Microbial Reduction of Fe(III) in Smectite Minerals by Thermophilic Methanogen Methanothermobacter Thermautotrophicus

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Geochimica et Cosmochimica Acta 106 (2013) 203–215 www.elsevier.com/locate/gca

Microbial reduction of Fe(III) in smectite minerals by thermophilic methanogen Methanothermobacter thermautotrophicus

Jing Zhang a,b, Hailiang Dong a,b,⇑, Deng Liu c, Abinash Agrawal d

a State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, PR China b Department of Geology and Environmental Earth Science, Miami University, OH 45056, USA c State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, PR China d Department of Earth and Environmental Sciences, Wright State University, OH 45435, USA

Received 15 May 2012; accepted in revised form 15 December 2012; available online 4 January 2013

Abstract

Clay minerals and thermophilic methanogens can co-exist in hot anoxic environments, including the continental subsurface, geysers, terrestrial hot springs, and deep-sea hydrothermal vent systems. However, it is unclear whether thermophilic methanogens are able to reduce structural Fe(III) in clay minerals. In this study, the ability of a thermophilic methanogen Methanothermobacter thermautotrophicus to reduce structural Fe(III) in iron-rich and iron-poor smectites, (nontronite NAu-2 and Wyoming montmorillonite SWy-2) and the relationship between iron reduction and methanogenesis were inves- tigated. M. thermautotrophicus reduced Fe(III) in nontronite NAu-2 and montmorillonite SWy-2 with H2/CO2 as substrate. The extent of bioreduction was 27% for nontronite and 13–15% for montmorillonite. Anthraquinone-2,6-disulfonate (AQDS) did not enhance the extent of bioreduction, but accelerated the rate. When methanogenesis was inhibited via addition of 2- bromoethane sulfonate (BES), the extent of bioreduction decreased to 16% for NAu-2 and 9% for SWy-2. These data suggest that Fe(III) bioreduction and methanogenesis were mutually beneﬁcial. The likely mechanism was that Fe(III) bioreduction lowered the reduction potential of the system so that methanogenesis became favorable, and methanogenesis in turn stimu- lated the growth of the methanogen, which enhanced Fe(III) bioreduction. NAu-2 was partly dissolved and high charge smectite and biogenic silica formed as a result of bioreduction. Ó 2012 Elsevier Ltd. All rights reserved.

1. INTRODUCTION environmental processes such as nutrient cycling, plant growth, contaminant migration, organic matter matura- Iron-bearing clay minerals are ubiquitous in soils, sedi- tion, and petroleum production (Stucki and Kostka, 2006; ments and sedimentary rocks (Stucki and Kostka, 2006; Dong et al., 2009; Dong, 2012). Dong et al., 2009; Dong, 2012). Iron redox state in these Reduction of iron in clay minerals can be achieved either clay minerals largely controls the physical and chemical chemically or biologically. Biologically, since the isolation properties of clay minerals, such as layer charge, degree of dissimilatory Fe(III) reducing bacteria (DIRB) in 1980s of swelling, and surface area. These properties aﬀect many (Arnold et al., 1986; Lovley and Philips, 1988; Myers and Nealson, 1988), numerous studies have demonstrated that various microbes living in moderate- (8–42 °C) and high- ⇑ Corresponding author at: State Key Laboratory of Biogeology temperature (42–121 °C) environments can perform dissim- and Environmental Geology, China University of Geosciences, ilatory Fe(III) reduction (Kostka et al., 1996; Lovley et al., Beijing 100083, PR China. 1998; Dong et al., 2003; Kasheﬁ and Lovley, 2003; Zhang E-mail addresses: [email protected], [email protected] et al., 2007a; Jaisi et al., 2011). In soils and sediments, (H. Dong).

