Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Arch. Microbiol. 114, 273-279 (1977)

Archives of

Microbiology �9 by Springer-Verlag 1977

Ammonia Oxidation by the Methane Oxidising Bacterium Methylococcus capsulatus Strain Bath

HOWARD DALTON

Department of Biological Sciences, University of Warwick, Coventry, Warwickshire CV4 7AL, England

Abstract. Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O2, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hy- droxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated Km value for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.

Key words." Ammonia oxidation - Methane mono- oxygenase - Hydroxylamine oxidation - Methylo- coccus capsulatus.

Ammonia is oxidised to nitrite by a number of different organisms which include the chemolithotrophs Nitrosomonas (Lees, 1952) and Nitrosocystis (Watson, 1965), the heterotrophs AspergiIlus flavus (Hirsch et al., 1961) and Arthrobacter (Verstraete and Alexander, 1972) and some methane oxidising bacteria (Hutton and Zobell, 1953; Whittenbury et al., 1970). It is with the chemolithotrophs, however, that most of our understanding of the mechanism of ammonia oxidation resides. Studies on whole cells have indicated that hydroxylamine is the only stable intermediate between ammonia and nitrite (Lees, 1952; Hofman and Lees, 1953) and that at least one atom of dioxygen is in- corporated into nitrite (Rees and Nason, 1966) in- dicating an oxygenase type reaction.

Inhibitor studies on ammonia and hydroxylamine oxidation by Nitrosomonas have strongly suggested

Abbreviations. PMS = Phenazine methosulphate; NADH = nicotinamide adenine dinucleotide, reduced form; Km = Michaelis con- stant; NOF = nitrite; NH2OH = hydroxylamine

the involvement of a metal ion such as copper, a P450- like protein and a functionally intact membrane in the initial hydroxylation of ammonia to hydroxylamine (Hooper and Terry, 197)). Such studies, however elegant, need to be verified in cell-free systems before a true interpretation of the mechanism of ammonia oxidation can be made. To this end a number of workers have isolated and purified the hydroxylamine oxidase from the autotrophic bacteria (Nicholas and Jones, 1960; Hooper and Nason, 1965; Rees, 1968). The enzyme will transfer electrons to a number of acceptors including phenazine methosulphate mammalian cytochromec, pyocyanine, methylene blue and benzyl viologen, with the former giving the highest specific activity.

Isolation of the enzyme responsible for the initial oxidative attack on ammonia has proven more difficult and it is only in recent years that preparations capable of ammonia oxidation in cell-free systems have been prepared. The successful preparation of extracts from Nitrosomonas has relied upon the addition of bovine serum albumin (Suzuki and Kwok, 1970; Suzuki et al., 1974, 1976) whereas extracts from Nitrosococcus oceanus required ATP, Mg 2+ and sea water for activity (Watson et al., 1970). In both instances the ammonia oxidising activity was associated with membrane-containing fractions of the cell and nitrite was the measured product of the reaction. NADH would support ammonia oxidation in Nitrosomonas but its role appeared to be catalytic rather that stoichiometric (Suzuki et al., 1976).

Ammonia, carbon monoxide and methane will specifically inhibit methane oxidation in the membrane bound methane mono-oxygenase from Methylomonas methanica (Colby et al., 1975; Ferenci et al., 1975) and in this respect the enzyme appears to have a degree of similarity with the ammonia oxidising enzyme from Nitrosomonas which is specifically inhibited by methane, carbon monoxide and methanol (Suzuki et al.,

274 Arch. Microbiol., Vol. 114 (1977)

1976). F u r t h e r m o r e , inh ib i tors o f the m e m b r a n e b o u n d me thane m o n o - o x y g e n a s e (Colby et al., 1975) will also inhib i t a m m o n i a ox ida t ion by Nitrosomonas cells ( H o o p e r and Terry , 1973).

W e have recent ly r epo r t ed the i so la t ion o f a complete ly soluble me thane m o n o - o x y g e n a s e system f rom Methylococcus capsulatus s t ra in Bath (Colby and Da l - ton, 1976) and this p a p e r repor t s on the ox ida t ion o f a m m o n i a and h y d r o x y l a m i n e by the soluble c rude extracts f rom this o rgan i sm and compares it wi th the systems f rom o ther a m m o n i a oxidisers.

