Thermophilic methane production and oxidation in compost

FEMS Microbiology Ecology 52 (2005) 175–184 www.fems-microbiology.org

Thermophilic methane production and oxidation in compost Udo Ja¨ckel *, Kathrin Thummes, Peter Ka¨mpfer Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany Received 11 March 2004; received in revised form 4 August 2004; accepted 2 November 2004 First published online 28 November 2004

Abstract Methane cycling within compost heaps has not yet been investigated in detail. We show that thermophilic methane oxidation occurred after a lag phase of up to one day in 4-week old, 8-week old and mature (>10-week old) compost material. The potential rate of methane oxidation was between 2.6 and 4.1 lmol CH4 (g dw)
1 h
1. Profiles of methane concentrations within heaps of different ages indicated that 46–98% of the methane produced was oxidised by methanotrophic bacteria. The population size of thermophilic methanotrophs was estimated at 109 cells (g dw)
1, based on methane oxidation rates. A methanotroph (strain KTM-1) was isolated from the highest positive step of a serial dilution series. This strain belonged to the genus Methylocaldum, which contains thermotolerant and thermophilic methanotrophs. The closest relative organism on the basis of 16S rRNA gene sequence identity was M. szegediense (>99%), a species originally isolated from hot springs. The temperature optimum (45–55
C) for methane oxidation within the compost material was identical to that of strain KTM-1, suggesting that this strain was well adapted to the conditions in the compost material. The temperatures measured in the upper layer (0–40 cm) of the compost heaps were also in this range, so we assume that these organisms are capable of effectively reducing the potential methane emissions from compost.  2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Thermophilic; Methane oxidation; Methane production; Compost; Methanotrophs; Methylocaldum szegediense

1. Introduction The most important anthropogenic methane sources are rice paddies, coal mining, enteric fermentation and landfills. The source strength of landfills is estimated to be 22–36 Tg CH4 a
1 [1–3]. In the last decades, methods of mitigation or controlling the methane emission from these sources have been investigated. Composting has been proposed as one method to reduce methane emission from landfills [4,5]. This method of processing organic waste has become an integral part of modern waste management, with the objective of reducing the mass and volume of the biodegradable portion of solid wastes. The resulting compost is mainly used as organic *

Corresponding author. Tel.: +49 641 99 37373; fax: +49 641 99 37359. E-mail address: [email protected] (U. Ja¨ckel).

fertilizer. At present, more than 600 full-scale composting plants produce nearly 6–7 million tons of biological waste in Germany [6] and measurements show considerable methane emissions from compost piles [7–10]. These emissions are influenced by the dimensions [10] and density [11] of the heaps, and by the input material [12]. Methane emissions from composting facilities were estimated to be 7.4 · 106 t CH4 a
1 [13], which amounts to 0.31–0.44% of the total methane emissions in Germany. Little is known about the thermophilic microorganisms involved in methane production and oxidation in these environments. In one study, it was shown that mushroom compost piles contain 2 · 108 thermophilic methanogens per gram dry matter, which were mainly identified as Methanobacterium thermoautotrophicum [14]. However, both methane production and oxidation determine the net methane emission to the atmosphere.

0168-6496/$22.00  2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.11.003

176

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

The oxidation is performed by methane-oxidising bacteria (MB). Phylogenetically MB belong to the Gammaproteobacteria (Methylococcaceae) and Alphaproteobacteria (Methylocystaceae and genera Methylocapsa and Methylocella) [15–19]. Many studies have addressed the balance between production and oxidation of CH4, for example in flooded rice fields [20,21], and have examined the CH4 oxidation process in the oxic soil surface layer and the rhizosphere [22–24]. These studies showed that up to 90% of the CH4 produced is oxidised before it can reach the atmosphere. Under mesophilic conditions, composted material used as landfill cover material showed a methane-consuming potential, or increased the methane oxidation potential of the landfill cover soil [25,26]. Due to self-heating, the temperature in compost is >55
C, and because the piles are regularly turned, these high temperatures act on the entire material. Mesophilic methanotrophs are therefore unlikely to mitigate methane emission from these piles. The existence of thermophilic methanotrophs in composting material, however, was indicated in one study [27]. In this, it was supposed that thermophilic methanotrophs were responsible for nitrification activity. Molecular analyses also indicated that Methylocaldum was present in landfill cover material that originated from compost material [28]. So far only few investigations have been carried out on methane oxidation under thermophilic conditions. On the basis of accepted definitions [29,30], the methanotrophs preferring hot conditions can be differentiated into thermotolerant (<45
C optimum, growth >45
C possible), moderately thermophilic (45–60
C) and strictly thermophilic (>60–70
C). Four strains of methane-oxidising bacteria, Methylococcus capsulatus, ‘‘Mc. Ucrainicus’’, Methylocaldum gracile and Md. tepidum, can be classified as thermotolerant; two strains, Mc. thermophilus and Md. szegediense, are moderately thermophilic; and one strain, ‘‘Methylothermus’’ sp. HB, is strictly thermophilic [31]. This paper reports about methane production and consumption in developing compost piles and the impact of thermophilic methanotrophs on the methane emission from these compost piles.

