29, 2009.
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The weather during both sampling events was as follows: air temperature, 9 °C; relative humidity, 90 %; overcast; no precipitation. On October 27, the wind was from the west at 2 m/s, whereas on October 29, it was from the east at 3 m/s. 5.2 Enrichment of the samples Enrichments were performed in 120-ml septum flasks containing 20 ml of modified Nitrate Mineral Salts Medium (NMS) (Whittenbury et al. 1970). The medium (pH 6.8) contained 10 mM KNO3, 4.4 mM MgSO4, 1.5 mM CaCl2, 1 lM Na2MoO4 and 10 lM ferric salt of EDTA. One ml of a trace element solution (Pfennig 1962) was added to 20 ml of medium. Ten ml of autoclaved phosphate buffer (0.2 M KH2PO4 and 0.4 M Na2HPO4, pH 6.8) was added to 1 l of the autoclaved medium. Bacteria were concentrated from rain and vortex chamber buffers by filtration of the whole sample onto a 0.22 lm Supor polyethersulfone filter (Pall Corporation, East Hills, New York). The collected microorganisms were re-suspended in autoclaved deionized water of a smaller volume, which resulted in a 100 times (rain samples) or 1,000 times (air samples) concentrated sample. Aliquots (200 ll) of concentrated samples were added to the serum bottles in quintuplicates. The filter was added to a separate bottle and later treated in the same manner as the replicates. Then, all bottles were sealed with rubber septa and aluminum crimps. Methane was injected at 2 % (v/v) using a gastight syringe and all enrichments were incubated at room temperature. Bacterial growth, decline in CH4 concentration and increase in CO2 concentration were used as criteria for the presence of methanotrophs in the serum bottles. Bacterial growth was determined by visual inspection comparing the optical density of the enrichment cultures to sterile NMS medium. The change in CH4 and CO2 concentrations in the headspaces was analyzed periodically (every 1–3 weeks) by gas chromatography (Shimadzu GC 8AIT, Kyoto, Japan) equipped with a flame ionization detector (FID), after separation on a PQ column (Porapak Q 50/20 mesh 8 feet 2–0330) with helium as the carrier gas. The injection port temperature was 50 °C and the oven temperature was 20 °C. Four negative controls, collected prior to each sample, were treated in the same way as the samples— they were both up-concentrated and enriched in
6 replicates—and yielded no bacterial growth or methane consumption. After 6 weeks of incubation, all enrichments were evaluated, with respect to bacterial growth, CH4 consumption and CO2 production. None of the replicates from the negative controls from the rain sample R2 and from the air sample A1 exhibited growth or CH4 consumption and were discontinued, leaving 6 replicate enrichments (Lpl.1–Lpl.5 real replicates and Lpl.F from filter incubation) of the landfill air plus 6 replicates (R1.1– R1.5 replicates and R1.F from filter incubation) of one of the rain samples for the remainder of the study. Twelve positive enrichments were diluted 20 times in 20 ml of fresh NMS medium and incubated with 2 % methane in the dark while being constantly shaken at 200 rpm. The residual methane concentration was determined after 3 months of incubation. Methane was analyzed on a SRI 310C GC (SRI, Torrance, CA) as described by Ba´rcena et al. (2010). Uninoculated NMS medium served as negative control in all experiments. One replicate from each environmental sample (rain sample enrichment R1.1 and air sample enrichment Lpl.5) were chosen for detailed studies of simulated cloud conditions on the oxidation of atmospheric methane.
