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Effects of added manganic and ferric oxides on sulfate reduction and sulfide oxidation in intertidal sediments
131
FEMS MicrobiologyEcology73 (1990) 131-138 Publishedby Elsevier FEMSEC00242
Effects of added manganic and ferric oxides on sulfate reduction and sulfide oxidation in intertidal sediments Gary M. King Institute of Ecology and Genetics. University of Aarhus~ Aarhus. Denwaark
Received26 April 1989 Revisionreceived13 July 1989 Accepted 18 July 1989 Key words: Manganese reduction; Iron reduction; Marine sediments
1. SUMMARY
2. INTRODUCTION
Freshly precipitated iron or manganese oxides were added to surface sediments from a salt marsh and from the intertidal region of Lowes Cove, Maine. in the presence of added manganese, sulfate was formed under anoxic conditions, suggesting a manganese dependent sulfide oxidation. Sulfate formation was not observed with iron additions. Sulfate reduction was substantially but not completely inhibited by either metal oxide, even though both were added at levels well in excess of natural concentrations. Manganesecatalysed sulfide oxidation was further documented using a combination of radiolabel, metal oxide, and inhibitor additions. Results from this study suggested that losses of radiolabelled sulfide could result in underestimates of gross sulfate reduction rates in the presence of significant manganic oxide concentrations, In addition, manganic oxides may facilitate the anaerobic regeneration of sulfate from sulfides.
Although both chemical and microbial manganese transformations have been documented for some time (e.g. refs. I-I0) a detailed understanding of manganese dynamics remains an unrealized goal [II]. For example, recent reports have extended the array of microbial transformations, thereby further complicating an already diverse set of processes (e.g. refs. 12, 13). In particular, Aller and Rude (14] have presented evidence indicating that manganic oxides serve as electron acceptors for the anaerobic oxidation of sulfide to sulfate. This process, thought to be microbial, could play an important role in anoxic sulfate regeneration; it may also represent a new form of lithotrophic metabolism by as yet uncharacterized organisms. However, as Ailer and Rude [14] have noted, many questions about the process remain unanswered. One area of uncertainty is the relationship between manganese oxides and sulfate reduction. For example, it is not clear if manganic oxides actually inhibit sulfate reduction as one might predict from the results of Lovley and Phillips [12], or if manganese oxidation of sulfide can prevent the depletion of sulfate, resulting in an
Correspondence to: G.M. King.Instituteof Ecologyand Genet-
ics, Uaivenityof Aarhus, Ny Munkegade.Bldg550. DK.8000 Aarhus C, Denmark.
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132 apparent inhibition. Likewise, it is not dear to what extent the presence of manganese oxides can affect the measurement of sulfate reduction rates using radiotracer techniques. The results presented here are from experiments designed to provide some insists for these questions. Additions of manganese oxides to several different marine sediments suggested the possibility that sulfate reduction may not be completely inhibited by manganese, even when present at relatively high concentrations. Radiotracer experiments have confirmed this possibility and provided further evidence for the role of manganic oxides in the oxidation of sulfide to sulfate. However, results from the radiotracer experiments have also indicated that the presence of manganese oxides can cause an underestimation of sulfate reduction rates, especially during long-term incubations. In contrast, iron oxides have an inhibitory effect similar to that of manganese oxides but do not appear to affect a conversion of sulfide to sulfate.
3. METHODS Sediments for this study were obtained from an intertidal region of Lowes Cove, Maine and a creekbank site on Skidaway Island, Georgia. The Lowes Cove site has been described previously [15]. After collection in 10-cm inner diameter core tubes, the 1-5 cm depth interval was processed as below. The Skidaway site and sediments were described by Aller and Rude [14]. These sediments were collected from the 2-5 cm interval, stored in a plastic 3.8-1 container at 4 ° C, and shipped to Maine by R. Aller. The sediments had been stored approximately 3 weeks prior to analysis. In general, sediments were processed by homogeniaing in an anaerobic gloveba8 (Coy lnc.) containing a gas phase of approximately 90~ nitrogen, and 5~ each of hydrogen and carbon dioxide. Sediments were not diluted in this case but the preparation could be considered a 'slurry'. For analyses of sulfate consumption and manganese reduction, screw-cap polypropylene centrifuge tubes (15 cm3) were completely filled with untreated, homogenized sediments (controls) or
with sediments containing 20 mg/cm 3 of a freshly prepared manganic oxide (prepared according to reL 16). This mineral phase was not characterized for crystallinity but an amorphous oxidized gel [MnO2] was expected. For analyses of sulfate reduction rates by a radiotracer method, homogenized sediments with or without added manganese were dispensed into cut-off 5-cm3 syringes that were sealed with butyl septa (Supelco, Inc.). Both tubes and syringes were incubated at room temperature for several weeks. During the initial experiments conducted with Priest Landing sediments, incubations involved storage of tubes in an anaerobic glovebag containing a gas phase with 5% hydrogen. Later experiments with Lowes Cove sediments were based on incubations of tubes or syringe.cores in sealed jars containing deoxygenated water. Time courses of activity were generated by sacrificing duplicate or triplicate samples as necessary. The radiotracer estimation of sulfate reduction rates was based on methods described by King et al. [17] and King [18]. Modifications involved the use of 5-cm3 syringes which were injected along their central axes with 10/~1 of a solution containing approximately 1 taCi of carrier-free 3SSO~-. The distillation of reduced radiolabelled sulfate reduction endproducts was based on either sequential acid and boiling chromous chloride digestions or on boiling chromous chloride digestions only. The efficacy of these distillations was determined by using a synthetic FeS in presence and absence of manganic oxide. Other analyses of sulfate reduction involved additions of manganese or iron oxides (20 mg/cm 3 sediment; prepared according to ref. 19; the expected mineral phase is an oxyhydroxidc gel) with or without sodium molybdate (final concentration 20 raM), Homogenized Lowes Cove sediments (4 cm~) in modified 5-cm~ syringes were used for these studies. The metal oxides or inhibitors were added after a pre-incubation of 16-20 h with 35SO~- to allow for accumulation of radiosulfide. The additions were made by injecting deoxygenated suspensions (1 cm3) of the metal oxides or solutions of sodium molybdate and by gently mixing the syringe contents. After an additional incubation sediments were processed as before
133
with the exception that sediments containing molybdate were distilled with boiling acidic titanous chloride. ]'he use of titanous chloride insured complete recovery of sulfide in presence of molybdate [20]. Time course analyses for dissolved manganese and sulfate were conducted by centrifuging sediments incubated in polypropylene tubes as above. Porewater from the tubes was used for a turbidimetric sulfate analysis [21]. Porewaters for manganese analysis were acidified with 6 N HCI and stored until assay by graphite-furnace atomic absorption spectrometry (Hitichai Perkin-Elmer). The output of the instrument was standardized with solutions of manganous chloride in an acidified sodium chloride matrix.
