Microbial oxidation of CH4 at high partial pressures in an organicland¢ll cover soil under di¡erent moisture regimes

Gunnar Boërjesson a;*, Ingvar Sundh a, Anders Tunlid b, Aî sa Frostegaîrd b,Bo H. Svensson c

a Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, S-750 07 Uppsala, Swedenb Department of Microbial Ecology, Lund University, S-223 62 Lund, Sweden

c Department of Water and Environmental Studies, Linkoëping University, S-583 81 Linkoëping, Sweden

Received 17 October 1997; revised 21 April 1998; accepted 24 April 1998

Abstract

The uptake and utilization of CH4 at high concentrations (5^18% vol.) and different soil moistures were followed for samplesfrom a landfill cover soil with a high organic matter content. Measurements of the utilization of CH4 and O2, and productionof CO2 indicated that the activity of methanotrophic organisms accounted for most of the O2 respiration. At water saturation,CH4 oxidation rates decreased with time, probably because NH�4 accumulated. Denitrification rates, estimated based on bothN2 and N2O production, were positively related to soil moisture and only slightly influenced by the extent of CH4 oxidation.Total phospholipid fatty acid concentrations increased, and concentrations of phospholipid fatty acids, typical for obligatemethanotrophic bacteria (e.g. 16:1g8 and 18:1g8), increased in the CH4-amended samples, indicating growth of both type Iand type II methanotrophs. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.All rights reserved.

Keywords: Ammonium; Denitri¢cation; Methanotroph; Methane oxidation; Nitrogen; Nitrous oxide; Phospholipid fatty acid

1. Introduction

In the greenhouse gas budget, biological methaneoxidation mediated by methanotrophic bacteria is animportant process mitigating CH4 £uxes to the at-mosphere [1]. Approximately half of the CH4 pro-duced or seeped from all sources is estimated to bedetained in this way [2]. Biological CH4 oxidationhas been found to restrict the £uxes of CH4 pro-duced in lakes and rice paddies by up to almost

100% [3,4], while the corresponding values for land-¢lls have been estimated to range from 10 to 70% [5^9]. Large quantities of CH4 are produced in land¢lls,and CH4 often appears at biogas concentrations(55% vol. [10]) in the surface of land¢ll cover soils.At such high partial pressures, O2 is absent, and CH4

oxidation therefore cannot be expected [9]. CH4 ox-idation rates are also dependent on soil moisture, asdemonstrated in laboratory incubations of land¢llcover soil samples, where CH4 oxidation rates weremuch higher under moderate moist conditions com-pared with under water-logged conditions [6,10].Thus, the di¡usion of CH4 and O2 through water

0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 3 6 - 1

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* Corresponding author. Tel. : +46 (18) 67 32 11; Fax:+46 (18) 67 33 92; E-mail: Gunnar.Borjesson@mikrob.slu.se

FEMS Microbiology Ecology 26 (1998) 207^217

may be rate-limiting for methane oxidation in soils[11]. The processes leading to oxidation of CH4 andNH�4 may also interact, since these compounds arecompetitive substrates for their respective enzymes[12], but it has also been shown that both nitri¢ca-tion and denitri¢cation were enhanced by CH4 addi-tions to sediment samples [13]. Interactions betweenCH4 and N cycling in land¢ll cover soils have notbeen reported previously.

Field observations at land¢lls have demonstratedthat organic cover soils have a high capacity to mit-igate CH4 emissions [14], which is also supported byresults from laboratory studies [15]. Furthermore,the CH4 oxidation potential in mineral soils can beenhanced by adding organic material, e.g. sewagesludge [8].

Methanotrophic bacteria seem to oxidize CH4

most e¤ciently when they occur in consortia amongother bacteria, where they may constitute ca. 90% ofthe microbial population [16]. In a methane-oxidiz-ing consortium isolated from a humisol, the uptakeof excess methanol, nitrite and hydroxylamine byaccompanying organisms was of great importancefor methanotrophic activity [17,18].

