Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers

Ammonia oxidizers (family Nitrobacteraceae) and methanotrophs (family Methylococcaceae) oxidize CO and CH4 to CO2 and NH4+ to NO2-. However, the relative contributions of the two groups of organisms to the metabolism of CO, CH4, and NH4+ in various environments are not known. In the ammonia oxidizers, ammonia monooxygenase, the enzyme responsible for the conversion of NH4+ to NH2OH, also catalyzes the oxidation of CH4 to CH3OH. Ammonia monooxygenase also mediates the transformation of CH3OH to CO2 and cell carbon, but the pathway by which this is done is not known. At least one species of ammonia oxidizer, Nitrosococcus oceanus, exhibits a Km for CH4 oxidation similar to that of methanotrophs. However, the highest rate of CH4 oxidation recorded in an ammonia oxidizer is still five times lower than rates in methanotrophs, and ammonia oxidizers are apparently unable to grow on CH4. Methanotrophs oxidize NH4+ to NH2OH via methane monooxygenase and NH4+ to NH2OH via methane monooxygenase and NH2OH to NO2- via an NH2OH oxidase which may resemble the enzyme found in ammonia oxidizers. Maximum rates of NH4+ oxidation are considerably lower than in ammonia oxidizers, and the affinity for NH4+ is generally lower than in ammonia oxidizers. NH4+ does not apparently support growth in methanotrophs. Both ammonia monooxygenase and methane monooxygenase oxidize CO to CO2, but CO cannot support growth in either ammonia oxidizers or methanotrophs. These organisms have affinities for CO which are comparable to those for their growth substrates and often higher than those in carboxydobacteria. The methane monooxygenases of methanotrophs exist in two forms: a soluble form and a particulate form. The soluble form is well characterized and appears unrelated to the particulate. Ammonia monooxygenase and the particulate methane monooxygenase share a number of similarities. Both enzymes contain copper and are membrane bound. They oxidize a variety of inorganic and organic compounds, and their inhibitor profiles are similar. Inhibitors thought to be specific to ammonia oxidizers have been used in environmental studies of nitrification. However, almost all of the numerous compounds found to inhibit ammonia oxidizers also inhibit methanotrophs, and most of the inhibitors act upon the monooxygenases. Many probably exert their effect by chelating copper, which is essential to the proper functioning of some monooxygenases. The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.


INTRODUCTION
An objective of this review is to summarize aspects of the biochemistry and physiology of methanotrophs and ammonia oxidizers which are relevant to the oxidizing activities of these organisms in their natural habitats. Ecological aspects have been extensively reviewed elsewhere (51. 128, 130). as have general aspects of methanotrophs (4. 5. 25, 55) and ammonia oxidizers (61,174). Pa rticular attention is given to the ammonia monooxygenase of ammonia oxidizers and the methane monooxygenase of methanotrophs since these are both key enzymes in the metabolism of CH4. NH4 , and Co.
relative contributions of these two kinds of organisms to in situ CO, CH4, and NH4+ metabolism.
NOJpeaks located at the boundary between anoxic and oxygenated waters are a common feature of moderately productive lakes (67). Several different processes, including nitrification and denitrification, could lead to the formation of such peaks (91). In the metalimnion of Lake Mendota (Wisconsin), an NO,maximum was found to occur at the depths where CH4 oxidation was most pronounced, prompting the suggestion that methanotrophs might be responsible for NO,accumulation in this environment (51,52). An alternative explanation (79) is that chemoautotrophic ammonia oxidizers were oxidizing CH4 in addition to NH4 + . Recently, it has been suggested that methanotrophs could be involved in the process of nitrification in lakes under winter ice (90). Methanotrophs may also be responsible for some of the nitrification observed in soils (130,159). S. R. Megraw and R. Knowles (unpublished data) have obtained strong evidence that, under certain conditions, essentially all of the NH4' oxidation measured in a particular humisol is carried out by methanotrophs. Biological oxidation is an important sink in the worldwide cycle of CO (104). Carboxydobacteria are aerobic organisms that can use this compound as sole source of carbon and energy (105). Most of the carboxydobacteria known at the present time exhibit half-saturation constants for CO oxidation which are considerably higher in value than naturally occurring CO concentrations. For this reason, it has been suggested that these organisms may not play a significant role in CO cycling. On the other hand, both methanotrophs and ammonia oxidizers exhibit high affinities for the compound, which has led some investigators to speculate about the importance of these organisms in the environmental metabolism of CO (27,80).
The participation of methanotrophs and nitrifiers in the atmospheric cycles of CH4, N2O, and CO can have increasingly important implications for global warming, due to the infrared absorption of CH4 and N2O (31), and for stratospheric chemistry (including ozone) since all three gases participate in vital reactions (78). An increased understanding of the relative roles of methanotrophs and nitrifiers in such processes is therefore desirable.
To determine the nature of the organisms responsible for an oxidation in an environment, one might use an inhibitor which selectively blocks only methanotrophs or only ammonia oxidizers. Several inhibitors which act upon ammonia oxidizers are used to delay nitrification in agricultural soils (135). Nitrification blockers have also have been used to quantify nitrification in aquatic environments (e.g., see ref- erence 137). However, it has been found that almost all agents that inhibit ammonia oxidizers also inhibit methanotrophs (129,155,156). This raises the possibility that in certain environmental studies activity attributed to ammonia oxidizers was in fact due to methanotrophs. We conclude by reviewing our present knowledge of inhibitors of these organisms and examine the limitations associated with the use of nitrification blockers in ecological studies.
OXIDATION OF CH4, NH4+, AND CO BY

Similarities between Methanotrophs and Ammonia Oxidizers
Methanotrophs (family Methvlococcaceae) can obtain all of their carbon and energy from CH4 under aerobic conditions (170). The pathway of methane oxidation is outlined in Fig. 1. Obligate methanotrophs grow only on CH4 and CH3OH. In addition to these substrates, facultative methanotrophs can also grow on multicarbon compounds. Methane oxidizers are grouped into two types based in part on their pathways of carbon assimilation (22). Type I methanotrophs fix formaldehyde by the ribulose monophosphate pathway, while type II organisms use the serine pathway to assimilate carbon dioxide and formaldehyde (4).