0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.12.031 204 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 certain mesophilic Fe(III)-reducing microorganisms can interactions are unclear. Thermophilic methanogens have couple oxidation of organic carbon with the reduction of only been shown to reduce soluble Fe3+ (Vargas et al., structural iron in clay minerals with important implication 1998). It is not known if they can reduce structural Fe(III) for bioremediation of organic and metal contaminants in clay minerals, and if so, what are consequences of this (Stucki et al., 1987; Zhang et al., 2007b; Bishop et al., 2011). reaction on methanogenesis and Fe(III) reduction? It is now known that in addition to dissimilatory iron- The objective of this study was therefore to investigate if reducing bacteria (DIRB), other metabolic types of anaero- a specific thermophilic methanogen is capable of reducing bic microorganisms, such as sulfate-reducing bacteria structural Fe(III) in clay minerals. We conducted labora- (SRB) (Li et al., 2004; Liu et al., 2012) and methanogens tory experiments to address the following questions: (i) is (Liu et al., 2011; Zhang et al., 2012), are capable of reduc- thermophilic methanogen capable of reducing structural ing structural Fe(III) in clay minerals either directly (DIRB Fe(III) in clay minerals? If so, what are the extents and and methanogens) or indirectly through H2S (SRB). Meth- rates of Fe(III) reduction and how do they compare with anogens are common archaea on both modern and ancient those by mesophilic methanogens; (ii) what is the relation- earth and they co-exist with iron oxides and clay minerals in ship between iron reduction and methanogenesis; (iii) what a variety of anoxic environments (Achtnich et al., 1995; van is the mechanism of bioreduction of structural Fe(III) in Bodegom and Stams, 1999; Liu et al., 2011). When poorly clay minerals by such methanogen; and (iv) whether a crystalline Fe(III) oxides are present in methanogenic sedi- thermophilic methanogen can promote the smectite–illite ments, CH4 production is inhibited (Lovley and Phillips, reaction. In this investigation, Methanothermobacter ther- 1987; Roden and Wetzel, 1996; Frenzel et al., 1999; Yao mautotrophicus, a thermophilic methanogen with the opti- and Conrad, 1999; Chidthaisong and Conrad, 2000), and mum temperature of 65 °C, and two different clay this inhibition has been ascribed to competition of DIRB minerals, iron-rich and iron-poor smectites, were selected over methanogens for the same substrate, i.e., H2 (Lovley for bioreduction experiments. Various geochemical and and Phillips, 1987; Achtnich et al., 1995; van Bodegom mineralogical methods were used to examine the reaction and Stams, 1999; Roden and Wetzel, 2003). However, stud- progress and to characterize mineralogical transformations. ies in the past 10 years have found that certain types of Our results demonstrated that thermophilic methanogen, methanogens could directly reduce ferric iron in iron oxides M. thermautotrophicus, was capable of reducing structural (Bond and Lovley, 2002; van Bodegom et al., 2004) or clay Fe(III) in clay minerals to various degrees. The results of minerals (Liu et al., 2011; Zhang et al., 2012). These results this study have important implications for the understand- suggest that the inhibition of methanogenesis may be due ing of the biogeochemical cycling of iron and CH4 in to diversion of electrons from CH4 production to Fe(III) clay-rich sediments under high temperature and anoxic reduction (Bond and Lovley, 2002; Liu et al., 2011; Zhang conditions. et al., 2012). Thus, Fe(III) as an alternative electron acceptor may be an important factor influencing the methane cycle. 2. MATERIALS AND METHODS To date, microbial reduction of structural Fe(III) in clay minerals by methanogens has only been studied at low tem- 2.1. Clay mineral preparation peratures. In the continental subsurface, geysers, terrestrial hot springs, and deep-sea hydrothermal vent systems, ther- Two clay minerals were selected: nontronite NAu-2 and mophilic microorganisms thrive (Rainey and Oren, 2006). Wyoming montmorillonite SWy-2. They have the similar Some thermophilic microorganisms have the ability to re- structure. Bulk materials of NAu-2 and SWy-2 were pur- duce Fe(III) in iron oxides (Vargas et al., 1998; Kashefi chased from the Source Clays Repository of the Clay Min- et al., 2002a,b; Kashefi and Lovley, 2003; Kashefi et al., erals Society (West Lafayette, IN). Previous studies have 2008) with important implications for early life on Earth shown the following formula for these two clay minerals: and life on other planets. However, relatively a small num- ber of thermophilic bacteria and archaea have been exam- NAu-2: M0:72ðSi7:55Al0:45ÞðFe3:83Mg0:05ÞO20ðOHÞ4, ined for their ability to reduce Fe(III) in clay minerals, all (Keeling et al., 2000) SWy-2: K Na Ca Al Fe3þ Fe2þ Mg of which are DIRB (Zhang et al., 2007a; Kashefi et al., ð 0:16 0:19 0:02Þð 3:29 0:34 0:01 0:28Þ Si Al O OH (Bishop et al., 2011) 2008; Dong et al., 2009; Jaisi et al., 2011). Similar to DIRB, ð 7:90 0:10Þ 20ð Þ4 thermophilic methanogens can certainly co-exist with where M represents the interlayer cation, such as Na, Ca, or Fe(III)-bearing clay minerals in high-temperature environ- K(Keeling et al., 2000). The total Fe in NAu-2 is 21.2%, of ments that can affect methanogenesis and global methane which 2% is Fe(II); and the total Fe in SWy-2 is 2.3%, of production. Recent discovery of methane on Mars and which 3% is Fe(II) (Bishop et al., 2011). the speculation on its biogenic origin (Atreya et al., 2007) The bulk clay minerals were manually ground and raises the possibility of finding methanogens even on Mars. soaked in 0.5 M NaCl solution overnight followed by Interestingly, Fe- and Mg-rich clay minerals, particularly centrifugation to harvest the 0.02–0.5 lm fraction. The smectite, are also detected on Mars (Poulet et al., 2005). Re- chloride anion was completely washed by deionized (DI) cent evidence (Ehlmann et al., 2011) suggests that these clay water, and its complete removal was tested with AgNO3. minerals likely exist in the subsurface of Mars which may After drying, these size fractions of clay minerals were also be suitable for thermophilic methanogens. However, ready for experiment. to date how thermophilic methanogens interact with Fe- For surface area measurement, the clay minerals were and Mg-rich clay minerals and the mechanisms of such homoionized with 0.1 M NaCl for 24 h in order to prevent J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 205 the interlayer collapse during the outgassing procedure. The (BES), an inhibitor of methanogenesis, was added to exam- BET surface area of NAu-2 and SWy-2 is 271 and 244 m2/ ine its effect on Fe(III) bioreduction. There were two con- g, respectively (Bishop et al., 2011). trol groups in this experiment. Control A group was identical to the experimental bottles except that cells were 2.2. Cell culturing replaced with an equal amount of the culture medium (bioreduction control), whereas Control B group