M A T E R I A L S A N D M E T H O D S

Phenazine methosulphate, NADH, e,c&dipyridyI, diethyl dithio- carbamate, thiourea, hydrazine sulphate and neocuproine were purchased from Sigma Chemical, Co., Kingston-upon-Thames, Surrey, U.K. Acetylene was supplied by Cambrian Chemicals, Croydon, Surrey, U.K. Research Grade CO was supplied by British Oxygen Co., London, S.W. 19, U.K. Methanol (Ultrar) was supplied by Hopkin and Williams, Chadwell Heath, Essex, U.K. The best available grade of all other chemicals were supplied by BDH Chemicals, Poole, Dorset, U.K.

Methylococcus capsulatus strain Bath (Whittenbury et al., I970) was grown at 45°C in a batch culture on ammonium mineral salts medium (Dalton and Whittenbury, 1976) either in a 100 1 stainless steel fermenter for the preparation of soluble extracts of the organism, or in a 2.5 1 chemostat culture for whole cell studies.

Soluble methane-oxidising extracts of the organism were prepared as described by Colby and Dalton (1976). The protein content of the extracts was determined by using the Folin-Ciocalteu reagent with crystalline bovine plasma albumin (Fraction 5) as standard (Lowry et al., 1951). Oxidation of NH3~ NO~- or NH2OH ~ NO~- was followed by determining nitrite in 100 gl aliquots of the reaction mixture by the sulphanilamide/N-(1-naphthyl) ethylene diamine hydrochloride method as described by Nicholas and Nason (1957). The protein precipitate formed in the nitrite assay was removed by centrifugation before reading the absorbance at 540 nm in a Pye Unicam SP 500 spectrophotometer. Standards of NaNO2 (up to 30 nmoles per assay) were also prepared and treated similarly.

NH3--+NH2OH oxidation was followed by determining hydroxylamine in a slightly modified version of the method described by Magee and Burris (1954). A 250 gl aliquot of reaction mixture was acidified with I N HC1 and to it was added 1 ml of 1 ~ (w/v) 8-hydroxyquinoline in ethanol followed by 1 ml of 1 M NazCOa with shaking. The "indo-oxine" product was measured after 2 h at 680 nm on an SP 500 Pye Unicam spectrophotometer. Standards of hydroxylamine (from 20 nmoles up to 300 nmoles) were similiarly treated and gave a linear response over the range indicated. No interference from ammonia, nitrite or nitrate was observed in the assay.

The enzyme reactions (1 ml volume) were done in 7 ml scin- tillation vials at 45 ° C in a shaking water bath. Reactions in which phenazine methosulphate (PMS) acted as electron acceptor were done in the dark by completely covering the vials with aluminium foil. When methanol or acetylene was added to the reaction the tops of the vials were closed with Suba seal stoppers.

Oxygen uptake by washed whole cell suspensions of M. capsulatus was measured in an oxygen electrode (Rank Bros., Bottisham, Cambs., U.K.) maintained at a temperature of 45 ° C.

All calculations of'g(m values from the (1/v)/(1/s) plots were done by least squares analysis Of the data obtained. A correlation coefficient of at least 0.99 was obtained for each plot.

In experiments in which the reaction was stopped after a specified time the reactions were found to be linear with respect to time and amount of extract as long as the latter was in excess of 3.5 mg/ml.

R E S U L T S

Oxidation o f Ammonia to Nitrite

Crude soluble me thane-ox id i s ing extracts o f Methylo- coccus capsulatus oxidise a m m o n i a to ni t r i te af ter an ini t ia l lag (Fig. l ) . The p r o d u c t i o n o f ni t r i te was enhanced by N A D H , a l though poss ible endogenous levels o f N A D H wou ld dr ive a m m o n i a ox ida t ion to a cer ta in extent. The add i t i on o f ca ta ly t ic a m o u n t s o f N A D H (0.01 gmole) gave no measu rab l e increase in ni t r i te p r o d u c t i o n over the endogenous p r o d u c t i o n level. Whereas a d d i t i o n o f 0.1 ~tmole N A D H sup- p o r t e d an increase in ni t r i te p r o d u c t i o n over endogenous levels.

The subs t i tu t ion o f bovine p l a s m a a lbumin for N A D H gave no enhancemen t in N O 2 p r o d u c t i o n above the level ob t a ined in the absence o f N A D H (c.f. Suzuki et al., 1976). D ioxygen was requi red for a m m o n i a ox ida t ion since no ni t r i te cou ld be de tec ted under anae rob ic cond i t ions ei ther in the presence or absence o f N A D H .

