Envlron. Sci. Technol. 1991, 25, 1062-1067
Enzymatic versus Nonenzymatic Mechanisms for Fe( I I I ) Reduction in Aquatic Sediments Derek I?.Lovley," Elizabeth J. P. Phillips, and Debra J. Lonergan Water Resources Division, US. Geological Survey, 430 National Center, Reston, Virginia 22092
The potential for nonenzymatic reduction of Fe(II1) either by organic compounds or by the development of a low redox potential during microbial metabolism was compared with direct, enzymatic Fe(II1) reduction by Fe(II1)-reducing microorganisms. At circumneutral pH, very few organic compounds nonenzymatically reduced Fe(II1). In contrast, in the presence of the appropriate Fe(II1)-reducing microorganisms, most of the organic compounds examined could be completely oxidized to carbon dioxide with the reduction of Fe(II1). Even for those organic compounds that could nonenzymatically reduce Fe(III), microbial Fe(II1) reduction was much more extensive. The development of a low redox potential during microbial fermentation did not result in nonenzymatic Fe(II1) reduction. Model organic compounds were readily oxidized in Fe(II1)-reducing aquifer sediments, but not in sterilized sediments. These results suggest that microorganisms enzymatically catalyze most of the Fe(1II) reduction in the Fe(II1) reduction zone of aquatic sediments and aquifers. W
Introduction
Many aquatic sediments and aquifers contain distinct zones in which the oxidation of organic matter is coupled to the reduction of Fe(II1) to Fe(I1) ( I , 2). This Fe(II1) reduction is of environmental significance as it can result in the release of trace metals ( 3 ) ,phosphate ( 4 ) ,and undesirably high concentrations of iron (2, 5 ) into water supplies. Fe(II1) may also serve as an important oxidant of contaminant organics (6-9). Furthermore, Fe(II1) reduction serves as a model for potential mechanisms for the reduction of other, more toxic, heavy metals such as uranium (IO). Numerous early studies suggested that microorganisms could enzymatically reduce Fe(II1) during organic matter metabolism (I, 11). However, it has generally been considered that much of the Fe(II1) reduction in sedimentary environments is the result of nonenzymatic reactions. This is evident from statements in authoritative texts in both the geochemical (12-16) and microbiological fields (11,17, 18). The conclusion that nonenzymatic Fe(II1) reduction is an environmentally significant process appears to be primarily based on three premises: (1)the fact that, under some conditions, some organic compounds can nonenzymatically reduce iron(II1) oxides (11, 19, 20); (2) the concept that Fe(II1) reduction is a reversible redox reaction that can be accurately modeled as a function of Eh and pH (12,14,15, 18); and (3) the fact that, until recently, there were no organisms known to effectively couple the oxidation of organic compounds to the reduction of Fe(II1) (18).
However, microorganisms that can effectively couple the oxidation of organic compounds to the enzymatic reduction of Fe(II1) have recently been described. These microorganisms can completely oxidize short-chain fatty acids and aromatic compounds to carbon dioxide with Fe(II1) as the sole electron acceptor (2,8,21-23). The cooperative activity of these Fe(II1)-reducing microorganisms and fermentative microorganisms can result in the complete ox1062 Environ. Scl. Technol., Voi. 25, No. 6, 1991
