RESEARCH ARTICLE

Hydrogen, acetate, and lactate as electron donors for microbial manganese reduction in a manganese-rich coastal marine sediment Verona Vandieken1,2, Niko Finke1 & Bo Thamdrup1 1

Department of Biology, Nordic Center for Earth Evolution, University of Southern Denmark, Odense M, Denmark; and 2Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, Oldenburg, Germany

Correspondence: Verona Vandieken, Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, Carl-von-Ossietzky-Straße 9-11, 26129 Oldenburg, Germany. Tel.: +49 441 793 5376; fax: +49 441 793 3404; e-mail: [email protected] Present address: Niko Finke, Department of Marine Sciences, University of Georgia, Athens, GA, USA

MICROBIOLOGY ECOLOGY

Received 12 August 2013; revised 20 October 2013; accepted 17 November 2013. Final version published online 11 December 2013. DOI: 10.1111/1574-6941.12259 Editor: Alfons Stams Keywords energy yield; fermentation products; volatile fatty acids; competition; anaerobic carbon degradation.

Abstract The role of hydrogen, acetate, and lactate as electron donors for microbial manganese reduction was investigated in manganese-rich marine sediment from Gullmar Fjord (Sweden). Here, manganese reduction accounted for 50% of the anaerobic carbon oxidation at 0–15 cm sediment depth. In anoxic incubations from 0 to 5 cm depth, where manganese reduction dominated completely as terminal electron-accepting process, the combined contribution of acetate and lactate as electron donors for manganese reducers corresponded to < ¼ of the electron flow. The concentrations, 14C-radiotracer turnover rates, and contributions to carbon oxidation of acetate and lactate associated with manganese reduction were similar to those found in deeper horizons dominated by concomitant iron and sulfate reduction and sulfate reduction alone, respectively. By contrast, hydrogen concentrations increased considerably with sediment depth, indicating thermodynamic control of the competition between the electron-accepting processes, and hydrogen may have contributed substantially to the > 75% of the electron flow that did not involve acetate and lactate. Alternatively, the oxidation of more complex organic substrates could be involved. Our study provides the first direct evidence of substrate utilization by a natural manganese-reducing community and indicates similar mechanisms of thermodynamic control and competition for electron donors as known from sediments dominated by iron reduction, sulfate reduction, or methanogenesis.

Introduction Microbial manganese reduction contributes significantly to carbon oxidation in manganese-rich marine sediments, with contributions of 25% to approximately 100% of anaerobic carbon oxidation attributed to manganese reduction as terminal electron-accepting process in surface sediments of the Panama Basin, deep parts of the Skagerrak, the Black Sea, and Northern Barents Sea in the Arctic Ocean (Aller, 1990; Canfield et al., 1993a,b; Thamdrup et al., 2000; Vandieken et al., 2006; Nickel et al., 2008). In these areas, high concentrations of manganese oxides (25–185 lmol cm 3) to depths of 1 to > 10 cm, resulting from geochemical focusing, favor microbial manganese reduction over iron and sulfate reduction as terminal electron-accepting process. The

FEMS Microbiol Ecol 87 (2014) 733–745

worldwide occurrence of locations with intermediate to high manganese oxide concentrations suggests that microbial manganese reduction plays an important role for carbon degradation. Examples include coastal fjords and basins (e.g. along the coast of Scotland or the North American Atlantic coast), the Bay of Biscay, deep parts of the Argentine Basin, the eastern tropical Pacific off the coast of Mexico and Panama, as well as further locations within the Arctic Ocean in the East Siberian, Chukchi, and Beaufort Seas (Sundby, 1977; Murray et al., 1984; Aller, 1994, Gobeil et al., 1997; Haese et al., 2000; Bartlett et al., 2007; Mouret et al., 2009; Macdonald & Gobeil, 2012). Oxidized manganese is, after oxygen and nitrate, a highly favorable electron acceptor for microorganisms. The reoxidation of reduced manganese relies on the

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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presence of oxygen in the surface water and can be facilitated by bioturbation and porewater irrigation to transport reduced manganese to oxic surface sediment. Manganese oxides are ubiquitous in aquatic sediments and have been recognized as being important for global biogeochemical cycles, for example, by controlling the availability and distribution of many essential and toxic trace metals. Nonetheless, manganese reduction is the least well-explored terminal electron-accepting processes. The complete degradation of organic material to CO2 in marine sediments proceeds roughly in a vertically ordered sequence of aerobic respiration, denitrification, manganese, iron, and sulfate reduction, and, finally, methanogenesis (Froelich et al., 1979). The depth distribution of terminal electron-accepting processes in anoxic sediments is thought to be regulated by competition for fermentation products such as volatile fatty acids (VFA) and hydrogen. This is most well-documented for hydrogen with iron and sulfate reduction or methanogenesis as terminal electron-accepting processes, which control the hydrogen concentration and deplete it to a level representing what appears to be a minimum energy yield (Lovley & Goodwin, 1988; Hoehler et al., 1998). The microorganisms using the electron acceptor with higher energy yield thereby competitively exclude electronaccepting processes with lower energy yield, for which the hydrogen concentration is too low to be thermodynamically favorable. While denitrifiers, typically being aerobes (Zumft, 1997), can use a greater variety of organic substrates than iron and sulfate reducers and likely do not completely rely on fermentation products as external electron donors, manganese reducers have not been investigated with respect to their in situ electron donor usage. As manganese and iron reduction are the only terminal electron-accepting processes using oxide particles, and manganese and iron oxides are mostly reduced by the same organisms in laboratory cultures, properties of the far better-studied iron reducers have often been assumed to apply to manganese reducers. However, a recent study in three manganese-rich sediments showed that the manganese-reducing, acetate-oxidizing community was taxonomically completely different from iron-reducing communities (Vandieken et al., 2012). This raises important questions about the functioning of manganese-reducing communities: Do manganese reducers use a different and possibly wider spectrum of electron donors than iron and sulfate reducers? Do manganese reducers compete with iron and sulfate reducers for fermentation products? Do manganese reducers control the concentrations of intermediates such as hydrogen and acetate in manganese reduction dominated sediment? As a contribution to filling this gap, we used a comparative approach with incubations of sediment from ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