4. RESULTS
4. L Effect of manganic oxides on sulfide distillation In order to measure sulfate reduction rates using radiotracer techniques, it was necessary to evaluate the efficacy of the distillation procedures used for recovering radiolabelled sulfides, This was done by determining recoveries for a synthetic iron monosulfide (FeS, about 20 mg) in the presence of excess manganic oxide, A distillation using 1 N HC! alone was ineffective (Table 1) due to the rapid formation of a yellow, carbon disulfide soluble precipitate which was presumably elemental sulfur. Elemental sulfur has been previously reported as the major oxidation product of sulfides in the presence of manganic ion at low pH [22]. Table l Effect of mangani¢oxide on recoveryof sutfides during acid and chromouschloridedistillations A. Recoveryof FeS std with ! N HCI: Added FeS: 226.3/tmol RecoveredSulfide: 228.8 ~mol:t: 10.3 (101.1~) B. Recoveryof FeS in the presenceof manganicoxide: Added FeS: 230,8 tsmol HCI recovery: 64,8 Fmol+ 15.3 (28.1~) CRS recovery: 150.5/tmol+20.0 (65.2~) Total: 215.3/tmo1:1:11,5 (93.3%) Values are means± 1 S,E. (n = 3),
Distillation with chromous chloride subsequent to an acid distillation resulted in a nearly complete recovery of mass, which indicated that the products of a chemical oxidation of sulfide did not include sulfate ('fable 1). These results were consistent with the initial formation of elemental sulfur by manganic ion and its subsequent reduction by chromous ion. A second evaluation of distillation methods involved a comparison of acidic solutions of chromous and titanous chlorides, each at 1 M concentrations. Titanous chloride, but not chromous chloride, effectively ,,;covered sulfide standards in the presence of molybdate. Recovery of radiolabelled sulfides from a sediment matrix in the absence of molybdate was not statistically different by ANOVA (P > 0.75, n = 6) when either titanous or cbromous chlorides were used for distillation. Therefore, titanous chloride was used for sediments amended with sodium molybdate while chromous chloride was used otherwise.
4.2, Effect of manganic oxides on sulfate depletion Time courses of sulfate exhibited two patterns. For the Priest Landing sediments, sulfate consumption proceeded without a lag in unamended controls (see Fig. 1: slope of regression analysis for first 7 days = 800 nmol/cm3/d; P ~ 0.002). In contrast, rates of sulfate consumption for the first 5 days in the presence of manganic oxide were not statistically different than zero (slope of regression analysis = 32 nmol/cm3/d; P ~ 0.221). Following this period, sulfate was depleted about an order of magnitude more rapidly in the presence of manganic oxide (slope of regression analysis = 375 nmol/cm3/d; P = 0.02) and at a rate equal to that of the controls. Although little sulfate depletion in manganeseamended sediments was observed dw-ing the initial 5-day incubation, a radiotracer analysis ef sulfate reduction indicated that actual depletion rates may have been higher than those calculated from changes in pool sizes. Average rates based on short-term (about 12 h) incubations with 35SO2were about 131 nmol/cm3/day at least 4-fold higher than rates from mass depletion. At a later point during the incubation (21 days), radiotracer-based rates were approximately equal
134 20
A
'
16
'
•
~......
B
4
e.
~;
24
+-~
18
8 6 03 I
0 [~+_ 0
6
12
18
24
30
Time (days} Fig. I. Time course of sulfate concentrations in sediments from a salt marsh creek bank (Priest Landing). • represent controls and • represent sediments amended with manganic oxide (see text for details). Concentrations are means of duplicate determinations. Trends in the data were determined by linear regression analysis. For the controls, region 'A' is described by: sulfate (raM) = 18A+0.049.time, r 2 = 0.9"/; for region 'B': sulfate (raM) = 13.73 :i: 0.023. time, r 2 = 0.94. For the manganese treatment, region 'A' is described by: sulfate (raM) = 18.2 + 0,002. time, r 2 = 0.01; for region 'B': sulfate (raM) = 21.32 +0.023, time, r 2 = 0,87.
to rates calculated by a regression analysis of stable sulfate losses (336 nmol/cm3/day vs. 375 nmol/cm3/day). Sediments from Lewes Cove exhibited a second pattern. In this instance, sulfate excesses (9.4 raM) were observed without lag after the addition of manganic oxide; in contrast, a rapid depletion was observed in controls (Fig. 2), Excess sulfate was formed for a period of about 11 days, after which a depletion was noted, During the period of sulfate production, dissolved manganese accumulated in the porewater of experimentals but not controls (Fig, 3), If the increase in sulfate was due to manganic oxide reduction by sulfides, a total of 37.6 mM manganese would have been expected, The observed concentration in the pore-water, 2,0 mM, was about 19-fold less, suggesting that the remainder of the reduced manganese was associated with the solid phase,
0.0
3.0
i
i
i
6.0 9.0 12.0 Time (days)
1
150
,, 18.0
Fig. 2. Time course of sulfate concentrations in sediments from the intertidal region of Lewes Cove, Maine. • represent controls and • represent sediments amended with manganic oxide (see text for details). Concentrations are means of duplicate determinations.
inhibited the accumulation of radiolabelled sulfide (Fig. 4a, b). lnhibitors were added after sediments were pre-incubated for 16 h. Sulfide accumulation relative to unamended controls was minimal in the presence of manganic oxide and molybdate (-0.6%) and otherwise exhibited the following pattern: molybdate only, 7.1-21.6% (Figure 4A and B respectively); ferric oxide + molybdate, 18.9%, ferric oxide only, 53.3%; manganic oxide, 58,0%. The differences between manganic oxide plus molybdate and molybdate only were statisti2400
1920
+Mn o x i d e /
1440
960
480 f
0 = 0,0
Ctrl
3,0
6,0 Time (days)
9.0
12.0
Fig, 3, Time course of total dissolved manganese concenlra-
4.3. Effect of manganic oxides on sulfide oxidation Additions of metal oxides with or without sodium molybdate and sodium molyhdate alone
lions in sediments from the intertidal region of Lowcs Cove+ Maine, • ~pre~nt controls and • represent sediments amended with manganic oxide, Concentrations are means of duplicate determinations.