Through the use of phospholipid fatty acid(PLFA) analysis, the microbial community structurein soil samples can be determined without cultivation[19]. Many isolated strains of obligate methano-trophic bacteria contain substantial amounts of theunusual PLFAs 16:1g8 and 18:1g8 [20^24]. Thequantitation of these two PLFAs has been used toestimate the abundance of methanotrophic bacteriain various types of environmental samples, e.g. innatural gas-enriched soil columns [25], peatlands[26], land¢ll covers [14], and halocarbon-degradingmethanotrophic mixed cultures [27]. In these studiesthe PLFAs suggested as biomarkers for methano-trophs, especially 18:1g8, were often strongly linkedto CH4 oxidation.

The aims of this experiment were to determine (1)the proportion of the total O2 respiration in an or-ganic land¢ll cover soil due to CH4 oxidation; (2)which N transformations (e.g. denitri¢cation) are af-fected by CH4 oxidation at di¡erent levels of soilmoisture, and (3) which methanotrophs contributemost to CH4 oxidation. A land¢ll cover soil with ahigh organic matter content and CH4 oxidation po-tential was incubated at various CH4 concentrations

and under di¡erent moisture conditions. Because ar-gon was used instead of ambient air in incubation£asks, it was possible to estimate denitri¢cation ratesbased on N2 production. Changes in the microbialcommunity were estimated based on changes inPLFA concentrations.

2. Materials and methods

2.1. Soil

Soil was collected from the cover of theHoëgbytorp land¢ll on 8 February 1994. Previous ex-periments have shown that this soil has a high ca-pacity for methane consumption [14]. It had a pH of7.3 (4 g soil in 10 ml 0.01 M CaCl2), a loss onignition at 550³C of 29.7%, and of the remainingmineral part 13.3^13.5% was clay, 36.5^27.8% siltand 48.7^49.2% sand. Total Kjeldahl N was 0.96^0.99% (n = 2). The water-holding capacity (WHC)was 62.1% w/ww (water of wet weight; or 164%water of dry weight soil). The soil was sieved (4mm), and after air-drying at 24³C a water contentof 44.5% w/ww (72% of WHC) was achieved. Thesoil was mixed and stored dark at 4³C.

Prior to the start of the experiment, the soil wassplit into four 300-g portions. To three of these, dis-tilled water was added to give moisture contents of80, 90 and 100% WHC. After storage in plastic bagsovernight (10 h), to obtain an even distribution ofmoisture, each of the four soils was divided into¢fteen 10-g d.w. samples and transferred to 134-mlglass £asks, which were then closed with gas-tightscrew caps [10]. Thus, the gas headspace volume inthe £asks ranged from 114 to 122 ml, depending onthe water content used.

2.2. Experimental design

Immediately prior to the experimental start, thegas phase of the incubation £asks was removedand replaced with Ar three times, leaving only traceamounts of N2 in soil water. (A test with sterilizedsoil showed that a minimum of 90% of the N2 thataccumulated in the £asks during the course of theexperiment was biologically produced.) At this stage,one triplicate of each soil moisture level (`time zero'

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samples) was processed for analyses of PLFAs andinorganic nitrogen (see below). To the other samples,30 ml pure O2 was added and the over-pressure ob-tained was released to achieve atmospheric pressure.Thereafter, 30 ml of mixtures of CH4+Ar in di¡erentratios were added, giving ¢nal concentrations of 0, 5,9.4 and 18.8% CH4 in gas headspaces together with16% O2 in all £asks. All additions were made within5 min after the change to Ar and repeated in tripli-cates for each CH4 level and each soil moisture level.The £asks were incubated at 25³C, and the durationof the experiment was set at 24 h for all samples.This duration was based on the results from pre-ex-periments showing that neither O2 nor CH4 wouldbecome limiting at this time, although both gaseswere consumed to a great extent. In a parallel experi-ment including six samples each of Ar+O2 and am-bient air atmospheres, no e¡ect of Ar on CH4 oxi-dation rates was noted (P = 0.51 in t-test).