different depth at the same site dominated by manganese reduction, concurrent iron and sulfate reduction, and sulfate reduction alone as terminal electron-accepting processes, respectively. We investigated hydrogen, acetate, and lactate as potential electron donors for these processes. We measured concentrations of hydrogen, VFA, products of manganese and iron reduction (Mn2+ and Fe2+), as well as sulfate reduction rates by radiotracer measurements. Furthermore, parallel incubations of the manganese-rich surface layer were amended daily with acetate to study the effect of additional substrate input on microbial manganese reduction. The turnover of acetate and lactate as electron donors for manganese, iron, and sulfate reducers was determined by 14C-tracer incubations. This study thus extends the identification of a unique bacterial manganese-reducing community in manganese-rich sediments (Vandieken et al., 2012) by investigating its electron donor usage and competition with other terminal electron-accepting processes.

Material and methods Study site and sediment sampling

Sediment was sampled in January 2009 with an Olausen box corer from the deepest part of the Gullmar Fjord on the Swedish west coast (58°19.35 N, 011°32.75 E; Vandieken et al., 2012; station S3 of Engstr€ om et al., 2005). The sediment depth was 119 m, and the sediment temperature 6.7 °C. Sediment was subsampled from the corer into plexiglass liners, transported back to the laboratory, and stored at 6 °C with the overlying water being aerated until further processing. Manganese and iron pools in sediment from the same cruise were analyzed in detail by Goldberg et al. (2012). The authors identified the major zones of net manganese and iron reduction based on dissolved Mn(II) and Fe(II) species at 0–5 and 4–10 cm sediment depth, respectively. Manganese oxides near the surface had an average oxidation state of 3.3, equivalent to a mix of 2/3 Mn(IV) and 1/3 Mn(II), whereas below 9 cm, the oxidation state was close to 2 (Goldberg et al., 2012). Iron oxides in the upper 12 cm were predominantly reactive and poorly crystalline (Goldberg et al., 2012). Stable isotope probing experiments for acetateoxidizing manganese-reducing bacteria and most probable number counts of manganese-reducing bacteria were conducted with surface sediment of Gullmar Fjord (Vandieken et al., 2012). Biogeochemical data of the first 13 days of incubation [DIC, Mn2+, Fe2+, and acetate concentrations, extractable manganese and Fe(III) oxide concentrations, sulfate reduction rates] from that study are also presented in this paper in conjunction with hydrogen concentration and turnover rate measurements. FEMS Microbiol Ecol 87 (2014) 733–745

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Electron donors for microbial manganese reduction

Sediment incubations

Sediment from 0 to 5, 5 to 10, and 10 to 15 cm depth was pooled from several cores, mixed with anoxic site water (3 : 1 sediment: site water w/w), and homogenized. Each slurry of c. 1000 mL was subsequently filled under N2 into glass bottles (1 L) and sealed with butyl rubber stoppers. From the slurries, c. 10 g of sediment each was filled under N2 into three 50-mL serum vials sealed with butyl rubber stoppers for hydrogen measurements. For sediment incubations of the 0–5 cm interval with acetate amendment (‘0–5 cm + acetate’), two parallel incubations in 1-L bottles and six incubations in 50-mL vials were conducted, of which one 1-L bottle and three 50-mL vials each were amended almost daily with 13C- or 12C-acetate (c. 170 lM final concentration), respectively, as described in Vandieken et al. (2012). The results of 13C- and 12Cacetate amendments were treated as replicates in this study. The sediment slurries were incubated at 6 °C for 30 days. The 1 L incubations were subsampled 11 times (day 0, 1, 2, 3, 4, 6, 9, 13, 17, 22, and 30) for porewater, solid phase, and sulfate reduction rates measurements, as well as three times (days 0, 4, and 9) for turnover rate measurements after addition of radiolabeled acetate and lactate. The headspace of the 50 mL incubations was sampled for hydrogen measurements. Porewater and solid phase analyses

Porewater for the analyses of pH, Fe2+, Mn2+, Ca2+, sulfide, sulfate, nitrate, nitrite, and DIC was retrieved by centrifuging sediment in 15-mL glass centrifuge tubes closed with rubber stoppers without headspace at 2500 g for 10 min and filtered through 0.2-lm filters. Dissolved Fe2+ was analyzed by filtering porewater directly into ferrozine solution (Stookey, 1970). Samples for Mn2+ and Ca2+ measurements were acidified by 6 M HCl and analyzed by inductively coupled plasma atomic emission spectrometry. Porewater for DIC analysis was added to glass vials with saturated HgCl2 solution, capped with Viton septa without headspace, and stored at 4 °C until analysis by flow injection (Hall & Aller, 1992). Sulfide samples were fixed with ZnCl2 and analyzed with methylene blue (Cline, 1969). Samples for sulfate were acidified, bubbled with N2 to drive out sulfide, and stored frozen until analysis by anion chromatography. Nitrate and nitrite samples were stored frozen until reduction with V(III) and detection of NO (Braman & Hendrix, 1989). Porewater for VFA analysis was centrifuged in combusted glass centrifuge tubes, filled into combusted glass vials, and stored frozen until analyzed. VFA (lactate, acetate, butyrate, isobutyrate, and propionate) were analyzed from parallels after derivatization with 2-nitrophenyl hydrazine by HPLC according to Albert & FEMS Microbiol Ecol 87 (2014) 733–745