idation of complex assemblages of organic matter to carbon dioxide in Fe(II1)-reducing sediments (23, 24). These studies have strengthened the previous suggestion ( I , 25-30) that enzymatic reduction of Fe(II1) has the potential to be an important mechanism for Fe(II1) reduction in aquatic sediments. The purpose of the study reported here was to assess the relative potential for nonenzymatic and enzymatic mechanisms for the reduction of Fe(II1) in sedimentary environments. The results demonstrate that Fe(II1)-reducing microorganisms can enzymatically couple the oxidation of a wide variety of organic compounds to Fe(II1) reduction, but, at the circumneutral pH typical of most aquatic sediments and groundwater, there is little potential for nonenzymatic Fe(II1) reduction in nonsulfidogenic environments. Materials and Methods
Fe(II1)-Reducing Cultures. As previously described (22),strict anaerobic culturing and sampling techniques
were used throughout. The anaerobic culture medium had the same inorganic constituents as previously described for the culture of the Fe(II1)-reducing microorganism, strain GS-15 (22). It contained (in grams per liter of deionized water) the following: NaHC03, 2.5; CaC1,.2H20, 0.1: KC1, 0.1; NH4Cl, 1.5; NaH2P04.H20,0.6; as well as a mixture of vitamins and trace minerals. The medium contained ca. 100 mmol of Fe(III)/L in the form of a poorly crystalline iron(II1) oxide. This was synthesized, as previously described (30),by neutralizing a solution of FeC13 with NaOH and washing the iron(II1) oxide precipitate with water until the chloride concentration in the associated water was less than 1mM. This procedure yields iron(II1) oxide particles and aggregates that range in size from 0.1 to 1pm in diameter and that do not give a detectable X-ray diffraction pattern (22,30). This iron(II1) oxide behaves similarly to the most chemically reactive, easily reducible fraction of the iron(II1) oxides in aquatic sediments (30-33). The gas phase was N2-C02 (80:ZO). The HC03--C02 buffer system buffered the pH of the medium at ca. 6.7. Anaerobic solutions of the organic substrates (0.5 or 1.0 mL) were added to the sterilized culture medium (10 mL) from sterile anaerobic stock solutions. All culture incubations were in the dark at 30 OC. Enrichment cultures on the various compounds were established by inoculating the media with freshwater sediments that had been collected from the same site in the Potomac River that yielded the Fe(II1)-reducing microorganism, strain GS-15 (30). The enrichment cultures were transferred (10% inoculum) a t least 10 times prior to conducting the studies described here. Escherichia coli (ATCC 4157) and Clostridium pasteurianum (ATCC 6013) were obtained from the American Type Culture Collection, Rockville, MD. Inocula of each organism were grown in the anaerobic medium described above with 10 mM glucose as the organic carbon source and with the iron(II1) oxide omitted. The E . coli culture (0.3 mL) was inoculated into iron(II1) oxide containing
Not subject to U.S. Copyright. Publlshed 1991 by the American Chemical SOClety
Table I. Fe(II1) Reduction in Enrichment Cultures (Inoculated) a n d Sterile Controls (Uninoculated) with Various Organic Compounds or Hydrogen as t h e Sole Electron Donor and Poorly Crystalline Iron(II1) Oxide as a Potential Electron Acceptor
electron donor' hydrogen formate pyruvate acetate ethanol glycerol lactate propionate butyrate malate succinate citrate glucose
(ino@ (uninoc) (inoc) (uninoc) (inoc)d (uninoc) ( inoc)e (uninoc) (inode (uninoc) (inoc) (uninoc) (inoc)d (uninoc) (inoc)e (uninoc) (inoc)' (uninoc) (inoc) (uninoc) (inoc) (uninoc) (inoc) (uninoc) (inoc) (uninoc)
Fe(I1) produced after incubation,b mmol/L 7 days 14 days 21 days 40.1 0.0 12.6 0.0 8.3 0.0 21.6 0.0 28.8 0.0 18.8 -0.3 3.6 0.0 10.3 0.0 10.4 0.0 16.3 -0.4 15.4 0.0 24.1 0.0 16.9 0.2
44.4 0.0 19.7 0.0 13.1 0.0 31.2 0.0 44.1 0.0 27.6 -0.3 7.5 0.0 18.3 0.0 20.7 0.0 18.5 -0.4 27.1 -0.1 30.4 0.0 19.8 0.0
45.8 0.0 19.2 0.0 14.9 0.0 33.3 0.0 45.5 0.0 25.4 -0.3 17.2 0.0 22.2 0.0 27.2 0.0 22.9 -0.4 23.9
electron donora fructose lysine serine aspartate g1ycine benzaldehyde benzyl alcohol p-hydroxybenzoate