2.3. Gas analyses

Samples of 1.5 ml were withdrawn from the gasheadspace of the incubation £asks with an Ar-£ushed syringe immediately after starting the experi-ment (within 5 min after O2 and CH4 addition, re-spectively), and every third hour thereafter. Concen-trations of O2, N2 and CH4 were immediatelydetermined on a gas chromatograph with a thermalconductivity detector according to methods previ-ously described [28], as modi¢ed by Boërjesson andSvensson [10]. Simultaneously, 1-ml samples werewithdrawn and transferred to 26-ml tubes with Aras the gaseous phase for later analysis of CO2 [10]and N2O [29].

Amounts of CO2 and N2O were adjusted for sol-ubility in the water phase according to Henry's con-stants kP= 31.7 and 43.1 atm mol31 kg H2O, respec-tively [30]. The solubility proportions of the otheranalyzed gases were treated as non-signi¢cant. Allgases used for the experimental additions and cali-bration mixtures were obtained from Air Liquide(Kungsaëngen, Sweden).

Estimates of the consumption and production ofgases and inorganic nitrogen, given in ¢gures andtables, were calculated as the di¡erence between ini-tial and 24-h concentrations, while initial rates ofconsumption and production were calculated from

second-degree functions for the ¢rst 8 h. Estimatesof growth were calculated from third-degree func-tions ¢tted to the changes in time-dependent meth-ane consumption during the whole experimental pe-riod (24 h).

2.4. PLFA analyses

At the end of the experiment, 1 g of soil was re-moved from each incubation £ask and frozen. Fromsamples incubated at 80, 90 and 100% WHC, PLFAswere extracted, methylated and derivatized accordingto methods previously described [26]. GC and GC-MS analyses were made according to Frostegaîrd etal. [31], as modi¢ed by Boërjesson et al. [14].

Fatty acids are designated in terms of the totalnumber of carbon atoms: number of double bonds,followed by the position of the double bond in rela-tion to the g (methyl) end of the molecule. Cis andtrans con¢gurations are indicated by `c' and `t', re-spectively. The pre¢xes `a' and `i' indicate anteisoand iso branching; `br' indicates unknown methylbranching position; `10Me' indicates a methyl groupon the tenth carbon atom from the carboxyl end ofthe molecule; and `cy' refers to cyclopropane fattyacids.

2.5. Inorganic nitrogen fractions

After removing samples for PLFA analyses, theremaining soil in the incubation £asks was immedi-ately extracted with 50 ml 2 M KCl on a rotaryshaker at 150 rpm for 1 h. After centrifugation at3000Ug, a 10-ml portion of the supernatant wasfrozen and stored at 320³C. Concentrations of in-organic N were later analyzed for NO3

2 with £owinjection analysis (FIA; Techator, Hoëganaës, Swe-den; application note ASN 51-01/84), while NO3

3

and NH�4 were determined colorimetrically on aTRAACS auto-analyzer (Bran and Luebke, Ger-many).

3. Results

3.1. CH4 utilization

Initial CH4 consumption rates were between 1 and

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2 Wmol CH4 g d.w.31 h31 in all samples. A smallincrease in CH4 oxidation rates occurred duringthe last part of the experimental period in samplesincubated at 90% WHC (Fig. 1), while the CH4 ox-idation rates in samples incubated at 100% WHCconstantly decreased (Fig. 2), although only a smallamount of oxygen had been consumed. For the for-mer, a doubling time of tgV20 h (speci¢c growthrate W= 0.035) for the last 6-h period of incubationwas calculated from third-degree functions (Fig. 1;9.4% and 18.8% CH4). In samples incubated at 100%WHC, only around half of the CH4 was oxidizedcompared with the drier samples (Fig. 3A,B). Atlower CH4 levels, the amount of O2 consumed inthe samples exceeded the consumption predicted bythe 1:1 relationship between CH4 and O2 expectedfor stoichiometric methanotrophic growth (Fig. 3C).Likewise, the relative CO2 production (Fig. 3D) was higher at the lowest CH4 levels, probably be-

cause O2 was utilized by organisms other than meth-anotrophs.