Martens (1997). Detection limits were 1.5 lM for acetate and 1 lM for other VFA. Production rates of porewater constituents were calculated from slopes of linear regressions of concentrations versus time and are reported with standard errors propagated from this analysis. DIC production rates were corrected for the precipitation of CaCO3 for incubations of 0–5 cm, 0–5 cm + acetate, and 5–10 cm as described by Thamdrup et al. (2000): DIC production = DIC accumulation + CaCO3 precipitation and CaCO3 precipitation = D[Ca2+]sol (1 + KCa), where KCa is the adsorption constant for Ca2+ (KCa = 1.6; Li & Gregory, 1974). For calculations of DIC production rates, only the early linear part of the production was used (for 0–5, 5–10, and 10–15 cm first 13 days and 0–5 cm + acetate first 9 days) as accumulation slowed down later in the incubations. Reactive manganese and iron oxides were extracted in duplicate from sediment subsamples with dithionite/citrate/acetic acid (Canfield, 1989) and HCl (Kostka & Luther, 1994), respectively. Manganese was analyzed by flame atomic adsorption spectrometry, Fe(II) with ferrozine and total iron with ferrozine plus 1% (w/v) hydroxylamine hydrochloride. Fe(III) concentrations were calculated by subtraction of Fe(II) from total iron concentrations. Sulfate reduction rates

At each of the 11 times, sulfate reduction rates were determined during the slurry incubations. Subsamples of c. 6 mL slurry were transferred to glass tubes closed with a plunger from disposable syringes and a rubber stopper from either side at each sampling time in duplicates. These were injected with 100 kBq 35S-sulfate. Samples were incubated between 4 and 15 h and stopped by injection into 20% Zn acetate and freezing. Total reduced sulfur was analyzed by hot Cr3+ distillation (Fossing & Jørgensen, 1989). Sulfate reduction rates were calculated as described by Jørgensen (1978). The contribution of sulfate reduction to carbon oxidation was calculated based on a stoichiometry of 2 : 1 DIC produced for sulfate reduced (Thamdrup & Canfield, 1996) using a time-weighted average of the sulfate reduction rates determined during the time interval used for the DIC rate calculations (see above). VFA turnover measurements

Turnover of 14C2-acetate and 14Cu-lactate was determined in subsamples of the slurries on days 0, 4, and 9 of the incubations from the production of 14C-labeled inorganic carbon as described by Finke et al. (2007). Tracer solutions were prepared with anoxic, sterile-filtered porewater at least one hour before the start of the incubation to allow for possible complexation reactions of the tracers (Finke et al., 2007). For each incubation with radiolabel, one incubation ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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per slurry was started and subsampled 6–7 times during 50– 80 min. Measurements of acetate turnover in the acetateamended slurries were started 24 and 29 h after acetate addition at days 4 and 9, respectively, when concentrations had decreased below 5 lM. Turnover rate constants were calculated from the slope of values of ln[(counts of VFA + counts of DIC)/counts of VFA] plotted versus time (Figs S1 and S2; Shaw et al., 1984), with the VFA activity counted as the 14C remaining in the porewater after expulsing of inorganic carbon (Finke et al., 2007). Hydrogen measurements

Hydrogen concentrations were determined from the 50mL vials at each sampling time by the headspace equilibration technique (Lovley & Goodwin, 1988; Hoehler et al., 1998). Hydrogen was analyzed with a reduced gas analyzer (Peak Performer 1; Peak Laboratories, Mountain View, CA) with a detection limit of 0.01 p.p.m.v., and concentrations calculated from the hydrogen solubility in seawater (Crozier & Yamamoto, 1974). Thermodynamic calculations

Gibbs free energies for terminal electron-accepting processes were calculated for manganite, pyrolusite, ferric hydroxide, goethite, and sulfate as electron acceptors (Table 1) at 6 °C and at the concentrations of reactants and products and pH measured at each sampling, using the thermodynamic constants compiled by Hanselmann (1991). When measured concentrations were below detection, the respective detection limits were used (hydrogen 0.013 nM, Fe2+ and sulfide both 1 lM).

poorly crystalline, HCl-extractable Fe(III) oxides (Fig. 1). Concentrations of manganese and Fe(III) in the anoxic slurry incubations (Table 2) were consistent with the depth-resolved profiles (Fig. 1). Nitrate and nitrite stayed below background concentrations of 3 lM after the first sampling in all incubations, and thus, reduction of these components was not important during the incubations. The biogeochemical analyses showed that different terminal electron-accepting processes dominated during the anoxic incubations of the three depth horizons. For the 0- to 5-cm incubation, the dominance of manganese reduction was demonstrated by Vandieken et al. (2012) based on the high concentration of manganese oxide and rapid accumulation of soluble Mn2+ (Figs 1 and 2). The addition of acetate stimulated DIC production and Mn2+ accumulation (Table 2, Fig. 2). Fe2+ did not accumulate, and sulfate reduction rates were low to nondetectable, so that microbial manganese reduction accounted for ≥ 98% of carbon oxidation in incubations with and without acetate addition (Table 2, Figs 2 and 3). In the incubation from 5 to 10 cm, manganese concentrations were low and dominated by reduced Mn(II) (Table 2, consistent with Goldberg et al., 2012), and dissolved Mn2+ increased only slightly at the beginning (Fig. 2), indicating that manganese oxide was not important as electron acceptor. Fe(III) was available for reducConcentration (μmol cm–3) 0

Terminal electron-accepting processes during the incubations

6

Electron acceptor Manganite Pyrolusite Ferric hydroxide Goethite Sulfate

Gf0 (kJ mol 1)

