Ecological Aspects of Methane Oxidation, a Key ... - Springer Link

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Ecological Aspects of Methane Oxidation, a Key Determinant of Global Methane Dynamics GARY M. KING 1. Introduction Methane oxidation became a subject of scientific inquiry when Alessandro Volta observed in 1776 that gas bubbles collected from a pond were combustible. Methane was subsequently exploited as a source of heat and light. However, in spite of its commercial significance, the biological and ecological aspects of methane oxidation were largely ignored until the pioneering work of S6hngen (1906), who first isolated methaneoxidizing bacteria (MOB). [Quayle (1987) notes that Lowe probably isolated the first MOB in 1892 without recognizing their ability to oxidize methane.] Little additional progress was made until the 1960s, at which time the systematic efforts of several groups provided methodological tools and details on the taxonomy, physiology, and biochemistry of C 1 metabolism. Aside from purely academic motivations, this work was stimulated by: (1) the potential use of methanotrophic bacteria as sources of "single cell protein"; (2) the role of methylotrophic bacteria in food spoilage; (3) the possible use of methanotrophs in the bioremediation of certain halogenated organic pollutants or as agents for commercial biotransformations (Higgins et at., 1980). Ecological studies were slower in development, but a number of important observations established the ubiquity of methanotrophs, the impact of methane oxidation in freshwater and some marine systems, and the potential for anaerobic as well as aerobic methane oxidation (see Hanson, 1980, and Rudd and Taylor, 1980, for earlier reviews). The critical role of methane in the atmosphere has stimulated more recent ecological research. The roles of methane in atmospheric chemistry and the Earth's heat budget are well documented (e.g., Ehhalt, 1985). Likewise, the trend for increasing atmospheric methane concentrations is clear (e.g., Blake and Rowland, 1988), as is the association of methane with past climate changes (Chappellaz et at., 1990). As a consequence, there is considerable interest in all aspects of the production, consumption, transport, and

GARY M. KING' Darling Marine Center, University of Maine, Walpole, Maine 04573. Advances in Microbial Ecology, Vol. 12, edited by K.C. Marshall. Plenum Press, New York, 1992.

431

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Gary M. King

chemistry of methane. Oremland (1988) and Cicerone and Oremland (1988) have summarized the results of many recent studies in excellent comprehensive reviews. The interest in methane dynamics has fostered consideration of the regulatory role of biological oxidation. Even so, much more is known about methanogenesis. Perhaps the focus on methanogenesis results from the search for atmospheric methane sources (e.g., Sheppard et aI., 1982; Cicerone and Oremland, 1988; Wahlen et al., 1989); perhaps biological oxidation has been discounted since the hydroxyl radical is the primary atmospheric methane sink (Khalil and Rasmussen, 1983; Ehhalt, 1985; Crutzen, 1991). Regardless, the available evidence indicates that biological oxidation is a major factor limiting fluxes from some of the most important methane sources (e.g., wetlands and rice paddies). In addition, biological oxidation is probably greater in magnitude than chemical oxidation if the total cycle of methane production and consumption is considered, and not just the fate of methane after transport to the atmosphere. The present review concentrates primarily on post-1980 evidence for the key role of biological oxidation. Oxidation in soils and sediments is emphasized since these systems are probably the most significant in terms of atmospheric methane fluxes. However, pertinent observations of water column studies and anaerobic oxidation are examined. Finally, relevant advances in the microbiology of MOB, symbiotic associations based on methane oxidation, and plant-associated methane consumption are considered. Others have examined in detail microbiological and biochemical aspects of methane oxidation (e.g., Higgins et aI., 1981; Haber et al., 1983; Anthony, 1982; Bedard and Knowles, 1989; Hanson et aI., 1990a, 1991).