was identical M. thermautotrophicus was kindly provided by Dr. Xiuz- to the experimental bottles but without any clay minerals hu Dong (Institute of Microbiology, Chinese Academy of (methanogenesis control). All treatments were performed Sciences, Beijing, China). This strain is routinely cultured in duplicates. All solutions and cultures were transferred in an enrichment culture medium (revised from Zehnder by using sterile needles and syringes. Experimental bottles and Wuhermann, 1977) under strictly anoxic condition. with 40 mL slurry (60 mL headspace) were incubated at In brief, the growth medium consists of (per liter) 0.54 g 65 °C in an incubator. KH2PO4, 2.14 g K2HPO43H2O, 1.6 g yeast extract, 0.5 g Bioreduction activity typically ceased after 40 days, but tryptone, 0.5 g peptone, 4 g NaHCO3, 0.29 g NH4Cl, the potential for bioreduction still persisted. In order to 0.096 g MgCl26H2O, 0.0096 g CaCl22H2O, 0.29 g NaCl, investigate the limiting factors of bioreduction, a fresh 1 mL vitamin solution (Kenealy and Zeikus, 1981), 1 mL batch of M. thermautotrophicus cells, filter-sterilized trace mineral solution (Zehnder and Wuhermann, 1977), H2:CO2 gas mix (80:20), autoclaved clay slurry (5 g/L) or and 1 mL 0.1% resazurin (redox indicator). The growth their various combinations were added to old cultures medium was made anoxic in glass serum bottles with O2- (10 months) following a similar idea developed previously free H2/CO2 (80:20) gas mix by passing through a hot cop- (Jaisi et al., 2007b). per column, and sterilized by autoclaving. The H2/CO2 (80:20) mix gas was injected into the headspace of the serum 2.4. Analytical methods bottles until a pressure of 140 kPa was reached. The initial amount of H2 in the serum bottles was approximately 2.4.1. Chemical analyses 0.6 mmol. Before cell inoculation, all the medium bottles In order to monitor the progress of Fe(III) reduction, to- were stored at 65 °C overnight to allow the sulfur func- tal Fe(II) concentration was measured at selected time tional group of yeast extract to react with any residual intervals by 1,10 phenanthroline method (Amonette and O2. Final pH of the medium was adjusted to 7.0 with Templeton, 1998). Aqueous Fe(II) concentration was mea- 0.1 N HCl. M. thermautotrophicus was cultured in this med- sured by Ferrozine assay (Jaisi et al., 2007a) after removing ium at 65 °C and transferred three times before bioreduc- solids by centrifugation of 0.5 mL cell–clay suspension in- tion experiments were initiated. side an anaerobic glove box (Coy Laboratory Products, Grass Lake, MI, USA). At the beginning and end of the 2.3. Bioreduction experiments and re-inoculation experiments experiments, pH values were measured by a pH meter inside glove box. The pH values in the incubation bottles Two clay minerals (final conc. = 5 g/L) were made into remained nearly constant at 6.4–6.8 throughout the experi- slurry with the culture medium in serum bottles (total vol- mental duration. ume, 100 mL). Because both K and Al are the limiting factors in smectite conversion to illite (Zhang et al., 2007a), 2.4.2. CH4 production external sources of K and Al were needed to promote this At given time points, 1 mL of headspace gas was col- reaction. The K concentration in the culture medium was lected from the experimental bottles, and analyzed for already adequate (>20 mM). An external Al source (final CH4 and H2 concentration. CH4 concentration was ana- conc. = 1 g/L) was provided in the form of amorphous lyzed by a HP 6890 series GC and separated on a capillary Al(OH)3nH2O to favor illite formation (Zhang et al., column (GS GasPro, 30 m 0.32 mm; J&W Scientific) 2007a). The bottles were purged with H2/CO2 (80:20), connected to a flame ionization detector. H2 was analyzed sealed with thick butyl rubber stoppers, and sealed with by a HP 5890 series GC system with a thermal conductivity aluminum crimps. After autoclaving, H2/CO2 (80:20) gas detector and a packed column (Shin Carbon 100/120, mix was added as a substrate into headspace and these bot- 2m 1 mm; 255 Restek, Belefonte, PA). Based on mea- tles were stored at 65°C overnight to achieve an anoxic con- sured partial pressures of CH4 and H2 in the headspace, dition. The amount of abiotic reduction of Fe(III) in the the aqueous CH4 and H2 concentrations at equilibrium clay minerals by the sulfur functional group of yeast extract were calculated by the Gas law and Henry’s law using their in control bottles was considered insignificant in compari- respective Henry’s constants (where KH for CH4 at son with the amount of bioreduction (see below). As such, 25 °C = 1.51 103 mol L1 atm1, Schwarzenbach et al., 4 1 1 appropriate corrections in measured Fe(II) values were 1995;KH for H2 at 25 °C = 7.8 10 mol L atm , made to calculate Fe(II) produced by bioreduction. Canfield et al., 2005). M. thermautotrophicus cells in their exponential phase were injected into the serum bottles with a final concentra- 2.4.3. Protein measurement tion of 108 cells/mL based on acridine-orange direct counts Because of the difficulty of enumerating cells in the (AODC). In select experiments, 0.1 mM Anthraquinone- presence of clay particles, protein concentrations were mea- 2,6-disulfonate (AQDS) was supplied as an electron shuttle. sured to evaluate change of total biomass over the course of In some experiments, 10 mM 2-bromoethane sulfonate the bioreduction experiments (Jaisi et al., 2007a). Cell–clay 206 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 suspension of 0.5 mL in volume was ultra-sonicated (Bran- for 24 h to allow further time for EG vapor to penetrate into son 2510MT ultrasonic bath with 2.84 L volume, 100 W the smectite interlayer. XRD patterns were obtained in a power output, and 42 kHz frequency) for 5 min and washed humidity-controlled laboratory immediately after EG satu- by phosphate-buffered saline (PBS) solution three times to ration without significant exposure to air. Powder XRD pat- remove any proteins from the medium (peptone, tryptone, terns were collected by a Scintag X-ray powder and yeast extract). After addition of 0.2 M NaOH, cell sus- diffractometer using CuKa radiation, a fixed slit scintillation pension was boiled 10 min to release protein and centri- detector, and a power of 1400 W (40 kV, 35 mA). The XRD fuged. Protein fraction in the supernatant was quantified slides were scanned in 0.02 two-theta steps with a count time with the Bradford assay using BSA as a standard (Jaisi of 2 s per step and a scanning range of 2–12° two-theta. et al., 2007a). Time course protein concentrations were only The XRD patterns of the glycolated samples were mod- measured successfully for the SWy-2 treatments and Con- eled with the SybillaÓ program developed by Chevrone trol B groups. The protein measurement was not successful Corporation (Aplin et al., 2006) to quantify the relative for the NAu-2 treatments because the fine-grained and well- proportions of high-charge smectite, mixed-layer illite– dispersed NAu-2 particles interfered with the protein smectite, and illite in both unreduced and bioreduced clay measurement. mineral samples.