Depth (cm)

4

Table 1. Equations for calculations of Gibbs free energies of the terminal electron-accepting processes and Gf0 values of the respective electron acceptors taken from Hanselmann (1991)

100

150

2

Results

Sediment of Gullmar Fjord is characterized by high concentrations and a deep distribution of manganese and

50

0

8

10

12 Terminal electron-accepting process

557.3 465.1 696.6

H2 + 2MnOOH ? 2Mn2+ + 4OH H2 + MnO2 ? Mn2+ + 2OH H2 + 2Fe(OH)3 ? 2Fe2+ + 2H2O + 4OH

490.4 744.6

H2 + 2FeOOH + 4H ? 2Fe + 4H2O 4H2 + SO24 + H+ ? HS + 4H2O +

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

2+

14

Mn Fe(III)

16 Fig. 1. Solid phase concentrations of Mn and Fe(III).

FEMS Microbiol Ecol 87 (2014) 733–745

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Electron donors for microbial manganese reduction

Table 2. Manganese and Fe(III) oxides concentrations at the beginning of the incubations. DIC production, sulfate reduction rates, and importance of sulfate reduction for the DIC production during the incubations. Average hydrogen concentrations of sediment were given for 0–9 and 13–30 days time intervals

0–5 cm 0–5 cm + acetate 5–10 cm 10–15 cm

Total manganese* (lmol cm 3)

Fe(III) (lmol cm 3)

DIC production (nmol cm 3 day 1)

37.3 37.3 13.8 20.8

45.3 45.3 16.5 5.5

99 314 58 26

†

   

10 25 9 7

Sulfate reduction rate (nmol cm 3 day 1) 0.9 0.7 13 13

   

0.7 0.8 6 4

Contribution of sulfate reduction (%) 2 0.5 44 98

Average hydrogen concentrations‡ (nM) 0–9 Days 0.026 0.030 0.077 0.19

   

13–30 Days 0.002 0.005 0.012 0.05

bd 0.044  0.008§ 0.020  0.007 0.21  0.05

bd, Below detection. *Total manganese oxides comprise dithionite-extractable Mn(II), Mn(III), and Mn(IV). † DIC production was calculated from slopes of the linear increase (first 13 days of incubation without acetate and first 9 days with acetate). ‡ Differences between the incubations were significant (P < 0.1, paired, two-tailed t-test) except for average concentrations of 0–9 days between 0–5 and 0–5 cm + acetate. § Excluding day 22.

tion at a concentration of 16.5 lmol cm 3, and iron reduction was indicated by increasing Fe2+ concentrations throughout the incubation (Table 2, Fig. 2). Concurrently, sulfate reduction occurred and, after an initial decrease in rates, rates increased with incubation time (Fig. 3). Elevated rates at the beginning of the incubations probably resulted from sediment mixing, which led to transient accumulation of organic substrates including VFA (Fig. 4). Sulfate reduction rates were equivalent to 44% of the total carbon oxidation rate as determined from DIC accumulation (Table 2), and we attribute the remainder to dissimilatory iron reduction (Canfield et al., 1993b; Thamdrup & Canfield, 1996). In the 10- to 15-cm incubation, Mn(IV) and Fe(III) oxide contents were low and likely represent unreactive oxides (Table 2; Goldberg et al., 2012), and Mn2+ or Fe2+ concentrations were relatively constant during the incubations (Fig. 2). Sulfate reduction rates were similar to the 5–10 cm interval, but did not increase with time (Fig. 3). The average sulfate reduction rates in the 10–15 cm incubation matched the anaerobic DIC production, and, accordingly, sulfate reduction was the dominant terminal electron-accepting process here (Table 2). Hydrogen and VFA concentrations

Hydrogen concentrations in the 0- to 5-cm incubations ranged from 0.034 nM after 3 days to below detection (detection limit 0.013 nM) at the end of the incubation (Fig. 4). At 5–10 cm, hydrogen concentrations decreased with time from 0.12 nM to below detection, while at 10– 15 cm, concentrations decreased during the first 3 days, similar to the 5- to 10-cm incubation and then rose to a transient maximum of 0.42 nM at day 9 (Fig. 4). Hydrogen concentrations in the 0–5 cm + acetate incubation stayed ≤ 0.07 nM except for a peak of 0.42 nM at day 22 FEMS Microbiol Ecol 87 (2014) 733–745

(Fig. 4). Despite the temporal variation, the hydrogen concentrations at any given day showed the general trend among incubations of 0–5 cm < 0–5 cm + acetate < 5– 10 cm < 10–15 cm (Fig. 4, Table 2). Acetate concentrations were elevated at the beginning of the unamended incubations, but decreased rapidly within the first 2 days, although all slurries showed small transient increases to 12–24 lM during the incubation (Fig. 4). Given this variability, there were no significant differences between the depth intervals. Other VFA (lactate, propionate, butyrate, and isobutyrate) followed a similar trend as acetate with elevated concentrations at the beginning in the 5- to 10- and 10- to 15-cm incubations and transient increases for 0–5 and 5–10 cm (Fig. 4). However, concentrations of these VFA were lower than acetate and mostly below the detection limit for the single VFA, and especially lactate was mostly below detection (Fig. 4). In the acetate-amended slurries, acetate concentrations, measured 1 day after each addition, were slightly higher only after the first addition with acetate (108 lM), but stayed below 11 lM at days 2–13 similar to the unamended incubation. After 13 days, the concentration increased steeply reaching 1.9 mM at the end of the incubation (Fig. 4). Other VFA increased simultaneously but only reached 9 lM (Fig. 4) with propionate, butyrate, and isobutyrate as the components of this increase. Turnover of acetate and lactate

Turnover rate constants of 14C-acetate and -lactate varied between the three times of measurement with up to ninefold difference for acetate and up to threefold for lactate in the incubations without acetate amendment, and in two cases, no turnover of acetate was detected (Table 3). Lactate turnover rate constants decreased with increasing ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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800

40

0–5 cm

0–5 cm 5–10 cm 10–15 cm

5–10 cm 0–5 cm + acetate

600

Mn2+ (µM)

SRR (nmol cm–3 day–1)

10–15 cm

400

30

20

10

0

200

0

0

5

10

15

20

25

30

Incubation time (day) Fig. 3. Sulfate reduction rates of the uninhibited slurry incubations. Error bars represent parallel incubations of slurries with acetate addition.

