Letters in Applied Microbiology 1995, 21, 16-19

Nitrification in vitro by a range of filamentous fungi and yeasts

A.M.K. Fallh and M. Wainwright Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

MOM/127: received 30 January 1995 and accepted 15 February 1995

A.M.K. FALIH AND M. WAINWRIGHT. 1995. A wide range of fungi including yeasts, growing on Czapek Dox medium, nitrified added ammonium and the ammonium released by urea hydrolysis. Phanerochaete chrysosporium and Hymenoscyphus ericiae were the only fungi tested which failed to nitrify. A soil yeast (isolate 1) was the most active nitrifier of ammonium in vi tro, forming 0.80 p g nitrate mg- ' biomass over the 7 d incubation period.

INTRODUCTION

Although the ability of fungi to hydrolyse urea and nitrify is well established (Killham 1986), most of the studies to date have been limited to species of Aspergillus, Fusarium and Penicillium (Eylar and Schmidt 1959; Hirsch et al. 1961; Wainwright and Grayston 1987). As a result little is known about the species diversity of nitrifying fungi, yeasts in particular having been neglected. In addition, workers have employed a variety of different media and growth con- ditions in their studies on fungal nitrification, making it impossible to determine the relative activities of individual species in these processes. The aim of this report was to attempt to correct these deficiencies by selecting a single medium (Czapek Dox) to study the ability of a range of fungi to: (1) nitrify ammonium; and (2) hydrolyse urea and nitrify the released ammonium to nitrate.

MATERIALS AND METHODS

Yeasts and filamentous fungi were grown on Czapek Dox agar (Oxoid) for 7 d at 25°C. Spore suspension (1 ml) was then inoculated into liquid Czapek Dox medium (Oxoid, 100 ml in 250 ml Erlenmeyer flasks), adjusted to pH 6.8 with 2 mol I - ' NaOH. The following fungi were included: Phanerochaete chrysosporium Burdsall, Hymenoscyphus ericiae (Read) Korf and Kernan, Pythium oligandrum Drechsler, Rhizomucor pusillus (Lindt) Schipper, Aspergillus oryzae (Ahlburg) Cohn, Mucor strictus Hagem, Fusarium solani (Martius) Saccardo, Penicillium chrysogenum Thom, Aspergillus niger Van Tieghem, Cladosporium herbarum (Persoon ; Fries) Link, Penicillium notatum Westling, Peni- cillium expansum Link ex S.F. Gray, Geotrichum candidum Link, yeast isolate 1, yeast isolate 2, Williopsis californica

Correspondence to: Dr A . M . K . Falih, Department of Molecular Biology and Btotechnology, Unrversity of Shefield, Shefield SIO ZUH, UK.

(Lodder) Krasil'nikov and Saccharomyces cerevisiae (Hansen).

The medium was amended with either urea (final con- centration IOOO pg m1-l) or ammonium (final concentra- tion 500 pg m1-I) as sole N-source. The flasks were then incubated with shaking (100 rev min-') at 25°C for 7 d. After filtration through pre-dried Whatman No. 1 filter paper the concentration of nitrate in the medium was deter- mined using the Orange 1 method (Middleton 1959) and medium pH was determined with a glass electrode. The weight of fungal mycelium retained by the filter paper was then determined (after drying to constant weight at 46°C) as a measure of fungal mycelium biomass. Flasks were set up in triplicate and uninoculated flasks were included to account for any non-biological urea hydrolysis or ammon- ium oxidation.

RESULTS AND DISCUSSION

Figure l(a) shows that, with the exception of Phanerochaete chrysosporium and Hymenoscyphus ericiae, all of the fungi oxidized ammonium to nitrate and nitrified the ammonium produced by urea hydrolysis to torm nitrate. Net nitrate production varied among the fungi depending on whether the ammonium was provided directly or as a hydrolysis product of urea. Generally, however, higher concentrations of nitrate were found when ammonium rather than urea was provided as the N-source.

Although, following fungal growth, the pH of media containing ammonium was generally lower than that of media containing urea (Fig. lb), the differences seen in nitrate formation do not directly correlate with pH. For example, while more nitrate was formed by yeast isolate 1 in urea containing medium, the pH of the medium was similar (at around pH 3) to the medium containing ammon- ium. The pH of the medium in which H. ericiae was growing was particularly high indicating active urea hydro- lysis.

6 1995 The Society for Applied Bacterology

FUNGAL NITRIFICATION 19

n c 0.6

0.5

0- C U E

n

5 p:

K

0 p:

u

Fig. 1 (a) Nitrification (net nitrate production) by a range of filamentous fungi and yeasts; 0, nitrification of ammonium; 4, nitrification of ammonium released by urea hydrolysis. (b) pH of medium containing ammonium 0 and urea

Yeast isolate 1 was the most active nitrifier of ammonium in vitro, forming 0.80 pg nitrate mg - ’ biomass over the 7 d incubation period. Most of the other species tested formed approximately 0.1-0.3 pg nitrate mg- biomass in the ammonium and urea amended media over this period (Fig. la). Pythium oligandrum, F. solani, P. chrysogenum, P. notatum and soil yeast W. californica produced 040, 0.40, 0.54, 0-50 and 0.44 pg nitrate mg-’ biomass respectively in the presence of added ammonium. The yeast-like (G. candidum) and yeast isolate 2 were average in this respect.

The most efficient oxidizers of ammonium were not nec- essarily the best oxidizers of ammonium released by urea hydrolysis and vice versa. The difference probably results from the higher pH in the medium in which urea hydro- lysis occurred.

Since most environments contain only small amounts of carbon, it is unlikely that the rates of nitrification observed using Czapek Dox medium would be relevant to nitrifica- tion in, for example, soil. However, in vitro studies such as this do at least indicate the potential ability of fungi to nitrify; a fungus which is incapable of nitrifying in vitro is unlikely to do so in the environment.

The results show that a wide range of fungi and yeasts are capable of nitrifying added ammonium, as well as nitrifying ammonium produced by urea hydrolysis, using Czapek Dox as a nutrient source. Of particular interest is the role of yeasts and yeast-like fungi like G. candidum in the process, since in the past only the role of mycelial fungi in nitrification has been emphasized. It is also noteworthy that two fungi P. chrysosporium and H. ericiae, although growing well in Czapek Dox medium, failed to nitrify. This observation could be useful in elucidating the biochemistry of fungal nitrification, since it would be interesting to determine what biochemical features these two fungi lack when compared with actively nitrifying fungi.

ACKNOWLEDGEMENT

AMKF wishes to thank the Government of the Kingdom of Saudi Arabia for financial support.

REFERENCES

Eylar, O.R. and Schmidt, E.L. (1959) A survey of heterotrophic microorganisms from soil for activity to form nitrite and nitrate. JournaI of General Microbiology 20,473481.

Hirsch, P., Overrein, L. and Alexander, M. (1961) Formation of nitrite and nitrate by actinomycetes and fungi. Journal of Bacte- riology 82, 442-448.

Killham, K. (1986) Heterotrophic nitrification. In Nitrification ed. Prosser, C.J.I. pp. 117-126. Oxford : Information Retrieval Limited Press.

Middleton, K.R. (1959) The use of the orange 1 method for determining nitrates and a comparison of the phenol-sulphuric acid method for certain soils in northern Nigeria. Journal of the Science of Food and Agriculture 10, 218-224.

Wainwright, M. and Grayston, S.J. (1987) Nitrification and sulphur oxidation by Aspergillus Javus growing in medium con- taining reduced nitrogen and sulphur. Transactions of the British Mycological Society 88, 309-31 5.

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21, 16-19