INTRODUCTION

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Humic substances constitute a chemically heterogeneous and very abundant class of organic compounds and are widely distributed on the Earth's surface (18). The interaction of humic substances with microorganisms has been a subject of research for the past 30 years. Initially, humic compounds were studied mostly as sources of carbon or micronutrients or for their general impact on the growth of microorganisms (14). Subsequently, however, it was discovered that natural organic matter, particularly humic acids, and the quinoid model compound 2,6-anthraquinone disulfonate (AQDS) can act as mediators in the chemical reduction of organic pollutants, such as nitroaromatic or halogenated compounds, if an appropriate electron donor (e.g., sulfide or cysteine) is provided (3, 5, 17). Most importantly, humic acids or reduced AQDS have also been shown to catalyze rapid chemical reduction of oxidized iron or manganese species (20, 22).

Recently, Lovley and coworkers (10, 11) reported that a wide range of iron-reducing bacteria can transfer reducing equivalents from acetate oxidation to various humic acid preparations, as well as to AQDS. All strains of acetate-oxidizing AQDS-reducing bacteria recovered from the highest dilutions of various sediments were capable of reducing both humic acids and Fe(III) citrate, and all of these organisms were members of the family Geobacteraceae (1).

Previous work in our laboratory revealed that reduction of AQDS is not restricted to iron-reducing bacteria since it also occurs with the fermenting bacterium Propionibacterium freudenreichii if the acceptor is continuously reoxidized at an appropriately polarized electrode (7). If this indicates a principal ability of fermenting bacteria to reduce humic acids, the spectrum of microorganisms that funnel electrons from oxidation of organic matter into iron reduction would be larger than expected. For this reason, we investigated several physiological groups of fermenting bacteria to determine their general abilities to reduce humic acids.

	MATERIALS AND METHODS

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Strains. Propionibacterium freudenreichii subsp. freudenreichii DSM 20271, Enterococcus cecorum DSM 20682, Lactococcus lactis subsp. lactis DSM 20481, Escherichia coli DSM 498, Clostridium homopropionicum DSM 5847, and Pelobacter propionicus DSM 2379 were obtained from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, or from the stock culture collection of our laboratory.

Media. All growth experiments were performed in anoxic bicarbonate-buffered mineral medium (27) supplemented with 1 mM sulfate as the sulfur source. After autoclaving and cooling under N₂-CO₂ (80:20, vol/vol), trace element solution SL9 (24) and a seven-vitamin solution (28) were added (each at 1 ml per liter) and the pH was adjusted to 7.2. In general, no reducing agent was added; only in experiments performed with C. homopropionicum and P. propionicus was the medium reduced with sodium cysteine (2 mM). The media used for P. freudenreichii, L. lactis, and C. homopropionicum were also supplemented with yeast extract (0.05%).

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Growth experiments. Precultures were centrifuged under axenic conditions at 4,000 × g for 10 min, resuspended in anoxic mineral medium twice, and adjusted to a final optical density at 578 nm of 0.07. Growth experiments were performed in bottles sealed with butyl rubber stoppers under an N₂-CO₂ (80:20, vol/vol) atmosphere at 30°C in the dark. Samples (1 ml) were taken with a syringe after thorough shaking and were transferred immediately into 2-ml polypropylene reaction vessels containing 1 ml of 1 M HCl to prevent autoxidation. Samples were kept at 4°C for 30 min to precipitate the humic acids and then centrifuged at 1,500 × g for 10 min; the supernatant was analyzed further. All experiments were performed in duplicate, unless noted otherwise.

Extraction of humic acids from sediment. Samples (80 ml) of the top 5 cm (above the black sulfidic layer) of profundal lake sediment from Lake Constance in Germany were suspended in 160 ml of 0.1 N NaOH in stainless steel centrifugation tubes under an N₂-H₂ (95:5, vol/vol) atmosphere in a glove box, sealed, and agitated on a rotary shaker (130 rpm) at 30°C for 24 h. The tubes were centrifuged at 20,000 × g for 50 min; the supernatant was acidified with 1 M HCl (final pH, <2) and kept at 4°C for 24 h to precipitate the humic acids. The humic acids were recovered by centrifugation, oven dried at 60°C, ground, and stored under a nitrogen atmosphere at 18°C until they were utilized. Commercial humic acids (technical grade) obtained from Sigma-Aldrich (Steinheim, Germany) were used as provided.

Iron analyses. To determine the total iron content, humic acids were dissolved in 0.1 M NaOH (1 mg of humic acids per ml), and the iron concentration was determined with an atomic absorption spectrophotometer (model 3030B; Perkin-Elmer, Norwalk, Conn.) at 284.3 nm. The acid-soluble ferrous iron concentration was determined by a photometric ferrozine assay; the total concentration of acid-soluble iron was determined by the same assay after reduction of the sample with hydroxylamine (19).

