Ammonia Fermentation, a Novel Anoxic Metabolism of Nitrate by Fungi*

The induction of fungal denitrification by Fusarium oxysporum requires a minimal amount of O 2 , although excess O 2 completely represses this process (Zhou, Takaya, Shoun, Arch. Microbiol. 175, 19–25). Here we describe another metabolic mechanism of nitrate in fungal cells, termed ammonia fermentation, that supports growth under conditions more anoxic than those of denitrification. The novel nitrate metabolism of eukaryotes consists of the reduction of nitrate to ammonium coupled with the cat-abolic oxidation of electron donors to acetate and sub-strate-level phosphorylation. F. oxysporum thus has two pathways of dissimilatory nitrate reduction that are alternatively expressed in response to environmental O 2 tension. F. oxysporum prefers O 2 respiration when the O 2 supply is sufficient. We discovered that this fungus is the first eukaryotic, facultative anaerobe known to ex-press one of three distinct metabolic energy mecha-nisms closely depending on environmental O 2 tension. We also showed that ammonia fermentation occurs in many other fungi that are common in soil, suggesting that facultative anaerobes are widely distributed among fungi

Rapid changes in O 2 supply are an ongoing challenge for many organisms living in environments such as soil. Facultative anaerobes are widely distributed among prokaryotes and can adapt immediately to rapid changes in aeration by altering their energy metabolism. On the contrary, much less is known about such adaptive techniques in eukaryotes, although many lower eukaryotes can survive under anoxic conditions (1)(2)(3). Most of these anaerobic eukaryotes have adapted permanently to extreme environments such as swamps and intestines where the O 2 supply is always poor. Thus, they are obligate, not facultative, anaerobes.
Nitrate is generally metabolized by organisms in assimilatory and dissimilatory reductive pathways. Bacteria, fungi, and plants reduce nitrate to ammonium to assimilate nitrogen into their biomass (Scheme 1). Dissimilatory reduction (nitrate respi-ration) is performed by many bacteria in which nitrate is used as an alternative electron acceptor for respiration when O 2 is not available. One form of nitrate respiration results in denitrification (Scheme 2), a strategy that is extensive in facultative anaerobic bacteria (4 -6). Another form (see Scheme 1) has been identified in enterobacteria and other proteobacteria (7).
Many fungi can perform denitrification (8 -10). Although the anaerobic process was initially thought to be only a prokaryotic feature (11), the fungal denitrifying system is localized to mitochondria where it acts as a mechanism for anaerobic respiration similar to that of bacteria (12). The finding of denitrification in fungi suggests that such organisms are eukaryotic facultative anaerobes. Here, we present evidence for ammonia fermentation, a second form of dissimilatory nitrate metabolism in denitrifying fungi, and for the alternative expression of ammonia fermentation and denitrification under anaerobic conditions in response to the O 2 supply. The results show that many fungi, which are common in soil and which have been considered aerobic organisms, should really be classified as facultative anaerobes.

EXPERIMENTAL PROCEDURES
Strains, Media, and Culture-Fusarium oxysporum MT-811 (8) was mainly studied throughout this investigation. Other fungal strains were obtained from Institute for Fermentation Osaka (IFO) 1 and Institute of Applied Microbiology, University of Tokyo. The culture medium consisted of basal mineral medium (8) supplemented with carbon (ethanol, unless otherwise stated) and nitrogen (indicated) sources. Timedependent changes in components (see Fig. 1) or effects of aeration (see Fig. 2) were determined using cultures at 30°C in a 1-liter volume jar fermentor containing 500 ml of medium (13). Aeration effects were examined in fed-batch cultures (13) in which nitrate was supplemented as soon as it was exhausted. After repeating two cycles of fed-batch cultures to determine adaptation, the effects of aeration on nitrate metabolism were determined in one cycle of the culture that continued from renewal of the nitrate supplement until its complete consumption.
Other cultures were maintained in flasks. Precultures were prepared semi-aerobically incubating the fungus at 30°C for 3 days in glycerol/ peptone/nitrate (5 mM) medium (13). The mycelia in the preculture, collected and washed three times with sterilized saline, were used to inoculate the culture to be examined. A portion of the mycelia (corresponding to the amount of cells obtained from a 20-ml preculture) was * This work was supported in part by the Cooperative Research Program from the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University and by the Program for the Promotion of Basic Research Activities for Innovative Biosciences and the Structural Biology Sakabe Project in the High Energy Accelerator Research Organization (KEK) (Tsukuba, Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed. E-mail: ahshoun@mail. ecc.u-tokyo.ac.jp. transferred to a 500-ml Erlenmeyer flask containing 100 ml of medium supplemented with 600 mM ethanol and 10 mM ammonium and/or nitrate. The flask was sealed with a rubber stopper after purging air from the medium and head space by flashing with argon gas (anoxic conditions) or sealed with a cotton plug without purging the air (aerobic conditions). Dry cell weight was determined after drying washed cells at 94°C for 24 h.