The ini t ia l lag phase observed with a m m o n i a ox ida t ion was e l imina ted by the add i t i on o f 5 gmoles h y d r o x y l a m i n e (Fig. 2) suggest ing tha t N H z O H was readi ly oxid ised to N O 2 in the presence o f NH3. This p r o d u c t i o n o f N O 2 f rom N H 2 O H and NH3 was a p p r o x i m a t e l y equal to the sum of the ind iv idua l a m o u n t s o f N O 2 p r o d u c e d by the reac t ion NH3 + N A D H and N H 2 O H + N A D H .

In an a t t emp t to resolve the ox ida t ion o f a m m o n i a to ni t r i te in to its c o m p o n e n t react ions , the prodjac t ion o f h y d r o x y l a m i n e dur ing the reac t ion was also meas- u red (Fig. 3). A g a i n there was an obvious lag before ni t r i te was p r o d u c e d in apprec iab le quan t i ty whereas the p r o d u c t i o n o f h y d r o x y l a m i n e f rom a m m o n i a was immedia te . W h e n the concen t ra t ion o f h y d r o x y l a m i n e a p p r o a c h e d 0.4 m M its u t i l i sa t ion ou t s t r ipped its p r o d u c t i o n and the concen t r a t i on decl ined. P resum- ab ly the abo l i t i on o f the lag phase in NO~- p r o d u c t i o n by h y d r o x y l a m i n e observed in F igure 1 was due to its immed ia t e convers ion to ni t r i te in the extract .

In the absence o f an exogenous supply, hydroxy l - amine has to be fo rmed f rom a m m o n i a before ni t r i te can be fo rmed in the react ion ,

NH3 ~ N H 2 O H ~ N O ~ .

The d a t a in F igure 3 suggest tha t the concen t ra t ion o f h y d r o x y l a m i n e in the reac t ion mus t be at least 0.25 m M before ni tr i te is formed. To test whether this in i t ia t ion of h y d r o x y l a m i n e ox ida t ion was subs t ra te

H. Dalton: Ammonia Oxidation by Methyl©coccus 275

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Fig. 1. The effect of NADH on nitrite productio n from ammonia. Each assay contained in 1 ml; 7 mg of Methyl©coccus capsulatus soluble extract, 5 ~tmoles ammonium sulphate, 20 ~tmoles phosphate buffer pH 7.0. • • no NADH, • • 0.1 gmole NADH, • • 5 gmoles NADH

Fig. 2. Effect of hydroxylamine on nitrite production. Each assay contained in 1 ml; 7 mg of Methyl©coccus eapsulatus soluble extract, 20 gmoles phosphate buffer pH 7.0. • • 5 gmoles hydroxylamine + 5 gmoles NADH, © © 5 gmoles ammonium sulphate + 5 gmoles NADH, • C 5 gmoles ammonium sulphate + 5 gmoles hydroxylamine + 5 gmoles NADH. The reactions were done in air

Fig. 3. Products of ammonia oxidation by Methyl©coccus capsulatus extracts. Each assay contained in 1 ml; 5 gmoles ammonium sulphate, 2.5 gmoles NADH, 20 gmoles phosphate buffer pH 7.0 and 7 mg extract. • • nitrite formation, • • hydroxylamine formation, © © nitrite -t- hydroxylamine formation. The reaction was done in air

Fig. 4. Lineweaver-Burk plot of hydroxylamine (NH2OH) formation from ammonia by Methyl©coccus capsulatus extracts at different concentrations of ammonium sulphate. The rates quoted were determined by measuring hydroxylamine formation after 4 rain. The reaction conditions were the same as those given in Figure 3

Fig. 5. Lineweaver-Burk plot of hydroxylamine (NH2OH) formation from ammonia by Methyl©coccus capsulatus'extracts at high concentrations of ammonium sulphate

concen t ra t ion dependent the Km values for hydroxylamine and a m m o n i a were measured. The Km for hydroxylamine oxidat ion was approximate ly 3 m M al though concent ra t ions above 8 m M were inhibi tory wherease that for a m m o n i a was 87 m M [the value for (NH4)2804 was 43.5 mM]. The kinetics of this latter react ion were rather complex and appeared only to have a Michaelis M e n t e n relat ionship at high substrate concent ra t ions (Fig. 4). The data for the reac- t ion velocity at the high substrate concen t ra t ion is shown in Figure 5 from which the Km was deduced.