Ammonia oxidizers are chemoautotrophs, members of the family Nitrobacteraceae (165). They oxidize NH4', which provides energy for the fixation of CO, via the Calvin cycle (167). The pathway of NH. oxidation is summarized in Fig.   1. In addition to their usual source ot energy, both methanotrophs and ammonia oxidizers can oxidize a variety of nongrowth substrates. Table 1 lists compounds which are oxidized by both groups of organisms. Methanotrophs can oxidize NH4 ' and ammonia oxidizers can oxidize CH. although neither type of organism can use the energy from the reaction for growth (79,127). Methane oxidizers can modify a great number of organic compounds in addition to those mentioned in Table 1 (47). The multiplicity of oxidations carried out by methanotrophs and ammonia oxidizers reflects the wide substrate specificities of the methane monooxygenase of the former organisms and the ammonia monooxygenase of the latter (34,56,70,71).
Other similarities between the two groups of organisms can be summarized as follows. (i) An NH.OH oxidase resembling that in ammonia oxidizers, and distinct from methanol dehydrogenase, may be present in at least some methanotrophs (136). (ii) A complex array of intracytoplasmic membranes is present in both types of organisms (28,107). (iii) Ammonia oxidizers and type I methanotrophs have predominantly 16:0 and 16:1 fatty acids (11,100,109). (iv) The type I methanotrophs possess a tricarboxylic acid cycle which lacks (x-ketoglutarate dehydrogenase (169). (v) Both groups of organisms tend to favor the same kinds of habitats: aerobic-anaerobic interfaces where CH4, NH4 and O. are readily available (70,127).
The possibility of an evolutionary link between methanotrophs and ammonia oxidizers has often been mentioned (86,123,169), and recent 5S and 16S ribosomal ribonucleic acid studies show the ammonia oxidizers in groups P-3 and y-l and a type I methanotroph in the y-I group of the purple bacteria (172,173). However, the elucidation of the relationship between methanotrophs and ammonia oxidizers may be rendered more difficult by recent discoveries which suggest that cross-species gene transfer contributes in a significant way to species evolution (150).
The present review concentrates on CH4 oxidation by ammonia oxidizers, NH4 ' oxidation by methanotrophs, and CO oxidation by both groups of organisms: activities which. as indicated earlier, may be of importance in the natural environment. First, the characteristics of the respective monooxygenases will be discussed.

Ammonia Monooxygenase
Although the enzyme involved has yet to be purified, it appears likely that the first step of NH4 'oxidation in Nitrosoinonas eiUlop(ae( is a monooxygenase-catalyzed oxidation of NH3 to NH,OH (35,59). Evidence that NH3 rather than NH4+ is the substrate for the monooxygenase comes from an examination of the effect of pH on NH4+ oxidation in cell-tree extracts of this organism (144). K,,1 values for the oxidation decreased markedly with increasing pH. However, the va_lues remalined essentially constant when they were recalculated in terms of unprotoncated NH, instead of total armmonia (NH, plus NH4 '). NH.OH is a probable intermediate in NHX oxidation (58,59,96,108). It is oxidized to NO. by whole cells and cell extracts of N. europaea and accumulcates during NH4' oxidation in the presence of hydrazine, a powerful inhibitor of NH,OH oxidation (58. 59, 96. 108). The inhibitor sensitivities of NH4' and NH.OH oxidation care very different (58. 62) see Table 3. Furthermore. NH.OH is the substrate of an oxido-reductaIse which hCas been isolated from N. eropae(l(l and extensively purified and characterized (for reviews, see references 32 and 61).
18O from SO, was incorporated into NO.during NH4 oxidation by N. europaea (126). Cell suspensions of this organism containing added hydriazine were used in isotopic studies of the NH,-to-NH,OH reaction step (35,59): the oxygenase nature of the reaction mechanism was demon- The immediate source of reductant "'A' has not been identified with any certcainty (61; see discussion below). rhe ability of ammonia oxidizers to oxidize small organic molecules provides further evidence for a monooxygenase mechanism (see section on CH4 oxidation by ammonia oxidizers).
Source of reducing power. Reducing power for the monooxygenase is apparently regenerfated via NH,OH oxidation (145). Close coupling of NH.OH and NH4+ oxidation has been found in resting cells (60), spheroplasts (145), and cell-free extracts of N. citropuae (63). The ammonia monooxygenase of N. europaea appears not to utilize NADH as a direct electron donor (148. 149). However, NADH can initiate NH4 oxidation in cell-fr-ee extracts (148,149), and it may do this by reducing cytochromes which are required for the reaction (149). Ascorbate is also ineffective as a source of reductant (148). A likelier candidate for the role may be cytochrome (554 (157). When added to a membrane fraction obtained from a cell-free extract of N. eiropaea containing NH4' and NH.OH oxidizing activities, as well as cytochrome oxidase, cytochrome (5 4. reconstituted NH4linked Or uptake (147). In this system, Tsang and Suzuki (157) found that NH4 ', NH,OH, and NH,NH, (a substrate for NH,OH oxidoreductase) all reduce the cytochrome.
Reduction by NH4t occurs at a slower rate than reduction by NH.OH and NH.NHH, and there is a lag between addition of NH4' and reduction of the cytochrome. Reduced cytochrome (554 in the presence of membrane fraction is immediately oxidized upon addition of NH4 or CO. The extent of the oxidation is dependent on the concentration of NH4+ or CO. These observations are consistent with a role as electron donor for cytochrome (554 (157). The cytochrome MlC ROR IOL RFV.
('s54 obtained from N. eiiropa(ea has a midpoint potential (E,i, 7) of +20 mV (106). It is a protein of M, 25,000, which contains four type c hemes and is located in the periplasmic space (3,32). Although it may bind substrate (e.g., O), its properties as determined by optical, Mossbauer, and electron paramagnetic resonance spectroscopies are more suggestive of an electron transfer role (3).
Triand tetramethylhydroquinone, two artificial electron donors, can support the activity of ammonia monooxygenase in whole cells of N. eurzopaea (133). These compounds may be useful in the eventual purification of the enzyme.
The structure and workings of the monooxygenase itself are not known in any detail. One component appears to be a protein of M, 28,000. This was identified by gel electrophoresis and autoradiography of proteins obtained from cells of N. euroopaea which had been incubated in the presence of 14C H, (73). CGH, appears to be a suicide substrate for the ammonia monooxygenase (73), and it is suggested that the attempted oxidation of C.H. by the monooxygenase produces a reactive compound which can bind to the enzyme (73).
Possible role of copper. That copper plays an important role in NH4' oxidation was first proposed more than 40 years ago by Lees (95). The presence of copper in ammonia monooxygenase is suggested by the ability of agents which bind the metal to block NH4 'oxidation in N. ciir-opaeai (62,97). Furthermore, NH4 'oxidation is stimulated by low levels of copper (99,153), although an absolute requirement for the metal has never been demonstrated. Finally, it has been suggested that ammonia monooxygenase possesses a photosensitive oxygenated state similar to that of other copper-containing enzymes (132).
Expanding on work done by Hooper and Terry (63), Shears and Wood (132) studied the light sensitivity of N. eliropaie(i. They found that illumination of cells with ultraviolet light leads to inactivation of NH4t but not NH.OH oxidation. Anaerobiosis, copper-chelating agents, and organic substrates of the monooxygenase tend to mitigate the effect. Difference absorption spectrophotometry of cell suspensions of Nitrosoinonas spp. shows that, upon exposure to ultraviolet light, there is a bleaching in the 350to 400-nm region. Two copper proteins, tyrosinase and hemocyanin, have similar spectroscopic characteristics. They are also sensitive to a number of chelating agents which also inhibit ammonia monooxygenase. Based on these findings, Shears and Wood have proposed a catalytic cycle for the monooxygenase (Fig. 2) modeled on the cycle known from tyrosinase (98). Two copper atoms are hypothesized to be at the active site as in tyrosinase. The ammonia monooxygenase could be present in one of three states: reduced (deoxy), oxygenated (oxy), and oxidized (met). 02 reacts reversibly with the deoxy form: the Cu(I) atoms are oxidized to Cu(II), while oxygen is bound as peroxide ion (O,). The enzyme. now in the activated oxy state, can be inactivated by ultraviolet light at a wavelength of 345 nm. This type of illumination causes the bond between copper and 022-to break, and release of O°can readily occur. Organic substrates protect the enzyme from inactivation by rapidly shifting it from the oxy to the met state. Oxy monooxygenase inserts one atom of oxygen into the substrate while the other is reduced to water. The enzyme, now in the met state, can be reduced by a two-electron transfer to the deoxy state. Chelating agents, such as thiourea, protect the monooxygenase by keeping it in this ultraviolet-insensitive form. Thiourea has a high affinity for Cu(I).
This initial step of CH4 oxidation has been most investigated in two species of methanotrophs: Methylococcus (Yapsiilatus Bath and Methylosinits trichospormiun OB3b. Both of these organisms produce a membrane-bound ("particulate") MMO and a cytoplasmic ("soluble") MMO. Because only the soluble enzymes of the two organisms have been extensively purified and characterized, it is not entirely clear whether soluble and particulate forms are distinct or whether they share components (26,41). In the case of Methy1lo(occl.us (eapsulaitis Bath, the former possibility appears likeliest (45). At least one other species, Methlylobacteriuiiml sp. strain CRL-26, can apparently express both a soluble and a particulate enzyme (115,116). Methvlomnonas (l/bits BG8 and Metlivlocystis parvits may possess only a particulate form (25). Dalton and Leak (25) suggested that soluble MMOs are restricted to only a few species of methanotrophs. There are no reports of organisms possessing this type of enzyme alone.
Role of copper. Copper may exert considerable influence on the nature of the monooxygenase expressed and on cell morphology (14,26,122). In both Methvlo(o Cllus ( apSi(lttS Bath and Methvlosinits tierihosporilon OB3b when Cu is present in limiting amounts, methane oxidation is carried out mostly by the soluble MMO. When Cu is present in nonlimiting quantities, it is the particulate MMO which dominates (14,26). Cu(I) and Cu(II) inactivate the soluble enzyme of Methvyl0o(o(( l(s ( apsultitiis Bath (44). Copper appears to be a requirement for particulate MMO activity in both this species (122) and MethYloba(cteriium sp. strain CRL-26 (115).
Increasing Cu(II) availability appears to lead to increased content of intracytoplasmic membranes in cells of Methylosinuts tricliosporilon and Methlylo(o(llus ( apsillatils (14,122,131).
Gel electrophoresis of cell extracts of Methvloc occuis (cipsulatlis Bath grown in continuous culture shows that, as copper supply increases, polypeptide bands corresponding to soluble MMO disappear while other bands possibly related to particulate MMO increase in intensity (122). A similar pattern was noted in Methylosinius triclhosporiuin OB3b (14).
Source of reductant. In an extensive review of the evidence, Anthony (5) concluded that, at least in cell-free systems, all forms of MMO can utilize NAD(P)H as electron donor. It has been suggested that, in a variety of methanotrophs, electrons from the oxidation of CH,OH by CH,OH dehydrogenase can be recycled directly to be particulate MMO via one or more electron transport proteins (93, 101. 154; Fig. 1). The existence of such a pathway is not certain. It could explain the greater carbon conversion efficiency of cells expressing the particulate MMO when compar-ed with that of cells expressing the soluble enzyme (94). Substrates. In Metlhylococcus capsiilatus and Metliilosinits trichosporium, the particulate MMO appears to have a narrower range of substrates than the soluble MMO. Both enzymes can oxidize alkenes and alkanes up to pentane (14,26,140). But, unlike soluble MMO, particulate MMO cannot oxidize aromatic and alicyclic compounds or higher (n > 5) alkanes (14,140). These differences could be due to dissimilar active sites or to disruption of the activity of the particulate MMO (26). It is interesting that ammonia monooxygenase differs from particulate MMO in that it can oxidize certain aromatic and alicyclic compounds (34,69). Inhibitors (122). When the organism was grown on CH,OH, particulate MMO activity was always present, whether or not copper had been added to the growth medium (122). Increasing copper concentrations led to increased particulate enzyme activity (122).