2. Materials and methods

of about 70% organic material from communal biowaste collection and 30% loppings (from tree-cutting) and grass. After storage under the gore-tex blankingcover, the piles were set up as trapezoid-shaped piles again (dimension: 30 m length, 3 m width and 1.8 m height). These piles were turned once or twice per week. The mature compost was sieved after 9–13 weeks of processing. Rough-textured material was recycled and transferred to the input material of a new compost process. 2.2. Vertical profiles of in situ methane concentration and temperature in compost piles The methane mixing ratio in different depths was measured by sampling gas from 20, 50, 80, 110 and 140 cm depth in 4-, 6- and 8-week old as well as in mature compost. Steel capillaries of different lengths (with an inner diameter of 1 mm) were fixed on a wooden post (Fig. 1), which was driven into the pile. Gas samples (30 ml) were then taken with plastic syringes connected to the steel capillaries via butyl tubing and a three-way valve. Syringes were closed with valves and gas samples were analysed as described below. Two profiles were analysed for each pile. Temperatures were measured using a NiCrNi-temperature sensor (1 m) connected to a digital thermometer (J. Dittrich Elektronic, BadenBaden, Germany). 2.3. Compost sampling Three sampling points were selected, one in the centre and one at each end of the pile. About 300 g of compost material was collected at each sampling point from a depth of 20 cm below the surface (at temperatures

Butyl tubing (2 m length)

Tree way cock syringe capillare

methane

Drill hole

2.1. Composting plant The composting plant examined is an open windrow composting system, with the modification that the composting process is carried out under a foil (Gore-tex) during the initial three weeks. The optimal oxygen supply for microorganisms was ensured via ventilation during this phase. The input material of the plant consisted

wooden post Fig. 1. Technical drawing of the gas sampler used for measuring the in situ profile of the methane concentration in compost piles.

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

between 45 and 55
C). The material was mixed well and sieved (4-mm mesh) in the lab. 2.4. Potential methane oxidation and methane production To measure the potential methane-oxidising activity, 2 g of mixed and sieved compost samples from heaps aged 4, 8 and 9–12 weeks were incubated under ambient air in 120-ml serum bottles closed with butyl stopper. Methane was added to a final mixing ratio of about 18,000–20,000 ppmv. Incubation was carried out at 25 or 50
C in the dark. Gas samples (0.3 ml) were taken repeatedly (5 times within 4 days) after the methane addition to determine the rate of potential methane oxidation. Apparent first-order oxidation rate constants were calculated from the exponential decrease of CH4 in time (MicrosoftExcel2000) and converted to CH4 oxidation rates by multiplication with the CH4 mixing ratio (20,000 ppmv). To measure the potential methane production, 2 g of compost material from heaps aged 3, 4, 5, 6, 7, 8 and 11 weeks were incubated at 50
C after flushing the head space with N2. Accumulated methane was quantified after a 5-day incubation period. Each experiment was carried out in triplicate. 2.5. Temperature effect on methane oxidation and methane production by composted material To examine the temperature effect on the potential methane oxidation of composting material, 2-g amounts of mature compost were incubated under ambient air in 120-ml serum bottles closed with butyl stoppers. Methane was added to a mixing ratio of about 20,000 ppmv. Samples were incubated at 25, 36, 45, 50, 55, 60 and 70
C, respectively, in the dark. Each temperature assay was carried out in triplicate. The amount of oxidised methane was quantified by gas chromatography after 28 h of incubation. To measure the temperature effects on the potential methane production, 2 g of a mixed compost sample from a six-week-old heap was incubated as described above under a dinitrogen atmosphere. Methane production rates were calculated by linear regression analysis using the software package MicrosoftExcel 2000. Rates are expressed per gram dry weight (g dw) of compost material. 2.6. Gas sampling The gas samples were taken with gas-tight pressurelock syringes (A-2 series; Dynatech, Baton Rouge, LA, USA). The amount of gas withdrawn for sampling was replaced by dinitrogen. Methane was analyzed by gas chromatography (Perkin Elmer

177

Autosystem XL B5902), using a flame ionization detector according to [32]. Detection signals were analysed with the integration program Peak Simple (SRI Instruments, CA, USA). Standard gases containing 10, 99 and 1000 ppmv methane (Messer, Germany) were used as reference. The FID-signal was linear up to 4 · 105 ppmv. 2.7. Methane emission estimation Methane mixing ratios at 20-cm depth and atmospheric mixing ratios (2 ppmv) at the surface were used to estimate methane emission rates from compost piles using FickÕs first law. F methane ¼
Dmethane=compost