The biphasic na ture of the 1/v versus l /s plot which is concave downwards suggests that negative cooperativity may be involved i n substrate b ind ing (Levitski and Kosh land , 1969), a l though this is no t a reliable indica t ion of this p h e n o m e n o n (Bardsley, 1977). A Hill plot of the data in Figure 4 gave an nu value slightly less than 1.

The Ana la r grade of a m m o n i u m sulphate con- ta ined 0.1 m o l e - ~ methano l so that the oxidat ion of a m m o n i u m sulphate concent ra t ions in excess of 200 m M would be confused by the propor t ionate ly

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The effect ofpH on ammonia and hydroxylamine oxidation by Methylococcus capsulatus extracts. Ammonia oxidation was measured by determining hydroxylamine formation ( e - - e ) after 5 min incubation. Reaction mixture contained 5 gmoles ammonium sulphate, 5 gmoles NADH, 7 mg extract and 20 ~tmoles buffer. Hydroxylamine oxidation was measured by determining nitrite formation (O---~O) after 5 rain incubation. Reaction mixture contained 5 gmoles hydroxylamine, 2.5 gmoles phenazine methosulphate, 7 mg extract and 20 Ixmoles buffer

h igher concen t r a t i on o f m e t h a n o l present . To test the e f fec t o f h igher a m m o n i a concen t ra t ions , a m m o n i u m ch lor ide was used as subs t ra te since this con ta ined no de tec tab le m e t h a n o l a t 2 M. A qua l i ta t ive ly s imi lar r e la t ionsh ip be tween 1/v and l / s was observed for the ch lor ide and su lpha te salts a t concen t r a t ions be low 200 m M , bu t above this a m a r k e d inh ib i t ion o f am-

m o n i a o x i d a t i o n was obse rved (Fig. 6). This inh ib i t ion at h igh subs t ra te concen t r a t i on was

�9 a lso obse rved in whole cells bu t on ly at a p H value o f 8. The Km and Vmax increased be tween p H 7 and p H 8 and , un l ike the sys tem f rom Nitrosomonas, am- m o n i a ox ida t i on was comple te ly inact ive at p H 9

(Table 1).

Arch. Microbiol., Vol. 114 (1977)

Table 1. Effect of pH on Km and Vmax for ammonia oxidation by Methylococcus capsulatus (Bath)

pH Km Vma x (nmoles 02 (mM) consumed/

rain �9 ceils)

7 31 37.6 8 66 86 9 no oxygen uptake with NHZ-

or CHr as substrate

Oxygen uptake rates were measured in an oxygen electrode system (see "Material and Methods") of 3 ml working volume. Varying amounts of 2 M ammonium chloride was added to the cell suspension and oxygen uptake was measured over a 4-min period using 3 different pH values. Calculation of K,, and Vm, x were made from l/v against 1/s plots

Table 2. The effect of methane on ammonia oxidation to hydroxylamine by extracts of Methylococcus capsulatus (Bath)

Methane Hydroxylamine formation (nmoles/min �9 mg protein)

(mM) (NH4)2SO4 (mM)

5.0 10.0 20.0 40.0 60.0

0 5.0 13.0 21.0 28.0 29.0 0.06 2.3 8.0 14.6 28.0 29.0 0.12 0.0 2.6 6.6 15.4 16.6 0.24 0.0 0.8 2.6 6.7 8.8

Hydroxylamine was determined as described in "Materials and Methods" after 4-min incubation�9 All reactions were done in flasks closed with Suba seal stoppers. The reaction mixture contained 20gmoles phosphate buffer pH 7, 1.5 gmoles NADH, 3,5 mg extract protein in 0.5 ml. Ammonium sulphate and methane were added as specified

M e t h a n e effectively inh ib i ted a m m o n i a ox ida t ion (Table 2) bu t the kinet ics o f the inh ib i t ion was such tha t the type o f the inh ib i t ion could no t be de te rmined

f rom l / v aga ins t 1/s plots . The reac t ions r epo r t ed above were done at a p H

value o f 7.0 which is the o p t i m u m value for the me thane mono-oxygenase . However , the o p t i m u m p H value for a m m o n i a ox ida t ion by Nitrosomonas is a r o u n d 8 and so the p H o p t i m a for the ind iv idua l reac t ions were de t e rmined (Fig. 7). A m m o n i a oxida- t ion was m a x i m u m a r o u n d p H 7.6 bu t hyd roxy l amine ox ida t ion was highest a t values be tween 6 and 6.5 which m a y have been due to the low level o f dissocia- t ion o f h y d r o x y l a m i n e above p H 6.5 (Suzuki et al. , 1974). Both react ions , however , p roceed with equal

veloci ty at the p H value in the r eac t ion so the lag is p r o b a b l y no t due to a p H effect.