2.4.4. XRD and Sybilla modeling 2.4.5. SEM Mineralogical changes after bioreduction were deter- Mineralogical changes were further characterized by mined by comparing XRD patterns of unreduced and biore- using scanning electron microscopy (SEM). Clay suspen- duced clay minerals. Clay mineral slurries were sampled sions (0.5 mL) were washed by anoxic DI water inside a with a syringe needle and smeared (Moore and Reynolds, glove box to remove extra Al and K in the medium, and 1997) onto petrographic slides and dried at 30 °C overnight mounted onto 15 mm cover slips pretreated with 1% in a glove box. XRD patterns were collected on both air- poly-L-lysine for 60 min. The sample-coated cover slips dried samples and those solvated with ethylene glycol were sequentially dehydrated using varying proportions of (EG) vapor at 60 °C overnight (Moore and Reynolds, ethanol followed by critical point drying with a Tousimis 1997) to expand smectite interlayers. The same slides were Samdro-780A Critical Point Dryer (CPD) (Dong et al., subsequently placed in a desiccator at room temperature 2003). The sample-coating cover slips were mounted on

Fig. 1. Production of total Fe(II) in NAu-2 (A) and SWy-2 (B) with time as measured by the 1,10-phenanthroline method. Initial cell concentration was 108 cells/mL. Averages of two measurements from duplicate experimental tubes were reported. The error bars, which are masked by the symbols, represent the diﬀerence between the higher and lower values. The arrows denote the time point at which a fresh batch of cells and H2/CO2 were added to the old culture tubes. Control A did not have any cells but otherwise was the same as the treatments. J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 207

SEM stub via clear double-sided sticky tape and coated creased again. For NAu-2, the rates for the second stage of with a Denton Desk II Gold Sputter Coater for SEM obser- bioreduction (9–15 days) were higher than the first stage vations. The samples were analyzed with a Zeiss Supra 35 (Table 1). For SWy-2, the second-stage bioreduction rates VP SEM with Genesis 2000 X-ray energy dispersive spec- (9–12 days) were lower (Table 1). troscopy (SEM/EDS). The EDS provided a means for min- Soluble Fe2+ concentrations exhibited similar time- eralogical identification. The SEM was operated at an course patterns as total Fe(II) (data not shown). The ratio accelerating voltage of 5–10 kV. A short working distance of aqueous Fe2+ to total Fe(II) was low (<1%), consistent (6–10 mm) and low beam current (30–40 lA) were used to with previous results of Fe(III) bioreduction in NAu-2 using achieve the best image resolution. A longer working dis- Shewanella putrefaciens CN32 (Jaisi et al., 2007a), suggest- tance (15–20 mm) and higher beam current (50–70 lA) ing that biologically produced Fe(II) either remained in were used for qualitative EDS. the structure, sorbed onto clay particle/cell surfaces, or inte- grated into secondary minerals (Zhang et al., 2012). 3. RESULTS 3.2. Re-inoculation experiment and the inhibition effect from 3.1. Microbial reduction of Fe(III) in clay minerals cell-associated Fe(II)

M. thermautotrophicus was capable of reducing struc- Addition of fresh cells, substrate (filter-sterilized tural Fe(III) in the clay minerals with H2/CO2 as substrate. H2/CO2 gas), or autoclaved clay slurry individually to the In general, the bioreduction experiments of structural old cell–clay cultures (10 months) did not result in any fur- Fe(III) in clays were complete in 20 days, but the experi- ther bioreduction. However, when fresh cells and H2/CO2 ments were allowed to continue for 40 days to ensure com- (80:20) were added together to the experimental bottles, pletion of bioreduction (Fig. 1). Minor amounts of Fe(III) additional Fe(III) bioreduction was observed (Fig. 1). reduction (<3%) occurred in the abiotic control. The net These data were consistent with previous research in biogenic Fe(II) production was calculated after correction showing that addition of fresh cells (S. putrefaciens for this abiotically produced Fe(II) and the initial amount CN32) into existing cell-NAu-2 cultures increased the of Fe(II) present at time zero. extent of bioreduction (Jaisi et al., 2007b). The bioreduction trends were different for the two clay minerals (Fig. 1). The extent of bioreduction for NAu-2 3.3. CH4 production and hydrogen production was virtually the same without and with AQDS (26.8% vs 26.9%), but significantly lower in the presence of a metha- In the absence of clay minerals (Control B), a significant nogenesis inhibitor, BES (15.6%). Similar trends were ob- amount of CH4 was produced with time (Fig. 2). However, served for SWy-2 but with lower extents (12.6%, 14.8%, addition of clay minerals (5 g/L) inhibited CH4 production. and 9.4% for the treatments without AQDS, with AQDS, Virtually no CH4 was produced in the NAu-2 group during and BES, respectively). the first phase of rapid Fe(III) bioreduction (Fig. 2). How- Although the bioreduction rates generally decreased ever, in the second phase of rapid Fe(III) bioreduction (9– over time, they fluctuated over time (Table 1). For both 15 days), methanogenesis was not inhibited. In fact, in the NAu-2 and SWy-2, the calculated initial rates (first 24 h) NAu-2 treatments, rapid methanogenesis occurred concur- were 0.06, 0.07, and 0.04 mmol/g/hr, for the treatments rently with rapid Fe(III) bioreduction (9–15 days). In the without AQDS, with AQDS, and with BES, respectively. SWy-2 group, CH4 was continuously produced (Fig. 2). When averaged over the first 5 days, the bioreduction rates By the end of the experiment, the final concentrations of were similar to the initial rates (Table 1). Bioreduction CH4 in the presence of the clay minerals were only slightly paused over the period of 5–9 days for both minerals. After lower than those in their absence. There was no CH4 pro- this “dormant” period, however, the bioreduction rates in- duction in the group with BES.