d½CH4  ; dz

where Fmethane is the emission density (g CH4 m
2 h
1), Dmethane/compost is the gas diffusion coefficient for CH4 within the compost (m2 h
1), and d[CH4]/dz is the concentration gradient (g CH4 m
3 compost m
1). The diffusion coefficient was obtained by Dmethane=compost ¼ DCH4 =air  a/bg ; where DCH4/air is the diffusion coefficient for CH4 in air (0.07056 m2 h
1 at 9.0
C [33], /g is the air-filled porosity (air m
3 of compost), and a and b are dimensionless coefficients accounting for the compost pore tortuosity and size distribution, respectively. The air-filled porosity /g is given by /g ¼ eð1
WFPSÞ; where e is the total porosity (m3 m
3) and WFPS the water-filled pore space (m3 H2O m
3) [34]. Dmethane/air is directly dependent on temperature (T) and inversely dependent on pressure (P) [35]. However, pressure effects do not need to be accounted for, since all of the gas samples were taken at the same site. The temperature correction was:  2 T ðKÞ DCH4 =air ðT Þ ¼ DCH4 =air ð9  CÞ  ; 282 K where DCH4/air (T) is the diffusion coefficient of CH4 at a particular temperature T (in K). To evaluate Dmethane/compost, approximate values were required for the dimensionless coefficients a and b. The most common values for these empirical coefficients used in soil CH4 oxidation studies are: a = 0.9 and b = 2.3 [35,36]. Methane emission rates were also calculated using CH4 mixing ratios measured at 60 or 80 cm depth and atmospheric mixing ratios (2 ppmv) at the surface (F60/80). The ratio of these two fluxes (F20/F60/80) was used to estimate the percentage of the total potential methane emission that was reduced by methanotrophic activity.

178

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

2.8. Extraction of microorganisms from compost material

3. Results and discussion

Ten grams of the freshly collected mixed and sieved compost material were diluted in 90 ml of a sodium pyrophosphate solution (0.18% w/v). The compost dilution was homogenized for 30 s in a sterile Waring-blender (Breda Scientific, Cenco, Meerbusch, Germany) at low power. Fifty millilitre of the homogeneous dilution was transferred to sterile polyethylene sampling tubes (Greiner, Nu¨rtingen, Germany). MPN-analysis or cell fixation was carried out using the supernatant after sedimentation of coarse material for 15 min according to [37].

3.1. Methane production and its regulation

2.9. MPN, cultivation and isolation of methanotrophs A serial decimal dilution up to 10
8 was made in glass tubes containing 4.5 ml of sodium chloride solution (0.18% w/v) for Most-Probable-Number counting according to [38] in quintuplicate. Mineral salts media used for cultivation were prepared according to [39] (Medium A) and [40] (Medium B). Tubes were incubated at 50
C in closed glass desiccators containing a gas mixture of 20% CH4, (v/v), 10% CO2 (v/v) and 70% (v/v) air. A tube from the highest positive dilution step (visible turbidity) was used as the inoculum for further cultivation and isolation on plates on the described media, which were solidified with 1.5% agar. Strain KTM-1 was isolated on medium A after repeated striking. 2.10. Temperature effect on growth and methane oxidation by strain KTM-1 To examine the temperature effect on the potential methane oxidation of the isolated strain KTM-1, sterile serum (120 ml) bottles were filled with agar-medium (B) up to a height of 0.5 cm. With an inoculating loop, one drop of pure liquid culture was spread over the surface. The bottles were then closed with butyl stoppers and filter-sterilised methane (80–100 lmol) was added. Duplicate samples were incubated at 25, 36, 45, 50, 55, 60, and 70
C, respectively, in the darkness. Growth was analysed visually and the amount of methane oxidised was measured by gas chromatography after eight days of incubation. 2.11. Phylogeny The 16S rRNA gene of strain KTM-1 was amplified by PCR using different ‘‘universal primers’’ [41] and sequenced. On the nucleic acid level, evolutionary distances between pairs of sequences were calculated by using the Kimura 2-parameter model provided in the MEGA 2.1 [42] software package. Phylogenetic trees were constructed by using the neighbour-joining method supplied by the MEGA 2.1 package.