0

10

20

30

40

of the incubation, when lactate turnover was determined with 2.8 nmol cm 3 day 1 (Table 3). The contribution of lactate oxidation to anaerobic carbon oxidation was constrained to ≤ 14% with most values below 10% (Table 3). The highest contributions of acetate oxidation of 14% and 47% were detected at the beginning of the incubation when VFA concentrations were elevated after sediment mixing (Fig. 4), while for the remainder of the incubation, the contribution was ≤ 9% similar to the contribution of lactate (Table 3).

30

Fe2+ (µM)

0–5 cm + acetate

20

Discussion

10

Thermodynamical control of the hydrogen concentration 0

0

10

20

30

Fig. 2. Dissolved Mn and Fe concentrations during the incubations. Error bars represent parallel incubations of slurries with acetate addition. Filled symbols denote concentrations below detection. Some data are taken from Vandieken et al. (2012).

sediment depth, while such a trend was not that obvious for the turnover of acetate. Turnover rate constants were in general higher for lactate than for acetate. Because concentrations were frequently below detection, turnover rates could only be calculated accurately in a few cases, and for the remainder rates were constrained using the detection limit (Table 3). The highest turnover rates in the unamended incubations were found in the incubations from 0 to 5 cm with 7.1 nmol cm 3 day 1 for acetate and ≤ 4.5 nmol cm 3 day 1 for lactate. Lactate concentrations were below detection except for the start ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

We found manganese reduction to dominate anaerobic carbon oxidation at 0 to 15 cm depth in Gullmar Fjord sediment with an integrated contribution of about half of the total, followed by sulfate reduction at approximately 30%, and iron reduction at approximately 20%. Thus, Gullmar Fjord is one of the few examples so far of marine sediments with high concentrations of manganese oxide and importance of microbial manganese reduction, similar to the Panama Basin, basins in the Skagerrak and the East Sea, the Black Sea, and the Northern Barents Sea, where microbial manganese reduction was found to account for 25–99% of carbon oxidation (Aller, 1990; Canfield et al., 1993a,b; Vandieken et al., 2006; Nickel et al., 2008). High concentrations of manganese and iron oxides as well as intermediate organic carbon reactivity lead to a relatively slow turnover and deep extension of the manganese and iron oxide pools, so that during our incubations, redox conditions and electron-accepting

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Electron donors for microbial manganese reduction

100 0–5 cm

0.5

10

0.4

8

0.3

10–15 cm

60 40

H2 (nM)

5–10 cm

VFA (μM)

Acetate (μM)

80

12

6 4

20

0.2 0.1

2 0

0 0

10

20

30

3000 0.… 0–5 cm + acetate

2000

VFA (μM)

Acetate (μM)

2500

1500

0

10

20

30

12

0.5

10

0.4

8

0.3

6

1000

4

500

2

0

0

0

10

20

30

0

10

20

30

–0.1

H2 (nM)

0

0.2 0.1 0

0

10

20

30

0

Incubation time (day)

10

20

30

–0.1

Incubation time (day)

Incubation time (day)

Fig. 4. Concentrations of acetate (left), other VFA (sum of lactate, propionate, butyrate, and isobutyrate; middle), and hydrogen (right) during the incubations. Error bars represent parallel incubations (0–5, 5–10, and 10–5 cm, hydrogen: three parallels; 0–5 cm + acetate, acetate and VFA: two parallels, and hydrogen: six parallels). Upper error bar for hydrogen 0–5 cm + acetate was cropped for reasons of comparison and is 0.49 nM in height. Filled symbols denote concentrations below detection. Some data of acetate concentrations are taken from Vandieken et al. (2012).

Table 3. Turnover rate constants, turnover rates, and the contribution of acetate and lactate oxidation to DIC production during incubations of sediment from 0 to 5, 5 to 10, 10 to 15, and 0 to 5 cm with acetate addition Acetate Day 0 Turnover rate constants (h 1) 0–5 cm 0.031  0.008 0–5 cm + acetate nd 5–10 cm bd 10–15 cm 0.004  0.0002 Turnover rate (nmol cm 3 day 1) 0–5 cm 7.1  1.9 5–10 cm – 10–15 cm 6.1  0.3 Contribution (%) to DIC production† 0–5 cm 14  4 5–10 cm – 10–15 cm 47  3

Lactate Day 4

Day 9

Day 0

Day 4

Day 9

0.052  0.020 0.16  0.03 0.020  0.006 bd

0.13  0.04 nd 0.074  0.015 0.021  0.003

0.064  0.010 nd 0.033  0.004 0.017  0.004

0.20  0.04 nd 0.10  0.040 0.024  0.004

1.5  0.7 ≤ 1.9*  0.6 ≤ 1.2*  0.4

2.4  0.9 ≤ 0.6*  0.21 –

2.8  0.8 ≤ 1.6*  0.3 ≤ 0.6*  0.08

≤ 1.4*  0.2 ≤ 0.7*  0.09 ≤ 0.4*  0.08

≤ 4.5*  0.9 ≤ 2.2*  0.9 ≤ 0.5*  0.09

31 ≤ 7*  2 ≤ 9*  3

52 ≤ 2*  1 –

92 ≤ 8*  2 7*  1

≤ 4*  1 ≤ 4*  0.4 ≤ 4*  1

0.018 0.29 0.059 0.037

   

