Short communication
Methane oxidation and phospholipid fatty acid composition in a
podzolic soil pro®le
Ingvar Sundh
a,*, Gunnar BoÈrjesson
b, Anders Tunlid
ca
Department of Microbiology, Swedish University of Agricultural Sciences, PO Box 7025, SE-750 07 Uppsala, Sweden
b
Department of Water and Environmental Studies, LinkoÈping University, SE-581 83 LinkoÈping, Sweden
c
Department of Microbial Ecology, Lund University, SE-223 62 Lund, Sweden
Accepted 16 November 1999
Abstract
We compared methane oxidation activity in laboratory incubations of samples from a podzolic soil pro®le to the microbial community structure of the soil, determined as the content and composition of phospholipid fatty acids (PLFAs). The abundances of two fatty acids considered unique for methanotrophs (and the abundances of all other quanti®ed PLFAs) were very weakly related to methane oxidation. This is in contrast to the situation in environments with much higher methane supply, indicating that these fatty acids should not be used as biomarkers for methanotrophs in upland forest soils. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Methane oxidation; PFLA composition; Podzols; Microbial community structure
1. Introduction
Microbially-mediated oxidation makes most aerated mineral soils act as sinks for atmospheric methane, a gas making a substantial contribution to the anthropo-genic greenhouse eect (Rodhe, 1990). However, it has not been established which organisms are responsible for the oxidation of methane in mineral soils. Notably, known isolates of methane-oxidizing bacteria are not able to grow on atmospheric methane concentrations (currently ca. 1.8 ml lÿ1) (Benstead et al., 1998; Con-rad, 1984). Ammonium-oxidizing bacteria (Bedard and Knowles, 1989), methanotrophs with high anities for methane (Bender and Conrad, 1992; Dun®eld et al., 1999) and mixotrophic methanotrophs (Benstead et al., 1998; Jensen et al., 1998) have been proposed as poss-ible candidate organisms.
Many methane-oxidizing bacteria contain unusual
phospholipid fatty acids (PLFAs) of 16 (Type I metha-notrophs) or 18 (Type II) carbons in length (Bowman et al., 1993). The abundances of these unusual PLFAs have been used to estimate the biomass distribution of Type I and II methanotrophic bacteria in environ-ments well supplied with methane (BoÈrjesson et al., 1998; Nichols et al., 1987; Sundh et al., 1995).
In the present study, we looked for correlations between methane oxidation activity and the microbial community structure (determined from analysis of PLFAs) in samples from a typical podzolic forest soil pro®le.
2. Materials and methods
Soil was collected in summer in a fairly dense spruce stand (Picea abies) on a forested moraine ridge, close to the Swedish University of Agricultural Sciences in Uppsala in the temperate zone of Sweden (59849'N, 17839'E). The soil is a Dystrocryept (according to the Soil Taxonomy system), i.e. it is an acidic but not fully
Soil Biology & Biochemistry 32 (2000) 1025±1028
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* Corresponding author. Tel.: +46-18-673210; fax: +46-18-673392.
developed podzol in a cool climate. Roughly, the O (organic) horizon extends from the surface to 5 cm depth, the A (eluviation) horizon from 5 to 20 cm, and the B (illuviation) horizon from 20 to 40 cm. Some basic characteristics of the soil are given in Table 1. Four cores (diameter 33 mm) down to 20 cm were taken within an area of approx. 0.3 m2. The cores were cut into 2-cm sections and the four replicates from each depth pooled and taken to the laboratory.
Soil used for measurements of the methane oxi-dation potential (the oxioxi-dation rate at saturating methane and oxygen concentrations, =Vmax) was col-lected at a later date. This time, bulk soil from the 0± 5, 5±10, and 10±20 cm intervals was collected.
For measurements of methane uptake at atmos-pheric concentration, duplicate 5-g (O horizon) or 10-g portions were transferred to serum bottles (118 ml), which were sealed and stored at 158C overnight. The bottles were then equilibrated with air and incubated at 208C. Samples for methane analysis were taken im-mediately and at intervals during ca. 7 h. The methane concentration in the bottles was analyzed by gas chro-matography, principally according to OÈrlygsson et al. (1993). The consumption of methane followed ®rst-order kinetics and oxidation rates at the initial atmos-pheric concentration were calculated from exponential equations.
The measurements of Vmax were similar, but methane was added to ca. 10, 100, and 1000ml lÿ1 to parallel bottles, so that zero-order kinetics (linear con-centration decrease of methane) was obtained. In a separate series, de-ionized water was added, and the bottles were subsequently shaken (200 rev minÿ1) during incubation. The incubation time was 22 h. Rates were slightly higher with water addition, and Vmax was calculated by linear regression for methane consumption in water-amended bottles with 1000 ml CH4lÿ
1 .