2. Microbiology 2.1. Phylogeny Although several new species have been described (e.g., Sieburth et aI., 1987; Lidstrom, 1988; Bowman et al., 1990), the overall taxonomic scheme for methanotrophs has changed relatively little since the initial proposals of Whittenbury and colleagues (e.g., Whittenbury etal., 1970a,b; but see Komagata, 1990, for descriptions of a new group of methylotrophs, the aerobic photosynthetic bacteria). Three groups are used to characterize the currently known isolates: type I, II, and X. These are distinguished by pathways of carbon assimilation, internal membrane structure, rosette formation, types of cysts, and catabolic enzyme suites (Table I; Hanson et al., 1990a, 1991). The general validity of Whittenbury's taxonomic scheme as well as relationships among the various MOB and other prokaryotes has been confirmed in general by analyses of 5 S and 16 S RNA (Bulygina et aI., 1990; Tsuji et aI., 1990). Tsuji et al. (1990) have published a largely complete sequence of the 16 S rRNA for a number of methylotrophic bacteria. They conclude that "phylogenetic relationships based on 16 S rRNA sequences reflect the classical taxonomic classification systems based on phenotypic characteristics." Their analyses place type I methylotrophs in the 13 subgroups of

Ecological Aspects of Methane Oxidation

433

Table I. General Diagnostic Characteristics of Methanotrophic Bacteriaa Character

Type I

Type II

Type X

Rods

Rods, vibrioid

Coccoid

Yes

No

Yes

No Cysts

Yes Exospores or cysts Yes

No Cysts

Morphology Membrane structure Bundles of vesciles located centrally or marginally Paired membranes aligned peripherally Resting stages Rosettes Carbon assimilation pathway Tricarboxylic acid cycle Nitrogenase Diagnostic fatty acid carbon length CO 2 fixation

No RMpb Incomplete No 16 No

Serine e Complete Yes 18 No

No RMP/serine Incomplete Yes 16 Yes

aSee text and Hanson et al. (1991) for additional details. blncorporation of formaldehyde via the ribulose monophosphate pathway, including the diagnostic enzyme, 3-hexulose phosphate synthase. cJncorporation of formaldehyde via serine, including the diagnostic enzyme, hydroxypyruvate reductase.

the Proteobacteria and type II methylotrophs in the IX subgroup. A somewhat different phylogeny results from 5 S rRNA analyses. Bulygina et ai. (1990) conclude that "the phylogenetic relations within groups of obligate methano- and methylotrophic bacteria revealed by comparative 5 S rRNA sequence analysis do not support the current rudimentary classification." Bulygina et ai. (1990) also conclude that the obligate methanotrophs are taxonomically distinct from all other methylotrophs; both obligate methanotrophs and methylotrophs form presumably independent monophyletic groups while the facultative methylotrophs are polyphyletic. However, with few exceptions the general groupings of methylotrophs within the Proteobacteria described by Bulygina et at. (I990) and Tsuji et at. (1990) are in agreement. Differences in phylogenetic placements and interpretation may result from both the lower resolution of 5 S versus 16 S rRNA and from the use of different algorithms for phylogenetic tree construction. Sequence analyses of methanol dehydrogenase (MDH) provide another useful perspective on genetic diversity. Machlin and Hanson (1988) have sequenced the structural gene for MDH from Methylobacterium organophilum XX. Comparisons of this sequence with the analogous genes of M. extorquens AMI and Paracoccus denitrificans reveal considerably similarity, in spite of numerous other physiological and genetic dissimilarities (Harms et ai., 1987; Machlin et at., 1987). More extensive comparisons among the methylotrophs may prove useful for establishing phylogenetic relationships and for reconstructing the evolution of methylotrophy. The genetics of methane monooxygenase, the initial catabolic enzyme in methane oxidation, have not been explored as completely (Lidstrom et ai., 1987; Hanson et at., 1990a; Lidstrom, 1990; Cardy et al., 1991) but may provide equally important taxonomic, evolutionary, and ecological in-