Preparation of Cells for Enzyme Analyses-Enzyme activities related to nitrate metabolism were determined with each subcellular fraction prepared from the fungal cells grown under various aeration conditions. F. oxysporum was rotary-shaken in a 5-liter Erlenmeyer flask containing 1 liter of medium containing 10 mM nitrate under each aeration condition. Anoxic (ammonia-fermenting) and aerobic (nitrate-assimilating) conditions were obtained, respectively, as described above. Sealing the flask with a rubber stopper without replacing head space air generated hypoxic (denitrifying) conditions (8). Non-induced cells were prepared by incubating under anoxic conditions in the absence of nitrate (nitrate was replaced with ammonium). Fungal cells were harvested at the early stage of exponential growth and then disrupted and fractionated into subcellular fractions as reported (12).
Enzyme Analyses-All enzyme activities were measured anaerobically at 30°C in subcellular fractions from fungal cells cultured under various conditions. Alcohol dehydrogenase (Ald) was assayed by following the production of NADH from 50 mM ethanol and 5 mM NAD ϩ . Acetaldehyde dehydrogenase (acylating) (AddA) was assayed by determining the amount of acetyl-CoA formed during an incubation with 50 mM acetaldehyde, 5 mM NAD ϩ , and 5 mM coenzyme A. Acetate kinase (Ack) was assayed by determining the amount of ATP formed from 5 mM ADP and 5 mM acetyl-CoA. Acetyl-CoA hydrolyase was assayed by determining the amount of acetate formed from 5 mM acetyl-CoA. Acetaldehyde dehydrogenase activity was measured by incubating with 50 mM acetaldehyde and 5 mM NAD ϩ , and the amount of formed acetate was measured. Acetyl-CoA synthetase was assayed by measuring the amount of acetyl-CoA formed during an incubation with 50 mM acetaldehyde, 5 mM coenzyme A, and 5 mM ATP. Nitrate reductase and nitrite reductase were, respectively, assayed by incubating with 10 mM sodium nitrate or sodium nitrite and 2 mM NADH, and the amount of generated nitrite or ammonium was determined. Buffers were as follows: 70 mM Tris-HCl (pH 7.2) for Ald, AddA, acetaldehyde dehydrogenase, and acetyl-CoA synthetase assays and 70 mM potassium phosphate (pH 7.2) for Ack, acetyl-CoA hydrolyase, nitrate reductase, and nitrite reductase assays.

RESULTS
Alternative Anaerobic Metabolism of Nitrate by F. oxysporum-We showed that the induction of denitrifying activity in the fungus F. oxysporum MT-811 requires a minimal amount of aeration (hypoxic conditions) and that the recovery of nitratenitrogen into N 2 O (denitrification yield) varies considerably depending on the extent of the O 2 supply (13). The coexistence of ammonium in the medium should improve the denitrification yield, because ammonium generally represses the assimilatory use of nitrate. Here we incubated hypoxic cultures (13) in the presence of both 10 mM nitrate and 10 mM ammonium to examine effects of ammonium on the denitrification yield (Fig.  1A). In agreement with prior observations (13), denitrification (N 2 O formation) was induced over a long time course (stage 3). This time, however, we observed an unusual phenomenon during the lag period before denitrification was induced. Ammonium is usually preferred over nitrate for use in assimilation, a characteristic of stage 1 cultures. At stage 2, nitrate levels began to decrease, and nitrite and ammonium concomitantly accumulated in the medium. At stage 3, nitrate decreased rapidly with concomitant evolution of N 2 O. Gas chromatography-mass spectrometry with the stable isotope species of nitrate, 14 NO 3 Ϫ and 15 NO 3 Ϫ , showed that ammonium ions, as well as N 2 O (8), were derived from nitrate during anoxic cul-ture (data not shown) (16). By contrast, the decrease or increase of each compound was linear over the entire course of the culture when ammonium was the sole nitrogen source (Fig. 1B).
