Physiology, Biochemistry, and Specific Inhibitors of ... · biochemistry and physiology of...

MICROBIOl OGICAL REVIFWS. Mar. 1989. p. 68-840146-0749/89/010068-17$02.00/()Copyright < 1989, American Society foI Microbiology

Physiology, Biochemistry, and Specific Inhibitors of CH4, NH4+,and CO Oxidation by Methanotrophs and Nitrifiers

CHARLES BEDARD-;- AND ROGER KNOWLES

Departmient of MicErobiology, Macdonald College of McGill Uni sve iit!, .Ste-An,ine-de-Belletiwe, Quebec, Cana(la H9X I CO

INTRODUCTION ....................................................................... 68

OXIDATION OF CH4, NH4+, AND CO BY METHANOTROPHS AND NITRIFIERS .........................69

Similarities between Methanotrophs and Ammonia Oxidizers ........................................................69

Ammonia Monooxygenase ....................................................................... 70

Source of reducing power ....................................................................... 70

Possible role of copper ....................................................................... 71

....................................................................... 71

Role of copper ....................................................................... 71

Source of reductant ....................................................................... 72

Substrates ....................................................................... 72

Inhibitors ....................................................................... 72

Regulation ....................................................................... 72

Soluble MMO ....................................................................... 72

Particulate MMO ....................................................................... 72

Methane Oxidation by Ammonia Oxidizers ....................................................................... 73

Products of CH4 oxidation ....................................................................... 73

Involvement of ammonia monooxygenase ....................................................................... 73

CH4 oxidation rates and affinities ....................................................................... 74

Ammonia Oxidation by Methanotrophs ....................................................................... 74

Involvement of MMO ....................................................................... 74

NH2OH oxidation ....................................................................... 75

NH4+ oxidation rates and affinities ....................................................................... 75

CO Oxidation by Methanotrophs and Ammonia Oxidizers ........................................................... 75

Involvement of methane and ammonia monoxygenases .............................................................75

CO oxidation rates and affinities ....................................................................... 75

INHIBITORS OF METHANOTROPHS AND AMMONIA OXIDIZERS ............................................ 76

Inhibitors of NH4+Oxidation in the Study of Nitrification ............................................................76

Differential Inhibition of Methanotrophs and Ammonia Oxidizers .................................................. 77

CONCLUDING REMARKS ....................................................................... 80

ACKNOWLEDGMENTS...80...............80LITERATURE CITED.................. 80

INTRODUCTION

An objective of this review is to summarize aspects of thebiochemistry and physiology of methanotrophs and ammo-

nia oxidizers which are relevant to the oxidizing activities ofthese organisms in their natural habitats. Ecological aspectshave been extensively reviewed elsewhere (51. 128, 130). as

have general aspects of methanotrophs (4. 5. 25, 55) andammonia oxidizers (61, 174). Pa rticular attention is given to

the ammonia monooxygenase of ammonia oxidizers and themethane monooxygenase of methanotrophs since these are

both key enzymes in the metabolism of CH4. NH4 , andCo.Many methanotrophs (family MetlhYloe oe(aee(eae) and

chemoautotrophic ammonia oxidizers (family Nitroha(ter-aceae) have been found to carry out the following reactions(25, 39, 61, 70, 110, 157):

CH4 + Or + AH -> CH-OH + H.O + A (1)

* Corresponding author.t Present address: National Research Council Canada. Biotech-

nology Research Institute, Montreal, Quebec, Canada H4P 2R2.

CO + )O + AH. CO.+ H,O A (2)

NH3 4-OA + AH-- NHOH + H.O + A (3)

T'he immediate source of reductant (AH.) can be reducednicotinamide adenine dinucleotide (phosphate) [NAD(P)H]in methanotr-ophs, but in ammonia oxidizeirs it may be a

cytochroome c (25. 157). All of the above oxidations are

pparently catalyzed by monooxygenase enzymes: methanemonooxygenase in methanotrophs and ammonia monooxy-

genase in ammonia oxidizei-s (25, 61). Only CH4 can supportgrowth in the former organisms. and only NH. can supportgrowth in the latter (39. 79. 80, 139). In both methanotrophsand ammonia oxidizers, there are repor-ts that CH,OH is

metabolized to CO, and cell C (25, 79, 164). While thepathway of CH,OH oxidation is well characterized in meth-anotrophs, in ammonia oxidizers it remains incompletelyunderstood (148, 149). In addition, during the production ofNO. from NH4' by both methanotrophs and ammoniaoxidizers, small amounts of N,O are evolved (174, 177, 178).Although there is evidence that, in certain environments,methanotrophs and armmonia oxidizers may carry out oxida-tion of the growth substrate of the other as well as CO, few,if any, attempts have been made to measure directly the

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NITRIFICATION BY METHANOTROPHS AND NITRIFIERS 69

Solublemethane Assimilated Into

cellular biornass

Methadshydrog

4 ,Cu H3OH

HOO

2 Particulate 2°methane e - J

monooxygenase

inolgenase

X HCHO

PQQH 2

HO2 Formaldehyde Formate

I.,derrydrogenase dehydrogenasef HCOOH C 2

NAD NADH 2 NAD NADH2

Ammonia oxidatlon

2 Ammonia 2 HroxylamInemonooxygenase ox idoreductase

NHJ -< NH OH so NO2N3 2 NO

** **

XH 2 X X XH 2

FIG. 1. Pathway of methane oxidation in methanotrophs and ammonia oxidation in ammonia oxidizers (after references 25 and 34). * PQQ,Pyrroquinoline quinone. **X and XH. are the oxidized and reduced forms of ain unknown electron donor.

relative contributions of these two kinds of organisms to insitu CO, CH4, and NH4+ metabolism.NOJ- peaks located at the boundary between anoxic and

oxygenated waters are a common feature of moderatelyproductive lakes (67). Several different processes, includingnitrification and denitrification, could lead to the formationof such peaks (91). In the metalimnion of Lake Mendota(Wisconsin), an NO,- maximum was found to occur at thedepths where CH4 oxidation was most pronounced, prompt-ing the suggestion that methanotrophs might be responsiblefor NO,- accumulation in this environment (51, 52). Analternative explanation (79) is that chemoautotrophic ammo-nia oxidizers were oxidizing CH4 in addition to NH4 + .Recently, it has been suggested that methanotrophs could beinvolved in the process of nitrification in lakes under winterice (90). Methanotrophs may also be responsible for some ofthe nitrification observed in soils (130, 159). S. R. Megrawand R. Knowles (unpublished data) have obtained strongevidence that, under certain conditions, essentially all of theNH4' oxidation measured in a particular humisol is carriedout by methanotrophs.

Biological oxidation is an important sink in the worldwidecycle of CO (104). Carboxydobacteria are aerobic organismsthat can use this compound as sole source of carbon andenergy (105). Most of the carboxydobacteria known at thepresent time exhibit half-saturation constants for CO oxida-tion which are considerably higher in value than naturallyoccurring CO concentrations. For this reason, it has beensuggested that these organisms may not play a significantrole in CO cycling. On the other hand, both methanotrophsand ammonia oxidizers exhibit high affinities for the com-pound, which has led some investigators to speculate aboutthe importance of these organisms in the environmentalmetabolism of CO (27, 80).The participation of methanotrophs and nitrifiers in the

atmospheric cycles of CH4, N2O, and CO can have increas-ingly important implications for global warming, due to theinfrared absorption of CH4 and N2O (31), and for strato-spheric chemistry (including ozone) since all three gases

participate in vital reactions (78). An increased understand-ing of the relative roles of methanotrophs and nitrifiers insuch processes is therefore desirable.To determine the nature of the organisms responsible for

an oxidation in an environment, one might use an inhibitorwhich selectively blocks only methanotrophs or only ammo-nia oxidizers. Several inhibitors which act upon ammoniaoxidizers are used to delay nitrification in agricultural soils(135). Nitrification blockers have also have been used toquantify nitrification in aquatic environments (e.g., see ref-erence 137). However, it has been found that almost allagents that inhibit ammonia oxidizers also inhibit methan-otrophs (129, 155, 156). This raises the possibility that incertain environmental studies activity attributed to ammoniaoxidizers was in fact due to methanotrophs. We conclude byreviewing our present knowledge of inhibitors of theseorganisms and examine the limitations associated with theuse of nitrification blockers in ecological studies.