434

Gary M. King

sights. Recently, Stainthorpe et ai. (1990) have shown that a gene cluster for the soluble methane monooxygenase (sMMO) can be used to prepare a probe suitable for detecting this enzyme in DNA extracts or colony hybridizations; comparative analyses of methanotrophs using such an approach are further facilitated by the highly conserved nature of the sMMO, at least among type II and X MOB (Pilkington and Dalton, 1991; Cardy et ai., 1991). The gene(s) which code for the particulate methane monooxygenase (pMMO) have been somewhat more elusive. However, several groups are pursuing the genetics of pMMO and a successful characterization is likely in the near future. This will be significant because the pMMO is common to all methanotrophs, unlike the sMMO. Whereas molecular phylogenies help clarify the evolution of and taxonomic relationships among methylotrophs, the basic sequence information from which phylogenies are constructed also provides an extremely powerful tool for exploring ecological problems. At present, analyses of MOB population structure depend primarily on traditional enrichment and isolation techniques. Such methods have proven valuable in establishing the ubiquity, and to some extent the diversity, of MOB in a wide variety of habitats (e.g., Whittenbury et ai., 1970a; Heyer et ai., 1984). However, the approach has obvious limitations, even though it has provided the raw material for classical as well as molecular taxonomy. Molecular probes provide an alternative for examining the distribution of extant species as well as changes in the composition of MOB populations within or among systems. Probes derived from 5 Sand 16 S rRNA sequence data or from the sequences of genes that are diagnostic for methylotrophs (e.g., MDH) allow species or even strain identification from relatively small samples in comparatively short periods of time. Numerous examples of the power of molecular approaches for the analysis of microbial populations are now available (e.g., Diels and Mergeay, 1990; Hahn et aI., 1990; Lee and Fuhrman, 1990; Piitz et ai., 1990; Torsvik et ai., 1990a,b). One particularly exciting possibility involves the use of sequence amplification by the polymerase chain reaction (PCR) to allow the detection of extremely small numbers of a given target sequence. This technique has been used to examine the competitiveness of strains of Frankia spp. for infecting the roots of Alnus spp. (Simonet et ai., 1990). Target sequences in the nifH gene of Frankia have been extracted from milligram amounts of root tissue and'detected after amplification by PCR. Simonet et ai. (1990) have reported detection limits of subnanogram quantities of target after a 30-cycle amplification. Application of this approach using primers prepared from existing sequence data (e. g. , Bulygina et ai., 1990; Machlin and Hanson, 1988; Tsuji et ai., 1990) could greatly facilitate the analysis of methylotrophic populations associated with microoxygen and methane gradients in sediments or plant tissues. Indeed, Hanson and colleagues (personal communication) have used both gene and rRNA probes to characterize the methanotrophic/methylotrophic populations of various reactors and soils. White and colleagues and others have demonstrated that methylotrophs possess diagnostic fatty acids or "biomarkers" which can be used to distinguish between type I and II organisms as well as other bacteria (Nichols et al., 1985; Bowman et ai., 1991). 'lYpe I methylotrophs contain novel 16-carbon monoenoic acids (16: ho8c, 16: lw8t, 16: lw7t, 16: lw5c, and 16: lw5t) while type II methylotrophs contain 18-carbon monoenoic acids (18: lw8c, 18: lw8t, 18: lw7t, and 18: lw6c). In fact, the distribution

Ecological Aspects of Methane Oxidation

435

of these "signature" fatty acids among methylotrophs is concordant with rRNA phylogenies (Guckert et al., 1991). These biomarkers have also been recovered from aquifer soils that were incubated with elevated methane or propane concentrations (Ringelberg et at., 1989). Further, the addition of methane to a soil microcosm results primarily in accumulations of the 18 : 1w8c fatty acid indicative of type II organisms (Nichols et al., 1987). Should the 16- and 18-carbon monoenoic acids prove specific for methylotrophs and should these acids occur in a relatively constant ratio to cell biomass, it would be feasible to estimate total microbial biomass (from total phospholipid or fatty acid concentrations; see, e.g., Brinch-Iversen and King, 1990), the fraction of biomass attributable to methylotrophs, methylotroph biomass dynamics, and in combination with molecular probes, methylotroph population structure. However, since biomarker analyses typically require relatively large amounts of material, the technique may not be suitable for environments with microgradients or systems where sample sizes are restricted. On the other hand, soils or sediments treated with methanotrophs in bioremediation programs represent ideal systems for analysis since sample availability is not necessarily limiting and since microscale sampling is less important. To date, a relatively small number of organisms have formed the basis for the microbiology of the MOB. Although numerous isolates have been obtained from diverse habitats (e.g., Whittenbury et al., 1970a; Heyer et al., 1984), the approaches used for isolation have been limited and used in common by many investigators. For example, the media most commonly employed for enrichment and isolation are based on the nitrate or ammonia mineral salts media reported by Whittenbury et al. (1970a); gas phase compositions of 30-50% methane have been routinely used. While such media have proven useful, they may have limited both the taxonomic and physiological diversity of the organisms isolated. Ecological studies have indicated that active MOB may be exposed to only low micromolar to submicromolar concentrations of methane and suboxic to microaerophilic oxygen concentrations (Kuivila et al., 1988; Frenzel et al., 1990; King, 1990b; King et at., 1990; Ward, 1990). Temperatures and pH regimes can also vary substantially in natural systems (Heyer and Suckow, 1985; Born et ai., 1990). None of these parameters have been duplicated well in attempts to characterize the diversity of MOB. Some exceptions include the isolations by Gal' chenko and colleagues of halotolerant and thermophilic MOB (see Gal'chenko et at., 1989, and references therein). In addition, Lees et al. (1990) have reported that some methanotrophs may not grow on solid media, a limitation that would certainly limit the diversity of typical enrichments. As a consequence, the current status of MOB microbiology may be analogous to that of the sulfate-reducing bacteria (SRB) during the 1970s. At that time, the SRB were considered a relatively simple group of organisms from both a taxonomic and physiological perspective (Widdel, 1988). The use of more varied isolation protocols by Pfenning, Widdel, Postgate, and co-workers has transformed this view almost entirely. The SRB are now considered highly diverse with characteristics and ecological roles that were unanticipated as little as 10-15 years ago (Widdel, 1988). A similar revision in the status of the MOB is not unlikely. Alternative approaches to enrichment and isolation, such as those of Lees et al. (1991) and Putzer et ai. (1991), provide promising directions for exploring MOB diversity.

