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Geochimica et Cosmochimica Acta 206 (2017) 57–72

Quantification and isotopic analysis of intracellularsulfur metabolites in the dissimilatory sulfate reduction pathway

Min Sub Sim a,b,⇑, Guillaume Paris a,c, Jess F. Adkins a, Victoria J. Orphan a,Alex L. Sessions a

aDivision of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125, USAbSchool of Earth and Environmental Sciences, Seoul National University, Seoul 08826, South Korea

cCentre de Recherches Petrographiques et Geochimiques, UMR 7358 CNRS-UL, Vandoeuvre-les-Nancy 54500, France

Received 30 July 2016; accepted in revised form 18 February 2017; Available online 27 February 2017

Abstract

Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in 34S and producing sulfidethat is depleted in 34S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfurisotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellularmechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopicratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis. Intracellular sulfateand APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulatesin the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in 34S.The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfateand sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the netfractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This ‘reservoir effect’diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations.Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the over-all (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for thewell-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while atthe same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in thisrelationship. Our approach, together with ongoing investigations of the kinetic isotope fractionation by key enzymes in thesulfate reduction pathway, should provide an empirical basis for a quantitative model relating the magnitude of microbialisotope fractionation to their environmental and physiological controls.� 2017 Elsevier Ltd. All rights reserved.

1. INTRODUCTION

Since Thode et al. (1951) first demonstrated that the H2Sproduced by microbial sulfate reduction is depleted in 34S

http://dx.doi.org/10.1016/j.gca.2017.02.024

0016-7037/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: School of Earth and EnvironmentalSciences, Seoul National University, Seoul 08826, South Korea.

E-mail address: mssim@snu.ac.kr (M.S. Sim).

relative to the reactant sulfate, isotopic fractionationbetween sulfate and sulfide has been widely used to tracethe activity of sulfate reducing microbes in modern andancient ecosystems (Fry, 1991; Canfield and Teske, 1996;Wortmann et al., 2001; Druhan et al., 2008). Given thelarge variations in natural abundance of sulfur isotopesup to several percent, numerous attempts have been madeto relate the magnitude of isotopic fractionation to their

58 M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72

genetic (Detmers et al., 2001), evolutionary (Pellerin et al.,2015), and environmental controls, including levels of elec-tron acceptor or donor (Harrison and Thode, 1958;Chambers et al., 1975; Habicht et al., 2005; Hoek et al.,2006; Sim et al., 2011a; Leavitt et al., 2013), limitation ofother nutrients (Sim et al., 2012), and temperature(Canfield et al., 2006; Mitchell et al., 2009). These studiesprovide a general consensus that limiting the supply of elec-tron donor to the sulfate reduction pathway leads to largersulfur isotope effects, while depletion of terminal electronacceptor, sulfate, decreases the magnitude of fractionation(see Figure 6 of Bradley et al., 2016). Such qualitativeframework helps to extract environmental informationfrom the sulfur isotope fractionation records preserved insediments and old rocks (Jones and Fike, 2013; Sim et al.,2015; Raven et al., 2016). Yet, the use of sulfur isotopesas a quantitative geochemical proxy is rather complicated,because all culture experiments show a non-linear andspecies-specific relationship between isotope effects andenvironmental stimuli (Ono et al., 2014; Wing andHalevy, 2014). These limitations are rooted, in part, inthe experimental shortcomings that microbial sulfur isotopefractionation has been explored primarily as a single neteffect, although it involves a series of enzymatic reactions.

So far, not much is experimentally known about theintracellular processes responsible for large variations inmicrobial sulfur isotope fractionations, spanning from �3to 66‰ (Harrison and Thode, 1958; Sim et al., 2011b). The-oretical models have provided a basis for linking isotopicfractionation to internal cellular processes, but remain lar-gely untested. The linear metabolic network model, firstproposed by Rees (1973), has been modified to meet newexperimental observations (Brunner and Bernasconi, 2005;Johnston et al., 2007; Wing and Halevy, 2014; Bradleyet al., 2016). Here the kinetic isotope effects assigned toeach enzymatic reaction define the possible range of sulfurisotope fractionation. These models invoke the reversibilityof each enzymatic reaction, that is, the ratio of backward toforward fluxes, as a mechanism underlying the varying frac-tionations (Fig. 1). Since reversibility is ultimately con-strained by the concentrations of substrates and products(Wing and Halevy, 2014), measuring the intracellular sulfurmetabolite levels and their isotopic compositions provides adirect means of bridging the gap between theoretical modeland experimental observations.

Here, we report a new analytical approach to determin-ing the concentrations and isotopic compositions of intra-cellular sulfur metabolites, sulfate, APS, and sulfite, insulfate-reducing microbes. Reliable and reproducible mea-surements are achieved via preparative ion chromatographycoupled to isotopic analysis by MC-ICP-MS (Paris et al.,2013). The 33S-dilution technique is employed to trackbackground contamination and validate the measurements.Using a series of batch cultures of Desulfovibrio alaskensis,we demonstrate that the intracellular sulfur metabolitelevels and their isotopic compositions fluctuate throughoutthe growth phase, and the relative limitation of intracellularsulfate (‘reservoir effect’) strongly constrain the expressedsulfur isotope fractionation during microbial sulfatereduction.

2. EXPERIMENTAL METHODS

2.1. Batch incubation

Three independent batch experiments were conductedwith a marine sulfate-reducing bacterium, Desulfovibrio

alaskensis. D. alaskensis was incubated in a chemicallydefined, carbonate buffered medium containing (per liter):NaHCO3, 5 g; Na2SO4, 3 g; KH2PO4, 0.2 g; NaCl 21 g;MgCl2�6H2O, 3.1 g; KCl, 0.5 g; CaCl2�2H2O, 0.15 g; resa-zurin, 1 mg; 1 ml of trace element solution SL-10 (Widdelet al., 1983); 10 ml of vitamin solution described as a partof DSMZ medium 141 (catalogue of strains 1993; DSMZ,Braunschweig, Germany); and 1 ml of tungsten-seleniumstock solution (4 mg of Na2WO4�2H2O and 3 mg ofNa2SeO3�5H2O per 1 L of 0.01 M NaOH). Cultures con-tained lactate (22 mM) as the sole organic electron donor,and sodium ascorbate (5 mM) and titanium (III) chelatedby nitrilotriacetate (NTA; 60 lM) as reducing agents. Thecompleted medium was titrated to pH 7.2 and sterilizedby filtration under 80% N2-20% CO2 gas. A preculturegrown in the same conditions was used as inoculum. Priorto inoculation, the cells were washed three times by anaer-obic centrifugation and resuspension in phosphate buffersolution (0.35 M NaCl, 0.05 M potassium phosphate, pHof 7.0) to minimize the carryover of sulfate and sulfide.For the first and third sets of experiments, three 500 ml cul-tures were inoculated in parallel, and each was sacrificed torecover intracellular sulfur compounds at different growthstages, while only stationary phase cells were tested forthe second experiment. Since the concentrations and sulfurisotope compositions of intracellular sulfur metabolitesturned out to be sensitive to the physiological status ofthe cell, biological replicates could be assessed by analyzingindependently-prepared samples only from a comparablegrowth stage. Here, two early stationary phase samples pro-vided a measure of biological reproducibility (series 1 and2). All cultures were incubated at 32 �C with 175 rpm agita-tion, and growth was monitored by measuring the opticaldensity at 660 nm (A660). A conversion factor for opticaldensity to dry weight was determined for A660 of 0.1 as40 lg/mL.