135 35000
36000 300O0
28000
1o
24000
21000 18000 14000
f
7000
I,
12000 6000
/ 9.0
0.0
Ci
18.0 27.0 Time (hrs)
36.0
45 0
0.0
10.0
20.0 30.0 Time (hrs)
40.0
50.0
Fig. 4. A. Effect of added mangaaic oxide (B), molylxlate (@), and molybdate+manganic oxide (El) on the accumulation of radiolahelled sulf'gl¢ versus um~,ated controls (o). See text for details. Data arc means of triplicate determinations; error bars arc + | standard error. B. Effect of added ferric oxide (13), molylxlate (IlL and molybdate + [erric oxide (@) on the accumulation of
radiolabclhal sulfide versus untreated controls (o). Data arc means of triplicate determinations; error bars are + 1 standard error.
cally significant (0.01 < P < 0.05, n = 6), but were not significant for ferric oxide plus molybdate versus molybdate only. The patterns of label distribution for manganic oxide plus molybdate and molybdat¢ only indicated that manganese facilitated the net consumption of radiolabelled sulfides. No net consumption was observed with ferric oxides. In a more detailed time course, extensive consumption of radio-labelled sulfides occurred in the presence of added manganic oxides (Fig. 5). In
this case sediments were pre-incubated with 35SO~- for 24 h, supplemented with sodium molybdate as above, incubated an additional 24 h, and then supplemented with manganic oxide. In the control sediments (molybdate only) radiolabelled sulfides did not accumulate after the first 48 h. In contrast, addition of manganic oxides resulted in rapid losses of label which amounted to 45.6~ of the increase in the controls after 72 h.
5. DISCUSSION 30000
i
....
[
. . . . .
24000
,
Cld
/
(~ 18000
¢.q I~000
6000
0.0
Molybdate
30.0
600
90.0
120,0
1500
Time (hrs) FiB. 5. Time course of radiolabcllcd sulfide in the presence of molybdate (13) or molybdat¢ + manganic oxide (o) after initial actumu}ation in unamended sedi~nts (se¢ text for details). Arrows d¢not¢ points of addition for molybdat© and mangani¢ oxide. Data arc means of triplicate determinations; error bars are :1:l standard error.
Radiotracer techniques can provide great sensitivity for the analysis of interactions between manganic oxides and sulfate reduction. However, these techniques require an accurate estimation of radiosulfide formation. Previous studies have shown that acidic chromous chloride distillations are the most reliable means for measuring radiosulfides [17,23,24]. Results from this study confirm that conclusion. Elemental sulfur is formed rapidly from sulfides in the presence of acidic solutions containing manganic ions [5]. This results in substantial losses of acid volatile radiosulfides, presumably from both tile dissolved and iron monosulfide fractions (Table 1). As a result, simple acid distillations are ineffective. In contrast, acidic chromous chloride is quite effective for recovering radiosulfides (Table 1). This is due to the well-
136 known ability of chromous chloride to reduce elemental sulfur and to the low potential of the chromous-chromic couple, which allows for manganic ion reduction as well [25]. Since patterns of label distribution from sequential acid and chromous chloride distillations are not likely to be meaningful [17,18,24], a single chromous chloride reduction is recommended for radiosulfide recovery. The analysis of both stable sulfate pools and radiosulfate reduction indicates that added manganic oxides facilitates the oxidation of sulfides, most likely in accordance with the mechanism proposed by Aller and Rude [14]: 4 MnO2 + S2- + 4 H + -~ 4 Mn2+ + SO~- + 4OH-
(1) Aller and Rude [14] suggested that the form of sulfide oxidized was an iron monosulfide, perhaps involving a dissolved intermediate. The same may have been true in this study, but the data are inadequate for drawing such a conclusion. Therefore the reaction stoichiometries are given for the sulfide ion only. This general reaction may explain the patterns observed for sulfate and sulfide in several different experiments. A comparison of sulfate reduction rates based on radiotracer reduction and changes in stable sulfate pools (Fig, 1) suggests sulfide recycling, the extent of which is greatest during the first 5 days of incubation. Recycling was probably minimal after this period since sulfate depletion was observed at rates comparable to controls (Fig. 1) and since radiosulfate consumption rates were similar to stable sulfate depletion rates. The period characterized by sulfide recycling was coincident with a period of manganese accumulation in the porewater while concentrations were more constant at later intervals. These results differ from those of Aller and Rude [14] who have used sediments from the same salt marsh. They have observed significant sulfate accumulation during short-term incubations. The differences may be explained by the fact :hat the sediments used for the work reported here were incubated in polypropyhne tubes in a glovebag containing approximately 5~ hydrogen. The pres-
ence of a hydrogen gas phase can stimulate sulfate reduction rates about 2-fold, due to diffusion of hydrogen through the tube walls (data not shown). Stimulation of sulfate reduction may have altered the balance between sulfate depletion and sulfide oxidation so that net excesses were not formed. In contrast, sulfate excesses similar to tho~ reported by Aller and Rude [14] were observed in sediments from l.owes Cove (Fig. 2). These sediments were incubated in sealed jars containing deoxygenated water. The absence of hydrogen may have contributed to the relative differences ~ t h the Priest Landing sediments. As with the Priest Landing sediments, porewater manganese accumulation corresponded to the period of sulfide oxidation (Fig. 3). However, manganese accumulation was not equivalent to that expected from the stoichiometry of reaction 1. Similar results were reported by Aller and Rude [14]. The discrepancy between dissolved manganese accumulation and sulfate excesses is mostly likely due to the accumulation of reduced manganese in the solid phase, both in an adsorbed form and as manganous carbonates [14,22]. The appearance of pink deposits in the Priest Landing and Lewes Cove sediments were evidence of the latter. Unfortunately, methods for extracting solid phase reduced manganese are subject to interferences from the dissolution and subsequent re. duction of oxidized species. For example, several methods exist for speciating manganic and manganous ions (e.g. ref. 26-28), but these involve acid extractions which can liberate ferrous iron and sulfide, both of which rapidly reduce manganic ions. Copper displacement can provide information on adsorbed pools [10], but does not mobilize minerals such as rhodochrosit¢ or kutnahorite. As a result, mass balances for manganese are difficult at best. The net loss of radiolabelled sulfide in the presence of molybdate plus manganese confirmed the role of manganic oxides as an oxidant for sulfide. The oxidation of sulfide in these experi. ments was presumably microbial, since the reported primary ,~ndproduct of chemical oxidation is demental sulfur (e.g refs, 10, 14). Formation of this or any other endproduct with the exception of sulfate would not have resulted in losses of radio-