OXIDATION OF CH4, NH4+, AND CO BYMETHANOTROPHS AND NITRIFIERS

Similarities between Methanotrophs and Ammonia OxidizersMethanotrophs (family Methvlococcaceae) can obtain all

of their carbon and energy from CH4 under aerobic condi-tions (170). The pathway of methane oxidation is outlined inFig. 1. Obligate methanotrophs grow only on CH4 andCH3OH. In addition to these substrates, facultative metha-notrophs can also grow on multicarbon compounds. Meth-ane oxidizers are grouped into two types based in part ontheir pathways of carbon assimilation (22). Type I methan-otrophs fix formaldehyde by the ribulose monophosphatepathway, while type II organisms use the serine pathway toassimilate carbon dioxide and formaldehyde (4).Ammonia oxidizers are chemoautotrophs, members of the

family Nitrobacteraceae (165). They oxidize NH4', whichprovides energy for the fixation of CO, via the Calvin cycle(167). The pathway of NH. oxidation is summarized in Fig.1.

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70 BEDARD AND KNOWLES

TABLE 1. Oxidations carried out by wvhole cells of bothmethanotrophs and ammonia oxidizers

SuLbstrate ProdUct Refef cnces

Methane Methanol 70. 169Ammonia Nitrite 164. 170Carbon monoxide Carbon dioxide 65. 80Methanol Formaldehyde 163. 169Bromomethane Formaldehyde 71. 143Ethylene Ethylene oxide 29. 72Propylene Propylene oxide 34. 56Cyclohexane Cyclohexanol 34. 56Benzene Phenol 34. 56Phenol Hydroquinone 56. 69

In addition to their usual source ot energy, both methan-otrophs 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 tothose mentioned in Table 1 (47). The multiplicity of oxida-tions carried out by methanotrophs and ammonia oxidizersreflects the wide substrate specificities of the methane mono-

oxygenase of the former organisms and the ammonia mono-

oxygenase of the latter (34, 56, 70, 71).Other similarities between the two groups of organisms

can be summarized as follows. (i) An NH.OH oxidaseresembling that in ammonia oxidizers, and distinct frommethanol dehydrogenase, may be present in at least some

methanotrophs (136). (ii) A complex array of intracytoplas-mic membranes is present in both types of organisms (28,107). (iii) Ammonia oxidizers and type I methanotrophs havepredominantly 16:0 and 16:1 fatty acids (11, 100, 109). (iv)The type I methanotrophs possess a tricarboxylic acid cyclewhich lacks (x-ketoglutarate dehydrogenase (169). (v) Bothgroups 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 methan-

otrophs and ammonia oxidizers has often been mentioned(86, 123, 169), and recent 5S and 16S ribosomal ribonucleicacid studies show the ammonia oxidizers in groups P-3 andy-l and a type I methanotroph in the y-I group of the purplebacteria (172, 173). However, the elucidation of the relation-ship between methanotrophs and ammonia oxidizers may berendered more difficult by recent discoveries which suggestthat cross-species gene transfer contributes in a significantway to species evolution (150).The present review concentrates on CH4 oxidation by

ammonia oxidizers, NH4 ' oxidation by methanotrophs, andCO oxidation by both groups of organisms: activities which.as indicated earlier, may be of importance in the naturalenvironment. First, the characteristics of the respectivemonooxygenases will be discussed.

Ammonia Monooxygenase

Although the enzyme involved has yet to be purified, itappears likely that the first step of NH4'oxidation in

Nitrosoinonas eiUlop(ae( is a monooxygenase-catalyzed ox-

idation of NH3 to NH,OH (35, 59). Evidence that NH3rather 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,,1values for the oxidation decreased markedly with increasingpH. However, the va_lues remalined essentially constantwhen they were recalculated in terms of unprotoncated NH,instead of total armmonia (NH, plus NH4 '). NH.OH is aprobable intermediate in NHX oxidation (58, 59, 96, 108). Itis oxidized to NO. by whole cells and cell extracts of N.europaea and accumulcates during NH4' oxidation in thepresence of hydrazine, a powerful inhibitor of NH,OHoxidation (58. 59, 96. 108). The inhibitor sensitivities ofNH4' and NH.OH oxidation care very different (58. 62) seeTable 3. Furthermore. NH.OH is the substrate of an oxido-reductaIse which hCas been isolated from N. eropae(l(l andextensively purified and characterized (for reviews, seereferences 32 and 61).

18O from SO, was incorporated into NO.- during NH4oxidation by N. europaea (126). Cell suspensions of thisorganism containing added hydriazine were used in isotopicstudies of the NH,-to-NH,OH reaction step (35, 59): theoxygenase nature of the reaction mechanism was demon-strated with 8(O, H,'8O, and iSNH4Cl. The oxygenase islikely to be of the mixed-function (monooxygenase) type(59): i.e., it inserts one 0 atom from dioxygen into ammoniawhile reducing the other to H,O: NH3- + AH. + O.NH,OH + A + H.O.The immediate source of reductant "'A' has not been

identified with any certcainty (61; see discussion below). rheability of ammonia oxidizers to oxidize small organic mole-cules provides further evidence for a monooxygenase mech-anism (see section on CH4 oxidation by ammonia oxidizers).

Cell extracts which oxidize NH4' have been preparedfrom N. eiir-opti(wa (146, 149) and Nitrosococcius oceanS(166). In the former species, bovine serum albumin, Mg-+,or spermine in the presence of adequate concentrations ofphosphate or other anions was required for activity (146,149). In Nitrosococcuts occonus, tris (hydroxymethyl) ami-nomethane, adenenosine triphosphate, phosphate, andMg" were requii-ements (166). In a cell-free extract of N.ceuroptiea, ammonia monooxygenase activity was associatedwith the membrane fraction (63).

Source of reducing power. Reducing power for the mono-oxygenase is apparently regenerfated via NH,OH oxidation(145). Close coupling of NH.OH and NH4+ oxidation hasbeen found in resting cells (60), spheroplasts (145), andcell-free extracts of N. citropuae (63). The ammonia mono-oxygenase of N. europaea appears not to utilize NADH as adirect electron donor (148. 149). However, NADH caninitiate NH4 oxidation in cell-fr-ee extracts (148, 149), and itmay do this by reducing cytochromes which are required forthe reaction (149). Ascorbate is also ineffective as a source ofreductant (148). A likelier candidate for the role may becytochrome (554 (157). When added to a membrane fractionobtained from a cell-free extract of N. eiropaea containingNH4' and NH.OH oxidizing activities, as well as cy-tochrome oxidase, cytochrome (5 4. reconstituted NH4 -linked Or uptake (147). In this system, Tsang and Suzuki(157) found that NH4 ', NH,OH, and NH,NH, (a substratefor NH,OH oxidoreductase) all reduce the cytochrome.Reduction by NH4t occurs at a slower rate than reductionby NH.OH and NH.NHH, and there is a lag betweenaddition of NH4' and reduction of the cytochrome. Reducedcytochrome (554 in the presence of membrane fraction isimmediately oxidized upon addition of NH4 or CO. Theextent of the oxidation is dependent on the concentration ofNH4+ or CO. These observations are consistent with a roleas electron donor for cytochrome (554 (157). The cytochrome

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NITRIFICATION BY METHANOTROPHS AND NITRIFlERS 71

('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, whichcontains four type c hemes and is located in the periplasmicspace (3, 32). Although it may bind substrate (e.g., O), itsproperties as determined by optical, Mossbauer, and elec-tron paramagnetic resonance spectroscopies are more sug-

gestive of an electron transfer role (3).Tri- and tetramethylhydroquinone, two artificial electron

donors, can support the activity of ammonia monooxygen-ase in whole cells of N. eurzopaea (133). These compoundsmay 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 electropho-resis and autoradiography of proteins obtained from cells ofN. euroopaea which had been incubated in the presence of14C H, (73). CGH, appears to be a suicide substrate for theammonia monooxygenase (73), and it is suggested that theattempted oxidation of C.H. by the monooxygenase pro-duces 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 40years ago by Lees (95). The presence of copper in ammoniamonooxygenase is suggested by the ability of agents whichbind the metal to block NH4 'oxidation in N. ciir-opaeai (62,97). Furthermore, NH4 'oxidation is stimulated by lowlevels of copper (99, 153), although an absolute requirementfor the metal has never been demonstrated. Finally, it hasbeen suggested that ammonia monooxygenase possesses a

photosensitive oxygenated state similar to that of othercopper-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 ultravi-olet light leads to inactivation of NH4t but not NH.OHoxidation. Anaerobiosis, copper-chelating agents, and or-

ganic substrates of the monooxygenase tend to mitigate theeffect. Difference absorption spectrophotometry of cell sus-

pensions of Nitrosoinonas spp. shows that, upon exposureto ultraviolet light, there is a bleaching in the 350- to 400-nmregion. Two copper proteins, tyrosinase and hemocyanin,have similar spectroscopic characteristics. They are alsosensitive to a number of chelating agents which also inhibitammonia monooxygenase. Based on these findings, Shearsand Wood have proposed a catalytic cycle for the monoox-

ygenase (Fig. 2) modeled on the cycle known from tyrosi-nase (98). Two copper atoms are hypothesized to be at theactive site as in tyrosinase. The ammonia monooxygenasecould be present in one of three states: reduced (deoxy),oxygenated (oxy), and oxidized (met). 02 reacts reversiblywith 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 byultraviolet light at a wavelength of 345 nm. This type ofillumination causes the bond between copper and 022-- tobreak, and release of O°- can readily occur. Organic sub-strates protect the enzyme from inactivation by rapidlyshifting it from the oxy to the met state. Oxy monooxygen-ase inserts one atom of oxygen into the substrate while theother 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 monooxygen-ase by keeping it in this ultraviolet-insensitive form. Thio-urea has a high affinity for Cu(I).