436

Gary M. King

Future isolation programs might profitably emphasize fungi and mixotrophic MOB. Zajic et at. (1969) have documented methane consumption by a fungus (Graphia spp.) isolated from soils associated with a gas pipeline. Later reports have documented methane oxidation by several yeasts (e.g., Wolfe and Hanson, 1980). Most recently, Jones and Nedwell (1990) have isolated methylotrophic species of Trichoderma and Penicillium from landfill soils. The implications of these observations are extremely important since it is conceivable that the oxidation of atmospheric methane in soils is carried out by fungi as well as the classically described MOB. An important role for fungi might explain apparent discrepancies between the kinetics of oxidation in soils and the kinetic properties of known bacterial isolates (but see Section 4). A role for fungi or mixotrophic MOB (see Jones and Nedwell, 1990, for indications of relative importance) is also consistent with predictions from a kinetic and maintenance energy of atmospheric methane oxidation in soils (Conrad, 1984).

2.2. Kinetics Km values for pure cultures, enrichments, cell-free extracts and purified methane monooxygenase (Table II) range from 1 to 92 J..LM and 0.1 to 37 J..LM for methane and oxygen, respectively (e.g., Nagai et at., 1973; Linton and Buckee, 1977; Lamb and Garver, 1980; Joergensen and Degn, 1983; Joergensen, 1985; Green and Dalton, 1986; Whalen et aI., 1990; Megraw and Knowles, 1987a,b; Oldenhuis et at., 1991). These ranges have no apparent taxonomic origin in that both high and low values have been recorded for the same species and for type I, II, and X organisms. The lowest and perhaps most accurate values for both methane and oxygen are those of Joergensen (1985), who used a membrane-inlet mass spectroscopic technique in which no diffusion limitations for gas transfer were apparent. The high values reported by Oldenhuis et at. (1991) for the same organism used by Joergensen (1985) may result from differences in culture conditions. Oldenhuis et at. (1991) report kinetic parameters for cells expressing only sMMO whereas data from Joergensen (1985) reflect the kinetics of pMMO (see Section 2.3). The half-saturation constants (Kapp) for methane uptake in natural samples (Table II) are comparable to values for cultures and pMMO. Ward (1990) has recently calculated submicromolar Kapp's from a four-point Lineweaver-Burke kinetic analysis. Yavitt et at. (1990a) have used a similar approach and reported Kapp's of about 5 11M for peat samples. Lidstrom and Somers (1984) and Kuivila et at. (1988) have estimated Kapp's of 5-10 11M for sediments from Lake Washington. King (1990b) has used progress curve analyses and reported Kapp's of about 2-4 J..LM for Danish wetland sediments. The lower Kapp's are generally consistent with the range of methane concentrations found in the oxic zones from which samples were collected. Few estimates of Kapp for oxygen uptake by MOB in natural samples are available; values of 10-20 J..LM have been obtained for sediments from Lake Washington (Lidstrom and Somers, 1984; Kuivila et al., 1988). Though few data are available, reported maximal uptake (or oxidation) rates (Vmax) correlate well with methane flux rates. This relationship suggests that the supply of