p-hydroxybenzylaldehyde p-hydroxybenzyl alcohol tyrosine
-0.1 29.9 0.0 20.0 0.0
Fe(1I) produced after incubation,b mmol/L 14 days 7 days 21 days
phenyl acetate
(inoc) (uninoc) (inoc) (uninoc) (inoc) (uninoc) (inoc) (uninoc) (inoc) (uninoc) (ino@ (uninoc) (ino$ (uninoc) (inoc)e (uninoc) (ino@ (uninoc) (ino+ (uninoc) (inoc) (uninoc) (inoc) (uninoc)
16.0 4.3 18.3 0.0 30.8 0.3 13.9 0.0 3.4 -0.8 7.0 -0.3 10.1 -0.4 3.3 -0.4 3.2
23.4 4.1 24.3 0.0 38.6 0.0 18.8 0.0 10.2 -0.8 9.8 0.3 19.1 -0.4 6.4 -0.1 5.6
NA
NA
4.1
9.0
NA NA NA NA NA
NA 6.4
NA 11.1
NA
25.6 6.2 25.0 0.0 40.3 0.0 20.9 0.0 14.6 -0.8 10.1 0.7 19.1 1.4 7.0 0.3 6.4 -0.7 10.1 -0.7 9.1 -0.8 12.3 0.4
'All organic electron donors were provided at 10 mM initial concentration except for aromatic compounds, which were provided at 0.5 mM. Hydrogen was provided as 0.5 atm in the headspace. bAmount of Fe(I1) produced was calculated by subtracting the initial concentration of Fe(I1) from the values on subsequent days. Values are means of duplicate determinations on duplicate culture tubes of each treatment. NA, not analyzed at that time point. Unless otherwise noted, inoculated with a purified Fe(II1)-reducing enrichment culture that had been established with the particular electron donor and transferred at least 20 times prior to the measurements reported here. Inoculated with Alteromonas putrefaciens. e Inoculated with strain GS-15.
medium (10 mL) with glucose as the organic carbon source. For growth of E. coli and the Fe(II1)-reducing microorganism, GS-15, in coculture, an inoculum of GS-15 was prepared as previously described (34) by growing GS-15 with nitrate as the electron acceptor and transferring the inoculum (0.3 mL) after the nitrate had been depleted. C. pasteurianum (3 mL) was inoculated into the anaerobic glucose medium (100 mL in a 160-mL serum bottle) that contained either no added iron(II1) oxide or ca. 100 mmol of iron(II1) oxide/L of medium. In some instances Fe(II1) was placed in dialysis tubing (7.6 cm diameter X 5 cm length; molecular weight cutoff 50000) and added under anaerobic conditions in order to provide an additional ca. 40 mmol of Fe(III)/L of medium. HC1-extractable Fe(I1) was measured by extracting subsamples with 0.5 N HCl as previously described (31). In order to measure dissolved Fe(II), a sample (0.5 mL) was taken with a sterile syringe and needle in an anaerobic glovebag filled with N2. The sample was filtered (Gelman Acrodisc filter, pore diameter, 0.2 pm) and 0.1 mL was added to 5 mL of the ferrozine-HEPES buffer solution to quantify Fe(I1) (31). Glucose, organic acids, ethanol, and aromatic compounds were quantified with HPLC techniques as previously described (24, 35). Hydrogen was quantified with gas chromatography and a reduction gas analyzer (36). Sediments in which Fe(II1) reduction was the predominant terminal electron-accepting process were collected from a shallow aquifer that has been contaminated with aromatic hydrocarbons and their decomposition products (8). As previously described (8) 10 mL of sediment was anaerobically incubated under N2-C02 (93:7) in anaerobic pressure tubes that were sealed with thick butyl rubber
stoppers. An anaerobic solution (0.1 mL) containing 0.5 pCi of [U-14C]glucose(280 mCi/mmol), [2-'*C]acetate (53 mCi/mmol), or [rir~g-~~C]benzoate (120 mCi/mmol) was injected into the sediments. Killed controls were treated with heat (121 "C, 1 h) prior to the addition of the I4Clabeled compounds. The sediments were incubated in the dark a t the in situ temperature of 9 "C. At appropriate time intervals, 1 mL of 2.5 N H2S04was injected into replicate tubes of sediment in order to stop microbial activity and the 14C02that had been produced was quantified (8).