H. Dalton: Ammonia Oxidation by Methylococcus 277

Hydroxylamine Oxidation to Nitrite

Hydroxylamine was readily oxidised to nitrite in crude extracts of M. capsulatus but its oxidation was stimulated more than 20-fold in the presence of the electron acceptor PMS (Table 3). Although mammalian cytochrome c acted as an acceptor of electrons from hydroxylamine in Nitrosomonas no nitrite was formed (Hooper and Nason, 1965; Rees, 1968). In M. capsulatus also, no nitrite could be detected from hydroxylamine with cytochrome c as acceptor.

The reason for an approximately 3-fold stimulation of nitrite formation by cyanide and the 2-fold stimulation by 8-hydroxyquinoline over the rate with NADH is unclear. Cyanide is a known inhibitor of methanol oxidase activity in M. capsulatus (Bath) (Colby and Dalton, 1976) and was added to see if it would also

Table 3. Oxidation of hydroxylamine to nitrite by crude extracts of Methylococcus capsulatus (Bath)

Reaction mixture Rate of NO£ production (nmoles/ min • mg protein)

No additions 0.77 + 5 ~tmoles NADH 1.66 + 5 lamoles NADH + 1 gmole cyanide 4.2 + 2.5 gmoles phenazine methosulphate 17.3 + 1 gmole 8-hydroxyquinoline 3.3 + 1 gmoles cytochrome c 0

The reaction mixture (1 ml) contained 20 gmoles phosphate buffer pH 7 and 5 gmoles hydroxylamine. All reactions were linear over a 10-min period

inhibit hydroxylamine oxidase activity. The un- expected stimulation of activity would suggest that the oxidase activity is not due to methanol oxidase. To determine whether hydroxylamine was oxidised by the methanol dehydrogenase in this organism, a purified preparation of this enzyme was tested for its ability to form NO~ from NHzOH using PMS as the electron acceptor. No nitrite was formed although PMS was rapidly reduced in the assay by hydroxylamine. Furthermore, 5 mM methanol did not inhibit hydroxylamine oxidation so it appears that hydroxylamine is not oxidised by the methanol oxidase/ dehydrogenase system.

Effect of lnhibitors on Ammonia and Hydroxylamine Oxidation

Oxidation of ammonia to nitrite via hydroxylamine is known to be susceptible to a number of inhibitors (Lees, 1952; Hofmann and Lees, 1953). In particular the involvement of metal ions in the ammonia and hydroxylamine oxidising systems in Nitrosomonas has been inferred from the results of Hooper and Terry (1973) and Hooper and Nason (1965). Metals are also presumed to be involved in the methane oxidising system from a number of methylotrophic bacteria (Colby et al., 1975; Hubley et al., 1975; Ribbons, 1975; Patel et al., 1976; Stirling and Dalton, 1977). In both groups of organisms inhibition by metal chelators has suggested that copper and iron are involved in the initial oxidation process and recently

Table 4. Effect of various compounds including metal binding agents on ammonia, hydroxylamine and methane oxidation in extracts of Methylococeus capsulatus (Bath)

Compound Concentration Inhibition (~)

NH3 (7.5 mM) NH2OH (1 mM) CH4 (0.45 mM)

Thiourea 1 mM 0 0 10 Hydrazine 1 mM 12 52 - Diethyldithiocarbamate 1 mM 9 98 0 c~c(-Dipyridyl 1 mM 3 52 I 8-Hydroxyquinoline 1 mM 85 stimulation 100

(see Table 1) Anaerobic 100 100 100 Acetylene (1 ~ v/v in air) 100 28 100 Methanol (5 mM) 100 0 50 Phenazine methosulphate a (2.5 mM) 100 stimulation - Nitrite 0 0 -