Table 1 Bioreduction rates for the ﬁrst 24 h and the two rapid stages of reduction. Phase Time Cell Cell + AQDS Cell + BES H L Ave. H L Ave. H L Ave. NAu-2 (mmol/g/hr) Initial 24 h 0.07 0.04 0.06 0.08 0.05 0.07 0.05 0.03 0.04 First 0–5 days 0.05 0.05 0.05 0.07 0.05 0.06 0.04 0.04 0.04 Second 9–15 days 0.11 0.09 0.10 0.11 0.10 0.11 0.06 0.05 0.06

SWy-2 (mmol/g/hr) Initial 24 h 0.06 0.05 0.06 0.08 0.06 0.07 0.04 0.04 0.04 First 0–3 days 0.04 0.03 0.04 0.03 0.02 0.03 0.04 0.03 0.04 Second 9–12 days 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.00 0.01 Duplicate incubation bottles generated two rates for each time period: H—high value, L—low value, Ave.—average of the duplicates. 208 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215

Fig. 2. Methane production and hydrogen consumption with time in NAu-2 (A and C), SWy-2 (B and D), and the Control B group. Averages of two measurements from duplicate experimental tubes were reported. The error bars represent the diﬀerence between the higher and the lower values. Control B did not have any clay minerals.

When no clay minerals were added, H2 was consumed in protein (Fig. 3). Protein production was the highest stoichiometric proportion to CH4 production. The amount (16.62 mg/L) when SWy-2 was absent (Control B, with no of H2 consumption in each serum bottle was 0.6 mmol clay), and the lowest (2.63 mg/L) when BES was added (Fig. 2), which was four times the amount of CH4 produc- to inhibit methanogenesis. Cell growth occurred in two tion, i.e., 0.15 mmol (Fig. 2) according to Eq. (1) (below): stages (Fig. 3), approximately corresponding to the two phases of Fe(III) bioreduction (Fig. 1). The trend of bio- 4H2 þ CO2 ! CH4 þ 2H2O ð1Þ mass production also paralleled the CH4 production. For With 5 g/L NAu-2 (with and without AQDS), however, example, fast cell growth occurred from day 1 to day 5 the amount of H2 consumption was 0.6 mmol, and corre- and from day 9 to day 15, which corresponded to the two spondingly 0.12 mmol CH4 and 0.2 mmol Fe(II) were pro- periods of rapid CH4 production (Fig. 2). After the 15th duced (Fig. 2). According to the 4:1 stoichiometric ratio of day, both the growth of M. thermautotrophicus and CH4 H2 and CH4, the amount of H2 required to produce production continued slowly. There was a small amount 0.12 mmol CH4 should be 0.48 mmol, leaving 0.12 mmol of initial growth in the bottles with BES-treatment, but for Fe(III) bioreduction. So the ratio of H2 consumption biomass remained low throughout the duration of the and Fe(III) bioreduction was approximately 0.12: 0.2, very experiments. Based on the similarity in the time-course close to the theoretical ratio of 1:2 according to Eq. (2) CH4 production pattern between the NAu-2 and SWy-2 (below): treatments (Fig. 2) and the fact that CH4 production is a reflection of methanogen growth (our own data for SWy- H2 þ 2FeðIIIÞþ2OH ! 2FeðIIÞþ2H2O ð2Þ 2 and also Maestrojuan and Boone, 1991), it was expected The trend of H2 consumption was similar for SWy-2. that time-course increase of protein concentration for the The total amount of H2 consumption in this system was NAu-2 treatments should be similar to the SWy-2 0.54 mmol, which produced 0.13 mmol CH4 and reduced treatments. 0.038 mmol Fe(III). Again there were no significant difference in the amounts of H2 consumption, CH4 production, 3.5. Mineralogical changes detected by XRD and Fe(III) bioreduction between the AQDS and no-AQDS treatments. The bioreduced samples by the end of 40 days were incubated for additional 60 days under the same conditions to 3.4. Protein production promote any mineralogical transformations. XRD patterns for the ethylene glycolated samples showed that NAu-2 and M. thermautotrophicus grew under all experimental SWy-2 remained after bioreduction for 100 days. For biore- conditions as shown by the time-course increase of total duced NAu-2, the d(001) spacing shifted from 17.40 to J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 209

Fig. 3. Time-course production of protein concentration produced by M. thermautotrophicus in SWy-2 and Control B group during Fe(III) bioreduction. Control B did not have any clay minerals. The error bars represent the diﬀerence between the higher and lower values from duplicate experimental tubes.