The in situ temperatures of developing compost are shown in Fig. 2(a). Corresponding to the pile age, the in situ temperature at a depth of 20 cm increased from 53
C in a 4-week old pile up to 65
C in a mature compost pile. With exception of the 4-week old pile, the temperatures increased also with increasing profile depth. The highest temperature was reached in the 6-week old pile, with almost 78
C at a depth of 1 m. In comparison to the temperature profiles of the various compost piles, Fig. 2(b) shows the corresponding methane mixing ratios for piles of different ages. The lowest methane mixing ratio of about 10 ppmv was observed in the 4-week old pile, while the highest one of >105 ppmv has been detected in the mature compost pile. Thereby the methane mixing ratios gradually increased from 8 ppmv in a 4-week old pile up to 104– 105 ppmv in a mature pile, respectively (Fig. 2(b)). Corresponding to the temperature profiles, the methane mixing ratios increased exponentially with the profile depth, starting from 20 cm, in all piles examined. Due to the initially high microbial activity the easily available organic compounds are converted into biomass and heat. The temperature rises dramatically, as generally observed in compost heaps [43]. This high temperature is maintained due to periodic turning and/or to the use of controlled air flow. The heat dissipation at the heap surface explains the decreasing temperature from the heap centre to the heap surface. After the easily degradable compounds are metabolized, the temperature is decreasing because heat dissipation surpasses the heat production. Composts are known to host thermotolerant fungi and a variety of thermotolerant and thermophilic bacteria. So far, these bacteria have been mainly investigated by cultivation studies and identified as aerobic bacteria like Thermus thermophilus, Bacillus sp., Hydrogenobacter sp., Thermoactinomyces sp. [43]. Due to the lower oxygen solubility in water at these high temperatures, the compost piles also seem to favour the development of anoxic niches during thermophilic phases, so that a basic requirement for thermophilic methanogenic activity is given as well. The incubation of compost material of a 6-week old pile at different temperatures under dinitrogen atmosphere showed the highest methane production rate at 60
C (0.9 lmol (g dw)
1 h
1), whereas at 36 and at 70
C the methane production potential was only about 18% of this value (Fig. 3(a)). These results indicate the presence of a highly adapted methanogenic population to this hot environment, as was also shown by [14], where 2 · 108 thermophilic methanogens per gram dry matter were detected

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

179

Temperature [˚C]

(a) 20

30

40

50

60

70

80

104

105

106

0 20

Depth [ cm]

40 60 80 100 120 140 160

Methane [ppmv]

(b)

1

10

10

2

103

0 20

Depth [ cm]

40 60 80 100 120 140 160

Fig. 2. Vertical profiles of the temperature (a) and the in situ CH4 mixing ratio (b) in 4-week (r), 6-week (n), 8-week (j) old compost piles, and in a pile of mature compost (h) from a municipal composting plant.

in mushroom compost and identified mainly as M. thermoautotrophicum. Further studies showed a simultaneous increase of methane emission from heaps and ether lipid contents in thermophilic compost material. It has been found [8,44] that methane is produced in compost only under high temperatures. These results indicate that the environmental conditions in compost induced the enrichment of anaerobic microorganisms, like thermophilic methanogens. The high in situ methane concentrations measured in this study facilitate earlier studies that showed considerable methane production in compost piles [6–9]. 3.2. Methane oxidation While at 25
C low or even no potential methane oxidation was observed (Fig. 4(a)), at 50
C all examined compost samples showed a methane-consuming activity, either immediately or after a lag-phase of up to 20 h (Fig. 4(b)). According to compost age and the increasing methane mixing ratio, repectively, the consumption rate slowly increased from 2.6 lmol CH4 (g dw)
1 h
1 to 4.1 lmol CH4 (g dw)
1 h
1(Figs. 2(b) and 4(b)). The measured methane oxidation rate was more than threefold higher compared to the highest detected methane pro-

duction potential, which was found at 60
C in 6-week old material (Fig. 3(a)). The temperature optimum of the methanotrophic community in the studied compost material was in the range of 45–55
C (Fig. 3(b)). The results show that, according to the high substrate concentrations and under the given environmental conditions, a moderate thermophilic methanotrophic community has been developed. This is astonishing, since the water solubility of methane and oxygen decreases with increasing temperatures, and thus limits methanotrophic growth [31]. However, the oxygen solubility in water at 60
C is still about 51% of the solubility at 20
C [45], and the existence of methanotrophs in thermophilic environments was shown earlier [46,47]. The first results of methanotrophs in compost material were presented by [48]. The potential for methane oxidation of compost material was shown and additional compost increased the methane oxidation potential of landfill cover soils to which it was added [25,26]. However, all these experiments were carried out under mesophilic conditions, in which we found comparatively low or even no potential for methane oxidation (Fig. 4(a)). So, the activity of mesophilic methanotrophs in this study can be excluded.

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

(a) 100

1 0.9

(a)

Methane [µmol gdw-1]

Methane production [µmol gdw—1h-1]

180

0.8 0.7 0.6 0.5 0.4 0.3 0.2

80

60

40

20 25˚C

0.1

0

0 20

30

40

50

60

0

70

20

40

Temperature [˚C]

80

100

(b) 100

2.5

(b)

Methane [molgdw-1]

Methane oxidation [µmol gdw-1h-1]

60

Time [h]

2.0

1.5

1.0

0.5

0.0 20

30

40

50

60

80

60

40

0

70

50˚C

20

0

20

40

60

80

100

Time [h]

Temperature [˚C] Fig. 3. Temperature effect on the potential methane production rates of 6-weekold compost material incubated under a dinitrogen headspace (a), and temperature effect on the methane oxidation potential of a mature compost material incubated under oxic conditions with 60–80 lmol CH4 (g dw)
1 compost
1 (b) Values represent mean of triplicate incubations ± SD.

Fig. 4. Time course of the methane concentration in compost incubation experiments under oxic conditions at 25
C (a) or 50
C (b). The compost material was sampled from 4-week old (r), 8-week old (j) and mature compost piles (m). Values are means of n = 3, error bars show one standard deviation.

