Nitrous Oxide Emission Associated with Autotrophic Ammonium

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1985, p /85/ $2./ Copyright 1985, Americn Society for Microbiology Vol. 5, No. 6 Nitrous Oxide Emission Associted with Autotrophic Ammonium Oxidtion in Acid Coniferous Forest Soil PERTTI J. MARTIKAINEN Deprtment of Generl Microbiology, University of Helsinki, SF-28 Helsinki 28, Finlnd Received 1 My 1985/Accepted 25 August 1985 Aerobic N2 production ws studied in nitrifying humus from ure-fertilied pine forest soil. Acetylene nd nitrpyrin inhibited both NH4' oxidtion nd N2 production, indicting tht N2 production ws closely ssocited with utotrophic NH4+ oxidtion. N2 production ws enhnced by low soil ph; it ws negligible bove ph 4.7. When soil ph decresed from 4.7 to 4.1, the reltive mount of N2-N produced from NH4+-N oxidied incresed exponentilly to 2%. There ws lso some evidence tht N2 formtion ws stimulted by slts (potssium sulfte nd sodium phosphtes). The mximum rte of N2-N production ws.17,ig of N2-N per g of soil per h. When humus ws treted with N2-, N2 evolved immeditely, indicting chemicl formtion, but no N2 ws formed on the ddition of N3-. The mount of N2-N evolved ws.6 to 4.6% of N2--N dded. A high concentrtion of N2- nd low soil ph enhnced chemicl production of N2. There ws no ccumultion of N2- during nitrifiction. The clcultions indicted tht chemicl formtion of N2 ws not the min source of N2 during NH4+ oxidtion. After the ddition of inhibitors of NH4+ oxidtion the soils contined N3, but no N2 ws produced. The results suggest tht enhnced utotrophic NH4+ oxidtion is potentil source of N2 in fertilied cid forest soil. Besides being produced during biologicl denitrifiction N2 is lso formed during NH4' oxidtion. Results of studies with pure cultures of NH4+ oxidiers hve reveled tht.5 to 25% of the nitrogen from N2- (NO2--N) produced evolved s N2-N when NH4+-N is used s substrte (13, 16, 17, 41). N2 production from NH2OH, which is n intermedite in NH4+-N oxidtion (1), cn be much higher. Nitrosomons europe cn produce over 9% of N2-N from NH2OH-N tht is oxidied (41). In well-erted soils N2-N production rnges from.1 to 2% of NH4+-N dded or N3 -N produced (2, 3, 5, 8, 12). When NO2-N ccumultes in soil during NH4+-N oxidtion, even higher mounts of N2-N my be formed (33) vi chemodenitrifiction (9), in ddition to direct N2 emission from cells of NH4+-oxidiing bcteri. Although N2 emission during nitrifiction my not be of generl gronomic importnce, it nevertheless is source of N2 postulted to be linked to the ctlytic destruction of strtospheric oone (1, 4). Most studies of N2 emission from soil, using erobic tmosphere, hve been mde with griculturl soils, nd only few results re vilble from cid forest soils. Goodrod nd Keeney (12) hve mesured N2 fluxes in deciduous nd coniferous forest sites nd hve found tht significntly more N2 is emitted from coniferous thn from deciduous forest floors. They concluded tht the lower ph of the coniferous forest soil might cuse the difference. Robertson nd Tiedje (3) hve studied N2 production in forest soils using intct soil cores in recirculting erobic tmosphere. Their results suggest tht in some cid forest soils mechnisms other thn denitrifiction might be importnt in N2 production. This report presents results from lbortory experiments in which N2 production ws studied in n erobic tmosphere