INTRODUCTION

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The free-living anaerobic flagellate Hexamita sp. is an unusual protozoon lacking both mitochondria and Golgi apparatus (2). Sequences of complete small-subunit rRNA coding regions place it as one of the deepest-branching eukaryotes (3, 20, 41). In the genus Hexamita, there exist both free-living and parasitic species. All species are believed to be anaerobic or microaerobic (depending on definition), with the free-living species reported only in reducing environments such as stagnant waters, wastewater treatment plants, and anoxic marine basins (7, 14, 30). However, in all of these environments, it is unlikely that permanent anoxia can be guaranteed and thus Hexamita undoubtedly experiences periodic fluctuations of O₂ tension. Recently, this free-living species of Hexamita has been shown to lack detectable cytochromes, but it nevertheless actively consumes O₂ both endogenously and in the presence of several substrates with an O₂ K_m of 13 µM (1). In addition, Hexamita was observed to withstand high O₂ tensions (up to 100 µM) by the adoption of several antioxidant defense strategies (1), making this organism a microaerobe rather than an anaerobe.

Metabolic studies on free-living anaerobic protozoa have been hampered by the limited number of organisms growing in axenic cultures; to date, only the ciliate Trimyema compressum has been studied in any depth (15, 17, 45, 46). Hence, detailed metabolic studies of anaerobic protozoa have been confined to rumen-dwelling and parasitic protozoa (6, 29, 44). These studies have revealed that these organisms, unlike their aerobic counterparts, do not generate energy by oxidative phosphorylation but rather have developed extended glycolytic metabolic profiles and derive their ATP from substrate-level phosphorylation.

Anaerobic protozoa are sensitive to the ambient O₂ tension. Like Hexamita sp., many have a high affinity for O₂ as well as high O₂ consumption rates (12, 23, 25, 31). It is not clear as yet whether this affinity is part of a protective strategy against O₂ toxicity or whether O₂ is beneficial. Trace amounts of O₂ have been demonstrated to enhance growth and yield of Trichomonas vaginalis (32) and Giardia lamblia (34) and to influence the flux of metabolic products of these and many other protozoa such as those found in the rumen (16, 39, 44).

Studies of the catabolism of amino acids by anaerobic protozoa have also been limited to a few anaerobic protozoa (22, 26, 37). In the natural environment, the uptake of amino acids, both free and as released from proteinase activity, may be a primary source of energy. With the aid of ¹³C nuclear magnetic resonance (¹³C NMR), high-performance liquid chromatography (HPLC), and mass spectrometry, it has been the aim of this study to elucidate the primary products of glucose and amino acid fermentation of Hexamita. In addition, the influence of different O₂ tensions on the fermentative metabolism of this primitive flagellate has been investigated.

	MATERIALS AND METHODS

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Isolation and culture. Hexamita sp. was isolated by Jaroslav Kulda from a Czechoslovakian lake. Axenic cultures were established by treatment with ciprofloxacin (5 µg ml¹) and colistin sulfate (100 µg ml¹). The culture medium contained 2% (wt/vol) Trypticase (BBL), 1% (wt/vol) yeast extract (Oxoid), 0.5% (wt/vol) maltose, 0.1% L-cysteine, 10 mM K phosphate buffer, 10% (vol/vol) fetal calf serum (heat inactivated), and gentamycin sulfate (50 µg ml¹) grown at pH 7.2 and 25°C. For experimentation, cultures were grown to late exponential phase (ca. 6.5 × 10⁵ cells ml¹), harvested by centrifugation at 650 × g (5 min), and washed twice in 100 mM K phosphate buffer (pH 7.2) sparged with N₂. Organisms were counted with a hemocytometer.

Incubation of Hexamita cells at desired O₂ tensions. Cell suspensions (5 ml) in 100 mM K phosphate buffer were incubated (25°C) in a stainless-steel open O₂ electrode system fitted with a Teflon membrane-covered O₂ electrode (Radiometer A/S, Copenhagen, Denmark) (9). With the aid of a digital gas mixer (8), gas mixtures of O₂ in N₂, humidified by passage through moist cotton wool, were passed over the surface of the stirred liquid vortex (stirring at 790 rpm), enabling the O₂ tension to be maintained at desired levels. Addition of substrates (e.g., glucose) and removal of metabolites for quantification were made through the gas exit port. The O₂ concentration of air-saturated buffer at 25°C was taken to be 253 µM (43). Each incubation was done in triplicate.