2.2. Extracellular metabolites

Samples for the quantification of organic acids were col-lected at intervals throughout incubation by filtering 1 mLof culture through a 33 mm-diameter, 0.2 lm-pore mem-brane (Millipore, Cork, Ireland) and stored at �80 �C untilanalysis. All collected samples were analyzed together usingan Agilent 1100 HPLC (Agilent Technologies, Santa Clara,CA, USA). Separation was achieved on an Aminex 87Hcolumn (300 mm � 7.8 mm, Bio-Rad, Hercules, CA,USA) with 8 mM sulfuric acid as an isocratic mobile phaseat 0.6 ml/min, and concentrations of lactate and acetatewere measured with both a UV–visible diode array detectorat 206 nm referenced to 260 nm and a refractive indexdetector. Acetate and lactate standard solutions were pre-pared by diluting stock solutions of 110 mM and 70 mM,respectively, and the calibration plot was obtained by linear

Fig. 1. Dissimilatory sulfate reduction pathway and sulfur isotope effects. (A) Classical Rees model for the sulfur isotope fractionation duringsulfate respiration (Rees, 1973). The reversibility of sulfate uptake (X1) and the sulfur isotope fractionation imparted by reductive enzymaticreactions (ered) primarily determine the overall fractionation (enet). Note that the sulfur isotope effect assigned to each enzymatic reaction wasinferred but not experimentally tested until recently. (B) Sulfur metabolites and enzymes in the dissimilatory sulfate reduction pathway (Wingand Halevy, 2014). This model was used here to quantify the intracellular ratio of sulfite to APS (see Fig. 7). SulP is sulfate permease. Atps isATP sulfurylase. Apr is APS reductase. Dsr is dissimilatory sulfite reductase. MKred and MKox stand for the reduced and oxidized forms ofmenaquinone, respectively. ETC refers to the electron transfer complex. (C) Recent advances in understanding the dissimilatory sulfitereduction and the associated sulfur isotope effect. Santos et al. (2015) revealed the role of DsrC, a small subunit of dissimilatory sulfitereductase, and Leavitt et al. (2015) conducted the first enzyme-specific sulfur isotope experiments, showing that the 34S/32S fractionationduring the first step of sulfite reduction was 15‰.

M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72 59

regression of peak-area against concentration. Dissolvedsulfate and sulfide in the medium were collected multipletimes during the incubation. A 1 mL aliquot of culture sam-ple was extracted, filtered through a 0.2 lm membrane fil-ter, and mixed with 0.2 ml of zinc acetate (1 M) solution.

Samples were stored at 4 �C until analysis. Sulfate concen-trations in the medium were determined on a Dionex DX500 ion chromatograph (IC) equipped with an AS11-HCcolumn (Dionex, Sunnyvale, CA, USA). The sulfate eluatewas collected for isotopic analysis. Sulfide concentration

60 M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72

was measured using a modified methylene blue assay (Cline,1969), and one lmol of precipitated zinc sulfide was con-verted to sulfuric acid for isotopic analysis on the MC-ICP-MS (see Section 2.4). Zinc sulfide was washed withdeionized water (DW) three times to remove residual sul-fate, and oxidized to sulfate in 15% H2O2 and 0.5 mM Fe(III)/NTA at 75 �C for 24 h. The amount of sulfate pro-duced from a known amount of zinc sulfide was examinedvia ion chromatography, confirming the complete conver-sion within the analytical error (5%). The oxidation of zincsulfide to sulfate by H2O2 was also described in Raven et al.(2016). After oxidation and drying, samples were dissolvedin 5 mM HCl, and any remaining precipitate was removedby centrifugation. The supernatant solution was thenloaded onto AG1-X8 anion exchange resin. Cations wereremoved by rinsing the resin with 3 ml of DW for fourtimes, and sulfate was eluted with 3.6 ml of 0.5 M HNO3

(Paris et al., 2014).

2.3. Intracellular metabolites

To recover intracellular metabolites, a whole culture(0.5 L) was harvested by centrifugation at 13,000g for15 min at 32 �C. Cells were then resuspended and washedthree times with anaerobic cold saline solution (2% NaCl,0 �C). For the early stationary phase samples, a cell suspen-sion was split into two aliquots before washing and pro-cessed in parallel throughout the following steps, whichserved as a measure of procedural reproducibility.Cypionka (1989) described that intracellular sulfate wasnot released by washing with the sulfate-free solution,although excess extracellular sulfate allowed the rapidexchange of sulfate across the membrane. Dissolved sulfurcompounds in the medium were removed by thoroughwashing with sulfate well below the levels of the fresh salinesolution (up to 0.2 lM) in the final wash. Washed cells weredistributed into microcentrifuge tubes, flash frozen in liquidN2, and stored at �80 �C until extraction. The use of cold

Fig. 2. Sequential elution of different sulfur compounds using the AS11-Hfrom a mixed standard solution. The separation was monitored by both

saline solution minimizes cellular damage (Wu and Li,2013) and sulfate reduction (Warthmann and Cypionka,1990) during the washing procedure. Intracellular sulfurmetabolites were extracted using a modified cold methanolprotocol after Maharjan and Ferenci (2003). The frozenpellet was thawed and resuspended in 0.6 mL DW in ananaerobic glove box (Coy Manufacturing Co., Ann Arbor,MI, USA). An equal volume of cold methanol was added tothe cell suspension, resulting in a final methanol concentra-tion of 50% v/v. 20 lL of ZnCl2 solution (50 mM) wasadded to precipitate dissolved sulfide as ZnS. Otherwise,dissolved sulfide can be oxidized to sulfur oxyanions whenexposed to air. It has been reported that ascorbate alsoserves as a sulfide anti-oxidant (Keller-Lehman et al.,2006), but this did not prevent the oxidation of sulfide tothiosulfate in our experimental conditions. After mixing,the sample was left on dry ice for 30 min and then thawedon ice for 10 min. The supernatant was then recovered bycentrifugation. 100 lL of 13 mM formaldehyde was added,which effectively suppresses the oxidation of sulfite duringthe subsequent anion-exchange chromatographic step(Lindgren and Cedergren, 1982; Keller-Lehman et al.,2006). APS is known to hydrolyze rapidly under acidic con-ditions (Kohl et al., 2012) but rather stable around neutralpH. Based on our tests, without an aid of enzymes, APSwas not decomposed in 2 h at 32 �C within the precisionof measurement (5%). Note that after harvesting cells, thetemperature was kept lower than 4 �C, and excess methanoland zinc helped quench the enzymatic activities(Kandlbinder et al., 2000; Nony et al., 2005). Sulfurmetabolites in the cell extract, including sulfate, sulfite,and APS, were quantified and collected by IC using a gra-dient elution with KOH as mobile phase. The optimizedgradient profile and the resulting separation of sulfur spe-cies are detailed in Fig. 2. Two detectors were used, conduc-tivity and UV absorbance. Samples were mixed with anequal volume of DW before loading into a 1.44 mL injec-tion loop, to avoid the high methanol content swelling the

C column with a KOH gradient. This chromatogram was obtainedconductivity (thin black line) and UV absorbance (thick gray line).