of fertilied cid coniferous forest humus. MATERIALS AND METHODS Soil smples were collected in 1983 from n experimentl Myrtillus pine forest site locted t Tmmel in southern Finlnd. The soil nd the fertilition tretments hve been 1519 described previously (18). The ure fertilition pplied in utumn 1978 ws 2 kg of N/h. Some soil chrcteristics re given in Tble 1. The smples were tken from the orgnic horion of both unfertilied nd ure-fertilied soils. The smpling techniques nd the soil homogenition method hve been described previously (18). Storge nd lbortory mnipultions re described below. Anlyticl methods. Soil ph ws mesured in soil-wter suspensions (1/2 [vol/vol]). When exchngeble mmonium, nitrte, nd nitrite were determined fter the ph ws mesured, 4 M KCl ws dded to the suspensions to obtin 2 M KCl for extrction. Ammonium nd combined nitrte plus nitrite were determined by stem distilltion (6). Nitrite ws mesured by the method of Montgomery nd Dymock (21). Totl N ws determined by the Kjehldhl method (7), nd totl C ws determined with Hewlett-Pckrd 185 C-H-N nlyer. N2 nd 2 were determined with Crlo Erb 42 gs chromtogrph equipped with n electron cpture detector nd Porpk Q 8/1-mesh column (length, 2 m). Detector, injector, nd oven tempertures were 2 to 3, 13, nd 5 C, respectively. The flow of crrier gs (either N2 or 9% rgon-1% methne) ws 3 ml/min. Anlyses re expressed on n oven dry weight bsis. The results were treted by the nlysis of vrince or correltion. The differences between mens of vrious tretments were tested, subsequent to nlysis of vrince, by the Tukey honestly significnt difference method (34). Soil incubtions for mesuring nitrifiction nd N2 production. Incubtions were performed t 14 C in 1-ml bottles, stopped with rubber sept, contining 1.5 to 2 g of soil (oven dry weight). Both fresh nd ir-dried soils were used. In ll experiments soil moisture content during incubtion ws 6% of the wter-holding cpcity (WH. Periodiclly, the bottles were erted fter determintion of the mount of N2 produced. At intervls three replicte bottles for every tretment were nlyed for nitrte nd nitrite (fter mesurement of soil ph; see bove). The prtil pressure of 2 ws lwys >.18. Autotrophic mmonium oxidtion ws repressed by dding nitrpyrin (31) in wter

2 152 MARTIKAINEN APPL. ENVIRON. MICROBIOL. TABLE 1. Description of soils Previous Soil prmeter fertilier tretment ph (in H2) Totl C (%) Totl N (%) C/N rtio None " b Ure ' C 35.1 ± 1.c UABM ± 5.8'.8 ±.11O 34.2 ± 4.6C For the mounts, see Mrtikinen (18). b Vlues re the men ± stndrd devition for nine determintions. C Vlues re the men + stndrd devition for three determintions. solution (5,ug/g of soil; Dow N-serve [18]) or C2H2 (1% [vol/vol]) (15, 16). All nutrients were dded in solution. Soil ph ws djusted by dding CCO3. In the first experiment smples were tken from the plot fertilied with ure, ptite, biotite, nd micronutrients (UABM). It hs lredy been shown tht, on fertilition, nitrifiction ctivity is highest in the plot fertilied with UABM (18). Soil smples for this experiment were tken in mid-jnury 1983 (soil temperture t depth of 5 cm ws to.5. After homogenition soil ws mended with ure (2,ug of N per g of soil) nd stored in polyethylene bgs for 3 weeks t 4 C to ctivte nitrifiction. Fresh soil (2 g oven dry weight) ws plced in bottles, some of which were supplemented with CCO3 (18 mg/g of soil) to obtin higher ph İn the second experiment the sme UABM soil s described bove ws used, but 4,ug of ure-n ws dded prior to 3-week storge t 4 C. The effects of the ddition of K2SO4 on nitrifiction nd N2O production were tested in this experiment. It previously hs been found tht in UABMfertilied soil nitrifiction is highly inhibited on the ddition of 53.5,umol of K2SO4 per g of soil (19). Here, K2SO4 ws dded t 5.35,umol per g of soil. The experiments described bove were done with UABM soil. N2 production during nitrifiction ws lso mesured in ure-fertilied soil not treted with ptite, biotite, nd micronutrients. Smples for this experiment were tken in My Lime, mmonium, phosphte, nd micronutrients were tested by the tretments shown in Tble 2. Becuse nutrients were dded in wter solutions, soil ws ir dried (fter homogenition with Moulinex homogenier), so tht ll solutions could be dded to obtin finl soil moisture content of 6% of the WHC. Soil ph ws djusted by dding CCO3, fter which the soil ws dded to 1-ml incubtion flsks, nd the nutrient solutions were then dded. N2 production from dded nitrite. Chemicl production of N2 from NO2- ws tested by dding NO2- to fresh humus smples. Aerobic soil incubtions were performed s described bove, except tht ech bottle contined 3 g of soil (oven dry weight). Soils were not sterilied before the ddition of NO2-. The possibility tht evolution of N2 ws due to biologicl denitrifiction ws tested by determining the mount of N2 produced from dded NO -. NO2- dditions were, 5, nd 2,ug of N per g of soil. Chemicl N2O evolution t low soil ph ws studied by supplementing smples with H2SO4. Additions of H+ were, 57, nd 21,xmol of H+ per g of soil. The highest H+ level ws equivlent to n oxidtion by utotrophic mmonium oxidiers of 1,5,ug of NH4+-N to NO2--N (2 mol of H+ is produced for every 1 mol of NH4' oxidied). An mount of 1,5,ug of N is equivlent to n NH4+-N ppliction of 2 - e CO 2. t..e e._ 2 _ 2._ S m Z O) :> +t xi _- Z Z o.c E._ 4). o _._w o CZ I- r 6. rsnwi) v ~ON NN l-, +1 +1± en ON WN 'n VI) -OOW)ON --VI ON.- N_O -.. ON. IR'N ~ " Cl- +1 tl ONC rn. ~ N_l_ ri eq en I l V ClC'^N'ON d N oo _ WI)CCl- ~N 'I ON C '. t Cl. Vl_ -,- +l CN cnc-lc t ON%Cr-N O N ON c OtN ON 'f ' VC +l C* Cl 'C e +1l ON-4 - _. +1 t +1 CN +1 ', Cl -_ +1 - NCN N N ON - e'n '. N '. Cl '.q N!' ON ON ^ un - N Cl'N Clx. s C' l Cll- Cl+ - +l - +l Cl-+ _ -. ON l l Cl /Nr'-n N - r~ ~ ZQQQQQm c CZZ ++ O c t C. e. tl O, L.

3 VOL. 5, 1985 N2 EMISSION IN FOREST SOIL 1521 kg/h to the horion (depth, 5 cm; bulk density,.26 g/cm3). RESULTS Nitrifiction nd N2 production. In the first experiment the lg period before nitrifiction ws shorter in soils treted with CCO3 thn in nontreted soils (Fig. 1). Also the mximum rte of nitrifiction nd the mount of (NO3- + NO2-)-N ccumulted ws higher in CCO3-mended soils (P <.1; Fig. 1). NO2-N did not ccumulte during incubtion (its concentrtion remined t <1 p.g/g of soil). After 2-week incubtion, when nitrifiction ctivity ws still low (Fig. 1), soil ph ws 5.2 nd 6.8 without nd with CCO3, respectively. During nitrifiction soil ph decresed but ws lwys higher in soils treted with CCO3 thn in nontreted soils (P <.1; Fig. 1). Although ccumultion of (NO3- + NO2)-N ws higher in soils with lime, production of N2 ws lower in these soils thn in soils without lime (P <.1; Fig. 1). The ddition of C2H2 inhibited nitrifiction nd N2 production (Fig. 1). The mximum rte of N2 production in soils without lime ws.64,ug of N2-N per g of soil per h. In the second experiment mmonifiction incresed soil ph (Fig. 2) during the lg phse in nitrifiction. When nitrifiction begn, soil ph decresed slightly less in soils receiving K2SO4 (P <.1), probbly becuse of the lower nitrifiction ctivity of these soils (P <.5; Fig. 2). In contrst, the ddition of K2SO4 incresed N2 production (P <.1; Fig. 2). Nitrpyrin inhibited both nitrifiction nd N2 formtion in K2SO4-treted soils (Fig. 2). Inhibition ws not tested in soils receiving no K2SO4. In nontreted nd K2SO4-treted soils the mximum rte of N2 production Z _X 6. C < cod soil ph in UABM soil without lime (-C) nd with lime (+C). Ech vlue is the men for three replicte incubtions t 14 C (6%o of the WH. The verge stndrd devitions re 4%, 2% (N2A-N), nd 1% (ph). Symbols: O, ph (-C); *, ph (+C); -, (NOf- + N2-)-N (-C); *-4, (NO3- + N2-)-N (+C), - --, N2-N (-C); *---, N2-N (+C). The ddition of C2H2 is shown by rrows. 7 Incubtion time (dys) FIG. 2. (NO3- + NO2)-N ccumultion, N2 production, nd soil ph in UABM soils treted or untreted with K2SO4 (5.35,umol/g of soil). Ech vlue is the men for three replicte incubtions t 14 C (6% of the WH. The verge stndrd devitions re 9%, 8% (N2-N), nd 1% (ph). Symbols: O, ph (-K2 S4); *, ph (+K2SO4), -, (NO3- + NO2)-N (-K2SO4); *-*, (NO3- + NO2-)-N (+K2SO4); ---, N2-N (-K2SO4); ---, N2-N (+K2SO4). The ddition of nitrpyrin is shown by rrows. ws.91 nd.121,ug of N2-N per g of soil per h, respectively. In the third experiment nitrifiction ctivity in soils without lime ws low, nd no N2 production ws found (Tble 2 nd Fig. 3). In soils with lime nitrifiction ctivity begn fter 15-dy incubtion (Tble 2). After incubtion for 39 dys the soils treted only with lime hd the highest (NO3 + NO2)-N content (P <.1) mong the soils with lime, but fter further incubtion no significnt differences in nitrte ccumultion were detected in these soils. There were differences (P <.1) in N2 production in soils with lime (Fig. 3). When N2 production begn fter 6 dys of incubtion (Fig. 3), soil ph ws different (P <.1) in these soils (Tble 2). In soils with lime the ddition of mmonium sulfte incresed the mount of N2 production (P <.1). The ddition of phosphtes incresed N2 formtion in soils treted with mmonium sulfte nd lime (P <.5) nd in those receiving only lime (not significnt; Fig. 3). Micronutrients did not hve sttisticlly significnt effect on N2 production. The rte of N2 production ws highest (.165,ug of N2-N per g of soil per h) in soils treted with lime, mmonium sulfte nd phosphtes (Fig. 3). Inhibition of both N2 production nd nitrifiction by C2H2 ws lso demonstrted in this experiment (Fig. 3 nd Tble 2). When N2 production begn (fter 65 dys of incubtion; Fig. 3) the ph ws lower in soils treted with mmonium thn in soils receiving only lime (P <.5; Tble 2). At the end of the experiment (fter 74 dys of incubtion; Tble 2) no sttisticlly significnt differences in ph were pprent in soils with lime. Micronutrients did not hve sttisticlly significnt effects on soil ph, nor did phosphtes fter 15 dys of incubtion. Since no nitrte or nitrite ws dded before soil incubtion, N2-N ws lso derived from oxidied mmonium. This is equl to (NO3- + NO2 + N2)-N, provided tht no other nitrogenous gs is produced besides N2. The gs chromtogrphic method used did not detect other N oxides.

4 1522 MARTIKAINEN APPL. ENVIRON. MICROBIOL.,1 cm A'A \ (I 65 7 Incubtion time (dys) FIG. 3. Effects of clcium crbonte (C), mmonium s (N), sodium phosphtes (P), nd micronutrients (M) on N2C duction in ure-fertilied soils. For ppliction rtes, see T Ech vlue is the men for three replicte incubtions t 14WC of the WH. The verge stndrd devition is 22%. Syn 9 /31 Tble 2), possibly by decresing soil ph nd not by stimuulfte lting nitrifiction (Tble 2; see bove). It hs lredy been pler2- found tht the ddition of K2SO4 nd (NH4)2SO4 to cid soils -(6% inhibits (NO3- + NO2-)-N ccumultion (19). In this study, nbols: sodium phosphtes hd no sttisticlly significnt effect on M- - M, no dditions; O-E, C; -M, C + P; --- -, C + N, soil ph nd nitrifiction during N2O production (see bove). -*, C + N + P; -, C + N + P + M. The ddition of C2H2 is shown by n rrow. Also NO2- ws not found. The mount of N2O-N prodluced ws.29 to 3.96% of the NH4+-N oxidied (Tble 2). N2 production nd soil ph. The results of the three experiments described bove suggest tht N2O productiion is enhnced by low soil ph. The rtio of N2-N evolvled to NH4+-N oxidied (see bove) ws plotted ginst soil p: H for different incubtion periods of the three experiments. The rtio of N2O-N evolved to NH4+-N oxidied ws deternnined becuse of vritions in the nitrifiction rtes. Avergm e soil phs for the mesurement period were used. N2 production ws negligible bove soil ph 4.7 (Fig. 4). The rtio of N2-N to NH4+-N oxidied incresed exponentilly with decirese in the soil ph from 4.7 to 4.1 (Fig. 4). Soils receiving K,2SO4 were excluded from this nlysis. The ddition of K enhnced N2O production, even though K2SO4-treted soils hd higher ph (Fig. 2). Chemicl production of N2 from dded NO2-. OIn the ddition of NO2-, N2O production ws mnifest in both unfertilied nd UABM-fertilied soils (Fig. 5). Soi 11 ph below 4.7 hd negligible effects on N2-N production t NO2--N concentrtion of 5,ug/g of soil but enhncedi it t concentrtion of 2,ug of NO2--N per g of soil (Fig. ' 5 nd 6). No N2O ws produced from dded NO3-. The mouint of N2O-N produced from dded NO2--N during the experiment (Fig. 5) rnged from.63 to 2.5% nd 2.55 to 4.58% when 5 nd 2,ug of NO2--N per g of soil, respectively, were dded. Mximum rtes of N2-N production occurred immeditely fter the ddition of NO2 (Fig. 5), nd these were much higher thn the mximum rtes in experiments in which N2O ws produced during nitrifiction (see bove). The rtes of chemicl N2 production decresed over the experimentl period (Fig. 5). After incubtion the mount of NO2-N remined below.5,ug/g of soil. DISCUSSION The results of this study show tht N2O my form in nitrifying forest humus under well-erted conditions. N2O production ws closely ssocited with utotrophic NH4' oxidtion nd ws enhnced by low soil ph. These results support the observtions of Robertson nd Tiedje (3), who found tht in some forest soils N2 might not be produced by denitrifiers. They hve found tht in some intct forest soil cores N2 is produced only in the bsence of C2H2. It is interesting tht these soils hd lower phs thn those tht produced N2 only in the presence of C2H2 (4.6 versus 5.2). Goodrod nd Keeney (12) hve found tht coniferous forest site produces more N2O in situ thn does deciduous site. Soil ph ws lower in the coniferous site (4.5 versus 5.). The effects of nutrients on N2 production must be considered in terms of nutrient effects on nitrifiction nd soil ph. A low soil ph fvored N2 production (Fig. 4). In K2SO4-treted soils N2 production nd soil ph were higher, but (NO3- + NO2)-N ccumultion ws lower thn tht in nontreted soils (Fig. 2). These results indicte tht 75 K2SO4 hs stimultive slt effect on N2 production. (NH4)2SO4 lso incresed N2-N production (Fig. 3 nd The slight increse in N2 production becuse of phosphtes might thus be slt or nutrient effect. N2O production by NH4+ oxidiers my vry with culturl I m EN 1-25~ 2_ S \,1 _ r, \ O4 4. e O\ 4.S 5 Soil ph Soil ph FIG. 4. Percentge production of N2-N from NH4+-N oxidied t ph 6.1 to 4.1. The vlues were clculted by the results shown in Fig. 1 (-), Fig. 2 (), Fig. 3 (l), nd Tble 2 (O). The insert shows the percentge of production of N2-N from NH4+-N oxidied t ph 4.6 to 4.1. In y = x , y = log% N2-N from NH4+-N oxidied, x = soil ph, r = (P <.1, df = 7).

5 VOL. 5, 1985 N2 EMISSION IN FOREST SOIL IU 3 2 ~~~~~~~~~~~~~~ 1~~~~~~~~~~~~~~~~~~~ FIG Incubtion time (hours) Cumultive production of N2-N from NO,--N in unfertilied (, ) nd UABM-fertilied (I, *) soils fter different dditions of H+ s H2SO4. Open symbols, 5,ug of N2--N per g of soil; closed symbols, 2,ug of NO2--N per g of soil. Added NO3--N (5,ug of N per g of soil) produced no N2. Ech tretment hd two replictes (mens re shown). Incubtions were t 14WC (6% of the WH. conditions, becuse it is dependent on the buffer systems used (16, 41) nd on cell ge. Old cells produce more N2 thn young ones (41). This observtion my be of some importnce in this study, in which N2 production occurred t the end of the experiments when most cells were ged. However, t or bove soil ph 4.7, N2 production ws negligible even fter long incubtion. Thus, low soil ph is essentil to N2 production. Autotrophic NH4' oxidiers hve been reported to produce N2 in both oxidtive nd reductive processes (16, 29). It hs been suggested tht N2 evolves from lbile intermedite between NH2OH nd NO2- (possibly N22H2) during NH4+ oxidtion (1, 29). A low oxygen level supports._ 'NI 8 2.5k h Soil ph FIG. 6. Mximum rtes of chemicl N2O-N formtion from dded NO2--N t different soil ph clculted by the results shown in Fig. 5. Symbols s described in the legend to Fig. 5. N2 formtion (13, 16, 17), indicting prticiption of reductive enyme system. Recently, Poth nd Focht (27) hve shown tht Nitrosomons europe produces N2 minly by NO2 reduction (nitrifier denitrifiction) nd hve pointed out tht simultneous NH4+ oxidtion is needed to trnsfer electrons from NH4' to NO2- (NO3- ws not reduced). This coupling of NO2- reduction to oxidtive processes cn explin why C2H2, which inhibits the first step in NH4' oxidtion, hs been reported to inhibit N2 production from NH4' in the presence of NO2- (16). C2H2 does not inhibit NH2OH oxidtion (15, 16), which explins why it does not inhibit N2 production from NH2OH (16). The observtions from this study tht no N2 is produced on the ddition of inhibitors, even when the soil contins NO3- for possible denitrifiction by heterotrophic bcteri, support nitrifier denitrifiction. However, the fvorble effects of low soil ph nd slts cnnot be unmbiguously explined by these results. Enhnced N2 production t low ph ws not detected in studies with pure cultures of Nitrosomons europe, but sterile soil ws found to gretly stimulte this process (16). It is not known wht effect low ph or slts hve on N2 production by Nitrosospir spp., which probbly is responsible for NH4' oxidtion in fertilied forest soil (2). It is possible tht Nitrosospir enymes tht ctlye the oxidtion of the intermedite between NH2OH nd NO2 re sensitive to low ph nd some slts t high concentrtions. If the intermedite (N22H2) ccumultes in cells, N2 my be formed during hydrolysis of N22H2 (29). The lbile intermedite theory is not supported by the results of Poth nd Focht (27). They hve pointed out tht the use of NO2- s terminl electron cceptor conserves 2 for the first step of NH4+ oxidtion. This oxidtion needs moleculr oxygen. Nitrite, which is toxic end product, cn lso be removed by its reduction to N2. In this study the oxygen hypothesis did not fit well into the observed N2 production pttern. For exmple, in the first experiment (Fig. 1), oxygen depletion probbly ws greter in soils with lime becuse of their higher level of nitrifiction

6 1524 MARTIKAINEN (nd respirtion of heterotrophic microbes?), nd N2 formtion thus must be higher in soils with lime thn in soils without lime. It is more likely tht under the extreme conditions cused by low soil ph (or slts) Nitrosospir spp. re sensitive to N2 nd will eliminte N2 by reducing it to N2. Besides direct evolution from cells of NH4' oxidiers, other mechnisms lso exist for the production of N2 in erted soils. Biologicl denitrifiction in nerobic microsites, even during incubtion of nonwterlogged soil in n erobic tmosphere, my occur. This possibility could not be totlly excluded in this study becuse nonsterile soil ws used. However, when nitrifiction ws inhibited by nitrpyrin or C2H2, N2 production cesed, lthough the soil still contined NO3. It ws to be expected tht NO3 would diffuse from soil solution into nerobic microsites. The possibility tht both C2H2 nd nitrpyrin inhibit denitrifiction is unlikely. It hs been shown tht the ddition of 5 pug of nitrpyrin per g of soil does not inhibit denitrifiction (14). One possible explntion is tht denitrifiers in the microsites use N2 but not NO3 s electron cceptor (37, 39). A Km vlue s low s.2 mg of 2 per liter t 27 C hs been found for Nitrosospir sp. in forest soil (2). It is possible tht N2 is produced by NH4+ oxidiers in microenvironment nd tht prt of it is reduced to N2 by denitrifiers in the sme niche. Denitrifiction is known to occur t 2 concentrtions of.1 to.2 mg/liter in sewge, ocen, nd pure cultures (11). Some denitrifiers cn use both 2 nd NO2 s electron cceptors simultneously (39). Png nd Cho (25) recently hve reported tht biologicl denitrifiction of dded NO2- cn occur in the L-H lyer of coniferous forest soil, even under erobic conditions. In soil, N2 is lso produced from NO2- by chemicl denitrifiction (9). High orgnic mtter content nd low soil ph fvor this process (22, 23); forest soil my thus be good environment for chemicl denitrifiction. In this study the mount of N2-N ws.6 to 4.6% of the mount of N2 -N dded. This grees with observtions mde with other types of soil (4, 23, 28, 32). In this study high concentrtion of N2 t low soil ph enhnced chemicl N2 formtion. The rtes of chemicl N2 formtion immeditely fter the ddition of NO2- were much higher thn those of N2 formtion during nitrifiction, but when NO2- concentrtions hd decresed (finl concentrtions, <.5 jig of N per g of soil) the rtes were of the sme mgnitude. In the second experiment (Fig. 2), chemicl N2 formtion could not explin the rte of N2 production. In soils tht received K2SO4, N2-N nd NO3-N productions verged 2.2 nd 7.,ug of N per g of soil per dy, respectively. The mount of N2-N produced from NO2-N did not exceed 5% (see bove). As estimted by the mounts of N2 nd NO3 formed, the verge rte of N2 -N production should be t lest (1/5 x 2.2) + 7. = 51,ug of N per g of soil per dy, to provide N2 for chemicl denitrifiction nd nitrite oxidtion during the whole 4-dy period. Over 2,,ug of NH4+-N thus must be oxidied during this period. In some K2SO4-treted soils mmonium oxidtion nd N2 production were inhibited by nitrpyrin during the 95 dys of incubtion (dt not shown in Fig. 2). The mmonium ccumultion rtes determined for these soils showed tht the highest possible mount of NH4+-N oxidtion ws bout 6 pg of NH4+-N per g of soil. Furthermore, fter 95 dys of incubtion the cumultive mount of (NH4+ + NO3- + NO2- + N2)-N ws higher in soils without nitrpyrin thn in soils with nitrpyrin (673 nd 69 jig of N per g of soil, respectively). With high chemicl N2 APPL. ENVIRON. MICROBIOL. production, the mount of N should be lower in the former thn in the ltter soils becuse N2 is minor component of the nitrogenous gses tht evolve during chemicl denitrifiction (4, 23, 28, 35). It must lso be mentioned tht the reduction of NO - to N2- could not provide enough N2- to mke the observed N2 formtion chemicl process. So, in these soils, the chemicl N2 formtion seems to be of reltively limited importnce in the totl N2 flux. The long-term lbortory incubtions in this study indicte potentil for erobic N2 production in nitrifying cid soil. A comprison of this potentil with the denitrifiction potentil in the UABM soil with nerobic incubtions nd n optimum NO3- level (38) showed tht the mximum erobic N2-N production (.17 Lg/g of soil per h) ws bout 5% of the denitrifiction potentil. The production of N2 by forest soil Nitrosospir spp. must be exmined t vrious conditions for better understnding of the mechnisms of N2 production. Additionl work must lso be done to evlute increses in in situ evolution of N2 in terms of fertilition, which stimultes nitrifiction in cid forest soil. Evidence from other types of soil with higher phs indicted tht mmonium or ure fertilition increses N2 emission in situ nd tht this N2 is produced by mmonium oxidiers (5, 9). Ure fertilition is likely to increse N2 evolution from coniferous forest soil becuse ure cn stimulte nitrifiction for mny yers (18). It recently hs been discussed how lime pplied to forest soil to neutrlie cid deposition ffects tree growth (26) nd soil microbes (24, 36). It is known tht the ddition of lime cn stimulte nitrifiction in cid forest soil (24). The results of this study indicte tht the ddition of lime my be environmentlly hrmful becuse of n increse in N2 evolution, especilly when nitrifiction is stimulted by lime but the soil ph remins below 5. LITERATURE CITED 1. Anderson, J. H The metbolism of hydroxylmine to nitrite by Nitrosomons. Biochem. J. 91: Aulkh, M. S., D. A. Rennie, nd E. A. Pul Acetylene nd N-serve effects upon N2 emission from NH4' nd NO3 treted soils under erobic nd nerobic conditions. Soil Biol. Biochem. 16: Blckmer, A. M., J. M. Bremner, nd E. L. Schmidt Production of nitrous oxide by mmoni-oxidiing chemoutotrophic microorgnisms in soil. Appl. Environ. Microbiol. 4: Bollg, J.-M., S. Dryml, nd L. T. Krdos Biologicl versus chemicl nitrite decomposition in soil. Soil Sci. 116: Breitenbeck, G. A., A. M. Blckmer, nd J. M. Bremner Effects of different nitrogen fertiliers on emission of nitrous oxide from soil. Geophys. Res. Lett. 7: Bremner, J. M Inorgnic forms of nitrogen, p In C. A. Blck (ed.), Methods of soil nlysis, prt II. Americn Society of Agronomy, Mdison, Wis. 7. Bremner, J. M Totl nitrogen, p In C. A. Blck (ed.), Methods of soil nlysis, prt II. Americn Society of Agronomy, Mdison, Wis. 8. Bremner, J. M., nd A. M. Blckmer Effects of cetylene nd soil wter content on emission of nitrous oxide from soils. 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