0.008 0.03 0.019 0.012

≤ 14*  3 ≤ 11*  4 ≤ 6*  1

bd, Below detection; nd, not determined. *Turnover rates and contributions are likely overestimated because turnover rates were calculated with concentrations of the detection limit for VFA measurements (acetate: 1.5 lM, lactate: 1 lM), but measured concentrations were below detection. † The contribution to DIC production was calculated based on a stoichiometry of 1 mol DIC formed from 1/2 moles acetate. The contribution was not calculated for 0–5 cm + acetate due to changing acetate concentrations and therefore changing contribution after each acetate addition.

FEMS Microbiol Ecol 87 (2014) 733–745

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pathways were likely not altered significantly from in situ conditions. Hydrogen concentrations differed significantly between the sediment horizons, increasing from the 0 to 5 cm interval dominated by manganese reduction, over the 5– 10 cm interval with concomitant iron and sulfate reduction to 10–15 cm depth where sulfate reduction prevailed (Table 2). This indicates a competition between the terminal electron-accepting processes, where the dominating process controls the hydrogen concentration, thereby inhibiting usage of the next most favorable electron acceptor until the dominating oxidant is depleted to limiting concentrations (Lovley & Goodwin, 1988; Hoehler et al., 1998). To our knowledge, these are the first measurements of hydrogen concentrations in sediment naturally dominated by manganese reduction. Lovley & Goodwin (1988) added manganese oxides to iron-reducing freshwater sediment and detected decreasing hydrogen concentrations to below their detection limit of 0.05 nM. In contrast, Hoehler et al. (1998) saw no effect on hydrogen when adding manganese oxide to a sulfate-reducing marine sediment, possibly due to a lack of manganese-reducing bacteria in substantial numbers or because the addition was consumed by abiotic reduction. Calculated Gibbs free energy yields for manganese reduction with different forms of manganese oxides and hydrogen were high (ΔG = 128 to 111 kJ per mol H2; Table 4) compared to the minimum energy yield of 6 to 5 kJ per mol H2 or 23 to 18 kJ per mol SO24 or CO2 estimated for hydrogenotrophic sulfate reduction and methanogenesis in marine sediment (Hoehler et al., 1998) and to the estimated minimum energy quantum for proton translocation of around 20 kJ mol 1 (Schink, 1997). High energy yields were also calculated for nitrate reduction in a marine sediment and seen as an indication that the nitrate-reducing organisms could not fully express their thermodynamic control on hydrogen concentrations

(Hoehler et al., 1998). The same seems to be the case for the manganese reducers in this study. For iron reduction, the energy yield depends strongly on the form of Fe(III) available. For crystalline goethite, hydrogen oxidation was not thermodynamically favorable in any of the incubations, whereas the ΔG of 34  3 kJ per mol H2 with amorphous ferric hydroxide at 0–5 cm suggests that iron reduction was not thermodynamically excluded here (Table 4). This is supported by the fact that the yield with amorphous ferric hydroxide at 5–10 cm, where our incubations indicated a substantial contribution of iron reduction to carbon oxidation, was close to the often suggested energy quantum of 20 kJ per reaction (Schink, 1997). Thus, iron reduction may have been active at 0–5 cm with the production of Fe2+ being masked by the rapid reoxidation with manganese oxide (Lovley & Phillips, 1988). By contrast, the low energy yield of sulfate reduction of 16  2 kJ per mol SO24 at 0–5 cm depth (Table 4) implies that this respiration was competitively inhibited, although the yield could be higher if sulfide concentrations were lower than the detection limit of 1 lM assumed in the calculations. In the 5- to 10-cm incubation, depleted in manganese oxide, iron and sulfate reduction co-occurred in the 5–10 cm incubation that was depleted in manganese oxide. As dissimilatory iron reduction in marine sediments is typically limited by availability of poorly crystalline Fe(III) at concentrations < 30 lmol cm 3 (Thamdrup, 2000; Jensen et al., 2003), this co-occurrence was in good agreement with the initial Fe(III) concentration of 16.5 lmol cm 3 (Table 2). Our free energy calculations (Table 4) also support the coexistence of the two pathways because the energy yields per unit reaction were very similar ( 21  3 kJ per mol H2 with ferric hydroxide and 22  6 kJ per mol SO24 , respectively). This comparison is based on the assumption that the unit reaction for iron reduction is the two-electron transfer from 1 H2 to 2

Table 4. Average Gibbs free energies (kJ per mol H2 or sulfate) for the dominating terminal electron-accepting processes during the incubations of sediment from 0 to 5, 5 to 10, 10 to 15, and 0 to 5 cm with acetate addition Manganese reduction

0–5 cm† 0–5 cm + acetate†,§ 5–10 cm† 10–15 cm

Iron reduction

Sulfate reduction*

With pyrolusite

With manganite

With ferric hydroxide

With goethite

128  2 121  4 No Mn(IV) No Mn(IV)

111  4 97  8 No Mn(IV) No Mn(IV)

34  3‡ 23  6‡ 21  3 No Fe(III)

13  3‡ 23  6‡ 25  3 No Fe(III)

16 17 22 34

   

2 2 6 5

*Some values calculated with detection limit for sulfide measurement. † Average Gibbs free energies were calculated only with data when hydrogen concentrations were above detection, that is, excluding days 13, 17, 22 and 30 for 0–5 cm, day 6 for 0–5 cm + acetate, and day 30 for 5–10 cm. ‡ Some values calculated with detection limit for Fe2+ measurement. § Calculations excluding day 22 with high hydrogen concentrations.