M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72 61

resin and building up high pressure in the column. The elu-atant fraction corresponding to each targeted sulfur analytewas collected for isotopic analysis. Collected analytes werequantitatively converted to sulfate by hydrolysis underacidic conditions (APS) or oxidation with H2O2 (sulfite)at 60 �C.

2.4. Isotopic analyses

Samples containing dissolved sulfate were dried on a hotplate and diluted in 5% HNO3 to a sulfate concentration of20 lM to match the in-house Na2SO4 working standard.NaOH was then added to yield equimolar Na and SO4

2�.Isotopic analysis was conducted on a Thermo Fischer Sci-entific Neptune Plus multi-collector inductively coupledplasma mass spectrometer (MC-ICP-MS), operated in med-ium resolution following the method described by Pariset al. (2013). Samples were introduced to plasma via anESI PFA-50 nebulizer and Cetac Aridus II desolvator. Sul-fur isotope ratios of the sample and working standard weremeasured in alternating 50 cycles of 4.194 s integrationtime, and instrumental blank was estimated after each sam-ple block. The mean blank value was subtracted from themeasured signal for each mass. The measured 34S/32S and33S/32S ratios were calibrated using a linear interpolationbetween the two bracketing standard values. Sulfur isotoperatios are reported here using the conventional deltanotation:

d xS ¼ xRsample=xRVCDT � 1 ð1Þ

where xRsample andxRVCDT are the isotopic ratios (33S/32S

or 34S/32S) of sample and Vienna-Canon Diablo Troilite(VCDT), respectively. Our working standard was calibratedagainst the IAEA S-1 reference material (d34SVCDT = -�0.3‰, d33SVCDT = �0.055‰) and has a d34S value of�1.55‰ ± 0.16 (2r) and d33S of �0.77 ± 0.17‰ on theVCDT scale. The d34SVCDT values of three IAEA standardBaSO4 solutions, SO5, SO6, and NBS127, measuredagainst this working standard agree within uncertainty withthe published values (Paris et al., 2013). Analytic repro-ducibility for d34S and d33S has been previously evaluatedas a 2r of 0.2‰ (Paris et al., 2013).

2.5. Blank detection by isotope dilution

The use of MC-ICP-MS lowers the detection limit ofsulfur isotope analyses down to a few nmol S (Paris et al.,2013), an essential capability for measuring trace intracellu-lar metabolites. At these levels, however, background con-tamination during sample work-up (i.e. the blank) can beproblematic. Incorporation of exogenous sulfate is the mostworrisome given its abundance in nature, and can be veryhard to detect based on d34S values alone. Highly variableconcentrations make the conventional approach of prepar-ing and analyzing method blanks perilous. Thus, an inter-nal control was required to assess the blank contributionto measured isotope ratios, especially for low-abundanceintracellular analytes. Given that the ICP-MS can accu-rately measure 32S, 33S, and 34S, we employed a 33S labelin the cultures for this purpose, as described below.

Changes in 34S/32S and 33S/32S ratios between species Aand B will be proportionate, depending on the mass differ-ence upon isotopic substitution. This yields the well-knownmass-dependent fractionation relationship

33RA

33RB¼

34RA

34RB

� �0:515

ð2Þ

Although described by a power law, the mass-dependentfractionation of sulfur isotopes is nearly linear in d for smallfractionations, such that sulfur from all modern terrestrialsources obeys the relationship d33S � 0.515�d34S. This rela-tionship is termed the terrestrial mass-dependent array(Farquhar et al., 2003). Addition of 33SO4

2� label into theculture medium shifts our experiments to a separate butparallel mass-dependent array (Fig. 3A), and provides aclear means to distinguish experimental analytes from lab-oratory blank. For the first and second sets of experiments,1 mL of 35 mM Na2

33SO4 solution was added per 1 Lmedium so that d33S of initial sulfate was adjusted upwardby �200‰ (Fig. 3A). The Na2

33SO4 was prepared from33S-elementals sulfur (99.8%, Trace Sciences InternationalInc., Wilmington, DE, USA), following the procedure out-lined in Dawson et al. (2016). S analytes derived entirelyfrom this supplied sulfate (e.g. APS, sulfite, sulfide) will nec-essarily have d33S and d34S values that stay on this sameexperimental array, regardless of how strongly they arefractionated. Introduction of contaminant sulfur, whichresides on the terrestrial array, will force the analyte d33Sand d34S values off the experimental array (Fig. 3B). In thisway we simultaneously used 34S/32S ratios to track sulfurisotope fluxes in the cell and 33S/32S ratios to assess poten-tial blank contributions. We note that this system does notprovide any visibility into contamination by other experi-mental species, for example extracellular sulfate that mightend up in the intracellular sulfate fraction. Instead, cross-contamination among sulfur metabolites could be limitedby manipulating the experimental conditions as describedin Section 2.3.

2.6. Data processing

Average specific growth rates (h�1) were derived fromthe slope of the natural log-transformed linear regressionof optical density, and growth yields were calculated asthe increase in optical density per sulfate consumed. Thespecific sulfate reduction rate (sSRR) during the same inter-val was calculated from the specific growth rate and growthyield (Sim et al., 2011a).

sSRR ðM �A660�1 � time�1Þ

¼ specific growth rate ðtime�1Þgrowth yield ðA660 �M�1Þ ð3Þ

To assess the sulfur isotope mass balance, theconcentration-weighted average of extracellular sulfateand sulfide isotope compositions, d34S, was evaluated andcompared with the d34S values of initial sulfate. When mul-tiple cultures were set up for the experiment, Na2

33SO4

solution was added together with the inoculum to eachincubation medium. The resulting bottle-to-bottle variation

Fig. 3. Schematic diagrams showing the data screening based on isotope dilution. (A) Due to the 33S-sulfate spike, sulfur isotopiccompositions of sulfur metabolites evolve along the new mass-dependent fractionation line, which is parallel to the terrestrial mass-dependentfractionation line (d33S � 0.515�d34S) but offset by about 200‰ in terms of d33S. (B) A deviation of measured sulfur isotopic ratios from thenew mass-dependent fractionation line indicates the degree of contamination, as illustrated by a series of parallel lines, with 0% meaning thatthere is not external contamination and 100% meaning that all measured sulfur is external contamination. In the absence of 33S tracer,contamination is difficult to detect because d34S values of most sulfur contaminants fall within the range produced by microbial processes.