The in situ activity of methanotrophs is indicated by the exponential decrease of methane mixing ratios from deeper layers to the pile surfaces in all compost heaps examined (Fig. 2(b)). This result was supported by the quantification of thermophilic methanotrophic bacteria via the MPNmethod. In the upper layer of the mature compost material, 1.8 · 106 to > 3.2 · 107 cultivable methanotrophic cells per g dw were detected. Methanotrophs contributed thus to 0.1% to >3% of the total DAPI-counted cells in cell extracts from mature compost (1 · 109 cells (g dw)
1). However, quantification of methanotrophs using the methane oxidation rates (Fig. 5) resulted in higher values of about 2 · 109 methanotrophs per g dw. This calculation assumes that the Vmax cell
1 of the methanotrophs is approximately 1.5 · 10
9 lmol h
1 cell
1 [49]. From these and the earlier results of [50], who detected 1010–1011 total cells g
1, we assume that the cell extraction by pyrophosphate yielded only <10% of the total cell numbers.

To our knowledge, these are the first results on quantification of moderately thermophilic methanotrophs in an anthropogenic environment. The abundance of 1000.00

10.00

-1

[µmol gdw ]

Methane production

100.00

1.00 0.10 0.01 0.00 0

5 10 Heap age (weeks)

15

Fig. 5. Methane production potential of compost material with different ages. Values represent the methane accumulation after an incubation of five days, given as a mean per gram dry weight in triplicate incubations.

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

From the last positive tube of the MPN dilution series we isolated one methanotrophic strain (KTM-1). The temperature optimum for methane oxidation by strain KTM-1 compared to the temperature optimum measured in the compost material was almost identical (Fig. 3 and Table 1). This isolate was identified by 16S rDNA sequence analysis as a strain of Md. szegediense (Fig. 7). From these results we suggest that strain KTM-1 was the abundant and active methanotroph in the compost environment. We conclude that KTM-1 is responsible for mitigating methane from the compost piles studied, especially because the temperatures in the upper surface layer (20–40 cm from the surface) ranged from 52 to 65
C, the optimal range for methane oxidation by this strain. The resistance of KTM-1 to a temperature of 80
C (data not shown), which is similar to Md. szegediense [52], shows that the highest observed temperature (78
C) within the compost piles (Fig. 2(a)) should not lead to a strong reduction of viable cells of this species and favour the development of a stable community. The increasing Cu++ concentration per mass of compost, following the microbial degradation of organic material, could be another factor enhancing the development of a Methylocaldum sp. population in compost piles. Growth stimulation for Md. szegediense was observed when additional Cu++ was added to the culture media [52]. Md. szegediense was previously isolated from a greenhouse heating system, fed directly from a hot spring connected to a natural gas field [52]. However, compost also seems to be a favourable environment for this species, since in another study [28] hints were found as well for

Temperature (
C)

Growth

Methane oxidised (lmol d
1 120 ml
1)

25 36 45 50 53 55 60


+ +++ +++ +++ +


0 0.5 ± 1.5 3 ± 2.1 11.5 ± 0.7 10.1 ± 0.1 8.25 ± 1.3 0


no growth; + weak growth; +++ strong growth.

the presence of Methylocaldum sp. in compost material. The habitat description for members of the genus Methylocaldum [52] therefore should be expanded to compost material. However, it will be no surprise to detect this genus in soils that have been fertilized with compost. 3.4. Methane emission Calculated methane emissions are shown in Fig. 6. The methane emission continuously increased with the pile age, from 2.4 mg CH4 m
2 d
1 from a 4-week old pile to 2500 mg CH4 m
2 d
1 from a mature pile, respectively. These calculated rates are comparable to the emission of 6000 mg CH4 m
2 d
1, measured via a closed chamber method (data not shown) and to earlier studies [8], or to the emission from small heaps [10]. Up to now, only few studies have been reported on methane emissions from composting systems, which makes extrapolation to national or international scales very difficult. Emission rates between 0 and 119 g CH4 m
2 d
1 from different heaps were shown in a study which documents the high variability due to 1000.00

120 100

100.00 80 10.00

60 40

1.00 20 0

0.10 4

6

8

Methane emission [µmol m-2d-1]

3.3. Methanotrophic isolate

Table 1 Growth and methane oxidation of strain KTM-1 incubated at different temperatures

Methane oxidized (%)

methanotrophic bacteria is comparatively high in the examined compost. Population sizes of 105–106 methanotrophs per g, for example, were found in paddy soils [39,51], sizes of 1 · 106 to 9 · 107 methanotrophs per g were found in sediments of a storage lake [39] and >106 methantrophic cells per g were found in acidic Sphagnum peat [17]. A similar high population compared to that in compost was detected in freshwater lake sediment (up to 109 cells (g dw)
1) [49]. Due to the only weak increase in potential oxidation rates with the compost age (Fig. 4(b)), it seems that the methanotrophic population is quite stable during the whole compost maturation. The organic material which was not completely degraded in one composting process was added to a new composting process, so that inoculation of fresh compost with thermophilic methanotrophic bacteria took place on a regular basis. Based on this knowledge, it is not surprising that a stable methanotorophic community, adapted to the given environmental conditions, can be developed.