2e- ffi ROH, H202+2+

RH, 2H +

+ + Cu -Cu2

DEOXY Cut-Cu 2-/

+thiourea U. V. light

(Cu ~- Cu )- thiourea Inactiveenzyme

°2FIG. 2. Proposed catalytic cycle for ammonia monooxygenase,

involving oxidation and reduction of a binuclear copper site. (Mod-ified from reference 172 with permission.) U.V., Ultraviolet.

MMO

The first step of CH4 oxidation is: CH4 + 02 + NADH,-CH3OH + H,O + NAD4 The catalyst for the reaction ismethane monooxygenase (MMO) (25). In cells of Methy-loinonas fiCnethanfico and Metlhvlononas filethaifo-oxid(ilns,'xO from added 18O but not H,18O was inserted into CH4,yielding CH318OH, demonstrating the monooxygenase na-ture of the reactions (57).

This initial step of CH4 oxidation has been most investi-gated in two species of methanotrophs: Methylococcus(Yapsiilatus Bath and Methylosinits trichospormiun OB3b.Both of these organisms produce a membrane-bound ("par-ticulate") MMO and a cytoplasmic ("soluble") MMO. Be-cause only the soluble enzymes of the two organisms havebeen extensively purified and characterized, it is not entirelyclear whether soluble and particulate forms are distinct orwhether they share components (26, 41). In the case ofMethy1lo(occl.us (eapsulaitis Bath, the former possibility ap-pears likeliest (45). At least one other species, Methlylobac-teriuiiml sp. strain CRL-26, can apparently express both asoluble and a particulate enzyme (115, 116). Methvlomnonas(l/bits BG8 and Metlivlocystis parvits may possess only aparticulate form (25). Dalton and Leak (25) suggested thatsoluble MMOs are restricted to only a few species ofmethanotrophs. There are no reports of organisms possess-ing this type of enzyme alone.

Role of copper. Copper may exert considerable influenceon the nature of the monooxygenase expressed and on cellmorphology (14, 26, 122). In both Methvlo(o Cllus ( apSi(lttSBath and Methvlosinits tierihosporilon OB3b when Cu ispresent in limiting amounts, methane oxidation is carried outmostly by the soluble MMO. When Cu is present in nonlim-iting quantities, it is the particulate MMO which dominates(14, 26). Cu(I) and Cu(II) inactivate the soluble enzyme ofMethvyl0o(o(( l(s ( apsultitiis Bath (44). Copper appears to be arequirement for particulate MMO activity in both this spe-cies (122) and MethYloba(cteriium sp. strain CRL-26 (115).Increasing Cu(II) availability appears to lead to increasedcontent of intracytoplasmic membranes in cells of Methy-losinuts 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

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copper supply increases, polypeptide bands correspondingto soluble MMO disappear while other bands possibly re-lated to particulate MMO increase in intensity (122). Asimilar pattern was noted in Methylosinius triclhosporiuinOB3b (14).

Source of reductant. In an extensive review of the evi-dence, Anthony (5) concluded that, at least in cell-freesystems, all forms of MMO can utilize NAD(P)H as electrondonor. It has been suggested that, in a variety of methan-otrophs, electrons from the oxidation of CH,OH by CH,OHdehydrogenase can be recycled directly to be particulateMMO 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 ofcells expressing the particulate MMO when compar-ed withthat of cells expressing the soluble enzyme (94).

Substrates. In Metlhylococcus capsiilatus and Metliilosi-nits trichosporium, the particulate MMO appears to have anarrower range of substrates than the soluble MMO. Bothenzymes can oxidize alkenes and alkanes up to pentane (14,26, 140). But, unlike soluble MMO, particulate MMO cannotoxidize aromatic and alicyclic compounds or higher (n > 5)alkanes (14, 140). These differences could be due to dissim-ilar active sites or to disruption of the activity of theparticulate MMO (26). It is interesting that ammonia mono-oxygenase differs from particulate MMO in that it canoxidize certain aromatic and alicyclic compounds (34, 69).

Inhibitors. The MMOs of Mcthlylococcls (apsIlatits andMethvlosinus trichosporilmtn share similar inhibitor profiles(26, 131, 141, 142). The particulate MMOs are inhibited by awide variety of compounds including thiol and metal chela-tors and electron transport inhibitors (26, 131). The solubleMMOs are sensitive only to acetylenic compounds and8-hydroxyquinoline (141, 142). C.H, inhibits both forms ofthe enzyme and apparently acts as a suicide substrate (121),thus resembling ammonia monooxygenase as mentionedearlier. Because of its inhibitor profile and membrane loca-tion, ammonia monooxygenase is thought to more closelyresemble the particulate MMO (70).

Regulation. The mechanisms of regulation of synthesis ofthe enzymes are not yet clear. The soluble MMO of Metli-vlococcus capsuluatis may be induced only in the presence ofCH4 since its activity could not be detected in cells grown onCH3OH (122). When the organism was grown on CH,OH,particulate MMO activity was always present, whether ornot copper had been added to the growth medium (122).Increasing copper concentrations led to increased particu-late enzyme activity (122).

Soluble MMO. The soluble MMO of Methylococcus(cp-siultlts has been purified and extensively characterized (19,20, 44, 45, 175). The picture that emerges from these studiesis briefly outlined below.

Soluble MMO consists of three components, labeled A, B,and C. Component A is a nonheme iron protein of Mr210,000 which binds CH4. Recent spectroscopic evidenceindicates the presence of a binuclear Fe center in Mceti lo-coccus capsulatus (40). Evidence regarding the presence ofa wt-oxo bridge associated with the Fe cluster in this andother organisms is conflicting (38, 42, 87, 119). Protein Aapparently uses reducing equivalents obtained from compo-nent C to activate 02, allowing insertion of an oxygen atominto CH4.Component B is a low-molecular-weight (Mr, 15,700)

protein which lacks any prosthetic groups. It converts theMMO 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 ofNAD. In the presence of protein B, NADH oxidationbecomes tightly coupled to CH4 oxidation. The oxidaseactivity is switched off.Component C is an iron-sulfur flavoprotein of M, 42,000. It

possesses a single polypeptide chain, one flavin adeninedinucleotide molecule, and an Fe,S, center. It is the reduc-tase component of the MMO, funnelling electrons fromNADH to component A.

Based on steady-state kinetic analyses, Green and Dalton(45) have proposed a mechanism of action for the solubleMMO of Metliylococcus (cpsIlatusI. CH4 binds to the mono-oxygenase, followed by NADH which reduces the enzyme.NAD' is released, and the reduced enzyme combines with0, to form a ternary complex which breaks down with therelease of CH3OH and HRO (a 'Bi Uni Uni Bi Ping Pong"mechanism). The K,,, values for CH4, NADH, and 02 were3, 55.8, and 16.8 [tM, respectively.