Results and Discussion Enzymatic Reduction of Fe(II1) versus Nonenzymatic Reduction by Organics. Hydrogen, as well as most of the organic compounds tested, could readily be oxidized with the reduction of poorly crystalline iron(II1) oxide in the presence of either an appropriate Fe(II1)-reducing isolate or a consortium of Fe(II1)-reducing organisms adapted to metabolize the organic compound (Table I). However, there were few compounds that reduced Fe(II1) at circumneutral pH under sterile conditions. Even some of the more reactive organic acids such as pyruvate, formate, and malate, which are frequently cited as being able to nonenzymatically reduce iron(1II) oxides at low pH (11, 37), did not reduce Fe(II1). Oxalate is another potential microbial metabolite that is frequently suggested to be a nonenzymatic reductant of iron(II1) oxides (11). Although we were unable to establish an Fe(II1)-reducing enrichment culture capable of using oxalate as the sole carbon and energy source, oxalate did not nonenzymatically reduce the iron(II1) oxide. This was in accordance with earlier findings that oxalate solubilized iron(II1) oxEnviron. Sci. Technol., Vol. 25, No. 6, 1991
1063
J
Table 11. Summary from Previous Studies of Aromatic Compounds That Can Be Oxidized to Carbon Dioxide with Fe(II1) as the Sole Electron Acceptor in the Presence of Fe(II1)-Reducing Microorganisms but That Are Not Nonenzymatically Oxidized in the Presence of Fe(II1) at pH 6.7 culture two-electron transfer t o Fe(II1) in 0.5 N HCI type GS-15 benzoic acid no GS-15 no toluene no GS-15 phenol no GS-15 p-cresol m-cresol enrichment no m-hydroxybenzoic acid enrichment no o-phthalic acid enrichment no syringic acid enrichment Yes ferulic acid enrichment yes nicotinic acid enrichment no compound
Sterile Control 0
10
20
30
Days of lncubatlon
Flgure 1. Dissolved and HCkxtractable Fe(I1) over time in anaerobic medium (pH 6.7) with 3 mM bcysteine as the sole electron donor and poorty crystalline iron(II1) oxkle as a potentiil electron acceptor. Note that the scale for dissolved Fe(I1) is one-tenth that for the HCI-extractable Fe(II), reflecting the fact that much of the Fe(I1) that is produced in the iron(II1) oxide medium is in solid form. Symbols: circles, enrichment culture; squares, sterile controls; closed symbols, HCI-extractable Fe(1I); open symbols, dissolved Fe(I1).
ides but did not reduce Fe(II1) a t pH 3.2 (38). Cysteine is known to nonenzymatically reduce Fe(II1) a t circumneutral pH with the oxidation of cysteine to cystine and the reduction of 1 mol of Fe(III)/mol of cysteine oxidized (19,39). Thus, the relative rate and extent of Fe(II1) reduction with cysteine in the presence and absence of microorganisms a t pH 6.7 was investigated (Figure 1). Small amounts of both dissolved and HC1extractable Fe(I1) were immediately detectable after addition of cysteine to sterile iron(II1) oxide media. There was no further increase in Fe(I1) under sterile conditions over time. However, in the Fe(II1)-reducing enrichment culture that was adapted to metabolize cysteine, the concentration of both types of Fe(I1) increased over time. The accumulation of HC1-extractable Fe(I1) in the enrichment cultures was consistent with complete oxidation of cysteine to carbon dioxide with Fe(II1) as the sole electron acceptor. These results indicate that although organic compounds with sulfhydryl groups can nonenzymatically reduce small amounts of Fe(III), the extent of the nonenzymatic Fe(II1) reduction is minor in comparison with the Fe(II1) reduction that is possible when microorganisms couple the oxidation of these organic compounds to Fe(II1) reduction. Fructose slowly reduced Fe(II1) over time in the absence of microorganisms (Table I). However, reduction of Fe(II1) was much faster and more extensive in the presence of microorganisms than under sterile conditions. Most types of the aromatic compounds that were tested in this (Table I) and previous studies (Table 11) did not reduce Fe(II1) under sterile