Nitrate did not seem to be converted to ammonium for assimilatory purposes, because a sufficient amount of ammonium remained in the medium when the conversion began. On the other hand, cell growth was significantly increased when nitrate was added (cf. Fig. 1, A and B), indicating that nitrate metabolism is an energy-yielding process. These findings show that nitrate is metabolized in a dissimilatory pathway to form ammonium at stage 2 and N 2 O at stage 3 (Fig. 1A). The evolution of N 2 O was accompanied by the accelerated evolution of CO 2 , indicating that the carbon source (ethanol) was decomposed as a respiration substrate for denitrification. In contrast, the rate of CO 2 evolution during stage 2 was much lower, suggesting that the reducing equivalent for the formation of ammonium was supplied by oxidizing ethanol to another C-2 compound.
Effects of Oxygen Supply on the Nitrate Metabolism-The effect of O 2 supply on nitrate metabolism by F. oxysporum MT-811 was examined using fed-batch cultures under various aeration conditions (0, 10, 20, or 30 ml O 2 /h) in which nitrate was the only nitrogen source (Fig. 2). Ammonium or N 2 O was determined for each culture when nitrate and formed nitrite disappeared. The rate of nitrate conversion to ammonium was highest in the complete absence of an O 2 supply. With increasing aeration, the recovery of ammonium declined, and conversely, the evolution of N 2 O increased. Less than 20% of nitrate was used for assimilation under these O 2 -limited conditions. With an excess O 2 supply, neither ammonium nor N 2 O was formed, although nitrate was consumed, indicating that nitrate is utilized only for assimilation, and that accelerated cell-growth (cf. Fig. 3, right) depends on O 2 respiration. Anoxic Cell Growth Coupled to the Ammonia Formation-The nitrogen source-dependent growth of flask cultures in the absence of O 2 (Fig. 3, left) indicates that nitrate supports more growth than ammonium. Because only slight denitrification occurs without O 2 (Fig. 2), nitrate-mediated anoxic growth must depend on nitrate metabolism to form ammonium. Aerobic cultures in the same medium increased enormously the yield of the cells (Fig. 3, right), supporting the conclusion derived from Fig. 2 that the anaerobic nitrate metabolism (am-monia formation and denitrification) is replaced by O 2 respiration when the O 2 supply is sufficient.
Morphology of Mitochondria- Fig. 4 shows transmission electron microscopy observations of mitochondria. Most mitochondria in the anoxic cells that formed ammonium (Fig. 4B) were immature and exhibited low electron density, in sharp contrast to the apparently intact mitochondria of cells grown under aerobic conditions (Fig. 4A) or in denitrifying cells (15). These results demonstrate that anoxic nitrate metabolism forms ammonium in a non-respiratory system that produces ATP. We term this method of eukaryotic nitrate metabolism "ammonia fermentation." Effect of Carbon Source on Ammonia Fermentation-We examined ammonia fermentation by F. oxysporum cultured in flasks under anoxic conditions with ethanol, glycerol, or glucose as the carbon source (Table I). Under these conditions, most nitrate-nitrogen was recovered into ammonium, consistent with the results shown in Fig. 2, and recovery of ammonium was high in the presence of ethanol. Ammonia fermentation was accompanied by acetate accumulation, as predicted above from the results in Fig. 1A. The stoichiometry of the formed ammonium and acetate was exactly 1:2 except for the culture with glucose. This

TABLE I Stoichiometry between the products because of ammonia fermentation by F. oxysporum
The cells were incubated by the anoxic flask culture as in Fig. 3 (left) in the medium containing only nitrate (10 mM, 1 mmol) for 5 days, and each product was determined. Nitrate was completely consumed by the end of each culture with the exception of that with glucose in which a half of nitrate still remained. indicates that the reducing equivalent derived from 4-electron oxidation to form acetate is utilized in the 8-electron reduction of nitrate to ammonium, consistent with the oxidation of ethanol to acetate. The recovery of ammonium was low in cells cultured with glucose, and instead, much more CO 2 evolved, suggesting that alcohol fermentation was predominant. Enzymes Involved in Ammonia Fermentation-The possible metabolic pathway described in Fig. 5 is based upon the above finding that ethanol is the best electron donor for ammonia fermentation and that the stoichiometry between the fermentation products, ammonium and acetate, is exactly 1:2. We determined the enzyme activities involved in each step using subcellular fractions prepared from cells cultured under aerobic (assimilatory, with respect to nitrate), hypoxic (denitrifying), or anoxic (ammonia-fermenting) conditions (Table II). AddA and the ATP-forming Ack activities were specifically induced only in anoxic cells that fermented ammonia, whereas other acetogenic activities (acetyl-CoA hydrolyase, acetaldehyde dehydrogenase, and acetyl-CoA synthetase) were particularly low (or absent) in the cells. These results demonstrated that ethanol is oxidized successively by Ald, AddA, and Ack to form acetate, coupled with ATP production and the reduction of nitrate to ammonia (Fig. 5), and that ammonia fermentation is an adaptive metabolism of the fungus F. oxysporum. These activities (Ald, AddA, and Ack) were recovered in the soluble and microsome fractions but not in the mitochondrial fraction, which would be consistent with the increasing population of damaged mitochondria in the anoxic cells (Fig. 4). NADH-dependent properties of nitrate reductase and nitrite reductase activities (Scheme 1) are quite distinct from those of the mitochondrial dissimilatory nitrate and nitrite reductases of the denitrifying cells (12) but are similar to the assimilatory reductases generally found among fungi.