3.2. Nitrogen fractions

Concentrations of ammonium at time zero rangedbetween 0.23 and 0.40 Wmol NH�4 g d.w.31 (Fig. 4a;0.21^0.32 mM in solution). At 70, 80 and 90%WHC, the NH�4 concentration decreased in all incu-bated samples to 0.004^0.064 Wmol NH�4 g d.w.31.At 100% WHC, a signi¢cant increase in NH�4 oc-curred in all incubated samples, up to concentrationsas high as 1.28 Wmol g d.w.31 measured in samplesfrom the highest CH4 level. This value was signi¢-cantly higher than those obtained in samples incu-bated in 0 and 5% CH4 (0.76 and 0.88 Wmol NH�4 gd.w.31).

For no obvious reason, time-zero nitrite concen-trations were higher, i.e. 12.5 and 32.0 nmol g d.w.31

NO32 (0.016 and 0.020 mM) at 72 and 100% WHC,

respectively, compared with values for the samples atintermediate moisture levels (Fig. 4b). During the24-h incubation NO3

2 decreased in all samplesdown to concentrations close to the detection limit(ca. 1 WM).

Time-zero concentrations of nitrate ranged be-tween 3.36 and 4.17 Wmol NO3

3 g d.w.31 (Fig. 4c;2.6^4.9 mM). A signi¢cant decrease in NO3

3 concen-trations occurred in samples incubated with CH4 (alllevels) at 100% WHC (down to 2.42^2.69 Wmol NO3

3

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Fig. 1. Cumulative consumption of O2 and CH4 and productionof CO2 for two of the samples incubated at 90% WHC soilmoisture. Plotted lines are third-degree functions. A: Initially9.4% CH4 in gas headspace: CCH4 (Wmol) = 0.0087666 t330.16041t2+16.676 t32.8008; which gives W= 0.0350 and tg = 19.8 for thelast 6 h. B: Initially 18.8% CH4 in gas headspace: CCH4 (Wmol) =0.011562 t330.28061 t2+21.995 t31.1376; which gives W= 0.0349and tg = 19.8 for the last 6 h.

Fig. 2. Cumulative consumption of CH4 for two of the samplesincubated at 100% WHC soil moisture. Plotted lines are third-degree functions. Initially 5.0% CH4 in gas headspace: CCH4

(Wmol) = 0.00389 t330.272 t2+10.26 t+2.82. Initially 9.4% CH4 ingas headspace: CCH4 (Wmol) =30.0051 t330.128 t2+15.93 t+0.85.

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Fig. 3. Comparison of CH4 and O2 utilization and CO2 production between samples incubated for 24 h with di¡erent levels of initialCH4 : 5.0% CH4 (white bars), 9.4% CH4 (diagonally striped bars), 18.8% CH4 (gray bars). A: Amounts of CH4 consumed. B: CH4 uti-lized of available amounts. C: Ratio between O2 and CH4 utilization. D: Ratio between CH4 utilization and CO2 production. Error barsare standard deviations for n = 3.

G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217 211

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Fig. 4. Amounts of inorganic nitrogen in soil samples at di¡erent moisture levels, including non-incubated (time-zero) samples (whitebars), and 24-h incubated samples with 0% CH4 (light gray bars), 5.0% CH4 (horizontally striped bars), 9.4% CH4 (diagonally stripedbars) and 18.8% CH4 (dark gray bars). Error bars are standard deviations for n = 3.

G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217212

g d.w.31) and with the highest initial CH4 level at90% WHC (down to 2.54 Wmol NO3

3 g d.w.31).The formation of N2O and N2 was primarily de-

pendent on the moisture content, with signi¢cant in-crease between all four soil moisture levels in increas-

ing order (Fig. 4d,e). The e¡ect of CH4 level wassmaller : Higher levels of initial CH4 resulted in in-creased N2O rates at lower soil moistures (72 and80% WHC), whereas the reverse tended to be trueat 100% WHC (Fig. 4d). By contrast, N2 production

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Table 1PLFAs (nmol g d.w.31) for di¡erent treatments, in order after mean percentual increase in CH4-treated samples compared to untreated(0% CH4) samples