Soluble MMO. The soluble MMO of Methylococcus(cpsiultlts has been purified and extensively characterized (19,20,44,45,175). The picture that emerges from these studies is briefly outlined below.
Soluble MMO consists of three components, labeled A, B, and C. Component A is a nonheme iron protein of Mr 210,000 which binds CH4. Recent spectroscopic evidence indicates the presence of a binuclear Fe center in Mceti lococcus capsulatus (40). Evidence regarding the presence of a wt-oxo bridge associated with the Fe cluster in this and other organisms is conflicting (38,42,87,119). Protein A apparently uses reducing equivalents obtained from component C to activate 02, allowing insertion of an oxygen atom into CH4.
Component B is a low-molecular-weight (Mr, 15,700) protein which lacks any prosthetic groups. It converts the MMO from an oxidase to an oxygenase. In its absence, proteins A and C catalyze the 4-e-reduction of 02 to H,O. Component B may play a role in controlling cellular levels of NAD. In the presence of protein B, NADH oxidation becomes tightly coupled to CH4 oxidation. The oxidase activity is switched off. Component C is an iron-sulfur flavoprotein of M, 42,000. It possesses a single polypeptide chain, one flavin adenine dinucleotide molecule, and an Fe,S, center. It is the reductase component of the MMO, funnelling electrons from NADH to component A.
Based on steady-state kinetic analyses, Green and Dalton (45) have proposed a mechanism of action for the soluble MMO of Metliylococcus (cpsIlatusI. CH4 binds to the monooxygenase, followed by NADH which reduces the enzyme. NAD' is released, and the reduced enzyme combines with 0, to form a ternary complex which breaks down with the release of CH3OH and HRO (a 'Bi Uni Uni Bi Ping Pong" mechanism). The K,,, values for CH4, NADH, and 02 were 3, 55.8, and 16.8 [tM, respectively.
Early efforts to purify the soluble MMO from Methylosinus trichlosporium failed due to the instability of enzyme preparations (142). However, the ability of purified components B and C from the soluble MMO of Methvlococcus (apsilatius to restore MMO activity to a fraction obtained from a soluble extract of Metliylosinus trichlosporium emphasizes the similarity between the enzymes of the two organisms. A binuclear Fe cluster is also reported for protein A from M. trichosporilm (42).
A soluble MMO obtained from Metli Iobac(teriunm sp. strain CRL-26 has been partially purified and characterized (113,115,120). It consists of three components named A, B, and C by analogy to the soluble MMO of Methvlococcus capsIlatius. Component B of Methylohacteriumn sp. was not required for MMO activity as measured by the oxidation of propylene to propylene oxide (113. 115). Component A of this organism is an iron protein similar in molecular weight to component A of Metlivllococcus (apsIlaItius (113), and it also contains a binuclear Fe-Fe cluster (119). Component C of Metlhilobacterium sp. is a flavoprotein which is reduced by NADH and the redox properties of which, as determined by optical and electron spin resonance spectra, are very similar to those of the corresponding protein in Methylococcus (apsldatlis (120). Components A and C of Methlilobacteri,mn sp. catalyze the epoxidation of propylene (113). An immunoglobulin fraction of antisera raised against these two purified components cross-reacted with certain proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from crude extracts of Mcthylobacterilm O/ oIgaiopliliun XX, a facultative type II organism; MethVlococcus sp. strain CRL-25, a type I organism; and Methylosinus sp. strain CRL-16, a type II organism (113). The molecular weights of reacting proteins corresponded to those of the purified components (113).
A type A protein has been purified from a soluble extract of an obligate type II methylotroph named SB-I (1). Antiserum raised against the protein cross-reacted with material in crude extracts from Metlhylobacteriiln o-ga,nophillum XX and Methyloinonas sp. strain 761 M, an obligate type I organism (1). All immunological studies cited point to a similarity between the soluble MMOs of what are, in some cases, seemingly very different organisms.
Particulate MMO. A particulate MMO was purified from cell extracts of Methvlosinus trichosporillm OB3b (154). Three components were present: a soluble CO-binding cytochrome c (Mr, 13,000), a copper protein (M,' 47,000), and a small protein (Mt', 9,400). The function of the different components and the manner in which they interact to MICROBIOL. REV. catalyze CH4 oxidation are not clearly understood. Ascorbate and the methanol-methanol dehydrogenase couple could both donate electrons to the system. However, a latereffort to duplicate these results was unsuccessfLul (131). In fact, on this occasion, only NAD(P)H was found to be an effective electron donor. A recent attempt to puLrify particulate MMO from Metlvilosinlus tricIlOSp)orium OB3b failed due to loss of activity of the enzyme upon solubilization (14).
The mechanism of action of the particulate MMOs may be different from that of the soluble MMOs. Joergensen (75) studied the kinetics of 0O and CH4 utilization by whole cells of an unidentified type II methanotroph. Growth conditions were such as to ensure that the only MMO expressed would be particulate. Intracellulalr NADH concentr-ations were apparently always above saturation. In this system, the kinetics are consistent with a random bireactant mechanism.
Either CH4 or Or binds to the enzyme, and this results in a decrease of affinity for the substrate which remains to be bound. The KM,s for 0 and CH4 were 0.14 and 1 F.M. respectively.

Methane Oxidation by Ammonia Oxidizers
Products of CH4 oxidation. Cell suspensions of a variety of ammonia oxidizers incubated in the presence of nanomolar concentrations of 14CH4 produced 14C0n and incorporated 14C into cellular material witb or without NH4 (79). Species and strains tested included Nitroso(coc(us oc()eanus, N. eiirop(ea, and Nitrosomionas marini s as well as a number of marine and freshwater isolates, suggesting that the ability to oxidize CH4 is widespread among the ammonia oxidizers. At the concentration (approximately 10 nM) used to screen their ability to oxidize CH4, none of the ammonia oxidizers tested incorporated more than 17% of the CH4 carbon utilized (79). The effects of CH4 and NH4 'levels on incorporation were examined in N. eCUi op(ie(i and Nitrosococcus oceanus (79). In general, the presence of NH4 ' appeared to decrease the proportion of CH4 carbon entering cell material. This proportion tended to increase with rising CH4 concentrations. The maximum observed value, 63%, occurred in N. eioop(a(i in the absence of NH4 and in the presence of approximately 120 F.M CH4. In the above study, the suspending medium was not examined for the presence of small organic intermediates of CH4 oxidation. However, Hyman and Wood (70) (163). Ward (164) confirmed the production of 14CO, and 14Clabeled particulalte matter from 14CH4 in N. oceanis. These products calso appeared on incubation of the organism with 14CH30H (164). Under the single set of incubation conditions used by Ward, approximately equal amounts of CO, and particulalte carbon were formed with either CH4 or CH,OH as substrate.