5.3 DNA extraction and clone library construction Bacterial cells from 20 ml of all 12 enrichment replicates were collected by centrifugation for 20 min at 43 0009g and 4 °C and consecutively washed in sterile deionized water. DNA was extracted using the FastDNA SPIN kit for soil (MP Biomedicals LLC, Illkirch, France) according to the instructions of the manufacturer. DNA was purified using the Geneclean DNA purification kit (Anachem Ltd., Bedfordshire, UK). This resulted in a total of 12 purified DNA samples. Nearly full length segments of 16S rRNA genes were amplified from all 12 DNA samples with Taq DNA Polymerase Master Mix RED (Ampliqon, Skovlunde, Denmark) using the universal bacterial primers 27f (50 -AGAGTTTGATCMTGGCTCAG-30 ) and 1492r (50 -GGYTACCTTGTTACGACTT-30 ) in presence of 10 mg/ml of BSA. The amplifications were performed by initial denaturation at 94 °C for 3 min, 30 cycles of 94 °C for 1 min, 55 °C for 30 s and 72 °C for 90 s and a final extension step at 72 °C for 15 min. PCR products were purified with a PCR
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clean-up Kit (Sigma Aldrich NA 1020-1, St. Louis, MO) just prior to cloning. The PCR products were ligated into the pGEMÒ-T vector system (Promega, Madison, WI) and transformed into chemically competent E. coli cells strain JM109 (Promega, Madison, WI). Twenty white transformants from each of the 12 samples were picked and a PCR was run with M13 forward and reverse primers using Taq DNA Polymerase Master Mix RED (Ampliqon, Skovlunde, Denmark) according to the instructions of the manufacturer. The sequencing was performed by GATC Biotech AG (Konstanz, Germany) using the T7 primer. A partial segment of the pmoA gene (around 500 bp) from 12 DNA samples was amplified using A189F (50 -GGNGACTGGGACTTCTGG-30 ) and A650R primers (50 -ACGTCCTTACCGAAGGT-30 ) (synthesized by Eurofins MWG Operon, Ebersberg, Germany) along with Taq DNA Polymerase Master Mix RED (Ampliqon, Skovlunde, Denmark). These amplifications were performed by initial denaturation at 96 °C for 10 min, 30 cycles of 94 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min and a final extension step at 72 °C for 15 min. The cloning and sequencing was carried out as with the 16S rRNA genes. The coverage of clone libraries for all enrichment replicates is presented in Table 1. 5.4 Phylogenetic analysis pmoA sequences from 12 clone libraries were converted into sense orientation using SequencherÒ version 5.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI USA). They were aligned with Muscle (Edgar 2004) and translated using BioEdit (Hall 1999). Distance matrices were generated in Phylip (Felsenstein 1993), and OTUs were constructed in Dotur (Schloss and Handelsman 2005) at an evolutionary distance of 7 % (Degelmann et al. 2010). The nearest neighbors were assigned to representatives of OTUs from GenBank (http://www.ncbi. nlm.nih.gov, BLASTX algorithm). All the sequences were aligned with Muscle (Edgar 2004). Maximum likelihood phylogenetic trees were constructed using the JTT model with gamma distributed rate heterogeneity and a proportion of invariable sites in MEGA (Tamura et al. 2011). Phylogeny was tested by running 1000 bootstrap replications. 16S rRNA gene sequences from 12 clone libraries were converted into sense orientation using OrientationChecker
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Version 1.0 (Bioinformatics Toolkit, http://www.bioin formatics-toolkit.org/) and aligned using Muscle (Edgar 2004). Distance matrices were generated in Phylip (Felsenstein 1993), based on the Kimura two-parametric evolutionary model, and used as input for Dotur (Schloss and Handelsman 2005), in which OTUs were constructed using the furthest neighbor algorithm at an evolutionary distance of 3 %. 5.5 Methane oxidation under simulated cloud conditions Microcosm incubations were performed to test the effect of low pH and abundant cloud water organic compounds on methane oxidation. NMS medium with pH values of 4.0, 5.0 and 6.0 was used to simulate pH conditions in clouds. Cloud water conditions were simulated in ‘‘artificial cloud water’’ (ACW) medium (Vaı¨tilingom et al. 2010), in order to test the effect of major chemicals dissolved in cloud water on methane oxidation. The solution mimicked the complex chemical composition of natural cloud water, and could thus be used to evaluate the influence of organic compounds (acetate, formate, succinate, and oxalate) on methane oxidation. Four concentrations of ACW (pH 6.8) were tested: 19 (corresponding to *40 lM concentration of dissolved organic matter (DOM)), 59 (*200 lM DOM), 109 (*400 lM DOM) and 409 (*1,600 lM DOM), covering the range of DOM concentrations reported for cloud water (Marinoni et al. 2004). To exclude that the absence of micronutrients, which are needed for pmoA expression and that are present in NMS medium, is responsible for differences in methane oxidation patterns, an additional microcosm with 59 ACW in combination with NMS medium was included. Methane oxidation was not affected by the presence of NMS medium (data not presented). The enrichments R1.1 and Lpl.5 were used for these two experiments. 100 ll each of the original enrichment cultures were diluted 20 times in fresh NMS medium and grown on 2 % methane. Methane concentrations were measured every 6–12 h until they reached the detection limit of the GC method. All experiments were performed in triplicate, and the average values of replicate measurements are presented. Triplicate negative controls, consisting of uninoculated NMS medium, were used and expressed neither bacterial growth nor methane consumption.