Soil moisture/PLFA Time zero After 24-h incubation Increase (%)

0% CH4 5% CH4 9% CH4 18% CH4

80% WHC18:1g8 6.94 ab 5.53 a 7.87 b 10.1 b 7.96 b 3.12 (56)16:1g6 7.26 a 6.53 a 8.31 ab 10.6 b 7.96 ab 2.44 (37)16:1g8 8.63 ab 8.09 a 9.12 ab 14.2 b 8.38 ab 2.47 (31)16:1g5 31.3 ab 25.7 a 31.5 ab 37.9 b 29.1 ab 7.16 (28)18:1g7 43.8 ab 40.9 a 50.0 c 52.6 c 48.0 bc 9.28 (23)14:0 16.8 a 17.3 ab 20.0 abc 22.4 c 20.5 bc 3.66 (21)cy19:0 10.1 ab 8.95 a 9.86 ab 11.9 b 10.0 ab 1.66 (19)18:2g6 16.1 abc 14.5 a 16.6 bc 17.1 c 15.1 ab 1.72 (12)19:1a 2.05 a 2.14 ab 2.28 ab 2.58 b 2.24 ab 0.23 (11)a17:0 7.65 a 7.99 a 9.00 b 8.46 ab 8.95 b 0.82 (10)18:1g9 53.1 ab 50.8 a 56.1 b 56.5 b 52.5 ab 4.30 ( 8)16:0 77.9 a 84.9 ab 90.4 b 91.8 b 85.9 b 4.49 ( 6)a15:0 38.8 a 41.1 a 44.7 a 42.4 a 44.0 a 2.61 ( 5)16:1g7 70.8 a 77.1 ab 75.7 a 87.3 b 74.7 a 2.12 ( 3)Total PLFAs 548 a 562 ab 609 bc 645 c 585 ab 23.2 ( 9)

90% WHC18:1g8 7.19 b 4.76 a 8.15 bc 8.09 bc 8.96 c 3.64 (76)16:1g5 24.7 b 15.7 a 23.1 b 24.1 b 28.9 b 9.62 (61)16:1g7 85.0 ab 68.1 a 94.3 bc 95.6 bc 108 c 31.4 (46)i16:1 1.02 a 0.76 a 0.96 a 1.02 a 1.14 a 0.28 (37)18:1g7 46.9 b 37.1 a 47.1 b 48.8 b 49.8 b 11.4 (31)14:0 15.5 a 16.7 a 20.1 b 20.2 b 23.3 c 4.52 (27)18:2g6 15.9 b 12.4 a 15.4 ab 15.6 ab 15.9 ab 3.24 (26)cy19:0 10.4 b 7.85 a 9.59 b 9.74 b 10.2 b 2.00 (25)16:1g8 7.45 a 9.65 ab 10.9 ab 10.4 ab 14.9 b 2.41 (25)18:1g9 54.7 b 44.5 a 50.7 b 52.9 a 55.0 a 8.41 (19)19:1a 2.02 ab 1.92 a 2.33 c 2.23 c 2.22 bc 0.34 (17)16:0 82.5 ab 79.0 a 88.9 bc 91.5 c 95.8 c 13.1 (17)17:1g8 5.11 b 4.16 a 4.52 ac 4.76 bc 5.09 b 0.63 (15)18:0 10.5 b 9.54 a 10.5 ab 10.8 b 11.3 b 1.33 (14)i17:1 9.24 a 9.23 a 10.3 b 10.2 b 10.5 b 1.13 (12)15:0 6.40 a 6.38 a 6.93 ab 7.21 b 7.25 b 0.75 (12)i17:0 7.02 a 7.10 a 7.73 b 7.92 b 8.10 b 0.82 (11)cy17:0 13.7 a 13.7 a 15.0 b 15.0 b 15.7 b 1.50 (11)10Me18:0 6.11 a 5.89 a 6.32 ab 6.52 b 6.74 b 0.64 (11)17:0 3.10 ab 2.89 a 3.03 ab 3.21 b 3.32 b 0.30 (10)10Me17:0 2.38 a 2.44 ab 2.49 ab 2.64 b 2.91 c 0.24 (10)i16:0 14.9 ab 14.4 a 15.1 abc 15.8 bc 16.3 c 1.32 (10)a15:0 37.1 a 36.5 a 38.3 a 39.8 a 40.3 a 3.00 ( 8)i15:1 8.32 a 8.45 a 9.11 b 8.89 ab 8.92 ab 0.52 ( 6)i15:0 49.4 a 55.2 b 57.5 bc 57.2 bc 59.1 c 2.78 ( 5)Total PLFAs 563 ab 516 a 597 bc 608 cd 652 d 103 (20)