181

mature

Heap age Fig. 6. Methane emission rates (d) from compost piles of 4-, 6-, and 8week old compost heaps and a mature compost pile, and % of methane produced (r) which was oxidized before it reached the atmosphere.

182

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184 78 100 84

Methylomicrobium albus (M95659) Methylomicrobium pelagica (X72775) Methylomonas methanica (L20840)

46

Methylococcus thermophilus (X73819) Methylococcus capsulatus (L20842)

76 100

Methylocaldum sp. (AF215632) Methylocaldum sp. (AF215633)

100

100

γ

Methylocaldum szegediense (U89300) KTM-1 (AJ627387)

thermophilic and thermotolerant methanotrohps

Methylocaldum tepidum (U89297)

99

Methylocaldum gracile (U89298)

98

“Methylothermus” sp. (89299) Methylocella palustris (Y17144) 100

Methylosinus sporium (M95665)

α

0.02

Fig. 7. Dendrogram showing the phylogenetic affiliation of strain KTM-1 to selected species of methanotrophs within the group of Gammaproteobacteria (gamma) and Alphaproteobacteria (alpha). The bracket encompasses thermotolerant to thermophilic methanotrophs. The dendrogram was constructed based on 16S rRNA gene sequences available from the European Molecular Biology Laboratory databank (accession numbers are given in brackets) and was constructed after multiple alignment of data by ClustalX [53]. Distances (distance options according to the Kimura-2 model) and clustering were calculated with the neighbour-joining method using the software package Mega (Molecular Evolutionary Genetics Analysis) version 2.1 [17]. Bootstrap values based on 1000 replications are given at the branching points.

different practical management of composting facilities [10]. So far, factors influencing the methane emissions from compost piles are pile dimension [10], pile density [11] and the consistence of input material [12]. From this study, methanotrophs seem to be an obvious biological factor regulating the emission from compost piles as well. Using the methane profile data, calculations showed that 46% of the methane produced in the 4-week old pile, and 84–98% of the methane produced in the 6and 8-week old and mature compost piles was oxidised by the present methanotrophic community before it reached the atmosphere (Fig. 6). The relevant methanotrophic organism, which we suggest to be species of Md. szegediense, has a high impact on the methane emission from the piles. This mitigation potential by methanotrophs is comparable to other biological methane sources which are also regulated by methanotrophic activity, such as paddy fields or landfills [22–24]. Our results indicate once more the importance of methanotrophic activity on the methane emission from anthropogenic methane sources.

4. Conclusion Composting has been proposed as a method to reduce methane emission from landfills [4,5]. Nevertheless, compost piles themselves were shown as sources for atmospheric methane. The extrapolation to national or international scales is very difficult due to the limited number of data. Our results show the activity of thermophilic methane producers and, as a first study, the activ-

ity of thermophilic methane oxidisers, which regulates the methane emission from compost heaps. These results should stimulate further studies on composting systems as an interesting environment for thermophilic methane cycles.

Acknowledgements We thank Katja Grebing for excellent technical assistance and Dr. Peter Dunfield for proofreading the manuscript.

References [1] Subak, S., Raskin, P. and von Hippel, D. (1993) National greenhouse gas accounts: current anthropogenic sources and sinks. Climate Change 25, 15–58. [2] Khalil, M.A.K. and Shearer, M. (1993) Sources of methane In: Atmospheric Methane: Sources, Sinks, and Role in Global Change (Khalil, M.A., Ed.), pp. 180–197. Springer-Verlag, Berlin. [3] Doorn, M.R.J., and Barlaz, M.A. (1995) Estimate of global methane emissions from landfills and open drumps. EPA-600/R95-019. US EPA Office of Research and Development, Washington, DC. [4] Fritz, J., Link, U. and Braun, R. (2001) Environmental impacts of biobased/biodegradable packaging. Starch/Sta¨rke 53, 105–109. [5] Ayalon, O. and Avnimenelech, Y. (2001) Solid waste treatment as a high-priority and low cost alternative for greenhouse gas mitigation. Environ. Manage. 27, 697–704. [6] Weißenfels, W. (2001) Verfahrenstechnik der aeroben Behandlung organischer Abfa¨lle In: Biologische Behandlung organischer Abfa¨lle (Ka¨mpfer, P. and Weißenfels, W.D., Eds.), pp. 81–98. Springer-Verlag, Berlin Heidelberg. [7] Schattner-Schmidt, S., Helm, M., Gronauer, A. and Hellmann, B. (1995) Composting biogenic wastes. Landtechnik 50, 364.