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5.6 Nucleotide accession numbers The pmoA and 16S rRNA gene clone sequences have been submitted to GenBank under accession numbers JX963057-JX963105 . Acknowledgments T.Sˇ.-T. was supported by a PhD fellowship granted by the Danish Agency for Science, Technology and Innovation (Forsknings- og Innovationsstyrelsen). Funding for the Stellar Astrophysics Centre is provided by The Danish National Research Foundation. The research is supported by the ASTERISK project (ASTERoseismic Investigations with SONG and Kepler) funded by the European Research Council (Grant agreement no.: 267864). The authors thank Lotte Frederiksen, Tove Wiegers and Fariba Barandazi for skilled technical assistance. We gratefully acknowledge the valuable advice of Teresa G. Ba´rcena and Svend Binnerup.
References Amato, P., Menager, M., Sancelme, M., Laj, P., Mailhot, G., & Delort, A. M. (2005). Microbial population in cloud water at the Puy de Dome: Implications for the chemistry of clouds. Atmospheric Environment, 39(22), 4143–4153. Ariya, P. A., Nepotchatykh, O., Ignatova, O., & Amyot, M. (2002). Microbiological degradation of atmospheric organic compounds. Geophysical Research Letters, 29(22), 2077–2081. Baani, M., & Liesack, W. (2007) Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2. Proceedings of the National Academy of Sciences, 105(29), 10203–10208. Ba´rcena, T., Yde, J. C., & Finster, K. W. (2010). Methane flux and high-affinity methanotrophic diversity along the chronosequence of a receding glacier in Greenland. Annals of Glaciology, 51(56), 23–31. Blando, J. D., & Turpin, B. J. (2000). Secondary organic aerosol formation in cloud and fog droplets: A literature evaluation of plausibility. Atmospheric Environment, 34, 1623–1632. Bull, I. D., Parekh, N. R., Hall, G. H., Ineson, P., & Evershed, R. P. (2000). Detection and classification of atmospheric methane oxidizing bacteria in soil. Nature, 405, 175–178. Burrows, S. M., Elbert, W., Lawrence, M. G., & Po¨schl, U. (2009). Bacteria in the global atmosphere—Part 1: Review and synthesis of literature data for different ecosystems. Atmospheric Chemistry and Physics Discussions, 9, 10777–10827. Christophersen, M., Linderød, L., Jensen, P. E., & Kjeldsen, P. (2000). Methane oxidation at low temperatures in soil exposed to landfill gas. Journal of Environmental Quality, 29, 1989–1997. Degelmann, D. M., Borken, W., Drake, H. L., & Kolb, S. (2010). Different atmospheric methane-oxidizing communities in European beech and Norway spruce soils. Applied and Environmental Microbiology, 76(10), 3228–3235. Dunfield, P. F., Liesack, W., Henckel, T., Knowles, R., & Conrad, R. (1999). High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph. Applied and Environmental Microbiology, 66(9), 4136–4138.
