Characterization of Methanotrophic Bacterial Populations in Soils Showing Atmospheric Methane Uptake

Andrew J. Holmes,^1,2 Peter Roslev,³ Ian R. McDonald,¹ Niels Iversen,³ Kaj Henriksen,³ and J. Colin Murrell^1,*

 

This article has been cited by other articles in PMC.

Original link:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC91497/

Abstract

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the ∼1% · year⁻¹ increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with ¹⁴CH₄ and analyzing a total soil monooxygenase gene library. Comparative analyses of [¹⁴C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the α subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of “atmospheric methane oxidizers” should be greatly facilitated by use of biomarkers identified in this study.

Methane is a radiatively active atmospheric trace gas whose concentration is increasing at a rate of ca. 1% · year⁻¹ (∼40 Tg · year⁻¹). Human activity is thought to be a causative factor in the rising methane concentration and, as such, may contribute to global warming (4, 8, 27). The global methane cycle consists of both atmospheric (mainly chemical) and terrestrial (mainly biological) processes (27). The observed increase in the methane concentration has been attributed to a combination of an increase in the number of sources of methane and a decrease in the number of sinks for methane (4).

The major sinks for methane are biological oxidation at or near the sites of production (∼700 Tg · year⁻¹), uptake of methane from the atmosphere by aerobic soils (20 to 60 Tg · year⁻¹), and photochemical oxidation in the atmosphere (∼450 Tg · year⁻¹) (27). Soil uptake of atmospheric methane is significant since the magnitude of the soil sink is equivalent to the observed annual increase in the methane concentration and it is more susceptible to disturbance by human activities (16, 21, 24, 34). A change in the soil sink can have a significant effect on the atmospheric mixing ratios of methane.

Biological methane oxidation consists of both aerobic and anaerobic processes. The global methane sink is dominated by aerobic methane-oxidizing bacteria (MOB). The biochemical basis of methane oxidation in all known MOB is similar (1, 9, 13, 20). All MOB possess a membrane-bound monooxygenase whose substrate range includes both methane and ammonia (note that some MOB contain an additional, biochemically distinct enzyme designated the soluble methane monooxygenase [sMMO]) (1). The membrane-bound monooxygenases are thought to be evolutionarily related (15). The MOB exhibit limited physiological, structural, and phyletic diversity compared to other functionally defined groups of bacteria (13, 25). Of particular significance are differences in the fate of carbon, the kinetic properties of the monooxygenase, and the evolutionary separation of the four major phyletic groups.

On the basis of cell physiology, the MOB can be divided into the methane-assimilating bacteria (MAB) (methanotrophs) and bacteria which cooxidize methane (autotrophic ammonia-oxidizing bacteria [AAOB]). The former organisms use methane as a sole source of carbon and energy and are characterized by the presence of a complete pathway for methane oxidation, the ability to assimilate cell carbon as formaldehyde, and apparent K_m values for methane in the micromolar range (1, 13). The AAOB use ammonia oxidation as an energy source for autotrophic growth; they are characterized by a complete pathway for oxidation of ammonia to nitrite and assimilation of cell carbon by the Benson-Calvin cycle. In most cases their apparent K_m values for methane are in the millimolar range and methane is cooxidized with no apparent benefit to the cells (1).

Both phenotypic and phylogenetic data can be used to subdivide the methanotrophs and AAOB into two additional groups that are defined on the basis of intracellular membrane type, major membrane fatty acids, and genetic comparison data (5, 13, 33). Thus, there is very strong support for the existence of four monophyletic groups of MOB, two MAB groups and two AAOB groups. The phyletic distinctiveness of these four groups from each other, combined with the relatively shallow phylogenetic depths of the groups, has allowed the use of various biomarkers as signatures in ecological studies. These biomarkers have included oligonucleotide probes and phospholipid ester-linked fatty acids (PLFA) (6, 12, 14, 23, 28, 29, 37, 38).

Soil methane uptake has been demonstrated to be biological. Methane uptake activity shares many features with the known MOB activity but also exhibits traits which do not occur during methane oxidation by extant organisms. The differences include a >100-fold-greater affinity for methane but an apparently poor capacity for growth on this substrate (2, 9, 20, 21, 31, 35). Perhaps the most significant difference is a much lower threshold concentration for sustained methane uptake. Several explanations have been proposed to account for this, including (i) mixotrophic growth of methanotrophs, (ii) cooxidation of methane by ammonia oxidizers, (iii) induction of a high-affinity enzyme system in response to starvation, and (iv) activity of novel methanotrophic bacteria (2, 7, 9, 20, 31, 40). More recently, workers have shown that methanotrophs in pure cultures can exhibit sustained uptake of atmospheric methane at normal atmospheric concentrations if the cultures are supplemented with methanol (3, 18). These workers proposed that the presence of methanol in soil may provide a physiological basis for methane uptake by conventional methanotrophs in soil.