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FEMS Microbiol Ecol 87 (2014) 733–745

Electron donors for microbial manganese reduction

Fe(III) and for sulfate reduction the eight-electron transfer from 4 H2 to 1 SO24 (Hoehler et al., 1998, 2001). These calculations also suggest that the form of Fe(III) utilized for dissimilatory iron reduction must have a free energy of formation similar to the ferric hydroxide used in our calculations, as the process would be energetically unfavorable with more stable forms. At 10–15 cm, the estimated energy yield for sulfate reduction coupled to hydrogen oxidation of 34  5 kJ per mol sulfate was clearly sufficient for ATP generation (Table 4). Controls on VFA concentrations and turnover

In contrast to hydrogen, acetate concentrations at different depths were similar and hence did not indicate thermodynamic control by manganese reducers (Fig. 4). Some previous studies found acetate concentrations to be 4- to 10-fold lower in freshwater sediments dominated by iron reduction relative to sulfate reduction or methanogenesis (Lovley & Phillips, 1987; Roden & Wetzel, 2003). Other investigations showed no clear trend of VFA concentrations between slurries with different terminal electron-accepting processes (Sansone & Martens, 1982; Achtnich et al., 1995). Given the difference in stoichiometry, acetate concentrations would need to vary more than hydrogen concentrations if they were governed by thermodynamic control (Hoehler et al., 1998, 2002). The temporal patterns also showed no co-variation in concentrations of hydrogen and acetate, further supporting the conclusion that the two species are regulated independently. In analogy to models of benthic iron and sulfate reduction, acetate would be expected to be an important electron donor for manganese reduction, because it is a major fermentation product of complex substrates such as algal detritus, amino acids, carbohydrates, and fatty acids in many marine surface sediments (Sørensen et al., 1981; Arnosti et al., 2005; Valdemarsen & Kristensen, 2010; Graue et al., 2012). The transient increase of acetate and other VFA after initial mixing (Fig. 4) shows that these compounds were produced in the sediment and that the terminal oxidizers were temporarily not able to keep up with the acetate production, as reported previously for sediments dominated by iron and sulfate reduction (Arnosti et al., 2005; Finke et al., 2007). The measured net decrease in acetate concentration of 3.3 nmol cm 3 day 1 during the first 22 h at 0–5 cm was broadly consistent with the relatively high 14C-acetate turnover rate of 7.1 nmol cm 3 day 1. Thus, the manganese-reducing community was able to respond quickly to the transient increase in electron donor concentration, likely by increasing their acetate oxidation rate. This is in line with the acetate-amended incubations from 0 to FEMS Microbiol Ecol 87 (2014) 733–745

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5 cm depth, where acetate turnover rates were five- and threefold higher at days 4 and 9, respectively, compared to the incubation without acetate (Table 3). The addition of acetate simulated an event of carbon input to the sediment. Thus, the manganese-reducing microbial community of Gullmar Fjord sediment seems to be well adapted to events of high organic carbon input. However, acetate and to lesser extent also other VFA accumulated toward the end of the incubation, indicating a nearly complete inhibition of acetate oxidation (Fig. 4). Depletion of manganese oxides could not explain this as the cumulated addition of acetate corresponded to only a fraction of the manganese oxide pool (Table 2), and neither reduction of iron nor sulfate was stimulated. Hydrogen concentrations were twice as high as in the incubation without acetate, but not as high as in the incubation from 5 to 10 cm (Fig. 4), indicating that hydrogen concentrations were likely still controlled by manganese reducers. Thus, we have no clear explanation for this delayed effect of acetate amendment. The precise determination of the respective VFA concentrations is very important for the calculation of turnover rates and probably the part of the rate determination that has the highest methodological and analytical error (Christensen & Blackburn, 1982; Shaw & McIntosh, 1990; Wellsbury & Parkes, 1995; Finke et al., 2007). As acetate and lactate concentrations were below detection most of the time, turnover rates are maximum values and, hence, likely overestimated. The turnover rate constants are independent of concentrations and were below detection only twice (Table 3). Turnover rate constants for 14C-acetate in the manganese-reducing incubation were between 0.018 and 0.052 h 1 and higher for lactate 0.064 and 0.20 h 1 (Table 3). Similar turnover rate constants in the other two incubations indicated no difference between terminal oxidizers. A similar range of turnover rate constants have been determined in Arctic sediments with iron and sulfate reduction as terminal electron-accepting processes (0.009–0.063 h 1 for acetate and 0.072–0.32 h 1 for lactate; Finke et al. 2007) and sulfate-reducing sediment from Skan Bay, Alaska (0.02 h 1 for acetate; Shaw & McIntosh, 1990). Acetate turnover rate constants of this study were at the lower end of ranges previously measured with 14C-tracer in coastal sediments characterized by high carbon mineralization rates (0.0046–0.16 h 1 in Cape Lookout Bight; Sansone & Martens, 1981, and 1.5–13 h 1 in Danish coastal sediments; Christensen & Blackburn, 1982). This suggests that higher turnover rate constants for VFA in sediments are associated with a faster turnover of organic carbon due to higher and more reactive carbon contents in general with no evident influence of the terminal electron-accepting processes. However, as direct ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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studies of electron donor usage by iron and manganese reducers in sediments are scarce, more investigations are needed. Lactate, acetate, and hydrogen as electron donors