Dunfield, P. F., Yimga, M. T., Dedysh, S. N., Berger, U., Liesack, W., & Heyer, J. (2002). Isolation of a Methylocystis strain containing a novel pmoA-like gene. FEMS Microbiology Ecology, 41, 17–26. Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797. Felsenstein, J. (1993). Phylogeny inference package (PHYLIP) version 3.5. Seattle: University of Washington. Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposium Series, 41, 95–98. Hanson, R. S., & Hanson, T. E. (1996). Methanotrophic Bacteria. Microbiological Reviews, 60(2), 439–471. Hueglin, C., Gehrig, R., Baltensperger, U., Gysel, M., Monn, C., & Vonmont, H. (2004). Chemical characterisation of PM2.5, PM10 and coarse particles at urban, near-city and rural sites in Switzerland. Atmospheric Environment, 39(4), 637–651. Isaksen, I. S. A., Granier, C., Myhre, G., Berntsen, T. K., Dalsøren, S. B., Gauss, M., et al. (2009). Atmospheric composition change: Climate–Chemistry interactions. Atmospheric Environment, 43, 5138–5192. Jensen, S., Prieme´, A., & Bakken, L. (1998). Methanol improves methane uptake in starved methanotrophic microorganisms. Applied and Environmental Microbiology, 64(3), 1143–1146. Knief, C., & Dunfield, P. F. (2005). Response and adaptation of different methanotrophic bacteria to low methane mixing ratios. Environmental Microbiology, 7(9), 1307–1317. Knief, C., Lipski, A., & Dunfield, P. F. (2003). Diversity and activity of methanotrophic bacteria in different upland soils. Applied and Environmental Microbiology, 69(11), 6703–6714. Kvenvolden, K. A., & Rogers, B. W. (2005). Gaia’s breath— global methane exhalations. Marine and Petroleum Geology, 22, 579–590. Marinoni, A., Laj, P., Sellegri, K., & Mailhot, G. (2004). Cloud chemistry at the Puy de Doˆme: Variability and relationships with environmental factors. Atmospheric Chemistry and Physics, 4, 715–728. Pfennig, N. (1962). Beobachtungen u¨ber das Schwa¨rmen von Chromatium okenii. Archives of Microbiology, 42(1), 90–95. Ricke, P., Kolb, S., & Braker, G. (2005). Application of a newly developed ARB software-integrated tool for in Silico terminal restriction fragment length polymorphism analysis reveals the dominance of a novel pmo a cluster in a forest soil. Applied and Environmental Microbiology, 71(3), 1671–1673. Schloss, P. D., & Handelsman, J. (2005). Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Applied and Environmental Microbiology, 71, 1501–1506. Staley, J. T., Brenner, D. J., & Krieg, N. R. (2005). Volume two: The proteobacteria. In G. M. Garrity (Ed.), Bergey’s manual of systematic bacteriology (2nd ed., pp. 411–476). Berlin: Springer. Svenning, M. M., Wartiainen, I., Hestnes, A. G., & Binnerup, S. J. (2003). Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system. FEMS Microbiology Ecology, 44, 347–354.
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Author's personal copy Aerobiologia Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731–2739. Temkiv, T. Sˇ., Finster, K., Hansen, B. M., Nielsen, N. W., & Karlson, U. G. (2012). The microbial diversity of a storm cloud as assessed by hailstones. FEMS Microbiology Ecology, 81(3), 684–695. Vaı¨tilingom, M., Amato, P., Sancelme, M., Laj, P., Leriche, M., & Delort, A. M. (2010). Contribution of microbial activity to carbon chemistry in clouds. Applied and Environmental Microbiology, 76(1), 23–29. Wang, J. S., McElroy, M. B., Logan, J. A., Palmer, P. I., Chameides, W. L., Wang, Y., et al. (2008). A quantitative assessment of uncertainties affecting estimates of global mean OH derived from methyl chloroform observations. Journal of Geophysical Research, 113(D12). doi:10.1029/2007JD008496. West, A. E., & Schmidt, S. K. (1999). Acetate stimulates atmospheric CH4 oxidation by an alpine tundra soil. Soil Biology & Biochemistry, 31(12), 1649–1655. Whittenbury, R., Phillips, K. C., & Wilkinson, J. F. (1970). Enrichment, isolation and some properties of methane-
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utilizing bacteria. Journal of General Microbiology, 61, 205–218. Wilkinson, T. G., & Harrison, D. E. F. (1973). The affinity for methane and methanol of mixed cultures grown on methane in continuous culture. Journal of Applied Bacteriology, 36, 309–313. Wise, M. G., McArthur, J. V., & Shimkets, L. J. (1999). Methanotroph diversity in landfill soil: Isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis. Applied and Environmental Microbiology, 65(11), 4887–4897. Wuebbles, D. J., & Hayhoe, K. (2002). Atmospheric methane and global change. Earth-Science Reviews, 57, 177–210. Yimga, M. T., Dunfield, P. F., Ricke, P., Heyer, J., & Liesack, W. (2003). Wide distribution of a novel pmoA-like gene copy among type II methanotrophs, and its expression in Methylocystis strain SC2. Applied and Environmental Microbiology, 69(9), 5593–5602. ˚ ., Holmfeldt, K., Thyrhaug, R., Zweifel, U. L., Hagstro¨m, A Geels, C., Frohn, L. M., et al. (2012). High bacterial 16S rRNA gene diversity above the atmospheric boundary layer. Aerobiologia, 28(4), 481–498. doi:10.1007/s10453012-9250-6.