However, no organism that has been isolated from soil has been conclusively demonstrated to account for soil methane uptake. Consequently, the biochemical, physiological, and phyletic relationships of soil high-affinity methane oxidizers to extant MOB are unknown. Demonstration and assessment of the biochemical and physiological relationships are important to the use of extant MOB as experimental models for soil methane uptake. The current mechanistic models for inhibition of high-affinity methane oxidation by soil additives are based on the assumption that there is biochemical similarity (7, 16, 21, 24, 34, 35). Understanding the phyletic relationships is important for using biomarkers to study the ecology of methane oxidizers.

ACKNOWLEDGMENTS

This work was supported by grant ERBIO4CT960419 from the European Union, by grant GST/02/622 from NERC, and by the Danish Research Council.

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REFERENCES

1. Bedard C, Knowles R. Physiology, biochemistry, and specific inhibitors of CH₄, NH₄⁺, and CO oxidation by methanotrophs and nitrifiers. Microbiol Rev. 1989;53:68–84. [PMC free article] [PubMed]

2. Bender M, Conrad R. Kinetics of CH₄ oxidation in oxic soils exposed to ambient air or high CH₄ mixing ratios. FEMS Microbiol Ecol. 1992;101:261–270.

3. Benstead J, King G M, Williams H G. Methanol promotes atmospheric methane oxidation by methanotrophic cultures and soils. Appl Environ Microbiol. 1998;64:1091–1098. [PMC free article] [PubMed]

4. Blake D R, Rowland F S. Continuing worldwide increase in tropospheric methane, 1978–1987. Science. 1988;239:1129–1131. [PubMed]

5. Bowman J P, Sly L I, Nicholls P D, Hayward A C. 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 Bacteriol. 1993;43:735–753.

6. Brusseau G A, Bulygina E S, Hanson R S. Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria. Appl Environ Microbiol. 1994;60:626–636. [PMC free article] [PubMed]

7. Castro M S, Peterjohn W T, Melilo J M, Steudler P A. Effects of nitrogen fertilization on the fluxes of N₂O, CH₄, and CO₂ from soils in a Florida slash pine plantation. Can J For Res. 1994;24:9–13.

8. Chappellaz J, Barnola J M, Raynaud D, Korotkevich Y S, Lorius C. Ice-core record of atmospheric methane over the past 160,000 years. Nature (London) 1990;345:127–131.

9. Conrad R. Soil microbial processes involved in production and consumption of atmospheric trace gases. Adv Microb Ecol. 1995;14:207–250.

10. De Rijk P, Van de Peer Y, Van den Broeck I, De Wachter R. Evolution according to large subunit ribosomal RNA. J Mol Evol. 1995;41:366–375. [PubMed]

11. Felsenstein J. PHYLIP—phylogeny inference package (version 3.2) Cladistics. 1989;5:164–166.

12. Guckert J B, Ringleberg D B, White C C, Hanson R S, Bratina B J. Membrane fatty acids as phenotypic markers for the polyphasic approach to taxonomy of methylotrophs within the Proteobacteria. J Gen Microbiol. 1991;137:2631–2641. [PubMed]

13. Hanson R S, Hanson T E. Methanotrophic bacteria. Microbiol Rev. 1996;60:439–471. [PMC free article] [PubMed]

14. Holmes A J, Owens N J P, Murrell J C. Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment. Microbiology. 1995;141:1947–1955. [PubMed]

15. Holmes A J, Costello A, Lidstrom M E, Murrell J C. Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett. 1995;132:203–208. [PubMed]

16. Hutsch B W, Webster C P, Powlson D S. Long-term effects of nitrogen fertilization on methane oxidation in soil of the Broadbalk wheat experiment. Soil Biol Biochem. 1993;25:1307–1315.

17. Hyman M R, Wood P M. Methane oxidation by Nitrosomonas europaea. Biochem J. 1983;212:31–37. [PMC free article] [PubMed]

18. Jensen S, Priemé A, Bakken L. Methanol improves methane uptake in starved methanotrophic microorganisms. Appl Environ Microbiol. 1998;64:1143–1146. [PMC free article] [PubMed]

19. Jones R D, Morita R Y. Methane oxidation by Nitrosococcus oceanus and Nitrosomonas europaea. Appl Environ Microbiol. 1983;45:401–410. [PMC free article] [PubMed]

20. King G. Ecological aspects of methane oxidation, a key determinant of global methane dynamics. Adv Microb Ecol. 1992;12:431–468.