Early efforts to purify the soluble MMO from Methylosi-nus trichlosporium failed due to the instability of enzymepreparations (142). However, the ability of purified compo-nents B and C from the soluble MMO of Methvlococcus(apsilatius to restore MMO activity to a fraction obtainedfrom a soluble extract of Metliylosinus trichlosporium em-phasizes the similarity between the enzymes of the twoorganisms. A binuclear Fe cluster is also reported for proteinA 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 MethvlococcuscapsIlatius. Component B of Methylohacteriumn sp. was notrequired for MMO activity as measured by the oxidation ofpropylene to propylene oxide (113. 115). Component A ofthis organism is an iron protein similar in molecular weight tocomponent A of Metlivllococcus (apsIlaItius (113), and it alsocontains a binuclear Fe-Fe cluster (119). Component C ofMetlhilobacterium sp. is a flavoprotein which is reduced byNADH and the redox properties of which, as determined byoptical and electron spin resonance spectra, are very similarto those of the corresponding protein in Methylococcus(apsldatlis (120). Components A and C of Methlilobacteri,mnsp. catalyze the epoxidation of propylene (113). An immu-noglobulin fraction of antisera raised against these twopurified components cross-reacted with certain proteins sep-arated by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis from crude extracts of Mcthylobacterilm O/oIga-iopliliun XX, a facultative type II organism; MethVlococcussp. strain CRL-25, a type I organism; and Methylosinus sp.strain CRL-16, a type II organism (113). The molecularweights of reacting proteins corresponded to those of thepurified components (113).A type A protein has been purified from a soluble extract

of an obligate type II methylotroph named SB-I (1). Antise-rum raised against the protein cross-reacted with material incrude extracts from Metlhylobacteriiln o-ga,nophillum XXand Methyloinonas sp. strain 761 M, an obligate type Iorganism (1). All immunological studies cited point to asimilarity between the soluble MMOs of what are, in somecases, seemingly very different organisms.

Particulate MMO. A particulate MMO was purified fromcell extracts of Methvlosinus trichosporillm OB3b (154).Three components were present: a soluble CO-binding cy-tochrome c (Mr, 13,000), a copper protein (M,' 47,000), anda small protein (Mt', 9,400). The function of the differentcomponents and the manner in which they interact to

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NITRIFICATION 13Y METHANOTROPHS ANt) NITRIFIERS 73

catalyze CH4 oxidation are not clearly understood. Ascor-bate and the methanol-methanol dehydrogenase couplecould both donate electrons to the system. However, a later-effort to duplicate these results was unsuccessfLul (131). Infact, on this occasion, only NAD(P)H was found to be aneffective electron donor. A recent attempt to puLrify particu-late MMO from Metlvilosinlus tricIlOSp)orium OB3b faileddue 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 cellsof an unidentified type II methanotroph. Growth conditionswere such as to ensure that the only MMO expressed wouldbe particulate. Intracellulalr NADH concentr-ations wereapparently always above saturation. In this system, thekinetics are consistent with a random bireactant mechanism.Either CH4 or Or binds to the enzyme, and this results in adecrease of affinity for the substrate which remains to bebound. 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 ofammonia oxidizers incubated in the presence of nanomolarconcentrations of 14CH4 produced 14C0n and incorporated14C into cellular material witb or without NH4 (79). Speciesand strains tested included Nitroso(coc(us oc()eanus, N. eii-rop(ea, and Nitrosomionas marini s as well as a number ofmarine and freshwater isolates, suggesting that the ability tooxidize CH4 is widespread among the ammonia oxidizers. Atthe concentration (approximately 10 nM) used to screentheir ability to oxidize CH4, none of the ammonia oxidizerstested incorporated more than 17% of the CH4 carbonutilized (79). The effects of CH4 and NH4 'levels on incor-poration were examined in N. eCUi op(ie(i and Nitrosococcusoceanus (79). In general, the presence of NH4 ' appeared todecrease the proportion of CH4 carbon entering cell mate-rial. This proportion tended to increase with rising CH4concentrations. The maximum observed value, 63%, oc-curred in N. eioop(a(i in the absence of NH4 and in thepresence of approximately 120 F.M CH4.

In the above study, the suspending medium was notexamined for the presence of small organic intermediates ofCH4 oxidation. However, Hyman and Wood (70) uLsedgas-liquid chromatography to monitor the accumulation ofCH3OH in the supernatant of cell suspensions of N. eiuro-porn incubated with CH4 in concentrations of 200 F.M andmore. Levels of CH,OH as high as 500 F.M were measur-ed(70). Voysey and Wood (163) used '3C nuclear magneticresonance spectroscopy to follow the fate of 13CH,OHadded to cell suspensions of N. europs(ae. In the superna-tant, the concentration of CH3OH decreased continuouslyover the period of incubation, while initially, both HCHOand HCOOH increased, the latter at a slower rate than theformer. After several hours, HCHO levels began to decreasewhile HCOOH continued to increase and did so over theduration of the experiment (30 h). Experimental conditionswere such that CO, release could not be adequately assessed(163). The distribution of intracellular 13C was not moni-tored.HCHO accumulation during CH,OH oxidation may have

important consequences since exogenously supplied HCHOstrongly inhibits NO- production in N. europucea (163).HCHO can react with NH,OH, an intermediate in NH4+oxidation, to form formaldoxime: HCHO + NH.OH >

H,C=NOH + HFO. Formaldoxime is a powerful inhibitor ofNH,OH oxidation in N. europa(ice and may be a substrate forNH.OH oxidoreductase (163). Cell suspensions of this or-ganism incubated in the presence ot [13C]-formaldoximeproduLced ['3C]formate, albeit at slow rates (163). The pro-duction of formaldoxime within cells has, however, neverbeen demonstrated (163).Ward (164) confirmed the production of 14CO, and 14C-

labeled particulalte matter from 14CH4 in N. oceanis. Theseproducts calso appeared on incubation of the organism with14CH30H (164). Under the single set of incubation condi-tions used by Ward, approximately equal amounts of CO,and particulalte carbon were formed with either CH4 orCH,OH as substrate.The findings of Jones and Morita (79), Hyman and Wood

(70), Voysey and Wood (163), and Ward (164) appear tocontradict previous reports that ammonia oxidizers cannotmetabolize CH4 or CH,OH. In their study of N. eiurop(iel,Hyman and Wood (70) measured an apparent half-saturationconstant (K,,,) for CH4 oxidation of 2 mM, leading them tosuggest that the concentration of CH4 used by Drozd (33)and Hynes and Knowles (74). 0.1 mM, was too low to allowthe detection of oxidation activity. Jones and Morita (79)pointed out that the measurement of 02 uptake as used bySuzuki et al. (148) and Drozd (33) is relatively insensitivewhen compared with methods in which radiolabeled sub-strates are used.The pathway of incorpor-ation of CH4 carbon into cellular

material is unknown. HCHO reacts reversibly with proteinsand nucleic acids to product additional compounds (117). Atthe present time, the possibility that at least part of theobserved incorporation of CH4 carbon occurs via this abioticroute cannot be dismissed. In N. eiurop(a(e, NH4' oxidationprovides energy for- CO, fixation via the Calvin cycle (124).NH4' stimulaltes H'4C0 uptake in this organism (124). InN. ieiropaicea and Nitrosococcus oceaunus, CH4 alone did notstimulate H14CO3 uptake (79). It should be noted, how-ever, that the maximum concentration of CH4 utilized inthese experiments did not exceed 150 F.M.

Attempts to gr-ow ammonia oxidizers on either CH4 orCH;OH have failed (79, 163), and 50 mM CH4 did notenhance the survival of NH4'-starved cells of a marinestrain of Nitrosomjonas sp. over a 25-week period (83). It isnot clear whether ammonia oxidizers derive any net benefitfrom CH4 oxidation.

Involvement of ammonia monooxygenase. Based on theirstudy of N. europaea, Hyman and Wood proposed thatammonia monooxygenase catalyzes CH4 oxidation toCH,OH (70). They found that CH4 inhibited NH ' oxidationto NO. in cell suspensions with a Ki of 2 mM (Suzuki et al.[148]. in a previous study on cell-free extracts of N. euro-p)(i(i, had measul-ed a K, of 50 FiM). Furthermore, CH4 aloneor with hydrazine, a source of reductant for ammoniamonooxygenase (62), stimulated 0 consumption. The stim-ulation was abolished in the presence of allylthiourea, aninhibitor of the monooxygenase (62). Finally, the stoichiom-etry of Or consumed to CH3OH produced was consistentwith the following reaction: CH4 + 2 [H] + Or > CH3OH +H.O. This is analogous to the first step of NH4+ oxidation inammonia oxidizers (35, 59). Similar evidence has been usedto implicate the ammonia monooxygenase of N. eiropac(l inthe oxidation of several other organic substrates includingbromomethane (71), ethylene (72), benzene (69), and, nota-bly, methanol (163). Other ammonia oxidizers may alsoutilize their ammonia monooxygenase to catalyze the firststep of CH4 oxidation. Ward (164) estimated a Ki of 6.6 p.M

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'IABLE 2. Kinetic characteristics of CH4, NIH-I, and CO oxidation by methatnotrophs and ammonia oxidizers"

Methianotr-ophs (references) Ammonia oxidizci-s (references)

Conipouiid MaximnUm oxidation rate (nimol of' Maximum oxidtationi r-atc (nmimolC or N g ol cells h ) A,,, (a-inp (PM) oft C or N g of cells - h ) K,,, ;app.) (PM)

Methane 10-31 (8. 53. 134)" 1-66 (77) 0.065-1.960) (70. 79) 6.6-2,000 (70, 163)Ammonium 0.03-1.05 (110) 600-87,00) (24, 11)) 24-62 (112, 130) 2-2,001) (85)

10" 0.((1-21"Carbon monoxide 0.27-7.74 (39) 2.7 (40) 0.015-1.95 (8() 0.1-14 (80. 157)

In most cases an interspecics range in valuICs is giVCe.Callculated fl-om yield and minimuIm doubling time.Calculated by using an average icld of(o t aitid the highest aInd lowest douibling tinmes r-eportei by Schmidt (13(1)KK,,,P.) valuies when the suLbstrate is NH,.

for the inhibition of NH4' oxidation to NO, - by CH4 in cellsuspensions of Nitroso(o(ccis oceanius. Consistent with thisfinding, Jones and Morita (79), working with the sameorganism, found that NH4' oxidation to NO,- was inhibitedby >80% in the presence of 119 FM CH4.