The findings of Jones and Morita (79), Hyman and Wood (70), Voysey and Wood (163), and Ward (164) appear to contradict previous reports that ammonia oxidizers cannot metabolize CH4 or CH,OH. In their study of N. eiurop(iel, Hyman and Wood (70) measured an apparent half-saturation constant (K,,,) for CH4 oxidation of 2 mM, leading them to suggest that the concentration of CH4 used by Drozd (33) and Hynes and Knowles (74). 0.1 mM, was too low to allow the detection of oxidation activity. Jones and Morita (79) pointed out that the measurement of 02 uptake as used by Suzuki et al. (148) and Drozd (33) is relatively insensitive when compared with methods in which radiolabeled substrates are used.
The pathway of incorpor-ation of CH4 carbon into cellular material is unknown. HCHO reacts reversibly with proteins and nucleic acids to product additional compounds (117). At the present time, the possibility that at least part of the observed incorporation of CH4 carbon occurs via this abiotic route cannot be dismissed. In N. eiurop(a(e, NH4' oxidation provides energy for-CO, fixation via the Calvin cycle (124).
NH4' stimulaltes H'4C0 uptake in this organism (124). In N. ieiropaicea and Nitrosococcus oceaunus, CH4 alone did not stimulate H14CO3 uptake (79). It should be noted, however, that the maximum concentration of CH4 utilized in these experiments did not exceed 150 F.M.
Attempts to gr-ow ammonia oxidizers on either CH4 or CH;OH have failed (79,163), and 50 mM CH4 did not enhance the survival of NH4'-starved cells of a marine strain of Nitrosomjonas sp. over a 25-week period (83). It is not clear whether ammonia oxidizers derive any net benefit from CH4 oxidation.
Involvement of ammonia monooxygenase. Based on their study of N. europaea, Hyman and Wood proposed that ammonia monooxygenase catalyzes CH4 oxidation to CH,OH (70). They found that CH4 inhibited NH ' oxidation to NO. in cell suspensions with a Ki of 2 mM (Suzuki et al. [148]. in a previous study on cell-free extracts of N. europ)(i(i, had measul-ed a K, of 50 FiM). Furthermore, CH4 alone or with hydrazine, a source of reductant for ammonia monooxygenase (62), stimulated 0 consumption. The stimulation was abolished in the presence of allylthiourea, an inhibitor of the monooxygenase (62). Finally, the stoichiometry of Or consumed to CH3OH produced was consistent with the following reaction: CH4 + 2 [H] + Or > CH3OH + H.O. This is analogous to the first step of NH4+ oxidation in ammonia oxidizers (35,59). Similar evidence has been used to implicate the ammonia monooxygenase of N. eiropac(l in the oxidation of several other organic substrates including bromomethane (71), ethylene (72), benzene (69), and, notably, methanol (163). Other ammonia oxidizers may also utilize their ammonia monooxygenase to catalyze the first step of CH4 oxidation. Ward (164) (62,148,163). The methanol dehydrogenase of conventional methylotrophs is characterized by a pyrroquinolinequinone prosthetic group (5). Efforts to detect the presence of this group in cells ot N. eluropucea were unsuccessful (163). Reaction steps beyond formaldehyde which lead to the formation of formate. cell C. and CO, remain unknown (163,164).
CH4 oxidation rates and affinities. There are few available estimates of CH4 oxidation rates and Iaffinities for ammonia oxidizers (70,79). What limited data exist are summarized in Table 2. The maximum methane oxidation rate for an ammonia oxidizer was still five times lower than the lowest value for a methanotroph. The apparent K,,, ( (68). NH4' , although it is oxidized by whole cells of mecthvlococcus capsulatits Bath (24). does not support CO, fixation in this organism (139).
Involvement of MMO. Cell-free extracts ot Methvlococcis capslllatus Balth containing soluble MMO activity oxidized NH4 'to NOr- (24). During the oxidation, NH.OH accumulated transiently and was the first detectable product of the reaction. On was a requirement for the reaction. The kinetics of NH4' oxidation to NH,OH were complex but conformed to the Michaclis-Menten model when NH4' concentrations fell between 20 and 200 mM. In this range, the calculated K,,, was 87 mM at pH 7. CH4 in all concentrations tested exhibited an inhibitor-y effect on NH4' oxidation. 8 (110). However, in cell suspensions of Methylococcus capsidatlits Bath, the K,,, for NH4+ oxidation increased from pH 7 to 8 and no activity could be observed at pH 9 (24). NH2OH oxidation. In crude extracts of Methvlococcits capsl(ulatis Bath, added NH,OH was readily oxidized to NO.and the rate of reaction was strongly enhanced by the addition of phenazine methosulfate (PMS) (24). The oxidation was not significantly inhibited by the addition of 8hydroxyquinoline or acetylene, agents which blocked the activity of the MMO present in the extracts (see above). Neither did methanol dehydrogenase appear to be responsible for NH,OH oxidation. Cyanide, a powerful inhibitor of methanol dehydrogenase in Methylococcus capsuldatis (24) actually stimulated NH,OH oxidation. The activity was not affected by the addition of CH3OH. The presence of a hydroxylamine oxidase was therefore postulated (24).
The CH3OH dehydrogenase and NH,OH oxidase activities present in soluble extracts of Methvlococcus tlermitioplhi/its, an obligate methane utilizer, were separated by ion-exchange chromatography (136). The NH,OH oxidase which was isolated shares a number of characteristics with the corresponding enzyme in ammonia oxidizers: a PMSstimulated ability to oxidize NH,OH to NO,-, NH,OH: cytochrome c oxidoreductase activity, a pH optimum of 9, and similar absorption spectra in the visible region (136). Upon addition of NH,OH, membrane preparations of Methylococcus ther-mophilius consumed O0, reduced NAD' and cytochrome c, and produced adenosine triphosphate (102). It is not known whether NH,OH oxidation can contribute significantly to the energy budget of methanotrophs which possess this activity. In fact, it remains unclear whether the ability to oxidase NH,OH is widespread among methanotrophs.