NMR spectroscopy measurements. Products of glucose fermentation were identified by incubating organisms in the open O₂ electrode system with 30 mM D-[1-¹³C]glucose. Proton-decoupled ¹³C NMR spectra were recorded at 67.5 MHz on a JEOL EX270 spectrometer equipped with a 5-mm multinuclear probe. Free induction decay was measured for a total of 32,000 data points covering a spectral width of 200 ppm with pulses of 7.4 µs (70°) at 29-s intervals. ²H₂O was used as the internal lock. Chemical shifts, in parts per million, were measured with respect to the C-1 resonance in the added D-glucose (97.0 ppm) (27).

Quantification of organic acids. At specific time intervals, samples of cell suspension were removed from the open O₂ electrode system and centrifuged immediately. Soluble metabolites present in the supernatant were identified and quantified by use of a HPLC coupled to a variable-wavelength UV detector (for examples, see references 13 and 34). Samples (20 µl) were separated by injection through a fermentation monitoring column packed with hydrogen sulfonated divinyl benzene-styrene copolymer resin (Bio-Rad) with a 1 mM H₂SO₄ mobile phase flowing at 0.6 ml min¹. Eluent streams were monitored at 210 nm and recorded with a potentiometric chart recorder. Metabolites were identified and quantified by using known standards.

Membrane inlet mass spectrometry. Dissolved-gas concentrations were monitored with a HAL series quadrupole gas analyzer (Hiden Analytical) linked to a temperature-controlled (25°C) incubation vessel (2 ml) by a stainless-steel probe (1.5-mm outside diameter; 0.5-mm inside diameter) with a 1-mm-diameter inlet covered by a silicone membrane (10, 24). Partial pressures of O₂ in the mobile phase were controlled with a digital gas mixer. Endogenous and substrate-supported CO₂ production rates (m/z = 44) were calibrated against those of standard solutions of NaHCO₃.

Amino acid analysis. Sulfosalicylic acid (10% [wt/vol]) was added to samples (1:1) for 1 h. Solutions were then centrifuged, and the supernatant was filtered (0.22-µm pore size). Amino acids were identified and quantified by ion-exchange chromatography with a Biochrom 20 amino acid analyzer (Pharmacia).

	RESULTS

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Identification of fermentation products by NMR. Proton-decoupled ¹³C NMR spectra of Hexamita incubated under microaerobic conditions (23 to 34 µM O₂) with D-[1-¹³C]glucose (30 mM) revealed that the primary products were acetate, ethanol, lactate, and alanine (Fig. 1). HPLC analysis also confirmed acetate and lactate (<0.1 mM) as products of glucose fermentation; however, in addition, propionate and n-butyrate (and iso-valerate, <0.1 mM) were also detected. The rate of acetate production was greatest under microaerobic conditions (Fig. 2a) compared to that under anaerobic (Fig. 2b) or aerobic (Fig. 2c) conditions. Since propionate and butyrate were not detected as products from labelled glucose by ¹³C NMR, it is suggested that these are the products of endogenous substrate fermentation.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Proton-decoupled 13C NMR spectra of Hexamita supernatant. Washed cells were incubated (25°C) for 6 h under a dissolved-O2 tension of 23 to 34 µM with D-[1-13C]glucose (30 mM). Chemical shifts in parts per million were as follows: C-1 glucose  peak, 93.2; C-1 glucose  peak, 97.0; C-2 acetate, 24.5; C-3 lactate, 21.2; C-2 ethanol, 18.0; C-3 alanine, 17.3.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Production of principle organic acids by washed cell suspension of Hexamita (1.95 × 105 cells ml1) at 25°C and pH 7.2 with added glucose (30 mM) under microaerobic (15 µM O2) (a), anaerobic (<0.25 µM O2) (b), and aerobic (200 to 253 µM O2) (c) conditions. Products were detected by HPLC. Symbols: , acetate; , propionate; , butyrate. Values are the means of the results of two separate experiments differing by <6%.

Influence of O₂ on the production of CO₂ with various substrates. CO₂ production by Hexamita cells was shown to be influenced by O₂ tension (Table 1). Pyruvate-, arginine-, and ethanol-supported CO₂ production rates were greatest under microaerobic conditions, and in all cases, a high O₂ tension inhibited CO₂ production by approximately one-third. The high rate of production of CO₂ from arginine suggests that this amino acid is rapidly catabolized.