Ecological Aspects of Methane Oxidation

437

Table II. Representative Kinetic Parameters for Methane Oxidation by Methane Monoxygenase, Various Pure and Mixed Cultures, and Natural Samples. Sample source or organism Methane monooxygenase (soluble, M. capsulatus)

Reference

3

56.0 a

Green and Dalton (1986)

Pure cultures M ethylosinus trichosporium

Strain OU-4-1 Landfill isolate Nitrosococcus oceanus Nitrosomonas europaea

Mixed cultures Sludge isolates Sediment isolates Sediments and soils Landfill soil Peat, 0-10 cm Lake Washington Lake Superior Danish wetland

2 0.8 9.3

26.0 b

5.3 c

6.6 d

2000

Lamb and Garver (1980) Linton and Buckee (1977)

1.7

32.0 2.5 3.7 8.3-10.7 5.1-10.0 4.6 2.2-3.7

Joergensen (1985) Joergensen and Degn (1983) Whalen and Reeburgh (1990) Ward (1987) Hyman and Wood (1983)

237'

1.91

38f 28f 0.71 662-14411

Whalen and Reeburgh (1990) Yavitt et al. (199Oa) Lidstrom and Somers (1984) Kuivila et al. (1988) Remsen et al. (1989) King (1990b)

anmole (mg protein A) - 1 min - 1 . bnmole (mg dry weight) - 1 min - I. cnmole (mg dry weight) - 1 min - I; calculated by assuming 5 X 10- 14 (g dry weight cell) dBut see also Ward (1990) for a discussion of methane uptake in Nitrosomonas. e ..mole liter - 1 hr - I; calculated assuming a soil density of I g em - 3. flJ.ffiole liter-

I

I.

hr- 1 .

methane to the zone of oxidation may determine Vmax (King, 1990b; King et al., 1990). Since Vmax is probably an indicator of active MOB biomass (Remsen et at., 1989), such a relationship is not surprising. Regardless, more extensive comparisons are necessary for determining if a general, predictable relationship exists among diverse sites. Additional kinetic evaluations are important because Km (Kapp) reflects the extent of adaptation for consumption of methane and oxygen at in situ concentrations. High micromolar Km's are inconsistent with the low micromolar to nanomolar concentrations observed in most oxic sediments and waters (e.g., Ward et al., 1987, 1989; Kuivila et al., 1988; Frenzel et al., 1990; King, 1990b); low nanomolar methane concentrations are also expected in soils at or near equilibrium with the atmosphere. Conrad (1984) has evaluated relationships among kinetic parameters, maintenance energy requirements, and population sizes and concluded that high-Km uptake systems are not likely to sustain oxidation rates sufficient to meet growth requirements at low in situ methane concentrations.