NH4' oxidation rates and affinities. The methanotroph's ability to nitrify is clearly not as developed as that of the ammonia oxidizers. The maximum rate of NOJproduction determined for a methanotroph is still more than 20 times lower than one of the lowest rates reported for an ammonia oxidizer ( Table 2). Although there is overlap in the K,,, values obtained in the two groups of organisms, the lowest K,,, (NH4+) for ammonia oxidizers is 300 times lower than the lowest K, for methanotrophs ( Table 2). Considerable interspecies variation in ability to nitrify appears to occur among the methanotrophs (110, 171).

CO Oxidation by Methanotrophs and Ammonia Oxidizers
A number of methanotrophs (39,40,65,143) and ammonia oxidizers (80,81,157) (70). Further evidence is required to clarify the issue. In both methanotrophs and ammonia oxidizers, CO stimulates Or uptake and oxidation of sources of reducing power for the monooxygenases (65,157). Stoichiometries are as expected for reactions catalyzed by mixed-function oxygenases: CO + AH, +-Or--CO, + A + H.O. In some cases (39,40,143,157), CO is oxidized only in the presence of a substrate which can directly, or via its own oxidation, provide reducing power to the monooxygenase.
CO is a powerful inhibitor of CH4 and NH4 ' oxidation. In N. eiuropaice extracts, the Kj for inhibition of NH4' oxidation by CO was measured at 3 FtM (148). NH4+ oxidation in two species of Nitr-osomiionatis and Nitrosococcus oceanits was inhibited by >50% in the presence of 6 F.M CO (82). CO at 100 FtM inhibited CH4 oxidation by 65Cc in Methl/losiniss trichosporim,n OB3b and by 100%t in Methylonolnlas mnethanica (39). The action of CO is apparently not at the level of cytochrome oxidase since the affinity of this enzyme for CO is apparently much less (6,176). NH4 ' at 3.6 mM inhibits CO oxidation in two species of Nitr-osomiionias, and Nitrosococcus oceanlits was inhibited by >50Cc (81). A significant inhibitory effect was seen at NH4 levels as low as 70 [tM (81). NH4 'inhibited CO oxidation by Methvlo4lon(is mllethlanlic(a with a Ki of 12 mM (40).
CO oxidation in ammonia oxidizers is blocked by numerous inhibitors of nitrification (82). When cyanide is added in concentrations low enough not to affect the terminal oxidases, it blocks CO oxidation in Metlivonioonas mnethanica (40).
CO oxidation rates and affinities. Maximum rates of CO oxidation by methanotrophs show considerable overlap with those of ammonia oxidizers ( Table 2). Both groups of organisms can oxidize CO when it is present in concentra-Vol. 53, 1989 76 BEDARD AND KNOWLES tions of <100 nM (84). Methane and ammonia oxidizer-s have K,,,(,,P,,, values for CO oxidation near those for theirrespective growth Substrates (Table 2).

INHIBITORS OF METHANOTROPHS AND AMMONIA OXIDIZERS
Inhibitors of NH,+ Oxidation in the Studv of Nitrification The activity of chemoautotrophic ammonia oxidizer-s is specifically blocked by numel-ouS compounds (62), and a number of these have received considerable attention for their ability to inhibit nitrification in agricultuLral soils (135).
Inhibitors have also been used to estimate nitrifierc activity in the environment (see references below). The method c(an be summarized as follows. A sample of soil, sediment, or wCater is divided equally between two sets of replicates. One set receives inhibitordissolved in a carrier; the other receives an equal amount of carrier alone. The disappearctnce of endogenous NH4' (49,50,54,90) and O. (90) or the appearance of NO- (16,48,49,50,54,90,168,179,180) can then be followed. Alternatively, a Substrate which can be metabolized by chemoautotrophic ammonia oxidizer-s is added and its utilization is monitored. Substrates used have included NH4+ (50, 180), 14CO, (10, 17, 37, 48, 90, 137, 160, 161. 162), 14CO, and 14CH4 (84). Compared with nonradioactive compounds, radiolabeled ones provide greatersensitivity and allow shorter (<24 h) incubation times (84. 152). When '4CO. is used, a ratio of nitrogen oxidized to carbon fixed is requir-ed to estimate rates of nitrification (10). An average ratio obtained from pul-e-cultur-e exper-iments is usually applied. Molar r-atios fall between 5 and 42 in ammonia oxidizers and 25 and 132 in nitrite oxidizers (43,85). Because of these wide variations, the validity of the 14CO, uptake method as a direct meCasuLre of nitrification has been questioned (48. 85,152).
The derivation of estimates ot nitrification lromn "4CO uptake measurements is even mor-e problemnatic. NH X4 depresses CO oxidation in nitrifiers (80. 81). As noted above, this is because both NH4' and CO acre substrates for ammonia monooxygenase (157).
although allylthiourea (49,50) and thioureal (18()) have talso been used. These compounds block the NH4'-to-NH,OH step of ammonia oxidation (15). The mode otf action of nitrification inhibitors is further discussed in the next section. Various problems associated with the use of these compounds in field studies are outlined in the following paragraphs.
Inhibitors may not act immediately upon addition. Powell and Prosser (118) noted a 10-h lag between amendnment with 6-chloropicolinic acid (2 FtM) and slowing of growth in a culture of N. euroopJaa. Although eventually effective, low nitrapyrin concentrations (<0.9 FtM) did not immediately block NH4+ oxidation by various strCains of nitrifiers (7). These problems may be alleviated by using higherlevels of inhibitor (7) or by preincubating samples with inhibitor before substrate addition (160)(161)(162)168).