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184 [8] Hellmann, B., Zelles, L., Palojarvi, A. and Bai, Q. (1997) Emission of climate-relevant trace gases and succession of microbial communities during open-windrow composting. Appl. Microbiol. 63, 1011–1018. [9] Hellebrand, H.J. (1998) Emission of nitrous oxide and other trace gases during composting of grass and green waste. J. Agric. Engng. Res. 69, 365–375. [10] Beck-Friis, B., Pell, M., Sonesson, U., Jo¨nsson, H. and Kirchmann, H. (2000) Formation and emission of N2O and CH4 from compost heaps of organic household waste. Environ. Monit. Assessm. 62, 317–331. [11] Sommer, S.G. and Moller, H.B. (2000) Emission of greenhouse gases during composting of deep litter from pig production – effect of straw content. J. Agric. Sci. 134, 327–335. [12] He, Y., Inamori, Y., Mizuochi, M., Kong, H., Iwami, N. and Sun, T. (2000) Measurements of N2O and CH4 from the aerated composting of food waste. Sci. Tot. Environm. 254, 65–74. [13] Hellmann, B. (1995) Freisetzung Klimarelevanter Spurengase in Bereichen mit hoher Akkumulation von Biomassen. Zeller Verlag, Osnabru¨ck, ISBN 3-535-02473-0. [14] Derikx, P.J.L., de Jong, G.A.H., Op den Camp, H.J.M., van der Drift, C., van Griesven, L.J.L.D. and Vogels, G.D. (1989) Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost. FEMS Microbiol. Ecol. 62, 251–258. [15] Dedysh, S.N., Liesack, W., Khmelenina, V.N., Suzina, N.E., Trotsenko, Y.A., Semrau, J.D., Bares, A.M., Panikov, N.S. and Tiedje, J.M. (2000) Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs. Int. J. Syst. Evol. Micr. 50, 955–969. [16] Dedysh, S.N., Horz, H.P., Dunfield, P.F and Liesack, W. (2001) A novel pmoA lineage represented by the acidophilic methanotrophic bacterium Methylocapsa acidophila B2. Arch. Microbiol. 177, 117–121. [17] Bowman, J.P., Sly, L.I., Nichols, P.D. and Hayward, A.C. (1993) Revised taxonomy of the methanotrophs: description of Methylobacter gen. nov., emendation of Methylococcus, validation of Methylosinus and Methylocystis species, and a proposal that the family Methylococcaceae includes only the group I methanotrophs. Int. J. Syst. Evol. Microbiol. 43, 735–753. [18] Hanson, R.S. and Hanson, T.E. (1996) Methanotrophic bacteria. Microbiol. Rev. 60, 439–471. [19] Bowman, J. (2000) The methanotrophs – the families Methylococcaceae and MethylocystaceaeThe Prokaryotes: A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. Springer-Verlag, New York, http://link.springerny.com/link/service/books/10125. [20] Holzapfel-Pschorn, A., Conrad, R. and Seiler, W. (1985) Production, oxidation and emission of methane in rice paddies. FEMS Microbiol. Ecol. 31, 343–351. [21] Denier-VanderGon, H.A.C. and Neue, H.U. (1996) Oxidation of methane in the rhizosphere of rice plants. Biol. Fertil. Soils 22, 359–366. [22] Conrad, R. and Rothfuss, F. (1991) Methane oxidation in the soil surface layer of a flooded rice field and the effect of ammonium. Biol. Fertil. Soils 12, 28–32. [23] Bodelier, P.L.E. and Frenzel, P. (1999) Contribution of methanotrophic and nitrifying bacteria to CH4 and NH4 oxidation in the rhizosphere of rice plants as determined by new methods of discrimination. Appl. Environ. Microbiol. 65, 1826–1833. [24] Ja¨ckel, U., Schnell, S. and Conrad, R. (2001) Effect of moisture, texture and aggregate size of paddy soil on production and consumption of CH4. Soil Biol. Biochem. 33, 965–971. [25] Humer, M. and Lechner, P. (1999) Alternative approach to the eliminiation of greenhouse gases from old landfills. Waste Manage. Res. 17, 443–452.