G. Boërjesson et al. / FEMS Microbiology Ecology 26 (1998) 207^217 213

was signi¢cantly higher in CH4-amended soil sam-ples only at 90% WHC (Fig. 4e). Accumulatedamounts of N2O and N2 were correlated, with N2-N g d.w.31 = 18.8 N2O-N g d.w.31+1.243 (r2 = 0.73,P6 0.0001).

3.3. PLFA contents

Most PLFAs occurred in signi¢cantly higheramounts in CH4-treated samples compared withnon-CH4 samples (0% CH4). These PLFAs are listedin Table 1, together with PLFAs that increased pro-portionally more than the sum of PLFAs. Amongthe 32 di¡erent PLFAs quanti¢ed, only the PLFAsi14:0, a15:0, 10Me16:0, and br18:1 never increasedsigni¢cantly at any water content. The total contentof PLFAs increased in most of the samples to whichCH4 had been added: (i) the increase in total PLFAcontent for these samples was 71.6 þ 33.4 nmolPLFA g d.w.31, which was 13 þ 6% of the PLFAcontent in non-incubated time-zero samples (t-test ;K= 0.05); and (ii) the CH4-incubated samples alsohad a higher PLFA content than the 0% CH4-incu-bated samples, with 60.6 þ 34.9 nmol PLFA gd.w.31, or 11 þ 6% di¡erence. The PLFAs 18:1g7and 18:1g8, the most common PLFAs in type II

methanotrophs [23], were well correlated with eachother (r2 = 0.61, P6 0.0001), and the content of bothdecreased in samples incubated for 24 h without CH4

(18:1g8 from 6.76 to 5.44 nmol g d.w.31, P = 0.0007for n = 9+9). Amounts of PLFAs typical for metha-notrophs were also those that increased their propor-tions most in CH4-incubated samples. For example,at 90% WHC the amount of 18:1g8 at 18% CH4 was1.88 times higher than at 0% CH4. Correspondingincreases for 16:1g8 were 1.91 times at 100%WHC, 9% CH4, and 1.76 times at 80% WHC, 9%CH4.

4. Discussion

The addition of water to the soil used in this ex-periment created two very di¡erent systems regard-ing CH4 oxidation rates and nitrogen transforma-tions. In the wettest soil treatment (100% WHC)CH4 oxidation rates were lowered, most likely owingto decreased rates of CH4 and O2 di¡usion throughwater, and NH�4 accumulated, whereas NH�4 wasconsumed in the drier soils.

Ratios between O2 and CH4 consumption (rang-ing from 1.00 to 1.88; Fig. 3C) indicate that meth-

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Table 1 (Continued).PLFAs (nmol g d.w.31) for di¡erent treatments, in order after mean percentual increase in CH4-treated samples compared to untreated(0% CH4) samples

Soil moisture/PLFA Time zero After 24-h incubation Increase (%)