Ammonia oxidation was followed by measuring NH2OH formation. Hydroxylamine oxidation was assayed by measuring NO; formation. Methane oxidation was assayed by measuring methanol formation except when methanol was the inhibitor in which case bromomethane was used as substrate The data for the effect of inhibitors on methane oxidation are taken from Stirling and Dalton (1977) a NADH rapidly reduces PMS non-enzymically so the inhibition of ammonia oxidation may have been due to lack of reducing power for the ammonia hydroxylase

278 Arch. Microbiol., Vol. 114 (1977)

(Tonge et al., 1977) copper has been detected in a purified fraction of the methane mono-oxygenase. Methane oxidation by the methane mono-oxygenase system from M. capsulatus (Bath) differs from that of other methane oxidisers so far identified in that it is soluble and does not appear to be readily inhibited by most metal binding agents (Colby and Dalton, 1976; Stirling and Dalton, 1977). If ammonia was oxidised by the methane mono-oxygenase then it would be expected that a similar pattern of inhibition of b o t h activities would result. The effect of metal binding agents on ammonia and methane oxidation is shown in Table 4.

DISCUSSION

Oxidation of ammonia to nitrite by Methylococcus capsulatus extracts appeared to be catalysed by the methane mono-oxygenase and a hydroxylamine oxidase. In the absence of purified preparation of the mono-oxygenase one cannot unequivocally state that ammonia is oxidised by this enzyme but much of the evidence points to this being so. This evidence is:

1. Both ammonia and methane oxidation required reduced pyridine nucleotide and dioxygen for activity.

2. The oxidations of methane to methanol and ammonia to hydroxylamine were specifically inhibited by acetylene, 8-hydroxyquinoline or methanol.

3. Neither methane nor ammonia oxidation were inhibited by thiourea, hydrazine, diethyldithiocarbamate or ee'-dipyridyl.

4. Methane was a good inhibitor of ammonia oxidation.

The inhibition pattern by compounds listed in Table 4 indicates that both ammonia and methane oxidation proceed via the same enzyme system whereas the effect of theinhibitors on hydroxylamine oxidation differs markedly from their effect on NH3 or CH4 oxidation. The partial inhibition of methane, and complete inhibition of ammonia oxidation by 5 mM methanol is presumably due to the vastly different Km values of the enzyme for methane (160 gM, Colby et al., 1977) and ammonia (96 mM).

The apparent K,, value for ammonia in the extracts and cells was very high and would suggest that ammonia oxidation by these organisms would only be significant when either the ammonia concentration was high or the methane concentration was low. Cer- tainly in some environments this situation could prevail and the methane oxidisers, which are present in high numbers in aquatic environments, could be the major nitrifying organisms.

An inhibition of hydroxylamine formation from ammonium sulphate solutions above 200 mM was

observed and presumed to be due to contaminating methanol, a potent inhibitor of ammonia oxidation (see Table 4), present in this salt. Repeating the experi- ment with ammonium chloride, in which methanol could not be detected, also showed a marked inhibition at this concentration (Fig. 6), and is presumed to be due to substrate inhibition that is a common feature of many enzyme reactions.

The oxidation of methane by washed whole cell suspension of M. capsulatus (Bath) is possible because further metabolism of the oxidation product methanol to CO2 provides the mono-oxygenase with a supply of NADH. Metabolism of other non-growth methane mono-oxygenase substrates such as carbon monoxide, which is oxidised to CO2 only, is only possible if an endogenous supply of NADH can be generated from an exogenously supplied substrate such as formate (Stirling and Dalton, 1976). The observation that ammonia was oxidised by washed whole cells in the oxygen electrode experiments in the absence of other substrates does suggest that further metabolism of the oxidation product(s) can supply the ammonia hydroxylase (sic methane mono-oxygenase) with the NADH required for the hydroxylation reaction.

One possible source of NADH from ammonia oxidation could be hydroxylamine which may give NADH by an ATP dependent reversal of electron flow as observed previously in Nitrosomonas (Aleem, 1966).

Preliminary experiments (H. Dalton, unpublished) do suggest that the oxidation of hydroxylamine by M. capsulatus extracts will reduce NAD + in the presence of PMS and ATP [under identical conditions prescribed by Aleem (1966)] and this will be investi- gated further.

Acknowledgements. The author would like to thank the Sciences Research Council for financial assistance, Dr. John Colby, for reviewing the manuscript and Dr. Jan. Drozd (Shell Research, Sittingbourne, Kent) for the reference on the determination of hydroxylamine.

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H. Dalton: Ammonia Oxidation by Methylococcus 279

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Received April 15, 1977

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Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath