Denitrification by fungi

FEMS MicrobiologyLetters 94 (1992) 277-282 © 1992 Federation of European MicrobiologicalSt~cieties1)378-11)97/92/$05.110 Published by Elsevier

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2015.08.05

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Denitrification by fungi H i r o f u m i S h o u n ~, D u - H y u n Kim ~, H i r o o U c h i y a m a h and J u n t a S u g i y a m a c "hlstitute of Applied Biochemistr)'. Unil'ersity of Z~ukuba. Tsukutnl. Jalnm. ~ National Inxtitute for Enciromnemal Re.~earch, Tsukuba. Ibaraki, Japan. and ' Institute of Applied Microhiok),(,~.'. Tile Unicersio"~t" Tokyo. Btmk.vo-ku. Tokyo, Jalnm

Received 21)April 1992 Revision received6 May 1992 Accepted 6 May 1902 Key words: Denitrification; Fungal denitrifier: Nitrous oxide 1. S U M M A R Y Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N_,O), a n d / o r dinitrogen (Nz). Several strains cou:d reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nttrite. Several fungi could also form N:. When ItS]nitrite was used as substrate for the N.,-forming denitrification, 15N20, 15NO, and 14NISN were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen. 2. I N T R O D U C T I O N It has long been believed that denitrification occurring in soil and other environments depends on bacteria oo!y [1]. We recently found, however, Correspondence to: H. Shoun. Institute of Applied Biochem-

istry. Universityof Tsukuba. Tsukuba. lbaraki 305. Japan.

that the hyphomycetous fungus Fusarium ox3"sporum (MT-811) exhibits a potent denitrifying activity [2]. Nitrate or nitrite was stoichiometricaUy converted to nitrous oxide (N,_O) under anaerobic conditions. This was the first demonstration of distinct denitrifying ability due to a fungus. We further showed that a cytochrome P-450 (P-450), tentatively termed P-450dN m [3], is involved in the process. The e D N A analysis revealed its unique features [3]. The epochal function of P450~N m is now being clariu,.d (manuscript submitted). In the present study we have investigated the occurrence of P-450~lNm (nitrate/nitrite-inducible P-450) in a range of fungi and we show that many fungal strains exhibit denitrifying activity.

3. M A T E R I A L S A N D M E T H O D S Each fungal strain was precultured as in the case of F. o a y s p o m m MT-811 [2] in liquid ntedium containing 1% glucose, 0.2% peptone, and inorganic salts [2] (pH 7.5). After 4 - 5 days 30 cm 3 the culture was transferred to 120 cm 3 of fresh medium containing 5 mM NaNO2 or 10 mM NaNO 3, in a 500 cm 3 Erlenmeyer flask with side

278 arms [2]. After inoculation, the flask was degassed and flushed by helium, sealed, and then incubated at 26.5°C on a rotary shaker. At each incubation time the upper space gas was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) as reported [2]. Most of strains were obtained from IFO (Institute for Fermentatl.on, Osaka) and IAM (Institute of Applied Microbiology, The University of Tokyo). P-450 was induced and detected spectrophotometrically, as reported [4]. [L~N]Nitrite and nitrate (99 atom%; sodium salts) were obtained from Cambridge Isotope Laboratories, USA.

4. RESULTS AND DISCUSSION Many fungal strains were shown to be capable of evolving N20, nitric oxide (NO), a n d / o r dinitrogen (N 2) when they were anaerobically incubated with nitrite, as shown in Table 1. In most cases the main product was N20. All strains of F. oxyspomm examined produced N20 from r,itrite with the exception of the strain IFO 9967. The following fungi also exhibited marked denitrifying activities: Gibberella fujikuroi, Fusarium linL Fusarium decemcelhdare, Fusarium solani, Cylindroce.rpon tonkinense, Trichoderma hamatum, Chaciomhun sp., and Talaromyces flavus. Some of these fungi also formed N,O from nitrite (Table 1). We previously observed that F. oxysporum MT-811 rapidly produced N20 from nitrate when the air in the incubation flask was not replaced by helium before incubation [2]. In contrast, a long lag time was required for N20 to be formed from nitrate when the air was replaced by helium (Tanimoto et al., unpublished observation). This situation seemed also to be the case with other strains of F. oxysporum, and was confirmed with F. oxysporum IFO 30705 (Table 1). The rapid nitrate utilization might depend on oxygen initially present in the flask, although the denitrification was completely inhibited by a continuous supply of oxygen [2]. The oxygen requirement prior to the initiation ot nitrate reduction was in contrast to the reduction of nitrite that did not

require oxygen. The difference in oxygen requirement between the reductions of nitrate and nitrite is to be elucidated. We previously showed that fungal taxa closely related to F. oxysporum, such as F. lini and G. fujikuroi, also produce a nitrate/nitrite-inducible P-450 that is immunologieallysimilar to P-450dNm [4]. These P-450-containing fungi were also shown to exhibit marked denitrifying activities (Table l). However, some denitrifiers did not produce P450,.INIR. On this basis fungal denitrifiers can be divided into two groups depending on the involvement of P-450 in their denitrifying process. The occurrence of P-450dNm among fungi is of evolutional interest, since horizontal transfer of the P-450 gene from bacteria to fungi has been suggested [3]. Figure 1 shows results on anaerobic incubation of nitrite with F. solani IFO 9425. N 2 was not formed when peptone was omitted from medium, and the N20 evolution was greater in the absence of peptone. Addition of ammonium ions instead of peptone did not increase N2 production (not shown). As shown in Fig. 2, Cylindrocarpon tonkineme IFO 30561 exhibited a potent denitrifying activity unlike the result in Table I. The activity was higher at pH 8.0 than at pH 6.0. It seems that the discrepancy between the results in Table l and Fig. 2 depends mostly on the difference in the growth of seed culture. Figure 3 shows a typical result on GC-MS analyses of NO, N20, and N2 that were formed during anaerobic incubations of [tSN]nitrite with F. solani or C. tonkb~ense. MS peaks with m / z of 46 and 31 were obtained, respectively, for N20 and NO, demonstrating that all nitrogen atoms in these denitrification products were derived from the [~SN]nitrite. N20 ( m / z = 46) was also obtained from [tSN]nitrate as the sole product (not shown). In contrast, m / z = 29 was the main peak for N 2, indicating that a hybrid species, 14N 15N, was formed. It would appear from results in Fig. 1 that nitrogen compounds in peptone, such as amino acids, supplied the second nitrogen atom. Such 'co-denitrification' has also been observed with F. oxyspomm where one nitrogen atom from azide or salicylhydroxamic acid and one from nitrite were used in the production of N20 [5].

279 Table I Nitrogen oxides and dinitrogen evolution by anaerobic incubations of nitrite of nitrate with fungi, and production of P-450d~ m in each fungus. Each fungus was incubated anaerobically in either nitrite or nitrate containing medium for 7 days. Total amounts of each gas evolved per each flask are indicated. The initial amounts of nitrite and nitrate added were 11.75 mmol and 1.5 mmol. P-450 was detected as reported [4]. The taxonomic arrangement for Fttsarium and its teleomorphs is based oa Fnsariam phylogenetic trees [61. Substrate

Strains

Nitrite product ( # m o l l

Nannizzia gypsea Talaromycesflm'as Fnsarium oxysport(m

IAM 12722 IAM 13768 I F O 30705

Fusariam oxysporu,n Fusariam oxysporam Fusarium oxysponon Fusarium oxysporum Fusarium oxysporum Fusariam o.wsponon GibbereUa fiejikaroi Fasarium lini Gibberella zeae Calonectria ilicicola Calonectria kyotensis Cakmectria rigidiuscula Fasariam decemcelhdare Nectria c#mabarina Nectria en~bescens Neetria peziza Fusariam solani Fusariam solani Fasariam solani Cylindrocarpon destnwtans Cylindroearpon selerotigenton Cylindroearpon tonkbwnse Hypoerea maroiatut Hypocrea nigricans Trichoderma hamamm Trichoderma harzianam Trichodenna ciride Chaetomh#n funicola Chaetomhon sp. Chaetomi~m~ sp. Hypomyces trichothecoides Nearospora erassa Glonium lineare Shiraia bambnsieola Botrytis cinerea Rhodosporidiam toraloides

I F O 9968 ! F O 30700 I F O 30710 IFO 9967 IAM 5(}{)9 IAM 5051 IAM 81148 IAM 511{18 I F O 9462 i F O 9131 I F O 8962 I F O 30918 I F O 31657 I F O 311679 I F O 31035 I F O 92118 I F O 9425 I F O 9955 1FO 311193 I F O 8239 1FO 31855 I F O 30561 IAM 125111 IAM 12499 IAM 125115 IAM 125116 1AM 61142 IAM 13491 IAM 8011 IAM 81117 I F O 6892 I F O 61)67 IAM 12749 IAM 12521 IAM 5126 IAM 13469

Nitrate

No

N20

N,

N20 "

143 - ~'

2 20 236

15 -

121 -

-

33{I 241 348

I

342 246 3711 311 65 3 6 3 1115 II

P-450

-

2(15 I0

-

14 26

7 11 27 -

15

9

14 -

146 13 258 7 12 69 4 15 153 4 4 126 15 166 I II 2 2 2 3

64 13 13 6 16

48

13

2411

12

178

24

18

N O and N 2 gases did not form from nitrate at all in every case. b not detected; Vacant space means "'not examined". ¢ Air in the upper space was not replaced by helium. Following strains did not evolve any nitrogen gases at all from nitrite: Mucor pasillus IFO 4579, Rhizopas jm'anicus IFO 6028, Rhizopus oryzae I F O 4706, Galactomyces reessii I A M 12479, Monascas anka 1AM 8001, Aspergillas nidnlans IAM 2155, EupeniciUium crutosunl 1AM 13739, Penicillium expansam 1FO 5854. Hyponlyces aarantias IFO 6847, Micronectriella cucumeris I F O 30005. Micronectriella nicalis I F O 7432, Micronectriella niL'alis I F O 7446. Filobasidinm floriforme YK 357.

280 !

200

NzO

400

IOO

i

N20

i

i

i

~..o.-.--o.

i

o

,o-

200

0

~ oc el

z

60

N2

c~._..~o"

~ 8c

2O 40 0

0

;

2 4 6 INCUBATION TIME (DAY)

Fig. I. N20 (upper) and Nz (lower) evolution during anaerobic incubation of nitrite with Fus*rium sohmi IFO t.1425. F. solalli was incubated with nitrite as described in MnTl~Rtat s ANt) MHHOl)S. Symbols: c,, complete medium; e. without peptone.

From the result it can be calculated that nitritenitrogen was stoichiometrically converted to N , O and N 2 u p o n incubation with C. tonkmense at pH 8.1) (Fig. 2). It was s h o w n that several fungal strains o t h e r than F. oaTsporum can evolve significant a m o u n t s of N_,O from nitrite, a n d a few strains could also utilize nitrate as a substrate. F u r t h e r , it was s h o w n that fungal denitrification exhibits a u n i q u e feature, i.e. utilization of nitrogen c o m p o u n d s o t h e r than nitrite or nitrate. V a r i o u s c o m p o u n d s , such as a m i n o acids, aniline, and azide, could serve as nitrogen d o n o r s (Kim et al., u n p u b l i s h e d observation). It is o f mechanistic interest that F. solani and C. tonkh~ense f o r m e d N 2 as the co-denitrification p r o d u c t w h e r e a s F. oxysporum forms N_,O [5]. T h e recovery of n i t r i t e / n i t r a t e - N in N 2 0 , N O , and N_, was lower in m a n y cases e x a m i n e d than that by F. o x y s p o n m l MT-811 [2]. However,

i

°o

i

2

,;

'

INCUBATION TIME (OAY) Fig. 2. N_,O (tipper) and N., (lower) evolulion from nitrite by ()'!indrocarpon tonkb~ense IF() 311561. C tonkbtense was incubated anaerobically with nitrite in a 3lX)-ml Erlenmeyer flask (space volume, 300 ml) containing 1811ml of the same medium as in Fig. I. htoculum size was I/4 against the final volume. "lk~tal amount of nitrite initially added was II.tJ(I retool. Tile medium contained I11 mM potassium phosphate. Symb~ls: o, pll 8: e. ptl 6.

NO

20

NzO

40 30

N2

50 20

40

m/z

Fig. 3. Mass spectra of NO (left). N20 (middle), and N, (right) evolved by anaerobic incubation of [tSN]nitn!'e with F." sohlni IFO 94?5. Incubations were done as in i-ig. 1. and evolved gases were analysed by GC-MS with a Shimadzu gas chromatograph-mass spectrometer GCMS-9000C. as reported [2]. N2 (m/z = 28) might be derived from contaminated air.

281 further studies will find more preferable conditions for the recovery and the utilization of nitrate in each case, as was the case with F. oxysporum or C. tonkinense. The best condition might vary depending on species. Most of the distinct den(triflers found in this study belong to Fusarium, and the related tcleomorphic genera (Gibberella and Nectria) and anamorphic genera (Cylindrocarpon and Trichotlerma). Our results seem to be consistent with the phylogeny of Fusarium species determined by r R N A sequence comparison [6]; i.e. Fusarium spp. and their related teleomorphs do denitrify, whereas Monographella nit'alis ( -~ Micronectriella nicalis) and its anamorph Gerlachia nicalis (=Fusarium nit'ale) t7,8] do not. The production of P-450dN m was restricted to a few, closely related species, i . e . G . ]iljikuroi, F. oxyspornm, and F.

lini. It is noteworthy that only Talaromyces flat'us, among distinct denitrifiers, belongs to a different class (Plectomycetes). It therefore seems possible that further screenings will find more fungal denitrifiers. It would appear from the present results that denitrifying activities are generally distributed among soil fungi, N_~O concentration is now increasing in the atmospheric air; however, the source of N~O evolution is not known. The present results have suggested that fungal denitri-

fication contributes, at least in part, to the increase of the greenhouse effect gas.

ACKNOWLEDGEMENTS We thank Dr. T. Hasegawa, Director of the Institute for Fermentation, Osaka, for supplying the fungal cultures. This work was supported by the University of Tsukuba Project Research (A), and Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

REFERENCES [I] Ferguson,S.J. (It)87) Trends Biochem.Sci. 12, 354-357. [2] Shoun, tl. and Tanimoto, T. lit)91) J. Biol. Chem. 266, 11078-11082. [3] Kizawa,tl., Tomura, D.. Oda, M., Fukamizu. A., tloshino. T., Gotoh, O., Yasui. T. and Shoun. tl. (1991) J. Biol. ('hem. 266, 10632-111637. [4] Shoun, H.. Suyama, W. and Kim, D.-H. (1991) Agric. Biol. ('hem. 55, 5q3-St)6. [5] Tanimoto, T., tlatano. K., Kim, D.-H., Uchiyama. tl. and Shoun, tl. I It~t,~2)FEMS Microbiol. Left.