Electron uptake capacity of humic acids. The electron uptake of humic acids by microbial reduction was calculated from the difference in electron recovery in the fermentation products between assay mixtures containing humic acids and the corresponding humic acid-free controls. The values obtained were compared to the electron uptake of humic acids during chemical reduction with H₂ in the presence of a Pd catalyst (26). For this procedure, aqueous suspensions of humic acids (1 mg ml¹) and a Pd catalyst (5% Pd on activated charcoal; 0.05 mg ml¹), adjusted to pH 7, were incubated under an H₂ atmosphere on a rotary shaker at 30°C for 24 h and then repeatedly degassed and flushed with N₂ with stirring to remove excess H₂. These preparations and untreated controls were titrated with potassium hexacyanoferrate(III), as described by Matthiessen (12); the difference between the reducing equivalents released by a hydrogen-reduced suspension and the reducing equivalents released by untreated controls was defined as the chemical (hydrogen) electron uptake capacity of the preparation.

Analytical procedures. Lactate, glucose, formate, and ethanol were analyzed by ion exclusion high-performance liquid chromatography on an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad, Hercules, Calif.) at 40°C by using a 5 mM H₂SO₄ mobile phase at a flow rate of 0.6 ml min¹ and a model ERC-7512 refractive index detector (Erma, Tokyo, Japan). Low concentrations of acetate and propionate (<2 mM) were quantified by gas chromatography by using a packed column (2 m by 2 mm; 60/80 Carbopack C-0.3% Carbowax 20M-0.1% H₃PO₄), an oven temperature of 100°C, and a flame ionization detector.

	RESULTS

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Humic acid reduction by P. freudenreichii. Cultures of P. freudenreichii incubated with lactate under anoxic conditions formed propionate and acetate at the expected 2:1 ratio (Fig. 1A). In the presence of humic acids, however, this ratio shifted significantly in favor of acetate formation (Fig. 1B). When amorphous ferric iron hydroxide was included in the medium together with humic acids, it was reduced to ferrous iron, and the final propionate-to-acetate ratio decreased even further (Fig. 1C). The amount of ferrous iron formed by chemical oxidation of humic acids in the absence of lactate was constant (0.182 ± 0.03 µmol per mg of humic acids), was independent of the presence of cells, and therefore was subtracted. The time course of lactate consumption was not significantly changed by the presence of humic acids or ferric iron, and no ferrous iron was formed in controls when only humic acids were omitted (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Typical time courses of lactate fermentation by P. freudenreichii in the absence (A) and in the presence (B) of humic acids (17 mg ml1). All assay mixtures contained lactate () and 0.05% yeast extract; propionate () and acetate () were the only products detected. When amorphous ferric iron (40 mM) was added in the presence of humic acids (C), ferrous iron () was formed almost stoichiometrically. The constant background level of ferrous iron resulting from the chemical reaction with humic acids (3.1 mM) was subtracted. For reproducibility and exact stoichiometries, see Table 1.

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Humic acid reduction by other bacteria. When lactic acid bacteria were grown in the presence of humic acids, the shift in the fermentation product pattern towards more oxidized products resembled the results obtained with P. freudenreichii (Table 2). Both E. cecorum and L. lactis formed significantly less lactate and more acetate from glucose in the presence than in the absence of humic acids, and the electron recovery in the fermentation products decreased significantly. In addition, no ethanol was formed by E. cecorum in the presence of humic acids. In the absence of substrate (glucose and yeast extract), no products were formed from humic acids by either organism. As observed with P. freudenreichii, the addition of ferric iron led to a shift towards more oxidized products and increased recovery of electrons in the form of ferrous iron with both L. lactis (Table 2) and E. cecorum (data not shown).

Electron uptake capacity of humic acids. The electron balances in Table 1 indicate that the average electron uptake is 0.191 meq per g of humic acids (Aldrich). Figure 2 shows that this value decreased slightly with increasing concentrations of humic acids and that the propionate-to-acetate ratio for lactate fermentation by P. freudenreichii could be shifted almost completely towards acetate, provided that enough electron acceptor was added. The low electron uptake capacities of humic acids observed during microbial reduction with P. freudenreichii were confirmed by chemical reduction of the same humic acids with H₂. Both values were virtually identical for the humic acids from Aldrich; for sediment humic acids, the chemical electron uptake capacity was slightly higher (Table 3). We calculated that on a scale between hydrogen-reduced and ferricyanide-oxidized humic acids, the humic acid preparations from Aldrich were 76% reduced, whereas the humic acid preparations from the oxidized layer of Lake Constance sediment were only 47% reduced. This was also reflected in the apparent redox potentials of the humic acid suspensions, which were 170 mV more negative for the commercial humic acids (87 ± 22 mV; n = 4) than for the humic acids from lake sediment (84 ± 12 mV; n = 3).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Influence of humic acid concentrations on the fermentation product pattern of P. freudenreichii. Cells were grown on lactate (3 mM) and yeast extract (0.05%); propionate () and acetate () were the only products detected. The specific electron uptake by the humic acids () was calculated from the amount of electrons recovered in the fermentation products.