14.67 A˚ (Fig. 4) and the peak became less intense and was typical of nontronite with a low Al/Si ratio, no K, and broader than the same peak for the abiotic control. XRD abundant Fe (A2, Fig. 5F), but no Fe was detected in the patterns of SWy-2 showed similar changes, and the net-shaped NAu-2 (A3, Fig 5F). Some plate-shaped NAu- d(001) spacing shifted from 16.15 to 13.66 A˚ . However, 2 and silica aggregates were observed in bioreduced NAu- no new mineral products were detected by XRD as a result 2(Fig. 5G–I). These plate-shaped NAu-2 particles exhibited of bioreduction (Fig. 4). a higher Al/Si ratio than the original NAu-2 with no K and The Sybilla modeling results for the ethylene glycolated Fe (Fig. 5G and I), probably representing high charge samples showed that there were two types of smectite layers smectite (possibly corresponding to 13 A˚ d-spacing in in the control and bioreduced samples, one with a spacing XRD patterns) (Zhang et al., 2007b; Liu et al., 2011). Sim- of about 17 A˚ , and the other with a spacing of about ilar mineralogical changes also took place for SWy-2, but 13 A˚ . The abundance of the 17-A˚ layer type decreased no plate-shaped SWy-2 and silica aggregates were observed. from 97% and 87% for the abiotic control of NAu-2 and SWy-2 to 41% and 24% for the bioreduced samples, respec- 4. DISCUSSION tively. In contrast, the 13-A˚ layer type increased in abundance from 3% and 13% for abiotic NAu-2 and SWy-2 to 4.1. Relationship between methanogenesis and Fe(III) 59% and 76% for bioreduced samples, respectively (Table 2). bioreduction This result was consistent with previous bioreduction data using mesophilic methanogen Methanosarcina barkeri (Liu This research demonstrated that thermophilic methano- et al., 2011), suggesting that bioreduction of structural gen can reduce structural Fe(III) in clay minerals. All stud- Fe(III) in clay minerals increased the layer charge of smec- ies to date (Bond and Lovley, 2002; van Bodegom et al., tite and caused layer collapse. 2004; Reiche et al., 2008; Liu et al., 2011; Zhang et al., 2012) have shown inhibition of methanogenesis by Fe(III) 3.6. SEM observations of mineralogical changes bioreduction using mesophilic methanogens. In our experiments, Fe(III) bioreduction inhibited methanogenesis only SEM images revealed that flaky texture dominated both at the beginning, but later enhanced it. There may be sev- NAu-2 and SWy-2 abiotic controls (Fig. 5A), and an EDS eral reasons for the initial inhibition of methanogenesis spectrum for abiotic NAu-2 showed a typical nontronite by Fe(III) bioreduction. Methanogens are strict anaerobes, composition (Fig. 5B). After bioreduction, there were abun- which require a low redox potential (Bond and Lovley, dant dissolution pits forming three-dimensional nets in 2002). At the beginning of the bioreduction experiments, both NAu-2 and SWy-2. Many microbial cells connected the presence of oxidized NAu-2 and SWy-2 would increase these net-shaped clay minerals. Some of these dissolution the redox potential of the system and resulted in an inhib- pits were similar to the size and shape of the cells iting effect on methanogenesis. The reduction potential of (Fig. 5C and D). There were some flaky-textured NAu-2 the Fe(III)-clay/Fe(II) couple for NAu-2 at neutral pH is remaining in the bioreduced NAu-2 (the left side on reported to be +600 mV (Jaisi et al., 2007a), which is much Fig. 5E), and some microorganisms appeared to be actively higher than the inhibitory threshold of 420 mV for metha- interacting with NAu-2 to form three-dimensional net (the nogenesis (Fetzer and Conrad, 1993). Similar inhibition of right side of Fig. 5E). The composition of the flaky NAu-2 methanogenesis has been observed previously (Liu et al., 210 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215

Fig. 4. XRD patterns showing change of the 001 peak of NAu-2 before and after bioreduction. The peak of NAu-2 became much wider and the intensity signiﬁcantly decreased after bioreduction.

Table 2 Sybilla modeling results based on the glycolated XRD patterns for abiotic and bioreduced NAu-2 and SWy-2 samples. Sample d-spacing (A˚ ) Interlayer cation content Proportion (%) NAu-2 Control 17.20 0.32 97 12.90 0.80 3 Cells only 17.19 0.30 73 13.76 0.80 27 Cells + AQDS 17.20 0.75 41 12.90 0.80 59 SWy-2 Control 17.20 0.38 87 12.90 0.80 13 Cells only 17.20 0.32 41 12.90 0.69 59 Cells + AQDS 17.16 0.45 24 12.90 0.76 76

2011) when NAu-2 was bioreduced by a mesophilic (Vargas et al., 1998; Lovley, 2000; Bond and Lovley, methanogen. 2002), and it is believed that the hydrogenase is responsible An alternative mechanism for inhibition of methanogen- for Fe(III) reduction (Bond and Lovley, 2002). In the nor- esis by Fe(III) bioreduction is through diversion of electron mal pathway of methanogenesis, hydrogenases can oxidize ﬂow from CO2 to Fe(III) reduction (Bond and Lovley, 2002; H2 to proton by releasing two electrons, and these two elec- van Bodegom et al., 2004). The capability of hydrogeno- trons should be utilized to reduce methyl-coenzyme M trophic methanogens to reduce Fe(III) has been reported (CH3-S-CoM) to CH4 (Deppenmeier et al., 1999). However, J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 211

Fig. 5. Secondary electron images showing dissolution of NAu-2 as a result of bioreduction after 100 days. (A) abiotic control NAu-2; (B) EDS spectrum of unreduced NAu-2 in the abiotic control; (C) cells and dissolution pits in bioreduced NAu-2; (D) cells and dissolution pits in bioreduced SWy-2; (E) ﬂaky textured and net-shaped particles in bioreduced NAu-2; (F) EDS spectra of ﬂaky-textured and net-shaped particles in bioreduced NAu-2; (G) high charge smectite in bioreduced NAu-2; (H) silica aggregates formed after bioreduction of Fe(III) in NAu-2; (I) EDS spectra of high charge smectite and silica aggregates after bioreduction of NAu-2. In all EDS spectra, the Au peak was from the Cu grid used to support the samples.