438

Gary M. King

2.3. Methane Monooxygenase and Dehalogenation The MOB are unique in many respects; one of the most remarkable aspects of these organisms is the enzyme, MMO. This copper-containing complex occurs in two forms, membrane-bound or particulate (pMMO) and soluble (sMMO). The former occurs in all MOB while the latter has a more restricted distribution (see Anthony, 1982; Dalton et ai., 1984; Dalton and Higgins, 1987). Aside from cellular location, these two enzymes differ in a number of respects, including substrate specificity, sensitivity to inhibitors, requirements for NAD(P)H, kinetics, and tertiary structure among others (e.g., Colby et ai., 1977; Dalton, 1980; Stanley et ai., 1983; Burrows et ai., 1984; Green and Dalton, 1986; Dalton and Higgins, 1987; Fox et ai., 1989). In at least some type II and X MOB, the expression of one form or the other is determined by the availability of copper (e.g., Stanley et ai., 1983; Burrows et ai., 1984; Pilkington and Dalton, 1991). At low copper concentrations (::; 1 fLM), the soluble form dominates activity; at concentrations> 1-5 fLM, MMO is exclusively particulate. Scott et ai. (1981) have also reported a sensitivity to other growth factors; however, their data are not inconsistent with copper regulation. Copper may also play an important role in type I MOB; Collins et ai. (1991) have reported that copper additions increased both cell yields and MMO activity in M. aibus BG8, an organism not known to produce sMMO. Recognition of the regulatory role of copper has provided a solution to some earlier discrepancies in the characteristics of certain MOB and clearly emphasized the need for detailed specification of growth conditions and trace metal availability in any comparative studies. The biochemical and physiological properties of the soluble and particulate MMO have significant ecological consequences. For example, the pMMO Km for oxygen appears considerably lower than that of the sMMO (0.1 versus 17 fLM; Joergensen, 1985; Green and Dalton, 1986). This difference, if consistent among all MOB, indicates that the capacity to compete for and remain active at low oxygen concentrations is determined in part by the active form of MMO. The more narrow specificity of the pMMO limits the effects of competitive substrates on rates of methane oxidation (Burrows et ai., 1984). The requirement of sMMO for NAD(P)H limits the extent to which co-oxidation of other substrates (e.g., ethanol) can sustain MMO activity; this requirement also limits the energetic efficiency of the sMMO versus pMMO system (Burrows et at., 1984; Green and Dalton, 1986; Leak and Dalton, 1986). In addition, there are commercial and environmental ramifications for the location of MMO activity. Unlike pMMO, sMMO oxidizes a variety of aromatic and alicyclic compounds, including aromatic alcohols, benzene, toluene, and cyclohexanol (e.g., Colby et at., 1977; Dalton, 1980; Burrows et at., 1984; Mountfort et at., 1990). Moreover, the sMMO, but not pMMO, of Ms. trichosporium also catalyzes the oxidative dehalogenation of various halomethanes, -alkanes, and -alkenes, with degradation of trichloroethylene (TCE) of particular interest for bioremediation (Oldenhuis et at., 1989, 1991; Tsien et ai., 1989; Brusseau et at., 1990; Fox et ai., 1990; Hanson et at., 1990b). Others have reported dehalogenation by soils exposed to methane (Wilson and Wilson, 1985; Strand and Shippert, 1986; Henson et ai., 1988; Lanzarone and McCarty, 1990), by pure and mixed methylotrophic cultures (Fogel et ai., 1986; Vogel et

439

Ecological Aspects of Methane Oxidation

al., 1987; Janssen et at., 1988; Little et al., 1988), by alkane (propane)-oxidizing mycobacteria (Wackett et al., 1989), by ammonia-oxidizing bacteria (Arciero et al., 1989), which contain an enzyme, ammonia monooxygenase, that is remarkably similar to MMO (e.g., Bedard and Knowles, 1989), and by methanogens (Vogel and McCarty, 1985). In the case of Ms. trichosporium, it is clear that copper availability plays a key role in TCE metabolism (Oldenhuis et al., 1989; Tsien et al., 1989; Brusseau et at., 1990; Fox et al., 1990), a fact consistent with involvement of sMMO (Fig. 1). Brusseau et al. (1990) have developed a rapid, sensitive assay for sMMO based on the oxidation of the polycyclic aromatic , naphthalene; this method has proven useful for optimizing

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o 0.1 0.25 0.5 1 Concentration Cu in Media in 11M Figure 1. Effects of copper concentration on rates of methane oxidation and trichloroethylene degradation in cultures of Methylosinus trichosporium OB3b. Cultures were incubated with various concentrations, harvested at the times indicated, and then assayed for methane oxidation or TCE degradation rates . Illustration courtesy of H.-C. Tsien and R. S. Hanson. Form Tsien et al. (1989).

440

Gary M. King

conditions for TCE degradation. In contrast, Henry and Grbic-Galic (1990) report that sMMO does not catalyze TCE dehalogenation by a Methytomonas sp. since the organism expresses only pMMO. They also demonstrate the sensitivity of methane oxidation, growth yield, and dehalogenation to medium composition, particularly copper and chelator concentrations. Alvarez-Cohen and McCarty (1991), Henry and Grbic-Galic (1991), and Oldenhuis et at. (1991) have all documented TCE toxicity, a general inactivation of cellular metabolism by TCE or TCE metabolites, and the significance of sources of reductant, such as formate, which provide NADH for sMMO without competing with TCE.