With the exception of a single measurement, the combined contribution of acetate and lactate oxidation to carbon oxidation with manganese, iron, and sulfate reduction as terminal electron-accepting processes during the incubations did not exceed 25% (Table 3). No obvious differences were indicated for acetate and lactate usage by different electron-accepting processes. Thus, acetate and lactate were used as electron donors by manganese reduction, and adaptation to increased concentrations was rapid. However, the contribution to carbon oxidation was clearly smaller than the contributions indicated in most previous studies with sulfatereducing sediments. Some of these showing acetate oxidation rates that often exceeded sulfate reduction rates (Ansbæk & Blackburn, 1980; Christensen & Blackburn, 1982; Shaw et al., 1984; Kristensen et al., 1994; Wellsbury & Parkes, 1995) were likely overestimated due to errors in acetate determination and overestimation of the biologically available acetate pool (Christensen & Blackburn, 1982; Shaw & McIntosh, 1990), but other studies also found that acetate alone contributed 40–65% to sulfate reduction (Sørensen et al., 1981; Christensen, 1984). In one study in an Arctic fjord sediment that also involved microbial iron reduction, acetate and lactate turnover accounted for 12% of iron reduction and 16–43% of sulfate reduction (Finke et al., 2007). This might, in analogy to differences in turnover rate constants between sediments, indicate that the importance of single VFA as electron donor is rather influenced by carbon content and mineralization rates in sediments than by the terminal electron-accepting processes. Whereas acetate and lactate clearly played a minor role as electron donor for manganese reducers in Gullmar Fjord sediment, hydrogen was evidently turned over and may have been an important electron donor in our incubations, as also suggested in a previous study where a similar fraction of the electron donors could not be identified (Valdemarsen & Kristensen, 2010). Other potential donors include fermentation products such as alcohols, which are produced and utilized in significant amounts in sulfate-reducing slurries and are used by iron and sulfate reducers in cultures (Lovley et al., 2004; Graue et al., 2012; Rabus et al., 2013). As an alternative to the paradigm that anaerobic mineralization is separated into fermentation and respiration by different organisms, the possibility of complete oxidation of some substrates by ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

V. Vandieken et al.

one organism must be considered. The direct usage of amino acids and sugars has been demonstrated for pure cultures of sulfate-reducing bacteria and has also been suggested from experiments with sediments (Stams et al., 1985; Burdige, 1989; Parkes et al., 1989; Hansen & Blackburn, 1995; Sass et al., 2002). The bacterial groups that were recently identified by stable isotope probing as acetate-oxidizing manganese reducers in Gullmar Fjord and two other manganese-rich sediments, Colwellia, Oceanospirillaceae, and Arcobacter (Vandieken et al., 2012), may potentially also have a broader substrate spectrum including complex organic compounds. Cultivated members of these clades were so far only described to grow by (micro-)aerobic respiration and nitrate reduction, and the extensive substrate spectrum of Colwellia and the genera Marinomonas and Neptunomonas within the Oceanospirillaceae include the degradation of starch and chitin (Bowman et al., 1998; Nogi et al., 2004; Gonzalez & Whitman, 2006; Jung et al., 2006). Similarly, Arcobacter-related species can utilize amino acids and additionally grow chemolithotrophically by oxidation of reduced sulfur species (McClung et al., 1983; Gevertz et al., 2000; Wirsen et al., 2002). The recent finding that these groups are also acetate oxidizers in manganese-poor coastal sediment of Aarhus Bay (Vandieken & Thamdrup, 2013) suggests that their electron acceptors may also include oxygen and nitrate, while their electron donor spectrum may include both complex organic substrates as well as fermentation products. Cultivation or metagenomic analysis of the manganese reducers from the manganese-rich sites could provide further insights into their metabolism. In summary, our results provide evidence that hydrogen, acetate, and lactate serve as electron donors for dissimilatory manganese reduction under in situ conditions and that this process is the dominant anaerobic pathway of carbon oxidation to 15 cm depth in the sediment of Gullmar Fjord. We found neither iron nor sulfate reduction by biogeochemical analyses in sediment with high manganese oxide concentrations, and the manganese reducers apparently controlled the low hydrogen concentration such that iron and sulfate reducers were competitively inhibited. Together with the identification of specialized acetate-oxidizing, manganese-reducing bacteria (Vandieken et al., 2012), this indicates that the manganese-reducing microbial assemblage in manganese-rich marine sediments is distinct from the better-known ironand sulfate-reducing communities and significantly influences energy and carbon conversion and biogeochemical conditions. Our finding of relatively small contributions from acetate and lactate as intermediates during carbon oxidation may be explained by a large contribution from hydrogen or the utilization of a more diverse spectrum of electron donors. The result emphasizes the need for idenFEMS Microbiol Ecol 87 (2014) 733–745

Electron donors for microbial manganese reduction

tification of the electron donors utilized during anaerobic respiration to improve our understanding of the structure and function of anaerobic microbial communities and their role in aquatic biogeochemistry.

Acknowledgements We thank Trine Gregersen for help with sampling at Gullmar Fjord as well as the captain and crew of the research vessel R/V Oscar von Sydow. We are grateful to Tim Ferdelman and Marcel Kuypers at the Max Planck Institute for Marine Microbiology in Bremen that we could measure VFA on their HPLC. We thank Heribert Cypionka and two anonymous reviewers for helpful comments on the manuscript. V.V. had been financed by a PostDoc fellowship of the Danish Natural Science Research Councils (FNU) and N.F. by a Marie Curie Outgoing International Fellowship. The study was further financed by grants from FNU and from the Danish National Research Foundation through Grant No. DNRF53.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Turnover of 14C-acetate measured at 3 days (days 0, 4 and 9) of unamended incubations of 0–5, 5–10 and 10–15 cm depth (right) and incubations of 0–5 cm amended with 12C- and 13C-acetate (right). Fig. S2. Turnover of 14C-lactate measured at 3 days (days 0, 4 and 9) of unamended incubations of 0–5, 5–10 and 10–15 cm depth.

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