137 sulfide, since the chromous chloride distillation technique used in this study recovers all major inorganic reduced and oxidized sulfur species other than sulfate. Several other aspects of these experiments deserve comment. First, the radiosulfide was very likely distributed by isotopic exchange among several phases within the sediment matrix including dissolved sulfides, polysulfides, elemental sulfur, iron monosulfides, and pyrite [24]. Which of these were oxidized is not clear, since the chromous chloride distillation does not distinguish among the various phases. However, in Lewes Cove sediments radiosulfides are typically highest in the acid volatile fractions (> 70~: King, unpublished data). These are the most probable sources of oxidizable label, since losses of the pyrite pool equivalent to the loss of label would have required an amount of manganic ion m excess of that added. As noted previously, oxida. tion of iron monosulfides has been suggested by others [14]. Second, it appears that added iron oxides do not facilitate the loss of sulfide by an oxidation to sulfate (Fig, 4). This is consistent with the known chemistry of ferric iron [14,29]. Under the conditions used in this study elemental sulfur is the expected endproduct, much as is the case with manganic oxides. In the absence of a biological mechanism to form sulfate, the conversion of sulfides to elemental sulfur does not result in a loss of mass or label as measured by the chromous chloride distillation, Third, the addition of either manganese or iron oxides inhibited sulfate reduction. In experiments with Priest Landing sediments, sulfate reduction rates from both mass depletion and radiotracer analyses were lower in the presence of manganic oxides by > g0~g. In the radiotracer experiments with Lewes Cove sediments, manganese addition resulted in an inhibition of about 42cg. This inhibition was in part due to the oxidation of radiolabel which can cause an underestimation of the true rate. In these experiments, oxidation may have accounted for as much as 53~ of the apparent inhibition. Iron oxides resulted in an inhibition of about 47~, all of which was presumably due to direct effects since no oxidation of label
by iron was observed. These patterns are similar to those of Lovley and Phillips [13] and Aller and Rude [14] who have reported significant inhibition of sulfate reduction by manganese and iron oxides, respectively. Direct inhibition of sulfate reduction by either metal oxide may have been due to competition for hydrogen between metal-reducing bacteria and sulfidogens [13]. Finally, both the mass and radiotracer data provide support for the contention that manganie oxides promote the oxidation or anaerobic recycling of sulfide. However, it must be noted that these results are based on the addition of relatively high concentrations of oxides which may alter the balance between carbon-based and sulfide-based manganese reduction. Since beterotrophic manganese reduction is characteristic of many sedimentary bacteria [11], the dynamics of the sulfide-manganese couple must be determined under in situ conditions before the relative importance of putative lithotrophic reactions can be established.
ACKNOWLEDGEMENTS The author wishes to thank Dr. R. Aller for discussions and advice which led to the origination of this study and for helpful input throughout. Ms. L. Hall is acknowledged for technical assis. tance. This work was supported in part by the National Science Foundation (OCE 8700873).
REFERENCES Ill Ghiorse,W.C.and Ehrlich,H.L.(1976)Electrontransport componentsof the MnO: reductasesystemand the location of the terminalreductas¢in a marineBacillus. Appl. Environ. Microbiol.31, 9"17-985. [2] Duinker,J,C., Wollast,g. and Billen,G. (1979)Bchaviour of manganesein the Rhine and Schddt estuaries. Est. Coastal Mar. ~i. 9. 727-738. [3] Froelich, P.N., Klinkhammer, G.P., Bender. M.L., Lucdtke, N,A.. Heath, G.R., Cullen. D, Dauphin. P, Hammond, D., Hartman, B. and Maynard. V. (1979) Early oxidationof organicmatterin pelagicsedimentsof the easlern equatorial Atlanticsuboxicdiagenesis.Geochim. Cosmochim.Acta43, 1075-1090.
138 [4] Yeats. P.A.. gundby, B. and Brewers, J.M. (1979) Manganese recycling in coastal waters. Mar. Chem. 8, 43-55. [5l Burdige, D.J. and Kepkay, P.E. (1983) Determination of bacterial manganeseoxidation rates in sediments using an in situ dialysistechnique. 1. Laboratory studies. Geochim. Cosmochim. Aeta 47, 1~07-1916. [6] l,axen, D.P. and Chandler, I.M. (1983) Size distribution of iron and manganesespecies in freshwater.Geochim. Cosmochim. Acta 47, 731-741. [7] Sonda, W.G,, Huntsman, 8,A. and Harvey, G.R. (1983) Photoreduction of manganese oxides in seawater and its geochemical and biological implications. Nature 301, 234-236. [8] Laxen, D.P.H.) Davison, W, and Woof, C. (1984) Manga. nes¢ chemistry in rivers and streams. Gcochim. Cosmochim. Acta 48, 2107-2111. [9] Rosson, R.A., Tebo, B M. and Nealson, K.H. (1984) Use of poisons in deter~,inationof microbial manganese binding rates in ~.awater. AppL Environ. Microbiol. 47, 740-745. [10] Burdige, DJ. and Nealson, K.H. (1985) Microbial manganese reduction by enrichment culturesfrom coastal marine sediments. Appl. Environ. Micrnbiol. 50, 491-497. [II] Ncalson, K.H. (1983) Microbial oxidation and reduction of manganese and iron, in Biomineralization and biological metal accumulation (P. Westbroek and E.W. de Jong, eds.),pp. 459-479. D. Reidel Publishing Co., Boston. [12] Meyers, C.R. and Nealson, K.H. (1988) Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science 240,1319-1321. [13] Lovley, D.R. and Phillips,E.J.P. (1988) Novel mode of energy metabolism: organic carbon oxidation coupled to dissimilatory reduction of iron or manganese. App. En,,'iron.Microbiol. 54,1472-1480. [14] Aller,R.C. and Rude, P.D. (1988) Complete oxidation of solid phase sulfides by manganese and bacteria in anoxic sediments. Geechim. Cosmochim.Acta 52, 751-765. [15] Anderson, F.E., Black, L., Watling, L., Mook, W. and Mayer, L. (1981) A temporal and spatial study of mudflat erosion and deposition. J. Seal. Pet. 51, 729-736. [16] Balistrieri, L.S. and Murray, J.W. (1982) The surface of ~-MnO2 in major ion seawater. Geochim. Cosmochim. Acta 46, 1041-1052.