62 M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72

in the d33S values of total sulfur was up to 6‰, so that 33Swas not used to measure the mass balance closure through-out the experiment. The isotope enrichment factor (34e) wascalculated using an approximate solution to the Rayleighdistillation equations:

lnðd34Sr þ 1Þ ¼ lnðd34So þ 1Þ � e � lnðf Þ ð4Þwhere f is the fraction of the remaining sulfate and d34Soand d34Sr are sulfur isotope compositions of the initial sul-fate and remaining sulfate, respectively (Mariotti et al.,1981). Plotting ln(d34Sr + 1) versus �ln(f) yielded a straightline, indicative of a constant fractionation throughout thecourse of exponential growth. The slope of a linear regres-sion through the data was taken as a measure of the averageenrichment factor for the batch culture experiment. As wehave defined it, positive values of e represent the depletionof heavy isotopes in the product (sulfide). All analyticalerrors were propagated via either Monte Carlo simulation(n = 5,000) or first-order Taylor series expansion(Bevington and Robinson, 2002).

3. RESULTS

Inoculated with 21 mM sulfate and 22 mM lactate, cul-tures of D. alaskensis reduced about a half of sulfate intosulfide and oxidized all lactate to acetate within 60 h,reflecting that the reduction of one sulfate to sulfiderequires the oxidation of two lactate molecules (Fig. 4;Table 1). After lactate depletion caused growth phase toend, concentrations of metabolic reactants and productsremained largely unchanged. The average growth rate andyield during the growth phase were 0.10 h�1 and 22.6A660/M SO4

2�, respectively, which implies a specific sulfatereduction rate of 4.4 mM SO4

2�/A660/h (Table 2). Forexperimental efficiency and precision, the optical densitywas used as a measure of cell abundance, while the rateof sulfate reduction was often normalized to the number

of cells in previous literature (Table 2). The average 34Senrichment factor (34e) was 3.8‰ (Table 2), and the d34Smass balance was always retained within the precision ofmeasurements (Fig. 4D). The minimal fractionation by D.

alaskensis is consistent with Leavitt et al. (2014) thatreported 34e values of 4.3‰ and 5.9‰ coupled with the oxi-dation of formate and lactate, respectively. A slight reduc-tion in growth rate occurred as lactate concentrationdropped below 5 mM (Fig. 4; Table 2). Gradual decreasein lactate concentration appeared to influence other growthkinetic parameters and sulfur isotope fractionation, butthese variations did not exceed analytical errors until cellsentered into stationary phase (Table 2), although a negativeshift in the d34Ssulfide value after the cessation of growth wasnotable (Series 1 in Table 1).

Intracellular metabolite samples were collected at differ-ent growth stages, from exponential growth through thedeath phase (Table 1). Intracellular sulfate content was lessthan 1 nmol/mg (dry weight) at the mid-log phase, but cellsrapidly accumulated above 30 nmol/mg sulfate as thegrowth rate declined. This accumulated sulfate was thenslowly depleted throughout the later stationary phase(Fig. 5). Intracellular APS contents ranged from belowdetection to 0.14 nmol/mg (Fig. 5). Although APS contentpeaked during the transitional phase, its ratio to intracellu-lar sulfate decreased over time. Intracellular sulfite contentswere more than an order of magnitude lower than those ofAPS throughout exponential and stationary phases (Fig. 5).However, when cells entered the death phase, APS contentdecreased rapidly, while sulfite showed little change. Thio-sulfate was detectable but could not be reliably measureddue to the interference with highly abundant substancessuch as phosphate.

Sulfur isotope ratios of intracellular metabolites weremeasured from the 33S-spiked experiments. Intracellularsulfate was always enriched in 34S relative to sulfate inthe medium (Fig. 6A). Although obtained in a single bio-

Fig. 4. Growth of D. alaskensis with lactate as a sole electron donor and the resulting sulfur isotope effect. Vertical broken lines indicate thetiming of lactate depletion. Blue, green, and red symbols represent experiment series 1, 2, and 3, respectively. (A) Optical density at 660 nm.(B) Lactate consumption and acetate evolution. (C) Sulfate consumption and sulfide evolution. (D) Sulfur isotopic composition of sulfate andsulfide, and their weighted average. The uncertainty of optical density (A660) is ±0.005, the concentrations determined by chromatography(lactate, acetate, and sulfate) and colorimetry (sulfide) are subject to an error of ±5%, and the analytical error in the isotope analysis is ±0.2‰for d34S. Propagated errors for the weighted average of d34Ssulfate and d34Ssulfide are smaller than the symbols. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.).

M.S. Sim et al. /Geochimica et Cosmochimica Acta 206 (2017) 57–72 63

logical replicate, this enrichment was as large as 49‰ at thelate exponential growth phase. When cells entered the sta-tionary phase, it gradually decreased down to 5‰. Triplesulfur isotope compositions of intracellular sulfate werealigned well along the 33S-enriched mass-dependent arraywith the mean deviation from the array being 4‰ (Fig. 6B).The d34S value of APS decreased from 22‰ to �2‰ overthe course of experiment. The overall pattern of d34SAPS

followed that of intracellular sulfate, but APS was alwaysestimated to be depleted in 34S relative to co-existing sul-fate. Also, the d33S values of APS deviated from the exper-imental mass-dependent array by up to 35‰ (Fig. 6B).Despite the limited data available, the d34S values of intra-cellular sulfate and APS showed an apparent linear rela-tionship. No sulfur isotope measurements could be madeon intracellular sulfite and thiosulfate due to their verylow cellular abundance.

4. DISCUSSION

4.1. Assessment of concentrations and isotope compositions

of intracellular sulfur metabolites

This is the first experimental measurement of intracellu-lar sulfur metabolite levels and their isotopic compositions,using actively respiring bacterial cells. Such novelty requiresus to carefully evaluate the quality and/or limitations ofthis method with several independent approaches. Procedu-ral and biological reproducibility is examined, and33S-sulfate is used as an internal control to monitor theincorporation of contaminant external sulfur. Our resultsare compared with previous experimental and theoreticaldata.

To assess procedural reproducibility, we split eachstationary-phase culture into two separate aliquots and

Table 1Growth, concentration and isotopic data from three batch culture experiments. Measured sulfur isotope compositions are presented as relative ratios to that of VCDT standard (d34S) or that ofinitial sulfate (d34Sx-SO4o). The uncertainty of optical density (A660) is ±0.005, the concentrations determined by chromatography and colorimetry are subject to an error of ±5%, and the 2ranalytical error in the isotope analysis is ±0.2‰ for d33S and d34S.