In cell suspensions of N. eiuropaiai the rate of CH,OHproduction from CH4 is stimulated by low concentrations ofNH4 and inhibited by higher concentrations (70). A similareffect was noted on the rate of production of CO, and cell-Cfrom CH4 in suspensions of N. (uropaceI and Nitrosococculsoceanuis (79). The same general pattern also occurred in theoxidation of ethylene and methanol by N. eur-opuia cells (72.163). The eflect of NH4' on the various oxid'ations listedabove can only be understood if it is remembered that.during the oxidation of NH14' to NH,OH, reducing powerrequired by ammonia monooxygenase comes from the fur-ther oxidation of NH,OH to NO>- (61) and that NH4 andCH4 are apparently competing for the same active site on themonooxygenase. It was proposed (70. 72. 163) that additionof small amounts of NH44 results in the production ofreducing power which can be used by ammoni'a monooxy-genase in the oxidation of CH4 and other organic substrates.Higher NH ' levels competitively inhibit CH oxidation.As indicated above, it appears that, in N. ei,, op(ieui

CH3OH can be oxidized to HCHO by ammonia monooxy-genase. It has been known for some time that CH,OHinhibits NH4' oxidation in N. emropa(ea (62, 148, 163). Themethanol dehydrogenase of conventional methylotrophs ischaracterized by a pyrroquinolinequinone prosthetic group(5). Efforts to detect the presence of this group in cells ot N.eluropucea were unsuccessful (163). Reaction steps beyondformaldehyde 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 ammoniaoxidizers (70, 79). What limited data exist are summarized inTable 2. The maximum methane oxidation rate for anammonia oxidizer was still five times lower than the lowestvalue for a methanotroph. The apparent K,,, (6.6 FM) forCH4 oxidation by Nitrosococcus o(eanlts is comparable tothat of methanotrophs (Table 2). Although in their work oncell-free extracts of N. euloptiea Suzuki et al. (148) mea-sured a Ki of 50 F.M for CH4 inhibition of NH14' oxidation, inwhole cells Hyman and Wood (70) noted a much highervalue (2 mM). There may thus be considerable interspeciesand interstrain variation in affinity for CH4 among ammoniaoxidizers.

Ammonia Oxidation by Methanotrophs

There is extensive evidence that many methanotrophs canoxidize NH4'to NO,-. This activity was exhibited by 21 of

67 enrichment cultures of marine methane oxidizers (68),atnd in another study all of the 104 isolates of methanotrophsobtained from a variety of environments produced NO-from NH4+ (171). Three species ot obligate methylotrophsoxidized NH4 to NO> in the presence of CH4 (127).Furthermore, during NH14' oxidation by some methaneoxidizers, including Metlhylosinis triclhosporiuimi OB3b,small amounts of N.O can be evolved (91a, 110, 171, 178).However, attempts to grow methanotrophs on NH4 in theabsence of CH4 have failed (68). NH4' , although it isoxidized by whole cells of mecthvlococcus capsulatits Bath(24). does not support CO, fixation in this organism (139).

Involvement of MMO. Cell-free extracts ot Methvlococciscapslllatus Balth containing soluble MMO activity oxidizedNH4 'to NOr- (24). During the oxidation, NH.OH accumu-lated transiently and was the first detectable product of thereaction. On was a requirement for the reaction. The kineticsof NH4' oxidation to NH,OH were complex but conformedto the Michaclis-Menten model when NH4' concentrationsfell between 20 and 200 mM. In this range, the calculated K,,,was 87 mM at pH 7. CH4 in all concentrations testedexhibited an inhibitor-y effect on NH4' oxidation. 8-Hydroxyquinoline and acetylene, well-established inhibitorsof the soluble MMO of Metliv-lo(coc((s (capsidIatils (141),strongly inhibited NH4' oxidation to NH.OH but notNH,OH oxidation to NO, Inhibitors specific to the partic-ulate MMO of the organism (26) had little effect on CH4 orNH4' oxidation in the extracts. Based on the above results,it w-as hypothesized that the soluble MMO of Meth1lo oc usapsi/(lltis catalyzes the oxidation of NH4 to NH,OH (24).A cell-fi-ee extract of Met olIvIon1(as mectaonic( containing

particulate MMO activity catalyzed NH4 -stimulatedNADH oxidation (21). The activity was blocked by a varietyof inhibitors, including metal-chelating agents, which werelater shown to be specific to particulate MMOs (26. 131). Inaddition. NH4 in high concentrations inhibited the oxida-tion by the extracts of bromomethane (an analog of meth-ane). Regrettably, this study and the one on Met hylococcuscapsulatius extracts cited above are the only investigations inwhich it can be determined, with some degree of certainty,whether a soluble or a particulate MMO was present in thesystem carrying out NH4' oxidation. Further efforts shouldbe made to characterize each type of enzyme with respect tothis activity.

Studies on whole cells also appear to implicate MMO inNH4' oxidation. NH4' stimulated 0O uptake by Methvlosi-misiitricohsporimun OB3b (110). The stoichiometry suggesteda reaction identical to that catalyzed by ammonia oxidizers:NH, + 1.50, HNO, + H,O. Formate and formaldehydeenhanced NH4' oxidation, presumably via NADH produc-tion. Except for a stimulatory effect at very low concentra-

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tions, CH4 generally inhibited NO,- production. The stim-ulation, confirmed in a later study (91a), may have been dueto the release of NADH during the oxidation of intermedi-ates in the pathway of CH4 metabolism. Presumably, CH4 inhigher concentrations excludes NH4, from the active site ofthe MMO. In this context, it is interesting to note that, insome methanotrophs, nitrification occurs only in the pres-ence of CH4 (68, 127).NH4+ can reduce the growth rate of many methanotrophs

by inhibiting CH4 oxidation (171). The degree of inhibitionvaries between species and strains. Ferenci et al. (40) notedthe competitive nature of the inhibition in cell suspensions ofMethylomnas menethanica and measured a K1 of 10 mMNH4 at pH 7. For Methvlosinus trichosporiumn OB3b, Kivalues varied between 17.5 mM at pH 6 and 0.2 mM at pH 8(110). The dependence on pH suggested that, similarly to theammonia monooxygenase of N. eurlopacea (144), NH, notNH4+ might be the substrate for the MMO present in thisculture of Methvlosinus trichlosporitum (110). However, incell suspensions of Methylococcus capsidatlits Bath, the K,,,for NH4+ oxidation increased from pH 7 to 8 and no activitycould be observed at pH 9 (24).NH2OH oxidation. In crude extracts of Methvlococcits

capsl(ulatis Bath, added NH,OH was readily oxidized toNO.- and the rate of reaction was strongly enhanced by theaddition of phenazine methosulfate (PMS) (24). The oxida-tion was not significantly inhibited by the addition of 8-hydroxyquinoline or acetylene, agents which blocked theactivity of the MMO present in the extracts (see above).Neither did methanol dehydrogenase appear to be responsi-ble for NH,OH oxidation. Cyanide, a powerful inhibitor ofmethanol dehydrogenase in Methylococcus capsuldatis (24)actually stimulated NH,OH oxidation. The activity was notaffected by the addition of CH3OH. The presence of ahydroxylamine oxidase was therefore postulated (24).The CH3OH dehydrogenase and NH,OH oxidase activi-

ties present in soluble extracts of Methvlococcus tlermitio-plhi/its, an obligate methane utilizer, were separated byion-exchange chromatography (136). The NH,OH oxidasewhich was isolated shares a number of characteristics withthe corresponding enzyme in ammonia oxidizers: a PMS-stimulated 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 Meth-ylococcus ther-mophilius consumed O0, reduced NAD' andcytochrome c, and produced adenosine triphosphate (102). Itis not known whether NH,OH oxidation can contributesignificantly to the energy budget of methanotrophs whichpossess this activity. In fact, it remains unclear whether theability to oxidase NH,OH is widespread among methano-trophs.NH4' oxidation rates and affinities. The methanotroph's

ability to nitrify is clearly not as developed as that of theammonia oxidizers. The maximum rate of NOJ- productiondetermined for a methanotroph is still more than 20 timeslower than one of the lowest rates reported for an ammoniaoxidizer (Table 2). Although there is overlap in the K,,,values obtained in the two groups of organisms, the lowestK,,, (NH4+) for ammonia oxidizers is 300 times lower thanthe lowest K, for methanotrophs (Table 2). Considerableinterspecies variation in ability to nitrify appears to occuramong the methanotrophs (110, 171).