Not all species and strains of nitrifiers are similarly affected by a given compound. For seven strains belonging to three separate genera of ammonia oxidizers, nitrapyrin concentrations required for complete inhibition ranged from 0.9 to 43 FiM (7). In a study of nine inhibitors, Jones and Morita (82) noted several differences in susceptibility among three species of nitrifiers. For example, 3-aminotriazole in a con-centraCtion of 1.19 mM completely inhibited NH4' oxidation by two species of Nitrosonioast.v but left Nitrosococcits O(ce(1111S unaffected. At this concentration most of the other inhibitor-s tested decrecased activity by >90%. The level of nitrapyrin originally recommended for use in the 14CO2 uptake assay was 22 FiM (10). It WalS Suggested that higher amounts would alffect other microorganisms. lThe extent of inhibition may depend on the substrate added. With many inhibitors, it appears thcat NH4 ' and CH4 oxidation are more affected than is the oxidation of CO (82).
TIhis could be related to the high affinity of ammonia monooxygenase for CO.
Nitrapyrin can be lost through volatilization or degrCadation (125). Both of these processes are probably of little importance in short-ter-m (<24-h) incubLtions or in assays on sealed water samples. No loss in effectiveness was seen in sealed lakewater samples incubated for more than 40 days (90). In sediments, the SituLation appecars to be different. Hall (49) noted relief of inhibition in freshwater sediment after 24 h of incubLation. TIo prevent nitrification from occurring in samples of marine sediment, Henriksen (54) found it necessary to add fresh nitrapyrin at intervals of 1 or 2 days. In the environment. nitrapyrin is hydrolyzed to 6-chloropicolinic acid (12. 125). Although this compound appears to inhibit nitrificattion (118), at equal concentration it is less effective than nitracpyrin (12,92,118). Little is known of the fate of the thioul-eats in the environment.
The influence of Vail-IOuS physicochemical characteristics of soil on the effectiveness of nitrapyrin has been extensively studied (135). Ihe etfects of these pCarameters on the thio-uL-eCas a1re poorly documented. It appears that, at least in some environments. these compounds may be less effective than nitriapyr-in. In puI-e cultures of N. euroop1ea, allylthiourea and nitrapyrin completely block NH4 oxidation at approximately the same concentration; 5 FiM (15). But, when added to soils in a concentration of 10 mg kg l, thioUrea and allylthiourea had no etfect on nitrification while, at an equivalent level, nitr-apyrin significantly inhibited this activity (13).
BecaLuse nitrapyrin is only sparingly soluble in water, it is uSuLlly dissolved in an organic solvent before addition to samples. TIhe carrier-itself mnay have an inhibitory eflect (12).
Ethanol has been used in a nunmberof studies (10,17,48,137,160). In a concentration of 90 mM, it blocks NH44 oxidation by N. europuaea (62). Hall (48) found that 1.2 mM ethanol had no effect on nitrifiers. In most of the studies citcd above, it appears that ethanol was near or below this level. Dimethyl sulfoxide has also been used as a carrier (84,90). When present at 1 ml liter 1, it had no effect on NH4 t, CH4. and CO oxidation by various nitrifiers (82). Potential problems with the carrier are avoided by using allythiourea and thiourea, which are soluble in water.
Studies designed to compare estimates of nitrification obtained with different inhibitors are almost nonexistent. Hall (49) found that measurements of nitrification in freshwater sediments, using allythiourea, were higher than those made with nitrapyrin. The author proposed that volatilization of the latter compound may have been an important MlC ROBIOI.. Rev.
factor. Billen (10) found nitrapyrin to be more satisfactory for determining 14CO, uptake by nitrifiers than either thiourea or hydrazine, although no specific data in support of this were provided. Despite all of the drawbacks listed above, inhibitor studies have yielded estimates of nitrification which agree within an order of magnitude with measurements made by using other approaches (10,17,37,48,90,137). This was the level of precision originally claimed for the method (10).
Since many of the inhibitors used in studies of nitrification also inhibit the oxidation of NH4 ' and CH4 by methanotrophs, as noted above, it is often not possible to conclude unequivocally that observed nitrification is due to the ammonia oxidizers alone. A differential inhibitor would therefore be useful in ecological studies.

Differential Inhibition of Methanotrophs and Ammonia Oxidizers
An inhibitor specific to either methanotrophs or ammonia oxidizers would be useful for distinguishing which of these organisms is responsible for NH4 , CH4, or CO oxidation in environments in which such activities occur (129). As noted above, nitrapyrin, thiourea, and allylthiourea all inhibit methanotrophs. Topp and Knowles (155) suggested that measurements of nitrapyrin-sensitive 14CO, uptake in sampies in which methanotrophs were present might yield overestimates of nitrifier activity. A similar problem exists with nitrapyrin-sensitive '4C0 uptake (84). Table 3 lists 35 agents which have been tested on methanotrophs and nitrifiers. The compounds inhibit one or the other or both types of organisms. Where pure culture data were not available, results from sewage sludge or soil studies are included. With very few exceptions, the inhibitors in Table 3 act on both groups of organisms.
In most of the studies of methanotrophs cited in Table 3, the nature of the monooxygenase, either particulate or soluble, was not monitored. Given what is now known about the inhibitor profiles of the two forms of monooxygenase (26), it appears that in a majority of the organisms investigated it was a particulate enzyme which was responsible for most of the observed methane oxidation. The results obtained on Methvlococc(s (cpslidatits Bath (24, 141) clearly do not fit this pattern. At the time of these studies, the monooxygenase under investigation was known to be soluble. With the possible exception of KCN, the inhibitor profile of Methvlococcus capsilatius Bath obtained by the investigators appears to be that which has now been recognized to be typical of the soluble MMOs (26). Table 3 have more than one effect. For example, KCN blocks both cytochrome oxidase and particulate MMO (75). But most of the compounds listed, when added in low enough concentration, affect mainly the CH4-to-CH3OH or NH4 t-to-NH,OH steps in the sequence of oxidations carried out by methanotrophs and ammonia oxidizers.

Some of the inhibitors in
A number of agents in Table 3 are thought to act by binding a metal (in all likelihood, Cu) which is essential for the activity of the particulate MMOs and ammonia monooxygenase. Such inhibitors would include those listed as "chelating agents" and "monodentate ligands" as well as nitrapyrin and histidine (15,62,66,97,141). Lees (97) examined the effect of eight different chelating agents on the oxidation of NH4' by Nitrosomonias spp. That the slope of the curve relating inhibitor concentration to the percent inhibition was the same for all the compound tested suggested that a common mechanism was involved. Inhibitor strength was closely related to the ability to chelate. Finally, in the cases of allylthiourea, guanidine, and L-histidine, the inhibition was noncompetitive.