Series Time(hr)

Timerelative toseries 1 (h)

OD(A660)

Concentration Sulfur isotope composition

Extracellular (mM) Intracellular (nmol/mgDW)

Extracellular Intracellular

Sulfate Sulfide Lactate Acetate Sulfate APS Sulfite Sulfate Sulfide Sulfate APS

d34S d34Sx-SO4o

d33S d34S d34Sx-SO4o

d33S d34S d34Sx-SO4o

d33S d34S d34Sx-SO4o

d33S

1 0 0 0.0124 21.1 0.0 22.0 0.0 �1.7 0.0 203.516.5 16.5 0.0061 21.1 0.0 22.0 0.029.5 29.5 0.0129 21.6 0.3 20.9 0.641 41 0.0694 19.3 2.3 16.8 4.250 50 0.1669 14.8 7.1 8.1 12.552 52 0.2097 13.0 8.5 4.8 17.2 8.7 0.09 0.00 �0.3 1.5 202.8 �4.4 �2.6 200.5 48.7 50.5 220.2 20.1 21.9 184.954 54 0.2312 11.8 9.0 1.9 18.155 55 0.2605 10.1 10.2 0.0 20.0 30.1 0.14 0.00 0.8 2.5 205.3 �4.3 �2.6 201.9 17.3 19.1 207.9 2.9 4.6 168.956.5 56.5 0.2605 10.5 9.9 0.0 20.363.5 63.5 0.2543 10.3 9.5 0.0 20.1 23.6 0.07 0.01 0.7 2.5 198.6 �4.8 �3.1 194.8 5.4 7.1 196.9 �3.4 �1.6 170.2

21.9 0.09 0.01 5.3 7.0 197.3

2 0 �9 0.0067 21.1 0.7 0.0 204.021 12 0.0074 21.2 0.7 0.0 204.344.5 35.5 0.0286 19.4 0.9 0.2 204.9 �2.0 �2.7 202.368.5 59.5 0.2615 10.6 19.7 0.07 0.01 3.3 2.6 205.2 �2.0 �2.7 203.2 7.7 7.0 206.0 1.7 1.1 181.1

20.1 0.07 0.01 7.8 7.1 206.0 2.4 1.8 175.6

3 0 3 0.0116 21.1 0.0 0.9 0.0 0.513.5 16.5 0.009226.5 29.5 0.01638.25 41.25 0.069845 48 0.160946 49 0.1554 16.1 4.3 0.8 0.02 0.00 2.0 1.1 1.1 �2.3 �3.2 �1.252.5 55.5 0.259870 75 0.2601 11.3 9.1 4.8 0.01 0.02 3.7 2.8 2.0 �2.1 �3.0 �1.079.5 84.5 0.250296 99 0.227 11.4 8.9 1.1 0.00 0.02 3.6 2.7 1.8 �2.3 �3.2 �1.0

64M.S.Sim

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Table 2Variations in growth kinetics and sulfur isotope fractionation from early to late exponential growth phases. Specific sulfate reduction rate herecould be compared with the cell-specific sulfate reduction rate in previous literatures. The size of D. alaskensis was reported to vary from 1 to5 lm in length and 0.5 to 1.2 lm in width (Feio et al., 2004), and assuming the average size of 3 by 0.85 lmwith spheroidal ends, the volume ofa single cell was estimated at 1.5 lm3. Then, the average cell-specific sulfate reduction rate was calculated to be about 290 fmol/cell/dayaccording to the cellular volume to dry weight ratio of 1.4 lL/mg (Varma et al., 1983) and the conversion factor for optical density to dryweight (see Section 2.1).

Average for the entire data set Series 1 (30–52 h) Series 1 (52–55 h)

Growth rate (h�1) 0.10 ± 0.00 0.11 ± 0.00 0.07 ± 0.02Growth yield (A660/mM sulfate) 22.6 ± 1.7 22.9 ± 2.7 17.3 ± 9.1Specific sulfate reduction rate (mM sulfate/A660/h) 4.4 ± 0.3 4.5 ± 0.5 4.0 ± 2.4Sulfur isotope fractionation (‰) 3.8 ± 0.4 2.9 ± 0.7 4.0 ± 1.5

Fig. 5. Dynamics of sulfur metabolites in the dissimilatory sulfatereduction pathway. A gray broken line presents the timing oflactate depletion. Both vertical axes are interchangeable forintracellular metabolites, but external sulfate and sulfide concen-trations should be read from the right vertical axis. Blue, green, andred symbols represent experiment series 1, 2, and 3, respectively.Procedural reproducibility is shown as vertical error bars whereavailable. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of thisarticle.)

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processed them in parallel. Biological reproducibility wasalso examined by comparing analyses of early stationaryphase cells collected from two independent cultures. Dupli-cate sample processing resulted in up to 15% variation inintracellular sulfur metabolite content, and similar repro-ducibility was observed when two independent cultureswere compared at early stationary phase (Table 1). Suchvariability is greater than expected due to analytical errorsin ion chromatography (5%), but still small relative to thescale of variations throughout the growth cycle. No intra-cellular sulfate shows a dilution of 33S spike greater than4%, and thus, assuming the isotope composition of contam-inant sulfur is near 0‰, the measured d34S value of intracel-

lular sulfate would deviate from its original isotopiccomposition by no more than 2‰ (Fig. 6). In contrast, sul-fur isotope compositions of APS deviate more substantiallyfrom the experimental mass-dependent array, indicating theincorporation of external sulfur contamination was as largeas 20%. Since reagents and blank samples contain nodetectable amount of APS, incorporation of exogenous sul-fate after the IC separation step seems more likely. It isnoteworthy that intracellular APS contents are about twoorders of magnitude lower than those of sulfate, comprisingjust a few nanomoles of APS per analysis. Consequently,these samples are much more susceptible to contamination.

Little is known about the intracellular concentrations ofsulfur metabolites in the actively respiring sulfate-reducingmicrobes, but the fact that sulfate-reducing bacteria canaccumulate sulfate inside the cell has been well known sincethe work of Furusaka (1961). Cypionka and his colleagues(Cypionka, 1989; Warthmann and Cypionka, 1990;Stahlmann et al., 1991; Krebe and Cypionka, 1992) exten-sively investigated the intracellular accumulation of sulfateby a wide range of sulfate-reducing microbes, showing thatsulfate concentrations were almost always higher inside theresting cell than outside. The accumulation factor (inside/outside ratio) decreased as the ambient sulfate concentra-tion increased. Here we calculate the concentration of intra-cellular sulfate using a conventional cellular volume to dryweight ratio of 1.4 lL/mg (Varma et al., 1983), used in theearlier sulfate accumulation studies (Cypionka, 1989;Warthmann and Cypionka, 1990; Stahlmann et al., 1991),and compare intracellular and extracellular concentrationsof sulfate (Fig. 5). In contrast to previous work, our datashow intracellular sulfate concentrations to be much lowerthan extracellular during exponential growth. Since theaccumulation experiments prevented active sulfate reduc-tion via low temperature or presence of O2 (Cypionka,1989; Warthmann and Cypionka, 1990; Stahlmann et al.,1991), relatively low intracellular sulfate levels during theexponential phase are presumably due to the rapid con-sumption of sulfate by vigorous sulfate reduction. Duringthe transition to stationary phase, however, intracellularsulfate concentrations exceed the external levels, resultingin accumulation factors similar to those reported by previ-ous experiments. Thus, drastic changes in the intracellularsulfate levels at different growth stages not only confirmthe previous findings, but describe the effect of active respi-ration on the intracellular sulfate accumulation.