The methods for PLFA analysis were described by
BoÈrjesson et al. (1998) and references therein. Forty dierent PLFAs were identi®ed and quanti®ed.
3. Results and discussion
The oxidation rate at atmospheric concentration was zero at the surface but increased to a subsurface maxi-mum around 15 cm, indicating highest biomasses of methanotrophs at this depth (Fig. 1). A subsurface maximum is very typical for temporal and boreal for-est soils (e.g. Roslev et al., 1997; Whalen et al., 1992).
In the Vmax estimates, there was no detectable oxi-dation down to 10 cm, regardless of methane concen-tration or water addition and agitation. However, at 10±20 cm oxidation was substantial (Table 1), and of the same order of magnitude as in other reports (Bender and Conrad, 1992; Benstead and King, 1997; Roslev et al., 1997; Whalen et al., 1992).
None of the PLFAs quanti®ed in this soil was well correlated with oxidation activity. Interestingly, the abundance of the two o8 fatty acids that worked so
well as biomarkers for methanotrophs in peatlands and land®lls (BoÈrjesson et al., 1998; Sundh et al., 1995), was totally unrelated to methane oxidation ac-tivity (Fig. 1). This indicates that methanotrophs are not a likely major source of these fatty acids.
Based on the calculations below, however, we con-clude that the dissimilarity between the distributions of oxidation and the o8 PLFAs in this soil pro®le is not
absolute proof that ``classical'', low anity methano-trophs similar to known isolates cannot be responsible for the oxidation. Based on Vmax (46 pmol gÿ1 d.w. soil minÿ1) we calculated the corresponding, expected, amounts of the o8 PLFAs if ``classical''
methano-trophs were responsible for the oxidation. The follow-ing assumptions were made: (1) The Vmax of methanotrophic bacteria is 500 nmol mgÿ1 d.w. cells minÿ1 (Carlsen et al., 1991; Sheehan and Johnson,
Table 1
Moisture and organic matter content, pH, andVmax(2SEM,n= 2) for methane oxidation in a podzolic soil pro®le
Horizon (cm) Moisture (% of f.w.) Organic matter (% of d.w.) pHa,b V
maxa(pmol CH4gÿ1d.w. minÿ1)
pH andVmaxwere measured on bulk soil samples from 0±5, 5±10, and 10±20 cm depth. bpH was measured in soil suspensions in de-ionized water.
I. Sundh et al. / Soil Biology & Biochemistry 32 (2000) 1025±1028
1971). (2) Methanotrophs contain 100 nmol PLFA mgÿ1 d.w. cells (Nichols et al., 1985; White et al., 1979a). (3) Half of the PLFAs in methanotrophs are
o8 (Bowman et al., 1993). The calculations revealed
that we could expect ca. 5 pmol o8 PLFA gÿ1 d.w. soil, which is less than 10% of the actual concen-trations in the mineral horizon (Fig. 1). Obviously, these amounts ofo8 PLFAs in methanotrophs may be
impossible to distinguish, if there are additional sources of these PLFAs.
Possible alternative sources of the PLFAs are dead cell material in organic detritus, or other living micro-organisms. Regarding phospholipids in dead cell ma-terial, they are degraded relatively rapidly in sediments (White et al., 1979b), but perhaps more slowly in soils (e.g. FrostegaÊrd et al., 1996). With the exception of some Bacillus species (Fulco, 1969), we know of no examples of the presence of 16:1o8 or 18:1o8 in
organisms other than methanotrophic bacteria. In methanotrophs, however, the o8 unsaturation always
occurs together with unsaturation in other positions (particularly o7 and o9), very common in many other
eubacteria. In this soil pro®le, the o8 isomers were
typically less than 1% of the total amounts of 16 and 18 carbon monounsaturated PLFAs. Additionally, their concentrations were positively correlated with those of several of the other isomers. Thus, maybe the main source of the o8 isomers was trace amounts in
non-methanotrophic organisms containing monounsa-turated 16- and 18-carbon PLFAs. The reason that they have not been found in other organisms may be that no-one has been actively looking for them.
From our results, we conclude that the major part of the o8 PLFAs in this podzolic forest soil does not
originate in methanotrophic bacteria. Therefore, these PLFAs should not be used as biomass indicators of methanotrophs in these types of soil, and maybe other upland forest soils.
Acknowledgements
We thank Mats OÈquist for assistance in ®eld and laboratory, Gunnel Fransson for lipid extractions, and Mats Linde (Department of Soil Science, SLU), who classi®ed the soil. This work was ®nancially supported by the Swedish Council for Forestry and Agricultural Research.
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