0% CH4 5% CH4 9% CH4 18% CH4

100% WHC16:1g8 7.31 a 6.38 a 9.22 ab 12.2 b 11.1 b 4.67 (70)18:1g8 6.16 ab 5.82 a 7.89 ab 8.47 b 6.89 ab 1.94 (46)16:1g5 25.9 a 26.8 a 32.6 ab 36.5 b 34.4 b 7.74 (29)16:1g6 6.11 a 7.18 ab 8.69 bc 9.25 c 8.52 bc 1.64 (23)br18:1 1.91 a 1.72 a 2.10 a 2.09 a 1.94 a 0.32 (19)16:1g9 5.11 a 4.86 a 5.66 a 5.68 a 5.48 a 0.75 (15)14:0 15.5 a 18.2 ab 21.2 b 21.5 b 18.4 ab 2.13 (12)cy17:0 12.3 a 13.5 ab 14.9 ab 15.6 b 14.5 ab 1.47 (11)16:1g7 70.2 a 70.9 a 79.3 a 82.9 a 73.3 a 7.58 (11)i17:1 8.77 a 9.19 a 10.8 b 10.3 ab 9.25 ab 0.92 (10)i15:0 49.3 a 54.8 ab 61.5 b 63.8 b 55.7 ab 5.50 (10)i15:1 8.86 a 9.15 a 10.1 a 10.0 a 9.26 a 0.64 ( 7)16:0 74.9 a 86.6 ab 95.9 b 94.3 ab 84.3 ab 4.90 ( 6)Total PLFAs 527 a 580 ab 648 b 638 ab 575 ab 40.5 ( 7)

Values with letters in common in horizontal lines are not signi¢cantly di¡erent (Ks 0.05). The Table accounts only for PLFAs where at leastone of the CH4 treatments has signi¢cantly higher amounts than samples incubated without CH4, where the increase is above 2 nmol gd.w.31, or where their proportional increase is above average. Values are means for n = 3.

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anotrophs dominated the consumption of O2 at thehigher CH4 levels. Moreover, the ratios between CO2

production and CH4 consumption, ranging from0.17 to 0.36 (Fig. 3D), indicate that most of thecarbon was incorporated into biomass. Utilizationof the gaseous substrates was governed by the initialamounts of CH4 ; thus the ratio between utilizedamounts of O2 and utilized amounts of CH4 waslower at high CH4 levels. These results are consistentwith the ¢ndings in another study of CH4 oxidationin an organic soil. Namely, Megraw and Knowles[32] reported a ratio of 1.0 mol O2 utilized per molCH4 for a humisol under concurrent production of0.27 mol CO2 [32]. Similarly, pure cultures used byWhittenbury et al. [33] had a O2/CH4 of 1.0^1.1 anda CO2/CH4 ratio of 0.2^0.3. In mixed methano-trophic continuous cultures, the corresponding ratioswere 1.24 and 0.40 when oxygen was limited [34],while it was estimated that up to 84% of addedCH4 was used for producing cell biomass whenCH4 was limited [35]. This is close to the lowesttheoretically possible CO2/CH4 ratio, which is 0.12according to Gommers et al. [36].

The sum of the analyzed N fractions (in Fig. 4)was much higher for the wettest soil conditions(100% WHC; 13.4^18.3 Wmol N g d.w.31) than forthe drier conditions (3.04^6.56 Wmol N g d.w.31).Most of this di¡erence was likely due to higher Nuptake due to higher growth of methanotrophs dur-ing dry conditions, since 1 mol N is assimilated con-comitantly with the assimilation of 4 mol C in meth-anotrophs [37].

The stagnation of CH4 oxidation rates underwater-saturated conditions (100% WHC) may havebeen due to the accumulation of NH�4 in these sam-ples. However, the concentration (max. 1.5 WmolNH�4 g d.w.31 or 0.91 mM) is within the range ofKi values for pure cultures of methanotrophs re-ported by O'Neill and Wilkinson [38]: 17.5 mMNH�4 at pH 6.0 and 0.2 mM NH�4 at pH 8.0. Thus,there are good reasons to believe that NH�4 had aninhibitory e¡ect in this soil, if the original pH of 7.3did not change considerably during the incubation.