Fig. 6. Sulfur isotopic compositions of intracellular sulfate andAPS. Blue and green symbols represent experiment series 1 and 2,respectively. (A) A strong enrichment of 34S in both intracellularsulfate and APS is relieved as cells enter the stationary phase. Avertical broken line indicates the timing of lactate depletion.Procedural reproducibility for d34S measurement is shown asvertical error bars where available. (B) Triple sulfur isotope ratiosof intracellular sulfate and APS. Upper and lower solid linesrepresent 33S-spiked and terrestrial mass-dependent fractionationlines, respectively. A dotted line exemplifies the mixing trendbetween the intracellular sulfate and the presumed contaminantwith the d34S value of 0‰. Note that sulfur isotopic compositionsof intracellular sulfate plot close to the 33S-spiked line, but those ofAPS deviate from the line by up to 35‰. As in panel (A),procedural reproducibility for d33S measurement is shown asvertical error bars where available. (For interpretation of thereferences to colour in this figure legend, the reader is referred tothe web version of this article.)

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Intracellular APS and sulfite concentrations have notbeen previously measured, but recent modeling work haspredicted sub-micromolar levels of APS and millimolarlevels of sulfite in the cell (Wing and Halevy, 2014). Such

high ratios of sulfite to APS, however, were not observedin this study. Instead, intracellular APS contents werehigher than those of sulfite except for late stationary phasecells. Because Wing and Halevy’s model assumes a steadystate condition, it might be misleading to compare theirmodel prediction directly with our results, where the con-centrations of intracellular sulfur metabolites changerapidly, especially during the late exponential growth phase(Fig. 5). However, it is also unlikely that a non-steady stateprocess is solely responsible for the several orders of magni-tude discrepancy between model and observations persist-ing from exponential growth to early stationary phases.In Wing and Halevy’s model, the reduction of APS and sul-fite is assumed to be coupled to oxidation of menaquinone.Since its midpoint potential (Eo

0 = �74 mV) might allowAPS reduction (Eo

0 = �60 mV) but not sulfite reduction(Eo

0 = �116 mV) at standard state, the ratio of reduced tooxidized menaquinone is assumed to be 100:1, generatinga more favorable redox potential (E0 = �129 mV). Underthese conditions, the reduction of APS is still favored rela-tive to sulfite, thereby resulting in a high sulfite/APS ratioof up to 1,000:1. Here we ran the same model, varyingredox potentials for the electron carriers coupled to APSand sulfite reduction individually (Fig. 7). These results pre-dict that the ratio of sulfite to APS is sensitive to the redoxpotential of the associated electron carriers. For example,with an electron donor for sulfite reductase having a redoxpotential of �200 mV, the resulting sulfite/APS ratio couldbe less than 0.1. When grown with lactate as an electrondonor, incompletely-oxidizing sulfate reducers like D.

alaskensis first oxidize lactate to pyruvate and then pyru-vate to acetyl-CoA. These two redox pairs have Eo

0 valuesof �190 mV and �480 mV, respectively, and sulfate-reducing microbes have other electron carriers withmidpoint potentials much lower than that of menaqui-nones, such as NADH (Eo

0 = �320 mV) or ferredoxin(Eo

0 = �398 mV). Thus, an electron donor with a redoxpotential of �200 mV or lower is entirely plausible in thepresence of a non-limiting supply of lactate (Fig. 7). In con-trast, the ratio of sulfite to APS increased considerably dur-ing late stationary phase, exceeding unity. Such an increaseis consistent with model predictions, because lactate deple-tion should decrease the ratio of reduced to oxidized elec-tron carriers. In summary, a variable intracellular redoxlevel can readily resolve the discrepancy between our mea-surements and model predictions, as long as the model con-straint of a menaquinone electron donor is relaxed.

4.2. Strong 34S enrichment of intracellular sulfate

An incomplete-oxidizing sulfate reducer, D. alaskensis isknown to fractionate sulfur isotopes by only a few permil(Leavitt et al., 2014), and the measured sulfur isotope frac-tionation in our study is also 3.8‰ (Table 2). When sulfatereduction occurs in the presence of a non-limiting supply oflactate, the resulting sulfur isotope fractionation tends to besmall, but rarely as low as 4‰ (Detmers et al., 2001; Simet al., 2011b). Since the dissimilatory sulfate reduction path-way, including sulfate transporters, ATP sulfurylase, APSreductase, and sulfite reductase, is highly conserved

Fig. 7. Influence of the electron donating reactions for APS and sulfite reduction on the relative abundance of APS and sulfite. Contoursrepresent the ratio of sulfite to APS. Intracellular APS and sulfite levels were calculated based on the model proposed by Wing and Halevy(2014), but the reducing potentials of both electron donating reactions were varied from �50 to �550 mV. Note that the original modelassumed that the reduction of APS and sulfite is coupled with the oxidation of menaquinone, and the ratio of reduced to oxidized forms is 100.All calculations were made using a specific sulfate reduction rate of 25 fmol/cell/day and an ambient sulfide concentration of 1 mM. An emptydiamond indicates the ratio of sulfite to APS predicted by the original model. In our experiment, the ratio of APS to sulfite increases from lessthan 0.1 to unity throughout the growth transition from exponential to stationary phases.

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(Pereira et al., 2011), D. alaskensis is an ideal model organ-ism for investigating the lower end of the dynamic range offractionation, in particular, whether or not the sulfur iso-tope fractionation imposed by intracellular enzymatic reac-tions diminishes as net fractionation decreases (Fig. 1).

Two series of batch culture experiments convincinglydemonstrate that sulfate inside the cell is enriched in 34S rel-ative to that outside of the cell. This pattern is consistentwith predictions from models that assume (1) exchange ofsulfate across the membrane is limited, and (2) fractiona-tion occurs primarily during intracellular enzymatic reac-tions (Rees, 1973; Brunner and Bernasconi, 2005;Johnston et al., 2007; Bradley et al., 2011; Sim et al.,2011a). In this situation, the preference of sulfate reductionfor light isotopes leaves the residual reactant (intracellularsulfate) enriched in 34S. Interestingly, the fractionationbetween sulfate inside and outside of the cell is up to49‰, demonstrating that the actual fractionation by intra-cellular enzymes in D. alaskensis remains large despite theminimal net fractionation (Fig. 6). Note that only two pre-vious pure culture studies have reported net fractionationsgreater than 50‰ (Sim et al., 2011b; Leavitt et al., 2013).Our results suggest that such large intracellular fractiona-tions may be intrinsic to the dissimilatory sulfate reductionpathway of most organisms, but in many cases that frac-tionation is not fully expressed due to closed-systembehavior.