conditions at pH 6.7, but the aromatics were readily oxidized to carbon dioxide with the reduction of Fe(II1) in the presence of Fe(II1)-reducing microorganisms. Of the wide variety of aromatic compounds tested, only catechol appeared to reduce the iron(II1) oxide at pH 6.7. The Fe(II1) was visibly reduced as catechol was added to the medium. The reduction of Fe(II1) in the presence of added catechol was accompanied by a decrease in catechol concentration from 5 mM to less than 1.25 mM in less than 30 min. As would be expected from previous studies (7), several other dihydroxyaromatic compounds such as hydroquinone and 2,3-, 2,5-, and 3,4hydroxybenzoate could transfer two electrons to Fe(II1) under the acidic conditions of the HC1-extractable Fe(I1) assay. When 5 mM of these compounds or catechol was added to sterile iron(II1) oxide medium and then assayed for HC1-extractable Fe(II), 8-10 mM HC1-extractable Fe(I1) was detected. Syringic and ferulic acids were pre1064 Environ. Sci. Technol., Vol. 25, No. 6, 1991
I
E&! -
ref 8 9 9 9
35 35 35 35 35 35
I
3,4-dihydroxybenzoate (enrichment culture) 10-
Sterile Controls
0
10
20
Days of Incubation
Flgure 2. Fe(II1) reduction in iron(II1) oxide enrichment cultures with 0.5 mM of either 3,4dihydroxybenzoate (closed circles) or 2,5dihydroxybenzoate (closed squares) as the sole electron donor. Open symbols are for the corresponding sterile controls.
viously found to transfer two electrons to Fe(II1) in the HC1-extraction procedure (35). However, a number of other dihydroxyaromatic compounds such as resorcinol, pholorglucinol, 2,4-dihydroxybenzoate, and 2,6-dihydroxybenzoate did not reduce Fe(III), even in the 0.5 N HCl. For the few aromatic compounds that did reduce Fe(II1) under acidic conditions, the extent of nonenzymatic Fe(II1) reduction, even under the acidic conditions, was minor in comparison with the Fe(II1) reduction that could take place at circumneutral pH in the presence of Fe(II1)-reducing microorganisms. For example when Fe(II1)-reducing enrichment cultures were established with 0.5 mM 2,5-dihydroxybenzoate or 3,4-dihydroxybenzoateas the electron donor, the production of HC1-extractable Fe(I1) over time was ca. 10-fold greater than the amount of Fe(I1) that was produced by acidic extraction of the sterile controls (Figure 2). The extent of Fe(II1) reduction in the enrichment cultures indicated that the 2,5-dihydroxybenzoate and 3,4-dihydroxybenzoate were being completely oxidized to carbon dioxide with Fe(II1) as the sole electron acceptor. Thus, although previous studies have emphasized the potential for aromatic compounds to nonenzymatically react with Fe(II1) (7,19,20,40,41),there are few aromatics that nonenzymatically react with Fe(II1) under conditions that would be expected in most aquatic sediments. Furthermore, the nonenzymatic oxidation of aromatic compounds with Fe(II1) is typically only a two-electron transfer, which results in the reduction of 2 mol of Fe(III)/mol of aromatic compound oxidized; no carbon dioxide is produced and the ring structure is not broken (7, 20). In contrast, Fe(II1)-reducing microorganisms can
Table 111. Fe(II1) Reduction during Glucose Metabolism by E . coli in the Absence and Presence of the Fe(II1)-Reducing Microorganism GS- 15"
organisms E . coli alone initial 2 days of incubtn 9 days of incubtn E . coli PIUS GS-15 initial 2 days of incubtn 9 days of incubtn f
Fe(II)b
glucoseb
0.0 f 0.0 0.0 f 0.0 0.0 f 0.0
9.4 f 0.1 0.4 f 0.1 0.0 f 0.0
0.0 f 0.0 0.7 f 0.9 145.7 f 15.9
8.8 f 0.4 0.4 & 0.1 0.0 f 0.0
H,*
formateb
ethanolb
acetateb
succinateb
NM
ND
ND
ND
ND
0.6 f 0.1 0.9 & 0.1
10.4 f 0.1 2.1 f 0.2
11.0 f 0.6 10.9 f 0.3
6.1 f 0.1 6.2 f 0.2
1.7 f 0.2 0.4 f 0.1
NM
ND
ND
ND
ND
0.9 f 0.1 1.1 f 0.1
9.4 f 0.2 2.1 f 0.2
10.1 f 0.2 1.6 f 0.6
6.2 f 0.3 5.7 f 0.2
4.6 i 4.8 0.9 f 0.1
pH NM NM 6.3 f 0.0
NM NM 7.3 f 0.1
"NM, not measured. ND, not detectable. bConcentrations in millimoles per liter except for H, which is in atmospheres. Values are mean standard deviation; n = 3.