183

[26] De Visscher, A., Schippers, M. and van Cleemput, O. (2001) Short-term kinetic response of enhanced methane oxidation in landfill cover soils to environmental factors. Biol. Fertil. Soils 33, 231–237. [27] Pel, R., Oldenhuis, R., Brand, W., Vos, A., Gottschal, J.C. and Zwart, K.B. (1997) Stable-isotope analysis of a combined nitrification-denitrification sustained by thermophilic methanotrophs under low-oxygen conditions. Appl. Environ. Microbiol. 2, 474–481. [28] Stralis-Pavese, N., Sessitsch, A., Weilharter, A., Reichenauer, T., Riesing, J., Csontos, J., Murrell., C. and Bodrossy, L. (2004) Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers. Environ. Microbiol. 6, 347–363. [29] Kristjansson, J.K. and Stetter, K.O. (1992) Thermophilic bacteria In: Thermophilic Bacteria (Kristjansson, J.K., Ed.), pp. 1–18. CRC Press, Boca Raton, FL, USA. [30] Wiegel, J. (1992) The obligatly anaerobic thermophilic bacteria In: Thermophilic Bacteria (Kristjansson, J.K., Ed.), pp. 105–184. CRC Press, Boca Raton, FL, USA. [31] Trotsenko, Y.A. and Kmelinina, V.N. (2002) Biology of extremophilic and extremotolerant methanotrophs. Arch. Microbiol. 177, 123–131. [32] Ja¨ckel, U., Schnell, S. and Conrad, R. (2004) Microbial ethylene production and inhibition of methanotrophic activity in a deciduous forest soil. Soil Biol. Biochem. 36, 835–840. [33] Ishizuka, S., Tsuruta, H. and Murdiyarso, D. (2002) Methane oxidation in Japanese forest soils. Soil Biol. Biochem. 32, 769– 777. [34] Keller, M. and Reiners, W.A. (1994) Soil atmosphere exchange of nitrous oxide, nitric oxide, and methane under secondary succession of pasture to forest in Atlantic lowlands of Costa Rica. Global Biogeochem. Cycl. 8, 399–409. [35] Campbell, G.S. (1985) Soil Physics wih BASIC. Transport Models for Soil–Plant systems. Devel. Soil Sci. 14, Elsevier, Amsterdam. [36] Billings, S.A., Richter, D.D. and Yarie, J. (2000) Sensitivity of soil methane fluxes to reduced precipitation in boreal forest soils. Soil Biol. Biochem. 32, 1431–1441. [37] Schacht, B., Neef, A. and Ka¨mpfer, P. (1999) Mikrobiologische Charakterisierung unterschiedlich genutzter landwirtschaftlicher Bo¨den in peripheren Regionen. Land use Devel. 40, 234–239. [38] De Man, J.C. (1983) MPN-Tables, Corrected. Eur. J. Appl. Microbiol. Biotechnol. 17, 301–305. [39] Heyer, J., Malashenko, Y., Berger, U. and Budkova, E. (1984) Verbreitung methanotropher Bakterien. Z. Allg. Mikrobiol. 24, 725–744. [40] Leadbetter, R.E. and Foster, J.W. (1958) Studies on some methane-utilizing bacteria. Arch. Microbiol. 30, 91–118. [41] Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R. (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington DC. [42] Kumar, S., Tamura, K., Jakobsen, I.B., and Nei, M. (2001) MEGA2: Molecular Evolutionary Genetics Analysis software. Bioinform, vol. 17(12), pp. 1244–1245. [43] Ja¨ckel, U., and Ka¨mpfer, P. (2003) Die Mikrobiologie der Kompostierung. In: Ho¨sel G., Bilitewski, B., Schenkel, W., Schnurer H. (Eds.) Mu¨llhandbuch. Erich Schmidt Verlag, Berlin, Germany, 5,5210, pp. 1–24. [44] Beck-Friis, B., Smars, S., Jo¨nsson, H., Eklind, Y. and Kirchmann, H. (2003) Composting of source-separated household organics at different oxygen levels: gaining an understanding of the emission dynamics. Compost. Sci. Utiliz. 11, 41–50. ¨ ber die Kompostierung von Siedlungsabfa¨llen [45] Niese, G. (1978) U unter Beru¨cksichtigung mikrobieller Gesichtspunkte. Grundl. Landtechn. 28, 75–81.

184

U. Ja¨ckel et al. / FEMS Microbiology Ecology 52 (2005) 175–184

[46] Bodrossy, L., Murerell, J.C., Dalton, H., Kalman, M., Puskas, L.G. and Kovacs, K.L. (1995) Heat-tolerant methanotophic bacteria from the hot-water effluent of a natural-gas field. Appl. Environ. Microbiol. 61, 3549–3555. [47] Bodrossy, L., Kovacs, K.L., McDonal, I.R. and Murrell, J.C. (1999) A novel thermophilic methane-oxidizing c-Proteobacterium. FEMS Microbiol. Ecol. 170, 335–341. [48] Sukesan, S. and Watwood, M.E. (1998) Effects of hydrocarbon enrichment on trichloroethylene biodegradation and microbial populations in mature compost. J. Appl. Microbiol. 85, 635– 642. [49] Costello, A.M., Auman, A.J., Macalady, J.L., Scow, K.M. and Lidstrom, M.E. (2002) Estimation of methanotoph abundances in a freshwater lake sediment. Environ. Microbiol. 4, 443–450.

[50] Ishii, K., Fukui, M. and Takii, S. (2000) Microbial succession during a composting process as evaluated by denatruing grandient gel electrophoresis analysis. J. Appl. Microbiol. 89, 768–777. [51] De Bont, J.A.M., Lee, K.K. and Bouldin, D.F. (1978) Bacterial oxidation of methane in a rice paddy. Ecol. Bull. 26, 91–96. [52] Bodrossy, L., Holmes, E.M., Holmes, A.J., Kovacs, K.L. and Murell, J.C. (1997) Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov. Arch. Mircrobiol. 168, 493–503. [53] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882.