CO Oxidation by Methanotrophs and Ammonia Oxidizers

A number of methanotrophs (39, 40, 65, 143) and ammoniaoxidizers (80, 81, 157) have been found to oxidize CO toCO,. Methanotrophs which exhibit this activity includeMethivlotnonas a/lits BG8 (65), MethYlosinits triclhosporillinOB3b (65), Metlivlononias ineCthlali(ca (39), and Metliviococ-(11s (apsidatalus Bath (143). Nitrifiers which oxidize COinclude all of the species tested by Jones and Morita (80),i.e., Nitrosococcus oceanus and various species and strainsof Nitrosomionas, including N. elirop(ea(.CO does not appear to support the gr-owth of either

methanotrophs or ammonia oxidizers (39, 80). No increasein survival rate was noted for cells of a marine Nitrosomnonasstarved of NH4 ' for 25 weeks in the presence of 50 nM CO(83). In ammonial oxidizers, carbon from CO is incorporatedinto cell material only in the presence of NH4 (80). Appar-ently, NH4 oxidation provides energy for the Calvin-cycle-mediated fixation of CO, produced from the oxidationof CO.

Involvement of methane and ammonia monooxygenases. Inthe ammonia oxidizers, it appecars that CO oxidation iscatalyzed by ammonia monooxygenase. Because the studieson CO metabolism by methanotrophs were carried out at atime when the fundamental distinction between particulateand soluble MMOs had not yet been made, there are, insome cases, no clues as to which type of enzyme wCasresponsible for an observed activity. However, the solubleMMO of Metli lococcus(apsid/aitus Bath was shown tocarry out CO oxidation (1128). Furthermore, the extremesensitivity to cyanide exhibited by the MMO extracts ofMethi/Sononas methanica used by Ferenci et al. (40) sug-gested that the enzyme present wcas of the particulalte type(70). Further evidence is required to clarify the issue.

In both methanotrophs and ammonia oxidizers, CO stim-ulates Or uptake and oxidation of sources of reducing powerfor the monooxygenases (65, 157). Stoichiometries are asexpected for reactions catalyzed by mixed-function oxygen-ases: CO + AH, +- Or-- CO, + A + H.O. In some cases(39, 40, 143, 157), CO is oxidized only in the presence of asubstrate 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' oxida-tion by CO was measured at 3 FtM (148). NH4+ oxidation intwo species of Nitr-osomiionatis and Nitrosococcus oceanitswas inhibited by >50% in the presence of 6 F.M CO (82). COat 100 FtM inhibited CH4 oxidation by 65Cc in Methl/losinisstrichosporim,n OB3b and by 100%t in Methylonolnlasmnethanica (39). The action of CO is apparently not at thelevel of cytochrome oxidase since the affinity of this enzymefor 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 NH4levels as low as 70 [tM (81). NH4 'inhibited CO oxidation byMethvlo4lon(is mllethlanlic(a with a Ki of 12 mM (40).CO oxidation in ammonia oxidizers is blocked by numer-

ous inhibitors of nitrification (82). When cyanide is added inconcentrations low enough not to affect the terminal oxi-dases, it blocks CO oxidation in Metlivonioonas mnethanica(40).CO oxidation rates and affinities. Maximum rates of CO

oxidation by methanotrophs show considerable overlap withthose of ammonia oxidizers (Table 2). Both groups oforganisms can oxidize CO when it is present in concentra-

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tions of <100 nM (84). Methane and ammonia oxidizer-s haveK,,,(,,P,,, values for CO oxidation near those for their- respec-tive growth Substrates (Table 2).

INHIBITORS OF METHANOTROPHS AND AMMONIAOXIDIZERS

Inhibitors of NH,+ Oxidation in the Studv of NitrificationThe activity of chemoautotrophic ammonia oxidizer-s is

specifically blocked by numel-ouS compounds (62), and anumber of these have received considerable attention fortheir ability to inhibit nitrification in agricultuLral soils (135).Inhibitors have also been used to estimate nitrifierc activity inthe environment (see references below). The method c(an besummarized as follows. A sample of soil, sediment, or wCateris divided equally between two sets of replicates. One setreceives inhibitor- dissolved in a carrier; the other receives anequal amount of carrier alone. The disappearctnce of endog-enous NH4' (49, 50, 54, 90) and O. (90) or the appearance ofNO- (16, 48, 49, 50, 54, 90, 168, 179, 180) can then befollowed. Alternatively, a Substrate which can be metabo-lized by chemoautotrophic ammonia oxidizer-s is added andits utilization is monitored. Substrates used have includedNH4+ (50, 180), 14CO, (10, 17, 37, 48, 90, 137, 160, 161. 162),14CO, and 14CH4 (84). Compared with nonradioactive com-pounds, radiolabeled ones provide greater- sensitivity andallow 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). Anaverage ratio obtained from pul-e-cultur-e exper-iments isusually applied. Molar r-atios fall between 5 and 42 inammonia oxidizers and 25 and 132 in nitrite oxidizers (43,85). Because of these wide variations, the validity of the14CO, uptake method as a direct meCasuLre of nitrification hasbeen questioned (48. 85, 152).The derivation of estimates ot nitrification lromn "4CO

uptake measurements is even mor-e problemnatic. NHX4depresses CO oxidation in nitrifiers (80. 81). As noted above,this is because both NH4' and CO acre substrates forammonia monooxygenase (157).

Nitrapyrin (2-chloro-6-trichloromethvl-pyr-idine) has beenthe inhibitor most often favored by investigators (152).although allylthiourea (49, 50) and thioureal (18()) have talsobeen used. These compounds block the NH4'-to-NH,OHstep of ammonia oxidation (15). The mode otf action ofnitrification inhibitors is further discussed in the next sec-tion. Various problems associated with the use of thesecompounds in field studies are outlined in the followingparagraphs.

Inhibitors may not act immediately upon addition. Powelland Prosser (118) noted a 10-h lag between amendnment with6-chloropicolinic acid (2 FtM) and slowing of growth in aculture of N. euroopJaa. Although eventually effective, lownitrapyrin concentrations (<0.9 FtM) did not immediatelyblock NH4+ oxidation by various strCains of nitrifiers (7).These problems may be alleviated by using higher- levels ofinhibitor (7) or by preincubating samples with inhibitorbefore substrate addition (160-162, 168).Not all species and strains of nitrifiers are similarly af-

fected by a given compound. For seven strains belonging tothree separate genera of ammonia oxidizers, nitrapyrin con-centrations required for complete inhibition ranged from 0.9to 43 FiM (7). In a study of nine inhibitors, Jones and Morita(82) noted several differences in susceptibility among threespecies of nitrifiers. For example, 3-aminotriazole in a con-

centraCtion of 1.19 mM completely inhibited NH4' oxidationby two species of Nitrosonioast.v but left NitrosococcitsO(ce(1111S unaffected. At this concentration most of the otherinhibitor-s tested decrecased activity by >90%. The level ofnitrapyrin originally recommended for use in the 14CO2uptake assay was 22 FiM (10). It WalS Suggested that higheramounts would alffect other microorganisms.

lThe extent of inhibition may depend on the substrateadded. With many inhibitors, it appears thcat NH4 ' and CH4oxidation are more affected than is the oxidation of CO (82).TIhis could be related to the high affinity of ammonia mono-oxygenase for CO.