Involvement of copper in CH4 and NH4 'oxidation has been deduced in part from the high affinities of inhibiting chelators for this metal (66,97). Hooper and Terry (62) found that, in N. clropaea, the inhibition of NH4 'oxidation caused by KCN or diethyldithiocarbamate could be reversed, at least in part, if cells were treated with a solution containing Cu' This same ion relieved the inhibition of CH4 oxidation by 8-hydroxyquinoline, ox-dipyridyl, and thiosemicarbazide in cells of Melthylosinus trichosporiiim OB3b (66). Cu'+ did not restore NH4 'oxidation in N. europcaea or CH4 oxidation in Metlhlosinus trichosporiiim OB3b when these organisms were exposed to allylthiourea (62,66). This may be because allylthiourea has a higher affinity for the Cu' ion (66). Addition of Cu2+ to cell suspensions of N. europaea treated with nitrapyrin restored the ammonia-oxidizing capability (15). Similar treatment did not relieve the inhibition of CH4 oxidation by nitrapyrin in Methllosinus trichlosporiumi OB3b (156). This suggests that nitrapyrin might function differently in methanotrophs and ammonia oxidizers.
It is possible that pyridine and its derivatives (Table 3) could act by binding key copper atoms, but a more direct interaction with the active site of the monooxygenases is suggested by the following findings. Benzene, an analog of pyridine, is actively oxidized by cell suspensions of N. eur(opCaIe (69). Pyridine is a substrate for soluble MMO (23) and inhibits particulate MMO (66). However, particulate MMO does not oxidize benzene or other aromatic compounds (14). Because of its resemblance to benzene and phenol, aniline, which inhibits NH4 oxidation in N. eiurop(aea (153), may also be a substrate for ammonia monooxygenase.
The target of some of the inhibitors may not be the ammonia monooxygenase or MMO. It has been suggested that, in methanotrophs, chelating agents may interact with an electron transport protein closely associated with the particulate MMO (26). Bhandari and Nicholas (9) proposed that NaN3, diethyldithiocarbamate, thiourea, nitrapyrin, and 2-trichloromethyl pyridine inhibit a copper-dependent translocase in ammonia oxidizers. However, the evidence advanced by these authors is also consistent with direct inhibition of NH4 oxidation (88). Furthermore, CH4 and CO oxidation in ammonia oxidizers is also inhibited by nitrapyrin (82). Since CO and CH4 must enter the cells by passive diffusion, it seems likely that the inhibitors listed above function on the oxidative machinery of ammonia oxidizers, not NH4 ' transport.
C,H, is a suicide substrate for both ammonia monooxygenase (73) and the MMO of Methylococcus capsulatiis (121). This may be the mechanism underlying the action of all acetylenic compounds which block CH4 and NH4 ' oxidation. The cytochrome P-450 monooxygenase of hepatic microsomes is subject to inhibition by a wide variety of acetylenic compounds (111). Here, too, a suicide mechanism is thought to be involved. Certain P-450 inhibitors have been shown to inhibit CH4 and NH4 'oxidation, although high concentrations are required ( Table 3). Inhibition of the growth of MVethvlococcus capsulatus (strain unspecified) by threonine and phenylalanine is believed to be due to end product inhibition of enzymes essential for the synthesis of certain key amino acids (36).  All inhibitor studies on pur-e cultur-es are listed. When suIch data were not available, results from soil exper-iments aire included. The commonly used appellation is given. When applicable, the co-rect terminology is given in pai-entheses. 'S, Solid; L, liquiid, G, gas.
The minimum tested concentration fouind to inhibit methaine/ammonium oxidation by >7(1% in at least one species of methanotr-oph ol-ammonia oxidizer. An inhibitor is juidged to show an effect if the r-ate of oxidation in its presence differ-ed from that in the control without inhibitorby more than 30%. ND. Not Of three ammonia oxidizer species tested, only one, Nitrosococcus oceanuis, showed no inhibition of NH4' CO, or CH4 oxidation in the presence of 1.190) ,uM 3-aminotriazole (82). 'CH4 oxidation was inhibited by 64%. See the discussion concer-ning pyridine in the text. Inhibition of NH4' oxidation in sludge wals approximately 50% (138).
No inhibition of short-term (3-h) NO,-produIction in concentrated cell suspensions of Nitrosoolionas spp. (97). AccLlmtilation of NO,over a 3-day period was inhibited by >70(% in cuIltuires of N. europae amended with amino acid in a concentr-ation of'4 mg liter ' (18 Threonine and lysine inhibit NO-production in growing cultures of N. eliriopueni (18) but the inhibition is, apparently. not immediate (97). Again, end product inhibition is suggested.
Hydrazine blocks NO-production by Nitrosomlonis spp. by acting as an alternative substrate for hydroxylamine oxidoreductase (58). The oxidation of hydrazine can actually stimulate the action of ammonia monooxygenrase by providing it with reducing power (70,174).
In high enough concentration, hydroxylamine inhibits its own production in cells of N. eiuropuiea (2). Hyman and Wood (70) proposed that this inhibition serves a regulatory purpose by helping to prevent accumulation of' NH.OH in cells. Salvas and Taylor (129) showed that picolinic acid in a concentration of 10 p.M blocks NH4' oxidation by Met/hylosiltiis tricliosporiumn OB3b but not in two species of marine ammonia oxidizers. Table 3 suggests that other compounds may selectively inhibit one or the other group of organisms. More specifically, the possible use of certain acetylenic compounds and pyridine derivatives should be examined.

CONCLUDING REMARKS
The question of the ecological roles of the methanotrophs and nitrifiers in the oxidation of CH4. NH4 and CO in nature remains perplexing. It will be resolved only by detailed physiological and biochemical studies of the mechanisms involved, coupled with the use of the more sophisticated ecological approaches. It is hoped that this review will provide some incentive for such studies.