Table IV. Glucose Metabolism and Fe(1II) Reduction by C . pasteurianum with and without Physical Contact between the Microorganisms and the Iron(II1) Oxide
treatment iron(II1) oxide iron(II1) oxide in dialysis tubing iron(II1) oxide inside and outside dialysis tubing
Fe(I1) produced" outside inside 18.3 0 12.8
NAc 0.3 0
fermentation productsb formate lactate butyrate
acetate 4.1 4.6 4.3
3.5 2.5 2.7
0 4.8 0
3.7 3.4 3.4
butanol
H2
0.9 1.5 1.0
0.20 0.23 0.25
GMillimolesof Fe(I1) per liter outside or inside the dialysis tubing after more than 20 days of incubation; mean of duplicate determinations on triplicate bottles of each treatment. Outside and inside designate whether the samples were taken from the bulk medium (outside) or inside the dialysis tubing. bAcids and alcohols in millimoles per liter after 8 days of incubation; H2 in atmospheres. Lactate may also include succinate as these comoounds cochromatoeraohed. NA. not amlicable.
completely oxidize a wide variety of aromatic compounds to carbon dioxide which, depending upon the aromatic compound, can result in ca. 30 or more moles of Fe(1II) reduced per mole of aromatic compound oxidized. Microbial activity was also necessary to bring about the oxidation of organic compounds in subsurface sediments in which Fe(II1) reduction was the terminal electron-accepting process (Figure 3). Acetate, glucose, and benzoate were chosen as representatives of a typical organic acid, fermentable compound, and aromatic compound, respectively. Trace quantities of these model compounds were readily oxidized to carbon dioxide in the nonsterile Fe(111)-reducingsediments. However, there was no oxidation of these compounds if microbial activity was inhibited. Previous studies have demonstrated that not even high concentrations (>1mM) of acetate, benzoate, or hydrogen could bring about the reduction of Fe(II1) in sterile sediments, but these compounds were readily oxidized with the reduction of Fe(II1) when the sterile sediments were inoculated with Fe(II1)-reducingmicroorganisms (2,8,36). Enzymatic Reduction of Fe(II1) versus Fe(II1) Reduction by a Low Redox Potential. In order to test the hypothesis that the generation of a low redox potential by microbial metabolism can result in the reduction of Fe(III), E. coli was grown in an anaerobic medium that contained glucose as the carbon source and iron(II1) oxide as a potential electron acceptor. E. coli is such a good scavenger for trace quantities of oxygen than even in medium that initially contains dissolved oxygen it will lower the redox potential to -600 mV (42). Under the anaerobic culture conditions, E. coli fermented the glucose with the accumulation of hydrogen, organic acids, and ethanol (Table 111). Despite the development of these highly reducing conditions, there was no Fe(II1) reduction, as indicated by a lack Fe(I1) accumulation (Table 111). However, when E. coli was cultured with the Fe(II1)-reducing microorganism strain GS-15, which cannot metabolize glucose but can metabolize some fermentation products, GS-15 metabolized the ethanol that was produced from E. coli fermentation with the reduction of large amounts of Fe(II1). Thus, even though the accumulation
-E '0
e
v)
4
Benzoate
cy
8
.-
Killed Controls
d
0 0
10
20
30
40
50
HOURS (X 0.1 for Benzoate)
Figure 3. Production of l4CO, over time when [2-I4C]acetate, [U14C]glucose,or [rinpi4C]benzoate was injected into Fe(II1breducing aquifer sediments. The benzoate data are from ref 8.