{171 King, G.M., Howes, B.L. and Dacey, J.W.H. (1985) Short-term endproducts of sulfate reduction in a salt marsh: formation of acid volatilesulfides,elementalsulfur, and pyrite. Geochim. Cosmochim.Acta 49,1561-1566. [18] King, G.M. (1988) Patterns of sulfate reduction and the sulfur cycle in a South Carolina salt marsh. Limnol. Oceanogr. 33, 376-390. [19] Parfitt, R.C., Atkinson, R.J. and Smart, R.St.C. (1975) The mechanism of phosphate fixation by iron oxides. Soil Sci. Soc. Am. Pro(:. 39, 837-841. [201 Banat, I.M., Lindstrom, E.B., Nedwell, D.B. and Balba, M.T. (1981) Evidence for coexistenceof two distinct functional groups of sulfate-reducing bacteria in salt marsh sediment. Appl. Environ. Microbiol.42, 985-992. [21] Tabatabai, M. (1974) Determination of sulfate in water samples. Sulphur Inst. J. 10, 11-13. [22] Burdige, D.J. and Neaison, K.H. (1986) Chemical and microbiologicalstudies of sulfide-mediatedmanganese reduction. Geomicrobiol.J. 4, 361-387. [23] Westrich, J.T. (1983) The consequences and controls of bacterial sulfate reduction in marine sediments. Ph.D. dissertation. Yale University,479 pp. [24] Fossing, H. and Jorgensen, B.B. (1989) Measurement of bacterial sulfate reduction in sediments: evaluation of a single-step chromium reduction method. Biogeochemistry, in press. [25] Cotton, F.A. and Wilkinson, G. (1962) Advanced inorganic chemistry: a comprehensive text. interscience Publishers, New York. [26] Kalhorn, S. and Emerson, S. (1984) The oxidation state of manganese in surface sediments of the deep sea. Geochim. Cosmochim. Acta 48, 897-902. [27] Lyle, M., Heath, G.R. and Robbins, J.M. (1984) Transport and release of transition elements during early diagenesis: sequential leaching of sediments from MANOP sites M and H. Part 1. pH 5 acetic leach. Geochim. Cosmochim. Acta 48, 1075-1715. [28] Murray, J.W., Balistrieri, L.S. and Paul, B. C984) The oxidation state of manganese in marine sedJmems and ferro-manganese nodules. Geochim. Cosmoc!,tm.Acta 48, 1237-1247. [29] Burner, R.A. (1970) Sedimentary pyr;*.eformation. Am. J. Sci. 268,1-23.
FEMS MicrobiologyEcology73 (1990) 131-138 Publishedby Elsevier FEMSEC00242
Effects of added manganic and ferric oxides on sulfate reduction and sulfide oxidation in intertidal sediments Gary M. King Institute of Ecology and Genetics. University of Aarhus~ Aarhus. Denwaark
Received26 April 1989 Revisionreceived13 July 1989 Accepted 18 July 1989 Key words: Manganese reduction; Iron reduction; Marine sediments
1. SUMMARY
2. INTRODUCTION
Freshly precipitated iron or manganese oxides were added to surface sediments from a salt marsh and from the intertidal region of Lowes Cove, Maine. in the presence of added manganese, sulfate was formed under anoxic conditions, suggesting a manganese dependent sulfide oxidation. Sulfate formation was not observed with iron additions. Sulfate reduction was substantially but not completely inhibited by either metal oxide, even though both were added at levels well in excess of natural concentrations. Manganesecatalysed sulfide oxidation was further documented using a combination of radiolabel, metal oxide, and inhibitor additions. Results from this study suggested that losses of radiolabelled sulfide could result in underestimates of gross sulfate reduction rates in the presence of significant manganic oxide concentrations, In addition, manganic oxides may facilitate the anaerobic regeneration of sulfate from sulfides.
Although both chemical and microbial manganese transformations have been documented for some time (e.g. refs. I-I0) a detailed understanding of manganese dynamics remains an unrealized goal [II]. For example, recent reports have extended the array of microbial transformations, thereby further complicating an already diverse set of processes (e.g. refs. 12, 13). In particular, Aller and Rude (14] have presented evidence indicating that manganic oxides serve as electron acceptors for the anaerobic oxidation of sulfide to sulfate. This process, thought to be microbial, could play an important role in anoxic sulfate regeneration; it may also represent a new form of lithotrophic metabolism by as yet uncharacterized organisms. However, as Ailer and Rude [14] have noted, many questions about the process remain unanswered. One area of uncertainty is the relationship between manganese oxides and sulfate reduction. For example, it is not clear if manganic oxides actually inhibit sulfate reduction as one might predict from the results of Lovley and Phillips [12], or if manganese oxidation of sulfide can prevent the depletion of sulfate, resulting in an
Correspondence to: G.M. King.Instituteof Ecologyand Genet-
ics, Uaivenityof Aarhus, Ny Munkegade.Bldg550. DK.8000 Aarhus C, Denmark.
0168-6496/90/$03.50 © 1990Federationof European MicrobiologicalSocieties
132 apparent inhibition. Likewise, it is not dear to what extent the presence of manganese oxides can affect the measurement of sulfate reduction rates using radiotracer techniques. The results presented here are from experiments designed to provide some insists for these questions. Additions of manganese oxides to several different marine sediments suggested the possibility that sulfate reduction may not be completely inhibited by manganese, even when present at relatively high concentrations. Radiotracer experiments have confirmed this possibility and provided further evidence for the role of manganic oxides in the oxidation of sulfide to sulfate. However, results from the radiotracer experiments have also indicated that the presence of manganese oxides can cause an underestimation of sulfate reduction rates, especially during long-term incubations. In contrast, iron oxides have an inhibitory effect similar to that of manganese oxides but do not appear to affect a conversion of sulfide to sulfate.