Screening of Other Ammonia-fermenting Fungi-Ammonia-fermenting activity was screened in 17 fungal strains closely or distantly related to F. oxysporum MT-811 cultured under anoxic flask conditions as shown in Fig. 3 (left). Only two among the 17 strains tested did not exhibit this activity, and the following 15 strains distinctly converted nitrate to ammonium under anoxic conditions: Talaromyces

DISCUSSION
The present study presents evidence that the denitrifying fungus F. oxysporum contains another anaerobic type of nitrate metabolism, which we refer to as ammonia fermentation. We also showed that ammonia fermentation and denitrification are alternatively expressed depending on the extent of the O 2 supply. Ammonia fermentation is expressed under the most anoxic conditions even in the complete absence of O 2 supply. Small but distinct cell growth during ammonia fermentation (see Fig. 1 and Fig. 3) should depend on substrate-level phosphorylation by Ack (acetate kinase), which is specifically induced in cells that ferment ammonia (Table II). Anoxic nitrate metabolism (ammonia fermentation) is replaced by denitrification ( Fig. 2) with concomitant formation of intact mitochondria when the O 2 supply is limited, and under a sufficient supply of O 2 , denitrification is further replaced by aerobic (O 2 ) respiration. Thus the fungus F. oxysporum that expresses diversified pathways of FIG. 5. Possible metabolic pathway for the fungal ammonia fermentation coupled to acetogenic oxidation of ethanol and substrate-level phosphorylation. Nir, nitrite reductase; Nar, Nitrate reductase; Add, Acetaldehyde dehydrogenase; Acs, Acetyl-CoA synthetase; Ath, Acetyl-CoA hydrolyase; Pi, inorganic phosphate. nitrate metabolism closely regulates energy metabolism in response to environmental O 2 tension, ammonia fermentation under anoxic conditions, denitrification when hypoxic, and oxygen respiration under aerobic conditions. This study is the first to show that an organism (or eukaryote) can use a multimodal type of respiration (or ATP-producing) system to rapidly adapt to changes in the oxygen supply. Ammonia fermentation was limited when glucose was the carbon source (Table I). The ammonium recovery was low, but far more CO 2 evolved, suggesting that alcohol fermentation predominates over ammonia fermentation when glucose is available. F. oxysporum ferments alcohol, which has been understood for some time (17). The recovery of ammonium was highest when ethanol was the electron donor (Table I), indicating that ammonia fermentation acts physiologically as a secondary fermentation method that replaces primary alcohol fermentation when nitrate is available.
This fungal process is similar to acetogenic fermentation in prokaryotes, a reaction that is coupled to nitrate reduction and substrate-level phosphorylation reactions (18,19). However, bacterial nitrate metabolism is highly restricted, arising only in a single genus of obligate anaerobes, Clostridium, and it is regarded as a primitive form of anaerobic respiration in these bacteria (20). It is therefore surprising to find anoxic nitrate metabolism (ammonia fermentation) in eukaryotes (fungi) that have been considered to date as aerobic organisms.
Recent advances in genome projects, along with our screening results (21), have revealed that cytochrome P450nor, a characteristic enzyme in fungal denitrifying systems that functions as nitric-oxide reductase, is expressed widely among fungi. This indicates that denitrification is a key mechanism of nitrate metabolism in fungi. We further demonstrate here that ammonia is also fermented in the denitrifying fungus F. oxysporum MT-811 and in many other fungi that are common to soil. These results demonstrated that many such fungi should really be classified as facultative, rather than as obligate aerobes, although most fungi are in fact so far recognized as obligate aerobes. Advances in soil microbiology have revealed that the microbial biomass of temperate soils is usually dominated by fungi (22). Our present conclusion that many soil fungi are facultative anaerobes is consistent with this fact. The fungal prosperity in soil should be supported by a multirespiratory system, because the natural environment in soil with respect to oxygen supply is highly variable, and the ability to immediately adapt to rapid changes should be a potent tool for survival. The present results should thus evoke interest in how eukaryotes have evolved such a variety of metabolic mechanisms to produce energy.