of reduced metabolic products was as great in the culture of E. coli alone as it was in the mixed culture, no Fe(II1) was reduced in the culture of E. coli alone because E. coli lacks the enzymes necessary to catalyze significant Fe(II1) reduction. Studies with C. pasteurianum further indicated that the development of a low redox potential during microbial fermentation cannot result in nonenzymatic Fe(I1I) reduction (Table IV). In contrast to E. coli, C. pasteuri a n u m reduces Fe(II1) while fermenting glucose (43). Within 8 days, the glucose was fermented to organic acids, butanol, and hydrogen in the presence or absence of Fe(II1) (Table 111). When there was the potential for contact between C. pasteurianum and the Fe(III), Fe(II1) was reduced. Formate did not accumulate in cultures in which Fe(II1) reduction was detected. As formate does not nonenzymatically react with iron(II1) oxides (see above), these results suggested that C. pasteurianum diverted metabolic electron flow from formate formation to Fe(II1) reduction (43). Fe(II1) reduction was inhibited when the iron(II1) oxide was contained within a dialysis tubing, which permitted the passage of dissolved compounds but excluded C. Environ. Sci. Technol., Vol. 25, No. 6, 1991
1065
pasteurianum (Table IV). The lack of reduction of Fe(II1) within the dialysis tubing could be attributed to the lack of contact between C. pasteurianum and the iron(II1) oxide because when cultures with Fe(II1)-containing dialysis tubing were also provided with iron(II1) oxide outside the dialysis tubing, the iron(II1) oxide outside the dialysis membrane was readily reduced (Table IV). The requirement for direct contact between C. pasteurianum and the iron(II1) oxide suggested that, as with E. coli, the accumulation of fermentation products was not sufficient to bring about Fe(II1) reduction. A similar requirement for contact between fermentative Fe(II1)-reducing microorganisms and Fe(II1) had been observed previously with hematite as the Fe(II1) source (29). However, hematite, a highly crystalline and stable iron(II1) oxide, is not considered to be an important source of Fe(II1) for Fe(II1) reduction in sediments ( I ) . Reactive, poorly crystalline iron(II1) oxides are the principal electron acceptors for Fe(II1) reduction in aquatic sediments (32). The results presented here demonstrate that even highly reactive iron(II1) oxides are not reduced by the development of reducing conditions during microbial fermentation. The concept that microbial metabolism lowers the redox potential of sediments and then the low redox potential brings about the reduction of Fe(II1) is probably the reverse of the actual order of events in sediments. In nonsulfidic environments, the platinum electrode typically used to measure the redox potential of sediments and groundwater responds primarily to the Fe(II1)-Fe(I1) couple (15,44). Thus, Fe(1I) must be generated before a low redox potential will be recorded. Therefore, it is more likely that microbially catalyzed Fe(II1) reduction generates the low measured redox potential rather than the low redox potential causing Fe(II1) reduction. Potential Special Instances of Nonenzymatic Fe(111) Reduction. It has been speculated that nonenzymatic reduction of Fe(II1) by organic compounds might be important in the special circumstance in which Fe(111)-reducing microorganisms release unique organic compounds that serve as an electron shuttle mechanism to permit Fe(II1)-reducing microorganisms to transfer electrons to iron(II1) oxides (45). However, this is not consistent with the finding that both fermentative Fe(111)-reducing microorganisms (see above) and the respiratory Fe(II1)-reducing microorganism GS-15 (22) require direct contact with Fe(II1) in order to reduce it. The requirement for contact with Fe(II1) in GS-15 results from the fact that the Fe(II1) reductase in this organism is membrane