2.4. Methylotrophic Symbioses Although MOB have been isolated routinely from a variety of sources during the last century, active symbioses involving MOB have only been described recently. Subsequent to reports of symbiotic associations between sulfide-oxidizing chemolithotrophs and the vestimniferan tube-worm, Riftia pachyptila (Cavanaugh et at., 1981), other symbioses were reported for various invertebrates (e.g., Southward et at., 1981; Ott et at., 1982; Cavanaugh, 1983, 1985; Dando et at., 1985; Dando and Southward, 1986). Based on stable isotope evidence, Southward et at. (1981) suggested a methane-based symbiosis for a pogonophoran; later efforts proved this suggestion incorrect (Southward et at., 1986). However, a variety of other observations confirmed that mollusks and pogonophorans did indeed harbor methane-oxidizing symbionts (Childress et at., 1986; Brooks et at., 1987; Cavanaugh et at., 1987; Fisher et at., 1987; Schmaljohann and Fliigel, 1987; Cary et ai., 1988, 1989; Wood and Kelly, 1989; Page et ai., 1990; Schmaljohann et at., 1990). For instance, numbers of MOB in gill tissues of a mollusk were about 3 x 108 cells (g tissue) - I, somewhat less than the 4 x 109 cells (g tissue) - I reported for sulfide-oxidizing bacteria associated with R. pachyptila, but still remarkable. To date the MOB symbionts have been either coccoid or rod-shaped cells with type I internal membranes (Fig. 2) and an active hexulose phosphate synthase pathway for carbon assimilation; thus far, type II organisms have not been observed (e.g., Cavanaugh et at., 1987; Wood and Kelly, 1989; Schmaljohann et at., 1990). Regardless of the taxonomic affinities of the MOB, these symbioses accounted for a large fraction of host carbon and energy requirements (e.g., Cary et at., 1988; Fisher et at., 1987) and apparently contributed to dramatic, productive benthic communities isolated from significant phytoplanktonic organic inputs (e.g., Paull et at., 1984, 1985; Kennicutt et at., 1985, 1989; Hovland and Thomsen, 1989; MacDonald et at., 1990; Dando et at., 1991). Symbiotic relationships between plants and MOB have received virtually no attention relative to animal symbioses, even though methanotrophs were first isolated from the leaves of the macrophyte Etodea (S6hngen, 1906). While MOB symbioses may be nonobligatory or even of minor benefit to plant hosts, the ecological ramifications of the relationship are substantial. DeBont et at. (1978) have reported methane oxidation in association with rice (Oryza sativa) roots. Holtzapfel-Pschorn et at. (1985, 1986) and Schiitz et at. (l989a) have made similar observations and concluded that much of the

Ecological Aspects of Methane Oxidation

441

Figure 2. Transmission electron micrographs of gill tissue from mussels collected at the Florida Escarpment. (A) Transverse section of a gill filament showing bacteria-containing host cells (bacteriocytes; nb, nucleus) separated by intercalary cells (ni , intercalary cell nucleus); bacterial cells are designated by arrows. Scale bar is 5 "",m. (B) Transverse section of bacterial cell showing apparently gram-negative ultrastructure and stacked internal membranes typical of type I methantrophs. Scale bar is 0 .3 J.Lm . Figure courtesy of C. Cavanaugh; from Cavanaugh et al. (1987).

442

Gary M. King

methane which diffuses to the rice rhizosphere is oxidized. This observation is extremely important because Cicerone and Shetter (1981), Schlitz et al. (1989b), Sass et al. (1990), and others have suggested that a large fraction of the methane efilux from rice paddies occurs through the plants. Consequently, methane oxidation is likely a major control of the significance of rice as a global methane source. Since rice accounts for about 25% of the current global flux estimate (Cicerone and Oremland, 1988; Wahlen et al., 1989), small changes in the rhizosphere oxidation term can have a globally important impact. Methane oxidation also occurs in association with the roots of other aquatic plants (King et al., 1990; Chanton et al., 1992). King et al. (1990) and King (unpublished data) have observed rapid methane oxidation by the sediment-free roots of a wide diversity of macrophytes, including species that are commonly distributed throughout northern wetlands. Several aspects ofthe observations are notable. First, methane oxidation by sediment-free roots occurs with no lag, implying that populations of MOB are exposed to conditions suitable for activity in situ. Second, thresholds for methane uptake are typically relatively high (>5 ppmv), implying that consumption of atmospheric methane is unlikely (King, unpublished data). A single exception for an aquatic grass has been observed, however, with threshold values 10 cm had an opposite effect (Moore and Knowles, 1989; Roulet et at., 1992). Seasonal decreases > 10 cm in the Great Dismal Swamp and the Florida Everglades were accompanied by decreased methane fluxes, even atmospheric methane