The increases in N2O and N2 formation observedwith increasing moisture content were expected, sincethe activity of denitri¢ers increases as a result of thedecrease in the oxygen supply that occurs as soilsbecome wetter [39,40]. N2O formation coupled to

autotrophic nitri¢cation may also contribute to thepattern observed. The positive in£uence of increasingCH4 levels on net N2O production at the low mois-ture contents in the experiment may have also beendue to oxygen limitation. The increase in oxygenconsumption caused by the methanotroph activityat higher mixing ratios of methane probably de-creased the availability of oxygen to the microbialpopulation in general. The same argument is validfor denitri¢cation as expressed in N2 formed at 90%WHC. There was a tendency for larger amounts ofN to be denitri¢ed in the presence of higher amountsof methane. At 100% WHC the e¡ect of methane asa factor regulating O2 availability seemed to havebeen negligible, since denitri¢cation was about equalat all initial CH4 levels. However, the possibility thatsome of the N2O formed was due to the activity bythe methanotrophs at low moisture levels cannot beruled out, since pure cultures of methanotrophs havebeen shown to be able to form N2O from NH�4[41,42].

The proportion of consumed CH4 that was usedfor biomass in the studied samples can be estimatedbased on changes in concentrations of PLFA andCH4 utilization. The average increase in total PLFAsfor the CH4-treated samples (n = 27) was 61 þ 35nmol compared with non-CH4 samples (n = 9; Sec-tion 3.3, Table 1). If 100 Wmol of PLFA correspondsto 1 g dry weight of methanotroph cells [43,44], andcarbon accounts for 47% of the dry weight in meth-anotrophic bacteria [33], then 285 þ 164 Wg C wasassimilated in biomass. This is similar to the di¡er-ence between utilized CH4 and produced CO2 (datain Fig. 3), which was 340 þ 173 Wg C (28.3 þ 14.4Wmol of C) in the corresponding samples.

Results of the PLFA analysis indicated that obli-gate methanotrophic bacteria had grown, as re£ectedin the increase in PLFAs speci¢c for methanotrophsin most of the CH4-treated samples. These results areconsistent with the ¢ndings of Nichols et al. [45] thatthe PLFAs 16:1g6, 16:1g8 and 18:1g8 were en-riched in soil columns exposed to natural gas. Inaddition to the more generic-speci¢c PLFA 18:1g8,type II methanotrophs also have signi¢cant propor-tions (up to 89%) of 18:1g7 [22]. Although 18:1g7 isubiquitous in bacteria [25] its increase in almost allCH4-amended soil samples further substantiates thatgrowth of type II methanotrophs occurred. Apart

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from the obvious e¡ects on 16:1g8 and other mono-unsaturated 16-C PLFAs, among the other PLFAsthat increased owing to the oxidation of CH4, severalsaturated PLFAs have also been documented in typeI methanotrophs. Thus, 14:0 (up to 24.6% of thetotal PLFAs), 15:0 (up to 12.7%) and 16:0 (up to56.0%) were all common in type I strains reported byBowman et al. [24]. An interesting observation isthat 18:1g8 had the highest percentual increase at80 and 90% WHC, while 16:1g8 showed the highestincrease at 100% WHC. This is in line with the sug-gestion of O'Neill and Wilkinson [38], that type Imethanotrophs are more tolerant to ammoniumthan type II.

An important conclusion of this experiment is thatunder water-saturated conditions in an organic soilCH4 oxidation is restricted not only by the di¡usionbarrier, but possibly also by the accumulation ofNH�4 under the reduced conditions. This has impli-cations for the use of this type of soil for bioreme-diation, covering of land¢lls etc. In the land¢ll case,the stoichiometrically unfavorable situation for CH4

oxidation (with 55% CH4 meeting 20% O2) could becounteracted by a prolonged retention time for CH4,thereby extending the period during which CH4 isexposed to methanotrophs. Land¢ll covering withorganic soils, which are nutrient-rich and have ahigh water-holding capacity, could be one way toachieve this.

Acknowledgments

John Stenstroëm suggested the use of argon in theexperimental setup. Rose-Marie Ericsson (Depart-ment of Soil Sciences) and Lena Funke carried outthe N analyses. Elisabet Wennberg and Maria Eriks-son provided additional laboratory assistance. Thisreport was part of a project funded by NUTEK(Swedish National Board for Industrial and Techni-cal Development) under Contract 706 005-1.

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