bound (46). Nonenzymaticreduction of Fe(II1) by sulfides is another potential mechanism for the reduction of Fe(II1) in sediments (47). However, this reaction has little significance for the coupling of organic matter oxidation to the reduction of Fe(II1) that is observed in the Fe(II1)-reducing zone of sediments. With the exception of highly eutrophic sediments, the zone of maximum Fe(II1) reduction is distinct from the zone of sulfide generation in both aquatic sediments and aquifers ( 3 6 , 4 8 4 0 ) . This is because Fe(111)-reducingmicroorganisms can outcompete sulfate-reducing microorganisms and maintain the concentrations of organic compounds and hydrogen too low for sulfate reducers to metabolize (33). Laboratory studies have demonstrated that, even in marine sediments, most of the Fe(II1) reduction may be independent of sulfide generation (27, 47). Summary The results demonstrate that, under the conditions typically found in nonsulfidogenic sedimentary environ1066 Environ. Sci. Technoi., Vol. 25, No. 6, 1991
ments, there is little potential for Fe(II1) reduction by frequently cited nonenzymatic mechanisms. &en in those rare instances in which organic compounds can nonenzymatically reduce Fe(III), the typical one- or two-electron transfer that results from nonenzymatic Fe(II1) reduction is minor in comparison to the complete oxidation of organic compounds in the presence of Fe(II1)-reducing microorganisms. The reducing conditions that develop as the result of microbial consumption of oxygen and nitrate and production of fermentation products do not result in nonenzymatic Fe(II1) reduction. Previous studies have also concluded that Fe(II1)-reducing microorganisms directly catalyze most of the Fe(II1) reduction in sediments (25,27-29,36,47). However, this conclusion was primarily based on the finding that when microbial activity in sediments was inhibited, Fe(II1) reduction was inhibited. This result is equivocal because it could also be interpreted as indicating that microbial metabolism was necessary for the generation of a low redox potential and/or for the production of reactive organic compounds capable of nonenzymatically reducing Fe(II1) ( 1 , 1 1 , 1 8 , 5 1 ) .Thus, many authoritative texts have continued to emphasize the importance of nonenzymatic Fe(111) reduction. However, the combination of previous sediment studies and the results presented here indicate that, in sediments in which oxidation of organic matter coupled to Fe(II1) reduction is the major process for organic matter decomposition, nonenzymatic Fe(II1) reduction is a trivial process. Thus, just as other anaerobic processes for organic matter oxidation such as nitrate reduction, sulfate reduction, and carbon dioxide reduction (methanogenesis) are enzymatically catalyzed, it appears that most of the oxidation of organic matter coupled to Fe(II1) reduction in aquatic sediments is the direct result of enzymatically catalyzed reactions. Acknowledgments
We thank Richard Smith and Frank Chapelle for helpful suggestions on the manuscript. Registry No. Fe, 20074-52-6; H, 1333-74-0;formate, 64-18-6; pyruvate, 127-17-3; acetate, 64-19-7; ethanol, 64-17-5; glycerol, 56-81-5; lactate, 50-21-5; propionate, 79-09-4; butyrate, 107-92-6; malate, 97-67-6; succinate, 110-15-6; citrate, 77-92-9; glucose, 50-99-7; fructose, 30237-26-4; lysine, 56-87-1; serine, 56-45-1; aspartate, 56-84-8;glycine, 56-40-6;benzaldehyde, 100-52-7; benzyl alcohol, 100-51-6;p-hydroxybenzoate, 99-96-7; p-hydroxybenzyl alcohol, 623-05-2; tyrosine, 60-18-4; phenyl acetate, 122-79-2.
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Pap. 1961, NO. 1469-H. Received for reuiew October 15, 2990. Revised manuscript received January 15,1991. Accepted January 15,1991. This study was supported by the US.Geological Survey Toxic WasteGround- Water Contamination Program.
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