Considering the cell as a partly closed-system, the mag-nitude of net 34S fractionation between sulfate and sulfide(34e) is strongly controlled by the extent to which intracellu-lar sulfate becomes 34S-enriched. That enrichment in turn

reflects a competition between sulfate exchange across themembrane, which will pull intracellular sulfate d34S downtowards that of extracellular sulfate, versus enzymatic sul-fate reduction, which will push intracellular sulfate d34Sup as a result of normal kinetic isotope effects (Fig. 8).Two bounding end-member scenarios can be considered.First, if the rate of sulfate reduction is low relative to thatof sulfate exchange across the membrane (‘open system’,Fig. 8C) via the reversible activity of sulfate permeases(Cypionka, 1989), intracellular sulfate will maintain ad34S value close to that of ambient sulfate, and the pro-duced sulfide will be strongly depleted in 34S (large 34enet).Conversely, if sulfate reduction is very rapid relative toexchange across the membrane (‘closed system’, Fig. 8A),then intracellular sulfate will be strongly 34S enriched andproduced sulfide will be only slightly depleted (small 34enet).A cell could operate anywhere between these two extremes(Fig. 8B), providing a means to explain nearly the entirerange of fractionations expressed in nature by sulfate-reducing microbes. The model is very much analogous tothose predicting C isotope fractionation in plant leaves asa function inside/outside PCO2 ratios (Farquhar et al.,1989).

Over the course of our batch experiments, sulfate levelsare not limiting growth, but the strong enrichment of34S-sulfate in the exponentially growing cells indicates thatmost intracellular sulfate is being reduced before it movesback outside the cell. Such distillation of the intracellularsulfate is similar to the mechanism suggested to accountfor the small sulfur isotope effect during sulfate-limitedgrowth (Habicht et al., 2005). Here we further test this

Fig. 8. A generalized scheme showing how 34S-enrichment in intracellular sulfate reflects a competition between sulfate exchange across themembrane and enzymatic reduction. Black arrows indicate SO4

2� uptake and leak, while white ones show the enzymatic reduction andsubsequent release of H2S out of the cell. Size of these arrows represents relative rate of sulfur flux, and the vertical position indicates sulfurisotope ratios (d34S). Note that the sulfur isotope mass balance is always maintained at a cellular level, and only reductive reactions areisotope-sensitive; Vertical double head arrows indicate either net fractionation (light grey, enet), the fractionation between external sulfate andsulfide, or enzymatic fractionation (dark grey, ered) the fractionation between internal sulfate and sulfide. The rate of sulfate uptake is assumedto be constant, but that of sulfate reduction decreases from left to right. In the ‘‘closed system” end-member (A), all sulfate moving into thecell is almost quantitatively reduced to sulfide, resulting in negligible isotope fractionation between external sulfate and sulfide (enet). Instead, asmall pool of residual intracellular sulfate becomes strongly enriched in 34S. In the ‘‘open system” end-member (C), since the rate of sulfateexchange across the membrane is much faster than that of reductive reactions, intracellular sulfate maintains a d34S value close to that ofextracellular sulfate. Here, sulfur isotope fractionation between external sulfate and sulfide (enet) reflects the fractionation imparted byintracellular enzymes (ered). (B) is intermediate between these two end-members.

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interesting coincidence with a simple model for thereversibility of sulfate transport. Reversibility of a general-ized enzymatic reaction is given as

reversibility ðX Þ ¼ bf¼ eDG=RT ð5Þ

where b and f denote backward and forward fluxes, R thegas constant, T the temperature, and DG the free energychange associated with the reaction (Van der Meer et al.,1980; Stoner, 1992; Beard and Qian, 2007). Values of X willvary between zero (unidirectional) and one (fully reversi-ble). The more negative the free energy change is, the lessreversible is the reaction. Since sulfate is transported acrossthe membrane simultaneously with protons (or sodiumions), DG for sulfate uptake is not a simple function of sul-fate concentration gradient across the membrane. Rather, itis also related to the transmembrane electrical potential andpH gradient (Wing and Halevy, 2014).

DG ¼ n � ðF � DWþ 2:3RT � DpHout�inÞ � 2F � DW

þ 2:3RT � log ½SO4;in�½SO4;out� ð6Þ

where n is the number of symported protons, DW is the elec-trical potential, and DpH is the pH gradient across themembrane. Under sulfate replete conditions, the low-accumulating symport with two protons is expressed(n = 2), while sulfate limitation increases the stoichiometryup to three protons per sulfate (n = 3) (Krebe andCypionka, 1992). Since DW and DpH possess negative val-ues (Cypionka, 1989), an increasing number of symported

protons (n) leads to less reversible sulfate uptake, reducingthe isotopic fractionation. It is unlikely that D. alaskensis

cells grown with 10–20 mM sulfate transport three protonsper sulfate, but the reduced accumulation of sulfate ([SO4,

in]/[SO4,out]) in the exponentially-growing cells could alsomake sulfate uptake more energetically favored, andthereby less reversible. Assuming typical values of�150 mV for DW, �0.5 for DpH (Cypionka,1989), andsymport with 2 protons, the estimated ratio of intracellularto extracellular sulfate concentrations at the late exponen-tial growth phase (�0.5, see the data at 52 h in Fig. 5)would result in a reversibility of X = 0.05. That is, duringexponential growth phase, �95% of transported sulfate isreduced to sulfide, rather than leaking back out (Fig. 1).Consequently, both sulfate supply and downstreamdemand have the same effect on the sulfur isotope fraction-ation by limiting the openness of intracellular sulfate pool,which tracks the reversibility of sulfate uptake (Fig. 1). Thisexplains why most batch culture experiments with excessorganic substrates have failed to reproduce the large sulfurisotope fractionation recorded in nature.

4.3. Metabolic response to lactate depletion

In the modern marine realm, sulfur isotopic offsetsbetween coeval sulfate and sulfide range up to about 70‰(Sim et al., 2011b). Such large sulfur isotope fractionationsby the natural population of sulfate reducing microbes havebeen related to slow in situ respiration rates resulting fromsubstrate limitation (Leavitt et al., 2013). The expression of

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sulfur isotope fractionation in a nutrient-replete batch cul-ture may not be the best analogue for those slow-respiringend members, but still, the physiological and sulfur isotopicresponse of D. alaskensis to starvation can provide impor-tant clues on the mechanism behind a wide range of frac-tionation in nature. A few previous studies haveinvestigated the effect of growth stages on isotopic fraction-ation at a culture scale (Davidson et al., 2009; Matsu’uraet al., 2016), but here we demonstrate how microbial sulfurisotope fractionation changes in response to energy limita-tion at a sub-cellular scale.