3. METHODS Sediments for this study were obtained from an intertidal region of Lowes Cove, Maine and a creekbank site on Skidaway Island, Georgia. The Lowes Cove site has been described previously [15]. After collection in 10-cm inner diameter core tubes, the 1-5 cm depth interval was processed as below. The Skidaway site and sediments were described by Aller and Rude [14]. These sediments were collected from the 2-5 cm interval, stored in a plastic 3.8-1 container at 4 ° C, and shipped to Maine by R. Aller. The sediments had been stored approximately 3 weeks prior to analysis. In general, sediments were processed by homogeniaing in an anaerobic gloveba8 (Coy lnc.) containing a gas phase of approximately 90~ nitrogen, and 5~ each of hydrogen and carbon dioxide. Sediments were not diluted in this case but the preparation could be considered a 'slurry'. For analyses of sulfate consumption and manganese reduction, screw-cap polypropylene centrifuge tubes (15 cm3) were completely filled with untreated, homogenized sediments (controls) or
with sediments containing 20 mg/cm 3 of a freshly prepared manganic oxide (prepared according to reL 16). This mineral phase was not characterized for crystallinity but an amorphous oxidized gel [MnO2] was expected. For analyses of sulfate reduction rates by a radiotracer method, homogenized sediments with or without added manganese were dispensed into cut-off 5-cm3 syringes that were sealed with butyl septa (Supelco, Inc.). Both tubes and syringes were incubated at room temperature for several weeks. During the initial experiments conducted with Priest Landing sediments, incubations involved storage of tubes in an anaerobic glovebag containing a gas phase with 5% hydrogen. Later experiments with Lowes Cove sediments were based on incubations of tubes or syringe.cores in sealed jars containing deoxygenated water. Time courses of activity were generated by sacrificing duplicate or triplicate samples as necessary. The radiotracer estimation of sulfate reduction rates was based on methods described by King et al. [17] and King [18]. Modifications involved the use of 5-cm3 syringes which were injected along their central axes with 10/~1 of a solution containing approximately 1 taCi of carrier-free 3SSO~-. The distillation of reduced radiolabelled sulfate reduction endproducts was based on either sequential acid and boiling chromous chloride digestions or on boiling chromous chloride digestions only. The efficacy of these distillations was determined by using a synthetic FeS in presence and absence of manganic oxide. Other analyses of sulfate reduction involved additions of manganese or iron oxides (20 mg/cm 3 sediment; prepared according to ref. 19; the expected mineral phase is an oxyhydroxidc gel) with or without sodium molybdate (final concentration 20 raM), Homogenized Lowes Cove sediments (4 cm~) in modified 5-cm~ syringes were used for these studies. The metal oxides or inhibitors were added after a pre-incubation of 16-20 h with 35SO~- to allow for accumulation of radiosulfide. The additions were made by injecting deoxygenated suspensions (1 cm3) of the metal oxides or solutions of sodium molybdate and by gently mixing the syringe contents. After an additional incubation sediments were processed as before
133
with the exception that sediments containing molybdate were distilled with boiling acidic titanous chloride. ]'he use of titanous chloride insured complete recovery of sulfide in presence of molybdate [20]. Time course analyses for dissolved manganese and sulfate were conducted by centrifuging sediments incubated in polypropylene tubes as above. Porewater from the tubes was used for a turbidimetric sulfate analysis [21]. Porewaters for manganese analysis were acidified with 6 N HCI and stored until assay by graphite-furnace atomic absorption spectrometry (Hitichai Perkin-Elmer). The output of the instrument was standardized with solutions of manganous chloride in an acidified sodium chloride matrix.
4. RESULTS
4. L Effect of manganic oxides on sulfide distillation In order to measure sulfate reduction rates using radiotracer techniques, it was necessary to evaluate the efficacy of the distillation procedures used for recovering radiolabelled sulfides, This was done by determining recoveries for a synthetic iron monosulfide (FeS, about 20 mg) in the presence of excess manganic oxide, A distillation using 1 N HC! alone was ineffective (Table 1) due to the rapid formation of a yellow, carbon disulfide soluble precipitate which was presumably elemental sulfur. Elemental sulfur has been previously reported as the major oxidation product of sulfides in the presence of manganic ion at low pH [22]. Table l Effect of mangani¢oxide on recoveryof sutfides during acid and chromouschloridedistillations A. Recoveryof FeS std with ! N HCI: Added FeS: 226.3/tmol RecoveredSulfide: 228.8 ~mol:t: 10.3 (101.1~) B. Recoveryof FeS in the presenceof manganicoxide: Added FeS: 230,8 tsmol HCI recovery: 64,8 Fmol+ 15.3 (28.1~) CRS recovery: 150.5/tmol+20.0 (65.2~) Total: 215.3/tmo1:1:11,5 (93.3%) Values are means± 1 S,E. (n = 3),
Distillation with chromous chloride subsequent to an acid distillation resulted in a nearly complete recovery of mass, which indicated that the products of a chemical oxidation of sulfide did not include sulfate ('fable 1). These results were consistent with the initial formation of elemental sulfur by manganic ion and its subsequent reduction by chromous ion. A second evaluation of distillation methods involved a comparison of acidic solutions of chromous and titanous chlorides, each at 1 M concentrations. Titanous chloride, but not chromous chloride, effectively ,,;covered sulfide standards in the presence of molybdate. Recovery of radiolabelled sulfides from a sediment matrix in the absence of molybdate was not statistically different by ANOVA (P > 0.75, n = 6) when either titanous or cbromous chlorides were used for distillation. Therefore, titanous chloride was used for sediments amended with sodium molybdate while chromous chloride was used otherwise.
4.2, Effect of manganic oxides on sulfate depletion Time courses of sulfate exhibited two patterns. For the Priest Landing sediments, sulfate consumption proceeded without a lag in unamended controls (see Fig. 1: slope of regression analysis for first 7 days = 800 nmol/cm3/d; P ~ 0.002). In contrast, rates of sulfate consumption for the first 5 days in the presence of manganic oxide were not statistically different than zero (slope of regression analysis = 32 nmol/cm3/d; P ~ 0.221). Following this period, sulfate was depleted about an order of magnitude more rapidly in the presence of manganic oxide (slope of regression analysis = 375 nmol/cm3/d; P = 0.02) and at a rate equal to that of the controls. Although little sulfate depletion in manganeseamended sediments was observed dw-ing the initial 5-day incubation, a radiotracer analysis ef sulfate reduction indicated that actual depletion rates may have been higher than those calculated from changes in pool sizes. Average rates based on short-term (about 12 h) incubations with 35SO2were about 131 nmol/cm3/day at least 4-fold higher than rates from mass depletion. At a later point during the incubation (21 days), radiotracer-based rates were approximately equal
134 20
A
'
16
'
•
~......
B
4
e.
~;
24
+-~
18
8 6 03 I
0 [~+_ 0
6
12
18
24
30
Time (days} Fig. I. Time course of sulfate concentrations in sediments from a salt marsh creek bank (Priest Landing). • represent controls and • represent sediments amended with manganic oxide (see text for details). Concentrations are means of duplicate determinations. Trends in the data were determined by linear regression analysis. For the controls, region 'A' is described by: sulfate (raM) = 18A+0.049.time, r 2 = 0.9"/; for region 'B': sulfate (raM) = 13.73 :i: 0.023. time, r 2 = 0.94. For the manganese treatment, region 'A' is described by: sulfate (raM) = 18.2 + 0,002. time, r 2 = 0.01; for region 'B': sulfate (raM) = 21.32 +0.023, time, r 2 = 0,87.
to rates calculated by a regression analysis of stable sulfate losses (336 nmol/cm3/day vs. 375 nmol/cm3/day). Sediments from Lewes Cove exhibited a second pattern. In this instance, sulfate excesses (9.4 raM) were observed without lag after the addition of manganic oxide; in contrast, a rapid depletion was observed in controls (Fig. 2), Excess sulfate was formed for a period of about 11 days, after which a depletion was noted, During the period of sulfate production, dissolved manganese accumulated in the porewater of experimentals but not controls (Fig, 3), If the increase in sulfate was due to manganic oxide reduction by sulfides, a total of 37.6 mM manganese would have been expected, The observed concentration in the pore-water, 2,0 mM, was about 19-fold less, suggesting that the remainder of the reduced manganese was associated with the solid phase,
0.0
3.0
i
i
i
6.0 9.0 12.0 Time (days)
1
150
,, 18.0
Fig. 2. Time course of sulfate concentrations in sediments from the intertidal region of Lewes Cove, Maine. • represent controls and • represent sediments amended with manganic oxide (see text for details). Concentrations are means of duplicate determinations.