Amino acid consumption by Hexamita. The amino acid composition of the culture media of Hexamita was analyzed before and after 6 days of growth, when stationary phase of growth was reached (Table 2). Confirmation of data from the ¹³C NMR measurements was obtained; alanine was again shown to be generated. Noticeably, asparagine was taken up, with almost the same amount of aspartic acid produced. The production of CO₂ from arginine indicated that arginine may be used as a substrate. Its uptake from the medium with the concomitant production of ornithine (and to a lesser extent citrulline) suggests the presence of the arginine dihydrolase pathway. Incubation of the Hexamita cell suspension with arginine resulted again in the uptake of arginine and the production of ornithine and citrulline (Table 3). The rate of arginine consumption was greater under anaerobic conditions than microaerobic conditions. The other amino acids were probably released from the intracellular amino acid pool. Alanine was again generated, its production being greatest under anoxia.

Growth of Hexamita in the absence of maltose. Maltose is the carbohydrate source in the growth medium of Hexamita. However, in the absence of maltose (ca. 1 mM glucose may be present in the serum), Hexamita cells were observed to grow, albeit at a lower growth rate and with a lower yield (Fig. 3), showing that alternative sources of energy had been utilized for growth.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Growth rate of Hexamita in culture medium with () and without () added maltose (0.5% [wt/vol]). The experiment was performed in triplicate; error bars indicate ± standard deviation.

	DISCUSSION

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Hexamita is shown to possess a fermentative metabolism; the principle products of glucose fermentation are acetate, ethanol, lactic acid, alanine, and CO₂. Other end metabolites include butyrate and propionate, which are not produced from glucose and therefore are products of an alternative substrate fermentation (amino acids and/or fatty acids).

Changes in O₂ concentration led to a marked alteration in the carbon balance of the metabolism. The production of organic acids and CO₂ was greatest under microaerobic conditions in comparison with that under aerobic and anaerobic conditions. The closely related diplomonad Giardia lamblia has been studied extensively and has also been shown to have a fermentative metabolism (18, 21, 42), generating acetate, ethanol, and CO₂ but, unlike Hexamita, not lactate. Similarly, all the anaerobic protozoa studied to date, e.g., the parasitic flagellates Trichomonas vaginalis (5, 28) and Tritrichomonas foetus (36), the rumen ciliates Dasytricha ruminantium (13, 40) and Isotricha sp. (35), and the free-living ciliate Trimyema compressum (15, 17, 46), metabolize glucose into a variety of organic acids.

The production of alanine was shown to be higher under anaerobic than microaerobic conditions; this is consistent with the results found for Giardia, where alanine is also produced (11) and is produced at increased rates under anoxia (33). In Giardia, alanine is thought to function as a major osmoregulator (19), and it is possible that it plays a similar role in Hexamita.

It is becoming increasingly apparent that glucose or other carbohydrates are not the sole energy sources of anaerobic protozoa (26, 38). Consistent with this view, Hexamita was shown to rapidly consume arginine, with the simultaneous production of CO₂, ornithine, and citrulline. Together with previous observations demonstrating arginine-supported respiration (1), these data suggest that a functional arginine dihydrolase pathway like that found in Giardia (37) and Trichomonas vaginalis (22) is present in Hexamita. The species of Hexamita we used is free-living, and thus it seems appropriate that it has alternative energy-yielding routes which best suit its mode of living. Certainly, it was shown that in the absence of an added carbohydrate source (Fig. 3), Hexamita was still able to grow, albeit with a lower growth rate and yield. Arginine uptake was highest under anaerobic conditions, which suggests that this pathway may be linked to the redox state of the NAD(P)H pool.

During growth, Hexamita was shown to take up a relatively large amount of asparagine, with stoichiometric production of aspartate. In bacteria under conditions where amino acids are consumed as carbon sources, asparaginase activity resulting in the deamination of asparagine to aspartic acid is stimulated more than a hundredfold under anaerobic conditions (4). The results reported here suggest that Hexamita also possesses asparaginase activity operating as a means of deamination under anaerobiosis. The possible involvement of asparaginase has also been invoked to explain the asparagine uptake and aspartic acid production observed in species of the anaerobic parasite Entamoeba (47).

Hexamita has been found in the water column of marine basins at depths where the O₂ tension ranged from 0 to 30 µM O₂ (14). We have shown that it is in this microaerobic realm that Hexamita optimally produces CO₂ and organic acids at the highest rates. Significantly, the rapid consumption of arginine suggests that carbohydrates are not its sole source of energy, a function which may be a result of its free-living mode.