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5

Figure 6. Effect of nitrogen fertilization on in situ rates of atmospheric methane uptake in soils of a pine forest. Soil temperatures Ce) are from the 0-2.5 cm interval. The low-nitrogen treatment C~I) represents an addition of 37 kg N ha - 1 year - 1while the high-nitrogen addition plots ICD)I received 120 kg N ha - 1 year - 1; rates of oxidation are compared to untreated or control plots Ci!!I). From Steudler et al. (1989).

The lack of a clear relationship between nitrogen addition and rates of oxidation suggests that the observed changes are the result of shifts in population or community structure or subtle changes in the kinetics of MOB extant prior to fertilization. Steudler et al. (1989) and Mosier et al. (1991) have suggested that changes in AOB may be causative. A role for AOB is questionable, however. To date, AOB have not been shown to oxidize atmospheric methane [though concentrations as low as 12 nM are consumed (Jones and Morita, 1983)] and ammonia, nitrite, and nitrate concentrations up to 0.7 mM stimulate methane oxidation in Nitrosococcus oceanus and Nitrosomonas europaea (Jones and Morita, 1983). Concentrations of 0.7 mM for dissolved inorganic nitrogen species are considerably higher than the values for KCl-extractable ammonia and nitrate reported by Mosier et al. (1991) and the probable free ammonia and nitrate levels in the soils examined by Steudler et at. (1989). Moreover, Jones and Morita (1983) do not show net inhibition of methane oxidation by 3.5 mM ammonia relative to 0.07 mM; nitrite and nitrate additions beyond 0.7 mM also have no inhibitory effect. Thus, it might be argued that nitrogen additions could stimulate, not inhibit atmospheric methane oxidation by AOB in soils. In contrast, inhibition of MOB by ammonia is consistent with culture data (e.g., O'Neill and Wilkinson, 1977) and offers at least a partial explanation of the field results; the response of MOB to ammonia can also be invoked to explain increased rates of nitrous oxide emission from fertilized soils (Yoshinari, 1985; Knowles and Topp, 1988), though this is probably better attributed to the activities of AOB and denitrifying bacteria. Of course, it is conceivable that the effects of nitrogen fertilization are expressed via indirect or secondary mechanisms rather than directly via substrate interactions with

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MOB. In support of this possibility, Ward and Kilpatrick (1990) have found that ammonia and methane oxidation rates in the water column of Saanich Inlet were independent of methane and ammonia, respectively. Obviously, the response of soil methane oxidation to nitrogen requires considerably more detailed field and culture analyses. Future efforts might emphasize not only the controls of methane oxidation by nitrogen additions, but other interactions with the nitrogen cycle, especially the production and consumption of N20 and NO (e.g., Kramer et al., 1990). The use of inhibitors, especially N-serv, is a common agricultural practice that may affect methane oxidation to a greater extent than fertilization. MOB are basically sensitive to the same inhibitors as AOB (reviewed in Bedard and Knowles, 1989). N-serv and related analogues are particularly effective inhibitors of methane oxidation in culture, soil, and sediments (Topp and Knowles, 1982, 1984; Salvas and Taylor, 1984; Bedard and Knowles, 1989; King, 1990b; Megraw and Knowles, 1990). The routine application of N-serv to agricultural soils may not only inhibit methane consumption by AOB, but may severely limit methane oxidation by MOB. The extent to which this aspect of agriculture, as opposed to nitrogen fertilization, has altered the soil methane sink has not been evaluated. In addition to emphasizing the response of MOB to nitrogen, it is important to understand basic kinetic phenomena. For example, Km and threshold values are critical determinants of methane consumption. The threshold for consumption, i.e., that level below which no uptake occurs, has not been systematically examined for cultures. However, in a limited survey of Methylosinus trichosporium OB3B, Methylomonas rubrum, M. albus BG8, Methylococcus capsulatus Bath, and an isolate from a lake sediment, all but M. Capsulatus could consume atmospheric methane; thresholds were