21. King G, Schnell S. Effect of increasing atmospheric methane concentrations on ammonium inhibition of soil methane consumption. Nature (London) 1994;370:282–284.

22. Leak D J, Dalton H. Growth yields of methanotrophs. Appl Microbiol Biotechnol. 1986;23:470–476.

23. McDonald I R, Holmes A J, Kenna E M, Murrell J C. Molecular methods for the detection of methanotrophs. In: Sheehan D, editor. Methods in biotechnology. 2. Bioremediation protocols. Totowa, N.J: Humana Press Inc.; 1997. pp. 111–126.

24. Mosier A, Schimel D, Valentine D, Bronson K, Parton W. Methane and nitrous oxide fluxes in native, fertilized and cultivated grasslands. Nature (London) 1991;350:330–332.

25. Murrell J C, Holmes A J, McDonald I R, Kenna E M. Molecular ecology of methanotrophs. In: Murrell J C, Kelly D P, editors. Microbiology of atmospheric trace gases. Berlin, Germany: Springer-Verlag; 1996. pp. 135–152.

26. Murrell J C, Holmes A J. Molecular biology of particulate methane monooxygenase. In: Lidstrom M E, Tabita F R, editors. Microbial growth on C₁ compounds. Dordrecht, The Netherlands: Kluwer; 1996. pp. 133–140.

27. Reeburgh W S, Whalen S C, Alperin M J. The role of methylotrophy in the global methane budget. In: Murrell J C, Kelly D P, editors. Microbial growth on C₁ compounds. Andover, England: Intercept; 1993. pp. 1–14.

28. Roslev P, Iversen N, Henricksen K. Oxidation and assimilation of atmospheric methane by soil methane oxidizers. Appl Environ Microbiol. 1997;63:874–880. [PMC free article] [PubMed]

29. Roslev P, Iversen N, Henricksen K. Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis. J Microbiol Methods. 1998;31:99–111.

30. Roslev, P., and N. Iversen. Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in soils. Submitted for publication. [PMC free article] [PubMed]

31. Schnell S, King G. Stability of methane oxidation capacity to variations in methane and nutrient concentrations. FEMS Microbiol Ecol. 1995;17:285–294.

32. Selenska S, Klingmuller W. DNA recovery and direct detection of Tn5 sequences from soil. Lett Appl Microbiol. 1991;13:21–24. [PubMed]

33. Stephen J R, McCaig A E, Smith Z, Prosser J I, Embley T M. Molecular diversity of soil and marine 16S rDNA sequences related to β-subgroup ammonia-oxidizing bacteria. Appl Environ Microbiol. 1996;62:4147–4154. [PMC free article] [PubMed]

34. Steudler P A, Jones R D, Castro M S, Melilo J M, Lewis D S. Influence of nitrogen fertilization on methane uptake in temperate forest soils. Nature (London) 1989;341:314–316.

35. Striegl R G, McConnaughey T A, Thorstenson D C, Weeks E P, Woodward J C. Consumption of atmospheric methane by desert soils. Nature. 1992;357:145–147.

36. Vannelli T, Bergmann D, Arciero D M, Hooper A B. Mechanism of N-oxidation and electron transfer in the ammonia oxidizing autotrophs. In: Lidstrom M E, Tabita F R, editors. Microbial growth on C₁ compounds. Dordrecht, The Netherlands: Kluwer; 1996. pp. 80–87.

37. Voytek M A, Ward B B. Detection of ammonium-oxidizing bacteria in the beta-subclass of the class Proteobacteria in aquatic samples with the PCR. Appl Environ Microbiol. 1995;61:1444–1450. [PMC free article] [PubMed]

38. Wagner M, Rath G, Koops H-P, Flood J, Amann R. In situ identification of ammonia-oxidizing bacteria. Syst Appl Microbiol. 1995;18:251–264.

39. Ward B B. Kinetics of ammonia oxidation by a marine nitrifying bacterium: methane as a substrate analog. Microb Ecol. 1990;19:211–225. [PubMed]

40. Whalen S C, Reeburgh W S. Consumption of atmospheric methane to subambient concentrations by tundra soils. Nature (London) 1990;346:160–162.

41. Wilkinson S G. Gram negative bacteria. In: Ratledge C, Wilkinson S G, editors. Microbial lipids. Vol. 1. London, United Kingdom: Academic Press; 1988. pp. 299–488.

42. Woese C R. Bacterial evolution. Microbiol Rev. 1987;51:221–271. [PMC free article] [PubMed]

43. Zahn J A, DiSpirito A A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath) J Bacteriol. 1996;178:1018–1029. [PMC free article] [PubMed]

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