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	DISCUSSION

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This is the first report of humic acid reduction by fermenting bacteria. We demonstrated that there were significant shifts towards more oxidized products in the fermentation product patterns of P. freudenreichii, E. cecorum, and L. lactis when humic acids were provided as electron acceptors. In the case of P. freudenreichii, even the fermentation end product propionate was oxidized in the presence of humic acids.

Humic acid reduction by fermenting bacteria. Lovley et al. (10) recently showed that iron-reducing bacteria reduce various humic acid preparations, as well as the low-molecular-weight model compound AQDS, with electrons derived from acetate, lactate, or H₂ oxidation. Since the quinoid moieties in humic acids (13) and low-molecular-weight quinones are considered redox mediators in the chemical reduction of iron or manganese species (20, 22) or of organic pollutants (3, 5, 17), the mechanisms of reduction of AQDS and humic acids may be related (1, 10, 11). Previous results from our laboratory and other laboratories showed that the ability to reduce AQDS or other external electron acceptors is not uncommon among fermenting bacteria and causes a significant shift in the fermentation product pattern towards more oxidized products (6, 7, 16, 23, 25). In this study, we observed similar effects on the fermentation product patterns of both propionic acid bacteria and lactic acid bacteria when humic acids were added.

Electron-accepting capacity of humic acids. During lactate fermentation by P. freudenreichii, the electron deficit in the fermentation products increased in proportion to the amount of humic acids added. The average specific electron uptake of humic acids is not very high (Table 3), and substantial amounts of humic acids were needed to shift the balance of lactate fermentation completely from propionate formation to acetate formation (Fig. 2). All attempts to recover the humic acids from the cultures by filtration at the end of the incubation period were unsuccessful; therefore, direct determination (by titration) of the amount of electrons transferred during microbial reduction was not possible. Nevertheless, the amount of electrons transferred to humic acids by the bacteria (as calculated from the electron deficit in the fermentation products) is in good agreement with the amount of electrons transferred to humic acids during chemical reduction with hydrogen in the presence of the Pd catalyst (Table 3). In general, it can be predicted that the electron uptake capacity of a humic acid preparation depends strongly on the nature of the humic acids (as shown by the results of Lovley and coworkers [10]) and their actual redox status (Table 3), which also depends on the isolation procedure (21).

Role of iron. It is known that humic substances are chemically oxidized by ferric iron or manganese oxides (10, 20, 22). Recently, it was demonstrated that humic acids and low-molecular-weight quinones can act as redox mediators in biological iron reduction (10) and greatly stimulate the reduction of structural Fe(III) in clay and crystalline iron minerals by iron-reducing bacteria (11). Since chemical reoxidation of humic acids should be independent of the biological process involved in humic acid reduction, it was not unexpected to observe enhanced acetate formation by P. freudenreichii when amorphous ferric iron was provided as an electron acceptor in the presence of humic acids (Table 1). The catalytic role of humic acids as mediators between substrate oxidation and ferric iron reduction was evident from the fact that exactly the same reaction stoichiometry was observed when the humic acid concentration was reduced to substoichiometric amounts (Table 1).

Mechanistic implications. Lovley and coworkers showed that the ability to reduce AQDS was correlated with the ability to reduce humic acids in all of the iron-reducing bacteria which they tested (1, 10, 11). Since quinoid moieties in the humic acids are considered responsible for the redox reactions (3, 5, 13, 18), it was postulated that these functional groups act as electron carriers between the biological and chemical partial reactions (substrate oxidation and iron reduction) (10). This hypothesis is certainly quite enticing, but the common biochemical basis for AQDS reduction and humic acid reduction still needs to be ascertained.

Ecological implications. The fact that humic substances are chemically oxidized by ferric iron (10, 20, 22) makes this abundant electron acceptor available not only to iron-reducing bacteria sensu stricto but also to all other microorganisms that are able to transfer electrons to humic acids. First, this has important implications for the autecology of anaerobic bacteria in soils and sediments. Increased formation of acetate is energetically favorable for fermenting bacteria, and the reduction of humic acids is in accordance with the general concept that the energetically most favorable available electron acceptors are utilized first (29). Such phenomena have been observed with P. freudenreichii and E. coli, which transfer electrons to an appropriately polarized electrode in the presence of a suitable redox mediator, such as AQDS or ferricyanide (6, 7), and for sulfate-reducing bacteria from sediments (4) or lactic acid bacteria from termite guts (23), both of which reduce oxygen when it is available.