inhibited the accumulation of radiolabelled sulfide (Fig. 4a, b). lnhibitors were added after sediments were pre-incubated for 16 h. Sulfide accumulation relative to unamended controls was minimal in the presence of manganic oxide and molybdate (-0.6%) and otherwise exhibited the following pattern: molybdate only, 7.1-21.6% (Figure 4A and B respectively); ferric oxide + molybdate, 18.9%, ferric oxide only, 53.3%; manganic oxide, 58,0%. The differences between manganic oxide plus molybdate and molybdate only were statisti2400
1920
+Mn o x i d e /
1440
960
480 f
0 = 0,0
Ctrl
3,0
6,0 Time (days)
9.0
12.0
Fig, 3, Time course of total dissolved manganese concenlra-
4.3. Effect of manganic oxides on sulfide oxidation Additions of metal oxides with or without sodium molybdate and sodium molyhdate alone
lions in sediments from the intertidal region of Lowcs Cove+ Maine, • ~pre~nt controls and • represent sediments amended with manganic oxide, Concentrations are means of duplicate determinations.
135 35000
36000 300O0
28000
1o
24000
21000 18000 14000
f
7000
I,
12000 6000
/ 9.0
0.0
Ci
18.0 27.0 Time (hrs)
36.0
45 0
0.0
10.0
20.0 30.0 Time (hrs)
40.0
50.0
Fig. 4. A. Effect of added mangaaic oxide (B), molylxlate (@), and molybdate+manganic oxide (El) on the accumulation of radiolahelled sulf'gl¢ versus um~,ated controls (o). See text for details. Data arc means of triplicate determinations; error bars arc + | standard error. B. Effect of added ferric oxide (13), molylxlate (IlL and molybdate + [erric oxide (@) on the accumulation of
radiolabclhal sulfide versus untreated controls (o). Data arc means of triplicate determinations; error bars are + 1 standard error.
cally significant (0.01 < P < 0.05, n = 6), but were not significant for ferric oxide plus molybdate versus molybdate only. The patterns of label distribution for manganic oxide plus molybdate and molybdat¢ only indicated that manganese facilitated the net consumption of radiolabelled sulfides. No net consumption was observed with ferric oxides. In a more detailed time course, extensive consumption of radio-labelled sulfides occurred in the presence of added manganic oxides (Fig. 5). In
this case sediments were pre-incubated with 35SO~- for 24 h, supplemented with sodium molybdate as above, incubated an additional 24 h, and then supplemented with manganic oxide. In the control sediments (molybdate only) radiolabelled sulfides did not accumulate after the first 48 h. In contrast, addition of manganic oxides resulted in rapid losses of label which amounted to 45.6~ of the increase in the controls after 72 h.
5. DISCUSSION 30000
i
....
[
. . . . .
24000
,
Cld
/
(~ 18000
¢.q I~000
6000
0.0
Molybdate
30.0
600
90.0
120,0
1500
Time (hrs) FiB. 5. Time course of radiolabcllcd sulfide in the presence of molybdate (13) or molybdat¢ + manganic oxide (o) after initial actumu}ation in unamended sedi~nts (se¢ text for details). Arrows d¢not¢ points of addition for molybdat© and mangani¢ oxide. Data arc means of triplicate determinations; error bars are :1:l standard error.
Radiotracer techniques can provide great sensitivity for the analysis of interactions between manganic oxides and sulfate reduction. However, these techniques require an accurate estimation of radiosulfide formation. Previous studies have shown that acidic chromous chloride distillations are the most reliable means for measuring radiosulfides [17,23,24]. Results from this study confirm that conclusion. Elemental sulfur is formed rapidly from sulfides in the presence of acidic solutions containing manganic ions [5]. This results in substantial losses of acid volatile radiosulfides, presumably from both tile dissolved and iron monosulfide fractions (Table 1). As a result, simple acid distillations are ineffective. In contrast, acidic chromous chloride is quite effective for recovering radiosulfides (Table 1). This is due to the well-
136 known ability of chromous chloride to reduce elemental sulfur and to the low potential of the chromous-chromic couple, which allows for manganic ion reduction as well [25]. Since patterns of label distribution from sequential acid and chromous chloride distillations are not likely to be meaningful [17,18,24], a single chromous chloride reduction is recommended for radiosulfide recovery. The analysis of both stable sulfate pools and radiosulfate reduction indicates that added manganic oxides facilitates the oxidation of sulfides, most likely in accordance with the mechanism proposed by Aller and Rude [14]: 4 MnO2 + S2- + 4 H + -~ 4 Mn2+ + SO~- + 4OH-
(1) Aller and Rude [14] suggested that the form of sulfide oxidized was an iron monosulfide, perhaps involving a dissolved intermediate. The same may have been true in this study, but the data are inadequate for drawing such a conclusion. Therefore the reaction stoichiometries are given for the sulfide ion only. This general reaction may explain the patterns observed for sulfate and sulfide in several different experiments. A comparison of sulfate reduction rates based on radiotracer reduction and changes in stable sulfate pools (Fig, 1) suggests sulfide recycling, the extent of which is greatest during the first 5 days of incubation. Recycling was probably minimal after this period since sulfate depletion was observed at rates comparable to controls (Fig. 1) and since radiosulfate consumption rates were similar to stable sulfate depletion rates. The period characterized by sulfide recycling was coincident with a period of manganese accumulation in the porewater while concentrations were more constant at later intervals. These results differ from those of Aller and Rude [14] who have used sediments from the same salt marsh. They have observed significant sulfate accumulation during short-term incubations. The differences may be explained by the fact :hat the sediments used for the work reported here were incubated in polypropyhne tubes in a glovebag containing approximately 5~ hydrogen. The pres-