in the presence of bioavailable Fe(III) these electrons could would compete against CH3-S-CoM for electrons and thus be diverted to Fe(III) (Bond and Lovley, 2002). Our data inhibit CH4 production. At the beginning of our experi- indicated that the consumption of H2 was stoichiometrically ments methanogenesis was inhibited due to the high redox proportional to the production of Fe(II) (Fig. 2), which was potential in the system. The growth of cells was minimal consistent with our previous research (Liu et al., 2011), (Fig. 3), possibly with a small amount of hydrogenase pro- implying that electrons that were transferred to Fe(III) duction. During the first phase of rapid bioreduction, all reduction were derived from H2. available electrons from hydrogenases were possibly trans- These two hypotheses were further confirmed by the ferred to Fe(III) resulting in similar reduction extents and BES treatment. BES is an analog of CH3-S-CoM, thus, it rates with and without BES (Figs. 1 and 2). As the Fe(III) 212 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 bioreduction continued in the serum bottles, the redox or incorporated into secondary minerals. Secondly, metha- potentials of the experimental systems slowly decreased to nogens only transfer a fraction of available electrons to low values that methanogenesis became favorable (Fig. 2). structural Fe(III), resulting in competition of H2 between By the second phase of Fe(III) bioreduction (Fig. 1), some methanogensis and Fe(III) bioreduction. According to the electrons were transferred to CH3-S-CoM to produce CH4, results from the re-inoculation experiment, the bioreduction and this methanogenesis would generate energy to support in our system might be limited by fresh surfaces of cells and the rapid growth of methanogens (Fig. 3). The rapid cell the supply of electron donor (H2). growth in turn promoted more Fe(III) bioreduction as long A previous study reported that AQDS can significantly as there was sufficient supply of electron donor (H2) in the enhance the extent of Fe(III) oxide reduction in high tem- system. Thus, in contrast to the first phase of Fe(III) biore- perature environments by Pyrobaculum islandicum (Lovley, duction where methanogenesis was inhibited, during the 2000). However, in our experiments, for NAu-2, AQDS second phase bioreduction and methanogenesis were mutu- only slightly enhanced the bioreduction rate during the first ally cooperative: Fe(III) bioreduction lowered the reduction phase of bioreduction, but did not make any difference in potential of the system to favor methanogenesis; methano- the ultimate extent of bioreduction. The possible reason genesis generated energy and supported the rapid growth of might be that M. thermautotrophicus could secrete their the cells which in turn favored Fe(III) bioreduction. own electron shuttling compounds similar to other metha- No methanogenesis was observed in the treatments with nogens such as M. mazei,(Abken et al., 1998). During BES (Fig. 2), and the amount of cell growth was minimal the first phase of rapid bioreduction, the microbial popula- (Fig. 3). Correspondingly, Fe(III) bioreduction was insig- tion was presumably low, and the amount of electron shut- nificant (Figs. 1 and 2). Eventually bioreduction gradually tling compounds, if any, could be low. In that case, AQDS stopped due to limiting factors. This pattern was in contrast functioned as an external shuttle and enhanced the rate of to the treatment without BES, where rapid methanogenesis bioreduction. However, during the second phase of biore- was observed until H2 was consumed completely. The duction, a greater amount of cells could have produced suf- amount of CH4 production in the system without BES ficient amounts of electron shuttling compounds to was comparable to Control B group (pure cultures without facilitate electron transfer to Fe(III). In this case, additional any clay minerals). electron shuttle such as AQDS would make no difference. However, further investigation is needed to confirm this 4.2. The rates and extents of Fe(III) bioreduction by hypothesis. thermophilic methanogen in comparison with other microorganisms 4.3. Two stage Fe(III) bioreduction mechanism in NAu-2 and SWy-2 Compared with previous reports using the same particle size of NAu-2, the initial bioreduction rate by M. thermau- The different extent and rate of bioreduction between totrophicus was nearly one order of magnitude higher than NAu-2 and SWy-2 may be explained by the different prop- that of NAu-2 by a mesophilic methanogen, M. barkeri erties of these two clay minerals. Previous studies (Bishop (0.005 mmol/g/hr) (Liu et al., 2011), and nearly twice the et al., 2011; Zhang et al., 2012) revealed that the larger reac- rate of NAu-2 by mesophilic Methanosarcina mazei tive surface area would expose more Fe(III) centers to ac- (0.027 mmol/g/hr) (Zhang et al., 2012). Similarly, the except more electrons, resulting in higher extent of Fe(III) tents of reduction by thermophilic methanogen are gener- bioreduction. However, there was no significant difference ally higher than those by mesophilic methanogens. The in the surface area between NAu-2 and SWy-2. Thus, higher extent and rate of bioreduction by thermophilic surface area cannot be the reason. The largest difference methanogen should be due to the effect of higher tempera- between NAu-2 and SWy-2 is the Fe(III) content. NAu-2 ture on the bioreduction kinetics, rather than temperature- contains 10 times more Fe(III) than SWy-2, however, the induced clay mineral dissolution (prior to bioreduction) bioreduction extent of NAu-2 was only two times greater because there was no clay mineral dissolution in abiotic than that of SWy-2. This result suggests that Fe(III) may controls. not be evenly distributed in their structures and is not The measured extents of bioreduction were in the range equally accessible for bioreduction. Thus, total Fe content of those observed for NAu-2 reduction by other thermo- may be an important reason for the different extent and rate philic bacteria (Zhang et al., 2007a; Kashefi et al., 2008; of bioreduction between NAu-2 and SWy-2. Jaisi et al., 2011). In all these bioreduction examples, the ex- One interesting feature observed in this study was the tent of Fe(III) bioreduction was far from complete (i.e., two-stage bioreduction pattern. These two-stage reduction 100% reduction). Numerous factors may limit the extent patterns have been observed for other minerals such as lep- of bioreduction, as discussed previously (Dong et al., idocrocite (O’Loughlin et al., 2010) and illite (Dong et al., 2009). Firstly, bioreduction may be inhibited by Fe(II), 2003). Although not explicitly stated in these studies, the which was released from reductive dissolution of clay min- reasons may be related to differential sorption strengths erals and subsequently sorbed onto NAu-2 and cell surfaces of anion and