Nitrapyrin can be lost through volatilization or degrCada-tion (125). Both of these processes are probably of littleimportance in short-ter-m (<24-h) incubLtions or in assays onsealed water samples. No loss in effectiveness was seen insealed 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 24h of incubLation. TIo prevent nitrification from occurring in

samples of marine sediment, Henriksen (54) found it neces-sary to add fresh nitrapyrin at intervals of 1 or 2 days. In theenvironment. nitrapyrin is hydrolyzed to 6-chloropicolinicacid (12. 125). Although this compound appears to inhibitnitrificattion (118), at equal concentration it is less effectivethan nitracpyrin (12, 92, 118). Little is known of the fate of thethioul-eats in the environment.The influence of Vail-IOuS physicochemical characteristics

of soil on the effectiveness of nitrapyrin has been extensivelystudied (135). Ihe etfects of these pCarameters on the thio-uL-eCas a1re poorly documented. It appears that, at least insome environments. these compounds may be less effectivethan nitriapyr-in. In puI-e cultures of N. euroop1ea, allylthio-urea and nitrapyrin completely block NH4 oxidation atapproximately 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 nitrificationwhile, at an equivalent level, nitr-apyrin significantly inhib-ited this activity (13).

Nitrification inhibitors can influence other biological pro-cesses. TIhe biological etfects of nitrapyrin are summarizedby Slangen and Ker-khotl (135). The possible effects of thethioureas have received little attention. Allylthiourea wasshown to stimulakte NO, disappearance and, in some cases,depr-ess almmonification in fi-eshwater sediments (49). Thio-urea and allylthiourea inhibit CH4 oxidation by methanotro-phs (66), cas does nitriapyrin (155, 156).

BecaLuse nitrapyrin is only sparingly soluble in water, it isuSuLlly dissolved in an organic solvent before addition tosamples. TIhe carrier- itself mnay have an inhibitory eflect (12).Ethanol has been used in a nunmber- of studies (10, 17, 48,137, 160). In a concentration of 90 mM, it blocks NH44oxidation by N. europuaea (62). Hall (48) found that 1.2 mMethanol had no effect on nitrifiers. In most of the studiescitcd above, it appears that ethanol was near or below thislevel. 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). Potentialproblems with the carrier are avoided by using allythioureaand thiourea, which are soluble in water.

Studies designed to compare estimates of nitrificationobtained with different inhibitors are almost nonexistent.Hall (49) found that measurements of nitrification in fresh-water sediments, using allythiourea, were higher than thosemade with nitrapyrin. The author proposed that volatiliza-tion of the latter compound may have been an important

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factor. Billen (10) found nitrapyrin to be more satisfactoryfor determining 14CO, uptake by nitrifiers than either thio-urea or hydrazine, although no specific data in support ofthis were provided.

Despite all of the drawbacks listed above, inhibitor studieshave yielded estimates of nitrification which agree within anorder of magnitude with measurements made by using otherapproaches (10, 17, 37, 48, 90, 137). This was the level ofprecision originally claimed for the method (10).

Since many of the inhibitors used in studies of nitrifica-tion also inhibit the oxidation of NH4 ' and CH4 by methan-otrophs, as noted above, it is often not possible to concludeunequivocally that observed nitrification is due to the am-monia oxidizers alone. A differential inhibitor would there-fore be useful in ecological studies.

Differential Inhibition of Methanotrophs andAmmonia Oxidizers

An inhibitor specific to either methanotrophs or ammoniaoxidizers would be useful for distinguishing which of theseorganisms is responsible for NH4 , CH4, or CO oxidation inenvironments in which such activities occur (129). As notedabove, nitrapyrin, thiourea, and allylthiourea all inhibitmethanotrophs. Topp and Knowles (155) suggested thatmeasurements of nitrapyrin-sensitive 14CO, uptake in sam-pies in which methanotrophs were present might yield over-estimates of nitrifier activity. A similar problem exists withnitrapyrin-sensitive '4C0 uptake (84).Table 3 lists 35 agents which have been tested on metha-

notrophs and nitrifiers. The compounds inhibit one or theother or both types of organisms. Where pure culture datawere not available, results from sewage sludge or soil studiesare included. With very few exceptions, the inhibitors inTable 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 orsoluble, was not monitored. Given what is now known aboutthe inhibitor profiles of the two forms of monooxygenase(26), it appears that in a majority of the organisms investi-gated it was a particulate enzyme which was responsible formost of the observed methane oxidation. The results ob-tained on Methvlococc(s (cpslidatits Bath (24, 141) clearlydo not fit this pattern. At the time of these studies, themonooxygenase under investigation was known to be solu-ble. With the possible exception of KCN, the inhibitorprofile of Methvlococcus capsilatius Bath obtained by theinvestigators appears to be that which has now been recog-nized to be typical of the soluble MMOs (26).Some of the inhibitors in Table 3 have more than one

effect. For example, KCN blocks both cytochrome oxidaseand particulate MMO (75). But most of the compoundslisted, when added in low enough concentration, affectmainly the CH4-to-CH3OH or NH4 t-to-NH,OH steps in thesequence of oxidations carried out by methanotrophs andammonia oxidizers.A number of agents in Table 3 are thought to act by

binding a metal (in all likelihood, Cu) which is essential forthe activity of the particulate MMOs and ammonia monoox-ygenase. Such inhibitors would include those listed as "che-lating agents" and "monodentate ligands" as well as ni-trapyrin and histidine (15, 62, 66, 97, 141). Lees (97)examined the effect of eight different chelating agents on theoxidation of NH4' by Nitrosomonias spp. That the slope ofthe curve relating inhibitor concentration to the percent

inhibition was the same for all the compound tested sug-gested that a common mechanism was involved. Inhibitorstrength was closely related to the ability to chelate. Finally,in the cases of allylthiourea, guanidine, and L-histidine, theinhibition was noncompetitive.

Involvement of copper in CH4 and NH4 'oxidation hasbeen deduced in part from the high affinities of inhibitingchelators for this metal (66, 97). Hooper and Terry (62) foundthat, in N. clropaea, the inhibition of NH4 'oxidationcaused by KCN or diethyldithiocarbamate could be re-versed, at least in part, if cells were treated with a solutioncontaining Cu' This same ion relieved the inhibition ofCH4 oxidation by 8-hydroxyquinoline, ox-dipyridyl, andthiosemicarbazide in cells of Melthylosinus trichosporiiimOB3b (66). Cu'+ did not restore NH4 'oxidation in N.europcaea or CH4 oxidation in Metlhlosinus trichosporiiimOB3b when these organisms were exposed to allylthiourea(62, 66). This may be because allylthiourea has a higheraffinity for the Cu' ion (66). Addition of Cu2+ to cellsuspensions of N. europaea treated with nitrapyrin restoredthe ammonia-oxidizing capability (15). Similar treatment didnot relieve the inhibition of CH4 oxidation by nitrapyrin inMethllosinus trichlosporiumi OB3b (156). This suggests thatnitrapyrin might function differently in methanotrophs andammonia oxidizers.

It is possible that pyridine and its derivatives (Table 3)could act by binding key copper atoms, but a more directinteraction with the active site of the monooxygenases issuggested by the following findings. Benzene, an analog ofpyridine, 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, particulateMMO does not oxidize benzene or other aromatic com-pounds (14). Because of its resemblance to benzene andphenol, aniline, which inhibits NH4 oxidation in N. eiuro-p(aea (153), may also be a substrate for ammonia monooxy-genase.The target of some of the inhibitors may not be the

ammonia monooxygenase or MMO. It has been suggestedthat, in methanotrophs, chelating agents may interact withan electron transport protein closely associated with theparticulate MMO (26). Bhandari and Nicholas (9) proposedthat NaN3, diethyldithiocarbamate, thiourea, nitrapyrin, and2-trichloromethyl pyridine inhibit a copper-dependent trans-locase in ammonia oxidizers. However, the evidence ad-vanced by these authors is also consistent with directinhibition of NH4 oxidation (88). Furthermore, CH4 andCO oxidation in ammonia oxidizers is also inhibited bynitrapyrin (82). Since CO and CH4 must enter the cells bypassive diffusion, it seems likely that the inhibitors listedabove function on the oxidative machinery of ammoniaoxidizers, not NH4 ' transport.C,H, is a suicide substrate for both ammonia monooxy-

genase (73) and the MMO of Methylococcus capsulatiis(121). This may be the mechanism underlying the action ofall acetylenic compounds which block CH4 and NH4 ' oxi-dation. The cytochrome P-450 monooxygenase of hepaticmicrosomes is subject to inhibition by a wide variety ofacetylenic compounds (111). Here, too, a suicide mechanismis thought to be involved. Certain P-450 inhibitors have beenshown to inhibit CH4 and NH4 'oxidation, although highconcentrations are required (Table 3).