ence of a hydrogen gas phase can stimulate sulfate reduction rates about 2-fold, due to diffusion of hydrogen through the tube walls (data not shown). Stimulation of sulfate reduction may have altered the balance between sulfate depletion and sulfide oxidation so that net excesses were not formed. In contrast, sulfate excesses similar to tho~ reported by Aller and Rude [14] were observed in sediments from l.owes Cove (Fig. 2). These sediments were incubated in sealed jars containing deoxygenated water. The absence of hydrogen may have contributed to the relative differences ~ t h the Priest Landing sediments. As with the Priest Landing sediments, porewater manganese accumulation corresponded to the period of sulfide oxidation (Fig. 3). However, manganese accumulation was not equivalent to that expected from the stoichiometry of reaction 1. Similar results were reported by Aller and Rude [14]. The discrepancy between dissolved manganese accumulation and sulfate excesses is mostly likely due to the accumulation of reduced manganese in the solid phase, both in an adsorbed form and as manganous carbonates [14,22]. The appearance of pink deposits in the Priest Landing and Lewes Cove sediments were evidence of the latter. Unfortunately, methods for extracting solid phase reduced manganese are subject to interferences from the dissolution and subsequent re. duction of oxidized species. For example, several methods exist for speciating manganic and manganous ions (e.g. ref. 26-28), but these involve acid extractions which can liberate ferrous iron and sulfide, both of which rapidly reduce manganic ions. Copper displacement can provide information on adsorbed pools [10], but does not mobilize minerals such as rhodochrosit¢ or kutnahorite. As a result, mass balances for manganese are difficult at best. The net loss of radiolabelled sulfide in the presence of molybdate plus manganese confirmed the role of manganic oxides as an oxidant for sulfide. The oxidation of sulfide in these experi. ments was presumably microbial, since the reported primary ,~ndproduct of chemical oxidation is demental sulfur (e.g refs, 10, 14). Formation of this or any other endproduct with the exception of sulfate would not have resulted in losses of radio-
137 sulfide, since the chromous chloride distillation technique used in this study recovers all major inorganic reduced and oxidized sulfur species other than sulfate. Several other aspects of these experiments deserve comment. First, the radiosulfide was very likely distributed by isotopic exchange among several phases within the sediment matrix including dissolved sulfides, polysulfides, elemental sulfur, iron monosulfides, and pyrite [24]. Which of these were oxidized is not clear, since the chromous chloride distillation does not distinguish among the various phases. However, in Lewes Cove sediments radiosulfides are typically highest in the acid volatile fractions (> 70~: King, unpublished data). These are the most probable sources of oxidizable label, since losses of the pyrite pool equivalent to the loss of label would have required an amount of manganic ion m excess of that added. As noted previously, oxida. tion of iron monosulfides has been suggested by others [14]. Second, it appears that added iron oxides do not facilitate the loss of sulfide by an oxidation to sulfate (Fig, 4). This is consistent with the known chemistry of ferric iron [14,29]. Under the conditions used in this study elemental sulfur is the expected endproduct, much as is the case with manganic oxides. In the absence of a biological mechanism to form sulfate, the conversion of sulfides to elemental sulfur does not result in a loss of mass or label as measured by the chromous chloride distillation, Third, the addition of either manganese or iron oxides inhibited sulfate reduction. In experiments with Priest Landing sediments, sulfate reduction rates from both mass depletion and radiotracer analyses were lower in the presence of manganic oxides by > g0~g. In the radiotracer experiments with Lewes Cove sediments, manganese addition resulted in an inhibition of about 42cg. This inhibition was in part due to the oxidation of radiolabel which can cause an underestimation of the true rate. In these experiments, oxidation may have accounted for as much as 53~ of the apparent inhibition. Iron oxides resulted in an inhibition of about 47~, all of which was presumably due to direct effects since no oxidation of label
by iron was observed. These patterns are similar to those of Lovley and Phillips [13] and Aller and Rude [14] who have reported significant inhibition of sulfate reduction by manganese and iron oxides, respectively. Direct inhibition of sulfate reduction by either metal oxide may have been due to competition for hydrogen between metal-reducing bacteria and sulfidogens [13]. Finally, both the mass and radiotracer data provide support for the contention that manganie oxides promote the oxidation or anaerobic recycling of sulfide. However, it must be noted that these results are based on the addition of relatively high concentrations of oxides which may alter the balance between carbon-based and sulfide-based manganese reduction. Since beterotrophic manganese reduction is characteristic of many sedimentary bacteria [11], the dynamics of the sulfide-manganese couple must be determined under in situ conditions before the relative importance of putative lithotrophic reactions can be established.
ACKNOWLEDGEMENTS The author wishes to thank Dr. R. Aller for discussions and advice which led to the origination of this study and for helpful input throughout. Ms. L. Hall is acknowledged for technical assis. tance. This work was supported in part by the National Science Foundation (OCE 8700873).
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{171 King, G.M., Howes, B.L. and Dacey, J.W.H. (1985) Short-term endproducts of sulfate reduction in a salt marsh: formation of acid volatilesulfides,elementalsulfur, and pyrite. Geochim. Cosmochim.Acta 49,1561-1566. [18] King, G.M. (1988) Patterns of sulfate reduction and the sulfur cycle in a South Carolina salt marsh. Limnol. Oceanogr. 33, 376-390. [19] Parfitt, R.C., Atkinson, R.J. and Smart, R.St.C. (1975) The mechanism of phosphate fixation by iron oxides. Soil Sci. Soc. Am. Pro(:. 39, 837-841. [201 Banat, I.M., Lindstrom, E.B., Nedwell, D.B. and Balba, M.T. (1981) Evidence for coexistenceof two distinct functional groups of sulfate-reducing bacteria in salt marsh sediment. Appl. Environ. Microbiol.42, 985-992. [21] Tabatabai, M. (1974) Determination of sulfate in water samples. Sulphur Inst. J. 10, 11-13. [22] Burdige, D.J. and Neaison, K.H. (1986) Chemical and microbiologicalstudies of sulfide-mediatedmanganese reduction. Geomicrobiol.J. 4, 361-387. [23] Westrich, J.T. (1983) The consequences and controls of bacterial sulfate reduction in marine sediments. Ph.D. dissertation. Yale University,479 pp. [24] Fossing, H. and Jorgensen, B.B. (1989) Measurement of bacterial sulfate reduction in sediments: evaluation of a single-step chromium reduction method. Biogeochemistry, in press. [25] Cotton, F.A. and Wilkinson, G. (1962) Advanced inorganic chemistry: a comprehensive text. interscience Publishers, New York. [26] Kalhorn, S. and Emerson, S. (1984) The oxidation state of manganese in surface sediments of the deep sea. Geochim. Cosmochim. Acta 48, 897-902. [27] Lyle, M., Heath, G.R. and Robbins, J.M. (1984) Transport and release of transition elements during early diagenesis: sequential leaching of sediments from MANOP sites M and H. Part 1. pH 5 acetic leach. Geochim. Cosmochim. Acta 48, 1075-1715. [28] Murray, J.W., Balistrieri, L.S. and Paul, B. C984) The oxidation state of manganese in marine sedJmems and ferro-manganese nodules. Geochim. Cosmoc!,tm.Acta 48, 1237-1247. [29] Burner, R.A. (1970) Sedimentary pyr;*.eformation. Am. J. Sci. 268,1-23.