natural organic matter onto lepidocrocite (Jaisi et al., 2007b). Our SEM evidence (Fig. 5) confirmed (O’Loughlin et al., 2010) and structural-textural heteroge- reductive dissolution of NAu-2 and SWy-2, but low aque- neities of illites (Dong et al., 2003). For the case of NAu- ous concentration of Fe(II)aq suggests that released Fe(II) 2 and SWy-2, the observed two-stage reduction pattern was either sorbed onto cell and NAu-2 particle surfaces may be related to Fe(III) distribution in the structure. Jaisi J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 213 et al. (2005) showed that tetrahedral Fe(III) of the NAu-2 4.5. Biogeochemical implications structure was more susceptible to reduction than cis-octahedral Fe(III). The bioreduction extent at the end of the first Iron-reducing bacteria may widely coexist with iron- stage (7.4% for NAu-2) was similar to the fraction of tetra- bearing clay minerals in high temperature environments. hedral Fe(III) in NAu-2 (9%) (Gates et al., 2002). There- It is speculated that the earliest forms of microbial life fore, it is possible that all bioreduced Fe(III) in the first may have lived in hot, anaerobic environments (Baross stage might be derived from the tetrahedral sites of NAu- and Hoffman, 1985; Pace, 1991; Holm, 1992; Bock and 2. In the second stage of NAu-2 bioreduction, however, Goode, 1996). If so, the metabolism of thermophilic micro- the reduction rate and extent were much greater than that organisms may provide insights into biogeochemical pro- from the first stage, presumably because Fe(III) was derived cesses on the early history of earth and other planets from octahedral sites. (Adams, 1994; Bock and Goode, 1996; Chastain and Kral, For SWy-2, there is no report of tetrahedral Fe(III) in its 2010). Fe(III) reduction by thermophilic microorganisms structure and it is assumed that Fe(III) is octahedral might be an important form of respiration in these ancient, (Bishop et al., 2011; Neumann et al., 2011). However, anaerobic, and hot environments (Vargas et al., 1998; Lov- Fe(III) in SWy-2 may be located in two different types of ley, 2000). octahedral sites and could result in different reduction rates; Some of the terrestrial hot spring sites, such as Lidy Hot this suggestion was supported by the fact that the rate of Spring, were considered to have similar habitats as the sub- the second stage bioreduction in SWy-2 was slower than surface of Mars and Europa (Chapelle et al., 2002). If so, that during the first-stage, which was in contrast to the ob- iron reduction by methanogens might be an important bio- served rates for NAu-2. It is then perhaps reasonable to geochemical process on other planets. Discovery of meth- speculate that Fe(III) located in some octahedral sites ane (Atreya et al., 2007) and Fe-rich clay minerals, such may be more reactive, and they were preferentially reduced as nontronite, on Mars (Poulet et al., 2005) raises this pos- followed by bioreduction of Fe(III) located in less reactive sibility. Recent evidence (Ehlmann et al., 2011) suggests octahedral sites. that Fe- and Mg-rich clay minerals may have formed in hydrothermal environments in the subsurface of Mars at 4.4. Clay mineral transformations as a result of bioreduction temperatures from ambient up to 400 °C. In such environ- by M. thermautotrophicus ments, thermophilic and hyperthermophilic methanogens could have existed and actively participated in reduction There are two proposed mechanisms for Fe(III) biore- of abundant structural iron in clay minerals. The results duction and mineral transformations: solid-state and disso- of this study showed that two important microbial pro- lution–precipitation (Dong et al., 2009). In the solid state cesses, iron reduction and methanogenesis may be corpora- model, Fe(III) in clay minerals is bioreduced in the solid tive or competitive, depending on specific conditions, which state, therefore, only small and reversible mineralogical would have important implications for our understanding changes occur (Gates et al., 1996; Favre et al., 2002; Lee of the methane and iron cycles on Earth and beyond. et al., 2006; Kashefi et al., 2008). However, in the dissolution–precipitation model, the dissolution of mineral occurs. 5. CONCLUSIONS As a result, mineralogical changes are irreversible and new minerals are formed (Dong et al., 2003; Kim et al., 2004; Li Methanogen M. thermautotrophicus reduced Fe(III) in et al., 2004; Jaisi et al., 2007b, 2011; Zhang et al., 2007a,b, nontronite NAu-2 and montmorillonite SWy-2 with H2/ 2012). Our data collectively suggested that the bioreduction CO2 as substrate. The extents of bioreduction were 27% of smectites by M. thermautotrophicus resulted in a small for nontronite and 13–15% for montmorillonite. AQDS amount of reductive dissolution. These data are consistent did not enhance the extent of bioredcution, only accelerated with published results on bioreduction of nontronite by the rate for NAu-2. When methanogenesis was inhibited mesophilic methanogens (Liu et al., 2011; Zhang et al., due to addition of BES, the extent of bioreduction de- 2012). creased to16% for NAu-2 and 9% in SWy-2. At the begin- While our XRD result did not show illite formation dur- ning of the experiment, methanogenesis was inhibited by ing smectite reduction, there were some changes to NAu-2. Fe(III) bioreduction, likely due to the high reduction poten- The Sybilla modeling results suggested formation of some tial of the system. At a later stage, the two processes were high charge smectite (Table 2) which was confirmed by hypothesized to have cooperated, i.e., Fe(III) bioreduction SEM observations (Fig. 5G and I). There may be two pos- may have lowered the reduction potential of the system to sible reasons for the lack of smectite conversion to illite. favor methanogenesis, and methanogenic activity stimu- First, the experimental duration may not be long enough. lated the cell growth, which would further enhance Fe(III) Jaisi et al. (2011) showed that six months may be needed bioreduction. NAu-2 was partly dissolved and high charge to observe the smectite and illite conversion as catalyzed smectite, and biogenic silica formed as a result of by thermophilic microbes at 65 °C. Second, the pH may bioreduction. be too low to form illite. In our experiment, CO2, as a substrate for the methanogen, should have continuously dis- ACKNOWLEDGMENTS solved into the medium. As a result, the working pH in the medium was slightly acidic (6.4–6.8), which would not We are grateful to Prof. Xiuzhu Dong for providing the strain favor illite formation (Zhang et al., 2007a; Jaisi et al., 2011). of M. thermautotrophicus. This work was supported by grants from 214 J. Zhang et al. / Geochimica et Cosmochimica Acta 106 (2013) 203–215 the National Basic Research Program of China (973 Program) Deppenmeier U., Lienard T. and Gottschalk G. 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