Inhibition of the growth of MVethvlococcus capsulatus(strain unspecified) by threonine and phenylalanine is be-lieved to be due to end product inhibition of enzymesessential for the synthesis of certain key amino acids (36).

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TABLE 3. Inhibitors of methanotrophs aind ammonia oxidizer-s

Inhibitol Etlect on Etlcct on ammonl oxld]ZClSmethtnotrophs

MinimumILin Effect on Refer-cncc(s)"State Effect on MinimlumIMo) wtt inhibitor'' hydoxlName' FormuLllMt at methanol inhibitor-v doxyl-10-) C'concnconann( Ml amine((AM )" oxidationcnn(m oxdtn

Chelating agentThiosemicarbazide (hydra-

zinecarbothiamide)Diethyl dithiocarbamnate

(diethylcarbamodithioicacid sodium salt)

ThioureaPotassium ethyl xanthate

(potassium xanthogenate)

Allylthiourea [(2-propenyl)thiourea]

ThioacetamideO-Phenanthroline (1,10-

phenanthroline)

8-Hydroxyquinoline(8-quinolinol

x(x'-Dipyridyl (2.2'-

bipyridine

Aminoguanidine (hydra-zinecarboximidamide)

NH,CSNNH.

(C,H.),NCS,Na

H,NCSNH.C,H5OCS,K

CH,=CHCH,NHCSNH,

CH.CSN H.

OH

NH.CNHNHNH.

91.14 S 100' No 10 No 62. 66, 114, 141

171.27 S 100 NoA 10( No 9, 62, 97, 141,151

76.12 S 1,()(( No 37.4160.30 S 100') No 9

No 62, 66, 97, 158ND 9, 64, 97, 151

116.19 S 10 No 1 No 15, 58, 62, 66,70, 82, 97

75.00 S 100' No 10 ND 15318(.0 s 100 No 50 ND 60,64, 66, 114,

141

145.16 S 100 No 10 No 62, 66, 114, 141

158.18 S 100') No 100 No 62, 64, 114, 141

74.09 S 10,((( No 1,01)O No 62, 66

Monodentate ligandSodium azide

Potassium cyanide

3-Aminotrialzole

NaN,

KCNH

N H2

65.02 X 1,0(( No 1,((( Yes (inhibi- 9. 62, 66tion)

65.11 S Ki = 0. 3 No S No

84.08 S 1((.(( No l1.00i Yes (stimu-lation)

62, 64, 66, 75,76, 114, 141

62, 66, 82

Pyridine compound

Pyridine

6-Chlor-o-2-picoline (2-chlo-ro-6-ethyl-pyridine)

Nitrapyrin (2-chloro-6-trichloromethyl pyridine)

Picolinic acid (2-pyridinecarboxylic acid)

Chloropicolinic acid

1-1 sonicotynil-2-isopropylhydrazide [4-pyridinecarboxylic acid 2-(1-methyl-ethyl)hydrazine]

Acetylenic compoundAcetylene (ethyne)Propyne2-Butyne

79.10 L 100'oi No 1,264k

"-ICI CH

3

Cl CCI

3

COOCH

CONHNHCH(CH3

HC-CHHC=CCH,CH C-CCH,

127.57 S 430' ND >1(

Yes 66, 129, 138,141

ND 129, 156

230.88 S 9 No 1 No 9. 15, 82, 129,155

123.11 S 10 ND >1( ND 129

158.56 S >1( ND 2.2"' ND 118, 129

179.22 S >1,(000...' ND 10,000

26.02 G 1,000 No 0.25 (K,)42.08 G 1.000 No 10 Pa"54.09 G 1,0(( No >10 Pa"

No 21, 62

No 30, 74, 82, 141ND 103, 141ND 103, 141

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TABLE 3-Conlitnuied

.. ~~~~~~~~ ~~~~Effecton ..Inhibitor- methan Eflect on ammonia oxidizersmethalnotroph s

Stat Minimum Eflect on Minimum Effect on Reference(s)'Mol wt inhibitor-v u hvdroxyl-Name') Formuilal A Iat methanol inhibitorv(103)) 'C concn omxidation25IC' (v)(t oxidation" concn )1.tm) oxdtn

1-ButynePropargylamine (2-propyn-

1-amine)2-Propyn-1-ol"

HC--CCH,CH3HC--CCH,NH.

HC=CCH,OH

54.09 G 1(00 No 10 Pa55.08 S >1.000 No 1,000 imol

kg56.06 L >1.(000 No 1.000()mol

kg I

NDND

ND

103. 141103, 141

103. 141

N N COOH

( HCHHNH2 2155.16 S 10,000 No 5,000 ND 18, 36, 66. 97

L-Threonine

L-Phenylalanine

Cytochrome P-450 inhibitorSKF 525-A (r-diethyl-amino ethyldiphenylpro-pyl acetate)

CH3CHCHNH,

OHCOOH(CH6H,)CH,CHNH,

COOHCOH16 5

H7C3COOCH2 CH2 N(C2 H5)2U6H5

119.12 S 1,((0" ND >5,000' ND 18. 36. 97

165.19 S 1,000" ND >5,000 ND 36, 97

353.5 S 5.000 No 50" No 62. 66

Lilly 18947 (2,4-dichloro-6-phenyl phenoxyethyl-diethylamine)

Cl

CI C C N H338.28 S 1((.(( No

C6H5

Lilly 53325 [2,4-dichloro(6-phenyl phenoxy)ethy-lamine hydrobromide]

Cl

CH H2 HBr

C H6 5

363.08 S 5.000 No 100 No 62. 66

Miscellaneous compoundsHydrazineAniline-Hydroxylamine

H,NNH,C6H,NH,

NH2OH

32.05 L >'20)093.12 L >10033.03 S 1(0

Yes 2.000 Yes 58, 62. 66No 100 ND 141, 153No 2.000 No 2. 66

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 inhibitor- by more than 30%. ND. Not

deter-mined.fDoes not inhibit Methvlococcus c(p.silatits Batth. See text fol- interpr-etation.

Stirling and Dalton (141) reported a 40%/r r-eduIction in the r-ate of CH,OH oxidation by whole cells of Meth/looc(cis cap.s/datois Baith in the presence of 100p.M diethy1dithiocarbamate. In cell-free extracts of this species (24) aind whole cells of' other species (see r-eterences 66 and 141). CH,OH oxidation was notaffected by the inhibitor.

"Inhibition of NH4' oxidation was 65%.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 NH4' oxidation at 10 ,uM (129).

"' Inhibition of NH4' oxidation was 60% (118)." CH4 oxidation in cell-free extracts (23)." Inhibition of nitrification in soils." 2-Propyn-1-ol is a substrate for methanol dehydrogenase in Methslococcus cap.sildtii.s Bath (141). At 10(0 ,uM, the compound inhibits methane oxidation by

40%.Inhibition of exponential growth over a 6- to 12-h period (36).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).

Amino acidL-Histidine

100 No 62. 66

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This leads to an imbalance in amino acid metabolism.Threonine and lysine inhibit NO- production in growingcultures of N. eliriopueni (18) but the inhibition is, apparently.not immediate (97). Again, end product inhibition is sug-gested.

Hydrazine blocks NO- production by Nitrosomlonisspp. by acting as an alternative substrate for hydroxylamineoxidoreductase (58). The oxidation of hydrazine can actuallystimulate the action of ammonia monooxygenrase by provid-ing it with reducing power (70, 174).

In high enough concentration, hydroxylamine inhibits itsown production in cells of N. eiuropuiea (2). Hyman andWood (70) proposed that this inhibition serves a regulatorypurpose by helping to prevent accumulation of' NH.OH incells.

Salvas and Taylor (129) showed that picolinic acid in aconcentration of 10 p.M blocks NH4' oxidation by Met/hy-losiltiis tricliosporiumn OB3b but not in two species of marineammonia oxidizers. Table 3 suggests that other compoundsmay selectively inhibit one or the other group of organisms.More specifically, the possible use of certain acetyleniccompounds and pyridine derivatives should be examined.

CONCLUDING REMARKS

The question of the ecological roles of the methanotrophsand nitrifiers in the oxidation of CH4. NH4 and CO innature remains perplexing. It will be resolved only bydetailed physiological and biochemical studies of the mech-anisms involved, coupled with the use of the more sophisti-cated ecological approaches. It is hoped that this review willprovide some incentive for such studies.

ACKNOWLEDGMENTS

We thank the Natural Sciences and Engineering Resear-ch Councilof Canada and the Canada Centre for Inland Water-s for support.

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NITRIFICATION BY METHANOCTROPHS AND NITRIFIERS 81

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