As discussed above, the low concentrations and strong34S enrichment of intracellular sulfate demonstrate that sul-fate reduction is as fast as sulfate uptake during the expo-nential growth, but once cells enter stationary phase, therate of sulfate reduction plummets to below detection limitwith decreasing 34S enrichment of intracellular sulfate. Dur-ing the early stationary phase, the intracellular quantity ofsulfate remained high, making the sulfate transport reversi-ble (Eq. (6)). All these results suggest that the rate of sulfateexchange across the membrane substantially exceeds thedemand of maintenance respiration. That is, the sulfur iso-tope fractionation associated with the enzymatic reactionsdownstream of intracellular sulfate should be fullyexpressed at the cell level (Fig. 8). Note that sulfur isotopefractionation between intracellular sulfate and sulfide wasas large as �50‰ even at the exponential growth phase.Unfortunately, however, since a negligible amount of sul-fide was produced during the stationary phase comparedto the exponential growth phase, it is challenging to showthe corresponding increase in sulfur isotope fractionationhere. In the first batch experiment, sulfide became isotopi-cally lighter by 0.5‰ after the cessation of growth (Table 1),which might underpin the increased fractionation by main-tenance respiration. It is also worth noting that Matsu’uraet al. (2016) convincingly showed increasing sulfur isotopefractionation upon the cessation of exponential growthusing batch cultures of Desulfovibrio desulfuricans. We fur-ther consider two different but not mutually exclusive mod-els describing the fractionation of sulfur isotopes in thestationary phase cells. First, according to the Rees modeland its later versions (Rees, 1973; Brunner andBernasconi, 2005; Wing and Halevy, 2014), sulfur isotopefractionation between intracellular sulfate and sulfidewould increase up to the equilibrium value (Sim et al.,2011b) with the reversibility reaching near unity at the ther-modynamic limit of microbial growth. Alternatively, theimportant enzymes in dissimilatory sulfate reduction path-way may act as a coherent respiratory complex, including arecently-characterized dissimilatory sulfite reduction system(Santos et al., 2015), where substrates are channeledthrough the multi-enzyme complex. Compared with reac-tions involving free intermediates, a substrate-channeledreaction would maintain a relatively constant isotope effectwith the reversibility being more tightly controlled. Measur-ing the isotopic composition of downstream intermediates(e.g. sulfite) may resolve which model is more appropriate,but in either case, slow maintenance respiration must leadto larger sulfur isotope fractionation corresponding to thedecrease in 34S enrichment of intracellular sulfate. Although

it deserves further investigation, preferably in continuousculture, this reservoir effect for sulfate – governed by thebalance between transmembrane sulfate exchange andenzymatic reduction – is likely responsible for the well-established inverse trend between the magnitude of sulfurisotope fractionation and the specific rate of sulfate reduc-tion (Chambers et al., 1975; Sim et al., 2011a; Leavitt et al.,2013; Ono et al., 2014).

Like intracellular sulfate, d34SAPS decreased as lactatewas depleted, but always, APS was depleted in 34S relativeto intracellular sulfate (Fig. 6). The sulfur isotope fraction-ation between APS and intracellular sulfate is unexpected,although it decreases to a few permil as growth ceases.Given the dilution of 33S-spike in APS, exogenous contam-ination can account for at most a few permil offset in d34S.APS in the late vegetative phase cells thus appears to bedepleted in 34S by �20‰ relative to intracellular sulfate(Fig. 6). Previously, a negligible equilibrium isotope effecthas been presumed between sulfate and APS because oftheir identical oxidation states (Rees, 1973; Brunner andBernasconi, 2005). Also, since no sulfur-oxygen bonds arebroken during APS formation, a large primary kinetic iso-tope effect is unlikely (Kohl et al., 2012; Parey et al., 2013).At this point, we cannot specify the mechanism of sulfurisotope fractionation between APS and intracellular sulfate,but possible explanations may include secondary isotopeeffects due to loosening of the S–O bonds in the transitionstate, or fractionation associated with the metabolicbranch-point between dissimilatory and assimilatory APSreduction. Alternatively, a non-steady state process couldpotentially account for the observed large isotopic offsetat the late exponential growth phase. For example, anincreasing backward reaction from sulfite to APS couldcontribute to the rapidly decreasing trend in d34SAPS

(Fig. 1). This explanation is however somewhat difficultto reconcile with the fact that APS was 34S-depleted relativeto sulfate at all measured time points.

5. CONCLUSIONS

We present an optimized analytical approach to measur-ing concentrations of intracellular sulfur metabolites andtheir isotopic compositions, providing a direct means ofprobing the cellular processes that shape sulfur isotope frac-tionation during dissimilatory sulfate reduction. Duringbatch incubations of D. alaskensis, intracellular sulfateand APS contents gradually increase and peak at the cessa-tion of exponential growth phase. Vigorous sulfate reduc-tion appears to enrich the internal pool of sulfate in 34S,but this enrichment diminishes once the cells enter station-ary phase. The large 34S enrichment of intracellular sulfate,up to 49‰ relative to extracellular sulfate, is counterbal-anced by an apparently large enzymatic fractionation suchthat the net sulfate/sulfide fractionation is small, only a fewpermil. We infer that the small net fractionation expressedby this (and probably other) sulfate reducers is a reflectionof a strong reservoir effect, rather than reduction of enzy-matic isotope effects. As the maintenance respiration abol-ishes this reservoir effect, the apparent discrepancy between

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the sulfur isotope fractionation in environmental and labo-ratory culture studies may reflect the in situ metabolic statesof microbes in nature.

Since Thode et al. (1951) first demonstrated the bio-genecity of sulfur isotope fractionation between sulfate andsulfide, many studies have linked sulfur isotope fractionationto various environmental factors (Harrison and Thode,1958; Chambers et al., 1975; Habicht et al., 2005; Canfieldet al., 2006; Hoek et al., 2006; Mitchell et al., 2009; Simet al., 2011a, 2012; Leavitt et al., 2013) and also to specificgroups of sulfur metabolizing microbes (Canfield andThamdrup, 1994; Zerkle et al., 2009), but cellular processeshave remained less explored. Here we describe tools to mon-itor the concentrations and isotopic compositions of intra-cellular sulfur metabolites. After the pioneering study ofdissimilatory sulfite reductase (Leavitt et al., 2015), kineticisotope effects imparted by other key enzymes are currentlyalso under investigation. Recently, Santos et al. (2015)revealed the role of DsrC, a small subunit of dissimilatorysulfite reductase, and proposed a new biochemical mecha-nism behind sulfite respiration. All these advances in molec-ular biology, biochemistry, and isotope biogeochemistryhave enabled investigations of microbial sulfur isotope frac-tionation at a sub-cellular level. In the near future, therefore,we expect to have a firm empirical basis for the quantitativemodel that links the magnitude of microbial isotopefractionation to their environmental and physiologicalcontrols.

ACKNOWLEDGEMENTS

This work was supported by an Agouron Geobiology Fellow-ship to MSS and the Gordon and Betty Moore Foundation GrantGBMF 3306 to VJO and ALS. The authors are indebted to Stepha-nie Connon, Silvan Scheller, and Derek Smith for their help anddiscussion. We also thank Boswell Wing, William Leavitt, and ananonymous reviewer for constructive comments on an earlier ver-sion of this manuscript.

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Associate editor: Ruth Blake